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)

Human preimplantation embryos and endometrium secrete platelet-activating factor (PAF). The mechanism of phosphatidylcholine (PC) degradation stimulated by PAF was investigated in endometrial explants prelabeled with [methyl-3H]choline or preincubated with [3H]butan-1-ol. Analysis of the water-soluble metabolites of PAF-induced PC hydrolysis in secretory endometrium demonstrated that the stimulated generation of [3H]choline ([3H]Cho) precedes that of [3H]choline phosphate ([3H]ChoP) and [3H]glycerophosphocholine ([3H]GPC). Within 30 sec there was a rapid rise in PAF-induced [3H]Cho generation and by 2 min this had increased to 59.9% +/- 10.6% (p less than 0.02), with no effect upon [3H]ChoP and [3H]GPC during this period. Both [3H]GPC and [3H]ChoP, however, were increased at a later time point. The slower [3H]ChoP generation may suggest that PC-specific phospholipase C activation as well as delayed [3H]GPC rise may be due to PC-specific phospholipase A2 and lysophospholipase activation. Phospholipase D activity was confirmed by the incorporation of high-specific-activity [3H]butan-1-ol into [3H]phosphatidylbutanol ([3H]PBut). The rapid generation of [3H]PBut, which paralleled the rise in intracellular [3H]Cho, strongly suggests that PC breakdown is catalyzed by the phospholipase D pathway. It is proposed that PAF induces PC hydrolysis as a consequence of an early phospholipase D-catalyzed breakdown of PC in human secretory endometrium. This may be an alternative source for prostaglandin synthesis and an important pathway essential for long-term activation of local cellular events at the time of implantation.
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PMID:Platelet-activating factor mediates phosphatidylcholine hydrolysis by phospholipase D in human endometrium. 163 48

The first step in the production of eicosanoids and platelet-activating factor is the hydrolysis of arachidonic acid from membrane phospholipid by phospholipase A2. We previously purified from the macrophage cell line RAW 264.7 an intracellular phospholipase A2 that preferentially hydrolyzes sn-2-arachidonic acid. The enzyme exhibits a molecular mass of 100 kDa and an isoelectric point of 5.6. When assayed for other activities, the phospholipase A2 was found to exhibit lysophospholipase activity against palmitoyllysoglycerophosphocholine, and both activities copurified to a single band on silver-stained sodium dodecyl sulfate-polyacrylamide gels. An antibody against the macrophage enzyme was found to quantitatively immunoprecipitate both phospholipase A2 and lysophospholipase activities from a crude cytosolic fraction. When the immunoprecipitated material was analyzed on immunoblots, a single band at 100 kDa was evident, further suggesting that a single protein possessed both enzyme activities. When assayed as a function of palmitoyllysoglycerophosphocholine concentration and plotted as a double-reciprocal plot, two different slopes were apparent, corresponding to concentrations above and below the critical micellar concentration (7 microM) of the substrate. Above the critical micellar concentration, lysophospholipase exhibited an apparent Km of 25 microM and a Vmax of 1.5 mumol/min/mg. Calcium was not required for lysophospholipase activity, in contrast to phospholipase A2 activity. The enzyme, when assayed as either a phospholipase A2 or lysophospholipase, exhibited nonlinear kinetics beyond 1-2 min despite low substrate conversion. Readdition to more substrate after the activity plateaued did not result in further enzyme activity, ruling out substrate depletion. Readdition of enzyme, however, resulted in another burst of enzyme activity. The results are not consistent with product inhibition, but suggest that the enzyme may be subject to inactivation during catalysis.
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PMID:Kinetic properties of a high molecular mass arachidonoyl-hydrolyzing phospholipase A2 that exhibits lysophospholipase activity. 164 32

