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)

Protein composition of cardiac sarcolemmal membranes was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Membranes were observed to contain about 20 polypeptide bands ranging from 18000 to 200 000 dalton mass. Out of these, six bands were prominent and together comprised 57% of the membrane protein. When sarcolemmal membranes, phosphorylated by [gamma-(32)P] ATP in the presence of Ca(2+) or Na+ with and without K+, were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at pH 2.4, the band III region (Mr 105 000) of gels was found to contain active sites of monomeric Ca-ATPase and (Na,K)ATPase. Bands I (Mr greater than 200 000), II (Mr 150 000), III (Mr 105 000), and VI (Mr 47 000) were accesible to trypsin; the extent of proteolysis was dependent on the time of exposure to, and the concentration of, trypsin (i.e, ratio of sarcolemmal protein/trypsin). Addition of molar sucrose protected sarcolemmal proteins from the tryptic proteolysis. Calcium transport was reduced by the action of trypsin; the degree of reduction was influenced by the time of exposure of membranes to trypsin as well as the concentration of trypsin. (Mg,Ca)ATPase activity, on the other hand, was elevated moderately at lower concentration and reduced at higher concentration of trypsin. Treatment with phospholipase C cium transport and (Mg,Ca)ATPase activity; electrophoretic patterns were unaffected by this treatment. Addition of lecithin to phospholipase C treated membranes produced a moderate increase in calcium transport. Exposure to Triton X-100 (1%) specifically solubilized three protein bands (Mr90 000, 67 000, and 57 000), whereas exposure to deoxycholate (1%) preferentially solubilized high-molecular-weight proteins, including band III (Mr 105 000); Lubrol-PX (1%) caused nonspecific solubilization of proteins, although the extent of solubilization with Lubrol-PX was considerably less than with either Triton or deoxycholate.
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PMID:Protein analysis of cardiac sarcolemma: effects of membrane-perturbing agents on membrane proteins and calcium transport. 21 4

Nicotinic acetylcholine receptor protein (nAChR) has been solubilized from rat cerebral cortices by extracting a crude membrane fraction with the nonionic detergent Triton X-100 (polyoxyethylene-p-t-octylphenol). The solubilized nAChR was partially purified by affinity chromatography (Naja naja siamensis alpha-toxin affinity arm, linked to Sepharose 4B) and characterized by binding of 125I-labeled alpha-bungarotoxin. The reaction of labeled toxin and nAChR appears to be second order with a rate constant (k1) equal to 0.38 X 10(5) M-1 S-1 at 20 degrees. The toxin-nAChR complex dissociates with a dissociation rate constant (k-1) of 1.23 X 10(-5) S-1 at 20 degrees (t 1/2 = 15.6 h). The kinetically determined dissociation constant (Kd) for the complex is 3.24 X 10(-10) M. A variety of cholinergic ligands were studied for their ability to inhibit binding of labeled toxin. The results indicate that the brain receptor is indeed nicotinic. The s20, w and v of the toxin-nAChR complex in 0.1% Triton were determined by velocity sedimentation in D2O and H2O sucrose gradients. The values are 12.9 S and 0.80 cm3 g-1. The Stokes radius of the complex determined by gel filtration equals 7.5 nm. The Mr of the complex calculated from the hydrodynamic parameters, and corrected for bound detergent, equals 357,000.
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PMID:Nicotinic acetylcholine receptor from rat brain. Solubilization, partial purification, and characterization. 97 72

The control of cytoskeletal actin and exocytosis was examined in intact and digitonin-permeabilized chromaffin cells. Cytoskeletal actin was assayed by determining the actin content of Triton-insoluble cytoskeletons. The secretagogues nicotine, high K+ and Ba2+ resulted in a rapid reduction in the amount of actin associated with the cytoskeleton. The effect of nicotine but not high K+ on cytoskeletal actin was independent of external Ca2+ and the reduction in cytoskeletal actin was mimicked by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate suggesting a role for protein kinase C. In digitonin-permeabilized cells micromolar calcium produced both catecholamine secretion and a reduction in cytoskeletal actin. The reduction in cytoskeletal actin was transient. Secretion was enhanced by the GTP analogue guanosine 5'-(3-O-thio)triphosphate and the analogue also reduced cytoskeletal actin at low calcium levels. The effects of guanosine 5'-(3-O-thio)triphosphate were inhibited by the phospholipase C inhibitor neomycin and were mimicked by 12-O-tetradecanoylphorbol-13-acetate. An additional GTP analogue, guanyl-5'-yl imidodiphosphate, had no effect on cytoskeletal actin. These results provide further evidence for a requirement for reorganisation of cortical actin in the secretory processes and suggest that the reduction in actin associated with the cytoskeleton may be mediated by protein kinase C and/or calcium in intact and permeabilized chromaffin cells.
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PMID:The control of cytoskeletal actin and exocytosis in intact and permeabilized adrenal chromaffin cells: role of calcium and protein kinase C. 251 77

