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)

Sarcolemmal Ca++-ATPase, Mg++-ATPase, and (Na+-K+)-ATPase activities were increased in late stages of heart failure in myopathic hamsters (BIO 14.6) without any changes in the adenylate cyclase activity. On the other hand, these hamsters at early and moderate stages of heart failure showed depressions in mitochondrial calcium binding and uptake and microsomal calcium binding. Sarcolemmal (Na+-K+)-ATPase was decreased in failing hearts because of substrate lack, oxygen lack, and perfusion with Ca++-free, Na+-free, or K+-free medium. Both Mg++-ATPase and Ca++-ATPase activities of sarcolemma did not change on perfusing the hearts with substrate-free, hypoxic, Na+-free, or K+-free medium. Adenylate cyclase activity decreased on substrate-free or Ca++-free perfusion. Intracellular calcium overload produced by perfusing the hearts with medium containing calcium after Ca++-free perfusion was associated with decrease in all the sarcolemmal-bound enzyme activities. All types of failing hearts employed in this study showed a dramatic shift in the electrolyte composition. Failure of the cardiac muscle to generate contractile force on treatment with trypsin was associated with defects in the functions of sarcolemma, mitochondria, and sarcoplasmic reticulum, whereas such an effect on treatment with phospholipase C was limited to alterations in the activities of sarcolemma. The data suggest that abnormality at the level of sarcolemma plays an important role in the pathogenesis of heart dysfunction; however, the degree and direction of alterations in the sarcolemmal functions seem to be dependent upon the type of heart failure.
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PMID:Role of sarcolemmal changes in cardiac pathophysiology. 13 Jun 63

Commercially available preparations of phospholipase C from Clostridium perfringens are commonly contaminated with theta haemolysin, one of a group of bacterial haemolysins called oxygen labile (O-labile) haemolysins. Treatment of erythrocyte ghosts and a mixed lipid dispersion containing cholesterol with commercially available phospholipase C in the absence of Ca-2+ and the presence of phosphate buffer and/or EDTA resulted in the formation and release of ring or arc-shaped structures. Highly purified phospholipase C, free of theta-haemolysin, produced no changes in the morphology of erythrocyte ghosts or lipid dispersions in the presence of phosphate or EDTA, but caused the formation of typical diglyceride droplets in the presence of Ca-2+ in the absence of these inhibitors. Ring structures, identical to those caused by commercial phospholipase C, were formed on addition of highly purified theta-haemolysin to erythrocyte ghost membranes, lipid dispersions containing cholesterol and cholesterol dispersions, but not on treatment of membranes from Micrococcus lysodeikticus. Heat-inactivated O-haemolysin (60 degrees C for 10 min) produced no such effects. The dimensions of rings and arcs displayed heterogeneity. The outside diameters in various preparations varied from approx. 27-58 nm with border thickness of 4.1-7.8 nm.
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PMID:Interaction of Clostridium perfringens theta-haemolysin, a contaminant of commercial phospholipase C, with erythrocyte ghost membranes and lipid dispersions. A morphological study. 16 11

The disruption of the molecular organization of the plasma membrane of leukocytes by phagocytosable particles, or by agents such as surfactants, antibodies, phospholipase C, fatty acids and chemotactic factors, leads to a stimulation of the phagocyte oxidative metabolism. Concanavalin A (Con A) has been used as a tool to study the mechanism of this metabolic regulation. The binding of Con A to the surface of polymorphonuclear leukocytes (PMNL) or macrophages produces a rapid enhancement of oxygen uptake and glucose oxidation through the hexose monophosphate pathway (HMP). This is explained by an activation of the granular NADPH oxidase, the key enzyme in the metabolic stimulation. The effect of Con A is not due to endocytosed lectin, since Con A covalently coupled to large sepharose beads still acts as stimulant. The metabolic changes caused by Con A are reversible. If, after the onset of stimulation, sugars with high affinity for Con A are added to the leukocyte suspension, the activity of granular NADPH oxidase and the rate of respiration and glucose oxidation return to their resting values. The metabolic burst, while partially supressed by treatment of PMNL with iodoacetate, sodium flouride and cytochalasin B, is slightly increased by colchicine. Con A induces a selective release of granular enzymes (beta-glucuronidase, peroxidase, alkaline phosphatase) from PMNL, whereas no leakage of cytoplasmic enzymes is observed. The enzyme release is inhibited by iodoacetate and by drugs known to increase cell levels of cyclic AMP. Based on a current view of the mode of interaction between Con A and cell surfaces, a model of the metabolic disruption of leukocytes is presented.
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PMID:Concanavalin A as a probe for studying the mechanism of metabolic stimulation of leukocytes. 16 45

