Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.1.5 (neuropathy target esterase)
 1,070 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phospholipases, a group of enzymes that catalyze the hydrolysis of membrane phospholipids, are classified according to the bond cleaved in a phospholipid into PLA1 (EC 3.1.1.3), PLA2 (EC 3.1.1.4), PLB (EC 3.1.1.5), PLC (EC 3.1.4.3), and PLD (EC 3.1.4.4). This paper reviews source and structure of PLA2 and the involvement of PLA2 and PLC in several biological phenomena, such as, signal transduction, photoreception, biosynthesis of lung surfactant, sperm motility, and fertilization. New assays for PLA2 activity and concentration in biological fluids are discussed. Phospholipases are involved in many inflammatory reactions by making arachidonate available for eicosanoid biosynthesis. The determination of PLA2 activity and mass concentration in plasma is useful in the diagnosis and prognosis of pancreatitis and of septic shock. Naturally occurring phospholipase inhibitors, such as lipocortins act as second messengers in the anti-inflammatory response to steroids. Lipocortins may be valuable therapeutic agents, because they are more specific in their anti-inflammatory action than glucocorticoids; therefore, they are less likely to produce harmful side effects.
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PMID:Phospholipases in biology and medicine. 225 31

Human cytosolic phospholipase A2 (cPLA2) is an 85-kDa protein which displays a preference for arachidonoyl phospholipids as substrates. This substrate preference and the assay characteristics of the enzyme are quite different from those of the smaller, more well-studied extracellular PLA2s. We now report the development of a nonradioactive, spectrophotometric, microtiterplate assay for human cPLA2 using a novel synthetic thio-phospholipid analog as a substrate. This substrate is a phosphatidylcholine derivative with an arachidonoylthioester in the sn-2 position and an alkyl-ether in the sn-1 position. The use of an sn-1 alkyl-ether in the substrate ensures that the assay will only measure PLA2 activity and will not be complicated by the metabolism of the lysophospholipid product by the enzyme's lysophospholipase activity. cPLA2 is assayed at pH 7.4 and 37 degrees C with a mixed micellar substrate consisting of 2 mM thio-phospholipid and 4 mM Triton X-100 in 30% glycerol. Under these conditions, the assay is fairly linear for over 1 h.
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PMID:1-Hexadecyl-2-arachidonoylthio-2-deoxy-sn-glycero-3-phosphorylcholine as a substrate for the microtiterplate assay of human cytosolic phospholipase A2. 820 36

The purpose of this study was to investigate in rats the effects of three anthracyclines, pirarubicin, doxorubicin and epirubicin on gastric prostaglandin E2 (PGE2) metabolism and phospholipase A2 (PLA2, EC 3.1.1.4) activity. The level of the membrane precursor, arachidonic acid, and the stability of the membrane were investigated by analysis of the composition of fatty acids. Enzymatic activities involved in the turnover of membrane phospholipids such as lysophospholipase (LPase, EC 3.1.1.5) and acyl-CoA lysophosphatidylcholine: acyltransferase (ACLAT, EC 2.3.1.23), and in the detoxification of lipid hydroperoxides, selenium-dependent glutathione-peroxidase (GSH-PX, EC 1.11.1.9) were measured after injection of the drugs for 4 consecutive days. Pirarubicin does not give rise to any changes in these activities but doxorubicin and epirubicin decreased PGE2 production and the activities of PLA2, LPase and ACLAT. GSH-PX activity was not changed by any of the drugs. The decrease in PLA2 activity does not seem to be related to variations in membrane lipid composition because the total phospholipids content was unchanged. The P/S (polyunsaturated/saturated) ratio increased in the doxorubicin group and decreased in the epirubicin group, and the unsaturation index was moderately modified. Arachidonic acid was increased only in the doxorubicin group. In vitro, PLA2 activity was not inhibited by the three drugs in the micromolar range. A marked inhibition was observed at 2.5 mM for pirarubicin and at 1.0 mM for doxorubicin and epirubicin. The Lineweaver-Burk representation showed that these inhibitions were of an uncompetitive type. Pirarubicin may therefore be considered to be an anthracycline without marked side-effects on gastric mucosa. However, the in vitro inhibition of PLA2 activity by anthracyclines does not fully explain the in vitro decrease in PLA2 specific activity observed after doxorubicin and epirubicin treatment, and in this context membrane structure modifications unconnected with the lipid composition can not be excluded. In vivo these phenomena may affect PGE2 synthesis, whose level was lower in the doxorubicin and epirubicin groups than in control group.
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PMID:Effect of anthracyclines on phospholipase A2 activity and prostaglandin E2 production in rat gastric mucosa. 834 60

