Dependence of the Activity of Phospholipase Cβ on Surface Pressure and Surface Composition in Phospholipid Monolayers and Its Implications for Their Regulation

Abstract

We have examined the influence of surface pressure and phospholipid composition on hydrolysis of phosphatidylinositol (4,5)-bisphosphate (PIP2) by phospholipase Cβ1 (PLCβ1) and PLCβ2 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 PLCβ1 and PLCβ2 by 4−6-fold, although PLCβ1 was more active than PLCβ2, even at high surface pressures. 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 PLCβ1 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, PLCβ1 activity increased 7-fold and PLCβ2 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 PLCβ 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 moleclues coalesce into enriched lateral domains which favor PLCβ activity.