COMPOSITIONS AND METHODS FOR IDENTIFYING MAMMALIAN MALONYL COA DECARBOXYLASE INHIBITORS, AGONISTS AND ANTAGONISTS

Abstract: The present invention relates to the area of fatty acid oxidation, generally to compositions and methods of identifying and testing fatty acid oxidation inhibitors, and in particular, compositions comprising a novel cardiac isoform of MCD, identified to be a key regulator of fatty acid oxidation in the heart. Additionally, nucleotide acid sequences of malonyl CoA decarboxylase genes from the rat pancreas, heart and liver are described. Such sequences and derived products are useful in screening methods for identifying and testing agonists and antagonists of the MCD pathway.

Description: COMPOSITIONS AND METHODS FOR DDENTTFYING MAMMALIAN MALONYL COA DECARBOXYLASE INHD3ITORS, AGONISTS AND ANTAGONISTS FJTELD OF THE INVENTION The present invention relates to the area of fatty acid oxidation, generally to compositions and methods of identifying and testing fatty acid oxidation inhibitors, and in particular, compositions comprising a novel cardiac isoform of Malonyl CoA decarboxylase (MCD), identified to be a key regulator of fatty acid oxidation in the heart. Additionally, this invention relates to compositions comprising the mammalian cDNA sequence of Malonyl CoA decarboxylase from rat pancreatic islet, heart and liver cells as well as the use of these sequences and derivatives for use in identifying and testing fatty acid oxidation agonists and antagonists. BACKGROUND OF INVENTION Most of the energy production in the heart, liver and other organs is known to be derived from the oxidation of fatty acids [Bing et al. Am J Med 15:284-296, (1953)]. The other important sources of energy are the oxidation of carbohydrates, and to a lesser extent ATP production from glycolysis. The contribution of these pathways to overall ATP production can vary dramatically, depending to a large extent on the carbon substrate profile delivered to the heart or other organ, as well as the presence or absence of underlying pathology within the target organ. Despite extensive research devoted to the study of the individual pathways of energy substrate metabolism, relatively few studies have been conducted examining the integrated regulation between carbohydrate and fatty acid oxidation in, for example, the heart. While the mechanisms by which fatty acids inhibit carbohydrate oxidation (i.e., the Randle cycle) have been characterized, much less is known about how carbohydrates regulate fatty acid oxidation in the heart. It is clear that an increase in intra-mitochondrial acetyl CoA derived from carbohydrate oxidation (via the pyruvate dehydrogenase complex) can down regulate β-oxidation of fatty acids, but it is not clear how fatty acid acyl group entry into the mitochondria is down-regulated when carbohydrate oxidation increases. An important protein known to be involved in the regulation of fatty acid oxidation in the heart, liver and pancreas is carnitine palmitoyltransferase 1 (CPTl). This protein is located within the outer mitochdndrial membrane and is a key regulatory enzyme involved in the first committed step of fatty acid oxidation [McGarry and Foster, Ann. Rev. Biochem. 49:395-420 (1980); McGarry et al., Diabetes 5:271-284 (1989)]. Malonyl-CoA, which is produced by acetyl-CoA carboxylase (ACC), is a potent inhibitor of CPTl [McGarry and Foster, Ann. Rev. Biochem. 49:395-420 (1980); McGarry et al, J. Biol. Chem. 253:4128-4136 (1978)]. Unlike liver, where a 88 kDa isoform of CPTl predominates, the heart predominantly expresses a 82 kDa isoform of CPTl [Esser et al, J. Biol. Chem. 268:5810-5816 (1993)] which is much more sensitive (10 to 50 times) to inhibition by malonyl CoA [McGarry and Foster, Ann. Rev. Biochem. 49:395-420 (1980); McGarry etal, J. Biol. Chem. 253:4128-4136 (1978); McGarry et al, Biochem. J. 214:21-28 (1983)]. Also, it has been observed that under conditions in which fatty acid oxidation can vary widely the IC50 of mitochondrial CPTl for malonyl CoA does not change [Kudo et al, J. Biol. Chem. 270:17513-17520 (1995); Lopaschuk et al, J. Biol. Chem. 269:25871-25878 (1994)]. For instance, in the post-ischemic rat heart, although fatty acid oxidation rates are very high, the sensitivity of CPTl to malonyl CoA inhibition does not change [Kudo et al, J. Biol. Chem. 270:17513-17520 (1995); Lopaschuk et al, J. Biol. Chem. 269:25871-25878 (1994). Rather, the actual levels of malonyl CoA drop, resulting in an increase in CPTl activity. Therefore, existing evidence suggests that actual changes in malonyl CoA levels appear to be the key factor regulating changes in fatty acid oxidation in the heart, as opposed to changes in sensitivity of CPTl to malonyl CoA inhibition. Current studies [Kudo et al, J. Biol. Chem. 270:17513-17520 (1995); Lopaschuk et al, J. Biol. Chem. 269:25871-25878 (1994); Kudo et al, Biochim. Biophys. Acta. 1301:67-75 (1996); Kudo et al, Circulation 92:1-771 (1996); Awan and Saggerson, Biochem. J. 295:61-66 (1993)] show that ACC in the heart functions as a key regulator of fatty acid oxidation, via the production of malonyl CoA [Lopaschuk et al, Biochem. Biophys. Acta. 1213:263-276 (1994)]. For example, in rabbit hearts the activity of ACC decreases between 1-day and 7-day following birth [Lopaschuk et al, J. Biol. Chem. 269:25871-25878 (1994)]. This is accompanied by a dramatic decrease in malonyl CoA levels and an increase in fatty acid oxidation rates [Lopaschuk et al, J. Biol Chem. 269:25871-25878 (1994)]. It is one ofthe key enzymes in a feedback loop that decreases acyl CoA transport into the mitochondria when carbohydrate oxidation rates are increased. ACC thus plays an important role in regulating the balance between carbohydrate and lipid metabolism in the heart. In the post-ischemic heart, a decrease in ACC activity is also accompanied by a decrease in malonyl CoA levels and an increase in fatty acid oxidation rates [Kudo et al, J. Biol. Chem. 270:17513-17520 (1995)]. This increase in fatty acid oxidation rates can provide from 90 to 100 % ofthe hearts energy requirements. Unfortunately, this occurs at the expense of glucose oxidation, which is critical for normal contractile function ofthe heart. Earlier reports indicated that low glucose oxidation rates during reperfusion of ischemic hearts contributes to contractile failure, and that by inhibiting fatty acid oxidation, glucose oxidation would thereby increase and lessen the severity of ischemic injury. A number of clinical trials have confirmed these observations and pharmaceutical agents that specifically inhibit fatty acid oxidation, such as ranolazine and trimetazidine, are presently being licensed for clinical use. Hence, in pathological situations characterized by abnormal fatty acid metabolism, enzymes regulating fatty acid or glucose oxidation could potentially be targeted for drug development. Also, fluorooxirane carboxylate as hypoglycemic agents have been used as fatty acid oxidation inhibitors. These fatty acid oxidation inhibitors operate by inhibiting CPTl, preventing the transport of the fatty acids into the mitochondria (See U.S. Patent No. 4,788,306 incorporated herein by reference). These compounds have particular utility in the treatment of glucose and fatty acid metabolism disorders, such as diabetes and appear to have significantly reduced potential for impairment of normal cardiac function. Although the liver is thought of as mainly a biosynthetic organ, it also oxidizes fatty acids as a source of energy [Goodridge, Fatty acid synthesis in eucaryotes. In: Biochemistry of lipids, liposomes and membranes. Ed: Vance and Vance. 111-139 (1991)]. Malonyl CoA is important in this process, since it inhibits carnitine palmitoyltransferase 1 (CPTl), the rate- limiting enzyme involved in the mitochondrial uptake of fatty acids [McGarry and Brown, EurJ Biochem 244:1-14 (1997); Ala and Saggerson, Biochem J334:233-241 (1998); Bird and Saggerson, Biochem 222:639-647 (1984)]. By inhibiting CPTl, mitochondrial uptake of fatty acids is decreased thereby reducing mitochondrial fatty acid oxidation [Lopaschuk et al, J Biol Chem 269 25871-25878(1994)] During times of nutritional deficiency or diabetes, decreases in malonyl CoA may result in limited synthesis of fatty acids, and an upregulation of fatty acid oxidation The question remains as to how malonyl CoA is degraded in the liver during the times when fatty acid synthase is not active Kolattukudy and co-workers [Kolattukudy et al, Methods Enzymol 71 150-153 (1981), Jang et al, J Biol Chem 264 3500-3505 (1989)] have previously identified an enzyme in both avian and mammalian tissues which is involved in the decarboxylation of malonyl CoA to acetyl CoA This enzyme, termed malonyl CoA decarboxylase (MCD), was first described as being a mitochondrial enzyme which protected certain mitochondrial enzymes such as methylmalonyl CoA mutase and pyruvate carboxylase from inhibition by mitochondrial derived malonyl CoA [Jang et al, JBiol Chem 264 3500-3505 (1989)] In the mitochondria, propionyl CoA carboxylase can, at low efficiency, use acetyl CoA as a substrate to produce malonyl CoA [Jang et al, J Biol Chem 264 3500-3505 (1989)] This, however, would seem to be only a small fraction of total cellular malonyl CoA produced The major source of malonyl CoA is thought to originate from the conversion of cytosolic acetyl CoA by ACC Recently, it has been suggested that MCD is able to regulate cytoplasmic as well as mitochondrial malonyl CoA levels [Alam and Saggerson, Biochem J 334 233-241 (1998), Dyck et al, Am J Physiology 275 H2122-H2129 (1998)] Our work has shown that an increase or maintained MCD activity in conjunction with a decrease in ACC activity is probably responsible for the decrease in malonyl CoA levels and increased fatty acid oxidation seen in both the post-natal heart and the reperfused ischemic heart [Dyck et al, Am J Physiology 275 H2122-H2129 (1998)] Similarly, Alam and Saggerson have provided indirect evidence of a cytosolic MCD activity which can alter cytosolic malonyl CoA levels in rat skeletal muscle [Alam and Saggerson, Biochem J 334 233-241 (1998)] These studies therefore implicate MCD as a regulator of fatty acid oxidation Whether the observed decreases in cytosolic malonyl CoA levels is due to a cytoplasmic form of MCD or to some unknown process is not clear There is a need for the identification of drugs that modulate MCD pathway activity whether in the heart, liver, pancreas or other organ, that do not have any side-effects Thus, what is needed are suitable assays to identify targets for pharmacological intervention that do not impair cardiac, liver or pancreatic function or confer toxicity as well as provide cost- effective, high throughput screening of fatty acid oxidation inhibitor compounds. SUMMARY OF THE INVENTION The invention generally relates to compositions and methods of identifying and testing Malonyl CoA decarboxylase (hereinafter "MCD") inhibitors, and in particular, compositions comprising a novel cardiac isoform of the MCD enzyme which is a key regulator of fatty acid oxidation. This enzyme is useful for identifying compounds that will inhibit the conversion of malonyl CoA to acetyl CoA which is then combined with R elabeled oxaloacetate to produce C-labeled citrate. Additionally, the invention relates to the isolation and determination of DNA sequences of the rat heart, liver and pancreatic genes encoding MCD. It is not intended that the present invention be limited to particular MCD proteins. A variety of closely related vertebrate homologues of MCD are contemplated that are involved in the regulation of fatty acid oxidation in various species. In one embodiment, the MCD enzyme is not the full-length native polypeptide. Rather, it is a portion of the full-length native enzyme. Preferably, this portion or fragment contains the active binding site. For example, but not by way of limitation, the present invention contemplates complexes of substrate and MCD enzyme wherein the MCD is partially purified. In yet another embodiment, the MCD have polypeptides that is mutated from the wild-type sequence. Similarly, the present invention is not limited to the native sequence of MCD. Even where portions or fragments are employed, these portions or fragments may have altered amino acid sequences. Thus, the present invention also contemplates substrate-enzyme complexes comprising MCD or its analogues. The present invention also contemplates compound screening using a variety of assay formats. In one embodiment, the present invention contemplates a method for compound screening, comprising: a) providing: i) a purified preparation comprising malonyl CoA decarboxylase, ii) a substrate, and iii) a test compound; b) mixing said malonyl CoA decarboxylase and said substrate under conditions such that said malonyl CoA decarboxylase can act on said substrate to produce product, wherein said mixing is done in the presence and absence of said test compound; and c) measuring directly or indirectly the amount of said product produced in the presence and absence of said test compound. Additionally, the present invention generally relates to compositions and methods of identifying and testing malonyl CoA decarboxylase pathway agonists and antagonists. Furthermore, the invention relates to methods to identify normal or mutant homologues of MCD which may be native to other tissue or cell types. The present invention also relates to methods of identifying tissues that may harbor homologous or mutant MCD genes. The present invention also relates to methods to generate reagents derived from the invention. The present invention contemplates as compositions the wild-type rat MCD gene from heart (SEQ ID NO:l), liver (SEQ LD NO:2) or pancreas (SEQ ID NO:3). The present invention also contemplates employing such sequences in screening methods. In one embodiment, the present invention contemplates utilizing such genes in the screening of compounds that are agonistic or antagonistic to MCD. In one preferred embodiment cells are transfected with constructs containing either sense or anti-sense MCD DNA, or portions thereof. Such DNA may readily be inserted into expression constructs and the present invention contemplates such constructs as well as their use. Compounds suspected of being either agonistic or antagonistic to MCD activity are then added and MCD activity is measured by methods known to those in the field. The present invention also contemplates RNA transcribed from the above-indicated cDNAs as well as protein (typically purified protein) translated from this RNA. Moreover, the present invention contemplates antibodies produced from immunizing with this translated protein. The present invention also contemplates transgenic animals