Patent Application: US-11960402-A

Abstract:
a highly conserved active site helix present within the p - 450 superfamily of proteins is found also in monoamine oxidase b , a major enzyme that catalyzes deamination of neuro - and vaso - active amines in the nervous system of mammals . mutation within the conserved region of the mao b enzyme directly reduces mao b &# 39 ; s activity and alters its ph profile , which allows for indirect regulation of the cellular neurotransmitters and vasoamines .

Description:
the present invention is directed toward the amino acid sequence and tertiary structure corresponding to the active site for the monoamine oxidase ( mao ) b enzyme and the engineering thereof to achieve neurotransmitter regulation . as used herein , the terms “ monoamine oxidase ” or “ mao ” refer to the enzyme responsible for the oxidative deamination , and hence deactivation , of the monoamine neurotransmitters 5 - hydroxytryptamine ( 5ht , serotonin ), noradrenaline , and dopamine . mao also has a role in providing protection from other exogenous ( i . e ., dietary or environmental ) amines that might otherwise have adverse effects on , for example , cardiovascular or neuronal function . mao exists in two main forms , types a and b ; mao - a ( specific inhibitor clorgyline ) is responsible for the oxidative removal of 5ht and noradrenaline , whereas in humans , mao - b ( specific inhibitor deprenyl ) is the enzyme by which dopamine is mainly metabolized . both forms of mao are encoded by separate genes on the x chromosome . mao - b has been been shown to activate a protoxin , mptp , that produces a parkinsons - like disorder in humans . as used herein , the term “ cytochrome p450 ” is a collective term for an extensive family of heme - containing electron - transport molecules present in liver microsomes and involved in enzymatic oxidation of a wide range of substrates and their conversion to forms that are more easily excreted . in some cases , the metabolites produced may be carcinogenic . cytochromes p450 are also involved in the synthesis of compounds such as steroid hormones and prostaglandins . many of the 200 or so genes that comprise this family are inducible by various exogenous agents . the inducibility shows genetic variability . in accordance with the present invention there may b e employed conventional molecular biology , microbiology , and recombinant dna techniques within the skill of the art . such techniques are explained fully in the literature . see , e . g ., maniatis , fritsch & amp ; sambrook , “ molecular cloning : a laboratory manual ( 1982 ); “ dna cloning : a practical approach ,” volumes i and ii ( d . n . glover ed . 1985 ); “ oligonucleotide synthesis ” ( m . j . gait ed . 1984 ); “ nucleic acid hybridization ” ( b . d . hames & amp ; s . j . higgins eds . ( 1985 )); “ transcription and translation ” ( b . d . hames & amp ; s . j . higgins eds . ( 1984 )); “ animal cell culture ” ( r . i . freshney , ed . ( 1986 )); “ immobilized cells and enzymes ” ( irl press , ( 1986 )); b . perbal , “ a practical guide to molecular cloning ” ( 1984 ). therefore , if appearing herein , the following terms shall have the definitions set out below . a “ vector ” is a replicon , such as plasmid , phage or cosmid , to which another dna segment may be attached so as to bring about the replication of the attached segment . a vector is said to b e “ pharmacologically acceptable ” if its administration can be tolerated by a recipient mammal . a “ dna molecule ” refers to the polymeric form of deoxyribonucleotides ( adenine , guanine , thymine , or cytosine ) in either single stranded form , or as a double - stranded helix . this term refers only to the primary and secondary structure of the molecule , and does not limit it to any particular tertiary forms . thus , this term includes double - stranded dna found , inter alia , in linear dna molecules ( e . g ., restriction fragments ), viruses , plasmids , and chromosomes . in discussing the structure herein according to the normal convention of giving only the sequence in the 5 ′ to 3 ′ direction along the nontranscribed strand of dna ( i . e ., the strand having a complementary sequence to the mrna ). a dna “ coding sequence ” is a double - stranded dna sequence which is transcribed into rna and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences . the boundaries of the coding sequence are determined by a start codon at the 5 ′ ( amino ) terminus and a translational stop codon at the 3 ′ ( carboxyl ) terminus . a coding sequence can include , but is not limited to , prokaryotic sequences , cdna from eukaryotic mrna , genomic dna sequences from eukaryotic ( e . g ., mammalian ) dna , and even synthetic dna sequences . a polyadenylation signal and transcription termination sequence will usually be located 3 ′ to the coding sequence . transcriptional and translational control sequences are dna regulatory sequences , such as promoters , enhancers , polyadenylation signals , terminators , and the like , that provide for the expression of a coding sequence in a host cell . a “ promoter sequence ” is a dna regulatory region capable of binding rna polymerase in a cell and initiating transcription of a downstream ( 3 ′ direction ) coding sequence . for purposes of defining the present invention , the promoter sequence is bounded at its 3 ′ terminus by the transcription initiation site and extends upstream ( 5 ′ direction ) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background . within the promoter sequence will be found a transcription initiation site ( conveniently defined by mapping with nuclease s1 ), as well as protein binding domains ( consensus sequences ) responsible for the binding of rna polymerase . eukaryotic promoters will often , but not always , contain “ tata ” boxes and “ cat ” boxes . various promoters may be used to drive vectors . an “ expression control sequence ” is a dna sequence that controls and regulates the transcription and translation of another dna sequence . a coding sequence is “ under the control ” of transcriptional and translational control sequences in a cell when rna polymerase transcribes the coding sequence into mrna , which is then translated into the protein encoded by the coding sequence . a “ selection gene ” refers to a gene that enables the discrimination of cells displaying a required phenotype upon implementation of certain conditions . for example , the growth of bacteria in medium containing antibiotics to select for the bacterial cells containing antibiotic resistance genes . the term “ oligonucleotide ” or “ probe ” as used herein , refers to a molecule comprised of ribonucleotides or deoxyribonucleotides . the exact size of the oligonucleotide or probe will depend upon many factors which , in turn , depend upon the ultimate function and use of the oligonucleotide . as used herein , the terms “ restriction endonucleases ” and “ restriction enzymes ” refer to bacterial enzymes , each of which cut double - stranded dna at or near a specific nucleotide sequence . a cell has been “ transformed ” by exogenous or heterologous dna when such dna has been introduced inside the cell . the transforming dna may or may not be integrated ( covalently linked ) into the genome of the cell . in prokaryotes , yeast , and mammalian cells for example , the transforming dna may be maintained on an episomal element such as a plasmid . with respect to eukaryotic cells , a stably transformed cell is one in which the transforming dna has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication . this stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming dna . a “ clone ” is a population of cells derived from a single cell or common ancestor by mitosis . a “ cell line ” is a clone of a primary cell that is capable of stable growth in vitro for many generations . two dna sequences are “ substantially homologous ” when at least about 75 % ( preferably at least about 80 %, and most preferably at least about 90 or 95 %) of the nucleotides match over the defined length of the dna sequences . sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks , or in a southern hybridization experiments under , for example , stringent conditions as defined for that particular system . defining appropriate hybridization conditions is within the skill of the art . see , e . g ., maniatis et al ., supra ; dna cloning , vols . i & amp ; ii , supra ; nucleic acid hybridization . a “ heterologous ” region of the dna construct is an identifiable segment of dna within a larger dna molecule that is not found in association with the larger molecule in nature . thus , when the heterologous region encodes a mammalian gene , the gene will usually be flanked by dna that does not flank the mammalian genomic dna in the genome of the source organism . in another example , a coding sequence is a construct where the coding sequence itself is not found in nature ( e . g ., a cdna where the genomic coding sequence contains introns , or synthetic sequences having codons different than the native gene ). allelic variations or naturally - occurring mutational events do not give rise to a heterologous region of dna as defined herein . transcriptional and translational control sequences are dna regulatory sequences , such as promoters , enhancers , polyadenylation signals , terminators , and the like , that provide for the expression of a coding sequence in a host cell . the amino acids described herein are preferred to be in the “ l ” isomeric form . however , residues in the “ d ” isomeric form can be substituted for any l - amino acid residue , as long as the desired functional property of immunoglobulin - binding is retained by the polypeptide . nh 2 refers to the free amino group present at the amino terminus of a polypeptide . cooh refers to the free carboxy group present at the carboxy terminus of a polypeptide . in keeping with standard polypeptide nomeclature , j biol . chem ., 243 : 3552 - 59 ( 1969 ), abbreviations for amino acid residues are shown in the following table of correspondence : table of correspondence 1 - letter 3 - letter amino acid y tyr tyrosine g gly glycine f phe phenylalanine m met methionine a ala alanine s ser serine i ile isoleucine l leu leucine t thr threonine v val valine p pro proline k lys lysine h his histidine q gln glutamine e glu glutamic acid w trp tryptophan r arg arginine d asp aspartic acid n asn asparagine c cys cysteine it should be noted that all amino acid sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino - terminus to carboxy - terminus . furthermore , it should be noted that a dash at the beginning or end of an amino acid sequence indicates a peptide bond to a further sequence of one or more amino - acid residues . the above table is presented to correlate the three - letter and one - letter notations which may appear alternately herein . the present invention is directed towards an isolated , genetically - engineered cellular mao b enzyme having at least one amino acid substitution for a wildtype amino acid in a mao b active site , where the wildtype amino acid is selected from the group of phe423 , glu427 and thr428 . the present invention also provides for an isolated dna encoding the isolated , genetically - engineered mao b enzyme . furthermore , the present invention provides for a plasmid containing the dna encoding the isolated , genetically - engineered mao b enzyme and regulatory elements necessary for expression . the present invention is specifically directed towards a n isolated , genetically - engineered cellular mao b enzyme , in which the wildtype amino acid , phe423 , has been substituted with alanine . the present invention is also specifically directed towards an isolated , genetically - engineered cellular mao b enzyme , in which the wildtype amino acid , glu427 , has been substituted with glutamine . the present invention is also specifically directed towards an isolated , genetically - engineered cellular mao b enzyme , in which the wildtype amino acid , thr428 , has been substituted with either serine or alanine . the present invention is also directed towards a pharmaceutical composition which interacts with an active site of mao b having the sequence seq id no . 1 . furthermore , the pharmaceutical composition may consist of derivative of fad , derivatives of acetylenic amine inhibitors , derivatives of cyclopropyl amine inhibitors , derivatives of deprenyl ( phenylisopropyl - methylproinylamine ) and derivatives of trans - phenylcyclopropylamine . the fad derivative may include 2 ′- deoxy fad or 3 ′- deoxy fad . furthermore , the present invention is directed to a method of regulating mao b , comprising the step of mutating an amino acid in the wild type active site shown in seq id no . 