1. Phospholipase C (EC 3.1.4.3) from Clostridium novyi (oedematiens) type A was purified 2000-fold by (NH4)2SO4 precipitation, DEAE-Sephadex treatment in a batchwise system and Sephadex G-100 column chromatography. 2. The purified preparation had a specific activity of 95 mumol per min per mg protein toward phosphatidylcholine. This preparation was free from protease, lipase and oxygen-labile delta-hemolysin. 3. Phosphatidylcholine was hydrolyzed at the highest rate, while sphingomyelin and lysophosphatidylcholine were hydrolyzed at much lower rates. 4. Sodium deoxycholate and divalent cations such as Mg2+ and Ca2+ were extremely effective in stimulating phosphatidylcholine-hydrolyzing activity of this enzyme. 5. This enzyme hemolyzed horse red cells by hydrolyzing phosphatidylcholine, spingomyelin and phosphatidylethanolamine.
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PMID:Phospholipase C from Clostridium novyi type A. I. 24 23

Highly purified bovine rod outer segment membranes show loss of structural integrity under an air atmosphere. Obvious ultrastructural changes are preceded by increases in absorbance below 400 nm. These changes are inhibited by Ar or N2 atmospheres and appear to be due primarily to oxidative damage to the polyunsaturated fatty acids of the membrane lipids. Loss of polyunsaturated fatty acids, formation of malonaldehyde and fluorescent products characteristic of lipid oxidation accompany the spectral alterations. The elevated ultraviolet absorbance can largely be removed from the membranes by gentle extraction of the lipids using phospholipase C and hexane without changing the visible absorbance of rhodopsin. We have found a large seasonal variation in the endogenous level of alpha-tocopherol (vitamin E) in the bovine rod outer segment preparations. For much of the year we find that the rod outer segment membranes contain higher levels of alpha-tocopherol than have been previously reported in biological membranes. Rod outer segments which are low in endogenous tocopherol can be protected from oxygen damage by adding exogenous tocopherol. The rod outer segments are extremely susceptible to oxygen damage due to the unusually high content of polyunsaturated fatty acids in the membrane lipids. The presence of tocopherol inhibits oxygen damage but does not eliminate it. The tocopherol in the rod outer segments is consumed in air, thus complete protection from peroxidation in vitro requires an inert atmosphere as well as high levels of tocopherol. This work suggests that extensive precautions against oxidative degradation should also be employed in studies of other membrane systems where important deleterious effects of oxygen may be less obvious.
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PMID:Oxidative damage of retinal rod outer segment membranes and the role of vitamin E. 96 69

