Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.1.5 (neuropathy target esterase)
1,070 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. Phospholipase B which hydrolyzes both the acyl ester bonds of diacylphospholipids (diacyl-hydrolase) and the acyl ester bond of monoacylphospholipids or lysophospholipids, [monoacyl-hydrolase or lysophospholipase, EC 3.1.1.5] was purified from Penicillium notatum about 2000-fold over the crude extract. The final preparation was homogeneous on disc electrophoresis. The apparent molecular weight, determined by gel filtration on Sephadex G-200, was about 116,000. The isoelectric point was pH 4.0. 2. The purified enzyme was a glycoprotein. The carbohydrate content was approximately 30%, consisting of mannose, glucose, and glucosamine. The amino acid composition was also determined. 3. The ratio of monoacyl-hydrolase to diacyl-hydrolase activities was influenced by the physical state of the substrate in the assay system. It was about 1 : 1 or 100 : 1 in the presence of absence of Triton X-100, respectively, and the latter value remained constant throughout the purification procedures. 4. Both enzyme activities had the same pH optimum, 4.0, and were heat-labile. None of the metals tested had any effect on either activity except for Fe2+ and Fe3+. Diisopropyl fluorophosphate at relatively high concentrations completely inhibited both enzyme activities. 5. The Michaelis-Menten constants (Km) of the enzyme for egg lecithin were about 1.5 and 25 mM in the absence and presence of Triton X-100, respectively. The Km value for dicaproyllecithin was 9.8 mM in the absence of Triton X-100. 6. Using a mixture of 1-[14C]stearoyl-lecithin and 2-[14C]oleoyl-lecithin in the presence of Triton X-100 as a substrate, it was found that the P. notatum phospholipase B attacked the acyl ester bonds sequentially, first the 2-acyl and then 1-acyl groups.
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PMID:Studies on a phospholipase B from Penicillium notatum. Purification, properties, and mode of action. 0 2

Phospholipase B has not yet been well defined. The most important points about this enzyme are its relationships with lysophospholipase and phospholipase A1. As reported [Saito, K., Sugatani, J. & Okumura, T. (1991) Methods Enzymol. 197, 446-456], Penicillium notatum phospholipase B is a glycoprotein with a molecular mass of 95 kDa and intrinsic lysophospholipase and phospholipase B activities; however, by endogenous proteolytic modification, its phospholipase B activity is lost almost completely, whereas its lysophospholipase activity remains unchanged. A cDNA library of P. notatum was screened by hybridization with two synthetic oligodeoxyribonucleotide probes, which corresponds to two different pentapeptides of the enzyme. A hybridization-positive clone, pPLB18, was isolated and its nucleotide sequence was determined. The deduced amino acid sequence was quite different from that found previously. Therefore, we rescreened the cDNA library with a Sau3AI fragment derived from pPLB18 and isolated a new clone, pPLB15. Comparison of the nucleotide sequences of pPLB15 and pPLB18 revealed that pPLB18 contained an insertion sequence of 53 bp. Consequently, the reading frame was open downstream for 603 amino acid residues. From the assigned sequence, it was deduced that the limited proteolysis occurred between Leu175 and Asp176; eight cysteine residues and 16 potential N-glycosylation sites were also found. No amino acid sequence similarity was found with other proteins, including other phospholipases.
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PMID:Primary structure of protein moiety of Penicillium notatum phospholipase B deduced from the cDNA. 172 56

