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)

IL-8 is a neutrophil-specific chemoattractant and cellular activator which exists in at least three forms, 69, 72, and 77 amino acids. The predominant monocyte product has 72 amino acids, whereas endothelial cells secrete the 77-amino acid form. The 72-amino acid form has been shown to increase intracellular calcium in neutrophils, but the exact biochemical pathways involved in stimulation of these cells is unknown. N-formyl peptide chemoattractants in neutrophils stimulate the formation of phosphatidylinositol-4,5-bisphosphate (PIP2), a reservoir for second messenger molecules and regulator of actin assembly through its association with the actin-binding proteins, profilin, and gelsolin. The present study examined whether IL-8 altered the enzyme which synthesizes PIP2, phosphatidylinositol-4-phosphate (PIP) kinase. Incubation of intact neutrophils with 10 nM IL-8 caused approximately a twofold increase in the activity of the enzyme. All forms of IL-8 stimulated PIP kinase activity in concentrations ranging from 1 to 50 nM, and the dose-response curves exactly correlated with the order of potency of these cytokines for interacting with the IL-8R on the surface of neutrophils. Lineweaver-Burk analysis of the kinetics of PIP kinase assayed in the presence of 0.03 to 0.7 mM ATP showed that 10 nM IL-8 increased the Vmax of the enzyme 38 to 70.5%, with no significant change in the apparent Km for ATP or for PIP. The stimulation of PIP kinase activity could not be explained by decreased degradation of PIP2 by phospholipase C or phosphomonoesterase activity in the membranes isolated from cells treated with IL-8 or by a decrease in the degradation of ATP. The microfilament disrupter, cytochalasin b, inhibited IL-8 induced stimulation of PIP kinase. These findings demonstrate that all forms of IL-8 stimulate PIP kinase in human neutrophils. This event may provide molecular signals to these cells that are necessary to maintain or change the state of microfilament assembly during cellular activation.
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PMID:IL-8 stimulates phosphatidylinositol-4-phosphate kinase in human polymorphonuclear leukocytes. 131 31

We have developed an experimental model to study in vivo inositol lipid metabolism in frog retinal pigment epithelial (RPE) cells, including the effect of light on phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate. RPE cells were rapidly isolated after either brief light or dark periods. Light and electron microscopy showed complete detachment of the retina from the RPE cells, and that the RPE cell suspensions were devoid of photoreceptor cell outer segments. Frog tissues were labeled in vivo for 20 hr by intravitreal injection of [3H]inositol (4 microCi, 4 microliters per eye) within a 24-hr constant illumination period. Following 1 hr of darkness (priming period), frogs were intravitreally injected with LiCl (0.5 M, 4 microliters per eye) 15 min before the onset of either 30-min light stimulation or an additional 30 min of darkness (controls). In order to preserve endogenous inositol phosphate pools present after dark and light exposure, the RPE cells were harvested in the shortest time possible, at low temperatures (18-20 degrees C), and in the presence of 10 mM LiCl. Total [3H]inositol-labeled water-soluble products (inositol plus inositol phosphates) were increased by 86% after 30 min of light. Inositol trisphosphate (IP3) showed the highest accumulation (a 5.5-fold increase), followed by inositol bisphosphate (1.9-fold increase) and inositol monophosphate (1.4-fold increase). Free [3H]inositol also accumulated (2.8-fold increase), reflecting only a partial inhibition of phosphomonoesterase by LiCl. These changes were paralleled by a 12% decrease in 3H-labeled phosphatidylinositol with no significant difference in the labeling of polyphosphoinositides.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Light stimulates in vivo inositol lipid turnover in frog retinal pigment epithelial cells at the onset of shedding and phagocytosis of photoreceptor membranes. 147 81