The NADPH oxidase is a multicomponent enzyme system that produces the reduced oxygen species essential for bacterial killing by polymorphonuclear leukocytes (PMN). Study of the oxidase has typically been carried out in cell-free systems in which Km values of 20-150 microM NADPH have been reported. However, when compared with affinities reported for other flavoprotein dehydrogenases and when considering the cellular concentration of NADPH/NADP+ of approximately 35 microM, the reported affinity of the oxidase for NADPH appears low. To investigate this apparent discrepancy we have studied the kinetics of NADPH oxidase activation in situ in human PMN permeabilized with Staphylococcus aureus alpha-toxin. alpha-Toxin permeabilization of human PMN did not initiate NADPH oxidase activation at physiologic concentrations of NADPH. If permeabilized cells were stimulated with 1 microM formyl-methionyl-leucyl-phenylalanine, 10 microM guanosine 5'-O-(3-thiotriphosphate), 0.5 mM Ca2+, 5 micrograms/ml cytochalasin B in the presence of varying concentrations of NADPH, we were able to demonstrate activation of the oxidase complex as shown by superoxide dismutase-inhibitable reduction of cytochrome c. In this system we determined that the Km for oxidase activation was 4-7 microM NADPH, a 4-10-fold decrease from reported values. The oxidase was the enzyme being studied as shown by the absence of enzymatic activity in patients with chronic granulomatous disease. In addition, if the enzyme was initially activated in permeabilized cells, the cells homogenized, and the Km for the oxidase determined in a cell-free system, the observed Km reverted to previously reported values (36 microM). These results indicate that NADPH oxidase, studied in situ, has a significantly higher substrate affinity than that observed in isolated membranes and, moreover, indicate that substrate affinity is optimal for catalysis at reported concentrations of cytosolic NADPH.
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PMID:Activation of NADPH oxidase in human neutrophils permeabilized with Staphylococcus aureus alpha-toxin. A lower Km when the enzyme is activated in situ. 130 41

A major function of human neutrophils (PMN) during inflammation is formation of oxygen radicals through activation of the respiratory burst enzyme, NADPH oxidase. Stimulus-induced production of both phosphatidic acid (PA) and diglyceride (DG) has been suggested to mediate oxidase activity; however, transductional mechanisms and cofactor requirements necessary for activation are poorly defined. We have utilized PMN permeabilized with Staphylococcus aureus alpha-toxin to elucidate the signal pathway involved in eliciting oxidase activity and to investigate whether PA or DG act as second messengers. PMN were permeabilized in cytoplasmic buffer supplemented with ATP and EGTA for 15 min before addition of NADPH and various cofactors. Oxidase activation was assessed by superoxide dismutase inhibitable reduction of ferricytochrome C; PA and DG levels were measured by radiolabeled product formation or by metabolite mass formation. Both superoxide (O2-) and PA formation were initiated by 10 microM GTP gamma S; addition of cytosolic levels of calcium ions (Ca2+, 120 nM) enhanced O2- and PA formation 1.5-2 fold. DG levels showed little change during these treatments. PA formation preceded O2- production and varying GTP gamma S levels had parallel effects on O2- and PA formation. However, while PA formation and oxidase activation occurred in tandem at Ca2+ levels of < 1 microM, higher calcium enhanced PA formation but inhibited O2- production. Removal of ATP completely blocked O2- production but had little effect on PA formation; in contrast, if ATP was replaced with ATP gamma S, parallel production of PA and O2- occurred in the absence of other cofactors. Finally, while inhibition of PA production by ethanol pretreatment led to inhibition of O2- formation in PMN treated with GTP gamma S alone, in cells stimulated with a combination of GTP gamma S and Ca2+, ethanol continued to inhibit PA formation but had no effect on O2- production. Our results do not support a role for DG in the signal transduction path leading to oxidase activation and, while we show a close correlation between oxidase activation and PA production under many physiologic conditions, we also demonstrate that PA is not sufficient to induce oxidase activation and O2- formation can occur when PA production is inhibited.
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PMID:Activation of NADPH oxidase and phospholipase D in permeabilized human neutrophils. Correlation between oxidase activation and phosphatidic acid production. 133 83

The functions of professional phagocytes depending on oxygen are briefly discussed. After appropriate stimulation, membrane-bound enzyme systems are activated--phospholipase C, protein-kinase C and NADPH-oxidase complex--and superoxide anion is produced. This process is called respiratory burst and is essential for killing of microorganisms but it may cause tissue damage and inflammation. The influence of superoxide anion on metabolism is reviewed. More attention is paid to modulating effects of superoxide anion in connection with the development inflammatory process.
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PMID:Reactive oxygen metabolites in inflammation. 133 88

