Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Acetyl CoA: deacetylcephalosporin C O-acetyltransferase, which catalyzes the final step of the biosynthetic pathway to cephalosporin C, was stabilized by a buffer solution containing 7-aminocephalosporanic acid and purified over 1300-fold from Acremonium chrysogenum. The purified enzyme has a molecular weight of 55,000 as measured by gel filtration. SDS-polyacrylamide gel electrophoresis showed two subunit bands corresponding to molecular weights of 27,000 and 14,000. The enzyme has an isoelectoric point at pH 4.0 and optimum activity at pH 7.5.
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PMID:Purification of acetyl coenzyme A: deacetylacephalosporin C O-acetyltransferase from Acremonium chrysogenum. 136 46

The present study provides strong evidence that the previously isolated hepatic microsomal beta-hydroxyacyl-CoA dehydrase (EC 4.2.1.17), believed to be a component of the fatty acid chain-elongation system, is derived, not from the endoplasmic reticulum, but rather from the peroxisomes. The isolated dehydrase was purified over 3000-fold and showed optimal enzymic activity toward beta-hydroxyacyl-CoAs or trans-2-enoyl-CoAs with carbon chain lengths of 8-10. The purified preparation (VDH) displayed a pH optimum at 7.5 with beta-hydroxydecanoyl-CoA, and at 6.0 with beta-hydroxystearoyl-CoA. Competitive-inhibition studies suggested that VDH contained dehydrase isoforms, and SDS/PAGE showed three major bands at 47, 71 and 78 kDa, all of which reacted to antibody raised to the purified preparation. Immunocytochemical studies with anti-rabbit IgG to VDH unequivocally demonstrated gold particles randomly distributed throughout the peroxisomal matrix of liver sections from both untreated and di-(2-ethylhexyl) phthalate-treated rats. No labelling was associated with endoplasmic reticulum or with the microsomal fraction. Substrate-specificity studies and the use of antibodies to VDH and to the peroxisomal trifunctional protein indicated that VDH and the latter are separate enzymes. On the other hand, the VDH possesses biochemical characteristics similar to those of the D-beta-hydroxyacyl-CoA dehydrase recently isolated from rat liver peroxisomes [Li, Smeland & Schulz (1990) J. Biol. Chem. 265, 13629-13634; Hiltunen, Palosaari & Kunau (1989) J. Biol. Chem. 264, 13536-13540]. Neither enzyme utilizes crotonoyl-CoA or cis-2-enoyl-CoA as substrates, but both enzymes convert trans-2-enoyl substrates into the D-isomer only. In addition, the VDH also contained beta-oxoacyl-CoA reductase (beta-hydroxyacyl-CoA dehydrogenase) activity, which co-purified with the dehydrase.
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PMID:Evidence that beta-hydroxyacyl-CoA dehydrase purified from rat liver microsomes is of peroxisomal origin. 141 96

In the enteric bacterium, Escherichia coli, acyl coenzyme A synthetase (fatty acid:CoA ligase (AMP-forming) EC 6.2.1.3) activates exogenous long-chain fatty acids concomitant with their transport across the inner membrane into metabolically active CoA thioesters. These compounds serve as substrates for acyl-CoA dehydrogenase in the first step in the process of beta-oxidation. The acyl-CoA synthetase structural gene, fadD, has been identified on clone 6D1 of the Kohara E. coli gene library and by a process of subcloning and complementation analyses shown to be contained on a 2.2-kilobase NcoI-ClaI fragment of genomic DNA. The polypeptide encoded within this DNA fragment was identified following T7 RNA polymerase-dependent induction and estimated to be M(r) = 62,000 using SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of acyl-CoA synthetase was determined by automated sequencing to be Met-Lys-Lys-Val-Trp-Leu-Asn-Arg-Tyr-Pro. Sequence analysis of the 2.2-kilobase NcoI-ClaI fragment revealed a single open reading frame encoding these amino acids as the first 10 residues of a protein with a molecular weight of 62,028. The initiation codon for methionine was TTG. Primer extension of total in vivo mRNA from two fadD-specific oligonucleotides defined the transcriptional start at an adenine residue 60 base pairs upstream from the predicted translational start site. Two FadR operator sites of the fadD gene were identified at positions -13 to -29 (OD1) and positions -99 to -115 (OD2) by DNase I footprinting. Comparisons of the predicted amino acid sequence of the E. coli acyl-CoA synthetase to the deduced amino acid sequences of the rat and yeast acyl-CoA synthetases and the firefly luciferase demonstrated that these enzymes shared a significant degree of similarity. Based on the similar reaction mechanisms of these four enzymes, this similarity may define a region required for the same function.
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PMID:Cloning, sequencing, and expression of the fadD gene of Escherichia coli encoding acyl coenzyme A synthetase. 146 45

