The substrate specificity of phosphoinositide-phospholipase C in rat heart sarcolemma.
In rat cardiac sarcolemmal membranes a phosphoinositide-specific phospholipase C (PLC) was found to be present. The enzyme hydrolysed exogenous [3H-]phosphatidylinositol 4,5-biphosphate ([3H-]PtdIns(4,5)P2) in an optimized assay mixture containing 15 micrograms SL protein, 100 mM NaCl, 1 mM free Ca2+, 14 mM Na-cholate and 20 microM [3H-]PtdIns-(4,5)P2 (400-500 dpm/microliter) in 30 mM HEPES-Tris buffer (pH 7.0). The average specific activity was 9.14 +/- 0.55 nmol.mg-1.2.5 min-1. The addition of Mg2+ to the assay mixture did not change PLC activity but increased the relative amounts of dephosphorylated inositol products. In the absence of Na+ and at a low Ca2+ concentration (0.3 microM), Mg2+ also enhanced the intraSL levels of PtdIns4P and PtdIns, and, moreover, inhibited PLC activity (IC50-0.07 mM). PtdIns4P seemed to be a good substrate for th rat SL PLC (23.07 +/- 1.57 nmol.mg-1.2.5 min-1) whereas PtdIns was hydrolysed at a very low rate (0.36 +/- 0.08 nmol.mg-1.2.5 min-1). Unlike PtdIns(4,5)P2, PLC-dependent PtdIns4P and PtdIns hydrolysis was not inhibited by Ca2+ concentrations over 1 mM. The possibility of distinct isozymes being responsible for the different hydrolytic activities is discussed.
Meij
J T
JT
Division of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, Winnipeg, Canada.
Panagia
V
V
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D009206:Myocardium / Q000201:enzymology*; D019269:Phosphatidylinositol 4,5-Diphosphate; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D051381:Rats / Q000378:metabolism; D017207:Rats, Sprague-Dawley; D012508:Sarcolemma / Q000201:enzymology*; D012964:Sodium / Q000494:pharmacology; D013379:Substrate Specificity
D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; C037178:phosphatidylinositol 4-phosphate; D012964:Sodium; D010727:Phosphoric Diester Hydrolases; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D008274:Magnesium; D002118:Calcium