Summary
The phosphoinositide-specific phospholipase C (PLC) activity present in the soluble and sarcolemmal enriched membrane fraction from guinea pig hearts was characterized using phosphatidyl [3H]inositol 4,5-biphosphate (PIP2) or phosphatidyl [3H]inositol 4-monophosphate (PIP) as substrates. The PLC activities (cytosolic and membrane associated) were specific for polyphosphoinositides (PIP2 and PIP) since no other phospholipids were hydrolyzed at pH 7.0 under various ionic conditions. Both enzymic activities were Ca2+-dependent (half maximal activities were achieved around pCa 5.0). The pH, detergent (deoxycholate), divalent (Ca2+ and Mg2+), and monovalent (Na+ and K+) cation dependencies were very similar between the cytosolic and membrane-associated enzyme activities, using either PIP2 or PIP as substrate. Hydrolysis of the polyphosphoinositides was inhibited in the presence of phosphatidylethanolamine, phosphatidylserine, or phosphatidylcholine.
Under optimal conditions (pH 7.0, 1 mM Ca2+, 2.5 mM Mg2+, 100 mM Na+ and 0.07% deoxycholate) the specific activities of the cytosolic and membrane-associated enzymes were 19.9±0.9 and 10.1±0.9 nmol/min/mg protein, respectively, using PIP2 as substrate. Under the same conditions these activitics were 18.1±1.0 and 8.0±0.8 nmol/min/mg protein for the cytosolic and membrane fractions, respectively, using PIP as substrate. Based on the similarity of the characteristics of these two PLC enzyme activities, it is suggested that the cytosolic and membrane-associated enzyme forms may be closely related.
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References
Bennett CF, Balcarek JM, Varrichio A, Crooke ST (1988) Molecular cloning and complete aminoacid sequence of form-I phosphoinositide-specific phospholipase C. Nature 334:268–270
Bennett CF, Crooke ST (1987) Purification and characterization of a phosphoinositide-specific phospholipase C from guinea pig uterus. J Biol Chem 262:13789–13797
Berridge MJ, Dawson RMC, Downes CP, Heslop JP, Irvine RF (1983) Changes in the levels of inositol phosphates after agonist dependent hydrolysis of membrane phosphoinositides. Biochem J 212:473–482
Berridge MJ (1984) Inositol triphosphate and diacylglycerol as second messengers. Biochem J 220:345–360
Berridge MJ (1988) Inositol triphosphate and dyacylglycerol: two interacting second messengers. Ann Rev Biochem 56:159–193
Jolles J, Zwiers H, Dekker A, Wirtz KWA, Gispen WH (1981) Corticotropin (1–24)-tetracosapeptide affects protein phosphorylation and polyphosphoinositide metabolism in rat brain. Biochem J 194:283–291
Jones LR, Besch JR Hr (1984) Isolation of canine cardiac sarcolemmal vesicles. Methods Pharmacol 5:1–12
Katan M, Kriz RW, Totty N, Philp R, Meldrum E, Aldape RA, Knopf JL, Parker PF (1988) Determination of the primary structure of PLC-154 demonstrates diversity of phosphoinositide-specific phospholipase C activities. Cell 54:171–177
Kawaguchi H, Yasuda H (1988) Prostacyclin biosynthesis and phospholipase activity in hypoxic rat myocardium. Circ Res 62:1175–1181
Lindemann JP (1986) α-adrenergic stimulation of sarcolemmal protein phosphorylation and slow responses in intact myocardium. J Biol Chem 261:4860–4867
Low MG, Carrol RC, Cox AC (1986) Characterization of multiple forms of phosphoinositide-specific phospholipase C purified from human platelets. Biochem J 237:139–145
Lowry OH, Rosenbrough NH, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275
Magnaldo I, Talwar H, Anderson WB, Pouyssegur J (1987) Evidence for a GTP-binding protein coupling thrombin receptor to PIP2-phospholipase C in membranes of hamster fibroblasts. FEBS Lett 210:6–10
Nishizuka Y (1986) Studies and perspectives of protein kinase C. Science 233:305–312
Poggioli J, Sulpice JC, Vassort G (1986) Inositol phosphate production following α-adrenergic, muscarinic or electrical stimulation in isolated rat hearts. FEBS Letter 206:292–298
Presti CF, Jones LR, Lindemann JP (1985) Isoproterenol induced phosphorylation of a 15-kilodalton sarcolemmal protein in intact myocardium. J Biol Chem 260:3860–3867
Robertson S, Potter JD (1984) The use of metal buffers in biological systems. Methods Pharmacol 5:63–75
Ryu SH, Cho KS, Lee K-Y, Suh P-G, Rhee SG (1987) Purification and characterization of two immunologically distinct phosphoinositide-specific phospholipase C from bovine brain. J Biol Chem 262:12511–12518
Sasaguri T, Hirata M, Kuriyama H (1985) Dependence on Ca2+ of the activities of phosphatidylinositol 4,5-biphosphate phosphodiesterase and inositol 1,4,5-triphosphate phosphatase in smooth muscles of the porcine coronary artery. Biochem J 231:497–503
Scholz J, Schaeffer B, Schmitz W, Scholz W, Steinfath M, Lohse M, Schwabe U, Puurunen J (1988) Alpha-1 adrenoceptor-mediated positive inotropic effect and inositol triphosphate increase in mammalian heart. J Pharm Exp Ther 245:327–335
Schwertz DW, Halverson JB, Palmer JW, Feinberg H (1987) Characterization of phospholipase C-mediated phosphatidylinositol degradation in rat heart ventricle. Arch Biochem Biophys 253:388–398
Stahl ML, Ferenz CR, Kelleher KL, Kriz RW, Knopf JL (1988) Sequence similarity of phospholipase C with the non-catalytic region or src. Nature 332:269–272
Suh P-G, Ryu SH, Moon KH, Suh HW, Rhee SG (1988) Cloning and sequence of multiple forms of phospholipase C. Cell 54:161–169
Taylor SJ, Exton JH (1987) Guanine-nucleotide and hormone regulation of polyphosphoinositide phospholipase C activity of rat liver plasma membranes. Biochem J 248:791–799
Unverferth DV, Lee SW, Wallick ET (1988) Human myocardial adenosine triphosphatase activities in health and heart failure. Am Heart J 115:139–146
Wang P, Toyoshima S, Osawa T (1987) Physical and functional association of cytosolic inositolphospholipid-specific phospholipase C of calf thymocytes with a GTP-binding protein. J Biochem 102:1275–1287
Wolf RA, Gross RW (1985) Identification of neutral active phospholipase C which hydrolyzes choline glycerophospholipids and plasmalogen selective phospholipase A2 in canine myocardium. J Biol Chem 260:7295–7303
Woodcock EA, White LBS, Smith AI, McLeod JK (1987) Stimulation of phosphatidylinositol metabolism in the isolated, perfused rat heart. Circ Res 61:625–631
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This work was supported by National Institutes of Health Grants HL26057 and HL22619.
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Edes, I., Kranias, E.G. Characterization of cytoplasmic and membrane-associated phosphatidylinositol 4,5-biphosphate phospholipase C activities in guinea pig ventricles. Basic Res Cardiol 85, 78–87 (1990). https://doi.org/10.1007/BF01907016
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DOI: https://doi.org/10.1007/BF01907016