The intestinal brush-border membranes of rats and guinea pigs possess a high molecular weight, calcium-independent phospholipase B (phospholipase A2 - lysophospholipase activities) with the characteristics of a digestive ectoenzyme. A combination of subcellular fractionation, Triton X-114 phase partitioning, chromatofocusing, and preparative sodium dodecyl sulphate - polyacrylamide gel electrophoresis was used to purify a full-length, although denatured, form of this enzyme from the rat. Renaturation of the gel-purified fraction confirmed that both enzyme activities were associated with this protein. Gel slices containing the purified phospholipase B were used to generate a polyclonal antiserum in rabbits that could be used for immunoblotting. The relative mobility of the phospholipase B during electrophoresis in sodium dodecyl sulphate gels was dramatically affected by the percentage of acrylamide and the presence or absence of reducing agents in the gels. This was true for both the purified protein visualized by silver-staining and following electrophoresis of the total proteins of the membrane, with the phospholipase visualized by immunoblotting. Estimates for the molecular mass of the enzyme varied from 130 to 170 kDa in 7.5% gels and from 120 to 130 kDa in 5-10% gradient gels (with a best estimate of 120 kDa). Upon solubilization from the brush-border membrane by papain digestion, the major immunoreactive band migrated with an apparent mass of 80 kDa in both the 7.5% and 5-10% gradient gels. A major cross-reactive band was detected at 97 kDa following immunoblotting of the papain-solubilized proteins from guinea pig brush-border membranes, in agreement with the size of the purified fragment reported in the literature and at 140 kDa following immunoblotting of the intact proteins. Similar immunoblotting produced reaction with a 135-kDa protein from the rabbit brush-border membrane, as well as 95-kDa protein following papain solubilization. These results suggest that while there are species-specific apparent molecular weights, the intestinal brush-border membrane phospholipase B is conserved among species.
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PMID:Further characterization of a novel phospholipase B (phospholipase A2--lysophospholipase) from intestinal brush-border membranes. 171 22

Phospholipase B inhibitor was found in the autolyzate of yeast cells, Torulaspora delbrueckii. The inhibitor was purified to homogeneity by ethanol precipitation, gel filtration with Sephadex G-10, ion-exchange chromatography with DEAE-Sephacel, and gel filtration with Asahipak GS-320. On thin-layer chromatography the purified inhibitor was detected with the Hanes-Isherwood reagent, which is used to detect phosphorus. The activity of the inhibitor was not affected by heat treatment at 100 degrees C for 1 h. Heating at 100 degrees C for 1 h in 1 M HCl and 1 M NaOH lowered activity to 76 and 80% of the original values, respectively, but heating at 110 degrees C for 24 h in 6 M HCl completely abolished activity. The inhibitor was highly soluble in water, but practically insoluble in alcohol, acetone, ether, and chloroform. The degree of inhibition of enzyme activity was not proportional to the concentration of inhibitor. The inhibitor inhibited both membrane-bound and water-soluble phospholipase B activity from T. delbrueckii at the same level; however, the inhibitor did not inhibit the activity of phospholipase A2 from snake venom (Naja naja).
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PMID:Isolation and fundamental characteristics of a phospholipase B inhibitor from autolyzed Torulaspora delbrueckii. 176 Jan 34

Lecithin-dependent haemolysin (LDH) of Vibrio parahaemolyticus was purified from Escherichia coli C600 transformed with a plasmid (pHL591) ligated with a 1.5 kb DNA fragment of V. parahaemolyticus. The final preparation comprised two LDH proteins with different molecular masses which were immunologically cross-reactive and had the same enzymic activity. The LDH was a phospholipase hydrolysing both fatty acid esters of phospholipid, i.e. it hydrolysed phosphatidylcholine (PC) to lysophosphatidylcholine (LPC) and then LPC to glycerophosphorylcholine (GPC). From this point of view, LDH should be classified as a phospholipase B. Phospholipase B, however, does not usually show haemolytic activity, because the intermediate (LPC), which is the actual haemolytic agent, is immediately hydrolysed to the final product (GPC). On the other hand, LPC formed by LDH action was comparatively stable, because the rates of the two reactions catalysed by LDH, PC to LPC and LPC to GPC, are almost the same. This is the reason that LDH shows haemolytic activity. Therefore, LDH of V. parahaemolyticus is an atypical phospholipase to be designated as phospholipase A2/lysophospholipase.
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PMID:Purification and characterization of a lecithin-dependent haemolysin from Escherichia coli transformed by a Vibrio parahaemolyticus gene. 179 26