comprising the above- indicated DNA (i.e., the "transgene") or portions thereof. In a particular embodiment, the transgenic animal ofthe present invention may be generated with the transgene contained in an inducible, tissue specific promotor. The present invention also contemplates using the above-named compositions in screening assays. The present invention is not limited by the particular method of screening. In one embodiment mammalian cells may be used. The present invention is not limited to the nature of the transfection construct. The transfection constructs utilized will be the optimal constructs available for the cell line chosen at the time of setting up the assay. In one embodiment, the present invention contemplates screening suspected compounds in a system utilizing transfected cell lines. In one embodiment, the cells may be transfected transiently. In another embodiment, the cells may be stably transfected. Constructs used to transfect cells stably may or may not utilize an inducible promoter. In yet another embodiment translation products of the invention may be used in a cell-free assay system. In yet another embodiment, antibodies generated to the translation products of the invention may be used in immunoprecipitation assays. The present invention may also be used to screen for tumors which manifest mutations in genes similar to the cDNA encoding MCD. In one embodiment cDNA encoding MCD may be used in microchip assays. The present invention contemplates a method of screening for tumors, said method comprising: a) providing in any order: i) microassay microchips wherein said microchip comprises cDNA encoding at least a portion ofthe oligonucleotide sequence of SEQ ID NOS: 1,2 or 3, ii) DNA from at least one tissue sample suspected of having mutations in genes similar to SEQ LD NOS:l, 2 or 3; b) contacting said microassay microchips with said DNA; and c) detecting hybridization of said cDNA with said tissue sample DNA. Isolated DNA would then be sequenced to assay for genetic mutations. The gene sequences of the present invention may also be used to screen for homologous. In, one embodiment cDNA encoding MCD may be used in microchip assays. The present invention contemplates a method of screening for homologues, said method comprising: a) providing in any order: i) microassay microchips wherein said microchip comprises cDNA encoding at least a portion of the oligonucleotide sequence of SEQ LD NOS:l, 2 or 3, ii) DNA from at least one tissue sample suspected of having a sequence in genes that is similar to SEQ ID NOS: 1, 2 or 3; b) contacting said microassay microchips with said DNA; and c) detecting hybridization of said cDNA with sample said tissue DNA. Isolated DNA would then be sequenced to assay for genetic mutations. The present invention may also be used to identify novel or mutant constituents ofthe MCD pathway. In one embodiment, antibodies generated to translation products of the invention may be used in immunoprecipitation experiments to isolate novel MCD pathway constituents or natural mutations thereof. In another embodiment, the invention may be used to generate fusion proteins that could also be used to isolate novel MCD pathway constituents or natural mutations thereof. In yet another embodiment, screens may be conducted using the yeast two-hybrid system. The present invention also contemplates screening for homologues using standard molecular procedures. In one embodiment screens are conducted using Northern and Southern blotting. The present invention contemplates a method of screening a compound, said method comprising: a) providing in any order: i) a first group of cells comprising a recombinant expression vector, wherein said vector comprises at least a portion of the oligonucleotide sequence of SEQ LD NOS:l, 2 or 3, ii) a second group of cells comprising a recombinant expression vector, wherein said vector comprises the plasmid used above without any portion ofthe oligonucleotide from SEQ ID NOS:l, 2, or 3, and iii) at least one compound suspected of having the ability to modulate MCD pathway activity; b) contacting said first and second groups of cells with said compound; and c) detecting inhibition or enhancement of MCD activity of said compound. The present invention also contemplates a method of screening for homologues, said method comprising: a) providing in any order: i) first nucleic acid comprising at least a portion ofthe sequence of SEQ LD NOS:l, 2 or 3, and ii) DNA libraries from cells or tissues suspected to comprise said homologue; and b) hybridizing said nucleic acid with said DNA of said library under conditions such that said DNA suspected of coding for said homologue is detected. The present invention also contemplates a method of screening for interactive peptides, said method comprising: a) providing in any order: i) a peptide comprising at least a portion of the peptide sequence encoded by SEQ LD NOS:l, 2 or 3 (including but not limited to portions that are part of fusion proteins, i.e., proteins that contain another portion, such as a portion useful for protein purification) and b) an extract from source (e.g., cells or tissues) suspected of having said interactive peptides; and c) mixing said peptide with said extract under conditions such that said interactive peptide is detected. The present invention contemplates another approach for screening for interactive peptides, said method comprising: a) providing in any order: i) antibodies reactive with (and usually specific for) at least a portion of a peptide encoded by SEQ LD NOS: 1, 2 or 3, and ii) an extract from a source (e.g., cells or tissues) suspected of having said interactive peptide(s); and b) mixing said antibody with said extract under conditions such that said interactive peptide is detected. DESCRD?TION OF THE DRAWINGS FIGURE 1 shows the biochemical characterization of the rat liver malonyl CoA decarboxylase (MCD). (A) is a representative photograph showing the immunoblot analysis of rat liver MCD (non-denaturing polyacrylamide gel electrophoresis: lane 1) and SDS-polyacrylamide gel electrophoresis: lane 2) with anti MCD antibody and irrelevant anti-catalase antibody: lane 3). (B) is a representative photograph of an immunoblot analysis using the anti MCD antibody, and compares MCD from rat liver (lane 1) and rat heart (lane 2). FIGURE 2 graphically depicts Malonyl CoA decarboxylase activity in 1-day and 7-day old rabbit hearts. Values are the mean ± S.E. of 8 hearts for both ages of rabbits. FIGURE 3 graphically depicts Malonyl CoA decarboxylase activity in aerobic, ischemic and reperfused ischemic rat hearts. Values are the mean + S.E. of 5-10 hearts for all groups. FIGURE 4 is a representative photograph of immunoblot analysis using anti MCD antibody depicting the levels of malonyl CoA decarboxylase protein in aerobic, ischemic and reperfused ischemic rat hearts. FIGURE 5 is a schematic depicting the role of 5'AMP-activated protein kinase, acetyl CoA carboxylase and malonyl CoA decarboxylase in the control of fatty acid oxidation in newborn (A) and reperfused ischemic hearts (B). (A) depicts both AMPK and MCD are activated during the newborn period. (B) shows ischemia activates AMPK while MCD activity is maintained. FIGURE 6 shows the DNA sequence encoding rat heart malonyl CoA decarboxylase, (SEQ LD NO:l). FIGURE 7 shows the chromatographic purification of rat liver malonyl CoA decarboxylase. Mitochondrial protein precipitated between 40 and 55 % of (NH ) 2 SO 4 was loaded onto a Butyl Sepharose 650 M column and eluted with a gradient of 1 to 0 M of (NH 4 ) 2 SO . (A) shows the fractions containing MCD activity were pooled and loaded onto a Phenyl Sepharose HP column arid proteins were eluted with the same gradient as above. (B) shows chromatography on a Q Sepharose HP column. (C) shows chromatography on a SP Sepharose HP column. (D) 7B and 7C were performed with the bound proteins being eluted with a gradient of 0 to 0.3 M and 0 to 0.4 M NaCl, respectively. MCD activity is expressed as change in relative fluorescence units/ minute and has not been standardized to protein concentration. FIGURE 8 shows the DNA sequence encoding rat liver malonyl CoA decarboxylase, (SEQ LD NO:2). FIGURE 9 shows kinetic properties of purified rat liver malonyl CoA decarboxylase. 9 A, the effect of malonyl CoA concentration on the activity of purified rat liver MCD (expressed as a percent of maximal MCD activity). 9B, The activity of MCD is effected by altering the pH ofthe assay buffer. FIGURE 10 shows the characterization of malonyl CoA decarboxylase antibodies. (A) the polyclonal antibodies directed against rat liver MCD were tested using protein from a rat liver mitochondrial fraction. The MCD239 antibodies (lane 1) recognized MCD only, while MCD240 (lane 2) recognized both catalase and MCD. MCD and catalase are indicated at the left. (B) immuno-inhibition studies were performed using both the MCD antibodies. The MCD antibody or pre-immune serum mixtures was measured for MCD activity and expressed as a % of MCD pre-incubated without serum for an identical period of time (30 minutes). Figure 11 shows the tissue distribution and cellular localization of malonyl CoA decarboxylase. (A) the protein levels of MCD were measured in a variety of rat tissues using MCD240 antibody. 150 μg of heart (lane 1), skeletal muscle (lane 2), liver (lane 3), kidney (lane 4), lung (lane 5), pancreas (lane 6), brain (lane 7) were boiled in a 2 % SDS gel loading buffer and subjected to western analysis. (B) a double labeling immunocytochemical technique was used to determine cellular localization of MCD in McArdle RH-7777 rat hepatoma cells. Images from MCD239 antibody (box 1) and anti Hsp 60 antibody (box 2) were combined to determine co-localization (box 3). Similarly, images from MCD240 antibody (box 4) and Mito-Tracker Red CMXRos (box 5) were combined to determine co- localization (box 6). FIGURE 12 shows rat liver malonyl CoA decarboxylase activity and protein levels in control and 6 week old streptozotocin rats. MCD activity (A) and MCD protein quantification (B) was performed on rat livers from 6 week old control rats and 6 week old streptozotocin treated rats. MCD protein is located in the bottom panel of each group and catalase protein levels are included as loading and transfer controls in the top panels. The Western blots underwent densitometry analysis and the levels of MCD protein were quantified (C). FIGURE 13 shows rat liver malonyl CoA decarboxylase activity and protein levels in control and JCRLA-corpulent rats. MCD activity (A) and MCD protein quantification (B) was performed on rat livers from JCRLA-corpulent lean controls and Cp/Cp rats. MCD protein is located in the bottom panel of each group and catalase protein levels are included as loading and transfer controls in the top panels. The Western blots underwent densitometry analysis and the levels of MCD protein were quantified (C). FIGURE 14 shows rat liver malonyl CoA decarboxylase activity and protein levels in control fasted, and refed rats. MCD activity (A) and MCD protein quantification (B) was performed on rat livers from control, fasted and refed rats. MCD protein is located in the bottom panel of each group and catalase protein levels are included as loading and transfer controls in the top panels. The Western blots underwent densitometry analysis and the levels of MCD protein were quantified (C). FIGURE 15 shows the DNA sequence encoding rat pancreatic malonyl CoA decarboxylase, (SEQ LD NO:3). FIGURE 16 shows Northern blot and RT-PCR analysis of MCD mRNA in various tissues. (A) shows Northern blot analysis of rat tissues with a rat MCD probe and an 18S ribosomal RNA cDNA fragment used as a control for RNA loading on the gel. (B) shows ethidium bromide stained agarose gels of RT-PCR experiments carried out with MCD (top) and β-actin (bottom) primers using total RNA isolated from various rat tissues and sorted pancreatic islet cells. DEFINITIONS To facilitate understanding ofthe invention, a number of terms are defined below. The abbreviations used herein are: MCD, Malonyl CoA decarboxylase; ATP, Adenosine Triphosphate; FA, Fatty Acid; FAS Fatty Acid Synthase; ACC, acetyl-CoA carboxylase; carnitine palmitoyltransferase 1, CPTl. The term "indicator" used herein refers not to the product, but indirectly indicates the presence of a product and more preferably the level of product. For e.g., in one embodiment, the "indicator" is citrate. The term "label" as used herein refers to any atom or molecule which can be used to provide a detectable (preferably quantifiable) signal, and which can be attached to a nucleic acid or protein. Labels may provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absoφtion, magnetism, enzymatic activity, and the like. The term "substrate" as used herein, refers to molecules on which an enzyme acts. For e.g., in one embodiment, the substrate is Malonyl CoA. The term "homologue" used herein encompasses molecules which differ from the parent molecule or reference molecule in structure but have similar function. For example, a homologue to an enzyme has the same general enzymatic activity (although the specificity and level of activity may be different) but differ in structure (For e.g., amino acid modifications or substitutions). Homologues can be generated by a variety of techniques, such as by using primers in a PCR reaction, that introduce new bases in the DNA sequence, thus resulting in amino acid substitutions. Such primers can be designed to hybridize with the cardiac isoform of MCD by terminal amino acid sequencing of the protein purified in the manner set forth herein. Alternatively, primers complementary to the non-human forms ofthe enzyme (For e.g., goose MCD [GenBank accession number is J04482] can be employed. The term "gene" refers to a DNA sequence that comprises control and coding sequences necessary for the production of a polypeptide or its precursor. The polypeptide can be encoded by a full length coding sequence or by any portion ofthe coding sequence. The term "nucleic acid sequence of interest" refers to any nucleic acid sequence the manipulation of which may be deemed desirable for any reason by one of ordinary skill in the art. The term "wild-type" when made in reference to a gene refers to a gene which has the characteristics of a gene isolated from a naturally occurring source. The term "wild-type" when made in reference to a gene product refers to a gene product which has the characteristics of a gene product isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designated the "normal" or "wild-type" form ofthe gene. In contrast, the term "modified" or "mutant" when made in reference to a gene or to a gene product refers, respectively, to a gene or to a gene product which displays modifications in sequence and or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally-occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product. The term "recombinant" when made in reference to a DNA molecule refers to a DNA molecule which is comprised of segments of DNA joined together by means of molecular biological techniques. The term "recombinant" when made in reference to a protein or a polypeptide refers to a protein molecule which is expressed using a recombinant DNA molecule. As used herein, the terms "vector" and "vehicle" are used interchangeably in reference to nucleic acid molecules that transfer DNA segment(s) from one cell to another. The term "expression construct", "expression vector" or "expression cassette" as used herein refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression ofthe operably linked coding sequence in a particular host organism. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. The terms "in operable combination", "in operable order" and "operably linked" as used herein refer to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced. The term "hybridization" as used herein refers to any process by which a strand of nucleic acid joins with a complementary strand through base pairing. As used herein, the terms "complementary" or "complementarity" when used in reference to polynucleotides refer to polynucleotides which are related by the base-pairing rules. For example, for the sequence 5'-AGT-3' is complementary to the sequence 5'-ACT-3'. Complementarity may be "partial," in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods which depend upon binding between nucleic acids. The term "homology" when used in relation to nucleic acids refers to a degree of complementarity. There may be partial homology or complete homology (i.e., identity). A partially complementary sequence is one that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid is referred to using the functional term "substantially homologous." The inhibition of hybridization ofthe completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a sequence which is completely homologous to a target under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction. The absence of non-specific binding may be tested by the use of a second target which lacks even a partial degree of complementarity (e.g., less than about 30 % identity); in the absence of non-specific binding the probe will not hybridize to the second non-complementary target. Low stringency conditions when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42° C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.1 % SDS, 5X Denhardt's reagent [50X Denhardt's contains per 500 ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)] and 100 μg/ml denatured salmon sperm DNA followed by washing in a solution comprising 5X SSPE, 0.1 % SDS at 42°C when a probe of about 500 nucleotides in length is employed. High stringency conditions when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42° C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaHJOfcO and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.5 % SDS, 5X Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followed by washing in a solution comprising 0.1X SSPE, 1.0 % SDS at 42° C when a probe of about 500 nucleotides in length is employed. When used in reference to nucleic acid hybridization the art knows well that numerous equivalent conditions may be employed to comprise either low or high stringency conditions; factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration ofthe salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of either low or high stringency hybridization different from, but equivalent to, the above listed conditions. D StringencyD when used in reference to nucleic acid hybridization typically occurs in a range from about T m -5°C (5°C below the T m ofthe probe) to about 20 °C to 25 °C below T m . As will be understood by those of skill in the art, a stringent hybridization can be used to identify or detect identical polynucleotide sequences or to identify or detect similar or related polynucleotide sequences. Under "stringent conditions" a nucleic acid sequence of interest will hybridize to its exact complement and closely related sequences. As used herein, the term "fusion protein" refers to a chimeric protein containing the protein of interest (i.e., MCD and fragments thereof) joined to an exogenous protein fragment (the fusion partner which consists of a non-MCD sequence). The fusion partner may provide a detectable moiety, may provide an affinity tag to allow purification of the recombinant fusion protein from the host cell, or both. If desired, the fusion protein may be removed from the protein of interest by a variety of enzymatic or chemical means known to the art. As used herein, the term "purified" or "to purify" refers to the removal of contaminants from a sample. The present invention contemplates purified compositions (discussed above). As used herein the term "portion" when in reference to a protein (as in "a portion of a given protein") refers to fragments of that protein. The fragments may range in size from four amino acid residues to the entire amino acid sequence minus one amino acid "Antibody" shall be defined as a glycoprotein produced by B cells that binds with high specificity to the agent (usually, but not always, a peptide), or a structurally similar agent, that generated its production. Antibodies may be produced by any of the known methodologies and may be either polyclonal or monoclonal. "Mutant" shall be defined as any changes made to a wild type nucleotide sequence, either naturally or artificially, that produces a translation product that functions with enhanced or decreased efficiency in at least one of a number of ways including, but not limited to, specificity for various interactive molecules, rate of reaction and longevity of the mutant molecule. GENERAL DESCRJIPTION OF INVENTION The present invention relates generally to compositions and methods of identifying and testing Malonyl CoA decarboxylase (MCD) inhibitors, and in particular, compositions comprising a novel cardiac isoform of MCD, identified to be a key regulator of fatty acid oxidation in the heart. Additionally, the invention relates to compositions and methods of identifying MCD pathway agonists and antagonists, and in particular, compositions comprising novel DNA sequences of rat heart, liver and pancreatic DNA The Description of the invention involves A) Alterations in Fatty Acid Oxidation during Reperfusion ofthe Heart after Myocardial Ischemia, B) Malonyl CoA in the Heart and C) Malonyl CoA in the liver and pancreas. A. Alterations In Fatty Acid Oxidation During Reperfusion Of The Heart After Myocardial Ischemia Energy substrate preference of the heart both during and after ischemia is an important determinant of the degree of functional recovery postischemia. For instance, high rates of fatty acid oxidation after ischemia can decrease cardiac function and efficiency during reperfusion. These high rates of fatty acid oxidation can be explained by a decrease in malonyl coenzyme-A (CoA) levels, a potent inhibitor of mitochondrial fatty acid uptake. In particular, activation of 5'-AMP-activated protein kinase and inhibition of acetyl CoA carboxylase appear to contribute to this decrease in malonyl CoA. As a result, it is likely that, inhibition of 5'-AMP-activated protein kinase and/or stimulation of acetyl CoA carboxylase may be a pharmacologic approach to inhibiting myocardial fatty acid oxidation during reperfusion. Decreasing fatty acid oxidation is accompanied by a parallel increase in glucose oxidation that results in an improvement in both cardiac function and efficiency in the reperfused ischemic heart [Lopaschuk, Am JCardiol 80:11A-16A (1997)]. B. Malonyl CoA In The Heart High rates of fatty acid oxidation in the post-ischemic heart are primarily due to a dramatic drop in myocardial levels of malonyl CoA. Malonyl-CoA is a potent inhibitor of carnitine palmitoyltransferase 1 (CPTl) and is important in regulating fatty acid uptake into the mitochondria. A decrease in malonyl CoA synthesis is partly responsible for the drop in malonyl CoA post-ischemia. Although the synthesis of malonyl CoA in the heart by a cardiac specific form of ACC has been well characterized, no information is available on how malonyl CoA is degraded in the heart. The present invention shows that the heart has an active malonyl CoA decarboxylase (MCD) that converts malonyl CoA to acetyl CoA. This was facilitated by the development of a novel and reliable assay to measure malonyl CoA decarboxylase (MCD) activity, that did not require extensive protein purification. Using this assay, MCD activity was characterized in rat hearts and identified to play an important role in regulating fatty acid oxidation. The MCD enzyme is under phosphorylation control, and can be activated under experimental conditions that result in enzyme dephosphorylation. The majority of MCD in the heart is associated with the mitochondria, with octylglucoside treatment of mitochondria resulting in 14 fold increase in MCD activity. Partial purification of cardiac MCD revealed a protein of approximately 45 kDa, that runs as a tetrameric complex when subjected to polyacrylamide gel electrophoresis. MCD has previously been described as a mitochondrial enzyme which is involved in protecting certain mitochondrial enzymes such as methylmalonyl CoA mutase and propionyl CoA from inhibition by mitochondrial derived malonyl CoA [Kim and Kolattukudy, Arch. Biochem. Biophys. 190:234-246 (1978); Scholte, Biochim. Biophys. Acta. 178:137-144 (1969); Landriscina et al, Eur. J. Biochem. 19:573-580 (1971); Koeppen et al, Biochemistry 13:3589-3595 (1974)]. While very little is known about mammalian MCD, two isoforms of MCD have been identified in the goose uropygial gland [Courchesne-Smith et al, Arch. Biochem. Biophys. 298:576-586 (1992)]. These proteins originate from the same gene although each have separate start sites of transcription and translation. These alternate start sites create two proteins, one targeted to the mitochondrial matrix and the other to the cytoplasm. In addition to the uropygial gland, non-mitochondrial levels of MCD have also been detected in low levels in the liver. To date, MCD activity and subcellular localization has not been characterized in the heart. The role of MCD in regulating fatty acid oxidation in the present invention, was also studied using isolated perfused hearts from newborn rabbits and adult rats. In newborn rabbit hearts, fatty acid oxidation increases dramatically between 1-day and 7-day following birth, which is accompanied by a decrease in both ACC activity and malonyl CoA levels. A parallel increase in MCD activity also was observed in 7-day old hearts compared to 1-day old hearts. If adult rat hearts are aerobically reperfused following a 30 minute period of no-flow ischemia, levels of malonyl CoA decreased dramatically, which was accompanied by an increase in fatty acid oxidation rates. The decrease in malonyl CoA during reperfusion could be explained by a decrease in ACC activity and a maintained MCD activity. This data suggested that the heart has an active MCD that has an important role in regulating fatty acid oxidation rates. Also, MCD appears to be a key enzyme which may be responsible for reducing malonyl CoA levels following birth in the rabbit heart, or following myocardial ischemia in the rat heart. Inhibition of this enzyme has considerable clinical potential as it represents a potentially important site for pharmacological intervention in pathological situations characterized by abnormal fatty acid metabolism Pharmacological inhibition of MCD would result in an increase in myocardial levels of malonyl CoA in the reperfused ischemic heart This would lower fatty acid oxidation rates, increase glucose oxidation and improve contractile function of reperfused ischemic hearts C. Malonyl CoA In The Liver And Pancreas The liver is thought of as mainly a biosynthetic organ, however, it also oxidizes fatty acids as a source of energy [Goodridge, Fatty acid synthesis in eucaryotes In Biochemistry of lipids, liposomes and membranes Ed Vance and Vance 111-139 (1991)] Malonyl CoA is important in this process, since it inhibits carnitine palmitoyltransferase 1 (CPTl), the rate- limiting enzyme involved in the mitochondrial uptake of fatty acids [McGarry and Brown, Eur J Biochem 244 1-14 (1997), Alam and Saggerson, Biochem J 224 233-241 (1998), Bird and Saggerson, Biochem 222 639-647 (1984)] By inhibiting CPTl, mitochondrial uptake of fatty acids is decreased, thereby reducing mitochondrial fatty acid oxidation [, Lopaschuk et al, J Biol Chem 269 25871-25878(1994)] During times of nutritional deficiency or diabetes, decreases in malonyl CoA may result in limited synthesis of fatty acids, and an upregulation of fatty acid oxidation The question remains as to how malonyl CoA is degraded in the liver during the times when fatty acid synthase is not active In β-cells in the pancreas, as in the heart and liver, the malonyl-CoA/CTPl interaction is a central element of a "fuel cross-talk" signaling network and may be implicated in the short and/or long term control of insulin secretion in the pancreatic β-cell [Chen et al, Diabetes 43 878-883 (1994), Prentki et al, J Biol Chem 267 5802-5810 (1992)] The intracellular concentration of malonyl-CoA in lipogenic tissues such as liver and adipose tissue is thought to result from its rate of formation by acetyl-CoA carboxylase (ACC) and its usage by fatty- acid synthase (FAS) However, other tissues like the pancreatic islet [Brun et al, Diabetes 45 190-198] express FAS at very low levels yet show rapid variations in malonyl-CoA under a number of experimental conditions which increase or decrease its concentration The fate of malonyl-CoA in the β-cell in unclear and it is attractive to believe that the intracellular concentration of this metabolic signaling molecule is controlled by an additional enzyme, at least in non-lipogenic tissue expressing low FAS A prime candidate for such function is MCD. DETAILED DESCRD?TION OF PREFERRED EMBODIMENTS Generally, the nomenclature used hereafter and the laboratory procedures in cell culture, molecular genetics, and nucleic acid chemistry and hybridization described below are those well known and commonly employed in the art. Standard techniques are used for recombinant nucleic acid methods, polynucleotide synthesis, and microbial culture and transformation (e.g., electroporation, lipofection). Generally enzymatic reactions and purification steps are performed according to the manufacturer's specifications. The techniques and procedures are generally performed according to conventional methods in the art and various general references [See, generally, Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., and Current Protocols in Molecular Biology (1996) John Wiley and Sons, Inc., N.Y.]