Alkaline phosphatase was the first zinc enzyme to be discovered in which three closely spaced metal ions (two Zn ions and one Mg ion) are present at the active center. Zn ions at all three sites also produce a maximally active enzyme. These metal ions have center-to-center distances of 3.9 A (Zn1-Zn2), 4.9 A (Zn2-Mg3), and 7.1 A (Zn1-Mg3). Despite the close packing of these metal centers, only one bridging ligand, the carboxyl of Asp51, bridges Zn2 and Mg3. A crystal structure at 2.0-A resolution of the noncovalent phosphate complex, E.P, formed with the active center shows that two phosphate oxygens form a phosphate bridge between Zn1 and Zn2, while the two other phosphate oxygens form hydrogen bonds with the guanidium group of Arg166. This places Ser102, the residue known to be phosphorylated during phosphate hydrolysis, in the required apical position to initiate a nucleophilic attack on the phosphorous. Extrapolation of the E.P structure to the enzyme-substrate complex, E.ROPO4(2-), leads to the conclusion that Zn1 must coordinate the ester oxygen, thus activating the leaving group in the phosphorylation of Ser102. Likewise, Zn2 appears to coordinate the ester oxygen of the seryl phosphate and activate the leaving group during the hydrolysis of the phosphoseryl intermediate. Both of these findings suggest that there may be a significant dissociative character to each of the two displacements at phosphorous catalyzed by alkaline phosphatase. A water molecule (or hydroxide) coordinated to Zn1 following formation of the phosphoseryl intermediate appears to be the nucleophile in the second step of the mechanism. Dissociation of the product phosphate from the E.P intermediate is the slowest, 35 s-1, and therefore the rate-limiting, step of the mechanism at alkaline pH. Since the determination of the initial crystal structure of alkaline phosphatase, two other crystal structures of enzymes involved in phosphate ester hydrolysis have been completed that show a triad of closely spaced zinc ions present at their active centers. These enzymes are phospholipase C from Bacillus cereus (structure at 1.5-A resolution) (43) and P1 nuclease from Penicillium citrinum (structure at 2.8-A resolution) (74). Both enzymes hydrolyze phosphodiesters. Substrates for phospholipase C are phosphatidylinositol and phosphatidylcholine, while P1 nuclease is an endonuclease hydrolyzing single stranded ribo- and deoxyribonucleotides. P1 nuclease also has activity as a phosphomonoesterase against 3'-terminal phosphates of nucleotides. The Zn ions in both enzymes form almost identical trinuclear sites.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Structure and mechanism of alkaline phosphatase. 152 73

1. Phosphatidylinositol 4-phosphate (PtdIns4P) is degraded by isolated membranes from Xenopus laevis oocytes. 2. Incubation of [4-32P]PtdIns4P with membranes yields only radioactive inorganic phosphate, indicating the presence of a phosphomonoesterase. 3. Membranes hydrolyze Ptd[2-3H]Ins4P to produce mainly Ptd[2-3H]Ins in the lipid phase. In this incubation [3H]inositol and inositol monophosphate appear in the water phase. 4. Membrane incubations of Ptd[2-3H]Ins4P carried out in the presence of excess non-radioactive Ins(1,4)P2 allows the trapping of small amounts of [3H]Ins(1,4)P2. These results demonstrate the presence of a phospholipase C. 5. Testing several phosphorylated analogs, it is determined that fructose 1,6-bisphosphate and alpha-glycerophosphate are potent inhibitors of the oocyte PtdIns4P phosphomonoesterase.
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PMID:The hydrolysis of phosphatidylinositol 4-phosphate in membranes of Xenopus laevis oocytes: characteristics of a phosphomonoesterase. 166 8

Doxorubicin, when incubated for 30 minutes with [32P]-labelled human erythrocyte membrane vesicles, produced an elevation of [32P]inositol-1,4,5-trisphosphate levels. The maximum rise was obtained with 10(-8) mol/l doxorubicin [132 (S.E. 13%) of control, n = 6, P = 0.001]. However, when the inositol lipids were examined, there was no evidence that doxorubicin stimulated the breakdown of [32P]phosphatidylinositol-4,5-bisphosphate under resting conditions, suggesting that the elevated levels of [32P]inositol 1,4,5-trisphosphate were not the result of the stimulation of phospholipase C. Instead, it was found that the dephosphorylation of inositol 1,4,5-trisphosphate by a 5'-phosphomonoesterase was partially inhibited by 10(-8) mol/l doxorubicin so that the rise in [32P]inositol 1,4,5-trisphosphate resulted from the inhibition of the breakdown of constitutively released [32P] inositol 1,4,5-trisphosphate. Similar data was also obtained with another aminoglycoside antibiotic, neomycin. The release of [32P] inositol 1,4-bisphosphate and [32P] inositol 1,4,5-trisphosphate and the breakdown of the inositol lipids in response to calcium (2.5 x 10(-4) and 10(-3) mol/l) stimulation was enhanced by doxorubicin (10(-6) to 10(-12) mol/l). These effects on resting and stimulated inositol lipid metabolism are discussed with reference to the paradoxical effects of doxorubicin to both stimulate and inhibit proliferation, according to concentration.
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PMID:Doxorubicin interactions at the membrane: evidence for a biphasic modulation of inositol lipid metabolism. 183 96

