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)

Activation of a variety of cell surface receptors results in the phospholipase C-catalyzed hydrolysis of the minor plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate, with concomitant formation of inositol 1,4,5-trisphosphate and diacylglycerol. There is strong evidence that inositol 1,4,5-trisphosphate stimulates Ca2+ release from intracellular stores. The Ca2+-releasing actions of inositol 1,4,5-trisphosphate are terminated by its metabolism through two distinct pathways. Inositol 1,4,5-trisphosphate is dephosphorylated by a 5-phosphatase to inositol 1,4-bisphosphate; alternatively, inositol 1,4,5-trisphosphate can also be phosphorylated to inositol 1,3,4,5-tetrakisphosphate by a 3-kinase. Although the mechanism of Ca2+ mobilization is understood, the precise mechanisms involved in Ca2+ entry are not known; the proposal that inositol 1,4,5-trisphosphate secondarily elicits Ca2+ entry by emptying an intracellular Ca2+ pool is considered.
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PMID:How do inositol phosphates regulate calcium signaling? 254 10

The kinetics of [3H]inositol phosphate metabolism in agonist-activated rat parotid acinar cells were characterized in order to determine the sources of [3H]inositol monophosphates and [3H]inositol bisphosphates. The turnover rates of D-myo-inositol 1,4,5-trisphosphate and its metabolites, D-myo-inositol 1,4-bisphosphate and D-myo-inositol 1,3,4-trisphosphate, were examined following the addition of the muscarinic receptor antagonist, atropine, to cholinergically stimulated parotid cells. D-myo-Inositol 1,4,5-trisphosphate declined with a t1/2 of 7.6 +/- 0.7 s, D-myo-inositol 1,3,4-trisphosphate declined with a t1/2 of 8.6 +/- 1.2 min, and D-myo-inositol 1,4-bisphosphate was metabolized with a t1/2 of 6.0 +/- 0.7 min. The sum of the rates of flux through D-myo-inositol 1,4-bisphosphate and D-myo-inositol 1,3,4-trisphosphate (2.54% phosphatidylinositol/min) did not exceed the calculated rate of breakdown of D-myo-inositol 1,4,5-trisphosphate (2.76% phosphatidylinositol/min). Thus, there is no evidence for the direct hydrolysis of phosphatidylinositol 4-phosphate in intact cells since D-myo-inositol 1,4-bisphosphate formation can be attributed to the dephosphorylation of D-myo-inositol 1,4,5-trisphosphate. The source of the [3H]inositol monophosphates also was examined in cholinergically stimulated parotid cells. When parotid cells were stimulated with methacholine, D-myo-inositol 1,4,5-trisphosphate, D-myo-inositol 1,3,4,5-tetrakisphosphate, D-myo-inositol 1,4-bisphosphate, and D-myo-inositol 4-monophosphate levels increased within 2 s, whereas D-myo-inositol 1-monophosphate accumulation was delayed by several seconds. Rates of [3H]inositol monophosphate accumulation also were examined by the addition of LiCl to cells stimulated to steady state levels of [3H]inositol phosphates. The sum of the rates of accumulation of D-myo-inositol 1-monophosphate and D-myo-inositol 4-monophosphate did not exceed the rate of breakdown of D-myo-inositol 1,4,5-trisphosphate or the sum of the rates of flux through D-myo-inositol 1,4-bisphosphate and D-myo-inositol 1,3,4-trisphosphate. These kinetic analyses suggest that agonist-stimulated [3H]inositol bis- and monophosphate formation in intact rat parotid acinar cells can be accounted for by the metabolism of D-myo-[3H]inositol 1,4,5-trisphosphate rather than by phospholipase C-catalyzed hydrolysis of phosphatidylinositol or phosphatidylinositol 4-phosphate.
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PMID:Source of 3H-labeled inositol bis- and monophosphates in agonist-activated rat parotid acinar cells. 254 8