1 . specifically , the wild type amino acid to be mutated may be phe423 , glu427 or thr428 . it will be apparent to one skilled in the art that various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention . the following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion : powell et al . ( 1 ) determined the amino acid sequence of bovine mao a and identified a short segment that exhibited high sequence identity to a region in cytochrome p - 450 in rat . fig1 shows the same region in human mao b aligned with other p - 450 enzymes , illustrating that all known p - 450 enzymes contain this conserved sequence . examination of this conserved sequence in a p - 450 enzyme of known structure , p . putida p - 450cam ( 12 ), revealed the location of this sequence within its three - dimensional structure . as seen in fig2 the sequence demonstrates helical three - dimensional structure and is located within the active site of p - 450cam in close contact to the camphor substrate . similarly , in the crystal structure of p - 450terp ( not shown ), the conserved sequence is in the active site and in close contact to the terpinol substrate ( 13 ). a third p - 450 enzyme in which the three - dimensional structure of the active site has been resolved is p - 450bm - 3 ( 14 ). although p - 450bm - 3 contains only 16 % sequence identity to p - 450cam and differs significantly from p - 450cam in tertiary structure , redox partner preferences and substrate specificities , the region of high sequence homology in the conserved sequence ( shown in fig1 ) is retained . in these p - 450 enzymes , the conserved sequence resides within an extended α - helix which traverses the active site . to determine whether mao b could adopt a similar helical structure in the conserved region of interest , secondary structure predictions were carried out using several different methods , including gibrat , levin , dpm , phd and sopma ( 15 ). all methods predicted the region in mao b from residues 410 - 455 to be primarily α - helical . the average of the five methods predicts that the region of mao b from residues 410 - 455 is 74 % helical . site - directed mutagenesis studies have been undertaken in numerous laboratories to identify specific residues that play a role in the active site ( 16 ). although the specific role of several amino acid residues in this region are being investigated , the residues which align with glu427 and thr428 in mao b are widely accepted as crucial for the activity of p - 450 enzymes ( 17 ). to examine the role of these two amino acid residues in mao b , point mutations were constructed at positions 427 and 428 within the homologous sequence ( fig3 ). glu427 was changed to gln ( e427q ), and thr428 was changed to ser ( t428s ) or ala ( t428a ), alterations which were consistent with mutagenesis studies carried out with the p - 450 enzymes ( 17 ). also , a phe residue at position 423 was changed to a nonaromatic ala residue ( f423a ). site - directed mutagenesis was performed by the method of deng and nickoloff ( 26 ) using a “ transformer site - directed mutagenesis ” kit ( clontech ) as described previously ( 27 ). the mutagenic primers and the corresponding amino acid changes are shown in fig3 . all mutagenic primers were designed to create a new restriction site , without altering the coding sequence of any other amino acids , for the purpose of screening . construction of mutants was carried out directly within the expression vector psvk3 with human mao b cdna inserted directly into the ecori site . base substitutions that do not alter the amino acid coding sequence were also included in each mutagenic primer to create a ngomi restriction site for the purpose of selection . the mutant clones were screened for the presence of the new restriction site created by the mutagenic primer . the presence of the correct mutations in all mutant cdnas were confirmed by double - stranded di - deoxy dna sequencing ( sanger et al ., 1977 ). both wild - type and mutant plasmid dnas were purified through cesium chloride gradients prior to transfection studies . the wild - type and mutant mao b enzymes were expressed in mammalian cos - 7 cells , and the enzymatic properties of the native enzyme and its mutant enzymes were determined and compared . wild - type and mutant mao b cdnas were transiently transfected into cos - 7 cells by electroporation . cells were harvested during late log phase growth and resuspended to a concentration of 3 . 