The effect of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) on PtdIns and PtdIns(4)P kinase activities was measured in rat liver plasma membranes. The addition of [32P]ATP resulted in the rapid incorporation of 32P into PtdIns(4)P and PtdIns(4,5)P2, with maximal levels reached within 30 s. GTP[S] (25-500 microM) increased the rate and magnitude of [32P]PtdIns(4)P and [32P]PtdIns(4,5)P2 formation by 50 and 120% respectively. Similar stimulatory effects were induced by guanosine 5'-[beta gamma-imido]triphosphate, GTP, GDP and guanosine 5'-[beta-thio]diphosphate. The stimulation of PtdIns phosphorylation by GTP[S] occurred in the presence of 2 mM-EGTA, a condition which fully inhibited phosphoinositide-specific phospholipase C. GTP[S] did not stimulate phosphomonoesterase activity, and its action was not due to the binding of magnesium. However, the overall ATP-hydrolysing activity of the membrane preparation was inhibited by GTP[S] and the other guanine nucleotides. There was a direct correlation between the extent of this inhibition and the stimulation of polyphosphoinositide formation. The results indicate that stimulation of polyphosphoinositide formation by guanine nucleotides in rat liver plasma membranes can be accounted for by an inhibition of ATP hydrolysis. These data are inconsistent with a specific GTP-binding protein (G-protein)-mediated stimulation of PtdIns or PtdIns(4)P kinase.
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PMID:Effect of guanine nucleotides on polyphosphoinositide synthesis in rat liver plasma membranes. 217 1

The involvement of inositol lipid metabolism in agonist-mediated Ca2+ signaling by Ins 1,4,5-P3 has become firmly established. Recent advances have led to a better understanding of the proteins associated with signal transduction in the plasma membrane. A number of specific receptors (G proteins, phospholipases and inositol lipid kinases) have now been purified and characterized. An Ins 1,4,5-P3 receptor has also been purified which is presumably involved in mediating Ca2+ efflux from intracellular stores. The morphological site of the hormone-sensitive Ca2+ pool has been tentatively identified as discrete, specialized intracellular structures (calciosomes), but further studies are required to demonstrate that these contain Ins 1,4,5-P3-gated Ca2+ channels and their possible functional relationship to the plasma membrane. Receptor occupancy by Ca2+ mobilizing agonists also stimulates Ca2+ entry into the cell, but the mechanism for activation of voltage insensitive Ca2+ channels and the possible involvement of Ins 1,4,5-P3, Ins 1,3,4,5-P4 and/or G proteins in this process has not been established. The Ca2+ signaling pathway is subject to multisite feedback regulation by Ca2+ itself and by a diacylglycerol-mediated activation of protein kinase C. Potential sites for Ca2+ interaction are displacement of Ins 1,4,5-P3 from its receptor by a Ca2+-dependent mechanism, promotion of Ins 1,3,4,5-P4 formation by the Ca2+/calmodulin-regulated Ins 1,4,5-P3 3-kinase, and efflux of Ca2+ from the cell or sequestration into intracellular Ca2+ stores by Ca2+/calmodulin-regulated Ca2+-ATPases. Protein kinase C activation potentially affects the rate of generation of Ins 1,4,5-P3 by negative feedback to the receptor-G protein-phospholipase C transduction system and possibly also the rate of Ins 1,4,5-P3 degradation by activation of an inositol polyphosphate 5-phosphomonoesterase. It may also attenuate the Ca2+ transient directly by increasing the activity of Ca2+-ATPases associated with the plasma membrane and the endoplasmic reticulum. Cell-to-cell heterogeneity in the relative control strengths of these different mechanisms may explain the differences in the Ca2+ signal in different tissues and even in different cells within a population. The ability of Ca2+ and protein kinase C to provide negative feedback at various points in the signal transduction pathway suggests that a complex mechanism involving multiple feedback loops is likely to regulate the generation of Ca2+ oscillations seen in some cells.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Hormone effects on cellular Ca2+ fluxes. 249 41

Incorporation of 32P from [gamma-32P]ATP into phosphatidylinositol 4,5-bisphosphate (PIP2) in membranes isolated from rat brain was enhanced in a concentration-dependent manner by the GTP analogue guanosine 5'-O-(thio)triphosphate (GTP gamma S). In contrast, neither the labeling of phosphatidylinositol 4-phosphate in the same membranes nor PIP kinase activity in the soluble fraction were stimulated by GTP gamma S. Synthesis of [32P]PIP2 was not stimulated by GTP, GDP, GMP, or ATP; however, the stimulatory effects of GTP gamma S were antagonized by GTP, GDP, and guanosine 5'-O-thiodiphosphate (GDP beta S). The nucleotide-stimulated labeling of PIP2 was not due to protection of [gamma-32P] ATP from hydrolysis, activation of PIP2 hydrolysis by phospholipase C, or inhibition of PIP2 hydrolysis by its phosphomonoesterase. Therefore, phosphatidylinositol 4-phosphate kinase activity in brain membranes may be regulated by a guanine nucleotide regulatory protein. This system may enhance the resynthesis of PIP2 following receptor-mediated activation of phospholipase C.
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PMID:Regulation of brain phosphatidylinositol-4-phosphate kinase by GTP analogues. A potential role for guanine nucleotide regulatory proteins. 253 38

