The substrate specificity of phosphoinositide phospholipase C in rat heart sarcolemma

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)P ₂) 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)P ₂ (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 μM), Mg²⁺ also enhanced the intraSL levels of PtdIns4P and PtdIns, and, moreover, inhibited PLC activity (IC₅₀∼0.07 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)P ₂, 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).