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)

Fatty acyl-ester hydrolase was not detectable in dry sunflower seeds using various p-nitrophenyl-acyl-esters, 1,2-O-didodecyl-rac-glycero-3-glutaric acid-resorufin ester or emulsified sunflower oil as substrate. After inhibition of the seeds, acyl-ester hydrolase activity slowly developed in cotyledon extracts and was maximal after 5 days. No activity was directly measurable on oil bodies. The enzyme was purified 615-fold to apparent homogeneity, as determined by performing SDS-PAGE electrophoresis, and biochemically characterized. With p-nitrophenyl-caprylate the optimum pH was around 8.0. The purification procedure involved an acetone powder from 5-day dark-germinated seedlings, chloroform-butanol extraction and three chromatography steps. We obtained 35 micrograms of pure enzyme from 250 g of fresh cotyledons with an activity yield of around 7%. It should be possible to subsequently improve this low recovery as we gave priority here, in the first instance, to purity at the expense of the yield. The enzyme consisted of one glycosylated polypeptide chain with a molecular mass of approx. 45 kDa and, as far as we could tell, it did not seem to require metal ions to be fully active, as it was not inhibited by EDTA or o-phenanthroline and not activated by various mono or bivalent metal ions. The amino acid composition showed the presence of four cysteine and four tryptophan residues. The enzyme was partially inhibited by dithiothreitol, DTNB and PCMB. The fact that high inhibition was observed in the presence of PMSF indicates that a serine residue may possibly be involved in the catalytic mechanism. The hydrophobicity index was about 53.6% which places this enzyme in the class of the soluble proteins in good agreement with the fact that it was mainly present in the soluble part of the crude extract. Partial characterization of glycan chains, using antiglycan antibodies, showed the presence of complex Asn-linked glycans. The enzyme was active on purified sunflower glycerol derivatives. It was also able to hydrolyze monooleyl and dioleyl glycerols, as well as phosphatidylcholine, but not cholesteryl esters. Using p-nitrophenyl-acyl-esters as substrate, the highest activity was observed with middle-chain derivatives (C6 and C8). Its maximum activity was about 0.015 units mg-1 with sunflower oil. It was not activated by an organic solvent such as isooctane. This enzyme probably is involved in acyl-ester hydrolysis which follows triacylglycerol mobilization by true lipases.
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PMID:Purification and characterization of a fatty acyl-ester hydrolase from post-germinated sunflower seeds. 769 23

An intracellular carboxylesterase from Pseudomonas sp. was overproduced in E. coli, and purified to homogeneity by a combination of hydrogen bond chromatography, gel filtration, and hydrophobic interaction chromatography. Gel filtration and SDS-PAGE suggested that the purified enzyme consisted of two subunits of molecular mass of 28 kDa. Its isoelectric point was 5.9. The enzyme was thermolabile, and showed its maximum activity at 22 degrees C (pH 7.5). Methyl propionate was hydrolyzed at the highest rate among the fatty acid methyl esters tested. PMSF, DFP, PCMB, and HgCl2 inhibited the enzyme markedly, suggesting that serine and/or cysteine is in or near the active site.
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PMID:Purification and characterization of a carboxylesterase from Pseudomonas sp. KWI-56. 776 64

D-Erythroascorbic acid was detected from the cell extracts of a dimorphic fungus, Candida albicans. Its concentration in yeast cells grown at 25 degrees C was estimated to be about 0.45 mumol/ml cell water. D-Arabinono-1,4-lactone oxidase, which catalyses the final step in the biosynthesis of D-erythroascorbic acid, was purified 639-fold from the mitochondrial fraction of C. albicans to apparent homogeneity, with an overall yield of 21.2%, by a purification procedure consisting of Triton X-100 solubilisation, ammonium sulphate precipitation, anion-exchange, hydrophobic-interaction, gel-filtration and dye-ligand chromatographies. Gel-filtration chromatography and polyacrylamide-gradient gel electrophoresis in the presence of deoxycholate gave apparent molecular masses of 110 kDa and 84.4 kDa, respectively. SDS/PAGE showed only one protein band corresponding to a molecular mass of 66.7 kDa. Considering the binding of detergents, the enzyme is suggested to be a single polypeptide. The enzyme showed a typical fluorescence excitation spectrum of a flavin-containing enzyme. The flavin was not released by treatment with SDS, CCl3CO2H or boiling, indicating that it may be covalently bound to the enzyme protein. The enzyme was optimally active at 40 degrees C and at pH 6.1. The enzyme was stable in the range pH 7.5-10. An apparent Km value for D-arabinono-1,4-lactone was 44.1 mM. L-Galactono-1,4-lactone, L-gulono-1,4-lactone and L-xylono-1,4-lactone could also serve as substrates. Competitive inhibition was demonstrated with D-glucono-1,5-lactone, L-arabinono-1,4-lactone, D-galactono-1,4-lactone and D-gulono-1,4-lactone. p-Chloromercuribenzoate, N-ethylmaleimide, iodoacetic acid, iodoacetamide and divalent metal ions such as Cd2+, Hg2+, Mn2+ and Zn2+ exhibited inhibitory effects on the enzyme.
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PMID:Characterisation of D-arabinono-1,4-lactone oxidase from Candida albicans ATCC 10231. 795 97