Antigen-mediated exocytosis in intact rat basophilic leukemia (RBL-2H3) cells is associated with substantial hydrolysis of membrane inositol phospholipids and an elevation in concentration of cytosol Ca2+ ([ Ca2+i]). Paradoxically, these two responses are largely dependent on external Ca2+. We report here that cells labeled with myo-[3H]inositol and permeabilized with streptolysin O do release [3H]inositol 1,4,5-trisphosphate upon stimulation with antigen or guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) at low (less than 100 nM) concentrations of free Ca2+. The response, however, is amplified by increasing free Ca2+ to 1 microM. The subsequent conversion of the trisphosphate to inositol 1,3,4,5-tetrakisphosphate is enhanced also by the increase in free Ca2+. Although [3H]inositol 1,4,5-trisphosphate accumulates in greater amounts than is the case in intact cells, [3H]inositol 1,4-bisphosphate is still the major product in permeabilized cells even when the further metabolism of [3H]inositol 1,4,5-trisphosphate is suppressed (by 77%) by the addition of excess (1000 microM) unlabeled inositol 1,4,5-trisphosphate and the phosphatase inhibitor 2,3-bisphosphoglycerate. It would appear that either the activity of the membrane 5-phosphomonoesterase allows virtually instantaneous dephosphorylation of the inositol 1,4,5-trisphosphate under all conditions tested or both phosphatidylinositol 4-monophosphate and the 4,5-bisphosphate are substrates for the activated phospholipase C. The latter alternative is supported by the finding that permeabilized cells, which respond much more vigorously to high (supraoptimal) concentrations of antigen than do intact RBL-2H3 cells, produce substantial amounts of [3H]inositol 1,4-bisphosphate before any detectable increase in levels of [3H]inositol 1,4,5-trisphosphate.
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PMID:Receptor-mediated release of inositol 1,4,5-trisphosphate and inositol 1,4-bisphosphate in rat basophilic leukemia RBL-2H3 cells permeabilized with streptolysin O. 264 90

Rat hippocampal formation slices were prelabelled with [3H]inositol and stimulated with carbachol for times between 7 s and 3 min. The [3H]inositol metabolites in an acid extract of the slices were resolved with anion-exchange HPLC. Carbachol dramatically increased the concentration of [3H]inositol monophosphate, [3H]inositol bisphosphate (two isomers), [3H]inositol 1,3,4-trisphosphate, [3H]inositol 1,4,5-trisphosphate, and [3H]inositol 1,3,4,5-tetrakisphosphate. The levels of [3H]inositol 1,4,5-trisphosphate rose most rapidly; they were maximally elevated after only 7 s and declined toward control levels in 1 min followed by a more sustained elevation in levels for up to 3 min. When [3H]inositol 1,4,5-trisphosphate was incubated with hippocampal formation homogenates in an ATP-containing buffer it was very rapidly metabolised. After 5 min [3H]inositol 1,4-bisphosphate, [3H]inositol 1,3,4-trisphosphate, and [3H]inositol 1,3,4,5-tetrakisphosphate could be detected in the homogenates. Under similar experimental conditions [3H]inositol 1,3,4,5-tetrakisphosphate is metabolised to [3H]inositol 1,3,4-trisphosphate and an inositol bisphosphate isomer that is not [3H]inositol 1,4-bisphosphate. We conclude that like other tissues the primary event in the hippocampus following carbachol stimulation is the activation of phosphatidylinositol 4,5-bisphosphate selective phospholipase C.
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PMID:Formation of [3H]inositol metabolites in rat hippocampal formation slices prelabelled with [3H]inositol and stimulated with carbachol. 278 59

One of the earliest actions of thrombin in fibroblasts is stimulation of a phospholipase C (PLC) that hydrolyses phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. In membranes prepared from WI-38 human lung fibroblasts, thrombin activated an inositol-lipid-specific PLC that hydrolysed [32P]PIP2 and [32P]phosphatidylinositol 4-monophosphate (PIP) to [32P]IP3 and [32P]inositol 1,4-bisphosphate (IP2) respectively. Degradation of [32P]phosphatidylinositol was not detected. PLC activation by thrombin was dependent on GTP, and was completely inhibited by a 15-fold excess of the non-hydrolysable GDP analogue guanosine 5'-[beta-thio]diphosphate (GDP[S]). Neither ATP nor cytosol was required. Guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) also stimulated polyphosphoinositide hydrolysis, and this activation was inhibited by GDP[S]. Stimulation of PLC by either thrombin or p[NH]ppG was dependent on Ca2+. Activation by thrombin required Ca2+ concentrations between 1 and 100 nM, whereas stimulation of PLC activity by GTP required concentrations of Ca2+ above 100 nM. Thus the mitogen thrombin increased the sensitivity of PLC to concentrations of free Ca2+ similar to those found in quiescent fibroblasts. Under identical conditions, another mitogen, platelet-derived growth factor, did not stimulate polyphosphoinositide hydrolysis. It is concluded that an early post-receptor effect of thrombin is the activation of a Ca2+- and GTP-dependent membrane-associated PLC that specifically cleaves PIP2 and PIP. This result suggests that the cell-surface receptor for thrombin is coupled to a polyphosphoinositide-specific PLC by a GTP-binding protein that regulates PLC activity by increasing its sensitivity to Ca2+.
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PMID:Stimulation of polyphosphoinositide hydrolysis by thrombin in membranes from human fibroblasts. 282 18