. Oligonucleotides can be synthesized on an Applied BioSystems oligonucleotide synthesizer [for details see Sinha et al, Nucleic Acids Res. 12:4539 (1984)], according to specifications provided by the manufacturer. Complementary oligonucleotides are annealed by heating them to 90° C in a solution of 10 mM Tris-HCl buffer (pH 8.0) containing NaCl (200 mM) and then allowing them to cool slowly to room temperature. For binding and turnover assays, duplex DNA is purified from native polyacrylamide (15 % w/v) gels. The band corresponding to double-stranded DNA is excised and soaked overnight in 0.30 M sodium acetate buffer (pH 5.0) containing EDTA (1 mM). After soaking, the supernatant is extracted with phenol/chloroform (1/1 v/v) and precipitated with ethanol. DNA substrates are 32 radiolabeled on their 5 -OH group by treatment with [g- P]ATP and T4 polynucleotide kinase. Salts and unincoφorated nucleotides are removed by chromatography on Sephadex G columns. The invention will be useful for, among other things, (1) the design and execution of screens to identify proteins or small molecules that inhibit MCD activity, and thus activate or prevent the degradation of malonyl CoA to acetyl CoA, (2) the development of high throughput screens for rational drug design and (3) the identification of MCD intra- and interspecific homologues or mutants. Additionally, the present invention contemplates assays for detecting the ability of agents to inhibit or enhance MCD activity where high-throughput screening formats are employed together with large agent banks (e.g., compound libraries, peptide libraries, and the like) to identify antagonists or agonists. Such MCD pathway antagonists and agonists may be further developed as potential therapeutics and diagnostic or prognostic tools. Screening Assays The screening assays ofthe present invention may utilize isolated or partially purified forms of the assay components (or MCD enzyme). "Purified" refers to the enzyme of the present invention which have been separated from their native environment (e.g., a cytoplasmic or nuclear fraction of a cell, yeast protoplasm, or by recombinant production). It is preferred that the enzyme is purified to at least about 10-50 % purity. A substantially pure composition includes such MCD enzyme(s) or complexes that are approaching homogeneity, i.e., about 70-90 % pure. A "pure" preparation refers to purity greater than 90 % and more preferably a purity greater than 95 %. Preferred embodiments include binding assays which use cardiac MCD which are produced by recombinant methods or chemically synthesized. In one embodiment, the methods of screening employs, in addition to MCD and the substrate malonyl CoA, the use of C- labeled oxaloacetate to produce C-labeled citrate. It is contemplated that the screening assays ofthe present invention will, among other things, identify agents that will inhibit MCD activity. Such inhibitors are contemplated to be useful in the treatment of ischemia (e.g., Pharmacological inhibition of MCD should result in an increase in myocardial levels of malonyl CoA in the reperfused ischemic heart), as well as other disorders. This will lower the fatty oxidation rates, increase glucose oxidation and improve contractile function of reperfused ischemic hearts. The present invention contemplates compound screening using a variety of assay formats. In one embodiment, the present invention contemplates a method for compound screening, comprising: a) providing: i) a purified preparation comprising malonyl CoA decarboxylase, ii) a substrate, and iii) a test compound; b) mixing said malonyl CoA decarboxylase and said substrate under conditions such that said malonyl CoA decarboxylase can act on said substrate to produce product, wherein said mixing is done in the presence and absence of said test compound; and c) measuring directly or indirectly the amount of said product produced in the presence and absence of said test compound. In one embodiment, inhibition is measured by detection of acetyl CoA formation as estimated by reduction of [ C] citrate levels. Malonyl CoA Decarboxylase Assay: To measure MCD activity, a novel MCD assay was developed in the present invention which detected the product of the MCD reaction, acetyl CoA. Acetyl CoA derived from MCD was incubated in the presence of [* Cjoxaloacetate and citrate synthase (0.73 μU/μL) to form citrate. The [ C]oxaloacetate was initially produced by a 20 minute transamination reaction performed at room temperature utilizing L-[ C(U)]aspartate (2.5 Ci/ml) and 2-oxoglutarate (2 mM). One of the advantages of the assay was that extensive purification of MCD was not required prior to assay, and that in vitro measurements of enzyme activity reflected the rate of enzyme activity in vivo. (Also, see Schematic A). 1. Screens To Identify Agonists Of Antagonists Of MCD There are several different approaches contemplated by the present invention to look for substances that specifically inhibit or enhance the MCD activity. One approach is to transfect cells with expression constructs comprising nucleic acid encoding the MCD and measure changes in MCD activity as compared to controls after the cells have been exposed to the compound suspected of modulating mediating MCD activity. Cells may be transiently transfected or stably transfected with the construct under control of an inducible promoter. Expression of supeφhysiological levels (supeφhysiological is defined as expression levels of the said gene product at levels greater than the cell would normally express without the transfection and expression of constructs containing the said gene into the cell) of MCD may enhance normal yet subtle cellular MCD interactions significantly to allow for the investigation of heretofore nonassayable phenomena. Other embodiments would include translation of the corresponding RNA and purification of the peptide. The purified peptide could then be used to test specific compound:protein interactions. Additionally, it is possible to generate antibodies to the translated invention allowing for the development of immunological assays such as, but not limited to, RIA, ELISA or Western blot. Furthermore, transgenic animal could be produced allowing for in vivo assays to be conducted. Tissue Homogenate (MCD) Malonyl-CoA > Acetyl-CoA (with or without the phosphatase inhibitors: NaF/NaPPl) aspartate aminotransferase L-[ C] aspartate + 2-oxoglutarate > [ C] oxaloacetate + L-glutamate citrate synthase [ 14 C]oxaloacetate + acetyl-CoA ► [ 14 C]citrate + CoASH Schematic A A. Transfection Assays Transfection assays allow for a great deal of flexibility in assay development. The wide range of commercially available transfection vectors will permit the expression ofthe MCD genes ofthe present invention in a extensive number of cell types. In one embodiment, cells are transiently transfected with an expression construct comprising in operable combination 1) nucleic acid encoding MCD and 2) an inducible promotor. Cells would be exposed to the agent suspected of modulating MCD activity, MCD expression would be turned on and MCD activity would be measured. Rates of MCD activity in cells expressing recombinant MCD are compared to rates of MCD activity in cells transfected with a control expression vector (e.g., an empty expression vector). Rates of MCD activity can be quantitated by any of a number of ways reported in the literature and known to those practiced in the art. In another embodiment, stably transfected cells lines are developed, i.e., cell lines stably expressing the MCD genes ofthe present invention. The use of an inducible promoter would be utilized in these systems. Screening assays for compounds suspected of modulating MCD activity would be conducted in the same manner as with the transient transfection assays. Using stably transfected cell lines would allow for greater consistency between experiments and allow for inter-experimental comparisons. 2. Screen To Identify Tissues Expressing Similar Or Homologous Gene Mutations In one embodiment tissue screens will be constructed using microassay microchip techniques. This will allow for the development of a high-through-put screen for the identification of tissues expressing mutant genes similar to, or homologous with, the sequenced MCD genes. 3. Screens To Identify MCD Signal Pathway Constituents a. In vitro Assays There are several different approaches to identifying MCD interactive molecules. The invention would allow the identification of proteins that may only associated with nonactive (or reduced activity) MCD or constitutively active MCD molecules. Such proteins may regulate MCD function. Techniques that may be used are, but not limited to, immunoprecipitation of MCD with antibodies generated to the transcription product ofthe invention. This would also bring down any associated bound proteins. Another method is to generate fusion proteins containing the mutant form of MCD connected to a generally recognized pull-down protein such as glutathione S-transferase. Bound proteins can then be eluded and analyzed. i) Immunoprecipitation After the generation of antibodies to wild type and mutant MCD, cells expressing transfected MCD are lysed and then incubated with one ofthe antibodies. Antibodies with the bound MCD and any associated proteins can then be pulled down with protein- A Sepharose or protein-G Sepharose beads, using standard techniques. ii) Fusion Protein Pull-down A method similar to immunoprecipitation is to construct fusion proteins of MCD and glutathione S-transferase (GST). The MCD fusion proteins are then incubated with cell extracts and then removed with glutathione Sepharose beads. Any bound, MCD proteins are then characterized. 4. Screens To Identify MCD Homologues Standard molecular biological techniques can be used to identify MCD homologues in rat, human or other species. For example, the present invention contemplates a variety of approaches including, but are not limited to, DNA-DNA hybridization techniques (e.g., Southern blots) and DNA-RNA hybridization techniques (e.g., Northern blots). Additional techniques may include, for example, immunoscreening of proteins made from library stocks with antibodies generated to translation products of SEQ ID NOS: l, 2 or 3. Furthermore, immunoprecipitation of known or suspected interactive proteins of MCD can be followed by the identification of possible mutant MCD homologues with antibodies generated to translation products of SEQ LD NOST, 2 or 3. EXPERIMENTAL The following examples serve to illustrate certain preferred embodiments and aspects ofthe present invention and are not to be construed as limiting the scope thereof. In the experimental disclosure which follows, the following methodology apply. Methodology Materials: L-[ C(U)]aspartate and [9,10- C]palmitate were purchased from Mandel, and hyamine hydroxide (methylbenzethonium; 1 M in methanol solution) was purchased from ICN. Bovine serum albumin (fraction V) was obtained from Boehringer Mannheim, West Germany. Dowex 50W-X8 cation exchange resin (100-200 mesh hydrogen form) was obtained from Bio-Rad Laboratories (Richmond, CA). ACS Aqueous counting scintillant was purchased from Amersham Canada (Oakville, Ontario). All other chemicals were reagent grade. 1. Preparation Of Rat Heart Malonyl CoA Decarboxylase Male Sprague-Dawley rats (300-350 g) were anesthetized with sodium pentobarbitol, and the heart quickly excised. Hearts were then either immediately homogenized for measurement of MCD, immediately used for preparation of mitochondria (see below), frozen in liquid N , or used for isolated working heart perfusions. Hearts were also obtained from anesthetized 1-day and 7-day old New Zealand White rabbits for isolated heart perfusions and are described as follows. Fresh or frozen hearts (10-15 mg of tissue) used for MCD measurements were homogenized for 2 x 15 seconds in a buffer consisting of KC1 (75 mM), Sucrose (20 mM), HEPES (10 mM), EGTA (1 mM), with or without NaF (50 mM) and NaPPi (5 mM). A fraction of this homogenate corresponding to 2 mg of tissue was used in the MCD assay. Isolation Of Rat Heart Mitochondria And Purification Of Malonyl CoA Decarboxylase: Rat hearts were excised and separated from their atrias. The ventricles were then minced into 1 mm cubes and rinsed with ice cold mannitol/sucrose/EGTA (MSE) buffer (225 mM mannitol; 75 mM sucrose; 1 mM EGTA, pH 7.5). A ratio of 2: 1 of MSE buffer to wet tissue weight was prepared and then homogenized using 5 strokes of a glass homogenizer with a teflon pestle. The homogenate was then diluted with MSE to 10 ml/g of wet tissue and centrifuged at 480 x g for 5 minutes. The supernatant was filtered through two layers of cheese cloth and then centrifuged at 10,000 x g for 30 minutes. The pellet was then resuspended in 10 mM sodium phosphate buffer, pH 7.6, containing 0.5 mM dithioerythritol (DTE) (50 mL/g of wet mitochondrial pellet). Triton X-100 was added to the suspension (0.1 %) and then the slurry was stirred at 4°C for 16 hours to lyse the mitochondria. The solution was then centrifuged at 24,000 x g for 10 minutes, the supernatant saturated to 40 % with powdered ammonium sulfate and stirred for 1 hour on ice. The precipitated protein was removed by centrifugation at 24,000 x g for 10 minutes. The supernatant was saturated to 55 % with powdered ammonium sulfate, stirred for 1 hour on ice, and centrifuged at 24,000 x g for 10 minutes. The pellet was resuspended in a 0.1 M potassium phosphate (ph 7.4), 1 mM DTT and 1 M ammonium SO 4 buffer. Chromatography On Butyl Sepharose 650M: The potassium phosphate/MCD solution was next loaded onto a 100 mL Butyl-Sepharose 650M previously equilibrated with a 0.1 M potassium phosphate buffer (pH 7.4) containing 1 mM DTT and 1 M ammonium SO 4 . The column was washed with 3 column volumes ofthe above buffer and proteins were eluted with a linear gradient of 1 to 0 M Ammonium SO 4 in a total volume of 500 mL with 10 mL fractions being collect. The fractions containing MCD activity were pooled and concentrated using ultrafiltration using a Millipore Ultrafree 15 mL centrifugal filter device, Biomax 10 K NMWL. The resulting 1 mL solution was brought to 50 mL in a 0.1 M potassium phosphate (pH7.4), 1 mM DTT and 1 M ammonium SO 4 buffer. Chromatography On Phenyl Sepharose HP: The enzyme solution was next loaded onto a 25 mL Phenyl Sepharose HP column previously equilibrated with a 0.1 M potassium phosphate buffer (pH 7.4) containing 1 mM DTT and 1 M ammonium SO 4 buffer. The column was washed