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 determination of the coenzymes NAD+, NADH, NADP+ and NADPH, by the use of a method of enzymatic cycling, demonstrates that the enzymes responsible for the stimulations found during the phagocytosis of Staphylococcus albus are NADH and NADPH oxidase of human leukocytes and NADPH oxidase in the case of guinea pig leukocytes. The effects of serum, of the bacterial strain used and of phospholipase C are also discussed.
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PMID:The energy metabolism of the leukocyte. IX. Changes in the concentration of the coenzymes NAD, NADH, NADP, and NADPH in polymorphonuclear leukocytes during phagocytosis of Staphylococcus albus and due to the action of phospholipase C. 1 47

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

By the binding of activators (f.e. complement factor C5a, leukotriene B4) to receptors of neutrophil granulocytes an activation of a GTP-binding protein is induced, that increases the activity of the phospholipase C in the membrane and thereby the liberation of inositol-1,4,5-phosphate (IP) and of diacylglycerol (DG). The IP effects the liberation of Ca from intracellular stores and an increase in the concentration of Ca-ions in the cytosol. The DG induces an activation of the phosphokinase C near the membrane, that phosphorylates a cytosolic protein, which takes part in the formation of the NADPH-oxidase. The mentioned reactions induce in the membrane the construction of the NADPH-oxidase from a NADPH-binding protein, a flavoprotein, a cytochrome b 558 and from 4 cytosolic proteins. When phagolysosomes are formed, the NADPH-oxidase is part of the membrane, so bactericidal superoxide-anions can be introduced in these. In the chronic granulomatous disease defects in the formation of cytochrome b or of 2 cytosolic proteins exist.
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PMID:[Current knowledge of the mechanism of neutrophil granulocyte and macrophage activation for the formation of superoxide anions and of H2O2 and for the causes of defects in the NADPH-oxidase enzyme system involved]. 164 88

Chronic granulomatous diseases (CGDs) are characterized by recurrent infections resulting from impaired superoxide production by a phagocytic cell, nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) oxidase. Complementary DNAs were cloned that encode the 67-kilodalton (kD) cytosolic oxidase factor (p67), which is deficient in 5% of CGD patients. Recombinant p67 (r-p67) partially restored NADPH oxidase activity to p67-deficient neutrophil cytosol from these patients. The p67 cDNA encodes a 526-amino acid protein with acidic middle and carboxyl-terminal domains that are similar to a sequence motif found in the noncatalytic domain of src-related tyrosine kinases. This motif was recently noted in phospholipase C-gamma, nonerythroid alpha-spectrin (fodrin), p21ras-guanosine triphophatase-activating protein (GAP), myosin-1 isoforms, yeast proteins cdc-25 and fus-1, and the 47-kD phagocyte oxidase factor (p47), which suggests the possibility of common regulatory features.
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PMID:Cloning of a 67-kD neutrophil oxidase factor with similarity to a noncatalytic region of p60c-src. 169 59

The phagocytosis of beta-glucan particles by human neutrophils and the associated activation of NADPH O2- forming oxidase were accompanied by an increased hydrolysis of phosphoinositides by phospholipase C, hydrolysis of phosphatidylcholine by phospholipase D, accumulation of diglyceride (DG) mass, and [Ca2+]i rise. The reaction of phospholipid hydrolysis played a minor role in the formation of DG, which was mainly formed by de novo synthesis from glucose. The activation of this pathway was shown by the stimulation of the incorporation of [U-14C]glucose into DG, which occurred very rapidly after the challenge of neutrophils with beta-glucan particles. This DG derived from glucose was found almost completely as 1-acyl-2-acyl-glycerol (DAG). On the basis of the finding that phosphatidic acid was the precursor of DAG, an increase in the incorporation of [U-14C]acetate into DAG did not occur, and the [14C]radioactivity was in the glycerol backbone, the synthesis of DAG from [U-14C]glucose occurred very likely via dihydroxyacetone phosphate and glycerol 3-phosphate, stepwise acylation to phosphatidic acid, and dephosphorylation by phosphatidate phosphatase.
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PMID:De novo synthesis of diacylglycerol from glucose. A new pathway of signal transduction in human neutrophils stimulated during phagocytosis of beta-glucan particles. 185 Jul 33