This study shows that the membrane-permeable stereospecific 1-oleoyl-2-acetyl-sn-glycerol (OAG), which is the analog of the natural 1,2-diacylglycerol (DAG), can stimulate the growth of ascites tumor cells. OAG can fully replace high serum concentrations in the culture medium and stimulates DNA synthesis in a dose-dependent manner. Investigation of the protein kinase C (PKC) isolated from a Triton extract of a 100,000g membrane pellet revealed that OAG can directly activate this enzyme. Concomitantly the phosphorylation of several cytosolic proteins with the molecular weights of 26, 33, 49, 55, 64, and 90 kDa is observed which is also found in serum-stimulated cells. Since DAG as a second messenger molecule originates from the hydrolysis of phosphoinositides we have investigated the metabolism of these lipids after labeling the cells with [3H]inositol. In detail, we have measured the amount of radioactive inositol trisphosphate (IP3) and the phosphodiesterase hydrolyzing phosphatidylinositol-4,5-bisphosphate (PIP2). The decreased radioactivity level of IP3 in OAG-stimulated cells as compared to non-growing cells (1-2% serum) indicates a feedback regulation of PIP2 hydrolysis which is substantiated by a profound reduction of PIP2-specific phospholipase C activity. The reduced IP3 formation has apparently no inhibitory effect on the cytoplasmic free Ca2+ concentration of OAG-stimulated cells, suggesting that the Ca2+ release is not directly correlated to the amount of IP3, which is also demonstrated for the non-growing cells. These data indicate that OAG apparently has a duel effect on the inositol phospholipid-mediated signal transfer system.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Differential effect on inositol-phospholipid hydrolysis, cytosolic-free Ca2+ concentration, protein kinase C activity and protein phosphorylation of 1-oleoyl-2-acetyl-sn-glycerol growth-stimulated ascites tumor cells. 284 1

Experimental modification of the membrane structure of rat liver microsomes affected the behavior of the 11-oxidase and 11-reductase components of 11 beta-hydroxysteroid dehydrogenase in different ways. 1) The latency of 11-oxidase was released by detergents, phospholipases, or elevated temperature; 11-reductase activity was not increased by these manipulations. 2) 11-Reductase was rapidly inactivated at 25 C and 37 C; 11-oxidase was stable at these temperatures. 3) Arrhenius plots of microsome bound 11-reductase between 5 C and 40 C showed discontinuity at 23 C. Activation energies above and below the critical temperature were 2 kcal and 16 kcal, respectively. Solubilized 11-reductase showed no discontinuity [activation energy (Ea) = 15 kcal]. Ea for 11-oxidase was 15 kcal at all temperatures for membrane bound or solubilized enzyme, with no discontinuities. 4) Phospholipases A2 and C rapidly inactivated 11-reductase. Triton DF-18 regenerated 50% of the reductase activity of phospholipase C-treated microsomes, but had no effect on phospholipase A2-treated microsomes. Phospholipases increased 11-oxidase activity. The independent behavior of corticosteroid 11-oxidase and 11-reductase are consistent with the properties of closely associated, independent enzymes.
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PMID:Evidence for independent 11-oxidase and 11-reductase activities of 11 beta-hydroxysteroid dehydrogenase: enzyme latency, phase transitions, and lipid requirements. 385 97

Permeability barriers must exist in transitional epithelium to prevent the free flow of water from underlying blood capillaries through the epithelium into the hypertonic urine, and such a barrier has now been demonstrated in isolated bladders. This barrier is passive in function and can be destroyed by damaging the luminal surface of the transitional epithelium with sodium hydroxide and 8 M urea solutions, by digesting it with trypsin, lecithinase C, and lecithinase D, or by treating it with lipid solvents such as Triton x 100 and saponin. From this it is concluded that the barrier depends on the integrity of lipoprotein cell membranes. The barrier function is also destroyed by sodium thioglycollate solutions, and electron microscope investigations show that sodium thioglycollate damages the thick asymmetric membrane which limits the luminal face of the superficial squamous cell. Cytochemical staining shows the epithelium to contain disulfide and thiol groups and to have a concentration of these groups at the luminal margin of the superficial cells. It thus appears that the permeability barrier also depends on the presence of disulfide bridges in the epithelium, and it is presumed that these links are located in keratin. Because of the effect of thioglycollates, both on the barrier function and on the morphology of the membrane, it is suggested that keratin may be incorporated in the thick barrier membrane. It is proposed that the cells lining the urinary bladder and ureters should be regarded as a keratinizing epitheluim.
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PMID:The permeability of rat transitional epithelium. Kertinization and the barrier to water. 590 98

