Characterization of cytoplasmic and membrane-associated phosphatidylinositol 4,5-biphosphate phospholipase C activities in guinea pig ventricles.
The phosphoinositide-specific phospholipase C (PLC) activity present in the soluble and sarcolemmal enriched membrane fraction from guinea pig hearts was characterized using phosphatidyl [3H]inositol 4,5-biphosphate (PIP2) or phosphatidyl [3H]inositol 4-monophosphate (PIP) as substrates. The PLC activities (cytosolic and membrane associated) were specific for polyphosphoinositides (PIP2 and PIP) since no other phospholipids were hydrolyzed at pH 7.0 under various ionic conditions. Both enzymic activities were Ca2(+)-dependent (half maximal activities were achieved around pCa 5.0). The pH, detergent (deoxycholate), divalent (Ca2+ and Mg2+), and monovalent (Na+ and K+) cation dependencies were very similar between the cytosolic and membrane-associated enzyme activities, using either PIP2 or PIP as substrate. Hydrolysis of the polyphosphoinositides was inhibited in the presence of phosphatidylethanolamine, phosphatidylserine, or phosphatidylcholine. Under optimal conditions (pH 7.0, 1 mM Ca2+, 2.5 mM Mg2+, 100 mM Na+ and 0.07% deoxycholate) the specific activities of the cytosolic and membrane-associated enzymes were 19.9 +/- 0.9 and 10.1 +/- 0.9 nmol/min/mg protein, respectively, using PIP2 as substrate. Under the same conditions these activities were 18.1 +/- 1.0 and 8.0 +/- 0.8 nmol/min/mg protein for the cytosolic and membrane fractions, respectively, using PIP as substrate. Based on the similarity of the characteristics of these two PLC enzyme activities, it is suggested that the cytosolic and membrane-associated enzyme forms may be closely related.
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Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Ohio.
Kranias
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D000818:Animals; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology*; D003593:Cytoplasm / Q000201:enzymology*; D003600:Cytosol / Q000201:enzymology; D006168:Guinea Pigs; D006352:Heart Ventricles / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols / Q000378:metabolism*; D012508:Sarcolemma / Q000201:enzymology; D010738:Type C Phospholipases / Q000378:metabolism*
D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D010738:Type C Phospholipases; D002118:Calcium