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)

Apparent values of Km and Vmax have been measured for catalysis of hydrolysis of unsonicated egg lecithin liposomes, activated through addition of 0.4 M n-hexanol, by phospholipases A2 from bee and snake venoms and by phospholipase C from Clostridium welchii as a function of the concentration of three surfactants: hexadecylamine, hexadecyltrimethylammonium bromide, and dihexadecyl phosphate. For all three enzymes, values of Km and Vmax show little or no dependence on the concentration of these ionic surfactants, demonstrating that the liposomal surface charge is not a crucial factor in determining susceptibility to phospholipase-catalyzed hydrolysis.
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PMID:Phospholipases. III. Effects of ionic surfactants on the phospholipase-catalyzed hydrolysis of unsonicated egg lecithin liposomes. 0 May 6

The fluorescent probes 8-anilino-1-naphthalenesulfonate (ANS) and 2-p-toluidinylnaphthalene-6-sulfonate (TNS) bind to highly purified myelin membranes obtained from bovine brain white matter. Binding of the dyes was markedly increased by environmental conditions which reduce the negative surface potential of the membrane, i.e., cations (La-3+ is greater than Ca-2+ is greater than Na-+,K-+), H-+, local anesthetics, and the antibiotic polymyxin B. Chemical alteration of accessible membrane charged groups affected dye binding in a manner consistent with the hypothesis that such binding is primarily dependent upon the membrane surface potential. Thus, binding was increased by blocking of carboxyl groups via carbodiimide activation and subsequent coupling with neutral amino acid esters, and even more so with a basic amino acid ester (e.g., arginine methyl ester). Dye binding was reduced by succinylation of amino groups, and by hydrolysis of choline and ethanolamine head groups of phospho- and sphingolipids by phospholipase C. Phospholipase C treatment of myelin, or sphingomyelin vesicles, reduced or abolished the augmentation of ANS and TNS binding due to cations, local anesthetics, or polymyxin B. Energy transfer from myelin tryptophan residues to bound ANS occurs, but with low efficiency. Oxidation of membrane tryptophan residues with N-bromosuccinimide, or alkylation with 2-hydroxy (or methoxy)-5-nitrobenzyl bromide, markedly reduced intrinsic membrane fluorescence and energy transfer to bound ANS, but did not significantly affect dye binding or the quantum yield of ANS fluorescence when excitation was at 380nm. Proteolytic digestion removed 6-30% of myelin protein, depending upon the enzyme used, but had no effect on fluorescent dye binding. It is concluded that the binding of the anionic fluorescent probes ANS and TNS to myelin is primarily a function of the membrane surface charge density and net surface potential, as is the case with other biological membranes. Conclusions about the degree of dye binding to membrane lipids or membrane proteins cannot be drawn unless additional studies are carried out on isolated water soluble membrane proteins.
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PMID:Reactions of fluorescent probes with normal and chemically modified myelin. 23 81

Phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) from Pseudomonas aureofaciens was purified 3600-fold from the culture filtrate with a recovery of 1.6%. Purification was performed with the useof (NH4)2SO4 precipitation, Sephadex G-100 gel filtration and by ion-exchange chromatography on DEAE-Sephadex A-50 and CM-Sephadex C-50. The purified enzyme appeared to be homogeneous as revealed by polyacrylamide disc gel electrophoresis at pH 9.3. The molecular weight was estimated to be 35 000 by gel filtration on Sephadex G-75. Under our experimental conditions, phosphatidylethanolamine was more rapidly hydrolysed than phosphatidylcholine. Lyso forms of these two phosphatides were poor substrates. Phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, cardiolipin and sphingomyelin were not hydrolysed. The enzyme activity with phosphatidylcholine as substrate was slightly stimulated by Ca2+, Mg2+, and Mn2+. However, these cations inhibited the activity with phosphatidylethanolamine as substrate. An anionic detergent, sodium deoxycholate, slightly enhanced the activity when phosphatidylcholine and phosphatidylethanolamine were used as substrates. A cationic detergent, cetyltrimethylammonium bromide, inhibited enzyme activity. EDTA and o-henanthroline inhibited the activity of the enzyme to a marked degree.
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PMID:Studies on phospholipase C from Pseudomonas aureofaciens. I. Purification and some properties of phospholipase C. 24 4

