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)

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

Phenylglyoxal (PG) is shown to be a cell surface probe specific for arginine moieties in protein: (1) It does not enter the cell as evidenced by lack of PG in the cytoplasm. (2) It does not cause excessive cell leakage as measured by release of 51Cr. (3) It reacts with positively-charged groups in proteins at the cell surface but not with those of phospholipids at the surface; since pronase removes PG from the surface, but phospholipase C does not. (4) Under the conditions used in these experiments, it reacts virtually exclusively with arginine moieties in protein (Freedman et al., '68; Takahashi, '68; Werber and Sokolovsky, '72). Synchronized cells were exposed to radioactive PG to assess quantity of arginine moieties in protein at the surface. There is a sharp decrease in arginine at the cell surface at entry into G1 phase from M and a 24-fold increase upon entry into S phase. There is a slight drop in exposed arginine in late S phase followed by an increase to 26 times the G1 level immediately prior to mitosis. Lactoperoxidase-catalyzed iodination of tyrosine moieties in protein at the surface of synchronized cells shows a very gradual increase in protein as the cells move through the cycle and increase in size. Since the increase in arginine moieties in protein at the surface does not reflect a similar increase in total protein at the surface, an arginine-rich protein appears to be exposed at the cell surface during the division-related phases of the cell cycle.
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PMID:Exposure of an arginine-rich protein at surface of cells in S, G2, and M phases of the cell cycle. 112 7

Endothelium-derived relaxing factor (EDRF) inhibits platelet function, but the mechanism underlying this inhibitory effect is not known. To examine this, cultured acetylsalicylic acid (ASA)-treated endothelial cells (EC) from bovine aorta (BAEC) or from human umbilical vein (HUVEC) were incubated with washed, ASA-treated human platelets. Incubation of platelets with either BAEC or HUVEC resulted in inhibition of thrombin-induced platelet aggregation that was dependent on the number of EC added. This effect was potentiated by superoxide dismutase and reversed by treating EC with NG-nitro-L-arginine or by treating platelets with methylene blue, indicating that the inhibition of platelet aggregation was due to the release of EDRF by EC. EC significantly blocked the thrombin stimulated breakdown of phosphatidylinositol-4,5-bisphosphate (PIP2) and the production of phosphatidic acid in [32P]orthophosphate-labeled platelets and of inositol trisphosphate in [3H]myoinositol-labeled platelets. In addition, the thrombin-mediated activation of protein kinase C (PKC) and phosphorylation of myosin light chain were inhibited in the presence of EC. Finally, thrombin stimulated an increase in cytosolic ionized calcium concentration ([Ca2+]i) in fura2-loaded platelets that was abolished by concentrations of EC which also blocked thrombin-induced aggregation. These data indicate that EDRF blocks thrombin-induced platelet aggregation by inhibiting the activation of PIP2-specific phospholipase C and thereby suppressing the consequent activation of PKC and the mobilization of [Ca2+]i.
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PMID:Endothelium-derived relaxing factor inhibits thrombin-induced platelet aggregation by inhibiting platelet phospholipase C. 130 23

Gq mediates hormonal stimulation of phosphoinositide-specific phospholipase C (PI-PLC). We mutated the alpha subunit of Gq (alpha q) to replace arginine 183 with cysteine. Mutations that substitute cysteine for the corresponding arginine residues of alpha s and alpha i2 constitutively activate their respective effector pathways, creating the gsp and gip2 oncogenes. Transient expression of alpha q-R183C in COS-7 and HEK-293 cells constitutively activates PI-PLC, but wild type (WT) alpha q does not. This suggests that the mutated arginines in alpha s, alpha i2, and alpha q share a common function in regulating the active state of these proteins and that the alpha q gene may serve as a target for oncogenic mutations in human tumors. In an attempt to develop an assay for receptor stimulation of recombinant alpha q, we co-expressed receptors with alpha q-WT. We found that the alpha 2-adrenoceptor stimulates PI-PLC activation in HEK-293 cells in a fashion that depends completely on co-expression of alpha q-WT. These findings create an experimental model, similar to that provided for alpha s by S49 cyc- cells, that should make it possible to analyze receptor and effector coupling by mutant alpha q against a null background.
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PMID:Recombinant Gq alpha. Mutational activation and coupling to receptors and phospholipase C. 130 40