The plasma-membrane receptors, coupling mechanisms, and effector enzymes that mediate target-cell activation by angiotensin II (AII) have been characterized in rat and bovine adrenal glomerulosa cells. The AII holoreceptor is a glycoprotein of Mr approximately 125,000 under non-denaturing conditions. Photoaffinity labeling of AII receptors with azido-AII derivatives has shown size heterogeneity among the AII binding sites between species and target tissues, with Mr values of 55,000 to 79,000. Such variations in molecular size probably reflect differences in carbohydrate content of the individual receptor sites. The adrenal AII receptor, like that in other tissues, is coupled to the inhibitory guanine nucleotide inhibitory protein (Ni). However, studies with pertussis toxin have shown that stimulation of aldosterone production by AII is not mediated by Ni but by a pertussis-insensitive nucleotide regulatory protein of unidentified nature. Although Ni is not involved in the stimulatory action of AII on steroidogenesis, it does mediate the inhibitory effects of high concentrations of AII upon aldosterone production. The actions of AII on adrenal cortical function are thus regulated by at least two guanine nucleotide regulatory proteins that are selectively activated by increasing AII concentrations. The principal effector enzyme in AII action is phospholipase C, which is rapidly stimulated in rat and bovine glomerulosa after AII receptor activation. AII-induced breakdown of phosphatidylinositol bisphosphate (PIP2) and phosphatidylinositol phosphate (PIP) leads to formation of inositol 1,4,5-trisphosphate (IP3) and inositol 1,4-bisphosphate (IP2). These are metabolized predominantly to inositol-4-monophosphate, which serves as a marker of polyphosphoinositide breakdown, whereas inositol-1-phosphate is largely derived from phosphatidylinositol hydrolysis. The AII-stimulated glomerulosa cell also produces inositol 1,3,4-trisphosphate, a biologically inactive IP3 isomer formed from Ins-1,4,5-trisphosphate via inositol tetrakisphosphate (IP4) during ligand activation in several calcium-dependent target cells. The Ins-1,4,5-P3 formed during AII action binds with high affinity to specific intracellular receptors that have been characterized in the bovine adrenal gland and other AII target tissues, and may represent the sites through which IP3 causes calcium mobilization during the initiation of cellular responses.
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PMID:Angiotensin II receptors and mechanisms of action in adrenal glomerulosa cells. 282 11

A soluble phospholipase C from rat liver was purified to homogeneity using phosphatidylinositol 4,5-bisphosphate (PIP2) as substrate. After ammonium sulfate fractionation, the purification involved chromatography on phosphocellulose, DEAE-Sepharose CL-6B, hydroxylapatite, Reactive Blue 2 dye-linked agarose, and Mono S cation exchanger. Under the conditions of the assay, the pure enzyme had a specific activity of 407 mumol/mg protein/min. It migrated as a single band with a molecular mass of 87 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The water-soluble product formed during the hydrolysis of PIP2 by the purified enzyme was inositol 1,4,5-trisphosphate. The enzyme shows one-half of maximum velocity at 2 microM Ca2+ with PIP2 as substrate. Between 0 and 100 microM Ca2+, the enzyme shows approximately the same activity with phosphatidylinositol 4-phosphate (PIP) as it does with PIP2, and very low activity with phosphatidylinositol. The enzyme is activated by low concentrations of basic proteins; for example, with PIP2 as substrate, 1 microgram/ml histone activates the enzyme 3.6-fold. The enzyme shows an almost absolute requirement for monovalent salts which can be met by different alkali metal halides. A second, minor peak of PIP2-hydrolyzing phospholipase C activity was resolved during chromatography of the enzyme on hydroxylapatite. The substrate specificity suggests that PIP and PIP2 are normal substrates of this enzyme. Under physiological conditions of activation, the enzyme may therefore generate inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate in amounts determined by the ratio of PIP and PIP2 present in the cellular membranes.
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PMID:Purification of a phospholipase C from rat liver cytosol that acts on phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate. 284 77

Exposure of rat brain or parotid gland slices to muscarinic receptor agonists stimulates a phospholipase C that degrades inositol phospholipids. When tissue slices were labelled in vitro with [3H]inositol, this response could be monitored by measuring the formation of [3H]inositol phosphates. Accumulation of inositol 1,4-biphosphate in stimulated brain slices suggests that polyphosphonositides are the primary targets for phospholipase C activity. Li+ (10 mM) in the medium completely blocked the hydrolysis of inositol 1-phosphate, partially inhibited inositol 1,4-bisphosphate hydrolysis, but had no effect on the hydrolysis of inositol 1,4,5-trisphosphate by endogenous phosphatases. Muscarinic receptor pharmacology was studied by measuring the accumulation of [3H]inositol 1-phosphate in the presence of 10 mM Li+. In experiments on brain slices, the response to carbachol was antagonised by atropine with an affinity constant of approximately 8.79 +/- 0.12. Dose-response curves to several muscarinic agonists were constructed using brain and parotid gland slices. The results are consistent with relatively direct coupling of low-affinity muscarinic receptors to inositol phospholipid breakdown in brain slices; full agonists were relatively more potent in the parotid gland compared with the brain. Explanations for these differences are suggested.
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PMID:Muscarinic receptors and hydrolysis of inositol phospholipids in rat cerebral cortex and parotid gland. 298 Dec 82