125 × 10 6 cells / ml in dmem supplemented with 10 % fetal bovine serum ( fbs ). fifteen μg of normal or mutant cdna were added to 0 . 8 ml ( 2 . 5 × 10 6 cells ) of cell suspension . electroporation was carried out in a bio - rad gene pulser with a setting of 0 . 25 kv and 500 μf . cells were resuspended in 15 ml of dmem / fbs and incubated at 37 ° c . with 5 % co 2 for 48 h . expression studies were carried out in mammalian cos - 7 cells , since these cells do not contain any detectable endogenous mao b , as determined by elisa , western blot and radiometric activity assays ( 28 ). aliquots ( 20 μl ) of transfected cos - 7 cell homogenates were incubated for 10 min at 37 ° c . in the presence of various concentrations of [ 14 c ] pea ( 20 mci / mmol ) or [ 14 c ] benzylamine ( 10 mci / mmol ) in 50 mm sodium phosphate buffer , ph 7 . 5 ( 200 μl total volume ). substrate concentrations ranged from 0 . 25 - 2 . 5 times the k m value ( determined in pilot experiments ) to yield 10 equally - spaced reciprocal increments . parallel assays were carried out with untransfected cos - 7 cell lysates as a control . reactions were stopped by addition of 25 μl of 6 n hcl and the deaminated products were extracted by vortexing for 30 seconds with 500 μl of toluene or toluene : ethyl acetate ( 1 : 1 ) for benzylamine or pea , respectively . samples were processed as described previously ( 28 ). apparent k m and v max values were determined by linear regression analysis of lineweaver - burk plots . k cat values were calculated from the v max values divided by the concentration of wild - type or mutant mao b determined in each experiment . expression levels of wild - type or mutant mao b were determined in triplicate for six separate experiments as follows : wild - type = 11 . 1 + 2 . 07 ; f423a = 9 . 30 + 1 . 89 ; e427q = 10 . 3 + 1 . 29 ; t428a = 10 . 2 + 2 . 07 ; t428s = 10 . 2 + 1 . 53 pmol mao b / mg protein . data are given as means + s . e . m . pmol / mg of protein determined using a subunit molecular weight of 58 , 800 . kinetic analysis of the e427q mutant enzyme showed a profound decrease in activity with either pea or benzylamine a s substrate ( tables 1 and 2 ), estimated at & lt ; 5 % of wild - type . since the e427q mutant replaces an ionizable carboxylate group with an amide group , the loss of activity may be due to an inability of the amide group to participate in a critical ionic bond or proton - dependent step in the reaction . furthermore , a mutation affecting a proton - dependent step in the reaction would be expected to change the ph profile of the enzyme . data are given as means ± s . e . m ., where n is equal to three separate experiments . dashed lines indicate that activity was too low to determine kinetic parameters . data are given as means ± s . e . m ., where n is equal to three separate experiments . dashed lines indicate that activity was too low to determine kinetic parameters . to examine the possibility that the mutation effects a proton - dependent step in the reaction , the enzymatic activity of the mutant and wild - type mao b enzymes was determined at different ph values from 6 . 0 to 9 . 0 ( fig4 ). although the activity of the e427q mutant was low (& lt ; 5 % of wild - type ), the ph profile appeared significantly different than the wild - type enzyme . the activity of e427q was essentially independent of ph , whereas the wild - type enzyme displayed a characteristic increase in activity with increasing ph . the dramatic loss of activity and the change in the ph profile are strikingly similar to results obtained by substitution of the corresponding residue in the p - 450 enzymes . for example , mutagenesis of the corresponding residue ( glu or asp ) in p - 450cam , p - 450d or aromatase also resulted in a dramatic decrease