A membrane fraction containing H,K-ATPase (EC 3.6.1.36) was prepared from pig gastric mucosa and found to contain phospholipase A2 (EC 3.1.1.4) and lysophospholipase (EC 3.1.1.5) activities. Washing the membranes decreased their protein content by 25%. Recovery profiles of H,K-ATPase, phospholipase A2 and lysophospholipase were similar for membranes washed either with water or with 0.15 or 1.5 M KCl. Nearly identical distribution profiles were obtained for the three enzyme activities after centrifugation of washed vesicle membranes on a linear sucrose gradient. The phospholipase A2 activity was stimulated by calcium and increased further in the presence of calmodulin. The amount of cellular radioactively labelled lysophosphatidylcholine was doubled upon cholinergic stimulation of isolated parietal cells prelabelled with [3H]glycerol or 32Pi. The liberated lyso[32P]phosphatidylcholine had its acyl chain in the sn-1 position, which implies an activation of a phospholipase A2. These findings indicate that secretagogues which increase the cytosolic Ca2+ concentration, i.e. acetylcholine, histamine and gastrin, may activate a phospholipase A2 in the parietal cell.
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PMID:Occurrence of phospholipase A2 and lysophospholipase in a gastric H,K-ATPase-containing membrane fraction, and the formation of lysophosphatidylcholine in stimulated pig parietal cells. 196 31

1. The action of crude venoms of four aculeate species: Apis mellifera, Vespa crabro, Vespula germanica and Vespula vulgaris on human erythrocytes was investigated in order to determine the lytic and phospholipase activity of different aculeate venoms and their ability to induce red blood cell hemolysis. 2. Bee venom was the only extract to completely lyse red blood cells at the concentration of 2-3 micrograms/ml. 3. Phospholipase activity in all of the examined vespid venoms was similar and the highest value was recorded in V. germanica. 4. Vespid venoms exhibited phospholipase B activity, which is lacking in honeybee venom. 5. In all membrane phospholipids but lecithin, lysophospholipase activity of vespid venoms was 2-6 times lower than the relevant phospholipase activity. 6. The incubation of red blood cells with purified bee venom phospholipase A2 was not accompanied by lysis and, when supplemented with purified melittin, the increase of red blood cell lysis was approximately 30%.
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PMID:Hemolytic potency and phospholipase activity of some bee and wasp venoms. 198 42

We have recently described a lysophospholipase A2 activity in guinea-pig heart microsomes that hydrolyses 2-acyl-sn-glycero-3-phosphocholine (2-acyl-GPC). The presence of a similar activity that hydrolyses 2-acyl-sn-glycero-3-phosphoethanolamine (2-acyl-GPE) was not known. In this study, a lysophospholipase A2 activity in guinea-pig heart microsomes that hydrolyses 2-acyl-GPE has been characterized. The enzyme did not require Ca2+ for activity and exhibited a high specificity for 2-arachidonoyl-GPE and 2-linoleoyl-GPE over 2-oleoyl-GPE and 2-palmitoyl-GPE. The specificity for these unsaturated substrates was observed in the presence and absence of detergents. Selective hydrolysis of 2-arachidonoyl-GPE over 2-palmitoyl-GPE was observed when equimolar quantities of the two substrates were incubated with the enzyme. There was no preferential hydrolysis of either 2-linoleoyl- or 2-arachidonoyl-GPE when presented individually or as a mixture. Significant differences in the characteristics of 2-acyl-GPE-hydrolysing and 2-acyl-GPC-hydrolysing activities included differences in their optimum pH, the effect of Ca2+ and their acyl specificities. Taken together, these results suggest that the two activities are catalysed by different enzymes. 2-Acyl-GPE lysophospholipase activity with a preference for 2-arachidonoyl-GPE over 2-oleoyl-GPE was observed in guinea-pig brain, liver, kidney and lung microsomes. Lysophospholipase A1 activity that catalyses the hydrolysis of 1-acyl-GPE was also present in guinea-pig heart microsomes and had different characteristics from the 2-acyl-GPE-hydrolysing activity, including a preference for saturated over unsaturated substrates. The 2-acyl-GPE lysophospholipase A2 activity appeared to be distinct from Ca(2+)-independent phospholipase A2. The characteristics of the 2-acyl-GPE lysophospholipase A2 suggest it could play a role in the selective release of arachidonic and linoleic acids for further metabolism in cells.
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PMID:2-acyl-sn-glycero-3-phosphoethanolamine lysophospholipase A2 activity in guinea-pig heart microsomes. 202 24