The urokinase-type plasminogen activator receptor (u-PAR) was demonstrated on cultured smooth muscle cells (SMCs) of bovine aorta. Binding of 125I-urokinase-type plasminogen activator (u-PA) was concentration dependent and saturable within 45-60 minutes. A similar concentration and time dependence was found in functional plasminogen activation studies. Human two-chain high-molecular-weight u-PA and its proenzyme (pro-u-PA) bound specifically with identical affinity (Kd). Activation of pro-u-PA was strongly accelerated on binding to SMCs and occurred only in the presence of plasminogen on the cell surface. A 100-fold molar excess of unlabeled high-molecular-weight u-PA effectively blocked binding of the radiolabeled ligands; tissue-type plasminogen activator, plasminogen, low-molecular-weight u-PA, and unrelated proteins did not. 125I-u-PA binding was abolished by a monoclonal antibody against the specific u-PA sequence responsible for u-PAR binding. Binding of u-PA sharply decreased on SMC exposure to phosphatidylinositol-specific phospholipase C, confirming the glycan phospholipid cell anchorage of u-PAR. Bovine and human alpha-thrombin (240 nM) increased the binding of 125I-u-PA fivefold, translating into an increase in the number of sites per cell from about 10(5) to 5 x 10(5) without significant change in the Kd (1.29 +/- 0.39 nM). Active site blockade of thrombin by D-Phe-Pro-Arg-chloromethyl ketone resulted in the total loss of stimulatory activity, as did the use of the inactive active site thrombin mutant, S205A. Hirugen (100 microM), which blocks the anion-binding exosite of thrombin, blocked u-PAR stimulating activity. Thus, both the catalytic activity and integrity of the exosite are important for thrombin's stimulatory activity. Other SMC mitogens (epidermal growth factor, transforming growth factor-beta 1, basic fibroblast growth factor, platelet-derived growth factor, and phorbol 12-myristate 13-acetate) increased u-PAR expression on SMCs six- to 20-fold while concomitantly increasing Kd four- to 10-fold. In all cases the induction of u-PAR was dependent on de novo protein synthesis. These observations assign a possible role for thrombin and other mitogens in u-PAR regulation, thereby influencing the pericellular proteolysis that is important in SMC migration and atheromatous plaque development.
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PMID:Regulation of the urokinase-type plasminogen activator receptor on vascular smooth muscle cells is under the control of thrombin and other mitogens. 132 97

Mechanisms of cathepsin B activation involved in methionine-enkephalin (ME) production induced by bradykinin (BK), des-Arg9-BK or L-arginine (L-Arg) were studied using cultured fibroblasts of the rat dental pulp, especially from a viewpoint of intracellular signal transduction. BK, des-Arg9-BK, L-Arg or cysteine enhanced the release of ME-like peptides from the cells, and the release of ME-like peptides induced by des-Arg9-BK was inhibited by des-Arg9-[Leu8]-BK (BK B1-receptor antagonist) and E-64 (a specific inhibitor of cysteine proteinases). The activation of cathepsin B by BK or des-Arg9-BK was inhibited by des-Arg9-[Leu8]-BK or islet-activating protein (IAP), and the activation of cathepsin B by L-Arg was inhibited by Leu-Arg (kyotorphin-receptor antagonist) or Botulinum C3-enzyme. The activation of cathepsin B by those stimulants was dependent on calcium ion. These results suggest that the ME production by BK or des-Arg9-BK may be mediated by Ca(2+)-dependent cathepsin B activation through B1-receptors and IAP-sensitive G-proteins, whereas the production by L-Arg may be mediated by Ca(2+)-dependent cathepsin B activation through kyotorphin-receptor and Botulinum C3-enzyme-sensitive G-proteins. On the other hand, the activation of cathepsin B was inhibited by neomycin B (phospholipase C inhibitor) and various serine/threonine kinase inhibitors. These results indicate that phospholipase C and serine/threonine kinases are involved in the activation of cathepsin B by BK, des-Arg9-BK or L-Arg. Genistein inhibited the activation of cathepsin B by des-Arg9-BK or L-Arg in a different fashion, suggesting that tyrosine kinase(s) is also involved in the activation. Cathepsin B activation by BK or L-Arg but not des-Arg9-BK was inhibited by L-NMMA (inhibitor of NO synthesis), and the activation by L-Arg was enhanced by beta-glycerophosphate (beta-GP: inhibitor of phosphatases), while the activation by BK or des-Arg9-BK was inhibited by beta-GP. These results suggest that BK-induced cathepsin B activation in the fibroblasts may be due to a combined effect of des-Arg9-BK and L-Arg.
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PMID:Activation of cathepsin B involved in enkephalin production by bradykinin and its cleavage products in cultured fibroblasts of the rat dental pulp. 134 8

Incubation of either C3a, C3ades Arg, or synthetic analogues of the C-terminal sequence of C3a with purified rat peritoneal mast cells resulted in a rapid and dose-dependent histamine release. The natural factors C3a and C3ades Arg were the most active of the factors tested exhibiting EC50 values of 3.3 and 2.2 microM, respectively. The corresponding 21- and 22-residue C-terminal analogues of C3a (Y21R and Y21) were less potent than intact factor exhibiting EC50 values of 10.9 and 25.1 microM, respectively. Histamine was released in a nonlytic manner and the mast cell stimulation by both natural and synthetic factors was sensitive to pertussis toxin, neuraminidase, benzalkonium chloride, and to an excess of calcium. C3a stimulated the generation of inositol polyphosphates that was inhibited by either pertussis toxin or benzalkonium chloride. The C3a anaphylatoxin also directly stimulates purified G proteins (i.e., GTPase activity) in a dose-dependent manner. The evident correlation between efficiency of C3a and C3a analogues to stimulate purified G proteins and their capacity to induce cellular histamine release led us to conclude that C3a fails to activate mast cells via a mechanism involving specific receptors on the cell. Instead, we propose that C3a either causes direct activation of G proteins of the Gi subtype, with a subsequent activation of phospholipase C, or interacts with a binding site of the cell surface specific for cationic molecules that is coupled to the G protein cascade.
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PMID:A mechanism of action for anaphylatoxin C3a stimulation of mast cells. 137 70

