Phosphatidylinositol 4,5-bisphosphate specific phospholipase C in Pharbitis nil membranes (original) (raw)
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An acid phospholipase C from Tetrahymena culture medium
Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, 1986
Al~traet--1. A phospholipase C in Tetrahymena thermophila culture medium was assayed with 1,2-di[1-J4C]stearoyl-sn-glycero-3-phosphorylcholine as substrate and purified by ammonium sulphate precipitation of the extracellular growth medium, DEAE cellulose ion exchange chromatography and gel filtration on Sepharose 6B.
Chemistry and Physics of Lipids, 2010
Eukaryotic phosphoinositide-specific phospholipases C (PI-PLC) specifically hydrolyze phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ], produce the Ca 2؉-mobilizing agent inositol 1,4,5-trisphosphate, and regulate signaling in multicellular organisms. Bacterial PtdIns-specific PLCs, also present in trypanosomes, hydrolyze PtdIns and glycosyl-PtdIns, and they are considered important virulence factors. All unicellular eukaryotes studied so far contain a single PI-PLC-like gene. In this report, we show that ciliates are an exception, since we provide evidence that Tetrahymena species contain two sets of functional genes coding for both bacterial and eukaryotic PLCs. Biochemical characterization revealed two PLC activities that differ in their phosphoinositide substrate utilization, subcellular localization, secretion to extracellular space, and sensitivity to Ca 2؉. One of these activities was identified as a typical membrane-associated PI-PLC activated by low-micromolar Ca 2؉ , modestly activated by GTP␥S in vitro, and inhibited by the compound U73122 [1-(6-{[17-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione]. Importantly, inhibition of PI-PLC in vivo resulted in rapid upregulation of PtdIns(4,5)P 2 levels, suggesting its functional importance in regulating phosphoinositide turnover in Tetrahymena. By in silico and molecular analysis, we identified two PLC genes that exhibit significant similarity to bacterial but not trypanosomal PLC genes and three eukaryotic PI-PLC genes, one of which is a novel inactive PLC similar to proteins identified only in metazoa. Comparative studies of expression patterns and PI-PLC activities in three T. thermophila strains showed a correlation between expression levels and activity, suggesting that the three eukaryotic PI-PLC genes are functionally nonredundant. Our findings imply the presence of a conserved and elaborate PI-PLC-Ins(1,4,5)P 3-Ca 2؉ regulatory axis in ciliates.
The EMBO Journal, 1994
The microorganism Dictyostelium uses extracellular cAMP to induce chemotaxis and cell differentiation. Signals are transduced via surface receptors, which activate G proteins, to effector enzymes. The deduced protein sequence of Dictyostelium discoideum phosphatidylinositol-specific phospholipase C (PLC) shows strong homology with the mammalian PLC-6 isoforms. To study the role of PLC in Dictyostelium, a plcmutant was constructed by disruption of the PLC gene. No basal or stimulated PLC activity could be measured during the whole developmental programme of the plccells. Loss of PLC activity did not result in a visible alteration of growth or development. Further analysis showed that developmental gene regulation, cAMP-mediated chemotaxis and activation of guanylyl and adenylyl cyclase were normal. Although the cells lack PLC activity, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was present at only slightly lower concentrations compared with control cells. Mass analysis of inositol phosphates demonstrated the presence of a broad spectrum of inositol phosphates in Dictyostelium, which was unaltered in the plcmutant. Cell labelling experiments with [3H]inositol indicated that [3H]Ins(1,4,5)P3 was formed in a different manner in the mutant than in control cells.
Comparative biochemistry and physiology. B, Comparative biochemistry, 1993
1. Polyphosphoinositide-specific phosphodiesterase (phospholipase C, PLC) activity against phosphatidylinositol 4,5-bisphosphate, present in gill and digestive gland homogenates of mussel (Mytilus galloprovincialis Lam.), has been biochemically characterized. 2. The enzyme was strictly modulated by free calcium ion concentration in both tissues and maximally activated at 10(-5) M Ca2+ (19 +/- 4 and 11 +/- 2 nmol phosphatidylinositol 4,5-bisphosphate hydrolysed/min/mg of protein for gill and digestive gland PLC, respectively, at 19 degrees C). Optimum pH at 10(-5) M Ca2+ was around 7.0 in both cases. The Ca(2+)-stimulated PLC activity showed high specificity for PIP2; the KMa for PIP2 were 150 and 170 microM for the gills and digestive gland, respectively. 3. Good substrate dispersion was obtained in the presence of sodium deoxycholate; the concentration routinely used in the assay (0.08%) produced a 9-fold activation of both gill and digestive gland PLC, consistent with previous rep...
