bluuebunny/crossref_metadata_2025 huggingface dataset:
Lumenal hydrolysis of menhaden and rapeseed oils and their fatty acid methyl and ethyl esters in the rat
Authors: Yang L.-Y. , Kuksis A. , Myher J. J.
Published: December 2009

Simple alkyl (ethyl) esters of polyunsaturated fish oil fatty acids have been proposed as dietary supplements, but their relative efficiency of digestion and absorption have not been determined. Using stomach tubes, we gave rats menhaden or rapeseed oils, or the corresponding methyl and ethyl esters, and determined by chromatographic methods the lipid classes and molecular species recovered from the lumen of the jejunum during the first 1 to 2.5 h of digestion. Hydrolysis of menhaden oil resulted in a preferential retention of a high proportion of the polyunsaturated long chain acids in the sn-2-monoacylglycerols and in the residual triacylglycerols, while digestion of rapeseed oil led to a preferential release of free long chain monounsaturated fatty acids. In contrast, hydrolysis of the alkyl (methyl and ethyl) esters of the fatty acids of either menhaden or rapeseed oil resulted in a composition of free fatty acids which was much more representative of the original esters. It was therefore concluded that the differential lumenal liberation of the long chain and polyunsaturated (three or more double bonds) fatty acids from fish and rapeseed oil is largely due to their characteristic distribution between the primary and secondary positions in the glycerol molecule, and to a much lesser extent to a chain length discrimination by pancreatic lipase. This study also shows that the methyl and ethyl esters are hydrolyzed about 4 times more slowly than the corresponding triacylglycerols, which is sufficient to maintain a saturated micellar solution of fatty acids in the intestinal lumen during absorption.Key words: gas chromatography, thin-layer chromatography, total lipid profiles, micellar and oil phases.
A simplified procedure for cDNA and genomic library construction using nonpalindromic oligonucleotide adaptors
Authors: D'Souza Cynthia R. , Deugau Ken V. , Spencer John H.
Published: December 2009

The properties and characteristics of oligonucleotide adaptors for use in a simplified procedure for the construction of cDNA and genomic DNA libraries are described. The adaptors are suitable for joining to blunt ended cDNA or sheared genomic DNA, and then to the cohesive ends of restriction sites in vectors. Each adaptor consists of two oligonucleotides with complementary but nonpalindromic sequences that include an internal restriction site, a 5′ phosphorylated blunt end, and an overlapping or staggered 5′ hydroxylated end corresponding to a restriction endonuclease site in a vector of choice. Ligation of the blunt end to high molecular weight target DNA proceeds efficiently and there is no tandem concatenation of the adaptor. Insertion into the appropriate vector only requires ligation of the cohesive ends. There is no requirement for methylation, restriction enzyme cleavage, G-C tailing, or denaturation after ligation of the adaptor to the target DNA, all characteristics of other procedures.Key words: library, genomic, cDNA, oligonucleotides, adaptors.
The activation of Dictyostelium discoideum alkaline phosphatase by carbohydrate binding proteins
Authors: Bhanot Pradeep , Weeks Gerald
Published: December 2009

Dictyostelium discoideum vegetative cell alkaline phosphatase was rapidly inactivated in the presence of 50 mM Tris–HC1, pH 7.4, when the incubation temperature was raised to 50 °C. In contrast, when extracts were preincubated in the presence of concanavalin A, alkaline phosphatase was rapidly activated when the temperature was raised to 50 °C. The alkaline phosphatase of culmination phase cell extracts was also inactivated at 50 °C, and although preincubation in the presence of concanavalin A reduced this inactivation, stimulation of activity was not observed. A mixed glycosidase preparation also activated the vegetative cell alkaline phosphatase and the binding of the glycosidases rather than their catalytic activity appeared to be the important event. In contrast, alkaline phosphatase from culmination phase cells was only marginally stimulated by glycosidase treatment. These results are discussed in relation to the unmasking of alkaline phosphatase activity that occurs during D. discoideum differentiation.Key words: concanavalin A, glycosidase, differentiation, Dictyostelium discoideum, alkaline phosphatase.
Structure of the pig pancreatic GP-2: role of intramolecular disulfides in the resistance to proteolysis
Authors: Lebel Denis , Paquette Jean
Published: December 2009

GP-2 is the major membrane glycoprotein characteristic of the pancreatic zymogen granule membrane. When granules are lysed in the presence of DTT, GP-2 becomes completely and specifically degraded. This proteolysis was reproducible with the same characteristics in the purified granule membrane. The protease was purified from this source using hydrophobic interaction chromatography. The proteolytic activity was identified as a 29-kDa protein because, in a reconstituted system containing both the purified GP-2 and the 29-kDa protein, the proteolytic degradation of GP-2 was sensitive to the same spectrum and concentrations of inhibitors or reducing agents as in the membrane. The activity was characteristic of a serine protease. It was also shown that GP-2 only becomes sensitive to proteolytic digestion when its disulfide bonds are reduced, and that DTT does not activate the protease. Seven intramolecular disulfide bonds were identified on GP-2. All of them are located in a 65-kDa tryptic fragment that is very resistant to exogenous proteases under nonreducing conditions. Because of the quite specific degradation of GP-2 under reducing conditions, we believe that the 29-kDa protease must be closely associated with GP-2 on the membrane. This protease could be responsible, in part, for the solubilization of the GP-2 from the membrane into the zymogen granule content and its resulting secretion by the pancreas.Key words: GP-2, zymogen granule, disulfide, exocrine, pancreas, secretion.
Glucocorticoid stimulation of tetrahydrobiopterin levels and phenylalanine hydroxylase activity in rat hepatoma cells
Authors: Parniak Michael A. , Pilkington Janice
Published: December 2009