In the yeast Saccharomyces cerevisiae, the enzymes which catalyse the synthesis of ethyl acetate, ethyl n-hexanoate and isoamyl acetate were partly resolved from a fraction containing slowly sedimenting lipoproteins released during cell disruption with glass beads. Solubilization with detergents and fractionation by affinity chromatography have demonstrated the presence of at least three, and probably four, ester synthases which differ in their catalytic properties. Isoamyl-acetate synthase was solubilized and extensively purified to apparent homogeneity by successive chromatographies on various columns. On the basis of its specific activity in cell-free extracts, the enzyme was purified 19,000-fold with a 5% activity yield. As judged by SDS/PAGE, it consists of a single polypeptide chain with a molecular mass of 57 +/- 3 kDa and its apparent pI is 5.5. The enzyme acetylates isoamyl alcohol, ethanol and 12-DL-hydroxystearic acid from acetyl-CoA but is unable to use n-hexanoyl-CoA as a cosubstrate. This enzyme, defined as an acetyl-CoA: O-alcohol acetyltransferase, could be the product of one of the anaerobically induced genes in S. cerevisiae.
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PMID:Short-chain and medium-chain aliphatic-ester synthesis in Saccharomyces cerevisiae. 148 49

1. Limited proteolysis of citrate synthase from Sulfolobus solfataricus by trypsin reduced the rate of the overall reaction (acetyl-CoA + oxaloacetate + H2O----citrate + CoASH) to 4% but did not affect the hydrolysis of citryl-CoA. Experimental results indicate that a connecting link between the enzyme's ligase and hydrolase activity becomes impaired specifically on treatment with trypsin. Other proteolytic enzymes like chymotrypsin and subtilisin inactivated catalytic functions of citrate synthase, ligase and hydrolase, equally well. 2. Tryptic hydrolysis occurs at the N-terminal region of citrate synthase, but a study by SDS/PAGE revealed no difference in molecular mass between native and proteolytically nicked citrate synthase. The peptide removed from the enzyme by trypsin, therefore, contains less than about 15 amino acid residues. 3. The Km values of the substrates for both native and nicked enzyme were identical, as was the state of aggregation (dimeric) of the two enzyme species. These could be separated by affinity chromatography on Blue-Sepharose and differentiated by their isoelectric points (pI = 6.68 +/- 0.08 and pI = 6.37 +/- 0.03 for native citrate synthase and the large tryptic peptide, respectively) as well as by the N-terminus which is blocked in the native enzyme only. 4. Edman degradation of the large tryptic fragment yielded the N-terminal sequence GLEDVYIKSTSLTYIDGVNGVLRY, which is 71% identical to the N-terminal region (positions 9-32) of citrate synthase from Thermoplasma acidophilum. 5. The conversion of citrate synthase into essentially a citryl-CoA hydrolase is considered the consequence of a conformational change thought to occur on tryptic removal of the N-terminal small peptide.
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PMID:Conversion, by limited proteolysis, of an archaebacterial citrate synthase into essentially a citryl-CoA hydrolase. 152 37

