Characterization of phospholipase C-mediated polyphosphoinositide hydrolysis in rat heart ventricles

Abstract

Phospholipase C (PLC)-mediated degradation of polyphosphoinositides (phosphatidylinositol 4,5-bisphosphate (PIP 2) and phosphatidylinositol 4-phosphate (PIP)) was found to be present in rat heart ventricular soluble and total membrane fractions (100,000 g supernatant and pellet). Distribution of polyphosphoinositide-specific phospholipase C activity between the membrane and soluble fraction was approximately 63 and 33% of total activity, respectively, whereas, phosphatidylinositol (PI) degradation could be detected only in the soluble fraction. Optimal PIP 2-PLC activity occurred at a pCa 2+ of 4.5. A similar peak in PIP-PLC activity could be demonstrated in soluble and membrane preparations; however, the rate of PIP degradation in the soluble fraction continued to increase at the highest calcium level tested ( pCa 2+ 3). With the exception of Sr 2+, other noncalcium polycations did not support homogenate PIP 2-PLC activity. In the presence of Ca 2+, addition of Mg 2+, La 3+, or Sr 2+ (10 −3 m) inhibited PIP 2-PLC while Mn 2+ and Gd 3+ stimulated activity. In both the total membrane and soluble fractions, maximal polyphosphoinositide degradation occurs at pH 5.5 and 6.8. The detergents deoxycholate, cholate, and saponin exert a biphasic effect on PIP 2-PLC activity (stimulating at lower concentrations and inhibiting at higher concentrations). The deoxycholate effect is observed in both the cytosolic and membrane fractions. Neutral and cationic detergents inhibit PIP 2-PLC activity in a concentration-dependent manner. Similar to cytosolic PI-PLC activity, PIP 2-PLC appears to depend on intact sulfhydryl groups. In the presence of a mixture of all three inositol phospholipids or the three phosphoinositides plus noninositol phospholipids, polyphosphoinositides are preferentially degraded.