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)

Phospholipase B (lysolecithin acyl-hydrolase, EC 3.1.1.5) from the mycelia of Penicillium notatum (Institute for Fermentation, Osaka, Japan; No.4640) was adsorbed from a crude solution to palmitoyl cellulose. Adsorption was efficient at pH 4 at low ionic strength (10 mM buffer), and at pH 4-9 at higher ionic strength (1-2M NaCl in 10 mM buffer). The adsorbed enzyme was eluted from the cellulose with a suitable detergent, such as Adekatol SO-120, Triton X-100, or deoxycholate. As an application of this procedure, the enzyme was purified from an extract of the mycelia by column chromatography on a palmitoylated textile (palmitoylated gauze) with an overall recovery of 59% and a 38-fold increase in specific activity. By subsequent column chromatographies on Amberlite XAD-2, Sephadex G-100 and G-150, and DEAE-Sephadex A-50, the enzyme was purified about 4,000-fold to a nearly homogeneous state from a mycelium extract with an overall recovery of 37%.
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PMID:Purification of phospholipase B from Penicillium notatum by hydrophobic chromatography on palmitoyl cellulose. 737 59

A phospholipase A2 bound tightly to the particulate fractions of rat ascites hepatoma cells was purified approximately 13,000-fold with a reasonably high yield (34%) by extraction with sodium cholate, ammonium sulfate fractionation, solubilization with sodium dodecyl sulfate, column chromatographies on Sephadex G-150 in the presence of sodium dodecyl sulfate, and on DEAE-cellulose and CM-cellulose in the presence of Triton X-100. The enzyme has a unique substrate specificity; namely, it preferentially hydrolyzes phosphatidylethanolamine and, to a lesser degree, phosphatidylglycerol. However, it does not attack phosphatidylcholine, phosphatidic acid or cardiolipin in the present experimental conditions. The final preparation shows both phospholipase A2 and lysophospholipase L2 activities, but neither lysophospholipase L1 nor lipase activity. The purified enzyme has a rather broad pH optimum ranging from 7 to 9, requires Ca2+, and is resistant to heat-treatment at 95 degree C for 5 min.
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PMID:Purification and properties of a membrane-bound phospholipase A2 from rat ascites hepatoma 108A cells. 739 Sep 73

Recent experiments in several laboratories have provided evidence that phosphatidic acid functions in cell signaling. However, the mechanisms that regulate cellular phosphatidic acid levels remain obscure. Here we describe a soluble phospholipase A1 from bovine testis that preferentially hydrolyzes phosphatidic acid when assayed in Triton X-100 micelles. Moreover, the enzyme hydrolyzes phosphatidic acid molecular species containing two unsaturated fatty acids in preference to those containing a combination of saturated and unsaturated fatty acyl groups. Under certain conditions, the enzyme also displays lysophospholipase activity toward lysophosphatidic acid. The phospholipase A1 is not likely to be a lysosomal enzyme because its optimum pH is 7.5-8.5. Furthermore, it is probably not a general lipid metabolic enzyme because high levels of activity are found in mature testis and brain but no measurable activity is seen in liver, spleen, or heart. The fact that the activity of the phospholipase A1 in mature bovine testis is > 10-fold higher than that in newborn calf testis raises the possibility that the enzyme may play a regulatory role in spermatogenesis or sperm function.
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PMID:Identification of a phosphatidic acid-preferring phospholipase A1 from bovine brain and testis. 793 8

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

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

The phospholipid deacylating enzyme was solubilized from the particulate (membrane) fraction of Mycobacterium lepraemurium with Triton X-100 and sodium cholate, and purified 1100-fold to homogeneous state by 5 steps of column chromatography: DE-52, PL-Sepharose (phosphatidylserine-attached sepharose), Mono P, heparin-Agarose and Mono Q column chromatography. The purified enzyme was composed of single polypeptide chain and molecular mass of 37 kDa was estimated for the protein by SDS-PAGE. The isoelectric point was determined about pH 4.6 and the protein was highly resistant to various kinds of proteolytic enzymes. The purified enzyme hydrolyzed both diacyl and monoacyl phospholipids showing that this enzyme was classified to phospholipase B (phospholipase A1/lysophospholipase). This phospholipase B had acidic pH optima and hydrolyzed both neutral phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE) and acidic phospholipids such as phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylglycerol (PG). Various fatty acids such as 12:0, 14:0, 16:0, 18:0 and 18:1 at sn-1 position, and 18:1, 18:2, 18:3 and 16:0 at sn-2 position were liberated from PC, suggesting no strict specificity toward the fatty acyl groups of phospholipids. From the comparison of degradation patterns of phosphatidylcholine with sn-1-[1-14C]- and sn-2-[1-14C]fatty acids, this enzyme was suggested to hydrolyze sn-1 position of phospholipid first and then sn-2 position, as the phospholipase B of M. phlei. This enzyme also attacked 1-acyl- and 2-acyl-lyso-PC at about same rates. The Km values for 1-acyl-2-oleoyl-PC and 2-oleoyl-lyso-PC were estimated 1.6 and 0.75 mM, respectively.
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PMID:Purification and properties of a membrane-bound phospholipase B from Mycobacterium lepraemurium. 881 50

