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:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previous studies have demonstrated that [3H]arachidonic acid is released from prelabeled human neutrophil phospholipids when the cells are stimulated by calcium ionophore A23187 or by opsonized zymosan. Neither lysophospholipid generated by phospholipase A2 activity, diacylglycerol nor monoacylglycerol produced via phospholipase C/diacylglycerol lipase action have been identified following neutrophil challenge. The inability to detect any intermediates during the release of arachidonate is due to either rapid reacylation of lysophospholipid or conversion of diacylglycerol (monoacylglycerol) to cellular acylglycerols. The addition of exogenous [14C]fatty acid at the time of challenge was employed to determine the involvement of either phospholipase A2 or phospholipase C activities. Neutrophil stimulation with calcium ionophore A23187 resulted in an incorporation of exogenous [14C]arachidonate into phosphatidylinositol and phosphatidylcholine, those phospholipids which specifically release arachidonate. When the saturated fatty acid, [14C]stearate, replaced [14C]arachidonate, very little [14C]fatty acid was incorporated into any of the phospholipid species. Lipid phosphorus measurements revealed no significant mass change in any phospholipid class following ionophore challenge. Production of [14C]phosphatidic acid was not detected, as would be expected if diacylglycerol kinase and de novo phospholipid metabolism were significantly involved.
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PMID:Mechanism of arachidonic acid release in human polymorphonuclear leukocytes. 640 27

The stability of and production of extracellular virulence factors by mucoid (M7) and nonmucoid (wild-type) strains of Pseudomonas aeruginosa were studied in batch culture and in chemostats. Chemostat cultures were nutrient limited by iron, carbon, nitrogen, phosphorus, magnesium, and sulfur at various growth rates. Both M7 and wild-type strains were relatively stable in simple salts media. The wild type gave rise to one variant and M7, to several. M7 was most stable under iron limitation. Chemostat production of extracellular polysaccharide, protease, elastase, lipase, and phospholipase C all varied in a complex manner with growth conditions.
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PMID:Influence of nutrient limitation of growth on stability and production of virulence factors of mucoid and nonmucoid strains of Pseudomonas aeruginosa. 641 14

When mouse pancreatic "minilobules" prelabeled with either [14C]arachidonic acid (AA), [14C]stearic acid (SA), or [3H]glycerol were stimulated with the secretogogue, caerulein, there was a 60-70% loss in radioactivity in phosphatidylinositol (PI) at 30 min. This loss was accompanied by the formation of [14C] phosphatidic acid (PA), [14C]diacylglycerol (DG), [14C] triacylglycerol (TG), and free [14C]AA, [14C]SA, and [3H]glycerol. The loss in radioactive PI was the same as the loss in chemically measured PI-phosphorus. Thirty to fifty per cent of the caerulein-induced loss of prelabeled PI could be accounted for as free [14C]AA, [14C]SA, or [3H]glycerol. Increased incorporation of fatty acid or glycerol residues into DG, PA, and TG accounted for the balance of the loss in PI. The specific DG-lipase inhibitor, RHC 80267, markedly inhibited the caerulein-stimulated release of [14C]AA, [14C]SA, and [3H]glycerol and roughly doubled the caerulein-induced increment in [14C]AA-, [14C]SA-, or [3H]glycerol-labeled DG, showing that the source of the caerulein-induced increment in fatty acids and glycerol was DG. When the PI was prelabeled with either [32P] orthophosphate, [3H]myoinositol, or [3H]glycerol, only 1% or less of the radioactivity in PI was in lysophosphatidylinositol (LPI), and there was no increase in radioactivity in LPI on stimulation with caerulein. These observations, taken together, argue strongly for a phospholipase C-catalyzed breakdown of PI followed by DG-lipase and argue against any significant involvement of phospholipase A2 in PI degradation in mouse pancreas. The formation of substantial amounts of free [14C]AA on stimulation supports the view that, among other things, the phosphoinositide effect in the exocrine pancreas serves to generate arachidonate (and its metabolites). The release of appreciable amounts of free fatty acids and glycerol shows that a significant portion of the DG formed as a result of caerulein-stimulated PI breakdown is not conserved in the phosphoinositide cycle.
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PMID:Secretogogue-stimulated phosphatidylinositol breakdown in the exocrine pancreas liberates arachidonic acid, stearic acid, and glycerol by sequential actions of phospholipase C and diglyceride lipase. 643 97