Cytokines, in particular IL-1, released mainly by infiltrating macrophages, can be one of the key mediators of immune-induced beta-cell destruction in IDDM. IL-1 is able to induce suppression of insulin release and biosynthesis in cultured rat pancreatic islets. In addition, the cytokine shows clear cytotoxic effects leading to beta-cell death. The proposed mechanisms of action of IL-1 after binding to the beta-cell receptors are varied. Concerning the cytotoxic effects of the cytokine, the role of oxygen free radicals, mainly derived from arachidonate metabolism (see Fig. 1) is clear, and possibly potentiated by a cytosolic Na(+)-mediated alkalinization of the beta-cell exposed to the cytokine. In fact, an increased influx of Na+ may explain some of the cytotoxicity since it results in concomitant water uptake leading to swelling of the endoplasmic reticulum. NO formation also seems to be related to the cytokine-induced cytotoxicity since inhibition of the NO synthase abolishes the effects of the cytokine (see Fig. 1). In relation to the inhibitory effects of the cytokine on the beta-cell, different studies point toward almost all known second messenger systems already described for several hormones, such as cAMP formation, increased phospholipase C activity, changes in cytosolic Ca++, and altered gene transcription (see Fig. 1). Of particular interest is the protease activation associated with IL-1 (a serine protease) that seems to be clearly connected with the effects of the cytokine upon the beta-cell. In conclusion, the different studies devoted to the problem of IL-1 signal transduction on the beta-cell seem to indicate that the action of the cytokine on the pancreatic insulin-secreting cells is not associated with an individual second messenger system but rather seems to be related to a plurifactorial transduction system.
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PMID:Interleukin-1 and beta-cell function: more than one second messenger? 142 86

Microsomal phospholipids (PL) are a good target for the reactive intermediates produced by either the oxidative or the reductive biotransformation of CHCl3 (Testai et al. (1990), Toxicol. Appl. Pharmacol. 104, 496-503). In order to preliminarily characterize the different PL with CHCl3 reactive intermediates, two common methods of PL breakdown have been exploited: the acid-catalyzed transmethylation and the enzymatic hydrolysis with phospholipase C. The results indicated that radioactivity derived from the adducts of PL with the oxidation metabolite, phosgene, partitioned preferentially in the aqueous phase (the ratio of aqueous to organic phase radioactivity contents was about 10); the opposite occurred (ratio about 0.1) when the PL adducts were produced by the reductive process metabolites (dichloromethyl radicals). Therefore, the two methods of PL adduct breakdown can be used to detect and quantitate selectively the two reactive intermediates of CHCl3 biotransformation. The use of phospholipase C, which specifically cleaves the bond between the glyceryl-oxygen and the phosphor atom of PL also gave some structural information. Indeed, the radioactivity partitioning in the aqueous phase after enzymatic hydrolysis of CHCl3 oxidation-associated PL adducts, indicated the selective covalent binding of phosgene residues with the PL polar heads. The clear-cut different partition of radioactivity observed after hydrolysis of PL adducts with CHCl3 reduction intermediates, analogously indicated that dichloromethyl radicals were selectively bound to the PL fatty acyl chains. Using this method we could confirm that in in vitro experimental conditions resembling the physiological status of the liver, both metabolic pathways were concurrently active in hepatic microsomes of B6C3F1 mice. Extents of reactive metabolites similar to those found in B6C3F1 mouse liver microsomes, could be measured in Sprague-Dawley rat liver microsomes only after pretreatment of the animals with PB and incubation with higher CHCl3 concentrations. The toxicological implications of these findings are discussed.
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PMID:The regioselective binding of CHCl3 reactive intermediates to microsomal phospholipids. 149 11


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