PHOSPHOINOSITIDE-SPECIFIC PHOSPHOLIPASE C-DELTA-1 - EFFECT OF MONOLAYER SURFACE PRESSURE AND ELECTROSTATIC SURFACE-POTENTIALS ON ACTIVITY

We added phospholipase C-delta1 (PLC-delta) to the aqueous subphase beneath monolayers formed from mixtures of phosphatidylinositol 4,5-bisphosphate (2% PIP2), phosphatidylserine (33% PS), and phosphatidylcholine (65% PC) and then measured the initial rate of hydrolysis Of PIP2 after addition of 10 muM free calcium. Increasing the surface pressure of the monolayer, pi, from 20 to 40 mN/m decreased the rate of hydrolysis 200-fold. The rate of hydrolysis depends exponentially on the surface pressure: rate is-proportional-to exp(-piA(p)/kT) where k is the Boltzmann constant, T is the temperature, and A(p) congruent-to 1 nm2. Similar results were obtained with different (1 and 100 muM) free [Ca2+] and with different mole fractions of PIP2. The results are consistent with a model in which PLC-delta binds to PIP2 with high affinity (K(a) = 10(6) M-1) in the absence of calcium ions [Rebecchi, M. J., Peterson, A., & McLaughlin, S. (1993) Biochemistry (preceding paper in this issue)], and a portion of PLC-delta of area A(p) inserts into the monolayer doing work = piA(p) prior to hydrolysis of PIP2. Removing the monovalent acidic lipid PS from the monolayer decreases the activity of PLC-delta 4-fold, this effect of PS on activity is similar to the effect of monovalent acidic lipids on the binding of PLC-delta to PIP2 in bilayer vesicles.