in activities ( 18 ). furthermore , gerber and sligar ( 18 ) observed a significant change in the ph profile of p - 450cam when asp251 was changed to asn ( from parabolic to descending slope ). they proposed a model in which asp251 and thr252 of p - 450cam provide a proton relay to protonate the heme - bound dioxygen prior to o - o bond scission and water release . a similar mechanism could occur in mao where glu427 and thr428 provide a proton relay to protonate a flavin - bound dioxygen in the reoxidation of fad . however , while dioxygen is involved in the reoxidation of fadh , a c ( 4αα ) flavin hydroperoxide anion intermediate such as that observed in the flavin monooxygenases ( 19 ), has never been observed with the mao enzymes . several observations indicate that glu427 in mao b may play a similar role as the corresponding asp residue in p - 450cam : 1 ) the region in mao b contains a high sequence identity to the p - 450 enzymes ; 2 ) the region in mao b is predicted to have a similar secondary structure to the p - 450 enzymes ( α - helix ); 3 ) a glu or asp is always observed at this position in mao b and in the p - 450 enzymes ; 4 ) mutagenesis of the residue results in near - complete loss of activity of mao b , as observed with the p - 450 enzymes ; and 5 ) mutagenesis of the residue results in an alteration of the ph profile for mao b , as observed with the p - 450 enzymes . substitution of the conserved thr to ala ( t428a ) or to ser ( t428s ) in mao b resulted in similar kinetic profiles . using pea as substrate , mutant enzymes showed a shift in the k m from 3 . 38 μm ( wild - type ) to greater than 11 μm , and approximately equal reductions (˜ 50 % of wild - type ) in k cat . using benzylamine as substrate , t428a and t428s showed k m values approximately equal to wild - type . however , the k cat was reduced by 44 % and 67 % for t428a and t428s , respectively . this was unexpected , since the ser side chain contains an hydroxyl group , as does the wild - type thr . p - 450 studies have also reported greater than 50 % reduction in activity using a conserved thr to ser substitution ( 24 ). these results suggest that the hydroxyl side chain must be present in a similar conformation as the wild - type thr . an hydroxyl group in the wrong conformation may cause a deleterious effect on catalysis by inducing a slight local conformational change , or by steric interference with the binding of substrate or o 2 . another mutant enzyme , f423a , was prepared to examine the role of the upstream aromatic moiety or moieties seen in many of the p - 450 enzymes and mao ( see fig1 ). one or two phe residues are present in the mammalian enzymes , but are absent in the p - 450cam and p - 450terp bacterial enzymes . furuya et al . ( 18 ) used site - directed mutagenesis to change phe3l3 of p - 450d to tyr , observing a decrease in the k cat of approximately 80 % or 25 % for benzphetamine or 7 - ethoxycoumarin , respectively . this suggests that the phe is conserved in this location to serve a role in packing , since this side chain extends into a hydrophobic portion of p - 450cam near the active site . in the studies leading to the present invention , the conserved phe residue was converted to ala ( f423a ). it was found that this mutant underwent a dramatic decrease in activity (& gt ; 95 % reduction ) compared to the wild - type enzyme , supporting the “ protein packing ” hypothesis . the enzymatic activity of f423a was determined over a ph range from 6 . 0 to 9 . 0 ( fig4 ). since the phe side chain is likely to be involved in protein packing and unlikely to donate a proton in a direct ph dependent reaction , a significant deviation from the wild - type ph profile was unlikely , and , as expected , the ph profile was fairly similar to wild - type mao b although the activity was largely reduced . only at ph 9 . 0 was a significant difference in the shape of the profile observed , where the activity of f423a continued to increase . thus , the decrease in activity may be attributed to disruption of a critical protein packing region in close proximity to the active site as hypothesized for p - 450d . 1 . human mao a and b - a . w . j . bach et al ., proc . natl . acad . sci . 