A novel racemase active toward 2-oxothiazolidine-4-carboxylic acid was purified 310-fold with 5% recovery to near homogeneity from a crude extract of Flectobacillus sp. B-1, which had been isolated as a bacterium being able to assimilate (S)-2-oxothiazolidine-4-carboxylic acid. The molecular weight was estimated to be 92,000 by gel filtration. The purified preparation migrated as a single band of molecular weight 49,000 upon SDS-polyacrylamide gel electrophoresis. The enzyme exhibited maximum activity at pH 8.0 and 45 degrees C. The enzyme also racemized 5-oxoproline but did not act on proline and 4-hydroxyproline. The enzyme apparently had no coenzyme requirement. The enzyme activity was inhibited to 62-100% by SH-blocking reagents such as HgCl2, AgNO3, PCMB, iodoacetamide, N-ethylmaleimide and N-bromosuccinimide.
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PMID:Purification and some properties of a novel racemase, which racemizes 2-oxothiazolidine-4-carboxylic acid and 5-oxoproline, from Flectobacillus sp. strain B-1. 818 29

Prolidase (iminodipeptidase, EC 3.4.13.9) was purified from an extract of Xanthomonas maltophilia, by ammonium sulfate fractionation and sequential chromatographies on DEAE-Toyopearl, Toyopearl HW65C, FPLC-Hiload Superdex 200 pg, and FPLC-Hitrap Q columns, which an activity recovery of 2.3%. The enzyme was the most active at pH 7.5 with Leu-Pro as substrate. It was stable between pH 6.0 and 8.5 for 60 min at 37 degrees C and retained half of activity after 60 min at 37 degrees C. The isoelectric point of the enzyme was 3.7. Its molecular weight was estimated to be 100,000 by gel filtration on FPLC-Hiload Superdex 200 and 51,000 by SDS-PAGE, suggesting that it is a dimer. It hydrolyzed dipeptides only if proline is located at the carboxyl terminal position. The enzyme was inhibited by PCMB and o-phenanthroline, and was activated by Mn2+.
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PMID:Prolidase from Xanthomonas maltophilia: purification and characterization of the enzyme. 854 47

An EDTA-insensitive prolidase (proline dipeptidase, EC 3.4.13.9) was isolated from a cell-free extract of Aureobacterium esteraromaticum IFO 3752. The enzyme was purified almost to homogeneity using acetone precipitation, hydrophobic chromatography, ion-exchange chromatography, and gel-permeation chromatography. The enzyme has a molecular weight of about 440,000 by gel permeation chromatography, and about 40,000 by SDS polyacrylamide gel electrophoresis. The isoelectric point was 4.6. The enzyme hydrolyzed aminoacylprolines such as Ser-Pro. Thr-Pro, Gly-Pro, Ala-Pro, Ile-Pro, Leu-Pro, and Pro-Pro. It also hydrolyzed Gly-Hyp and Pro-Hyp. The rate of hydrolysis for Pro-Hyp was the highest among the substrates tested. Optimum pH for hydrolyzing Pro-Hyp was 9.0 and the enzyme was stable in the pH range from 5 to 10. The optimum temperature was estimated to be 45 degrees C using 10 min of reaction. At least 90% of the initial activity remained after 30 min of incubation at 60 degrees C. p-Chloromercuribenzoic acid and o-phenanthrolin inhibited the enzyme's activity while EDTA did not. Addition of Mn2+ ion did not stimulate activity. These results suggest either that the metal ion in the enzyme may be tightly bound to the polypeptide chain, or that the enzyme is not a metallo-enzyme but a thiol-enzyme.
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PMID:Purification and characterization of a prolidase from Aureobacterium esteraromaticum. 878 7

Lactobacillus (L.) gasseri JCM1031 has 2 kinds of 6-phospho-beta-galactosidase (P-beta-gal) in the cytosol. P-beta-gal I was already isolated and characterized for our previous paper [Biosci. Biotech. Biochem., 60, 139-141 (1996)]. Another, P-beta-gal II, was purified to homogeneity through several liquid chromatographic steps for this paper. The molecular mass of the purified enzyme was estimated to be 58 kDa by SDS-PAGE. The Km and V(max) for ONPGal-6P were 0.76 mM and 86.0 mumol/min/mg, respectively. Reducing reagents and Fe2+ ion activated the enzyme, but PCMB, Zn2+, and Hg2+ ions, and alkylation reagents strongly inhibited it. Twenty-five residues of the N-terminal amino acid sequence of the enzyme were identified. P-beta-gal II was different from P-beta-gal I in several characteristics.
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PMID:Coexistence of two kinds of 6-phospho-beta-galactosidase in the cytosol of Lactobacillus gasseri JCM1031--purification and characterization of 6-phospho-beta-galactosidase II. 882 45

