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)

This study demonstrates that p-bromophenacyl bromide irreversibly inhibits, in a time- and dose-dependent manner, yeast alcohol dehydrogenase, bovine pancreatic alpha-chymotrypsin, human platelet phosphatidylinositol (PI)-specific phospholipase C, in addition to the neutral-active and calcium-dependent phospholipase A2 of human platelets. The PI-specific phospholipase C has maximal activity at pH 5,5 is calcium-dependent, and is strongly inhibited by sulfhydryl reagents 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and methylmethane thiosulfonate . Increasing concentrations of DTNB produced concomitant inhibition of phospholipase C activity and titration of sulfhydryl groups. In contrast, human platelet phospholipase A2 activity was unaffected by concentrations of DTNB that titrated sulfhydryl groups, and completely inhibited PI-specific phospholipase C activity. Treatment of cysteine with p-bromophenacyl bromide resulted in modification of the amino acid as demonstrated by paper chromatography, and loss of titratable sulfhydryl groups. These data show that p-bromophenacyl bromide inhibits a wide spectrum of enzymatic activities including PI-specific phospholipase C. This reagent modifies amino acid residues other than active-site histidines and therefore has a broader reactivity than previously considered. Thus, it should not be used as a selective inhibitor of enzymes in crude cellular experiments.
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PMID:Nonspecific inhibition of enzymes by p-bromophenacyl bromide. Inhibition of human platelet phospholipase C and modification of sulfhydryl groups. 673 33

The rate of phospholipid hydrolysis in erythrocyte ghosts by Bacillus cereus phospholipase C was markedly decreased by the presence of NaCl at concentrations between 25 and 200 mM. The inhibition seemed to be due to Cl- and was unaffected by the type of cation present. The larger univalent anions such as HCO3-, Br-, Cl-, NO3-, CNO- and I- seemed most effective, whereas the bivalent anion SO42- was relatively ineffective at 0.1 M, as were acetate and formate. Tris buffers at 0.1 M caused marked inhibition. With bovine brain myelin, phospholipid hydrolysis by phospholipase C was also much more strongly inhibited by I- and Cl- than by SO42- or acetate. NaCl inhibited the hydrolysis by the enzyme of the soluble substrate dihexanoylglycerophosphocholine, thereby suggesting that the inhibiton did not arise simply from substrate effects.
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PMID:Inhibition of Bacillus cereus phospholipase C by univalent anions. 681 Aug 75

Phospholipase C (heat-labile hemolysin) was purified from Pseudomonas aeruginosa culture supernatants to near homogeneity by ammonium sulfate precipitation followed by a novel application of DEAE-Sephacel chromatography. Enzymatic activity remained associated with DEAE-Sephacel even in the presence of 1 M NaCl, but was eluted with a linear gradient of 0 to 5% tetradecyltrimethylammonium bromide. Elution from DEAE-Sephacel was also obtained with 2% lysophosphatidylcholine, and to a lesser extent with 2% phosphorylcholine, but not at all with choline. The enzyme was highly active toward phospholipids possessing substituted ammonium groups (e.g., phosphatidycholine, lysophosphatidylcholine, and sphingomyelin); however, it had little if any activity toward phospholipids lacking substituted ammonium groups (e.g., phosphatidylethanolamine, phosphatidylserine, and phosphaditylglycerol). Collectively, these data suggest that phospholipase C from P. aeruginosa exhibits high affinity for substituted ammonium groups, but requires an additional hydrophobic moiety for optimum binding. The specific activity of the purified enzyme preparation increased 1,900-fold compared with that of culture supernatants. The molecular weight of the phospholipase C was estimated to be 78,000 by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Sephacryl S-200 column chromatography and was 76,000 by high-performance size exclusion chromatography. The isoelectric point was 5.5. Amino acid analysis showed that phospholipase C was rich in glycine, serine, threonine, aspartyl, glutamyl, and aromatic amino acids, but was cystine free.
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PMID:Phospholipase C (heat-labile hemolysin) of Pseudomonas aeruginosa: purification and preliminary characterization. 681 52

Mepacrine and p-bromophenacyl bromide, in addition to their inhibitory effect on lipolysis, are also potent inhibitors of fatty acid acylation into renal medullary lipids. Significant qualitative and quantitative differences in the inhibition by the two drugs were seen. p-Bromophenacyl bromide exerted a non-selective effect inhibiting the incorporation of saturated and unsaturated fatty acids into all phospholipid classes and triacylglycerols. In contrast, mepacrine selectively inhibited the incorporation of both saturated and unsaturated acids into phosphatidylcholine, phosphatidylethanolamine and triglycerides, and concurrently markedly enhanced their incorporation into phosphatidylinositol. Quantitative analysis of these mepacrine effects, together with the known inhibitory effects of this compound on phospholipase A2 and phosphatidylinositol-specific phospholipase C, suggests that mepacrine also inhibits phosphatidic acid phosphatase, thereby shunting the flux of phosphatidic acid away from diglyceride formation and into synthesis of phosphatidylinositol.
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PMID:Phospholipase A2 inhibitors. Differential inhibition of fatty acid acylation into kidney lipids by mepacrine and p-bromophenacyl bromide. 687 Sep 35