A cDNA encoding a rat intestinal Ca(2+)-independent phospholipase B/lipase (PLB/LIP) was cloned from an ileac mucosa cDNA library using a probe amplified by polymerase chain reaction based on the purified enzyme's sequence. PLB/LIP consists of an NH2-terminal signal peptide, four tandem repeats of about 350 amino acids each, and a hydrophobic domain near the COOH terminus. The enzyme purified previously was found to be derived from the second repeat part. To examine the function of each domain, the full-length PLB/LIP, individual repeats, and a protein lacking the COOH-terminal hydrophobic stretch were expressed in COS-7 cells. The results showed that the second repeat, but not the other repeats, had all the activities (phospholipase A2, lysophospholipase, and lipase) found in the purified natural and expressed full-length enzymes, suggesting repeat 2 is a catalytic domain. The full-length enzyme was mainly present in membrane fractions and efficiently solubilized by treatment with 1% Triton X-100, but not with phosphatidylinositol-specific phospholipase C. Deletion of the COOH-terminal hydrophobic stretch caused the secretion of > 90% of synthesized PLB/LIP into culture media. These results suggest the hydrophobic domain is not replaced by a glycosylphosphatidylinositol anchor but serves as a membrane anchor directly. A message of the full-length PLB/LIP was abundantly expressed in the ileum and also, in a smaller, but significant amount, in the esophagus and testis. Immunohistochemistry showed that PLB/LIP is localized in brush border membranes of the absorptive cells, Paneth cells, and acrosomes of spermatid, suggesting its roles related and unrelated to intestinal digestion.
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PMID:Identification of functional domains of rat intestinal phospholipase B/lipase. Its cDNA cloning, expression, and tissue distribution. 944 65

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

We identified a unique phospholipase A (PLA) with relatively low heparin affinity, which was distinguishable from the heparin-binding secretory PLA2s, in rat, mouse, and bovine brains and testes. The partially purified enzyme was Ca2+-independent at neutral pH but Ca2+-dependent at alkaline pH. It predominantly hydrolyzed phosphatidic acid (PA) in the presence of Triton X-100 and phosphatidylethanolamine (PE) in its absence. When rat brain-derived endogenous phospholipids were used as a substrate, the enzyme released saturated fatty acids in marked preference to unsaturated ones. Consistent with this observation, the enzyme hydrolyzed sn-1 ester bonds in the substrates about 2,000 times more efficiently than sn-2 ones, thereby acting like PLA1. The enzyme also exhibited weak but significant sn-1 lysophospholipase activity. On the basis of its limited tissue distribution, substrate head group specificity and immunochemical properties, this enzyme appears to be identical to the recently cloned PA-preferring PLA1.
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PMID:Characterization of heparin low-affinity phospholipase A1 present in brain and testicular tissue. 1034 99

A recent study demonstrated that phospholipase B (PLB), lysophospholipase (LPL) and lysophopholipase transacylase (LPTA) are secreted by Cryptococcus neoformans var. neoformans and showed that the amount of enzyme production correlated with virulence in mice. The present study characterised the extracellular enzyme activities further by radiometric assays and 31P nuclear magnetic resonance spectroscopy (NMR). All three enzymes were most active between 25 and 40 degrees C. Bovine lung surfactant and its major lipid components, disaturated phosphatidylcholine and phosphatidylglycerol, were the optimal substrates for PLB. Lysophosphatidylcholine was the favoured substrate for LPL and LPTA. PLB and LPL/LPTA were differentially affected by Triton X-100, and palmitoyl carnitine was a potent inhibitor of the three phospholipases. LPL and PLB activities were inhibited by dithiothreitol; N-ethylmaleimide inhibited LPL and LPTA activities. None of the enzymes was inhibited by N-bromosuccinimide or p-bromophenacyl bromide. Cellular disruption experiments indicated that >85% of the phospholipase activities were cell-associated, with LPL and LPTA being more easily released than PLB. At pH 5.5 and 7.0, the heat-inactivated secreted enzyme preparations decreased the viability of human neutrophils. This effect was attenuated by active supernates. The relative activities of the PLB, LPL and LPTA in the environment of neutrophils are likely to determine the fate of these cells in vivo. Both phospholipases and heat-stable substances secreted by C. neoformans at 37 degrees C could contribute to membrane degradation and virulence.
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PMID:Biochemical and functional characterisation of secreted phospholipase activities from Cryptococcus neoformans in their naturally occurring state. 1045 Sep 96


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