The phosphoinositides are metabolized by phospholipase C in response to hormone or agonist stimulation in many cell types to produce diglyceride and water-soluble inositol phosphates. We have recently shown that the phospholipase C reaction products include cyclic phosphate esters of inositol. One of these, inositol 1, 2-cyclic 4,5-trisphosphate, is active in promoting Ca2+ mobilization in platelets and in inducing changes in conductance in Limulus photoreceptors similar to those produced by light (Wilson, D. B., Connolly, T. M., Bross, T. E., Majerus, P. W., Sherman, W. R., Tyler, A., Rubin, L. J., and Brown, J. E. (1985) J. Biol. Chem. 260, 13496-13501. In the current study, we have examined the metabolism of the inositol phosphates. We find that both cyclic and non-cyclic inositol trisphosphates are metabolized by inositol 1,4,5-trisphosphate 5-phosphomonoesterase, to inositol 1,2-cyclic bisphosphate and inositol 1,4-bisphosphate, respectively. However, the apparent Km of the enzyme for the cyclic substrate is approximately 10-fold higher than for the non-cyclic substrate. These inositol bisphosphates are more slowly degraded to inositol 1,2-cyclic phosphate and inositol 1-phosphate, respectively. Inositol 1,2-cyclic phosphate is then hydrolyzed to inositol 1-phosphate, which in turn is degraded to inositol and inorganic phosphate by inositol 1-phosphate phosphatase. The human platelet inositol 1,2-cyclic phosphate hydrolase enzyme and a similar rat kidney hydrolase do not utilize the cyclic polyphosphate esters of inositol as substrates. These results suggest that the inositol cyclic phosphates and the non-cyclic inositol phosphates are metabolized separately by phosphatases to cyclic and non-cyclic inositol monophosphates. The cyclic monophosphate is then converted to inositol 1-phosphate by a cyclic hydrolase. We suggest that the enzymes that metabolize the inositol phosphates may serve to regulate cellular responses to these compounds.
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PMID:Isolation and characterization of the inositol cyclic phosphate products of phosphoinositide cleavage by phospholipase C. Metabolism in cell-free extracts. 300 Oct 44

Human platelet plasma membranes incubated in the presence of [gamma-32P]ATP and 15 mM MgCl2 incorporated radioactivity mostly into phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4-phosphate (PIP), which represented together over 90% of the total lipid radioactivity. After washing, reincubation of prelabelled membranes revealed some hydrolysis of the two compounds by phosphomonoesterase(s), as detected by the release of radioactive inorganic phosphate (Pi) from the two phospholipids. This degradation attained 40%/30 min for PIP in the presence of 2 mM calcium and cytosol. The effect of calcium was observed at concentrations equal to or greater than 10(-4) M. In no case did calcium alone facilitate the formation of inositol 1,4,5-trisphosphate (IP3) and inositol 1,4-bisphosphate (IP2). In contrast, simultaneous addition of 2 mM calcium and 2 mg/ml sodium deoxycholate promoted the formation of IP3 and IP2, indicating phosphodiesteratic cleavage of PIP2 and PIP. Phospholipase C activity was detected at calcium concentrations as low as 10(-7) M, in which case PIP2 hydrolysis was slightly more pronounced compared to PIP. Addition of cytosol increased to some extent the phospholipase C activity, suggesting that the low amount of enzyme remaining in the membrane is sufficient to promote submaximal degradation of PIP2 and PIP. We conclude that platelet polyphosphoinositides are present in the plasma membrane in a state where they remain inaccessible to phospholipase C, which is still fully active even at basal calcium concentrations, i.e., 10(-7) M. These results support the view that phosphodiesteratic cleavage of PIP2 promotes and thus precedes calcium mobilization brought about by IP3. The in vitro model presented here may prove very useful in future studies dealing with the mechanism rendering polyphosphoinositides accessible to phospholipase C attack upon agonist-receptor binding.
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PMID:Studies of endogenous polyphosphoinositide hydrolysis in human platelet membranes. Evidence that polyphosphoinositides remain inaccessible to phosphodiesterase in the native membrane. 300 80


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