Long-chain acyl-CoA thioesterases, which catalyze the cleavage of acyl-CoA's to free fatty acids and CoASH, are abundant in animal cells. However, in yeast little is known about presence and function of acyl-CoA thioesterase activity. Therefore a commercial lipase preparation from the yeast Candida rugosa was investigated and found to contain high myristoyl-CoA thioesterase activity. Hydrophobic interaction chromatography separated the activity into three peaks, of which two enzymes (YTE-1 and YTE-2) were purified to apparent homogeneity with molecular masses of about 40 kDa as determined by size-exclusion chromatography and SDS-PAGE. The employed purification protocol resulted in final preparations with specific activities of about 90 micromol/mg/min with myristoyl-CoA as substrate. YTE-1 and YTE-2 showed similar kinetic properties and YTE-1 was characterized in detail. Acyl-CoA chain-length specificity showed that YTE-1 was not active on acyl-CoAs shorter than decanoyl-CoA, at the substrate concentrations tested. The best substrates were C14-C18 acyl-CoAs with Vmax values of about 150 micromol/mg/min and Km values of 15-46 microM. The enzyme was very active with lauroyl-CoA (Vmax about 400 micromol/mg/min) although the Km was high (about 325 microM). The purified enzyme was also active on short-chain nitrophenyl esters but inactive with tributyrin. Treatment of the protein with N-glycosidase F decreased the molecular mass about 1-2 kDa, indicating the presence of carbohydrate of the high mannose type. Diisopropyl fluorophosphate (DFP) inhibited the enzyme activity efficiently and the protein was covalently labeled with [3H]DFP. p-Chloromercuribenzoic acid inhibited the thioesterase activity but did not affect carboxylesterase activity. N-terminal sequence analysis and labeling by DFP suggest that these long-chain acyl-CoA thioesterases belong to a novel group of yeast serine esterases.
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PMID:Isolation and characterization of novel long-chain acyl-CoA thioesterase/carboxylesterase isoenzymes from Candida rugosa. 883 45

A highly stable cysteine protease was purified to homogeneity from the latex of Ervatamia coronaria by a simple purification procedure involving ammonium sulfate precipitation and ion-exchange chromatography. The molecular mass was estimated to be approximately 25,000 Da by SDS-PAGE and gel filtration. The extinction coefficient (epsilon 280 nm 1%) of the enzyme was 24.6. The enzyme hydrolyzed denatured natural substrates like casein, hemoglobin, azoalbumin, and azocasein with a high specific activity but showed low specific activity towards synthetic substrates. The pH and temperature optima were 7.5-8.0 and 50 degrees C respectively. The activity of the enzyme was strongly inhibited by thiol-specific inhibitors like leupeptin, iodoacetamide, PCMB, NEM, and mercuric chloride. The striking property of this enzyme was its stability over a wide pH range (2-12) and other extreme conditions of temperature, denaturants, and organic solvents. The N-terminal sequence showed marked similarity to known cysteine proteases.
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PMID:Purification and characterization of a highly stable cysteine protease from the latex of Ervatamia coronaria. 983 31

Miltpain (EC.3.4.22.-) is a cysteine proteinase that preferentially hydrolyzes basic proteins, previously found in the milt of chum salmon. Here we report a similar cysteine proteinase in the milt of the marine Pacific cod. The enzyme was isolated and purified 6900-fold and with an estimated mass of 63 kDa by gel filtration chromatography and 72 kDa by SDS/PAGE. Cod miltpain has an optimum pH of 6.0 for Z-Arg-Arg-MCA hydrolysis, and Km of 11.5 microM and kcat of 19.0 s-1 with Z-Arg-Arg-MCA. It requires a thiol-inducing reagent for activation and is inhibited by E-64, iodoacetamide, CA-074, PCMB, NEM, TLCK, TPCK, ZPCK and o-phenanthroline. This proteinase strongly hydrolyzes basic proteins such as salmine, clupeine and histone, and exhibits unique substrate specificity toward paired basic residues such as Lys-Arg, Arg-Arg on the substrates of P2-P1. The isoelectric point is 5.2 by isoelectric focusing. N-Terminal sequencing gave a sequence of < EVPVEVVRXYVTSAPEK. The cysteine proteinase from Pacific cod very closely matches the previously reported miltpain from chum salmon.
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PMID:Miltpain, a cysteine proteinase, from milt of Pacific cod (Gadus macrocephalus): purification and characterization. 1090 66


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