A remarkable and immediate decrease in GDP-mannose:retinyl phosphate mannosyltransferase activity was found on pre-incubation of rat liver postnuclear membranes with phospholipase A2 or phospholipase C. Under the same conditions of pre-incubation (1 min at 37 degrees C) trypsin did not affect the enzyme activity, whereas pre-incubation for 30 min with trypsin and Pronase abolished enzyme activity. The lipid extract of untreated rat liver membranes partially restored enzyme activity after phospholipase treatment. Sphingomyelin was as active as the endogenous lipids. Other phospholipids were less active in the following order: phosphatidylcholine greater than phosphatidylethanolamine greater than phosphatidylinositol = phosphatidylserine. Dolichyl phosphate mannose synthesis was inhibited less (33%) by phospholipase C than was Ret-P-Man synthesis (98.5%) under identical conditions of incubation, which included 0.025% Triton. However, retinyl phosphate mannose synthesis by purified endoplasmic reticulum was found to be resistant to phospholipase C. Mixing experiments failed to demonstrate an inhibitory effect of the phospholipase-treated postnuclear membrane fraction on the synthetic activity of the endoplasmic reticulum, thus excluding the release of an inhibitory factor from the postnuclear membranes.
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PMID:Retinyl phosphate mannose synthesis in rat liver membranes. Phospholipase sensitivity and phospholipid requirement. 619 16

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

Two soluble forms of AChE from lymphocyte membrane have been obtained, the Triton solubilized Sd form and the high molar salt solubilized Ss form. They present similar Km (0.10 mM). Hydrodynamic properties of these forms have been studied on saccharose gradients with and without detergent or salt. A similar sedimentation coefficient has been found for these two forms (5.7 S). Lymphocyte plasma membrane AChE is a dimeric form (G2). Without detergent, the Sd form shows multiple secondary forms due to main form polymerization. Increase of NaCl concentration (2M) gives rise to a partial dissociation of these polymers. In the same conditions, the Ss form is not affected. The Ss form centrifugated on cesium chloride gradient has a higher density than the Sd form. These two forms have been treated by HPLC: the Stokes radii are respectively 7.1 nm for the Sd form and 4.5 nm for the Ss form. The molecular weights have been estimated at 175 000 for the Sd form and 105 000 for the Ss form. Pronase enzymatic digestion shows that the Ss form is more rapidly inactivated than the Sd form. Phospholipase C inhibits the Ss form and indicates that this form is a lipid-enzyme complex. The Sd form presents a different behaviour: this form is first activated, and afterwards inhibited by phospholipase C. This behaviour could be due to a more preponderant lipidic environment for the Sd form. The Sd form is probably a detergent-lipid-enzyme complex with an important hydrophobocity. These two forms can be explained by a different association between the enzyme and the phospholipids at the plasma membrane.
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PMID:[Properties and characterization of soluble forms of lymphocyte acetylcholinesterase from an ox]. 674 96

Genistein, a potent tyrosine kinase inhibitor, inhibits contraction of several types of smooth muscle, suggesting that protein tyrosine phosphorylation may be an important regulatory mechanism for smooth muscle contraction. We suspected that one site between activation of smooth muscle and contraction which might be modulated by protein tyrosine phosphorylation involved mechanisms for control of Ca2+ sensitivity. Since smooth muscle permeabilized with staphylococcal alpha-toxin permits direct assessment of agonist-induced Ca2+ sensitivity, we studied the effects of genistein on potential coupling between tyrosine phosphorylation and Ca2+ sensitivity in permeabilized ileal smooth muscle. Results show that contraction of intact preparations with carbachol is markedly and reversibly inhibited by 40% at 4 micrograms genistein/ml and by 60% at 20 micrograms genistein/ml. Permeabilized preparations that are contracted with a submaximal [Ca2+] in the presence of GTP relax when genistein is added to the medium. Genistein also reversibly inhibits contractions induced in permeabilized muscle with either a submaximal or maximal [Ca2+] in the presence of GTP, as well as receptor-coupled activation of Ca2+ sensitization with 10 microM carbachol/10 microM GTP. Activation of permeabilized preparations at pCa 4.6 in the presence of 100 microM GTP promotes time-dependent tyrosine phosphorylation of several substrates. Both phosphorylation and force are inhibited by genistein. However, relatively high levels of myosin light chain phosphorylation persist during genistein-induced inhibition of Ca2+ sensitivity. In contrast, genistein has no effect on Ca(2+)-activated contraction in Triton-skinned preparations in either the presence or the absence of GTP. This shows that it does not directly inhibit actin-myosin interaction and suggests that its target(s) may be a cytosolic or membrane-bound regulatory protein(s) that is leached from the preparations during Triton-skinning. Taken together, these new data suggest that (a) tyrosine phosphorylation of one or more substrates may be coupled to mechanisms which regulate Ca2+ sensitivity and (b) the inhibitory effects of genistein are probably due to inhibition of agonist-induced Ca2+ sensitivity.
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PMID:Modulation of Ca2+ sensitivity in smooth muscle by genistein and protein tyrosine phosphorylation. 762 29


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