with 3 column volumes ofthe above buffer and proteins were eluted with a linear gradient of 1 to 0 M ammonium SO 4 in a total volume of 500 mL with 10 mL fractions being collected. The fractions containing MCD activity were pooled and concentrated using ultrafiltration as above. The resulting 1 mL solution was brought to 50 mL in a 20 mM Bis-Tris, pH 7 buffer containing 1 mM DTT. Chromatography On Q Sepharose HP: The enzyme solution was next applied to a 10 mL Q Sepharose HP column previously equilibrated with a 20 mM Bis-Tris, pH 7 buffer containing 1 mM DTT. The column was washed with 3 column volumes ofthe same buffer and bound proteins were eluted with a linear gradient of 0 to 0.3 M NaCl in a total volume of 100 mL. 5 mL fractions were collected and the fractions containing MCD activity were pooled and concentrated as above. The concentrated pool was resuspended in 50 mL of a 50 mM malonate (pH 5.6) buffer containing 1 mM DTT. Chromatography On SP Sepharose HP: The enzyme solution was next applied to a 5 mL SP Sepharose HP column previously equilibrated with a 50 mM malonate (pH 5.6) buffer containing 1 mM DTT. The column was washed with 3 column volumes of the same buffer and bound proteins were eluted with a linear gradient of 0 to 0.3 M NaCl in a total volume of 50 mL. 2 mL fractions were collected and the fractions containing MCD activity were pooled, neutralized to pH 7.0, and concentrated as above. The concentrated pool was resuspended in 5 mL of 50 mM malonate (pH 5.6) buffer containing 1 mM DTT. Malonyl CoA Affinity Elution From SP Sepharose HP: The enzyme solution was next reapplied to a 0.5 mL SP Sepharose HP column previously equilibrated as above. The column was washed to remove unbound proteins and then eluted with a 50 mM malonate (pH 5.7) buffer containing 1 mM DTT and 10 μM malonyl CoA. This allowed for highly specific elution of MCD. The eluted protein fraction form the second SP Sepharose HP column was resolved on a 10 % SDS- polyacrylamide gel and stained for visualization using a Coomassie solution. The MCD band of protein was excised from the gel and subjected to an "in the gel" tryptic digest. Once digested, the tryptic peptides were separated on a microbore HPLC and selected peptides were subjected to Edman degradation for protein sequencing. Malonyl CoA Decarboxylase Assay: To measure MCD activity, a novel MCD assay was developed in the present invention, which detected the product ofthe MCD reaction, acetyl CoA. Acetyl CoA derived from MCD was incubated in the presence of [ C] oxaloacetate and citrate synthase (0.73 μU/μL) to form citrate. The [ Cjoxaloacetate was initially produced by a 20 minute transamination reaction performed at room temperature utilizing L-[ C(U)]aspartate (2.5 μCi/ml) and 2-oxoglutarate (2 mM). One ofthe advantages ofthe assay was that extensive purification of MCD was not required prior to assay, and that in vitro measurements of enzyme activity reflected the rate of enzyme activity in vivo. To initiate the MCD assay, heart homogenates or mitochondrial preparations were incubated in a 210 μl reaction mixture (0.1 M Tris, pH 8.0; 0.5 mM dithiothreitol; 1 mM malonyl CoA) for 10 minutes at 37°C, in the presence or absence of NaF (50 mM) and NaPPi (5 mM). The reaction was stopped by the addition of 40 μl of perchloric acid (0.5 mM), neutralized with 10 μL of 2.2 M KHCO 3 (pH 10) and centrifuged at 10,000 x g for 5 minutes to remove precipitated proteins. The incubation ofthe heart sample with malonyl CoA allowed for the conversion of malonyl CoA to acetyl CoA which was then combined with [ Cjoxaloacetate (0.17 μCi/ml) to produce [ Cjcitrate. Unreacted [ C] oxaloacetate was then removed from the reaction mixture by the addition of sodium glutamate (6.8 mM) and aspartate aminotransferase (0.533 μU/μl), followed by a 20 minute incubation at room temperature. This allowed for transamination of unreacted [ C] oxaloacetate back to [ C]aspartate. The resulting solution was then stirred in a 1 :2 suspension of Dowex 50W-8X (100-200 mesh) and centrifuged at 400 x g for 10 minutes. The pelleted Dowex fraction removed [ Cjaspartate, while the supernatant contained [ C]citrate. The supernatant fraction was then counted for C present in the form of [ C]citrate. The amount of acetyl CoA produced by MCD was then quantified by comparison to acetyl CoA standard curves which had been subjected to the identical assay conditions as described above. A standard acetyl CoA concentration curve was run with each experiment. These curves were always found to be linear (r = 0.99). Determination Of CoA Esters: CoA esters were extracted from powdered tissue into 6 % perchloric acid and measured using the modified high performance liquid chromatography (HPLC) procedure described earlier [Lopaschuk et al. , J. Biol. Chem. 269:25871-25878 (1994)]. Essentially, each sample (100 μl each) was run through a precolumn cartridge (C18, size 3 cm, 7 μm) and a Microsorb short-one column (type C18, particle size 3 μm, size 4.6 x 100 mm) on a Beckman System Gold HPLC. Absorbance was set at 254 mM and flow rate at 1 ml/minute. A gradient was initiated using buffer A (0.2 M NaH2PO , pH 5.0) and buffer B (0.25 M NaH2PO and acetonitrile, pH 5.0) in a ratio of 80:20 (v/v). Initial conditions (97 % A and 3 % B) were maintained for 2.5 minutes and were changed thereafter to 18 % B over 5 minutes using Beckman's curve 3. At 15 minutes the gradient was changed linearly to 37 % B over 3 minutes and subsequently to 90 % B over 17 minutes. At 42 minutes the composition was returned linearly back to 3 % B over 0.5 minutes, and at 50 minutes column equilibration was complete. Peaks were integrated by Beckman System Gold software package. Acetyl-CoA Carboxylase Assay: Approximately 200 mg of frozen heart tissue was homogenized, centrifuged and dialyzed as previously described [Lopaschuk et al, J. Biol. Chem. 269:25871-25878 (1994)] 25 μl of dialyzate was added to a reaction mixture (final volume 160 μl) containing 60.6 mM Tris acetate, pH 7.5; 1 mg/ml bovine serum albumin; 1.32 μM β-mercaptoethanol; 2.12 mM ATP; 1.06 mM acetyl CoA; 5.0 mM magnesium acetate; 18.2 mM NaHCO 3 ; and 10 mM magnesium citrate. Samples were incubated at 37°C for either 0, 1, 2, 3, or 4 minutes and the reaction stopped by the addition of 25 μl of 10 % perchloric acid. Samples were then spun for 2 minutes at 13, 000 rpm and the malonyl CoA concentration in the supernatant measured using the HPLC procedure described above. Western Blot Analysis: Samples were subjected to either non-denaturing polyacrylamide gel electrophoresis or to SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose as described [Lopaschuk et al, J. Biol. Chem. 269:25871-25878 (1994)]. Membranes were immunoblotted with either rat anti-MCD antibody or to bovine anti-catalase antibody (Chemicon) in 1 % milk powder. The antibodies were visualized using the Amersham Enhanced Chemiluminescence Western blotting and Detection System as described by the manufacturer. Heart Perfusions: Isolated perfused hearts were obtained either from newborn New Zealand White rabbits (1-day and 7-days following birth) or from adult Sprague-Dawley rats. One-day old rabbit hearts were isolated and perfused, as described previously, at a coronary perfusion pressure of 30 mm Hg [Lopaschuk et al, J. Biol. Chem. 269:25871-25878 (1994)]. Seven-day old hearts were perfused, as described previously, at a 7.5 mm Hg left atrial preload and 30 mm Hg aortic afterload [Lopaschuk et al, J. Biol. Chem. 269:25871-25878 (1994)]. Rat hearts were perfused at a 11.5 mm Hg left atrial preload and 80 mm Hg aortic afterload [Lopaschuk et al, J. Biol. Chem. 269:25871-25878 (1994)]. The newborn rabbit hearts were perfused with Krebs'-Henseleit solution containing 3 % bovine serum albumin, 0.4 mM [9,10- C]palmitate, 11 mM glucose and 100 μU/ml of insulin. Hearts were perfused for a 40 minute period and fatty acid oxidation was measured as described below. The rat hearts were perfused with Krebs'-Henseliet solution containing 11 mM glucose, 1.2 mM palmitate, and 100 μU/ml insulin. Hearts were perfused for either a 60 minute aerobic period, a 30 minute aerobic and 30 minute global ischemic period, or a 30 minute aerobic, 30 minute global ischemia and 60 minute reperfusion period. At the end of all perfusions, hearts were frozen with tongs cooled to the temperature of liquid N 2 . Frozen ventricular tissue from perfused hearts was weighed and powdered in a mortar and pestle cooled to the temperature of liquid N 2 . A portion ofthe powdered tissue was used to determine the dry-to-wet weight ratio ofthe ventricles. The atrial tissue remaining on the cannula was removed, dried in an oven for 12 hours at 100°C, and weighed. With the dried atrial tissue, total frozen ventricular weight, and the ventricular dry-to-wet weight ratio, the total dry weight ofthe heart was determined. Measurement Of Palmitate Oxidation: Steady state rates of palmitate oxidation were measured in both newborn rabbit hearts and in aerobic and reperfused ischemic hearts by quantitatively collecting ι 4 CO 2 produced from hearts perfused with [ " C]palmitate (approximately 50,000 dpm/ml buffer). Collection of CO 2 released as gas in the oxygenation chamber and the CO 2 trapped in the NaHCO 3 in the perfusate was performed as described previously [Lopaschuk et al, J. Biol. Chem. 269:25871-25878 (1994)]. Statistical Analysis: The unpaired t-test was used for the determination of statistical difference of two group means. For groups of three, analysis of variance followed by the Nemine-Keels test was used. A value of p < 0.05 was considered significant. All data presented are represented as mean ± standard error ofthe mean (S.E.M.). 2. Preparation Of Rat Liver/Pancreas Malonyl CoA Decarboxylase Male Sprague-Dawley rats (300-350 g) were anesthetized with sodium pentobarbitol, and the liver/pancreas were quickly excised. 50 rat livers were minced into 1 mm cubes and rinsed with ice cold MSE buffer (225 mM mannitol; 75 mM sucrose; 1 mM EGTA, pH 7.5). A ratio of 2:1 of MSE buffer to tissue was used per liver and then homogenized using 5 strokes of a glass homogenizer with a teflon pestle. The homogenate was then diluted with MSE to 10 ml/g of wet tissue and centrifuged at 480 x g for 5 minutes. The supernatant was filtered through two layers of cheese cloth and then centrifuged at 10,000 x g for 30 minutes. The pellet was then resuspended in 10 mM sodium phosphate buffer, pH 7.6, containing 0.5 mM dithioerythritol (DTE) (5 mL/g of wet mitochondrial pellet). Triton X-100 was added to the suspension (0.1 %) and then the slurry was stirred at 4°C for 16 hours to lyse the mitochondria. The solution was then centrifuged at 24,000 x g for 10 minutes, the supernatant saturated to 40 % with (NH 4 ) 2 SO 4 and stirred for 1 hour on ice. The precipitated proteins were removed by centrifugation at 24,000 x g for 10 minutes. The supernatant was saturated to 55 % with (NH 4 ) 2 SO 4 , stirred for 1 hour on ice, and centrifuged at 24,000 x g for 10 minutes. The pellet, enriched in MCD activity, was resuspended in a 0.1 M potassium phosphate (pH 7.4), 1 mM DTT and 1 M (NH 4 ) 2 SO 4 buffer in preparation for the first column. Malonyl CoA Decarboxylase Assay: A fluorometric assay which follows the formation of acetyl CoA from malonyl CoA in a coupled assay using citrate synthase and malate dehydrogenase was used to measure MCD activity. Reaction mixtures of 1.4 mL contained 0.1 M Tris-HCL, pH 8.0, 1 mM DTE, 0.01 M malic acid, 0.17 mM NAD + , 0.136 mM malonyl CoA, 11 U malate dehydrogenase, 0.44 U citrate synthase. The reaction was initiated by the addition of varying amounts of MCD depending on enzyme activity (10 - 50 μL). The reaction measured the formation of NADH from NAD + over a 4 minute time period using a Shimazdu spectrofluorometer RF-5000. The formation of NADH was measured at an excitation wavelength of 340 nm and an emission wavelength of 460 nm [Sherwin and Natelson, Clin Chem 21 :230-234 (1975)]. A radiometric MCD assay was also used for MCD activity measurements in whole tissue [Dyke et al, Am J Physiology 275:H2122-H2129 (1998)]. Acetyl CoA derived from MCD was incubated in the presence of [ Cjoxaloacetate and citrate synthase (0.73 μU/μL) to form citrate. The [ C]oxaloacetate was initially produced by a 20 minute transamination reaction performed at room temperature utilizing L- [ 14 C(U)]aspartate (2.5 μCi/ml) and 2-oxoglutarate (2 mM). To initiate the MCD assay, preparations were incubated in a 210 μl reaction mixture (0.1 M Tris, pH 8; 0.5 mM dithiothreitol (DTT); 1 mM malonyl CoA) for 10 minutes at 37° C, in the presence or absence of NaF (50 mM) and NaPPi (5 mM). The reaction was stopped by the addition of 40 μl of perchloric acid (0.5 mM), neutralized with 10 μL of 2.2 M KHCO 3 (pH 10) and centrifuged at 10,000 x g for 5 minutes to remove precipitated proteins. The incubation ofthe heart sample with malonyl CoA allowed for the conversion of malonyl CoA to acetyl CoA which was then combined with [ 14 C]oxaloacetate (0.17 μCi/ml) to produce [ C] citrate. All reactions were carried out in the presence of N-ethylmaleimide which removes excess CoA remaining in the latter stages ofthe reaction so that the citrate present cannot generate non-MCD derived acetyl CoA. Unreacted [ C] oxaloacetate was removed from the reaction mixture by the addition of sodium glutamate (6.8 mM) and aspartate aminotransferase (0.533 μU/μl), followed by a 20 minute incubation at room temperature. This allowed for transamination of unreacted [ C] oxaloacetate back to [ Cjaspartate. The resulting solution was then stirred in a 1 :2 suspension of Dowex 50W-8X (100-200 mesh) and centrifuged at 400 x g for 10 minutes. The pelleted Dowex fraction removed [ C]aspartate, while the supernatant contained [ Cjcitrate. The supernatant fraction was then counted for C present in the form of [ C] citrate. The amount of acetyl CoA produced by MCD was then quantified by comparison to acetyl CoA standard curves which had been subjected to the identical assay conditions as described above. A standard acetyl CoA concentration curve was run with each experiment. These curves were always found to be linear (r = 0.99) (data not shown). Chromatography On Butyl Sepharose 650M: The MCD solution adjusted to 1 M (NH ) 2 SO 4 was loaded onto a 100 mL Butyl-Sepharose 650 M column ( 2.6 cm x 40 cm) previously equilibrated with a 0.1 M potassium phosphate buffer (pH 7.4) containing 1 mM DTT and 1 M (NH ) SO 4 . The column was washed with 3 column volumes ofthe above buffer and proteins were eluted with a gradient of 1 to 0 M (NH 4 ) 2 SO 4 in a total volume of 150 mL with 9 mL fractions being collected. The fractions containing MCD activity were pooled and brought to 100 mL in a 0.1 M