85 , 4934 ( 1988 ); human mao b - y - p . p . hsu et al ., j . neurochem . 51 , 1321 ( 1988 ); rat mao a - t . kuwahara , s . takamoto , a . ito , agric . biol . chem . 54 , 253 ( 1990 ); rat mao b - a . ito , t . kuwahara , s . inadome , y sagara , biochem . biophys . res . commun . 157 , 970 ( 1988 ); bovine mao a - j . f . powell et al ., biochem . j . 259 , 407 ( 1989 ) 2 . j . grimsby , et al ., proc . natl . acad . sci . 8 , 3637 ( 1991 ); z .- y . chen et al ., nucleic acid res . 19 , 4537 ( 1991 ); s .- w . kwan , et al ., psychopharmacology 106 , 51 ( 1992 ). 3 . p . l . dostert , et al ., med . res . rev . 9 , 45 ( 1989 ). 4 . westlund , et al ., science 230 , 181 ( 1985 ); thorpe , et al ., j . histochem . cytochem . 35 , 23 ( 1987 ); westlund , et al ., neurosci . 25 , 439 ( 1988 ). 5 . r . m . denney , et al ., science 215 , 1400 ( 1982 ); p . levitt , j . e . pintar , x . 0 . breakefield , proc . natl . acad . sci . 79 , 6385 ( 1982 ); r . m . denney , l . m . kochersperger et al ., j . neurosci . 11 , 2874 ( 1985 ). 6 . nagy , et al ., arch . biochem . biophys . 208 , 388 ( 1981 ); dostert , et al ., biochem . soc . trans . 19 , 207 ( 1991 ); r . b . silverman biochem . soc . trans . 19 , 201 ( 1991 ); efange , et al ., j . med . chem . 36 , 1278 ( 1993 ). 7 . b . h . nicholson biochem . soc . trans . 19 , 24s ( 1991 ). 8 . wouters et al ., biochem . biophys . res . commun . 208 , 773 ( 1995 ). 9 . h .- f . wu , k . chen , j . c . shih mol . pharmacol . 43 , 888 ( 1993 ); j . gottowik , a . m . cesura , p . malherbe , g . lang , m . da prada , febs lett . 317 , 152 ( 1993 ); s .- w . kwan , et al ., archs . biochem . biophys . 316 , 385 ( 1995 ); b . p . zhou , d . a . lewis , et al ., j . biol . chem . 270 , 23653 ( 1995 ); b . p . zhou , d . et al ., biochemistry 34 , 9526 ( 1995 ). 10 . j . gottowik , et al ., febs lett . 317 , 152 ( 1993 ); f . ogata , et al ., in flavins and flavoproteins ( walter de gruyter & amp ; co ., berlin n . y . ), pp . 795 - 798 ; j . gottowik , et al ., eur . j . biochem . 230 , 934 ( 1995 ). 11 . j . mitoma , a . ito , j . biochem . 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( walter de gruyter , berlin n . y ., 1984 ) pp . 605 - 618 . 21 . t . l . poulos , b . c . finzel , j . j . howard , j . mol . biol . 195 , 687 ( 1987 ); r . raag , t . l . poulos , biochemistry 28 , 7586 ( 1989 ). 22 . r . raag , s . a . martinis , s . g . sligar , t . l . poulos , biochemistry 30 , 11420 ( 1991 ). 23 . y . imai , m . nakumura , febs lett . 234 , 313 ( 1988 ); y . imai , m . nakamura , biochem . biophys . res . commun . 158 , 717 ( 1989 ); n . hanoika et al ., biochemistry 31 , 3364 ( 1992 ); t . fukuda et al ., j . biochem . ( tokyo ) 113 , 7 ( 1993 ). 24 . s . chen , d . zhou , j . biol . chem . 267 , 22587 ( 1992 ); y . he , et al ., biochemistry 33 , 419 ( 1994 ). 25 . d . r . nelson , h . w . strobel , biochemistry 28 , 656 ( 1988 ). 26 . w . p . deng , j . a . nickoloff , anal . biochem . 200 , 81 ( 1992 ). 27 . b . p . zhou , et al ., biochemistry 34 , 9526 ( 1995 ). 28 . kwan , et al ., archs . biochem . biophys . 316 , 385 ( 1995 ) any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains . further , these patents and publications are incorporated by reference herein to the same extent as if each individual publication was specifically and individually indicated to b e incorporated by reference . one skilled in the art will appreciate readily that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned , as well as those objects , ends and advantages inherent herein . the present examples , along with the methods , procedures , treatments , molecules , and specific compounds described herein are presently representative of preferred embodiments , are exemplary , and are not intended as limitations on the scope of the invention . changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined by the scope of the claims . asp arg ile tyr phe ala gly thr glu thr ala thr his trp ser arg ile tyr phe ala gly thr gln thr ala thr his trp ser arg ile tyr phe ala gly thr glu ala ala thr his trp ser arg ile tyr phe ala gly thr glu ser ala thr his trp ser arg ile tyr phe ala gly thr glu thr ala thr his trp ser