Cultured aortic fibroblasts express high affinity Et-1 binding sites that poorly discriminate between Et-1 and Et-3. Both endothelins activate phospholipase C hence indicating the presence of ETB receptors. Fibroblasts respond to bradykinin by large activations of phospholipase C and increases in [Ca2+]i in a manner that was abolished by D-Arg, [Hyp3,Thi5,8,D-Phe7]-bradykinin, thus indicating the presence of B2 kinin receptors. Finally, ATP, UTP and ADP increases [Ca2+]i in aortic fibroblasts via a nucleotide receptor that has a higher affinity for ATP and UTP (3 microM) than for ADP (50 microM) and that is distinct from P2x and P2y purinoceptors.
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PMID:High reactivity of aortic fibroblasts to vasoactive agents: endothelins, bradykinin and nucleotides. 141 43

1. In canine antrum, rhythmic electrical activity consists of a rapid upstroke phase followed by a plateau depolarization. In response to slow waves, cytosolic Ca2+ ([Ca2+]cyt) and tension increased. 2. Addition of sodium nitroprusside (SNP, 0.5 microM) decreased the amplitude of the plateau phase of slow waves without significant effects on the upstroke depolarization. SNP also inhibited changes in [Ca2+]cyt and tension associated with the plateau potential. SNP induced a negative chronotropic effect at concentrations above 0.1 microM. 3. Similar to the effects of SNP, illumination of muscles during slow waves with ultraviolet (UV) light caused premature repolarization. UV illumination is known to release NO in some tissues. 4. L-NG-monomethyl-arginine (L-NMMA, 300 microM), Methylene Blue (MB, 5 microM) and oxyhaemoglobin (oxy-Hb, 5 microM) increased the force of contractions. In contrast, L-arginine (L-Arg, 300 microM) decreased contractile force and antagonized the effects of L-NMMA. 5. During the upstroke phase, SNP caused a small reduction in [Ca2+]cyt and a large reduction in force, suggesting that SNP caused a decrease in Ca2+ sensitivity. 6. In muscles permeabilized by alpha-toxin, cyclic GMP (100 microM) and UV illumination inhibited Ca(2+)-induced contraction (at pCa 5.5). 7. These data suggest that NO or NO-related compounds are spontaneously released in gastric muscles. These agents have two effects on excitation-contraction coupling: (i) inhibition (directly and/or indirectly) of the voltage-dependent Ca2+ channels that participate in the plateau phase of slow waves, and (ii) reduction in the Ca2+ sensitivity of the contractile element.
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PMID:Spontaneous release of nitric oxide inhibits electrical, Ca2+ and mechanical transients in canine gastric smooth muscle. 150 Nov 33

The membrane-bound form of aminopeptidase P (aminoacylprolyl-peptide hydrolase) (EC 3.4.11.9) was purified to apparent homogeneity from bovine lung microsomes. The enzyme was solubilized using phosphatidylinositol-specific phospholipase C (Bacillus thuringiensis), indicating that bovine lung amino-peptidase P is attached to membranes via a glycosylphosphatidylinositol anchor. The enzyme was purified 1900-fold with a yield of 25% by chromatography on decyl-agarose, omega-aminodecyl-agarose, a second decylagarose column, DEAE-Sephacel, and an ultrafiltration step. Native gradient polyacrylamide gel electrophoresis revealed a single stained protein band whose position in the gel corresponded to cleavage of the Arg1-Pro2 bond of bradykinin. The Mr was 360,000 by gel permeation chromatography and 95,000 by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The substrate specificity of aminopeptidase P was determined using approximately 50 peptides with proline in the second position. The enzyme could hydrolyze lower NH2-terminal homologs of bradykinin, including Arg-Pro-Pro, which was used as the routine substrate in a rapid fluorescence assay performed in the absence of added Mn2+. Some peptides having NH2-terminal amino acids other than arginine were also cleaved. Aminopeptidase P appeared to favor peptides that had 2 proline residues or proline analogs in positions 2 and 3 of the substrate. In general, tripeptides having a single proline residue in position 2 were poor substrates. Aminopeptidase P was inhibited by a series of peptides, 3-8 residues long, having an NH2-terminal Pro-Pro sequence. The enzyme was also inhibited by metal-chelating agents, 2-mercaptoethanol (4 mM), p-chloromercuribenzenesulfonic acid, and NaCl at concentrations greater than or equal to 0.25 M. The purified enzyme had a pH optimum of 6.5-7.0 and was most stable in the basic pH range. A role for membrane-bound aminopeptidase P in the pulmonary inactivation of circulating bradykinin is proposed.
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PMID:Membrane-bound aminopeptidase P from bovine lung. Its purification, properties, and degradation of bradykinin. 153 67


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