potassium phosphate (pH 7.4), 1 mM DTT and 1 M (NH 4 ) 2 SO buffer. Chromatography On Phenyl Sepharose HP: The enzyme solution was loaded onto a 25 mL Phenyl Sepharose HP column (2.6 cm x 20 cm) previously equilibrated with a 0.1 M potassium phosphate buffer (pH 7.4) containing 1 mM DTT and 1 M (NH ) 2 SO buffer. The column was washed with 3 column volumes ofthe above buffer and proteins were eluted with a gradient of 1 to 0 M (NH 4 ) 2 SO 4 in a total volume of 175 mL with 9 mL fractions being collected. The fractions containing MCD activity were pooled and concentrated by ultrafiltration. The resulting 1 mL solution was brought to 50 mL in a 20 mM Bis-Tris, pH 7 buffer containing 1 mM DTT. Chromatography On Q Sepharose HP: The enzyme solution was applied to a 10 mL Q Sepharose HP column ( 1.5 cm x 20 cm) previously equilibrated with a 20 mM Bis-Tris, pH 7 buffer containing 1 mM DTT. The column was washed with 3 column volumes ofthe same buffer and bound proteins were eluted with a gradient of 0 to 0.3 M NaCl in a total volume of 80 mL. 4 mL fractions were collected and the fractions containing MCD activity were pooled and concentrated by ultrafiltration. The concentrated pool was resuspended in 50 mL of a 50 mM MES (pH 5.5) buffer containing 1 mM DTT. Chromatography On SP Sepharose HP: The enzyme solution was applied to a 5 mL SP Sepharose HP column (1.5 cm x 20 cm) previously equilibrated with a 50 mM MES (pH 5.6) buffer containing 1 mM DTT. The column was washed with 3 column volumes ofthe same buffer and bound proteins were eluted with a linear gradient of 0 to 0.4 M NaCl in a total volume of 150 mL. 4 mL fractions were collected and the fractions containing MCD activity were pooled, neutralized to pH 7.0, and concentrated by ultrafiltration. The concentrated pool was resuspended in 5 mL of 50 mM malate (pH 5.6) buffer containing 1 mM DTT. Malonyl CoA Affinity Elution From SP Sepharose HP: The enzyme solution was re-applied to a 0.5 mL SP sepharose HP column (0.5 cm x 10 cm) previously equilibrated as above. The column was washed with 50 mM malate (pH 5.6) buffer containing 1 mM DTT to remove unbound proteins and then eluted with a 50 mM malate (pH 5.6) buffer containing 1 mM DTT and 10 μM malonyl CoA. The eluted fractions were neutralized to pH 7.0. The affinity elution protocol allowed for highly specific elution of MCD. Protein Sequencing: The affinity eluted protein fraction from the second SP Sepharose HP column was resolved on a 9 % SDS- polyacrylamide gel and stained for visualization using a Coomassie blue solution. The major protein of approximately 52 kDa was thought to be MCD and was excised from the gel and subjected to an endoLys C digest. The protein digest was subjected to HPLC and the appropriate peptides underwent amino acid N-terminal sequencing. The amino acid sequences of two internal peptides were obtained. Protein digestion, and amino acid N-terminal sequencing was performed by Eastern Quebec Peptide Sequencing Facility (Quebec, Canada). Antibody Production Against Rat Liver MCD: The protein which was affinity eluted from the SP-sepharose column was subjected to SDS-PAGE, Coomassie stained and the 52 kDa band was cut from the gel. The protein (3 μg) was eluted from the gel fragment and injected into rabbits. Similarly, another fraction from the affinity elution of the S-sepharose column protein (6 μg) was injected into rabbits without undergoing the gel separation step. The rabbits were injected at 2 week intervals for 2 months before the serum was used. 3. Probe Synthesis, Library Screening And DNA Sequencing Of Rat Pancreatic β-cell MCD RT-PCR was made on INS-I cells total mRNA with the Superscript reverse transcriptase and Taq polymerase (GLBCO) according to the supplier's protocols. The annealing temperature was 55 °C. For PCR, degenerated oligonucleotides were designed from the goose MCD sequence [Jang et al, JBiol Chem 264:3500-3505 (1989)]. The sense primer positioned at base 547 ofthe goose sequence was 5'GANTSTGARGCTGTGCAYCCTGT3' (SEQ LD NO:4); the antisense primer, starting at base 1102 was 5 ACARRTACCAGGCRCACARYCTCAT3* (SEQ LD NO:5). The 580 base pair fragment was subcloned in pBluescript (Stratagene), sequenced, restriction-enzyme digested and purified to be used as a probe. A pancreatic β (INS) cell cDNA library [Bonny et al, JBiol Chem 273 : 1843- 1846 (1998)] constructed in Lambda ZAPExpress (Stratagene) was screened using the purified 580 bp PCR fragment labeled with -[ P]-dCTP by random priming. After two rounds of screening, four clones were in vivo excised and assayed for MCD activity in 293 cells 24 h after DNA transfection (5 μg/10 cells) with the calcium phosphate precipitate technique [24]. The one bearing the highest enzymatic activity was sequenced in both directions using the deletion technique (Erase-a-Base, Promega) and the dye-primer sequencing strategy (Autoread Sequencing Kit and ALF DNA sequencer from Pharmacia). The size ofthe DNA was 2020 bp. An additional positive clone was sequenced to confirm the sequence obtained with that bering high enzymatic activity upon transfection in 293 cells. DNA transfection ofthe β-galactosidase gene under the control ofthe CMV promoter served as a negative control for MCD activity and as positive control for the efficiency of transfection (about 20 %). Ins-1 cells [Asfarai et al, Endocrinology 130:167-178 (1992)]and human kidney 293 cells [Becker et al, J Biol Chem 269:21234-21238 (1994)] were cultured as described in the quoted references. β-cell MCD mRNA Measurements: Northern blot analysis was carried out using standard techniques for the gel preparation and transfer [Sambrook et al, Molecular Cloning, Cold Spring Harbor Laboratory Press, New York (1989)]. Briefly, 20 μg of total mRNA per lane were separated on a 1 % agarose gel in the presence of formaldehyde, transferred by capillarity onto Zeta probe QT membrane (Biorad), fixed with UV light, prehybridized, hybridized in the presence ofthe labeled 580 bp MCD cDNA fragment described above, and washed according to the supplier's protocol. The signals were revealed with a Fuji phosphorimager. Control hybridization with an 18S ribosomal precursor probe (0.77 kb EcoRl-BamHl fragment, position 1-1780, of mouse 18S ribosomal rRNA cDNA subcloned in pUC830) was done on the same membrane confirming the ethidium bromide staining ofthe RNA. RT-PCR was performed using total RNA obtained from rat tissues by the guanidinium isothiocyanate acidic phenol technique [Chomczinsky and Sacchi, Anal. Biochem 162: 156-160 (1987)]. Reverse transcription was done with 5 μg RNA as described above. cDNAs from sorted pancreatic α- and β-cells were prepared as described in reference [Heimberg et al, Proc Nat Acad Sci USA 93:7036-7041 (1996)]. PCR reactions were performed with one third ofthe RT reaction using the same primers and conditions as described above. The conditions were compared to PCR made on the same material with rat β-actin oligonucleotides primers [Heimberg et al, Proc Nat Acad Sci USA 93:7036-7041 (1996)]. Reactions were monitored after 15, 25 and 35 cycles and analyzed on 1 % agarose gels stained with ethidium bromide. Cloning Of Rat Liver/Heart MCD: Oligonucleotides based on the cloned sequence ofthe rat β-cell MCD [Voilley et al, Biochem J Submitted (1999)] were designed to amplify a 900 base pair product of MCD from rat liver cDNA using polymerase chain reaction (PCR). The forward primer, PMCDF1 (5TGGTCGACGGCTTCCTGAACCTG; SEQ ID NO:6) was used with the reverse primer, PMCDR (5'TCCCTAGAGTTTGCTGTTGCTCTG; SEQ ID NO:7) in a PCR reaction. This 900 bp fragment was used as a probe for screening a Superscript rat liver cDNA library (Gibco, Life Technologies), The full length cDNA clone was sequenced in both directions using internal primers and compared to the rat islet cDNA sequence. Rat heart MCD DNA was cloned in a similar manner using techniques known to those skilled in the art. Immunocytochemistry: To determine the cellular localization of MCD, a double labeling immunocytochemical technique was used. McArdle RH-7777 hepatoma cells were grown on sterilized glass coverslips (60 % confluency) in α-MEM supplemented with 20 % Fetal Bovine Serum, pH 7.4 and incubated in a humidified atmosphere supplemented with 5 % CO 2 at 37 °C. Anti-MCD antibody raised in rabbit was used against the MCD antigen and anti-heat shock protein (Hsp) 60 antibody, raised in mouse, was used against the Hsp 60 antigen. The Hsp 60 is predominantly a mitochondrial specific matrix chaperon protein [Stuart et al, Trends Biochem Sci 19:87-92 (1994)]. At 60 % confluency, the coverslips were washed 3 times in 1 x phosphate buffered saline (PBS), pH 7.4 followed by fixation in 4 % paraformaldehyde for 10 minutes. The fixation reaction was terminated by replacing the fixative with 100 mM glycine in 1 x PBS for 15 minutes. The cells were then washed with two changes of 0.1 % Triton-X 100 and 0.1 % BSA in 1 x PBS (TA-PBS) for 1 minute each, and then permeabilized by incubating in the same medium for 30 minutes. The coverslips were then washed with 3 changes of TA-PBS and then blocked in 5 % fetal bovine serum for 20 minutes to prevent non-specific binding of antibodies of choice. Following 3 washes in TA-PBS, coverslips were incubated for 2 hours at room temperature (RT) with rabbit polyclonal anti- MCD antibody (dilution 1:100). At the end of this reaction, the coverslips were washed with 3 changes of TA-PBS and incubated in mouse monoclonal anti Hsp 60 antibody (dilution 1 : 1000; Stress Gen Biotechnologies Corp., Canada) for 1 hr at RT. Following this, coverslips were washed 3 times with 1 x PBS, pH 7.4 and reacted with goat anti-mouse rhodamine (Rh; Jackson Immuno Research Laboratories, U.S.A.) conjugate (dilution 1 :200) and goat anti-rabbit fluorescein isothiocyanate (FITC; Jackson Immuno Research Laboratories, U.S.A.) conjugate (dilution 1 :200) for 1 hour at RT. At the end ofthe secondary antibodies reaction, the cover slips were washed in 1 x PBS, pH 7.4 and mounted in 50 % glycerol containing 1 % propyl gallate, an anti-fluorescence photobleacher, on microscope slides and stored in the dark. All slides were examined with an Olympus fluorescent microscope and/ or with a laser confocal microscope (Leica) connected to a Silicon Graphics computer. In other experiments, Mito-Tracker Red CMXRos (Molecular Probes), a novel mitochondria-selective dye, was used to label mitochondria in cultured cells. At 60 % confluency, the medium was replaced with prewarmed medium (37°C) containing 200 nM of Mito-Tracker Red CMXRos and incubated for 20 min in a humidified atmosphere supplemented with 5 % CO at 37° C. The cells were then washed in 1 x PBS and fixed in 4 % paraformaldehyde for 10 min and processed for probing with anti-MCD antibodies to localize MCD within the cell as described above. Western Blot Analysis: Samples were subjected to SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose as described [Dyck et al, Am J Physiology 275:H2122-H2129 (1998)]. Membranes were immunoblotted with anti- MCD antibodies (1 :500 dilution) in 1 % milk powder. The antibodies were visualized using the Pharmacia Enhanced Chemiluminescence Western blotting and Detection System. Statistical Analysis: The unpaired t-test was used for the determination of statistical difference of two group means. For groups of three, analysis of variance followed by the Neuman-Keuls test was used. A value of p < 0.05 was considered significant. All data are presented as mean + standard error ofthe mean (S.E.M.). EXAMPLE 1 In this example, MCD was characterized in the rabbit heart. Use of previously described assays, such as the spectrophotometric assay to study the activity of heart MCD activity proved to be extremely variable, and high background values were obtained with heart tissues [Kolattukudy et al, Meth. Enzymol. 71 : 150-163 (1981)]. The assay also did not have the sensitivity to accurately measure MCD in the heart tissue, with the spectrophotometric readings routinely being very close to high background absorbance values. Also, the radiotracer assay which utilizes C-malonyl CoA is an expensive assay. Hence, a new assay was developed which quantified the amount of acetyl CoA formed by MCD (see Methodology section). The assay was optimized for use in heart homogenates, as well as for isolated mitochondrial preparations. A tissue protein standard curve using heart homogenates demonstrated that 2 mg of tissue produced MCD activity which was linear for up to 20 minutes of incubation. Similarly, a time course standard curve using a 10 minute incubation time produced rates that were linear when 0 to 10 mg of tissue were used. Therefore 2 mg of tissue homogenate and a 10 minute incubation period was used. This gave acetyl CoA values that were in the middle ofthe acetyl CoA standard curve. Similar experiments for time and protein dependence were also performed for the isolated mitochondrial preparations. Using the MCD assay, the existence of MCD activity was identified in heart homogenates obtained from rats (Table 1). Rates of MCD activity were found to be significantly higher than previously observed rates of ACC [Lopaschuk et al, J. Biol. Chem. 269:25871-25878 (1994)]. Since malonyl CoA synthesis from ACC is under phosphorylation control, experiments were also performed to determine if MCD was under phosphorylation control. Isolation of tissue under conditions that preserved the phosphorylated state ofthe enzyme did not alter rates of MCD activity compared to enzyme isolated under conditions which did not attempt to preserve the phosphorylation state seen in vivo (Table 1). However, incubation of heart homogenates in vitro with alkaline phosphatase resulted in an increase in MCD activity. This suggested that MCD is under phosphorylation control, with phosphorylation ofthe enzyme resulting in a decrease in enzyme activity. Crude homogenates, isolated mitochondria or the post mitochondrial supernatant were also assayed for MCD activity (see Table 2). Measurable levels of MCD activity were detected in all these heart fractions. To determine whether the levels of MCD activity observed in these fractions was due to the MCD being localized to the mitochondrial matrix as previously suggested [Jang, S-H. et al, J. Biol. Chem. 264:3500-3505 (1989)], the same fractions were treated with octylglucoside. With the addition of octylglucoside, the relative level of MCD activity TABLE 1 Malonyl CoA Decarboxylase Activity In Aerobically Perfused Rat Hearts Values are the mean ± S.E. of at least 4 hearts. Heart homogenates were assayed for MCD activity in the presence or absence of NaF and NaPPi and with or without alkaline phosphatase treatment.