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)

The effects of several kinds of carbohydrate oxidase, SH-inhibitors and some other chemical reagents on the activities of von Willebrand factor, factor VIII procoagulant and factor VIII-related antigen were studied. Factor VIII procoagulant and von Willebrand factor activities were both inhibited by galactose oxidase, p-hydroxymercuribenzoic acid, 2,4,6-trinitrobenzensulfonic acid and sodium periodate, alpha-Mannosidase, N-ethylmaleimide and phospholipase C inactivated factor VIII procoagulant but not von Willebrand factor activity. Dithiothreitol had little effect on factor VIII procoagulant activity but reduced significantly that of von Willebrand factor. It is suggested that galactose and the thiol and epsilon-aminogroup groups of lysine may play an important role in both factor VIII procoagulant and von Willebrand factor activity. Mannose may be responsible for the factor VIII procoagulant activity but not for the von Willebrand factor activity. The Laurell rocket heights of factor VIII-related antigen rose with increasing concentration of galactose oxidase, 2,4,6-trinitrobenzenesulfonic acid or sodium periodate. Gel filtration experiments showed that factor VIII-related antigen may be dissociated into subunits by galactose oxidase but not by 2,4,6-trinitrobenzenesulfonic acid or sodium periodate.
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PMID:Studies of von Willebrand factor: effects of different kinds of carbohydrate oxidases, SH-inhibitors and some other chemical reagents. 30 2

Biotin derivatives of methotrexate (biotin-SS-MTX) and folate (biotin-SS-folate), in which the functional components are joined by a dissociable disulfide-containing spacer, have been synthesized, purified by DEAE-Trisacryl chromatography, and characterized by HPLC, elemental analysis and mass spectrometry. These compounds provide a convenient means for the single-step purification of the folate transporters from L1210 cells. Parental L1210 murine leukemia cells, which contain only the microM transporter (the reduced folate/MTX transport protein) were treated with the N-hydroxysulfosuccinimide ester of biotin-SS-MTX, and a detergent extract of the plasma membranes was exposed to streptavidin-agarose beads to adsorb the labeled protein. Dithiothreitol cleavage of the disulfide linkage released the transporter, which migrated as a well-defined component (43 kDa) on SDS-PAGE gels; no other proteins were present. An L1210 subline (JF), obtained by adapting cells to grow on nanomolar concentrations of folate, contains both the microM transporter and the nM transporter (high-affinity folate binding protein). When these cells were treated with the N-hydroxysulfosuccimide ester of biotin-SS-folate and processed as described above, analysis on SDS-PAGE gels revealed the presence of two proteins, the microM transporter (43 kDa) and the nM transporter (39 kDa). Both transporters were characterized with respect to amino acid content; blocked N-termini precluded Edman sequencing. Treatment of the nM transporter with peptide:N-glycosidase F produced a smaller component (32 kDa); the microM transporter, conversely, was unchanged by this procedure. When the microM transporter in parental L1210 cells was labeled with fluorescein-MTX and then treated with phosphoinositol-specific phospholipase C (PI-PLC), no change in fluorescence was detected. Alternatively, when the nM transporter in the JF subline was labeled with fluorescein-folate and then treated with PI-PLC, complete loss of fluorescence was observed. These results indicate that the L1210 microM transporter is a non-glycosylated, integral membrane protein, while its nM counterpart is heavily glycosylated and anchored exofacially to the membrane by a glycosylphosphatidylinositol component.
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PMID:Multiple folate transport systems in L1210 cells. 132 5

Sheep lung dipeptidase was released from a lung membrane preparation by digestion with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis. The total enzyme activity released into the supernatant was 4- to 5-fold greater than that measured in the intact membrane prior to solubilization. The release of the peptidase from the membrane by this treatment is typical of proteins anchored to the lipid bilayer by a covalent attachment of phosphatidylinositol via a C-terminal glycolipid extension. The solubilized lung peptidase was further purified by ammonium sulfate fractionation followed by affinity chromatography and high-pressure liquid chromatography. A linear relationship between log molecular weight and elution volume for proteins of known molecular weight was established using a Toya Soda TSK 3000 high-pressure liquid chromatography column, and the molecular weight of the lung dipeptidase was estimated at 105,000. The peptidase activity against glycyldehydrophenylalanine of the purified enzyme co-chromatographed in high-pressure liquid chromatography with the activity that converted leukotriene D4 to leukotriene E4. In kinetic studies using leukotriene D4 as substrate, the relationship between the rate of hydrolysis and enzyme concentration was shown to be linear over the range 20 ng to 98 ng enzyme. Values of Km and Vmax for the dipeptidase using leukotriene D4 as substrate were 43 +/- 6 microM and 11,200 +/- 400 nmol/min per mg, respectively. Inhibition of the conversion of leukotriene D4 to leukotriene E4 was observed with a series of inhibitory agents. Cilastatin, bestatin and chloracetyldehydrophenylalanine were all effective at the micromolar level with cilastatin proving to be the most effective inhibitor. Dithiothreitol was effective within the millimolar range.
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PMID:Bioconversion of leukotriene D4 by lung dipeptidase. 215 8