The [3H]tetradecylglycidyl-CoA (TDG-CoA)-binding protein (Mr approx. 88,000) of purified outer membranes from rat liver mitochondria was identified by SDS/PAGE. The region in which it migrated was shown to contain another protein which stained strongly with periodic acid-Schiff reagent and could be removed from membrane extracts by incubation with Sepharose-concanavalin A. Amounts of TDG-CoA-binding protein were prepared from lectin-treated extracts using preparative SDS/PAGE and used to raise a polyclonal antibody in a sheep. The IgG fraction purified from this anti-serum reacted strongly with a protein of Mr approximately 88,000 on Western blots, and much more weakly with two other proteins of Mr approximately 76,000 and Mr approximately 53,000 in extracts of rat liver mitochondrial outer membranes. The crude IgG fraction and immunopurified IgG both removed carnitine palmitoyltransferase (CPT) I activity from very pure outer membrane extracts, suggesting that the TDG-CoA-binding protein against which the antiserum was raised also expresses CPT I activity. This was confirmed by the demonstration of a strong positive correlation between CPT I activity and the amount of immunoreactive protein of Mr approximately 88,000 in mitochondria prepared from rats in different physiological states. By contrast, the antibody did not react with CPT II either in mitochondria or in purified form. Similarly, an anti-(CPT II) antibody did not cross-react with CPT I on Western blots, proving conclusively that CPT I and CPT II are immunologically distinct proteins, as well as being of different functional molecular sizes [Zammit, Corstophine & Kelliher (1988) Biochem. J. 250, 415-420]. Immunoblots of mitochondrial proteins obtained from different tissues indicated that, of the rat tissues tested, only kidney cortex mitochondria contain the same isoform of CPT I as that in liver. Heart, skeletal muscle and brown adipose tissue mitochondria contain a slightly smaller isoform which was only weakly reactive with anti-(rat liver CPT I) antibody, indicating that these tissues contain a molecularly quite distinct isoenzyme. This would explain the previous observations that CPT I in these tissues has markedly different kinetic characteristics from the isoenzyme present in liver mitochondria.
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PMID:Development and characterization of a polyclonal antibody against rat liver mitochondrial overt carnitine palmitoyltransferase (CPT I). Distinction of CPT I from CPT II and of isoforms of CPT I in different tissues. 154 54

3-Oxoacyl-[ACP] reductase (E.C. 1.1.1.100, alternatively known as beta-ketoacyl-[ACP] reductase), a component of fatty acid synthetase has been purified from seeds of rape by ammonium sulphate fractionation, Procion Red H-E3B chromatography, FPLC gel filtration and high performance hydroxyapatite chromatography. The purified enzyme appears on SDS-PAGE as a number of 20-30 kDa components and has a strong tendency to exist in a dimeric form, particularly when dithiothreitol is not present to reduce disulphide bonds. Cleveland mapping and cross-reactivity with antiserum raised against avocado 3-oxoacyl-[ACP] reductase both indicate that the multiple components have similar primary structures. On gel filtration the enzyme appears to have a molecular mass of 120 kDa suggesting that the native structure is tetrameric. The enzyme has a strong preference for the acetoacetyl ester of acyl carrier protein (Km = 3 microM) over the corresponding esters of the model substrates N-acetyl cysteamine (Km = 35 mM) and CoA (Km = 261 microM). It is inactivated by dilution but this can be partly prevented by the inclusion of NADPH. Using an antiserum prepared against avocado 3-oxoacyl-[ACP] reductase, the enzyme has been visualised inside the plastids of rape embryo and leaf tissues by immunoelectron microscopy. Amino acid sequencing of two peptides prepared by digestion of the purified enzyme with trypsin showed strong similarities with 3-oxoacyl-[ACP] reductase from avocado pear and the Nod G gene product from Rhizobium meliloti.
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PMID:3-Oxoacyl-[ACP] reductase from oilseed rape (Brassica napus). 156 81

