DEPENDENCE OF THE ACTIVITY OF PHOSPHOLIPASE C-BETA ON SURFACE PRESSURE AND SURFACE-COMPOSITION IN PHOSPHOLIPID MONOLAYERS AND ITS IMPLICATIONS FOR THEIR REGULATION

We have examined the influence of surface pressure and phospholipid co mposition on hydrolysis of phosphatidylinositol (4,5)-bisphosphate (PI P2) by phospholipase C beta 1 (PLC beta 1) and PLC beta 2 in mixed com position phospholipid monolayers. Increasing the monolayer surface pre ssure from 15 to 36 mN/m reduced the rate at which PIP2 was hydrolyzed by PLC beta 1 and PLC beta 2 by 4-6-fold, although PLC beta 1 was mor e active than PLC beta 2, even at high surface pressures. Reduced enzy me activity was accompanied by an increase in reaction induction times , suggesting that increasing surface pressure reduced the penetration rate of the enzymes into the monolayer. Quantitation of interfacial en zyme concentration using S-35-labeled PLC beta 1 confirmed that less e nzyme was associated with the monolayer at higher pressures. The relat ionship between PLC activity and substrate concentration was examined at a single surface pressure of 30 mN/m. This relationship was not hyp erbolic, and increases in the mole percentage (mol %) of PIP2 in the m onolayer resulted in an upwardly-curving increase in PLC activity. Thu s, PLC beta 1 activity increased 7-fold and PLC beta 2 activity increa sed 4-fold when the mol % of PIP2 in the monolayer increased from 17.9 % to 29%, increasing further thereafter. Paradoxically, increasing the mol % of PIP2 from 0 to 60% was accompanied by a 3-fold decrease in i nterfacial enzyme concentrations. Taken together, these data show that the catalytic activity of PLC beta involves some element of penetrati on of lipid interfaces, and suggest that the organization of the subst rate facilitates PLC activity, giving credence to the substrate theory of interfacial activation of phospholipases. We present a hypothesis suggesting that PIP2 molecules coalesce into enriched lateral domains which favor PLC beta activity.