Characterization of phospholipase C-mediated polyphosphoinositide hydrolysis in rat heart ventricles.
Phospholipase C (PLC)-mediated degradation of polyphosphoinositides (phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4-phosphate (PIP] was found to be present in rat heart ventricular soluble and total membrane fractions (100,000g 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 PIP2-PLC activity occurred at a pCa2+ 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 (pCa2+ 3). With the exception of Sr2+, other noncalcium polycations did not support homogenate PIP2-PLC activity. In the presence of Ca2+, addition of Mg2+, La3+, or Sr2+ (10(-3) M) inhibited PIP2-PLC while Mn2+ and Gd3+ 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 PIP2-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 PIP2-PLC activity in a concentration-dependent manner. Similar to cytosolic PI-PLC activity, PIP2-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.
Schwertz
D W
DW
Department of Pharmacology, University of Illinois, Chicago.
Halverson
J
J
D000818:Animals; D002118:Calcium / Q000502:physiology; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D003902:Detergents; D006352:Heart Ventricles / Q000201:enzymology / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008297:Male; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility; D013324:Strontium / Q000502:physiology; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds; D010738:Type C Phospholipases / Q000502:physiology*
D003902:Detergents; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D013438:Sulfhydryl Compounds; D010738:Type C Phospholipases; D002118:Calcium; D013324:Strontium