Stimulation of phosphatidylinositol 4,5-bisphosphate phospholipase C activity by phosphatidic acid*1
Archives of Biochemistry and Biophysics, 1989
Phosphatidic acid was a potent activator of the phosphatidylinositol 4,5-bisphosphate (PtdIns-P2) phospholipase C activity associated with human platelet membranes. Lysophosphatidic acid was half as active as phosphatidic acid, and shortening the fatty acid chain reduced the effectiveness of the corresponding phosphatidic acid. Compounds lacking either the phosphate group (diacylglycerol or phorbol ester) or the fatty acid (glycerol phosphate) were not activators. When the negative charge was contributed by a carboxyl group (fatty acid or phosphatidylserine), stimulation of phospholipase C was weak but detectable. Structural analogs of phosphatidic acid (lipopolysaccharide, lipid A, and 2,3-diacylglucosamine 1-phosphate) were less effective but also enhanced Ptdlnsp2 hydrolysis. Phosphatidic acid potentiated the activation of phospholipase C by α-thrombin, chelators, and guanine nucleotides. Phosphatidylinositol 4-phosphate and PtdIns-P2 were also effective activators of PtdIns-P2 degradation. Other phospholipids were without effect. The production of inositol 1,4,5-trisphosphate and diacylglycerol via the activation of phospholipase C provides a rationale for the cellular responses evoked by phosphatidic acid and the ability of this phospholipid to potentiate and initiate hormonal responses.
Chemistry and Physics of Lipids, 1997
Phosphatidylinositol-specific phospholipase C (PI-PLC) was studied with sonicated dispersions of a thiophosphate analog of phosphatidylinositol, 1,2-dimyristoyloxypropane-3-thiophospho(1D-1-myo-inositol) (D-thio-DMPI). Kinetic parameters were derived from the rate as a function of bulk lipid concentration at constant saturating surface concentration of substrate (case I), and as a function of surface concentration of substrate at a constant saturating bulk concentration of lipid (case II). The substrate, D-thio-DMPI, was diluted with L-thio-DMPI or dimyristoyl phosphatidylmethanol (DMPM). In the presence of L-thio-DMPI, values for V max = 133 vmol min − 1 mg − 1 , K s % (the apparent dissociation constant for the enzyme-interface complex) = 0.097 mM, and K m * (the apparent interfacial Michaelis constant) = 0.22 mol fraction were obtained. DMPM caused enzyme inhibition in case I but no inhibition in case II. L-Thio-DMPI is an ideal neutral diluent with which to study the kinetics of PI-PLC.
Some Characteristics of Phospholipase C from Bacillus cereus
European Journal of Biochemistry, 1977
1. The amino acid composition of purified Bacillus cereus phospholipase C is reported. The enzyme contains one methionine residue and two fragments are obtained after cyanogen bromide cleavage. The sequence of the amino-terminal fragment (25 residues) is reported.
Proceedings of the National Academy of Sciences, 1970
Phospholipase D (cabbage) inhibits the vectorial phosphorylation of a-methylglucoside by isolated membrane preparations from Escherichia coli NIL 308-225 without increasing the efflux of intramembranal a-methylglucoside-P. This effect is shown to be related to the ability of phospholipase D to hydrolyze membrane phosphatidylglycerol specifically. After treatment with phospholipase D, the membranes resynthesize phosphatidylglycerol with a return in their ability to take up a-methylglucoside. Since proline uptake by the same preparations is only slightly inhibited by phospholipase D, the data indicate that phosphatidylglycerol is. required specifically for transport processes which are mediated by the P-enolpyruvate-P-transferase system.
Membrane-associated phosphoinositidase C activity in Dictyostelium discoideum
FEBS Letters, 1990
Membrane-associated phosphoinositidase C activity has been identified in Dictyostelium disco&an using phosphatidylinositol 4,5-bisphosphate as exogenous substrate. Maximal activity was observed with 0.4 mM phosphatidylinositol4,5-bisphosphate at pH 7.0. The enzyme was stimulated by micromolar concentrations of free calcium with maximal activity at 100 PM.