Iodination of staphylococcal alpha-toxin by the lactoperoxidase method resulted in the maximal incorporation of about 2.5 atoms of iodine per molecule of alpha-toxin. The iodination primarily involved a single tyrosine residue as shown by analysis of both cyanogen bromide and tryptic peptides. Iodination at a level of 1.2 iodine atoms per alpha-toxin molecule led to a dramatic decrease in the hemolytic and lethal activities, although no decrease in the binding of iodinated toxin to rabbit erythrocytes was observed (Cassidy and Harshman (1976), Biochemistry, the following paper in this issue). Monoiodinated alpha-toxin was found to have 15% of the specific hemolytic activity of native alpha-toxin. Incubation of rabbit erythrocytes with iodinated alpha-toxin led to a significant protection from the hemolytic activity of native alpha-toxin added later. The results show the modification of a single unique tyrosyl residue in alpha-toxin permits the resolution of alpha-toxin's biological activities from its cell binding activity.
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PMID:Iodination of a tyrosyl residue in staphylococcal alpha-toxin. 127 41

We recently reported that prostaglandin E2 (PGE2) and arachidonic acid (AA) each induced a gradual secretion of catecholamines from cultured bovine adrenal chromaffin cells in the presence of ouabain by stimulation of phosphoinositide metabolism. In the present study, we examined the relationship between phospholipase A2 and C activation and catecholamine secretion by PGE2 in chromaffin cells. The phospholipase A2 inhibitors p-bromophenacyl bromide and mepacrine did not affect the basal and ouabain-induced release, but dose-dependently blocked PGE2-evoked phosphoinositide metabolism and the consequent catecholamine release at an IC50 value of 3 microM. PGE2 induced rapid hydrolysis of [3H]AA from prelabeled phospholipid pools: the release of [3H]AA could be detected at as early as 15 sec and reached a plateau after 1 min. While the phospholipase C inhibitor neomycin did not inhibit PGE2-induced AA release, phospholipase A2 inhibitors dose-dependently inhibited it at IC50 values comparable to those for catecholamine release. Pretreatment of intact cells with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, but not with pertussis toxin, prevented AA release by PGE2. These results demonstrate that PGE2 activates phospholipase A2 as well as phospholipase C in a pertussis toxin-insensitive manner and suggest that the released arachidonic acid may be involved in PGE2-induced catecholamine release from chromaffin cells.
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PMID:Prostaglandin E2-induced arachidonic acid release and catecholamine secretion from cultured bovine adrenal chromaffin cells. 133 53

The method of DNA binding to nitrocellulose filters was applied to DNA isolated from mouse liver and Ehrlich ascite carcinoma (EAC), calf thymus, and lymphocytes from patients with chronic lymphoid leukemia. In those and phage PM2 DNA the increase in the DNA binding to the filters with a rise in NaCl concentration from 0.5 up to 4.5 M was sigmoidal being suggestive of a conformational transition. No such activity was found in the case of phage lambda or single-stranded DNA. The binding decreased dramatically after mild cleavage of DNA with DNAase I or treatment with phospholipase C or Eco RI and Hin PI restrictases. Incubation of DNA with ethidium bromide led to decrease in the amount of bound DNA. This effect was enhanced with a rise in the dye concentration. The isotherms of ethidium bromide binding to eukaryotic DNA obtained in Scatchard plots by optic titration had a component with a positive slope at low values of r. Bivalent ions (Mg2+, Zn2+) shifting the equilibrium towards the Z-form increased the proportion of macromolecules retained on the filters at NaCl concentrations of 1-3 M. Local changes in the helix conformation were studied with the help of chemical probes: diethylpyrocarbonate (guanine Z-DNA) and osmium-pyridine reagent (pyrimidines of boundary B-Z sites). These probes incorporation into samples of liver DNA, EAC, and lymphocytes resulted in chemical modification of all these samples. Modification of DNA by osmium-pyridine reagent led to inhibition of subsequent restriction by Eco RI restrictase. The data obtained are suggestive of the presence of Z-regions in the B-helix of eukaryotic DNA. A topological model of Z-site stabilization in small superhelical loops of DNA fixed by protein or lipoprotein molecules is proposed.
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PMID:[Detection of left-helical segments in eukaryotic DNA]. 148 26