Nitrogen dioxide (NO2), an environmental oxidant, is known to activate phospholipase A1 and modulate the plasma membrane structure of porcine pulmonary artery endothelial cells. We evaluated the effects of exposure to NO2, purified phospholipase B (which acts as phospholipase A1 and A2), or phospholipase A2 on 125I-angiotensin II (Ang II) receptor binding, internalization, or both in pulmonary endothelial cells. Exposure to 5 ppm NO2 for 48 hr at 37 degrees C or 0.075 U each of phospholipase B or A2 in phosphate-buffered saline (PBS) for 30 min at 24 degrees C resulted in an increase in total Ang II binding (i.e., cell surface bound and internalized) by 45% (p less than 0.05), 50% (p less than 0.05), and 85% (p less than 0.001), respectively, compared to controls. An Ang II receptor antagonist, [Sar1 Ile8] Ang II, competitively displaced Ang II binding to control, NO2-, phospholipase B-, and phospholipase A2-exposed cells. Dissociation of bound Ang II in the presence of PBS was less than 1% of total bound Ang II in control, NO2-, and phospholipase B-exposed cells and was 50% of total bound Ang II in phospholipase A2-exposed cells. In the presence of isotonic acetic acid/NaCl, in excess of 90% of cell surface-bound Ang II was dissociated from control, NO2-, and phospholipase B-exposed cells, and there was less than 2% of Ang II detectable when acid-treated cells were subjected to NaOH solubilization. In cells exposed to phospholipase A2, acetic acid treatment did not release cell-bound Ang II, and the remaining Ang II was recovered in the NaOH solubilized fraction.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Oxidant injury increases cell surface receptor binding of angiotensin II to pulmonary artery endothelial cells. 209 20

Human umbilical vein endothelial cells (HUVEC) in culture synthesize prostacyclin (PGI2) as the predominant metabolite of arachidonic acid which is derived from the deacylation of phospholipids. Under basal-unstimulated condition, PGI2 release from HUVEC is extremely low; however, when endothelial monolayers were preincubated with the natural vitamin E (R,R,R-alpha-tocopherol), we found a dose-dependent potentiation of basal PGI2 release. When HUVEC were stimulated with arachidonate or ionophore A23187, there was a dose-dependent increase of PGI2 release in response to tocopherol enrichment. When HUVEC were labelled with [Me-3H]choline followed by A23187 stimulation, a significantly higher lysophosphatidylcholine was found in the tocopherol-enriched cells, suggesting a change in enzymes involved in phosphatidylcholine metabolism. Analysis of these enzymes revealed that phospholipase A2 activity was enhanced by tocopherol enrichment, whereas lysophospholipase and acyl-CoA acyltransferase were unaffected. To determine the specificity of the tocopherol molecule, different analogues were tested for their PGI2 potentiating activity. Results showed that the free hydroxyl group on the chromanol ring as well as the phytyl side-chain are absolutely required to stimulate PGI2 release, whereas, different methyl locations and substituents on the chromanol ring had no effect. These studies demonstrated that tocopherol potentiates basal PGI2 release in HUVEC and in contrast to its reported inhibitory role in rat platelets, myocardium and neutrophils, tocopherol stimulates phospholipase activity in HUVEC.
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PMID:R,R,R-alpha-tocopherol potentiates prostacyclin release in human endothelial cells. Evidence for structural specificity of the tocopherol molecule. 210 79


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