Phospholipase B bound tightly to the membrane fraction of baker's yeast (Saccharomyces cerevisiae) was purified approximately 226-fold by extraction with sodium deoxycholate (DOC), acetone precipitation, ammonium sulfate fractionation, and column chromatographies on Phenyl-Sepharose CL-4B, DEAE-Sepharose CL-6B, and Sepharose 4B. Only one major activity peak with an apparent molecular weight of 330,000 was detected by gel filtration on Sepharose 4B. However, two glycoprotein bands, one major and one minor, were evident on SDS-polyacrylamide gel electrophoresis in the absence of 2-mercaptoethanol. Isoelectric focusing also revealed two activities, pI 3.4 (major) and pI 3.0 (minor). The purified enzyme had phospholipase B activity (in the presence of DOC) and lysophospholipase activity in a ratio of 1:4.8. Both activities had an optimum pH of 3.5-4.0. Phospholipase B activity was appreciably stimulated only by DOC among bile acids, but lysophospholipase activity was markedly inhibited by them. Both activities were not stimulated by Ca2+ and inhibited by SDS, Triton X-100, Fe3+ and Al3+. The purified enzyme hydrolyzed the acyl ester bonds of phosphatidylcholine sequentially, first the 2-acyl and then the 1-acyl groups. It preferentially hydrolyzed 1-acyl-sn-glycero-3-phosphocholine and, to a lesser degree, 2-acyl-sn-glycero-3-phosphocholine.
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PMID:Purification and properties of a membrane-bound phospholipase B from baker's yeast (Saccharomyces cerevisiae). 636 32

Phospholipase B and lysophospholipase activity is secreted from yeast cells (Saccharomyces cerevisiae) growing aerobically in batch cultures during the exponential phase. A glycoprotein with both activities running on SDS-polyacrylamide slab gels as a broad band between 200 000 and 280 000 Da was purified about 2500-fold by gel filtration, chromatofocusing and hydrophobic interaction chromatography with octyl-Sepharose. The secreted phospholipase has a slightly higher carbohydrate content of 41 mumol/mg protein compared to a form of the enzyme associated to the plasma membrane described in the previous communication (Witt, W., Schweingruber, M.E. and Mertsching, A. (1984) Biochim. Biophys. Acta 795, 108-116) and exerts very similar enzymatic properties. Fatty acids are set free from lysophosphatidylcholine with a 68-fold higher rate than from phosphatidylcholine with a concomitant generation of the corresponding diacyl compound. pH optima of 3.0 and 3.5 were determined with phosphatidylcholine and lysophosphatidylcholine, respectively. During the enzymatic degradation of the cell wall, high amounts of phospholipase activity were released, indicating that the enzyme is present in the periplasmatic space or associated to cell wall components.
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PMID:Secretion of phospholipase B from Saccharomyces cerevisiae. 638 May 92

A lysophospholipase-transacylase (h-LPTA) was purified to homogeneity from a clinical isolate of Candida albicans (C. albicans) that had high extracellular phospholipase activity (strain 16240). The purified enzyme was a glycoprotein with molecular mass of 84 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activities of the enzyme were 117 mumol/min per mg protein for fatty acid release and 459 mumol/min per mg protein for phosphatidylcholine (PC) formation. An apparent Km of the hydrolase activity of the enzyme for 1-palmitoyl-sn-glycero-3-phosphocholine (1-palmitoyl-lyso-PC) was 60.6 microM. The enzyme had a pH optimum at 6.0. Transacylase activity of the enzyme was partially inhibited by palmitoylcarnitine (35% inhibition) and N-ethylmaleimide. In contrast, the hydrolase activity of the enzyme was stimulated by palmitoylcarnitine but was partially inhibited by N-ethylmaleimide. The enzyme exhibited broad specificity to lyso-phospholipids. The h-LPTA activity was not dependent on divalent cations (Ca2+ and Mg2+) and was not inhibited by addition of EDTA or EGTA. These results show that C. albicans strain 16240 with high extracellular phospholipase activity produced h-LPTA in large amount. This enzyme is biochemically distinct from the LPTA enzyme previously isolated from C. albicans 3125.
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PMID:Purification and characterization of lysophospholipase-transacylase (h-LPTA) from a highly virulent strain of Candida albicans. 761 59