Incubation of H4-II-E-C3 rat hepatoma cells with either hydrocortisone or dexamethasone resulted in 3- to 5-fold increases in the levels of both phenylalanine hydroxylase and its essential cofactor, tetrahydrobiopterin. Maximum elevation of phenylalanine hydroxylase was noted after 24 h of incubation, whereas significant increases in tetrahydrobiopterin were found only after 48 h exposure of the cells to glucocorticoids. Removal of hormone from the culture medium resulted in rapid loss of cell tetrahydrobiopterin, but a much slower decline in the level of phenylalanine hydroxylase. Thus, although the levels of both phenylalanine hydroxylase and tetrahydrobiopterin in rat hepatoma cells are regulated by glucocorticoids, this regulation is apparently not strictly coordinated. Nevertheless, control of cellular tetrahydrobiopterin levels may be an important regulator of hepatic phenylalanine catabolism since significant increases in the ability of intact rat liver cells to hydroxylate phenylalanine were observed only after 48 h exposure to glucocorticoids, in correlation with increases in cell tetrahydrobiopterin content.Key words: phenylalanine hydroxylase, tetrahydrobiopterin, glucocorticoid, hepatoma cells.
Investigation of adenylate kinase from Escherichia coli and its interaction with nucleotides by Fourier transform infrared spectroscopy
Authors: Arrondo J. L. R. , Gilles A. M. , Bârzu O. , Fermandjian S. , Yang P. W. , Mantsch H. H.
Published: December 2009

The secondary structure of adenylate kinase (EC 2.7.4.3) from E. coli was investigated under various conditions using Fourier transform infrared spectroscopy. The overall band contour of the conformation-sensitive amide I mode indicates that in HEPES buffer (pH 7.4) the major structure of the protein is α-helical. A more detailed estimate obtained from decomposition of the amide I band into its constituent component bands gives 50% α-helix, 26% β-structure, 15% turns and loops, and about 9% nonrepetitive unordered structures. Binding of nucleotide (e.g., ATP) to the donor site decreases the β-content and shifts the amide I band to higher wavenumbers, whereas binding of nucleotide (e.g., AMP) to the acceptor site does not produce any change in conformation of the protein. These results agree with the protection by ATP and lack of protection by AMP when adenylate kinase is digested with trypsin. The effect of protein denaturing agents and conditions (temperature, high pH, sodium dodecyl sulfate) on changes in the protein conformation as revealed by the conformation-sensitive amide I bands is discussed.Key words: adenylate kinase, infrared, conformation, denaturation.
The reaction of S-mercuric-N-dansylcysteine with acetylcholinesterase and butyrylcholinesterase
Authors: Tomlinson George , Kinsch Evelyn M.
Published: December 2009

S-mercuric-N-dansylcysteine was investigated as a potential probe of protein sulphydryl groups using bovine serum albumin, S-carboxymethyl – bovine serum albumin, lysozyme, and partially reduced lysozyme as test proteins. Criteria used to assess covalent binding through mercury-bridged mercaptide linkages include a finite reaction time (minutes to hours), abolition of the characteristic fluorescence spectrum following addition of a reducing agent, and failure to separate probe and protein after chromatography or electrophoresis. By these criteria, both Torpedo californica acetylcholinesterase and human serum cholinesterase (butyrylcholinesterase) contain four free sulphydryl groups per tetrameric enzyme molecule whereas Electrophorus electricus acetylcholinesterase has none. Labeled acetylcholinesterase and butyrylcholinesterase remain active and responsive to the inactivator Zn2+. Zn2+ promotes an increase in the fluorescence of bound S-mercuric-N-dansylcysteine, whereas activators such as Mg2+ or gallamine promote a decrease, suggesting that the label may be a useful probe of ligand-induced conformational changes. With T. californica acetylcholinesterase, but not with human serum cholinesterase, Zn2+ also promotes access to two additional groups that are reactive towards the sulphydryl reagent.Key words: acetylcholinesterase, serum cholinesterase, sulphydryl groups, S-mercuric-N-dansylcysteine.
Overproduction and domain structure of the glutamyl-tRNA synthetase of Escherichia coli
Authors: Brisson Anne , Brun Yves V. , Bell Alexander W. , Roy Paul H. , Lapointe Jacques
Published: December 2009

The charging of glutamate on tRNAGlu is catalyzed by glutamyl-tRNA synthetase, a monomer of 53.8 kilodaltons in Escherichia coli. To obtain the large amounts of enzyme necessary for the identification of structural domains, we have inserted the structural gene gltX in the conditional runaway-replication plasmid pOU61, which led to a 350-fold overproduction of glutamyl-tRNA synthetase. Partial proteolysis of this enzyme revealed the existence of preferential sites of attack that, according to their N-terminal sequences, delimit regions of 12.9, 2.3, 12.1, and 26.5 kilodaltons from the N- to C-terminal of the enzyme. Their sizes suggest that the 2.3-kilodalton fragment is a hinge structure, and that those of 12.9, 12.1, and 26.5 kilodaltons are domain structures. The 12.9-kilodalton domain of the glutamyl-tRNA synthetase of E. coli is the only long region of this enzyme displaying a good amino acid sequence similarity with the glutaminyl-tRNA synthetase of Escherichia coli.Key words: glutamyl-tRNA synthetase, structural domains, overproduction, runaway-replication plasmid, Escherichia coli.
Low resolution three-dimensional models of the 7SL RNA of the signal recognition particle, based on an intramolecular cross-link introduced by mild irradiation with ultraviolet light
Authors: Zwieb Christian , Schüler Dierk
Published: December 2009