Human cytosolic phospholipase A2 (cPLA2) is an arachidonic acid specific enzyme which may play a role in arachidonic acid release, eicosanoid production, and signal transduction. The PLA2 activity of this enzyme is stimulated by microM levels of Ca2+. Using a pure recombinant enzyme, we have confirmed that cPLA2 is not absolutely dependent on Ca2+, since Sr2+, Ba2+ and Mn2+ also gave full enzyme activity. Heavy metals, in contrast, inhibited enzyme catalysis suggesting the involvement of an essential cysteine residue. In the absence of Ca2+, high salt concentrations overcame the requirement for divalent metals, indicating that Ca2+ is not required for PLA2 catalytic activity. cPLA2 also displays a lysophospholipase (lyso PLA) activity with lysophosphatidylcholine micelles as a substrate. Unlike the PLA2 activity, the lyso PLA activity toward these micelles is not stimulated by Ca2+. However, upon the addition of glycerol or Triton X-100 to the assay, Ca2+ activation is observed, indicating that substrate presentation can affect the apparent Ca2+ dependence. Glycerol was found to be a potent stimulator of lyso PLA activity and specific activities up to 50 mumol min-1 mg-1 were observed. In addition to the PLA2 and lyso PLA activities, we report that cPLA2 displays a relatively low, CoA-independent transacylase activity which produces phosphatidylcholine from lysophosphatidylcholine substrate. The observation of this novel transacylase activity is consistent with the formation of an acyl-enzyme intermediate.
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PMID:Metal ion and salt effects on the phospholipase A2, lysophospholipase, and transacylase activities of human cytosolic phospholipase A2. 848 88

A high activity lysophospholipase A (lysoPLA) was purified from the soluble fraction of bovine brain. The separation included sequential DEAE-Sephacel, phenyl-Sepharose FF, heparin-Sepharose CL-6B, and Q-Sepharose FF column chromatography. Mono Q, Sephacryl S300HR, and hydroxylapatite column chromatography in the presence of the detergent CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate) and glycerol further purified the activity to 17,000-fold. The enzyme was purified to homogeneity by polyacrylamide gel electrophoresis using nondenaturing conditions. The pure enzyme migrated as a single polypeptide of 95 kDa mass by SDS-polyacrylamide gel electrophoresis and deacylated arachidonoyl-lysophosphatidylcholine (ara-lysoPC) at rate of 70 micromol/(min mg). The enzyme showed selectivity for arachidonoyl-substituted lysoPC, since palmitoyl-lysoPC was deacylated at a much lower rate (7 micromol/(min mg)). LysoPLA activity was maximal at pH 7.4-8.0 and was increased 1.3-fold by MgCl2 (5 mM). By including MgCl2, however, the range of optimal activity was expanded to pH values up to 9.0. The 95-kDa protein also deacylated arachidonoyl groups from 1-O-hexadecyl-2-arachidonoyl-PC (PLA2 activity) at a rate of 15 micromol/(min mg). Moreover, the deacylation of arachidonoyl groups from diacylPC was greatly increased by including purified bovine brain PLA1 in the reaction mixture. Thus, the same 95-kDa polypeptide catalyzed both lysoPLA and PLA2 activities, but the rate of arachidonoyl group deacylation was increased by prior sn-1 deacylation. Finally, the 95-kDa polypeptide cross-reacted with antibodies raised against a human recombinant cPLA2, implying that the 95-kDa protein is structurally similar to cPLA2. Additionally, these data suggest that the combined actions of PLA1 and the 95-kDa protein generate significant amounts of free arachidonic acid in the brain.
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PMID:Purification of a lysophospholipase from bovine brain that selectively deacylates arachidonoyl-substituted lysophosphatidylcholine. 866 71

