The substrate specificity of phosphoinositide phospholipase C in rat heart sarcolemma |
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Authors: | Johanna T. A. Meij Vincenzo Panagia |
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Affiliation: | (1) Division of Cardiovascular Sciences, St. Boniface General Hospital Reserach Centre and Department of Anatomy, Faculty of Medicine, University of Manitoba, Winnipeg, Canada |
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Abstract: | 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 leg SL protein, 100 mM NaCl, 1 mM free Ca2+,14 mM Na-cholate and 20 AM [3H-]PtdIns (4,5)P2 (400–500 dpm/gm-l) 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 M), Mg2+ also enhanced the intraSL levels of PtdIns4P and PtdIns, and, moreover, inhibited PLC activity (IC500.07 mM). PtdIns4P seemd to be a good substrate for the 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. (Mol Cell Biochem116: 27–31, 1992).Abbreviations DAG sn-1,2-diacylglycerol - EGTA ethyleneglycol-O,O-bis(aminoethyl)-N,N,N,N,-tetraacetic acid - Ins(1,4,5)P3 inositol 1,4,5-trisphosphate - InsP inositol monophosphate (unidentified isomer) - InsP2 inositol bisphosphate (unidentified isomer) - InsP3 inositol trisphosphate (unidentified isomer) - InsPx any inositol phosphate - PLC phospholipase C - PtdIns phosphatidylinositol - PtdIns(4,5)P2 phosphatidylinositol 4,5-bisphosphate - PtdIns4P phosphatidylinositol 4-monophosphate - SL sarcolemma |
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Keywords: | phospholipase C phosphoinositides phosphatidylinositol 4,5-bisphosphate phosphatidylinositol 4-phosphate rat heart sarcolemma |
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