A number of intramolecular RNA-RNA cross-links were introduced into the human 7SL RNA by mild irradiation of a reconstituted signal recognition particle with ultraviolet light. Synthesis of radioactively labeled RNA was initiated in vitro from the T7 promoter. Smaller cross-linked complexes were generated by digestion of the RNA with RNase H in the presence of complementary deoxyoligonucleotides. Cross-linked complexes were separated from noncross-linked fragments by two-dimensional Polyacrylamide gel electrophoresis. A close proximity between the stemloop around position 200 and the nucleotides at positions 67 to 76 was revealed by the absence of a characteristic oligonucleotide from the fingerprint of one of the complexes, suggesting a close spatial neighborhood between these two regions of the RNA. This and previous results, which described two different conformers of the RNA, were used to deduce two preliminary three-dimensional structure models of the 7SL RNA. The models differ in the base pairing scheme of the conserved core of the 7SL RNA.Key words: signal recognition particle, 7SL RNA, cross-linking, three-dimensional model.
Asparagine synthetase from root nodules of alfalfa
Authors: Ta T. C. , Macdowall F. D. H. , Faris M. A.
Published: December 2009

Asparagine synthetase, the enzyme which catalyzes the formation of asparagine from aspartate and glutamine (the preferable N donor), was partially purified to 300-fold from root nodules of alfalfa (Medicago sauva L.). The enzyme has Km values for aspartate, glutamine, and ammonium of 1.25, 0.16, and 2.70 mM, respectively. The ratio of glutamine- to ammonium-dependent activity is 2.5 and the pH optimum is between 7.8 and 8.2. Its subcellular location is the cytoplasm. The activity of asparagine synthetase increased in parallel with that of glutamine synthetase and the amounts of organic N in the nodules during their development. Maximum levels were formed at about 3 weeks following innoculation with Rhizobium meliloti. These values decreased rapidly after removal of the shoots: asparagine synthetase reached its lowest value (10% of original) while glutamine synthetase retained 45% of its activity at day 7. Treatment with an argon atmosphere (80% Ar + 20% O2, v/v) to prevent N2 fixation by nodules, and the resulting inhibition of NH4+ production, caused a decrease in asparagine synthetase activity to 10% of control, while this treatment only slightly affected glutamine synthetase (80% of control activity remained).Key words: alfalfa nodules, asparagine synthetase.
Production and interconversion of 1,2-propanediol and hydroxyacetone by Escherichia coli
Authors: Commodari Fernando , Hunter Brian K.
Published: December 2009

The anaerobic metabolism of marginally lethal levels of [13C]formaldehyde by Escherichia coli (K12, MU352, CRB, and CR63) was followed in vivo by 13C NMR. The products include 1, 2-propanediol. Under aeration, the 1, 2-propanediol is converted to hydroxyacetone. The hydroxyacetone is reconverted to 1, 2-propanediol when aeration is stopped. The process can be cycled by varying the rate of aeration.Key words: hydroxyacetone, 1, 2-propanediol, Escherichia coli, production.
Thioredoxin and glutaredoxin enhance the binding of L-triiodothyronine to its hepatic nuclear receptors
Authors: Takagi Shinko , Bhat Ganesh B. , Hummel Brian C. W. , Walfish Paul G.
Published: December 2009

The influence of thioredoxin and glutaredoxin on binding of L-triiodothyronine (T3) to the rat hepatic nuclear T3 receptor was compared with that of the exogenous activator dithiothreitol. Specific [125I]T3 binding, the affinity constant, Ka, and the maximal binding capacity, MBC, were measured using whole nuclei, solubilized preparations of receptor, and chromatographed nuclear receptor. Both the thioredoxin system (thioredoxin, thioredoxin reductase, and NADPH) and the glutaredoxin system (glutaredoxin, glutathione reductase, glutathione, and NADPH) increased specific binding of T3 to nuclei, solubilized receptor, and chromatographed receptor significantly. Compared with the values obtained in the absence of added thiol (Ka = 1.6 ± 0.1 × 109 M−1 MBC = 1.7 ± 0.06 pM), the thioredoxin and glutaredoxin systems increased Ka by 147 and 112%, respectively, while decreasing MBC by 51 and 45%, respectively, when chromatographed receptor was used. The same tendency was observed with solubilized receptor. However, dithiothreitol increased Ka without affecting MBC when solubilized receptor was used. These results, the first demonstration of endogenous disulphide reductant systems enhancing binding of T3 to its receptor, suggest that the thioredoxin and (or) glutaredoxin systems may modulate the physiological effects of thyroid hormone.Key words: nuclear triiodothyronine (T3) receptor, thioredoxin, glutaredoxin, Scatchard analysis.
Molecular biology of Leishmania
Authors: Bard Enzo
Published: December 2009