The breakdown of exogenously added [3H]inositol-labeled phosphoinositides by rat brain cortical membranes was stimulated by the muscarinic cholinergic agonist carbachol. The stimulation required the presence of guanine nucleotide. Optimal conditions were similar to those described for guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) + carbachol stimulation of phosphoinositide breakdown in [3H]inositol-prelabeled brain membranes (Claro, E., Garcia, A., and Picatoste, F. (1989) Biochem J. 261, 29-35). Carbachol stimulated [3H]phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown was inhibited by atropine and guanosine 5'-O-(2-thiobisphosphate). The magnitude of the stimulation of exogenous PIP2 breakdown by carbachol and GTP gamma S (2- to 3-fold) was little affected over a PIP2 concentration range of 0.03-100 microM. Phosphatidylinositol 4-phosphate (PIP) was as good a substrate at all concentrations as PIP2 for carbachol stimulation of phospholipase C activity. There was appreciable phosphomonoesterase degradation of PIP to phosphatidylinositol (PI) over 10 min. There was also some conversion of added PIP to PIP2 in the presence of added ATP. The effect of calcium on PIP breakdown was similar to that on PIP2 breakdown, with an apparent EC50 for Ca2+ stimulation of 0.74 and 0.72 microM, respectively, under basal conditions. The stimulation of PIP2 and PIP breakdown by carbachol in the presence of GTP gamma S was greatest on a percentage basis at the lowest free Ca2+ concentrations. Above 1 microM free Ca2+, the stimulatory effect was lost, whereas 10 microM free Ca2+ gave a maximal stimulation of basal phospholipase C activity. Degradation of added PI was also stimulated by carbachol in the absence of ATP. PI breakdown had an EC50 for Ca2+ stimulation of 1.07 microM. The best stimulation of PI breakdown due to carbachol plus GTP gamma S was seen with 0.3 microM free Ca2+ and 100 microM PI. Maximal activation of PI breakdown was seen at 1 mM deoxycholate as was true for PIP2 and PIP breakdown. There was little effect, even of 30 microM GTP gamma S alone or of carbachol alone, on PI breakdown. Half-maximal activation of the carbachol response required only 0.2 microM GTP gamma S. These results indicate that the phospholipase C enzyme(s) activated by carbachol in the presence of GTP gamma S in rat brain cortical membranes can degrade PIP2, PIP, and PI to inositol phosphates and diacylglycerol.
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PMID:Carbachol in the presence of guanosine 5'-O-(3-thiotriphosphate) stimulates the breakdown of exogenous phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol 4-phosphate, and phosphatidylinositol by rat brain membranes. 255 3

Phosphatidylinositol 3-phosphate (PtdIns(3)P), a recently described phospholipid, has been linked to polyoma virus-induced cellular transformation and platelet-derived growth factor-mediated mitogenesis. PtdIns(3)P, in contrast to phosphatidylinositol, phosphatidylinositol 4-phosphate (PtdIns(4)P), and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), is resistant to hydrolysis by bovine brain phospholipase C gamma. We present here the identification of a phosphomonoesterase activity from the soluble fraction of NIH 3T3 cells which removes the phosphate from the D-3 position of PtdIns(3)P. This enzyme is specific as it has little or no activity on the monoester phosphates of PtdIns(4)P, PtdIns(4,5)P2, or inositol 1,3-bisphosphate and is tentatively designated phosphatidylinositol 3-phosphatase (PtdIns 3-phosphatase). The enzyme does not require added metal ions for activity and is maximally active in the presence of EDTA. It is inhibited by Ca2+, Mg2+, Zn2+, and the phosphatase inhibitor VO4(3-). In addition, there is no phospholipase C activity toward PtdIns(3)P in the soluble fraction of NIH 3T3 cells. In view of the absence of a phospholipase C activity that hydrolyzes PtdIns(3)P, we propose that PtdIns(3)P is not a precursor for a soluble inositol phosphate messenger but that it instead may act directly to control certain cellular processes or as a precursor for other phosphatidylinositols. PtdIns 3-phosphatase may thus terminate a metabolic signal or regulate precursor levels for other phosphatidylinositols that are phosphorylated in the D-3 position.
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PMID:The discovery of a 3-phosphomonoesterase that hydrolyzes phosphatidylinositol 3-phosphate in NIH 3T3 cells. 255 36


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