Calcium binding to leaky erythrocyte plasma membranes was measured by three different procedures: Millipore filtration, equilibrium dialysis, and partition centrifugation. The curve derived from the binding equation, which best fit the means of the raw data, was used to estimate the association constants and capacities of the binding sites. A computer program (Gaushaus) which uses a nonlinear, least-squares regression protocol was also used to confirm these estimates. On the basis of these analyses we propose the presence of three classes of calcium-binding sites with the following apparent association constants and capacities: site 1, Ka = 3 X 10(4) M-1 and n = 30 nmol/mg protein; site 2, Ka = 3 X 10(3) M-1 and n = 200 nmol/mg protein; site 3, Ka = approximately 10(2) M-1 and n = approximately 200 nmol/mg protein. Calcium binding to erythrocyte membranes sealed in a high-salt solution showed the presence of site 3, but not site 2. The influence of phospholipids on the binding of calcium was evaluated by pretreating ghosts with phospholipase C (Clostridium welchii, EC 3.1.4.3). Treatment with this enzyme removed 80% of the total membrane phosphorus, predominantly from sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine. By the method of partition centrifugation two classes of binding sites were identified by computer analysis. Their association constants and capacities are, respectively, 1.1 X 10(5) M-1 and 20 nmol/mg protein for site 1 and 4.4 X 10(3) M-1 and 200 nmol/mg protein for site 2. We speculate that calcium-binding site 1 is composed of acidic phospholipids, calcium-binding site 2 is composed of spectrin and actin, and calcium-binding site 3 is composed of sialic acid.
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PMID:The determination of calcium-binding sites of human erythrocyte membranes. 646 80

Triton-insoluble cytoskeletons prepared from thrombin-activated platelets were found to potentiate the activation of prothrombin (prothrombinase activity). Cytoskeletons prepared from red cells or lymphoblasts contained no prothrombinase activity. The platelet prothrombinase activity was dependent on cytoskeletal-associated Factor Va, and exogenously added Factor Xa and prothrombin. Cytoskeletons contained 38% of the total platelet prothrombinase activity. Both platelets and cytoskeletons displayed half-maximal activities at similar prothrombin concentrations. The role of lipids in the cytoskeletal prothrombinase activity was investigated. Cytoskeletons were found to contain 3.8% of the total platelet phospholipids, consisting of the following lipids expressed as percentage of total present in platelets: 6.0% sphingomyelin, 3.8% phosphatidylcholine, 2.9% phosphatidyl-ethanolamine, 4.4% phosphatidylinositol, and 2.2% phosphatidylserine. The cytoskeletal prothrombinase activity and the lipid phosphorus content of cytoskeletons decreased after treatment of cytoskeletons with various doses of phospholipase C. Incubation of cytoskeletons with the highest concentrations tested (10 micrograms/ml) resulted in a 72% loss of phosphatidylserine and 84% loss of cytoskeletal prothrombinase activity. Cytoskeletal prothrombinase activity destroyed by phospholipase C treatment could be restored to control levels by treatment of hydrolyzed cytoskeletons with total cytoskeletal lipid or mixtures of phosphatidylserine/phosphatidylcholine (25:75% by weight). These results suggest that the cytoskeletal prothrombinase complex in addition to containing Factor Va, as has been previously shown (15), contains a lipid cofactor activity consisting in part of phosphatidylserine.
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PMID:The platelet cytoskeleton contains elements of the prothrombinase complex. 653 31

The thiophospholipid 1,2-dipalmitoyl-sn-glycero-3-thiophosphocholine (DPPsC) was shown to be a mixture of two diastereomers by 31P nuclear magnetic resonance. The isomer that resonates at the lower field in CDCl3 (56.12 ppm) was designated as isomer A and the other (resonates at 56.07 ppm) as isomer B. Phospholipase A2 from four different sources (bee venom, Naja naja venom, Crotalus adamanteus venom, and porcine pancreas) was shown to hydrolyze the isomer B of DPPsC specifically, whereas phospholipase C from two different sources (Bacillus cereus and Clostridium perfringens) hydrolyzes isomer A specifically. So that the two diastereomers could be separated, DPPsC(A + B) was first digested with phospholipase A2 to give 1-palmitoyl-sn-glycero-3-thiophosphocholine (MPPsC) (which is designated as isomer B of MPPsC) and the unreacted DPPsC(A). Reacylation of MPPsC(B) gave pure DPPsC(B). The properties of DPPsC(A) and DPPsC(B) were investigated by 31P, 13C, 1H, and 14N nuclear magnetic resonance (NMR). 1H and 13C NMR showed that both isomers in methanol solution have conformational properties similar to those of the natural phospholipid, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. On the other hand, the two isomers A and B showed small but significant differences in the chemical shifts of the carbon in the chiral carbon center and the phosphorus in the chiral phosphorus center.
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PMID:Phospholipids chiral at phosphorus. Preparation and spectral properties of chiral thiophospholipids. 668 28