Leishmania is a trypanosomatid protozoa with a digenetic life cycle. Sandflies inject promastigotes, the free living form present in their salivary glands, into mammals where the parasite colonizes macrophages, transforming into intracellular amastigotes. The cycle is completed when during a blood meal the insect ingests infected macrophages, the amastigotes are released in the gut where they transform back into promastigotes. Leishmania has to adapt to the changing life conditions, from free-living forms in the poikilothermic insect vector to obligatory intracellular parasite in the homeothermic mammalian host. It also has to adapt to the acidic pH of the macrophage's phagolysosome where amastigotes multiply. The adaptative response of Leishmania includes morphological, physiological, and biochemical changes. Promastigotes can be grown in culture medium. Studies of changes taking place during adaptation have been facilitated by the establishment of in vitro conditions that allow the transformation of amastigotes into promastigotes and vice versa. The system is well suited for studying regulation of gene expression during adaptative differentiation. Some mechanisms of mRNA processing are unique to these protozoa: trans-splicing and RNA editing. Several genes that are differentially expressed in the two stages have been studied. No obvious cis regulatory motifs have been found in the DNA.Key words: Leishmania, genes, differentiation, regulation.
Ultrastructural localization of nuclear antigens during interphase in mouse 3T3 fibroblasts
Authors: Chaly Nathalie , St. Aubin Gerald , Brown David L.
Published: December 2009

Thin sections of mouse 3T3 fibroblast nuclei labelled by immunoperoxidase with anti-nuclear antibodies I1, PI1, PI2, anti-peripherin, -lamin, and -centromere have been examined in the electron microscope. Staining was compared with the corresponding immunofluorescence labelling patterns, and was correlated with nuclear ultrastructure in conventionally fixed and uranyl-lead stained samples and in unlabelled immunoperoxidase controls. Peripherin was detected at the nuclear rim in a band broader and more irregular than the lamins/lamina. The peripheral components of PI1 and PI2 appear to be localized at nuclear pores and the nuclear envelope, respectively. The internal component of PI1 staining consisted of irregular patches and strands in the nucleoplasm, closely resembling snRNP staining as reported by others. Internal PI2 labelling consisted of spots distributed apparently at random in interchromatinic regions. The spots resembled labelling by antibody I1, but were fewer and more irregular in size. Neither the PI2 nor the I1 spots were centromere associated, nor could they be correlated with specific interchromatinic structures in conventional preparations and in unlabelled controls. The results support the hypothesis that the nucleus is segregated into function-specific domains, distinguished by morphology and (or) composition from surrounding regions of the nucleus.Key words: nuclear matrix, peripherin, immunoperoxidase, electron microscopy, nuclear antigens.
Cytochrome c methylation
Authors: Pair Woon Ki , Cho Young-Bong , Frost Blaise , Kim Sangduk
Published: December 2009

In this review, protein methylation is outlined in general terms, highlighting the major amino acids that are methylated and some of the proteins in which they are found. The majority of the review examines the methylation of cytochrome c at Lys-77 of lower eukaryotes as a possible model for methylation studies. Early work involving the purification and characterization of the methyltransferase responsible for this methylation indicated cytochrome c was methylated posttranslationally, yet prior to import into the mitochondria. Methylation in vitro occurred only at the in vivo methylation site and only on cytochrome c. Later studies using in vitro translated apocytochrome c revealed that methylated, as compared with unmethylated, apocytochrome c was imported preferentially into yeast, but not rat liver, mitochondria. Efforts to discover the reasons for this preference have shown that methylation of apocytochrome c dramatically lowers its isoelectric point (against a predicted increase) and decrease its Stokes radius. A possible mechanism for these differences involving the disruption of hydrogen bonds is presented here with space-filling models. Finally, the in vivo significance of this modification is also discussed.Key words: yeast iso-1-apocytochrome c, enzymatic methylation, in vitro translation, protein methylase III, pI change.
Pyrimidine biosynthesis in Saccharomyces cerevisiae: the ura2 cluster gene, its multifunctional enzyme product, and other structural or regulatory genes involved in de novo UMP synthesis
Authors: Denis-Duphil Michèle
Published: December 2009

There are six enzymatic steps in the de novo biosynthesis of uridine monophosphate (UMP). In yeast, six structural genes (ura2, ura4, ura1, ura5, ura10, and ura3) and one regulatory gene (PPR1) are involved in this metabolic pathway. Gene ura2 codes for a multifunctional protein that carries the first two enzymatic activities of the pathway, i.e., carbamylphosphate synthetase (CPSase) and aspartate transcarbamylase (ATCase). Gene ura2 has been cloned and sequenced, revealing the presence of three open reading frames, one of which codes for the multifunctional protein, a polypeptide of 2212 amino acids, with a mRNA of 7 ± 0.3 kilobases. Expression of gene ura2 is regulated at the transcriptional level. As I indicate here, it could also be controlled at the posttranscriptional level since all the consensus sequences for a 1.2-kilobases intron are present in the coding sequence of the open reading frame. The deducted amino acid sequence has allowed the identification of four domains. Starting from the amino terminus of the protein, these are glutamine amido transferase, CPSase, a domain that resembles dihydroorotase (DHOase-like) but does not have DHOase activity, and ATCase. There are also two sites of interest that match known concensus phosphorylation sites; one is located in the distal part of the CPSase domain, the other in the connector region between DHOase-like and ATCase domains. The protein has been purified as a multienzyme aggregate and as a multifunctional protein. The latter form, when isolated from a protease B deficient strain of Saccharomyces cerevisiae, contained mostly polypeptide chains of 220 kilodaltons. Work is currently in progress to determine the site(s) of phosphorylation of this protein in vitro. ATCase activity of both wild-type and protease-deficient strains has been found to be localized in the nucleus. Channeling of carbamyl phosphate, the first intermediate in the pathway, has been demonstrated both in vitro and in permeabilized cells. The other genes of UMP biosynthesis, except for ura5, are regulated by induction of their transcription by the combined action of the product of the ppr1 gene and the inducer, dihydroorotate. Dihydroorotate dehydrogenase activity was found in the cytoplasm. Two isoenzymes of orotate phosphoribosyl transferase have been found, coded for by ura5 and ura10. The products of genes ura10 and ura3 are proposed to participate in the channeling of orotidine monophosphate. The discussion considers the problem posed by the isolation of both multienzyme complexes and multifunctional proteins resulting from the expression of the same cluster genes. I suggest that regulation by processing at the posttranscriptional and posttranslational levels could be regarded as an alternative explanation for these observations, which were previously explained in terms of proteolysis.Key words: yeast, pyrimidines, multifunctional enzyme, phosphorylation, proteolysis.
Specific inhibitors of Neurospora endo-exonuclease
Authors: Hatahet Zafer , Fraser Murray J.
Published: December 2009

