Dependence of the activity of phospholipase C beta on surface pressure and surface composition in phospholipid monolayers and its implications for their regulation.Biochemistry. 1997 Jan 28; 36(4):848-55.B
We have examined the influence of surface pressure and phospholipid composition on hydrolysis of phosphatidylinositol (4,5)-bisphosphate (PIP2) by phospholipase C beta 1 (PLC beta 1) and PLC beta 2 in mixed composition phospholipid monolayers. Increasing the monolayer surface pressure 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 more active than PLC beta 2, even at high surface pressure. Reduced enzyme 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 enzyme concentration using 35S-labeled PLC beta 1 confirmed that less enzyme was associated with the monolayer at higher pressures. The relationship between PLC activity and substrate concentration was examined at a single surface pressure of 30 mN/m. This relationship was not hyperbolic, and increases in the mole percentage (mol %) of PIP2 in the monolayer resulted in an upwardly-curving increase in PLC activity. Thus, PLC beta 1 activity increased 7-fold and PLC beta 2 activity increased 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 interfacial enzyme concentrations. Taken together, these data show that the catalytic activity of PLC beta involves some element of penetration of lipid interfaces, and suggest that the organization of the substrate 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.