The Group VI 80-kDa Ca2+-independent phospholipase A2 (iPLA2) has been purified from murine P388D1 macrophages and Chinese hamster ovary (CHO) cells. The amino acid sequence of the iPLA2 has been determined and shown to contain a lipase consensus sequence and eight ankyrin repeats, which makes it distinct from Group I-V PLA2s. This enzyme appears to play a key role in mediating basal phospholipid remodeling. We now report that the Group VI iPLA2 displays interfacial activation toward short chain phospholipids, 1-octanoyl-2-heptanoyl-sn-glycero-3-phosphocholine, 1,2-diheptanoyl-sn-glycero-3-phosphocholine, and 1,2-dihexanoyl-sn-glycero-3-phosphocholine micelles. ATP protects the iPLA2 from a loss in activity as a result of prolonged incubation during the assay. Hence higher enzyme activity is observed in the presence than in the absence of ATP. Similar protection was obtained with glycerol. In addition, the iPLA2 exhibits multiple activities which are strongly dependent on substrate presentation. The lysophospholipase activity of this enzyme was diminished by Triton X-100 and stimulated by glycerol. With the combination of 50 microM Triton X-100 and 50% glycerol, the enzyme's lysophospholipase activity achieved equivalent activity to its PLA2 activity. The iPLA2 displayed both lysophospholipid/transacylase and phospholipid/transacylase activity, supporting the conclusion that the mechanism of action of iPLA2 proceeds through an acyl-enzyme intermediate as proposed for the Group IV cPLA2.
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PMID:Interfacial activation, lysophospholipase and transacylase activity of group VI Ca2+-independent phospholipase A2. 963 Jul 2

Previous studies have suggested that activation of calcium-independent PLA2 (CaIPLA2) is an early event in cell death after hypoxic injury in proximal tubule cells. An approximately 28-kD CaIPLA2 with preferential activity toward plasmalogen phospholipids has been recently purified from rabbit kidney cortex (D. Portilla and G. Dai, J Biol Chem 271, 15,451-15,457, 1996). Their report describes the cloning of a full-length rat cDNA encoding CaIPLA2, using sequences derived from the purified rabbit kidney cortex enzyme. In addition, cDNA from rabbit kidney that encode the rabbit homologue of the enzyme and a closely related isoform were isolated. The rat cDNA is predicted to encode an approximately 24-kD protein, and each cDNA contains the sequence G-F-S-Q-G, which fits the active site consensus sequence G-X-S-X-G of carboxylesterases. Several lines of evidence (DNA sequence comparison, Southern blot analysis, and examination of the expressed sequence tag database) show that CaIPLA2 enzymes are encoded by a multigene family in rats, mice, rabbits, and humans. Northern analysis of various tissues from the rat indicated that the CaIPLA2 gene is ubiquitously expressed, with highest mRNA abundance observed in the kidney and small intestine. The rat CaIPLA2 cDNA, when expressed in a baculovirus expression system, and the purified rabbit kidney cortex protein exhibit both CaIPLA2 and lysophospholipase activities. The cloned CaIPLA2 cDNA are expected to aid in understanding the role of CaIPLA2 in cell death after hypoxic/ischemic cell injury.
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PMID:cDNA cloning and expression of a novel family of enzymes with calcium-independent phospholipase A2 and lysophospholipase activities. 964 27