A heat-stable, trypsin-sensitive cytosolic inhibitor of Neurospora crassa endo-exonuclease has been purified 300-fold in 90% yield from crude extracts of mycelia. On electrophoresis in sodium dodecyl sulfate – polyacrylamide gels, it showed a relatively broad band corresponding to polypeptides with an average molecular mass of about 24 kDa. The protein inhibited the single strand specific endonuclease activity of endo-exonuclease noncompetitively, but the inhibition was not complete (maxima of 70–95%) in the presence of excess inhibitor. The inhibition of the double strand exonuclease activity was competitive and complete when inhibitor was present in excess. In addition, the inhibitor completely blocked the formation of site-specific double strand breaks and nicking by endo-exonuclease in linearized pBR322 DNA. The ribonuclease activity of endo-exonuclease was also inhibited by this protein. The inhibition of Neurospora endo-exonuclease was very specific. Although inhibition was seen also for an immunochemically related nuclease derived from mycelia of Aspergillus nidulans, no inhibition was observed for two other nucleases of Neurospora, nor for a wide variety of commercially available nucleases. A bound form of inhibitor was also partially purified from the cytosol of Neurospora in association with inactive, but trypsin-activable endo-exonuclease. Activity gel analysis showed that this was an endo-exonuclease–inhibitor complex. On heating at 80 °C, inhibitor was released from the complex with the same first order kinetics as inactivation of the trypsin-activable endo-exonuclease and in approximately equivalent amounts. The bound inhibitor was also 24 kDa in size, but completely inhibited both the endo- and exonuclease activities of endo-exonuclease. It was found unexpectedly that heat-inactivated endo-exonuclease stimulated the exonuclease activity of the active enzyme without affecting the endonuclease activity. The stimulated exonuclease activity was also completely blocked by inhibitor.Key words: Neurospora crassa endo-exonuclease, inhibitor, constitutive, cytosolic.
Characterization of the fragmented forms of calcineurin produced by calpain I
Authors: Wang Kevin K. W. , Roufogalis Basil D. , Villalobo Antonio
Published: December 2009

Calcineurin, a calmodulin-stimulated phosphatase from bovine brain, was hydrolyzed by calpain I from human erythrocytes. In the absence of calmodulin, calpain rapidly transformed the 60-kilodalton (kDa) catalytic subunit of calcineurin into a transient 57-kDa fragment and thereafter a 43-kDa limit fragment. In the presence of calmodulin, the 60-kDa subunit was sequentially proteolysed to a 55-kDa fragment and then a 49-kDa fragment. Upon proteolysis in the absence or presence of calmodulin, the p-nitrophenyl phosphatase activity (assayed in the presence of calmodulin) was increased by 300%. The 43- and the 49-kDa fragments were found to (i) remain associated with the small subunit (17 kDa), (ii) have lost the ability to bind and to be activated by calmodulin, and (iii) have phosphatase activity that was still stimulated by Mn2+ or Ni2+. The 43- + 17-kDa form had similar Km values for various substrates, but the Vmax values were increased compared with the native enzyme. It is proposed that (i) a 43-kDa core segment of the 60-kDa subunit of calcineurin contained the catalytic domain, the small subunit-binding domain, and the metal ion (Mn2+ and (or) Ni2+) binding site; and (ii) two distinct types of inhibitory domains exist near the end(s) of the large subunit, one of which is calmodulin regulated, while the other is calmodulin independent.Key words: calcineurim, calpain, calmodulin, phosphatase, protease.
Lipoprotein-mediated efflux of radiolabeled cholesterol from cells does not indicate net removal of cellular cholesterol mass
Authors: Ranganathan Subramanian , Gasman John D. , Matsuura Hideo , Kottke Bruce A.
Published: December 2009

The efflux of cholesterol from human skin fibroblasts was determined using radioisotope techniques and mass measurements. When the cells were labeled with [14C]- or [3H]-cholesterol and then incubated with very low density, low density, or high density lipoproteins or with serum, 20 to 30% of the label was released into the medium in 20 h. However, when the cellular cholesterol content was determined after incubation with various lipoproteins under identical conditions, only the heavier subfraction of high density lipoproteins (HDL3) caused a significant decrease in cellular cholesterol. This net removal of cholesterol can be observed in the cells without overloading them with cholesterol, by incubation with low density lipoproteins. Time studies indicated that at least 24 h of incubation is required to detect significant removal of cellular cholesterol. These experiments show that methods using the release of labeled cholesterol from cultured cells to determine net cholesterol removal mediated by high density lipoprotein, although currently used by many investigators, can lead to erroneous conclusions when employed without the measurement of cholesterol mass.Key words: low-density lipoproteins, high-density lipoproteins, fibroblasts, cholesterol, bidirectional flux.
Purification and kinetics of phenylpropanoid O-methyltransferase activities from Brassica oleracea
Authors: Carolis Emidio De , Ibrahim Ragai K.
Published: December 2009