A mechanism of selective localization of membrane-bound enzymes was examined by studying the interaction between D-beta-hydroxybutyrate dehydrogenase (EC 1.1.1.30) and native cellular membranes in which the lipid components were altered. (1) The catalytic activity of the purified lipid-free enzyme could be restored by the re-interaction with microsomal and mitochondrial membranes, whereas with erythrocyte membranes or liposomes from lipids of erythrocyte membranes this activity could not be restored (Miyahara, M., Utsumi, K. and Deamer, D.W. (1981) Biochim. Biophys. Acta 641, 222-231). In the erythrocyte lipid components, only lysophosphatidylcholine markedly inhibited the enzyme reactivation. (2) The inhibitory effect of lysophosphatidylcholine was confirmed in microsomes in which the lysophosphatidylcholine contents had been increased, by phospholipase A2 treatment, to the levels in erythrocyte membranes. (3) Selective digestion by phospholipase C of phosphatidylcholine in the microsomes was accompanied by a lowering of the level of reactivation in the membranes. (4) The presence of lipophilic alkyl compounds such as cetylamine and cetyltrimethylammonium bromide, which contain the ammonium group, in the membranes also inhibited the enzyme reactivation. However, negatively charged and neutral alkyl compounds were less suppressive. The results above suggested that the interaction of D-beta-hydroxybutyrate dehydrogenase with native cellular membranes is dependent on the amounts of phosphatidylcholine and lysophosphatidylcholine exposed on the membrane surface. It was also suggested that the presence of the ammonium group of non-diacyl compounds is unfavorable for the effective interaction of the enzyme.
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PMID:Lipid-dependent interaction of D-beta-hydroxybutyrate dehydrogenase with cellular membranes. 721 15

The modulation of a brain phosphoinositide-specific phospholipase C-alpha activity was studied using a variety of compounds of different charge. Detergents such as sodium deoxycholate and cetyltrimethylammonium bromide stimulated the phospholipase C activity when used alone but when used together the effects were not additive. Spermine was an effective inhibitor of the enzyme activity while the cationic peptide, Melittin, had no effect. The inositol phosphates produced by hydrolysis with phosphoinositide-specific phospholipase C were inhibitory while diacylglycerol and inositol did not affect the phospholipase activity. Myelin basic protein, which was previously shown to stimulate phospholipase C activity by 2.5-fold, did not interact with the anionic inositol phosphatases to any significant extent. Thus we concluded that the mechanism of stimulation was not due to relief of product inhibition. Crosslinking studies with the photoactivatable reagent, N-hydroxysuccinimidyl-4-azidosalicylic acid, showed that peptide 24-33 of myelin basic protein, which stimulated the activity almost as much as the native protein, interacted specifically with the phospholipase C. Thus the mechanism by which myelin basic protein stimulated the enzyme appeared to be through specific protein-protein interaction.
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PMID:The mechanism of stimulation of brain phospholipase C-alpha by myelin basic protein involves specific interactions. 751 86

As was shown in our previous work, the intracellular pH (pHi) of cultured human fibroblasts depends on cell density. The pHi is low in single cells, higher in cells, forming small groups and maximal in a sparse monolayer. On the other hand, the pHi is low in areas of confluent monolayers. In the present work, we show that the effects of inhibitors of various pH-controlling mechanisms as well as inhibitors of key enzymes in signal transduction pathways depend on the local cell density. We have found that N-ethylmaleimide and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, known as inhibitors of V-type H+ ATPase, inhibit the elevation of pHi induced by cell-cell contact interactions; meanwhile Cd2+ ions, which inhibit H+ conductive pathway, cause an increase of pHi in a confluent monolayer. Our data revealed also that the Na+/H+ antiporter does not play an essential role in the pHi regulation by intercellular contacts. Inhibitors of phospholipase A2 (4-bromophenacyl-bromide), phospholipase C (neomycin) and protein kinase C (H-7) dramatically change the way the pHi is modulated by local cell density. It is suggested that cell-cell interactions regulate cell activities via modulation of pHi, which is under positive control from phospholipase A2 and under negative control from protein kinase C.
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PMID:Regulation of intracellular pH by cell-cell adhesive interactions. 758 3