We have examined the direct effects of oxidant metabolites on cardiac sarcolemmal phosphoinositide phospholipase C which transduces signals from various receptors for the modulation of intracellular Ca2+ levels. The enzyme activity in rat cardiac sarcolemmal membranes that had been preincubated (10 min; 37 degrees C) with xanthine-xanthine oxidase, a superoxide anion generating system, was not significantly affected. The addition to this system of superoxide dismutase, which converts superoxide anion to hydrogen peroxide (H2O2), resulted in a significant decrease of the enzyme activity in comparison with control values. Such decrease was fully prevented by catalase. Preincubation of sarcolemma with hypochlorous acid also gave a significant inhibition of phospholipase C, which was counteracted by the synthetic thiol reducer dithiothreitol. H2O2-pretreatment induced a concentration-dependent inhibition of the enzyme which was prevented by catalase but not by the iron chelator deferoxamine. Dithiothreitol was able to protect against, as well as to recover the enzyme activity from the H2O2 effects. These data suggest that superoxide anions and hydroxyl radicals did not interfere with phospholipase C activity, and that the nonradical oxidants, H2O2 and hypochlorous acid, may have acted through oxidation of thiol (SH) groups. The existence of reactive SH groups associated with the enzyme was confirmed by the inhibitory effects of SH modifiers (p-chloromercuriphenylsulfonic acid, 5'5'-dithio-bis(2-nitrobenzoic acid), N-ethylmaleimide and methyl methanethiosulfonate), which were prevented and in some cases also reversed by dithiothreitol. The biological reducer glutathione (GSH) was not able to recover the H2O2-induced inhibition of phospholipase C, whereas its oxidized form (GSSG) decreased the enzyme activity both in control and H2O2-pretreated membranes. The enzyme was active in a wide range of GSH/GSSG redox states, but H2O2 pretreatment narrowed this range. The results showed that oxidative stress changed the redox state of sarcolemmal phospholipase C, and this deactivated the enzyme. The oxidants' concentrations that significantly impaired phospholipase C in this study were compatible with those occurring in vivo during ischemia-reperfusion [Am. J. Med. 91(Suppl. 3C):235, 1991]. This supports the possibility that alteration of the receptor-associated phospholipase C may be a factor in the oxidant-related dysfunction of the ischemic-reperfused heart.
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PMID:Oxidative stress modifies the activity of cardiac sarcolemmal phospholipase C. 828 Jul 55

Neutrophils (PMN) contain two types of phospholipase A2 (PLA2), a 14 kDa 'secretory' Type II PLA2 (sPLA2) and an 85 kDa 'cytosolic' PLA2 (cPLA2), that differ in a number of key characteristics: (1) cPLA2 prefers arachidonate (AA) as a substrate but hydrolyzes all phospholipids; sPLA2 is not AA specific but prefers ethanolamine containing phosphoacylglycerols. (2) cPLA2 is active at nM calcium (Ca2+) concentrations; sPLA2 requires microM Ca2+ levels. (3) cPLA2 activity is regulated by phosphorylation; sPLA2 lacks phosphorylation sites. (4) cPLA2 is insensitive to reduction; sPLA2 is inactivated by agents that reduce disulfide bonds. We utilized PMN permeabilized with Staphylococcus aureus alpha-toxin to determine whether one or both forms of PLA2 were activated in porated cells under conditions designed to differentiate between the two enzymes. PMN were labeled with [3H]AA to measure release from phosphatidylcholine and phosphatidylinositol; gas chromatography-mass spectrometry was utilized to determine total AA release (mainly from phosphatidylethanolamine) and to assess oleate and linoleate mass. A combination of 500 nM Ca2+, a guanine nucleotide, and stimulation with n-formyl-met-leu-phe (FMLP) were necessary to induce maximal AA release in permeabilized PMN measured by either method; AA was preferentially released. [3H]AA and AA mass release occurred in parallel over time. A hydrolyzable form of ATP was necessary for maximum AA release and staurosporin inhibited PLA2 activation. Dithiothreitol treatment had little affect on [3H]AA release and metabolism but inhibited AA mass release. Assay of cell supernatants after cofactor addition did not detect sPLA2 activity and the cytosolic buffer utilized did not support activity of recombinant sPLA2. These results strongly suggested that cPLA2 was the enzyme activated in the permeabilized cell model and this is the first report which unambiguously demonstrates AA release in response to activation of a specific type of PLA2 in PMN.
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PMID:Activation of cytosolic phospholipase A2 in permeabilized human neutrophils. 855 68