Neutrophils pretreated with phorbol 12-myristate 13-acetate (1-10 nM) and stimulated with low concentrations of chemotactic agonists (1-10nM) exhibited a marked increase in respiratory burst activity that was characterized by regular oscillations. These were accompanied by parallel oscillations in turbidity having the same phase and period. Four different agonists, f-Met-Leu-Phe, complement fragment C5a, platelet-activating factor, and leukotriene B4, induced virtually identical oscillations, with mean periods of 7.9 +/- 0.6 s (respiratory burst) and 7.9 +/- 0.8 s (turbidity) at 37 degrees C. No burst oscillations were observed at high agonist concentrations (50-100 nM) unless the fungal metabolite 17-hydroxywortmannin was added prior to stimulation. In the absence of phorbol 12-myristate 13-acetate, the respiratory burst activity was inhibited by 17-hydroxywortmannin, the protein kinase C inhibitor staurosporine, and calcium depletion, while agonist-dependent turbidity changes including the oscillations were unaffected. Turbidity changes reflect corresponding changes in cell size and/or shape, suggesting that cyclic alterations in morphology such as lamellipod extension and retraction physically affect the catalytic efficiency of the membrane-bound burst enzyme NADPH-oxidase. The oscillations appear to be controlled via receptor-dependent activation mechanisms which do not involve PKC activation or the rise in internal calcium presumably derived from phospholipase C activation.
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PMID:Respiratory burst oscillations in human neutrophils and their correlation with fluctuations in apparent cell shape. 277 66

The effects of 17-hydroxywortmannin (HWT), a powerful inhibitor of the respiratory burst associated with phagocytosis (Baggiolini, M., Dewald, B., Schnyder, J., Ruch, W., Cooper, P. H., and Payne, T. G. (1987) Exp. Cell Res. 169, 408-418), were studied in human neutrophils stimulated with chemotactic agonists or phorbol myristate acetate. At nanomolar concentrations HWT inhibited superoxide production and the release of granule contents induced by N-formyl-Met-Leu-Phe, C5a, platelet-activating factor, and leukotriene B4, but not by phorbol myristate acetate, indicating that it interferes with receptor-mediated activation of the neutrophils, without directly affecting protein kinase C (Ca2+/phospholipid-dependent enzyme), the NADPH-oxidase, or the process of granule exocytosis. Moreover, HWT did not influence agonist-induced [Ca2+]i changes, indicating that it does not interfere with the function of agonist receptors, G-proteins or the phosphatidylinositol-specific phospholipase C. By studying the effect of HWT on the respiratory burst elicited in normal and Ca2+-depleted cells by combined stimulation with N-formyl-Met-Leu-Phe and phorbol myristate acetate, evidence was obtained that two transduction sequences, both of which are G-protein-dependent, are necessary for the induction of the response by receptor agonists. One sequence is Ca2+-dependent, HWT-insensitive, and leads to activation of protein kinase C, the other is Ca2+-independent and HWT-sensitive. Ca2+ depletion, which blocks the first, and HWT, which blocks the second, can be used to show that both processes must be functional for the transduction of agonist signals into a respiratory burst response.
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PMID:Two transduction sequences are necessary for neutrophil activation by receptor agonists. 284 36

The stimulation of the human neutrophil NADPH-oxidase is initiated by a variety of agonists, which appear to utilize more than one activation pathway. We have discerned that opsonized zymosan (OZ) stimulates O2- release by a mechanism distinct from that of phorbol myristate acetate (PMA). PMA differs from OZ stimulation in its susceptibility to H-7 (a protein kinase inhibitor) inhibition of O2- release and the lack of PMA-initiated release of radiolabeled arachidonic acid ([3H]AA) from prelabeled cells. That AA release was linked to O2- generation in OZ-stimulated cells was suggested by the finding that mepacrine, a phospholipase inhibitor, exhibits parallel dose response inhibition for both O2- generation and [3H]AA release, whereas mepacrine did not significantly inhibit the O2- generation induced by PMA. The specific involvement of phospholipase A2 (PLA2) in the release of AA was indicated by the lack of release of [3H]oleate, which is not released by PLA2 in intact cells; [3H]AA released from phosphatidylinositol and phosphatidylcholine and not accompanied by the formation of [3H]-arachidonyl phosphatidic acid, thus eliminating the involvement of phospholipase C; and the inhibition of [3H]AA release by p-bromophenacyl bromide, a specific PLA2 inhibitor. The reduction of O2- formation by inhibitors of AA metabolism (BW755C, acetylsalicylic acid, and indomethacin) further supports a linkage between AA release and O2- generation. That [3H]AA release, like O2- generation, in OZ-stimulated cells was calcium dependent further differentiates OZ from calcium-independent PMA activation. These studies in toto suggest that OZ stimulation of the NADPH-oxidase differs from PMA, in that the particulate stimulus is PLA2 mediated and independent of protein kinase C.
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PMID:Identification of distinct activation pathways of the human neutrophil NADPH-oxidase. 302 Jan 28


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