The group VIA PLA2 is a member of the PLA2 superfamily. This enzyme, which is cytosolic and Ca2+-independent, has been designated iPLA2beta to distinguish it from another recently cloned Ca2+-independent PLA2. Features of iPLA2beta molecular structure offer some insight into possible cellular functions of the enzyme. At least two catalytically active iPLA2beta isoforms and additionalsplicing variants are derived from a single gene that consists of at least 17 exons located on human chromosome 22q13.1. Potential tumor suppressor genes also reside at or near this locus. Structural analyses reveal that iPLA2beta contains unique structural features that include a serine lipase consensus motif (GXSXG), a putative ATP-binding domain, an ankyrin-repeat domain, a caspase-3 cleavage motif DVTD138Y/N, a bipartite nuclear localization signal sequence, and a proline-rich region in the human long isoform. iPLA2beta is widely expressed among mammalian tissues, with highest expression in testis and brain. iPLA2beta prefers to hydrolyze fatty acid at the sn-2 fatty acid substituent but also exhibits phospholipase A1, lysophospholipase, PAF acetylhydrolase, and transacylase activities. iPLA2beta may participate in signaling, apoptosis, membrane phospholipid remodeling, membrane homeostasis, arachidonate release, and exocytotic membrane fusion. Structural features and the existence of multiple splicing variants of iPLA2beta suggest that iPLA2beta may be subject to complex regulatory mechanisms that differ among cell types. Further study of its regulation and interaction with other proteins may yield insight into how its structural features are related to its function.
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PMID:The molecular biology of the group VIA Ca2+-independent phospholipase A2. 1152 80

The phospholipase B family (PLB) are enzymes sharing phospholipase (PL), lysophospholipase (LPL) and lysophospholipase-transacylase (LPTA) activities. They have been shown to be important virulence factors in several human fungal pathogens including Candida albicans and Cryptococcus neoformans. Aspergillus fumigatus, a human opportunistic fungal pathogen leading to a high rate of mortality in immunosuppressed patients is known to possess an extracellular phospholipase B activity. In this paper, we report the molecular characterisation of three PLB genes from A. fumigatus (afplb) using degenerate primers in PCR amplification and data from the A. fumigatus genome project. They are expressed at 37 degrees C, and two of them (afplb1 and afplb3) are induced by lecithin. They encode proteins of 633, 588 and 630 amino acids, respectively, presenting together a T-Coffee score of 81. They also possess the amino acid triad responsible for enzymatic activity in the mammalian cytosolic PLA2 and other fungal PLBs. AfPLB1 and afPLB3 are secreted with a cleaved signal peptide. The complete cDNA sequences were obtained by RACE-PCR for the two secreted afPLBs and probably account for the extracellular phospholipase activity previously reported in the culture media of A. fumigatus.
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PMID:Characterisation and expression of phospholipases B from the opportunistic fungus Aspergillus fumigatus. 1545 Nov 5

The phospholipase A2s (PLA2s) are a diverse group of enzymes that hydrolyze the sn-2 fatty acid from phospholipids and play a role in a wide range of physiological functions. A 61-kDa calcium-independent PLA2, termed cPLA2gamma, was identified as an ortholog of cPLA2alpha with approximately 30% overall sequence identity. cPLA2gamma contains a potential prenylation motif at its C terminus, and is known to have PLA2 and lysophospholipase activities, but its physiological roles have not been clarified. In the present study, we expressed various forms of recombinant cPLA2gamma, including non-prenylated and non-cleaved forms, in order to investigate the effects of C-terminal processing. We examined the expression of the wild type and non-prenylated (SCLA) forms of cPLA2gamma, and found that the SCLA form was expressed normally and retained almost full activity. Expression of the prenylated and non-cleaved form of cPLA2gamma using yeast mutants lacking prenyl protein proteases AFC1 (a-factor-converting enzyme) and RCE1 (Ras-converting enzyme) revealed decreased expression in the mutant strain compared to that in the wild type yeast, suggesting that complete C-terminal processing is important for the functional expression of cPLA2gamma. In addition, cPLA2gamma was found to have coenzyme A (CoA)-independent transacylation and lysophospholipid (LPL) dismutase (LPLase/transacylase) activities, suggesting that it may be involved in fatty acid remodeling of phospholipids and the clearance of toxic lysophospholipids in cells.
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PMID:Roles of C-terminal processing, and involvement in transacylation reaction of human group IVC phospholipase A2 (cPLA2gamma). 1594 8


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