The role of adenosine 3',5'-cyclic monophosphate (cAMP) in generating the osmo-dependent slow inward membrane currents (S(in)) elicited by activation of follicle stimulating hormone (FSH) or acetylcholine (ACh) receptors was studied in voltage-clamped, follicle-enclosed oocytes of Xenopus laevis (follicles). Forskolin (FSK) also generated S(in) currents, and in low concentrations it potentiated the S(in) currents elicited by FSH but not those elicited by ACh. Moreover, intra-oocyte injections of cAMP elicited similar slow inward currents (cAMP-S(in)) that: (i) were carried mainly by chloride ions; (ii) were abolished by defolliculating the oocytes; and (iii) were dependent on the osmolarity of the external medium. Compared with the Ca(2+)-dependent chloride channels that are located in the oocyte membrane; the cAMP-activated S(in) channels were less permeable to I- and Br-, and their current-voltage relation did not rectify strongly at negative potentials. Generation of cAMP-S(in) desensitized the FSH-S(in) currents, but did not have effects on both the S(in) and the fast chloride current (F(in)) specifically elicited by ACh. Furthermore, follicular phospholipase C activation through stimulation of angiotensin II (AII) receptors failed to generate the current responses elicited by ACh. We conclude that cAMP acts as a potent second messenger in generating the osmo-dependent Cl- currents elicited by FSH but not those elicited by ACh. The mechanisms underlying the ACh responses remain unknown. The osmo-dependent chloride channels activated by cAMP may play a role in the control of volume of the follicular cells-oocyte complex.
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PMID:Osmo-dependent Cl- currents activated by cyclic AMP in follicle-enclosed Xenopus oocytes. 788 64

The molecular basis of the interaction of apolipophorin III (apoLp-III), an exchangeable apolipoprotein from hemolymph of the sphinx moth. Manduca sexta, with lipoprotein surfaces and phospholipids was studied by investigating the structural and binding properties of the C-terminal fragment of the native protein. A 4K peptide, corresponding to the terminal helical segment of the native protein, was generated by cyanogen bromide treatment, purified by gel filtration and reverse-phase HPLC, and characterized by N-terminal sequencing and amino acid and mass spectrometric analysis. Circular dichroism (CD) spectroscopy of the peptide in buffer indicated a predominantly unstructured state while addition of trifluoroethanol (TFE), a helix-inducing agent, resulted in an alpha-helical structure. Sedimentation equilibrium studies revealed that the 4K peptide was monomeric in buffer. The 4K peptide assumed an alpha-helical conformation in the presence of sodium dodecyl sulfate (SDS) and lysolecithin, but was unstructured in the presence of dimyristoylphosphatidylcholine, either when added to preformed vesicles or upon cosonication, indicating an ability to bind to detergent micelles but not to phospholipid bilayers. Unlike native apoLp-III, the 4K peptide did not confer protection against turbidity development to human low density lipoprotein upon incubation with phospholipase C, indicating an inability to interact with the surface of lipoproteins. Upon interaction with SDS micelles, both the 4K peptide and apoLp-III were resistant to urea-induced denaturation when compared to free apoLp-III, as evaluated by CD spectroscopy. The structural stability conferred upon interaction with detergents was similar for both the peptide and the native protein.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Structural and binding characteristics of the carboxyl terminal fragment of apolipophorin III from Manduca sexta. 794 39

We sought to evaluate the mechanisms by which mechanical perturbation elevates intracellular calcium in endothelial cells. We report that the transient elevation in intracellular calcium in cultured bovine aortic endothelial cells (BAEC) in response to gentle perturbation with the side of a micropipette was not blocked by depolarization (external K+, 130 mmol/L), nifedipine (10 mumol/L), or Bay K 8644 R(+) (10 mumol/L). Thus, voltage-dependent calcium channels were not involved in the response. Also, amiloride (10 mumol/L) and tetraethylammonium (1 mmol/L) had no effect on calcium mobilization, indicating that Na+ and K+ transporters were not involved. Pretreatment of the cells with the phospholipase C and phospholipase A2 inhibitor manoalide (10 mumol/L) for 10 minutes at 37 degrees C completely abolished the calcium response, as did a 10-minute pretreatment with the inhibitor of actin polymerization, cytochalasin B (1 mumol/L). We observed an inhibitory effect of the phospholipase A2 and phospholipase C inhibitor 4-bromophenacyl bromide (10 mumol/L) on the mechanical response of BAEC that was not as potent as that observed with manoalide. To examine the role of arachidonic acid (AA) and subsequent metabolites that may be released after a putatively mechanical activation of phospholipase A2, we exposed BAEC to exogenous AA. We found that continued exposure of BAEC for 5 minutes to 10 nmol/L to 10 mumol/L AA caused no elevation of intracellular calcium. If mechanical stimulation activates phospholipase A2, the liberated AA and subsequent metabolites do not appear to have much effect on BAEC intracellular calcium.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mechanically induced calcium mobilization in cultured endothelial cells is dependent on actin and phospholipase. 798 Nov 91


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