Two phenylpropanoid O-methyltransferase isoforms were purified to homogeneity from young cabbage leaves. They catalyzed the meta-O-methylation of caffeic and 5-hydroxyferulic acids to ferulic and sinapic acids, respectively. Both isoforms I and II exhibited different elution patterns from a Mono Q column, distinct apparent pIs on chromatofocusing, different product ratios, and stability on adenosine–agarose affinity column. On the other hand, both isoforms had similar apparent molecular masses (42 kilodaltons) and a pH optimum of 7.6. They exhibited no requirement for divalent cations and were both irreversibly inhibited by iodoacetate. Substrate interaction kinetics of the more stable isoform I, using the 5-hydroxyferulic acid and S-adenosyl-L-methionine, gave converging lines. Product inhibition studies showed competitive inhibition between S-adenosyl-L-methionine and S-adenosyl-L-homocysteine and non-competitive inhibition between the phenylpropanoid substrate and its methylated product. The kinetic patterns are consistent with an ordered bi bi mechanism, where S-adenosyl-L-methionine is the first substrate to bind and S-adenosyl-L-homocysteine is the last product released.Key words: phenylpropanoid O-methyltransferase, purification, isoforms, adenosine–agarose affinity chromatography, kinectic mechanism.
Unusual properties of a cold-labile fraction of Atlantic cod (Gadus morhua) brain microtubules
Authors: Strömberg E. , Serrano L. , Avila J. , Wallin M.
Published: December 2009

A cold-labile fraction of microtubules with unusual properties was isolated from the brain of the Atlantic cod (Gadus morhua). The yield was low, approximately six times lower than that for bovine brain microtubules. This was mainly caused by the presence of a large amount of cold-stable microtubules, which were not broken down during the disassembly step in the temperature-dependent assembly–disassembly isolation procedure and were therefore lost. The isolated cold-labile cod microtubules contained usually only a low amount of microtubule-associated proteins (MAPs). Three high molecular mass proteins were found, of which one was recognized as MAP2. Cod MAP2 differed from mammalian brain MAP2; it was not heat stable and had a slightly higher molecular mass. In contrast to mammalian MAPs, MAP1 was not found in the cold-labile fraction of microtubules. A new heat-labile MAP of higher molecular mass (400 kilodaltons) was however present, as well as a heat-stable protein of slightly lower molecular mass than MAP2. These MAPs showed similar tubulin-binding characteristics as bovine brain MAPs, since they coassembled with taxol-assembled bovine brain microtubules consisting of pure bovine tubulin. In spite of the fact that Ca2+ bound equally to cod and porcine tubulins, it did not inhibit cod microtubule assembly even at high concentrations (> 1 mM). In contrast, rings, spirals, and macrotubules were formed. The results show that there are major differences between this fraction of cod microtubules and microtubules from mammalian brain.Key words: microtubules, microtubule-associated proteins, calcium, cod.
Selective metal ion binding at the calcium-binding sites of the sea urchin extraembryonic coat protein hyalin
Authors: Robinson John J.
Published: December 2009

The interaction of metal ions with the sea urchin extraembryonic coat protein hyalin was investigated. Hyalin, immobilized on nitrocellulose membrane, bound Ca2+ and this interaction was disrupted by ruthenium red and selective metal ions. The divalent cations Cd2+ and Mn2+, when present at a concentration of 30 μM, displaced hyalin-bound Ca2+. In competition assays, 1 mM Cd2+ or 3 mM Mn2+ were effective competitors with Ca2+ for binding to hyalin. Cobalt, at a concentration of 30 μM, was unable to displace protein-bound Ca2+, but was effective in competition assays at a concentration of at least 10 mM. Magnesium and the monovalent cation Cs+ were unable to disrupt Ca2+–hyalin interaction. Interestingly, Cd2+, Mn2+, and Co2+ mimicked the biological effects of Ca2+ on the hyalin self-association reaction. These results clearly demonstrate that the Ca2+-binding sites on hyalin can selectively accommodate other divalent cations in a biologically active configuration.Key words: calcium, metal ion, binding, hyalin.
Acetylcholinesterase and nonspecific cholinesterase activities in rat liver: subcellular localization, molecular forms, and some extraction properties
Authors: Berninsone Patricia , Katz Eleonora , Napp Monica , Azcurra Julio
Published: December 2009

