Calcium-requirement and its pH dependency of lysophosphoinositide-specific phospholipase C in porcine platelet membranes.
The lysophosphoinositide-specific phospholipase C (lysoPI-PLase C) in porcine platelet membranes had an optimal pH of 9.2 and the activity at a physiological pH of 7.3 was 20% of the maximum in the absence of added divalent metals (Murase, S. et al. (1985) J. Biol. Chem. 260, 262). The activity was completely inhibited by 1 mM EGTA in the assay mixture but was restored by addition of excess Ca2+ or Mn2+, indicating that this is a metalloenzyme. However, membranes pretreated with 1 mM EGTA and washed with buffer retained full activity at a free Ca2+ concentration of 5 nM and no stimulation was observed by added Ca2+ at pH 9.2. In contrast to the results obtained at pH 9.2, addition of Ca2+ stimulated lysoPI-PLase C activity severalfold at pH 7.3, apparently by shifting down the optimal pH and broadening the pH profile. The effect of Ca2+ at pH 7.3 was to enhance Vmax with no significant change in Km value. The stimulatory effect of Ca2+ at pH 7.3 alone did not appear to be of physiological significance since millimolar concentrations of Ca2+ were necessary to reach the maximum activity. However, a shift in pH had a profound effect on the Ca2+-dependency of the activity. A rise in 2 pH units increased the apparent affinity for Ca2+ 10,000-fold. These results indicate that the alkalinization and the rise in free Ca2+ concentration known to occur in stimulated platelets could synergistically provide conditions under which the lysoPI-PLase C exerts its activity when the substrate lysoPI is generated by phospholipase A.
D000818:Animals; D001792:Blood Platelets / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology*; D002413:Cations, Divalent; D002462:Cell Membrane / Q000201:enzymology; D004533:Egtazic Acid; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010716:Phosphatidylinositols / Q000378:metabolism*; D013552:Swine; D010738:Type C Phospholipases / Q000097:blood*
D002413:Cations, Divalent; D010716:Phosphatidylinositols; D004533:Egtazic Acid; D010738:Type C Phospholipases; D002118:Calcium