The lethal alpha-toxin was isolated from the culture filtrate of Clostridium novyi type B using ammonium sulfate precipitation and ion exchange chromatography. The alpha-toxin has a mol. wt of 190,000 and does not contain any disulfide cross-linkages. It consists of a single polypeptide chain. The peptide fragments resulting from the cyanogen-bromide cleavage were isolated using reversed phase and gel filtration HPLC. The immunogenic actions of these peptides and peptide mixtures were studied in Balb/c mice. Three polyclonal antisera recognizing the uncleaved native toxin could be found using an ELISA test (Br3, Bro2, Bro5). One peptide mixture (Tx5), which was proved lethal in shell-less quail eggs (in vitro), was rechromatographed with gel filtration HPLC that resulted in one peptide with mol. wt 3000 (Txleth), which again proved lethal in the shell-less quail egg lethality test. The immunogenic peptides differ from the lethal one, therefore we assumed different locations on the polypeptide chain. The separation of the immunogenic, non-toxic fragment from the lethal one may allow the production of a highly specific non-toxic vaccine. By using synthetically produced immunogenic peptides, time-consuming purification methods and working with the whole toxin will become unnecessary.
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PMID:Isolation of immunogenic and lethal peptides of alpha-toxin from Clostridium novyi type B. 151 54

1. Calcium currents (ICa) were measured in frog ventricular myocytes using the whole-cell patch clamp technique and a perfused pipette. The effect of internal perfusion with the hydrolysis-resistant GTP analogue, GppNHp (5'guanylylimidodiphosphate), on basal ICa and ICa stimulated with forskolin or isoprenaline was examined to gain insight into the role of G proteins in ICa regulation. 2. Without added guanine nucleotides, isoprenaline stimulated ICa approximately 14-fold with an EC50 of 0.09 microM. Forskolin stimulated ICa approximately 10-fold with an EC50 of 0.30 microM. 3. Internal 30 microM-GppNHp produced an approximately 80% decrease in ICa elevated by 0.3 microM-isoprenaline or 3 microM-forskolin. The inhibition of isoprenaline stimulation was due to a decrease in the maximal stimulation from approximately 14-fold to approximately 14-fold without a significant change in the EC50. In contrast, the reduction in forskolin stimulation was due to a 22-fold increase in the EC50 to 11.4 microM, with little change in maximal stimulation. 4. The inhibition of stimulated ICa by GppNHp is likely to be mediated by a G protein, because the effects of GppNHp are irreversible, and are blocked by excess GTP. ICa is affected similarly by GppNHp and by ACh. This suggests that GppNHp activates the same G protein that is normally activated by ACh, but activation by GppNHp occurs in the absence of agonist occupation of the muscarinic receptor. 5. The increase in the EC50 for forskolin produced by internal GppNHp was reversed by exposure to isoprenaline, which itself did not affect ICa amplitude. On average, exposure to isoprenaline in the presence of GppNHp caused an irreversible 81-fold decrease in the EC50 for forskolin to 0.14 microM. Stimulation of ICa by forskolin after internal GppNHp and exposure to isoprenaline was completely blocked by the protein kinase A inhibitor PKI(5-22). 6. These effects do not involve the phospholipase C system, because they are not mimicked by phorbol esters or internal inositol 1,4,5-trisphosphate (IP3) and are not blocked by bromophenacyl bromide or neomycin. 7. Direct effects of G proteins on ICa were not evident, because internal perfusion with PKI(5-22) completely inhibited isoprenaline- or forskolin-stimulated increases in ICa, and neither ACh nor internal GppNHp (30-500 microM) affected basal ICa or ICa elevated by internally perfused cyclic AMP. 8. These results suggest that the predominant site of action of the inhibitory G protein activated by either GppNHp or ACh is adenylyl cyclase. Furthermore, the internally perfused frog cardiomyocytes may provide a useful approach for probing the detailed interactions of G proteins, forskolin, and adenylyl cyclase in an intact cell.
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PMID:Regulation of Ca2+ current in frog ventricular cardiomyocytes by 5'-guanylylimidodiphosphate and acetylcholine. 165 25

