Purification and characterization of bovine heart phosphoinositide-specific phospholipase C: kinetic analysis of the Ca2+ requirement and La3+ inhibition.
Bovine heart contains multiple phosphoinositide-specific phospholipase C (PIC) activities separable by ion-exchange chromatography. One PIC activity was purified to apparent homogeneity and migrated as a single band of Mr 85,000 on SDS-PAGE. The purified PIC was characterized with sonicated suspensions of either pure phosphatidylinositol 4,5-bisphosphate (PIP2) or phosphatidylinositol (PI) as substrates. At pH 7, apparent Vmax and Km values were higher for PIP2 than for PI, but the value of Vmax/Km was similar for the two substrates. PIC required Ca2+ for the hydrolysis of either PI or PIP2, and increasing free Ca2+ concentrations from 20 to 300 nM saturated PIC activity. The requirement of Ca2+ for PIC activity and the sensitivity of PIC to Ca2+ concentrations in the physiological range suggested the ion may be a cofactor. The PIC reaction mechanism was determined by two-substrate kinetic analysis; the data fit a model in which PIC contained single sites for Ca2+ and phosphoinositide, and utilized a rapid-equilibrium, random-order ternary mechanism for phosphoinositide hydrolysis. The KCa value for either PI or PIP2 hydrolysis was approximately 30 nM, suggesting resting intracellular free Ca2+ concentrations are sufficient to saturate the Ca2+ site of PIC. La3+ was used as a calcium analogue to modulate PIC activity. Low concentrations of LaCl3 (0.01-0.3 microM) inhibited PIC activity competitively with respect to calcium, consistent with a Ca2+ binding site on the enzyme.
McDonald
L J
LJ
Department of Biological Sciences, Wright State University, Dayton, Ohio 45435.
Mamrack
M D
MD
D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D006868:Hydrolysis; D007700:Kinetics; D007811:Lanthanum / Q000378:metabolism; D009206:Myocardium / Q000201:enzymology*; D019269:Phosphatidylinositol 4,5-Diphosphate; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010716:Phosphatidylinositols / Q000378:metabolism; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification*
D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D010726:Phosphodiesterase Inhibitors; D007811:Lanthanum; D010727:Phosphoric Diester Hydrolases; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D002118:Calcium