Subcellular distribution and some extraction properties of acetylcholinesterase (AchE) (EC 3.1.1.7) and nonspecific cholinesterase (ChE) (EC 3.1.1.8) were studied in rat liver employing subcellular fractionation techniques. All purified subcellular fractions were enriched in total cholinesterase activity over the homogenate. Plasma membrane and Golgi fractions showed a significant enrichment in AchE activity, while ChE activity was enriched in both rough and smooth endoplasmic reticulum. Subcellular fractions were subjected to conditions that selectively release proteins having varying degrees of association to membranes. High-pH treatment (known to release peripheral and soluble proteins) extracted ChE activity, but more than 90% of AchE activity remained associated to the pellet. Solubility properties and molecular forms of AchE and ChE in this tissue were studied by extraction in high-salt medium with and without Triton X-100, followed by velocity sedimentation centrifugation. Most of AchE activity (88%) (41% G4 and 59% G2 + G1) was detergent soluble; 42% of ChE activity (detected only as G2 + G1) was high-salt soluble, whereas remaining ChE activity was detergent soluble. These results indicate not only a different subcellular location for both enzymes, but also point to a differential association to membranes. AchE behaves as an integral membrane protein and ChE behaves as a peripheral or a luminal soluble protein.Key words: acetylcholinesterase, membrane association, molecular forms, nonspecific cholinesterase, rat liver, subcellular distribution.
Relationship of phospholipid hexagonal phases to biological phenomena
Authors: Epand Richard M.
Published: December 2009

Phospholipid bilayers can undergo morphological rearrangements to other phases. The formation of one of these nonbilayer phases, the hexagonal phase, is preceded by an increase in the hydrophobicity of the bilayer surface and a destabilization of the bilayer structure. Certain membrane additives promote, while others inhibit, the formation of the hexagonal phase. Many of the molecular features that determine this phase preference are understood. Some of the properties of membranes are modulated by agents that affect the relative stability of the bilayer and hexagonal phases. Addition of bilayer stabilizers to a membrane decreases its fusogenic behaviour. One such bilayer stabilizer is cholesterol sulfate, which may function physiologically to inhibit the fusion of sperm cells. Several antiviral agents are also found to be bilayer stabilizers and some have been shown to inhibit membrane fusion phenomena. Another biological property that is modulated in a predictable manner by agents which affect the bilayer–hexagonal phase equilibrium is insulin-promoted glucose uptake in adipocytes. Bilayer stabilizers inhibit this process showing that the effects of insulin can be modulated by the bulk biophysical properties of the membrane. The activity of a number of membrane-bound enzymes is also lowered by bilayer stabilizers. Neutral and zwitterionic bilayer stabilizers are inhibitors of protein kinase C. Thus, the alteration of the bilayer–hexagonal phase transition by drugs may provide a useful parameter for predicting their effects on biological membranes.Key words: hexagonal phase, phosphatidylethanolamine, membrane fusion, virus, insulin, protein kinase C.
Antibodies to liposomal phosphatidylserine and phosphatidic acid
Authors: Banerji Benoy , Alving Carl R.
Published: December 2009

Polyclonal antisera to phosphatidylserine or phosphatidic acid were induced in rabbits by injecting liposomes containing phosphatidylserine or phosphatidic acid and lipid A. Adsorption of antisera with liposomes containing different phospholipids revealed that some degree of reactivity with one or more phospholipids other than the immunizing phospholipid was often observed. However, cross-reactivity with other phospholipids was not a universal phenomenon, and one antiserum to phosphatidylserine failed to cross-react (i.e., was not adsorbed) with liposomes containing other phospholipids. All of the antisera were inhibited by soluble phosphorylated haptens (e.g., phosphocholine but not choline), but one of the antisera to phosphatidylserine was inhibited both by phosphoserine and by serine alone. Liposomal membrane composition influenced the activity of antiserum to phosphatidylserine. Regardless of whether unsaturated (beef brain) or saturated (dimyristoyl) phosphatidylserine was used in the immunizing liposomes, the antisera reacted more vigorously with liposomes containing unsaturated than saturated phosphatidylserine. We conclude that liposomes containing lipid A can serve as vehicles for stimulating polyclonal antisera to phosphatidylserine and phosphatidic acid. Although cross-reactivity with certain other phospholipids can be observed, sera from selected animals apparently can exhibit a high degree of specific activity to the immunizing phospholipid antigen.Key words: liposomes, antibodies, phospholipids, phosphatidylserine, phosphatidic acid.
Transfer of arachidonic acid from phosphatidylcholine to phosphatidylethanolamine during storage of human platelets for 5 days
Authors: Lacasse Julie , Labow Rosalind S. , Kates Morris , Adams George A.
Published: December 2009

Human platelets are routinely stored for 5 days prior to transfusion, but they deteriorate during storage. Since very little information is available concerning the effect of storage on platelet phospholipid metabolism, the biosynthesis and remodelling of platelet phospholipids were studied. Platelets were incubated separately with [14C]glycerol, [14C]arachidonic acid, or a mixture of [14C]glycerol and [3H]arachidonic acid, and stored in a platelet storage medium at 22 °C. Maximum glycerol uptake (20%) was attained after 6 h. [14C]Glycerol was incorporated into phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol, and to a much lesser extent phosphatidylserine, under storage conditions for 5 days. The distribution of the initial arachidonic acid uptake was not as would be expected based on the molar composition of endogenous phospholipids. The arachidonic acid (75%) which was taken up within 10 min of incubation distributed 55% into the phosphatidylcholine and only 14% into the phosphatidylethanolamine; the molar composition is actually 18% phosphatidylcholine and 47% phosphatidylethanolamine. During storage, there was a continuous transfer of the radiolabeled arachidonic acid from phosphatidylcholine to phosphatidylethanolamine until, after 5 days, the distribution of arachidonic acid was identical to the endogenous distribution. In contrast, no change in the glycerol incorporation pattern was detected during storage. This suggested that the mechanism for arachidonic acid redistribution was not through exchange of polar head groups, but through acyl transfer of arachidonic acid from phosphatidylcholine to phosphatidylethanolamine.Key words: human, platelet, storage, arachidonate, phospholipids.
Cloning and characterization of the N-acetylglucosamine operon of Escherichia coli
Authors: Peri Krishna G. , Goldie Hughes , Waygood E. Bruce
Published: December 2009