Snake venom cardiotoxin (CTX) fractions induce contractures of skeletal muscle and hemolysis of red blood cells. The fractions also contain trace amounts of venom-derived phospholipase A2 (PLA2) contamination and activate tissue phospholipase C (PLC) activity. The present study examines the mechanisms of action of a CTX fraction from Naja naja kaouthia venom in skeletal muscle. Sphingosine competitively antagonized CTX-induced red blood cell hemolysis, but not skeletal muscle contractures. CTX rapidly lowered the threshold for Ca(2+)-induced Ca2+ release in heavy sarcoplasmic reticulum fractions, as monitored with arsenazo III. There was also a slower time-dependent reduction of Na+ currents, as assessed by whole cell patch-clamp techniques. The CTX fractions elevated levels of free fatty acids and diacylglycerol for 2 hr in primary cultures of human skeletal muscle by a combined action of venom-derived PLA2 contamination in the fraction and activation of endogenous PLC activity. The activation of tissue PLC activity could be readily distinguished from the contribution of the venom PLA2 by p-bromophenacyl bromide treatment of CTX fractions. The mechanism of action involved in contractures of skeletal muscle appears to be related to the immediate and specific effect of CTX (Ca2+ release by the sarcoplasmic reticulum), while the mechanisms involved in hemolysis of red blood cells and decreased Na+ currents in skeletal muscle most likely relate to long-term effects on lipid metabolism.
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PMID:Effects of a cardiotoxin from Naja naja kaouthia venom on skeletal muscle: involvement of calcium-induced calcium release, sodium ion currents and phospholipases A2 and C. 166 2

1. Intracellular mechanisms and second messengers involved in chloride current activation by intracellular GTP gamma S (guanosine 5'-O-(3-thiotriphosphate] in bovine chromaffin cells were studied using the whole-cell patch-clamp technique combined with measurements of intracellular calcium [Ca2+]i. 2. No correlation between the time of current activation and the appearance of [Ca2+]i transients was observed; intracellular introduction of sufficient EGTA (10 mM) to suppress the [Ca2+]i transients did not affect the current activation by GTP gamma S. 3. The cyclic nucleotides, cyclic AMP or cyclic GMP, did not activate the current when introduced intracellularly (50-250 microM). The ability of GTP gamma S to activate the current decreased when cyclic GMP (250 microM), together with MgATP (2 mM), was added to the perfusate. 4. Neomycin (0.5-1 mM), a presumed inhibitor of phospholipase C effectively prevented the current activation by GTP gamma S but it did not prevent [Ca2+]i transients. 5. Modulation of protein kinase C activity using specific inhibitors (H-7, 300 microM; polymyxin B, 400 U/ml) or activators (phorbol ester PMA, 100 nM, 20-90 min at 37 degrees C) did not affect the current activation by GTP gamma S nor did it cause current activation in the absence of GTP gamma S. 6. Activation of the current by GTP gamma S could be prevented by incubating the cells for 10-15 min with 2.5 microM p-bromophenacyl bromide (p-BPB), an inhibitor of phospholipase A2 activity. Exogenous arachidonic acid (5-10 microM), applied extracellularly or intracellularly, neither activated the current itself nor did it interfere with its activation by GTP gamma S. 7. Activation of the current by GTP gamma S could also be prevented by incubating the cells with 1 microM-nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, but not with indomethacin (2 microM), an inhibitor of cyclo-oxygenase pathway of arachidonic acid metabolism. 8. It is suggested that chloride current activation by GTP gamma S in bovine chromaffin cells involves G protein-mediated stimulation of phospholipase A2 activity and subsequent formation of lipoxygenase product(s) of arachidonic acid metabolism.
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PMID:Second messengers mediating activation of chloride current by intracellular GTP gamma S in bovine chromaffin cells. 171 42


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