S-PLI, an inhibitor of phospholipase C (PLC) produced by Streptomyces sp. strain No. 6288, was purified from the culture filtrate by salting-out with solid ammonium sulfate, column chromatography on CM-cellulose and gel filtration on Sephadex G-75. The molecular weight of S-PLI was estimated to be 65,000 by SDS-polyacrylamide gel electrophoresis. The inhibitor was found to be a glycoprotein with a composition of 609 amino acids and 19 glucose residues having an isoelectric point at 7.8. S-PLI was stable from pH 3 to 10 at 37 degrees C and up to 40 degrees at pH 6.0. The inhibitory activity showed pH- and temperature-dependence with a maximum around pH 7.0 at 50 degrees C. S-PLI inhibited phospholipase C in a competitive manner (Ki value; 9.5 x 10(-6) mM), but did not inhibit S-Hemolysin, phospholipase A2; phospholipase B, phospholipase D and phosphatases. S-PLI is the first reported example of a glycoproteinaceous inhibitor of microbial origin which is able to specifically inhibit phospholipase C.
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PMID:A novel phospholipase C inhibitor, S-PLI produced by Streptomyces sp. strain no. A-6288. 883 43

Guinea pig intestinal phospholipase B is a calcium-independent phospholipase hydrolyzing sequentially the acyl ester bonds at sn-2 and sn-1 positions of glycerophospholipids, promoting the formation of sn-glycero-3-phosphocholine from phosphatidylcholine. This 140-kDa glycoprotein from the brush border membrane of differentiated enterocytes contributes to lipid digestion as an ectoenzyme. The cDNA coding for guinea pig phospholipase B was revealed to be the homologue of AdRab-B, an mRNA appearing in rabbit upon intestine development. The sequence predicts a polypeptide of 1463 amino acids displaying four homologous repeats, two of them containing the lipase consensus sequence GXSXG. A 5-kilobase transcript was particularly abundant in mature ileal and jejunal enterocytes but was also detected in epididymis, where phospholipase B displayed a higher molecular mass (170 kDa versus 140 kDa in intestine), with no obvious evidence for enzyme activity. Trypsin treatment of phospholipase B immunoprecipitated from epididymal membranes reduced its size to 140 kDa, coinciding with the appearance of a significant phospholipase A2 activity. The same results were obtained in COS cells transfected with phospholipase B cDNA. Since sn-glycero-3-phosphocholine present at high concentrations in seminal plasma mainly stems from epididymis, this suggests a possible role of phospholipase B in male reproduction. This novel localization also unravels a mechanism of phospholipase B activation by limited proteolysis involving either trypsin in the intestinal lumen or a trypsin-like endopeptidase in the male reproductive tract.
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PMID:Ectopic epididymal expression of guinea pig intestinal phospholipase B. Possible role in sperm maturation and activation by limited proteolytic digestion. 959 72

An enzyme with lipase and esterase activity was purified from bovine pancreas. Furthermore, a non-radioactive lipase assay was developed which is 100 times more sensitive than the conventional methods and allowed the characterization of the lipase activity of the enzyme. The lipase activity increased 42 times in the presence of 10 mM sodium taurocholate, which for the first time provides direct evidence that a bile salt-activated lipase (bp-BAL) was isolated from bovine pancreas. This conclusion is further supported by the fact that the N-terminal amino acid sequence of this lipase/esterase is 88% homologous to human milk BAL and human pancreatic BAL. Staining with various lectins showed that bp-BAL is a glycoprotein which contains fucose residues. Previously from bovine pancreas a lysophospholipase has been purified and a gene was cloned and sequenced encoding an enzyme with cholesterol esterase/lysophospholipase activity. Comparison of the N-terminal amino acid sequence of bp-BAL with the deduced amino acid sequence of the latter revealed that they are identical. Furthermore, the molecular weight of the purified bp-BAL of 63,000, as estimated by SDS-PAGE, is very similar to that of the purified lysophospholipase (65,000) and to the theoretical molecular weight of 65,147 of the cholesterol esterase/lysophospholipase. These data suggest that these three enzymes are one and the same.
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PMID:Purification and characterization of bovine pancreatic bile salt-activated lipase. 1022 May 79