2-Bromopalmitate and 2-bromopalmitoyl-CoA have been shown to inhibit a variety of enzymes and proteins associated with lipid metabolism. We found that both of the brominated compounds were non-competitive inhibitors of two microsomal activities of triacylglycerol biosynthesis, the mono- and diacylglycerol acyltransferases. With both compounds, the calculated Ki values were lower than the Km value for the palmitoyl-CoA substrate. In addition to inhibiting two other lipid synthetic activities, fatty acid CoA ligase and glycerol-3-P acyltransferase, 2-bromopalmitate and 2-bromopalmitoyl-CoA also inhibited two microsomal enzyme activities that are not related to lipid metabolism, NADPH cytochrome-c reductase and glucose-6-phosphatase. Inhibition of the three acyltransferases and fatty acid CoA ligase could be overcome by the addition of phospholipid vesicles, and 2-bromo[14C]palmitate readily labeled a large number of membrane-bound proteins as well as cytosolic proteins that had been solubilized in SDS. Thus, it appears likely that the inhibitory properties of the brominated compounds strongly depend on the effective concentration of the inhibitor within membranes rather than on any specific affinity for an acyl-chain binding region of the enzyme.
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PMID:2-Bromopalmitoyl-CoA and 2-bromopalmitate: promiscuous inhibitors of membrane-bound enzymes. 157 64

1. A new two-step method for purifying component E II of lactyl-CoA dehydratase was developed. The source of the enzyme was Clostridium propionicum grown on either D,L-alanine or L-threonine. No difference in these preparations was observed whether during purification or by SDS/PAGE of the pure enzymes. Both preparations exhibited similar activities towards (R)-lactyl-CoA as well as towards (R)-2-hydroxybutyryl-CoA, the latter being the superior substrate. 2. Three species of (2R)-2-hydroxybutyrate labelled with 3H at C3 were prepared containing 96%, 37% and 63% of the 3H in the 3S-position. By incubation of these species with acetyl-CoA, propionate CoA-transferase and lactyl-CoA dehydratase 104%, 32% and 70% of the 3H, respectively, was release as 3HOH. The data indicate that stereospecific abstraction of the 3Si hydrogen of (2R)-2-hydroxybutyryl-CoA during the dehydration. 3. The identity of the product of the dehydration as crotonyl-CoA was established by the combined action of the enzymes crotonase and (S)-3-hydroxyacyl-CoA dehydrogenase. The results indicate that the elimination of water from (R)-2-hydroxybutyryl-CoA occurs in a syn mode. 4. All enzyme activities necessary for the conversion of L-threonine via (R)-2-hydroxybutyryl-CoA to butyrate were detected in cell-free extracts of C. propionicum. 5. A new mechanism for the dehydration of lactyl-CoA is proposed.
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PMID:(R)-lactyl-CoA dehydratase from Clostridium propionicum. Stereochemistry of the dehydration of (R)-2-hydroxybutyryl-CoA to crotonyl-CoA. 159 94

A 52 kDa protein could only be co-purified with the CoA-modified forms of acetyl-CoA acetyltransferase (acetoacetyl-CoA thiolase) (EC 2.3.1.9) from rat liver mitochondria. Immunoprecipitations of these modified forms with anti-(acetyl-CoA acetyltransferase) IgG or anti-(52 kDa protein) IgG yielded, in addition to the appropriate proteins, the 52 kDa protein or the CoA-modified form of acetyl-CoA acetyltransferase (41 kDa) respectively. This was demonstrated by SDS/PAGE and immunoblots. The modified forms containing the 52 kDa protein could be cross-linked by 1,5-difluoro-2,4-dinitrobenzene to a high-molecular-mass complex containing both the 41 kDa and 52 kDa proteins. The 52 kDa protein was identified as mitochondrial glutamate dehydrogenase (EC 1.4.1.3) by amino acid sequence analysis. The results of co-immunoprecipitation and cross-linking characterize the CoA-modified forms of acetyl-CoA acetyltransferase and the glutamate dehydrogenase as nearest-neighbour proteins.
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PMID:Identification of the CoA-modified forms of mitochondrial acetyl-CoA acetyltransferase and of glutamate dehydrogenase as nearest-neighbour proteins. 168 1


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