*, indicates significant differences between groups of hearts. TABLE 2 Values are the mean ± S.E. The malonyl CoA content and ACC activity were measured as descπbed previously (7) and the palmitate oxidation rates were determined as outlined in the "Methods" section. *, indicates significant differences between appropriate groups. TABLE 3 Malonjl CoA Content, Acetyl CoA Carboxylase Activity, Cardiac Work, Palmitate Oxidation Rates And Palmitate Oxidation Rates/Cardiac Work In Aerobic And Reperfused Adult Rat Hearts and cardiac work were measured as described λ'alues for malonyl CoA content and ACC indicates significant differences between appropπate groups co = cardiac wor , psp = pea sjsto ic pressure an cw = car iac wor in the crude homogenate increased approximately 6 fold In isolated mitochondrial fractions a 14 fold increase in MCD activity was seen compared to the untreated isolated mitochondrial fractions This increase in MCD activity strongly suggested that the solubilization ofthe mitochondrial membrane allows the MCD to be released from the matrix or exposed to the extra-mitochondrial space This confirmed that a large proportion ofthe heart MCD is localized to the mitochondrial matrix To characterize the physical properties of MCD, Western blot analysis was performed using an MCD antibody [Kim and Kolattukudy, Arch. Biochem. Biophys. 190 234-246 (1978)] In rat heart and liver tissue, this antibody cross reacted with high affinity with at least one other protein Since this other protein was found to be catalase, the results were further clarified with the MCD antibody using an antibody directed against catalase Rat liver MCD was partially purified using the procedure described above The partially purified protein was loaded onto two lanes of a non-denaturing gel and subjected to electrophoresis After electrophoresis, one lane was transferred to nitrocellulose and blotted with MCD antibody, while the other lane was stored in water for future use In Western blots from a non-denaturing gel (see Figure 1 A), the MCD antibody reacted with one large band which is approximately 160-190 kDa as predicted by Kim and Kolattukudy, Arch. Biochem. Biophys. 190 234-246 (1978) The position ofthe band was aligned with the non transferred lane ofthe non-denatured gel and the region containing MCD was extracted from the gel The protein from the gel slice was eluted (so as to create a substantially pure preparation) and either assayed for MCD activity or subjected to SDS-PAGE, transferred to nitrocellulose, and blotted with the anti-MCD antibody (see Figure 1 A, lane 2) The eluted protein exhibited quite large MCD activity and when subjected to Western analysis, reacted with the MCD antibody The blot was then stripped and re-probed with anti catalase antibody to ascertain which band corresponded to the catalase protein(s) (Figure 1, lane 3) This experiment determined that the molecular weight of rat liver MCD is approximately 45 kDa This result is consistent with previous conclusions that the native enzyme is a tetramer Kim and Kolattukudy, Arch. Biochem. Biophys. 190 234-246 (1978) Western blot analysis was also performed on denatured semi-purified heart samples (see Figure IB) Under these conditions the 45 kDa band was identified in both liver and heart tissue samples (Figure IB, lanes 1 and 2). If samples were treated with alkaline phosphatase and then subjected to SDS-PAGE and western blot analysis, there was no shift in molecular weight ofthe protein. This example clearly demonstrates MCD activity in control hearts, and identifies a cardiac isoform of MCD, of approximately 45 kDa in size, that can be phosphorylated and inhibited by a yet unidentified kinase. EXAMPLE 2 In this example, the role of MCD in regulating fatty acid oxidation is described. Previous studies have demonstrated that myocardial fatty acid oxidation increases dramatically between 1-day and 7-day rabbit hearts [Lopaschuk et al, J. Biol. Chem. 269:25871-25878 (1994)]. As shown in Table 3, a significant increase in fatty acid oxidation rates is seen following birth in the 7-day old hearts compared to 1-day old hearts. This was accompanied by a decrease in malonyl CoA levels and an decrease in ACC activity. However, to decrease malonyl CoA levels in the heart, a decreased rate of synthesis would have to be accompanied by a simultaneous degradation of malonyl CoA or metabolic utilization. MCD activity was therefore measured in 1-day and 7-day old rabbit hearts. As shown in Figure 2, the MCD activity in 7-day old rabbit hearts was significantly elevated compared to 1-day old hearts. In light ofthe amount of malonyl CoA present, these high rates of MCD activity suggested a rapid turnover of malonyl CoA. If heart extracts were dephosphorylated in vitro with alkaline phosphatase no change in MCD activity was observed. This suggested that unlike adult rat hearts, MCD activity is not under the same degree of phosphorylation control as seen in newborn rabbit hearts. Reperfusion of adult rat hearts following a 30 minute period of global no-flow ischemia (global no-flow ischemia shall be defined as the cessation of blood flow to all parts ofthe rat heart for the specified time period) resulted in a dramatic drop in malonyl CoA levels (data not shown). A parallel decrease in ACC activity was observed as well as a slight increase in fatty acid oxidation rates. However, since cardiac work is significantly inhibited during reperfusion of ischemic hearts, a large increase in fatty acid oxidation per unit work was observed. Measurement of MCD activity in aerobic, ischemic and reperfused ischemic hearts is graphically shown in Figure 3. Levels of MCD activity were maintained at the end of ischemia and/or reperfusion. Treatment of samples with alkaline phosphatase also resulted in an increase in MCD in all groups. Figure 4 shows the MCD protein levels in samples extracted from aerobic, ischemic and ischemic/reperfused rat hearts. Levels of MCD protein were not altered during the perfusion protocols, nor did the molecular weights ofthe MCD shift between the different groups, suggesting that MCD is not modified post-translationally by phosphorylation. Also, when whole heart extracts were used for Western analysis, another protein of a slightly larger molecular weight was detected than that seen with semi-purified MCD from mitochondria (see Figure 4, lanes 3-8). It is likely that samples which contain cytoplasmic extracts, instead of purely mitochondrial extracts, may also possess the cytoplasmic form of MCD. In the goose uropygial gland, the cytoplasmic form of MCD is approximately 55 kDa while the mitochondrial form is processed post-translationally into a 50 kDa molecular weight protein [Courchesne-Smith et al, Arch. Biochem. Biophys. 298:576-586 (1992)]. Western blot analysis suggested that similar processing may occur in the rat heart. EXAMPLE 3 In this example, based on the results shown in Figure 3, a model which may contribute to enhanced myocardial ischemic injury has been proposed in Figure 5B. During ischemia AMPK is activated thereby inactivating ACC upon reperfusion, resulting in a decrease in malonyl CoA synthesis [Lopaschuk et al, J. Biol. Chem. 269:25871-25878 (1994)]. In the presence of a maintained MCD activity a reduction of malonyl CoA levels occurs in the heart. With the fall in malonyl CoA levels, CPTl becomes more active due to the removal of its inhibitor. This results in an increase in fatty acid oxidation rates, which leads to an enhanced ischemic injury. This model implicates MCD as being an important contributing factor to injury during reperfusion of ischemic hearts. As a result, pharmacological modification of MCD may also prove to be a beneficial approach to treating ischemic heart disease. Thus, the present invention demonstrates that the heart expresses a 45 kDa isoform of MCD which is important in regulating myocardial malonyl CoA levels. An increase or maintained MCD activity in conjunction with a decrease in ACC activity can result in a decrease in malonyl CoA levels and an increase in fatty acid oxidation in the heart. It is contemplated that drug screening by using the novel MCD composition and assay ofthe present invention will help to identify such potential compounds. Such inhibitors are contemplated to be useful in the treatment of ischemia. EXAMPLE 4 In this example a purification scheme for rat liver MCD was developed which is substantially different from that originally described by Kolattukudy et al. [Kolattukudy et al, JBiol Chem 71 : 150-163 (1981)]. The butyl sepharose and the phenyl sepharose columns are hydrophobic resins which will separate proteins based on hydrophobicity. Since MCD is eluted from the column quite late, it suggests that the interaction between the column and MCD is quite strong. The Q-sepharose column is a strong anion exchange resin and as the salt gradient is increased two peaks of MCD activity separate at different ionic strengths (Figure 7B). The MCD contained in these two peaks possess identical kinetic characteristics but are distinctly separated. Since our goal was to purify large enough quantities of MCD for specific down stream applications, we pooled all the active fractions from both peaks to run on the next column. These fractions were applied to a SP-sepharose cation exchange column. In this case, MCD eluted relatively early in the salt gradient, indicating that the interaction with the resin was weak (Figure 7C). Due to this weak interaction with the SP-sepharose column, MCD could easily be eluted from this resin by its negatively charged substrate, malonyl CoA. This presumably altered the conformation and/or the net charge of MCD such that it could no longer associate with the SP-sepharose resin at pH 5.5. A summary ofthe purification scheme is shown in Figure 7. This newly designed scheme allowed MCD to be purified more than 1200 fold from the initial 55 % ammonium sulfate pellet (Table 4). Due to the inability of our fluoromertric assay to detect MCD activity from whole cell extracts the purification profile only represents the increase in purity beginning from the mitochondrial preparation. For this reason we are confident that we have actually purified MCD activity to a much greater extent than we have indicated. When pooled fractions from the columns were subjected to SDS-PAGE analysis and stained using a sensitive Coomassie dye, we detected two proteins with molecular weights of 52 and 65 kDa. Previous work on mammalian MCD has suggested that the 52 kDa protein we observed was MCD [Dyck et al, Am J Physiology 275:H2122-H2129 (1998); Voilley et al, Biochem J Submitted (1999)]. Immunoblotting experiments revealed that the second protein (which co-purifies with MCD) was catalase, suggesting that our initial mitochondrial isolation also contained peroxisomes. EXAMPLE 5 In this example oligonucleotides based on the cloned sequence of rat islet MCD cDNA [Noilley et al, Biochem J Submitted (1999)] where designed to amplify a 900 base pair product of MCD from rat liver cDΝA using PCR. This 900 bp fragment was used as a probe in the screening of a rat liver cDΝA library. A full length (2.2 Kb) clone was obtained, sequenced in both directions and compared to the rat islet MCD sequence. The cD A sequence encoding rat liver MCD is shown in Figure 8. Sequence comparison between liver and islet forms of MCD reveals that the two forms are identical (not shown). The potential phosphorylation sites are still present (Figure 8) along with the potential peroxisomal targeting sequence (SKL). The two peptide sequences obtained from amino acid sequence analysis are numbered and underlined in bold. A leucine zipper motif has also been identified (underlined and black dots) which may be responsible for the protein-protein interaction required for MCD tetrameric structure similar to phenylalanine hydroxylase [Hufton et al, Biochem Biophys Acta 1382:295-304 (1998)]. EXAMPLE 6 In this example rat liver MCD was characterized. The fractions from the affinity elution ofthe SP-sepharose column which demonstrated the highest MCD specific activity were pooled and aliquoted. The first aliquot was subjected to SDS-PAGE. The 52 kDa band was cut from the gel, digested with endoLys C, subjected to HPLC and the appropriate peptides underwent amino acid N-terminal sequencing. The amino acid sequences of two internal peptides were obtained and compared to the cDNA sequence obtained from the rat liver cDNA clone for confirmation. The second aliquot was used for kinetic analysis. MCD activity was measured at various concentrations of malonyl CoA to determine the Km. Figure 9A demonstrates that two Km's may exist, one at approximately 1-2 M and the other at 30-40 μM as determined by a Lineweaver-Burke plot. This latter value is similar to the published Km obtained from a crude rat liver MCD preparation [Kim and Kolattukudy, Arch Biochem Biophys 190:234-246 (1978)]. A pH dependence curve was also performed to determine the optimum pH at which liver MCD is most active (Figure 9B). In our fluorometric assay the optimum pH is between 7 and 8. Both these results are similar to the existing information available on rat liver MCD [Kim and Kolattukudy, Arch Biochem Biophys 190:234-246 (1978)]. Our earlier work has indicated that cardiac MCD is under phosphorylation control [Dyck et al, Am J Physiology 275:H2122-H2129 (1998)]. Using our purified liver MCD and a wide variety of kinases and phosphatases we were only able to identify alkaline phosphatase as a potential regulator of MCD activity (Table 5). Similar experiments were performed which dephosphorylated and activated MCD using alkaline phosphatase and then tried to inactivate the enzyme using various kinases. All of these experiments proved to be unsuccessful. The kinases or phosphatases tested were casein kinase II, cyclic AMP dependent protein kinase protein kinase C, 5'AMP-activated protein kinase, protein phosphatase 2 A and protein phosphatase 2C. Another group of pooled fractions from the affinity elution ofthe SP-sepharose column was subjected to SDS-PAGE, coomassie stained and the 52 kDa band was cut from the gel. The protein was eluted from the gel fragment and injected into rabbits for the generation of MCD antibodies. Similarly, another fraction from the affinity elution of the S-sepharose column was injected into rabbits without undergoing the gel separation step. This procedure could ensure that we obtained antibodies which were generated against both a denatured and a non-denatured MCD protein. Western blot analysis using rat liver mitochondria revealed that both antibodies recognized a protein with a molecular weight of 52 kDa (Figure 10A). The antibody generated using non-denatured MCD (MCD240) also recognized catalase, while the antibody raised against denatured MCD (MCD239) appeared to be more specific for MCD (Figure 