Three enzymes are required for N-acetylglucosamine (NAG) utilization in Escherichia coli: enzyme IInag (gene nagE), N-acetylglucosamine-6-phosphate deacetylase (gene nagA), and glucosamine-6-phosphate isomerase (gene nagB). The three genes are located near 16 min on the E. coli chromosome. A strain of E. coli, KPN9, incapable of utilizing N-acetylglucosamine, was used to screen a genomic library of E. coli for a complementing recombinant colicin E1 plasmid that allowed for growth on N-acetylglucosamine. Plasmid pLC5-21 was found to contain all three known nag genes on a 5.7-kilobase (5.7-kb) fragment of DNA. The products of these nag genes were identified by complementation of E. coli strains with mutations in nagA, nagB, and nagE. The gene products from the 5.7-kb fragment were identified by [35S]methionine-labelled maxicells and autoradiography of sodium dodecyl sulphate – polyacrylamide electrophoresis gels. The gene products had the following relative masses (Mrs: nagE, 62 000; nagA, 45 000; nagB, 29 000. In addition, another product of Mr 44 000 was detected. The genes have been sequenced to reveal an additional open reading frame (nagC), a putative catabolite activator protein binding site that may control nagB and nagE, putative rho-independent terminator sites for nagB and nagE, and sequence homologies for RNA polymerase binding sites preceding each of the open reading frames, except for nagA. The calculated molecular weights (MWs) of the gene products derived from the sequence are as follows: nagA, 40 954; nagB, 29 657; nagC, 44 664; nagE, 68 356. No role is known for nagC, although a number of regulatory roles appear to be plausible. No obvious transcriptional termination site distal to nagC was found and another open reading frame begins after nagC. This gene, nagD, was isolated separately from pLC5-21, and the sequence revealed a protein with a calculated MW of 27 181. The nagD gene is followed by repetitive extragenic palindromic sequences. The nag genes appear to be organized in an operon: [Formula: see text]Key words: N-acetylglucosamine, N-acetylglucosamine-6-P deacetylase, glucosamine-6-P isomerase, repetitive extragenic palindromic sequences, catabolite repression.
Influence of biotin on the lipid composition of plasma membranes from Hypomyces chlorinus
Authors: Touze-Soulet J.-M. , Weete J. D. , Sancholle M. , Rami J. , Dargent R.
Published: December 2009

Subfractions of microsomal membranes from the biotin heteroauxotroph Hypomyces chlorinus Tul., cultured in media containing optimal or suboptimal levels of the vitamin, were isolated using a two-phase polymer system and characterized as plasma membrane-enriched on the basis of absence of Zn2+-induced aggregation, enrichment of appropriate enzyme markers, and phosphotungstic acid – chromic acid staining. The protein/lipid ratio of membranes from the biotin-sufficient and -deficient cultures was 2; the phospholipid content was 126 and 181 μg/mg protein; and the sterol/phospholipid molar ratios were 0.38 and 0.16, respectively, with ergosterol as the predominant sterol. The lipids were qualitatively similar in the plasma membrane fractions from the two treatments. Ergosterol content of the membrane fractions from mycelium cultured in low biotin medium was 40–100% less than that from the corresponding biotin-sufficient cultures. A nonphosphorus glycolipid was the predominant lipid of the plasma membranes from both treatments.Key words: Hypomyces, plasma membrane, sterols, biotin deficiency, ergosterol.
Dynamic structure of the lower density lipoproteins. II. Deuterium NMR studies of the monolayer of very low and low density lipoproteins
Authors: Chana Ravinder S. , Treleaven W. Dale , Parmar Yashpal I. , Cushley Robert J.
Published: December 2009

The order of phosphatidylcholine (PC) acyl chains in the surface monolayer of very low density lipoproteins (VLDL) and low density lipoproteins (LDL) has been determined from 2H nuclear magnetic resonance order parameters, SCD, using selectively deuterated PC or palmitic acids. From the computer simulated line shapes, we find two distinct phospholipid domains within the amphiphilic monolayer of both VLDL and LDL. In the more ordered domain of LDL, SCD was ≈ 0.3 for the "plateau" chain region. The SCD values of VLDL particles are similar to those of LDL for the 5,6- and 11,12-positions, hence we suggest the organization of the more ordered region of VLDL and LDL are similar. The domain of low order in LDL comprises < 10% of the phospholipid molecules (we do not distinguish between PC and sphingomyelin), having approximately the same order (SCD < 0.1) as egg PC - sphingomyelin unilamellar vesicles. In VLDL, the domain of low order comprises between ≈ 10 and ≈ 20% of the phospholipid molecules and the entire acyl chain is in an essentially isotropic environment (SCD < 0.02). We prepared VLDL-sized microemulsions composed of egg PC, deuterated PC, and triolein to test whether the apoproteins were responsible for creating the two differently organized domains in VLDL and LDL. Surprisingly, these protein-free particles also showed two domains of different order at two temperatures. The high order region, however, is less ordered than in VLDL and LDL. We explain two surface domains of PC in terms of lipid organization and the unique interactions of lipids in the various lipoprotein particles.Key words: lipoproteins, deuterium NMR, phospholipid organization, microemulsions, surface diffusion.