Infection caused by the fungus Cryptococcus neoformans is potentially fatal. A highly active extracellular phospholipase, demonstrating phospholipase B (PLB), lysophospholipase (LPL) and lysophospholipase/transacylase (LPTA) activities, was purified to homogeneity from C. neoformans using (NH(4))(2)SO(4) fractionation, and hydrophobic-interaction, anion-exchange and gel-filtration chromatography. All three enzyme activities co-purified as a single protein with an apparent molecular mass of 70-90 kDa by SDS/PAGE and 160-180 kDa by gel filtration. The ratio of the three activities remained constant after each purification step. The amino acid composition, as well as the sequences of the N-terminus and of five internal peptide fragments were novel. The protein was an acidic glycoprotein containing N-linked carbohydrate moieties, with pI values of 5.5 and 3.5. The apparent V(max) values for PLB and LPL activities were 12.3 and 870 micromol/min per mg of protein respectively; the corresponding K(m) values were approx. 185.3 and 92.2 microM. The enzyme was active only at acidic pH (pH optimum of 4.0 for PLB and 4.0-5.0 for LPL and LPTA). Enzyme activity did not require added cations, but was inhibited by Fe(3+). LPL and LPTA activities were decreased by 0.1% (v/v) Triton X-100 to 50% of the control value. Palmitoylcarnitine (0.5 mM) inhibited PLB (97% inhibition) and LPL and LPTA activities (35% inhibition) competitively. All phospholipids except phosphatidic acid were degraded by PLB, but dipalmitoyl phosphatidylcholine and dioleoyl phosphatidylcholine were the preferred substrates. This is the first complete description of the purification and properties of a phospholipase, which may be involved in virulence, from a pathogenic fungus.
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PMID:Purification and characterization of secretory phospholipase B, lysophospholipase and lysophospholipase/transacylase from a virulent strain of the pathogenic fungus Cryptococcus neoformans. 1074 72

The pathogenic fungus Cryptococcus neoformans produces an extracellular PLB1 (phospholipase B1), shown previously to be a virulence factor. A novel phospholipase (LPL1) with only LPL (lysophospholipase) and LPTA (transacylase) activities has now been characterized in C. gattii, and found to be a 66-kDa glycoprotein (by SDS/PAGE), with a native molecular mass of 670 kDa. The pI was 6.3, and it was active at high temperatures (to 70 degrees C), as well as at both acidic and neutral pH values. It was stimulated by calcium and palmitoyl carnitine at pH 7.0, but not at pH 5.0, and palmitoyl lysophosphatidylcholine was the preferred substrate. Sequencing indicated that LPL1 is a novel cryptococcal lysophospholipase, and not the gene product of CnLYSO1 or PLB1. A protein with only LPL and LPTA activities was subsequently isolated from two strains of C. neoformans var. grubii. A PLB1 enzyme was isolated from both C. gattii and a highly virulent strain of C. neoformans var. grubii (H99). In both cases, all three enzyme activities (PLB, LPL and LPTA) were present in one 95-120 kDa glycoprotein (by SDS/PAGE) with pI 3.9-4.3. Characterization of PLB1 from C. gattii showed that it differed from that of C. neoformans in its larger native mass (275 kDa), high PLB activity relative to LPL and LPTA, and preference for saturated lipid substrates. Differences in the properties between the secreted phospholipases of the two cryptococcal species could contribute to phenotypic differences that determine their respective environmental niches and different clinical manifestations.
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PMID:Cryptococcal phospholipases: a novel lysophospholipase discovered in the pathogenic fungus Cryptococcus gattii. 1532 Aug 65


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