Measurements of phosphatidylinositol (PI) and phosphatidic acid (PA) by phosphorus assays and by radioactivity ([14C]arachidonate) indicate that thrombin induces the degradation of a given fraction of the total PI to PA. The maximal conversion of PI to PA represents approximately one-third of the total PI which can be degraded by thrombin. This same amount of PI is converted to PA even in the presence of 1 mM quinacrine, which completely inhibits the release of arachidonic acid from phospholipids and which reduces by two-thirds the loss of labeled PI. In this case the fall in PI is equal to the amount of PA formed. If thrombin is added to platelets previously maximally stimulated by ionophore A23187, PA is produced from PI in amounts equal to those produced by thrombin in the absence of other stimuli. Furthermore, the resynthesis of PI from PA is also unaffected by quinacrine, and thus the entire thrombin-stimulated PI-cycle is maintained. The data thus indicate the existence of a quinacrine-insensitive phospholipase C which can initially convert a given amount of PI to PA and which is closely associated to the thrombin receptor. The further breakdown of PI and production of arachidonic acid might result from the action of quinacrine-sensitive activities (i.e. phospholipase A2). The simplest scheme is one in which thrombin specifically produces an active fraction of PA which in some way results in the subsequent production of arachidonic acid from various phospholipids (including PI), perhaps by activation of quinacrine-sensitive phospholipase A2.
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PMID:The initial action of thrombin on platelets. Conversion of phosphatidylinositol to phosphatidic acid preceding the production of arachidonic acid. 678 76

In stimulated platelets phosphatidylinositol is degraded by a phosphatidylinositol-specific phospholipase C to 1,2-diacylglycerol which is then phosphorylated to phosphatidic acid. Thrombin stimulation of horse and human platelets prelabeled with [32P]orthophosphate induces the formation of [32P]lysophosphatidylinositol, suggesting that phosphatidylinositol is also degraded by a phospholipase of A type activity. Stimulation of platelets prelabeled with 32P or with 32P plus [3H]inositol produces a lysophosphatidylinositol which has a 32P-specific activity and a 3H/32P ratio which has a 32P-specific activity and a 3H/32P ratio identical with those of phosphatidylinositol. These results suggest that the lysophosphatidylinositol derives from phosphatidylinositol. Thrombin stimulation of platelets double label with 32P and [3H]arachidonate induces loss of [3H]arachidonate from phosphatidylinositol and formation of [32P]lysophosphatidylinositol, suggesting the involvement of a phospholipase A2 activity. Ionophore A23187 also induces the formation of lysophosphatidylinositol in horse and human platelets. With either stimulus, [32P]lysophosphatidylinositol appears within seconds after stimulation and parallels the loss of [3H]arachidonic acid from phosphatidylinositol. The lysophosphatidylinositol produced by thrombin or by ionophore A23187 represents 40% of the degraded phosphatidylinositol as assessed by lipid phosphorus. Quinacrine, which inhibits the liberation of arachidonic acid from phospholipids, also blocks the formation of lysophosphatidylinositol. The results presented here indicate that phosphatidylinositol is degraded by both phospholipases, C and A2, in stimulated platelets.
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PMID:Formation of lysophosphatidylinositol in platelets stimulated with thrombin or ionophore A23187. 680 48

A fibrogenic factor which stimulates collagen production without cell proliferation of rat skin fibroblast cultures was isolated from CCl4-damaged rat liver. (1) The factor was isolated from saline extracts of CCl4-induced fibrotic rat liver and fractionated by Sephadex G-50S gel filtration and DEAE-cellulose chromatography. The original extract produced a 6-fold increase in collagen synthesis and the active factor eluted from gel filtration columns in a region corresponding to 5000 daltons. (2) The active factor was destroyed by heat (57 degrees C, 30 min), phospholipase C digestion, but was insensitive to proteolytic enzymes or phospholipase A. Chemical analysis of the partially purified factor revealed relatively high quantities of phosphorus (3%) and low quantities of protein (13.3%), neutral sugar (1.9%) and uronic acid (4.9%). The possibility of this component being a complex phospholipid containing polypeptide is suggested. (3) Fibrogenic properties of the isolated factor was enhanced by apparent oxidation in air, to a more active, yet insoluble complex. Attempts to solubilize the oxidized product completely destroyed its biological activity.
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PMID:Isolation and characterization of a fibrogenic factor from CCl(4)-damaged rat liver. 711 58

A simple method is described for the determination of serum glycerophosphatides, with the exception of lyso-phosphatide. Two determinations are necessary. The usual "triglyceride" determination is performed on native serum, and a further determination is performed after incubation of the serum with phospholipase C. Glycerophosphatides are hydrolysed to diglycerides and phosphorylcholine by phospholipase C. The resulting diglycerides are determined in addition to the mono-, di-, triglycerides and free glycerol of the native serum. The difference between the two glyceride determinations corresponds to the concentration of glycerophosphatides. Involvement of glycerophosphatides in blood coagulation and their increased levels in certain liver illnesses are two reasons for the importance of the present method. Compared with the usual method of measuring phospholipid phosphorus, the present method involves considerably less work, and, as a fully enzymatic method, it has a higher specificity.
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PMID:[Enzymatic determination of serum glycerophosphatides, using phospholipase C (author's transl)]. 720 62


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