10A). MCD 240 could immunoprecipitate a protein with a molecular weight of 52 kDa, although, MCD activity could not be detected in this immunoprecipitate. To confirm that the antibodies raised against purified MCD were indeed able to recognize MCD in solution, we performed immuno-inhibition studies (Figure 10B). Purified rat liver MCD activity was measured using the fluorometric MCD assay described in the Methods section. The purified MCD was pre-incubated with either MCD239, MCD240 or the appropriate pre-immune serum. After 30 minutes, the mixture was measured for MCD activity and expressed as a percent of MCD pre- incubated without serum for an identical period of time. Figure 10B indicates that only MCD240 was able to inhibit MCD activity. Since MCD240 inhibits MCD activity, the results would seem to explain why MCD240 can immunoprecipitate MCD while not demonstrating MCD activity in the precipitated pellet. EXAMPLE 7 In this example, using Western blot analysis and the MCD240 antibody, the distribution of MCD in a variety of rat tissues was determined. All tissues tested expressed relatively high levels of MCD protein. Oxidative tissues such as liver and heart express the highest levels of MCD protein supporting the concept that MCD may play a role in controlling fatty acid oxidation [Dyck et al, Am J Physiology 275:H2122- H2129 (1998)]. These experiments used detergent solubilized whole tissue homogenate to measure total MCD levels. This prevented a loss of MCD protein based on cellular localization. To determine the subcellular localization of liver MCD we performed double labeling immunocytochemistry on rat hepatoma McArdle RH-7777 cells using two separate approaches. First, antibodies directed against either MCD or a mitochondrial matrix specific protein, Hsp 60 [Stuart et al, Trends Biochem Sci 19:87-92 (1994)] were used. Both fluorescent and confocal analysis showed that MCD co-localized with Hsp 60 (Figure 11B, 1-3). Similar studies using a mitochondrial specific dye (MitoTracker Red CMXRos) arid MCD antibody confirmed these results (Figure 1 IB, 4- 6). It is not clear whether MCD is localized to other organelles other than the mitochondria. It is clear, however, that the much ofthe liver MCD is mitochondrial in nature. EXAMPLE 8 In this example MCD expression and activity were measured in three animal models where alterations in fatty acid metabolism have been previously characterized. Two of these are diabetic models where hepatic fatty acid biosynthesis is inhibited (6 week old streptozotocin diabetic rats) or elevated (JCR:LA-corpulent insulin resistant rats). Table 6 shows the changes seen in both plasma glucose and fatty acid levels in the various rat models. Serum levels of fatty acids rose 3 fold in the 6 week old streptozotocin diabetic rat and 1.75 fold in the JCR:LA-corpulent rat (Cp/Cp) as compared to their lean control groups. Similarly, in the 6 week old streptozotocin diabetic rat, serum glucose levels increased 2.2 fold above their controls. Contrary to this, the levels of glucose in the serum were unaltered in the Cp/Cp rat as compared to the lean controls. These changes in glucose and fatty acid levels in the serum are consistent with the pathophysiology of these animals [Russell et al, Metabolism 43:538- 543 (1994); Topping and Targ, Horm Res 6:129-137 (1975); Mathe Diabete Metab 21: 106-111 (1995)]. MCD activity rose approximately 2 fold in the 6 week old streptozotocin diabetic animal as compared to control rats (Figure 12) while MCD activity was unaltered in the Cp/Cp rats (Figure 13). To determine if changes in activity were due to dephosphorylation of MCD and/or to an increase in the relative abundance ofthe enzyme, western blot analysis was performed using our MCD antibody. The level of MCD protein increased approximately 2 fold in the 6 week streptozotocin diabetic rat livers over control livers, while no changes were observed in the levels of MCD in the livers ofthe Cp/Cp rats (Figure 13). Catalase antibody was used as a control to demonstrate that similar amounts of protein were loaded and transferred in all lanes. Western blot analysis, however, does not reveal any alterations in the level of MCD protein in the Cp/Cp rat (Figure 13). This suggests that changes seen in MCD activity in the Cp/Cp rat may be due to alterations in the phosphorylation state ofthe enzyme and not to the amount of protein. Both phosphorylation control and increased MCD protein levels seem to be involved in regulating MCD activity in the diabetic rat liver. When liver MCD from both 6 week old streptozotocin and Cp/Cp rats are treated with alkaline phosphatase we demonstrated an increased activity ofthe enzyme for all groups (Table 5). Alkaline phosphatase treatment of liver MCD appears to consistently produce MCD enzyme activity at approximately 11,000 - 15,000 nmol/g dry wt " /min " , indicating that alkaline phosphatase treatment is sufficient to produce a maximally active enzyme. Only the streptozotocin diabetic animals have higher MCD activity, which is probably due to a 2- fold increase in MCD protein expression. Another well established protocol for altering fatty acid biosynthesis and oxidation in the liver is to fast and refeed rats [Porter and Swenson, Mol Cell Biochem 53:307-325 (1983); Clarke et al, JNutr 120:218-224 (1990); Witters et al, Arch Biochem Biophys 308:413-419 (1994)]. Table 6 demonstrates the significant decrease in glucose and the increase in serum fatty acid levels at this time. Similarly, MCD activity was also increased during the 48 hour fasted period (Figure 14A). When rats have been deprived of food for 48 hours and then refed for 72 hours, there was a decrease in MCD activity (Figure 14A). The levels of MCD activity begin to approach that of control rats which have not undergone the same fasting or refeeding protocol (Figure 14 A). Although MCD activity was altered during these various nutritional states, the level of MCD protein was not significantly altered (compare to Figure 12). Once again this suggest that the activity of MCD is under post-translational regulation, most probably via phosphorylation. EXAMPLE 9 The cloning ofthe rat pancreatic β-cell cDNA was performed in a two step approach using degenerated oligonucleotides. One ofthe selected pairs tested on INS cells mRNA generated a single product of an expected size of 580 bp as predicted by comparison with the goose cDNA (Figure 15). Sequencing of this fragment indicated 67% identity with the goose nucleotide sequence and no match with any other sequence available on the blast server. This fragment was then used as a probe to screen an INS-1 cell cDNA library. Among the 4 in vzvo-excised clones, only one showed substantial MCD activity after transfection into 293 cells. 293 cells did not express the enzyme at a detectable level either under untransfected conditions or following transfection with the β-galactosidase gene. Sequencing of this clone of 2020 bp revealed an open reading frame of 1473 bases corresponding to an amino acid sequence of 491 residues and a predicted protein of about 52 KDa. A poly-A tail with a polyadenylation signal at the end ofthe 3' non-coding region was also present. Previous studies with the MCD enzyme(s) purified from various mammalian tissues evaluated a global molecular weight of about 170 KDa suggesting that the enzyme might multimerize in vivo. The deduced rat protein sequence shows 69 % identity with the goose sequence from amino acid 39, a methionine, to the end. The nucleotide sequence surrounding this methionine (AGCGCCATGG; SEQ ID NO:8) fairly fits a Kozak consensus site (GCCA GCCATGG; SEQ ID NO: 9), suggesting that it may be a site of translation initiation. A sequence of 38 amino acids in the same reading frame is present on the N- terminal side of this methionine. The first amino acids of this sequence (MRGL), which starts with a methionine, are identical to the goose sequence. However the remaining part of this sequence shows relatively little homology with the goose sequence from amino acid 5 to 38. This portion ofthe rat protein has the features of a mitochondrial targeting sequence since it is rich in positively charged (8 arginines) and hydroxylated amino acids and lacks acidic amino acids. Therefore, it is likely a cleavable NH 2 -terminal targeting sequence belonging to a larger MCD precursor. No common cleavage site (R/K-R-A-X-S-S/T; SEQ ID NO: 10) in the presequence is present around Met39. However another cleavage-site motif (PRLCSG; SEQ ID NO: 11), as defined by Y. Gavel is predicted around position 31, suggesting that the presequence may be removed by matrix proteases and that β (INS) cell MCD is, at least in part,a mitochondrial matrix enzyme. Motif analysis of rat MCD done with the web PSORT and PROSITE programs revealed interesting features. In addition to the NH 2 -mitochondrial matrix targeting sequence, the C-terminal part ofthe enzyme ends with a peroxisomal targeting motif characterized by the SKL signature. Potential protein kinase C and casein Kinase II phosphorylation sites (3 and 7, respectively) are present along the protein. Thus, MCD might be regulated by (de)phosphorylation reactions since both kinases are present in mitochondria. Rat MCD does not contain acylation sites (N-myristoylation, glycosylphosphatidylinositol, isoprenylation, farnesylation) excluding MCD as a potential membrane-bound protein modified by one of these lipid anchors. EXAMPLE 10 Tissue distribution of MCD mRNA was investigated by Northern blot analysis and complemented by a semi-quantitative RT-PCR study (Figure 16). A mRNA of about 2.2 kb is present in all tested rat tissues and is expressed at relatively high levels in liver, kidney, heart and adipose tissues. The diffuse signal obtained is due to the fact that the MCD transcript co-migrates with the 18S-ribosomal RNA. To confirm this ubiquitary distribution, we performed RT-PCR on the same tissues and on sorted pancreatic α- and β-cells. After 25 cycles of PCR, the relative signals among tissues were similar to that at 35 cycles except that the band intensities were weaker. Comparisons were made possible thanks to a β-actin control PCR on the same samples. A control PCR of non-reverse transcripted material gave no signal, excluding genomic DNA contamination ofthe RNA preparations. Thus, the RT-PCR results confirm the Northern blot analysis and furthermore indicate that MCD mRNA is expressed also in the α- and β-cells of islet tissue to an extent similar to that of β(INS) cells. EXAMPLE 11 The presence ofthe active enzyme was measured in tissue extracts. The results show a broad range of activities from high levels in liver, heart and pancreatic β-(INS)- cells to low activity in the brain and spleen as well as undetectable MCD activity in the duodenum, untransfected 293 cells and 293 cells transfected with RSV-βgal. The tissue distribution of MCD mRNA (Fig. 15) did not closely correlate with enzymatic activity measurements. This discrepancy may be explained by differences among tissues in the translational control ofthe MCD gene. Another possibility to consider is that MCD is an allosteric enzyme regulated by covalent modification(s) and that the enzyme is differentially phosphorylated in various tissues. Consistent with this view, the rat MCD sequence shows many potential phosphorylation sites and alkaline phosphatase treatment of tissue extracts prior to performing the activity assay resulted in higher MCD activity.

Claims: CLAIMS 1. A method for compound screening, comprising: a) providing: i) a purified preparation comprising malonyl CoA decarboxylase, ii) a substrate, and iii) a test compound; b) mixing said malonyl CoA decarboxylase and said substrate under conditions such that said malonyl CoA decarboxylase can act on said substrate to produce product, wherein said mixing is done in the presence and absence of said test compound; and c) measuring directly or indirectly the amount of said product produced in the presence and absence of said test compound. 2. The method of Claim 1, wherein said substrate is malonyl CoA. 3. The method of Claim 1, wherein said preparation is purified from heart tissue. 4. The method of Claim 1, wherein said product comprises acetyl CoA. 5. The method of Claim 4, wherein said acetyl Co A is measured directly. 6. The method of Claim 4, wherein said acetyl CoA is measured indirectly by detecting the amount of an indicator. 7. The method of Claim 6, wherein said indicator comprises citrate. 8. The method of Claim 7, wherein said citrate is labelled. 9. The method of Claim 8, wherein said citrate is radiolabelled. 10. The method of Claim 9, wherein said radiolabelled citrate is [ C] citrate. 11. A method for compound screening, comprising: a) providing: i) a malonyl CoA decarboxylase homologue, ii) a substrate, and iii) a test compound; b) mixing said malonyl CoA decarboxylase homologue and said substrate under conditions such that said malonyl CoA decarboxylase homologue can act on said substrate to produce product, wherein said mixing is done in the presence and absence of said test compound; and c) measuring directly or indirectly the amount of said product produced in the presence and absence of said test compound. 12. The method of Claim 11, wherein said substrate is malonyl CoA. 13. The method of Claim 11 , wherein said homologue differs from malonyl CoA decarboxylase purified from heart tissue by an amino acid substitution. 14. The method of Claim 11, wherein said product comprises acetyl CoA. 15. The method of Claim 14, wherein said acetyl CoA is measured directly. 16. The method of Claim 14, wherein said acetyl CoA is measured indirectly by detecting the amount of an indicator. 17. The method of Claim 16, wherein said indicator comprises citrate. 18. The method of Claim 17, wherein said citrate is labelled. 19. The method of Claim 18, wherein said citrate is radiolabelled. 20. The method of Claim 19, wherein said radiolabelled citrate is [ C]citrate. 21. A composition comprising isolated and purified DNA having an oligonucleotide sequence selected from the group consisting of: a) Heart MCD having the nucleotide sequence of SEQ ID NO: 1 ; b) Liver MCD having the nucleotide sequence of SEQ ID NO:2; c) Pancreas MCD having the nucleotide sequence of SEQ ID NO:3; 22. RNA transcribed from the DNA of Claim 21. 23. Protein translated from the RNA of Claim 22. 24. Antibodies produced from the protein of Claim 23. 25. Expression constructs comprising DNA of Claim 21.

Inventors: DYCK JASON, LOPASCHUK GARY D, DYCK, JASON, LOPASCHUK, GARY, D.
Applicants: DYCK JASON, LOPASCHUK GARY D, UNIV ALBERTA, DYCK, JASON, LOPASCHUK, GARY, D., THE GOVERNORS OF THE UNIVERSITY OF ALBERTA
Application year: 1999
Main classification: C12N15
IPC codes: C07K16, C12N9, C12N15, C12Q1