Phosphoinositide-specific phospholipase C-delta 1: effect of monolayer surface pressure and electrostatic surface potentials on activity.
We added phospholipase C-delta 1 (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 microM 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 alpha exp(-pi Ap/kT) where k is the Boltzmann constant, T is the temperature, and Ap congruent to 1 nm2. Similar results were obtained with different (1 and 100 microM) 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 (Ka = 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 Ap inserts into the monolayer doing work = pi Ap 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.
Rebecchi
M
M
Department of Physiology and Biophysics, State University of New York, Stony Brook 11794-8661.
Boguslavsky
V
V
Boguslavsky
L
L
McLaughlin
S
S
D004563:Electrochemistry; D006868:Hydrolysis; D007700:Kinetics; D010713:Phosphatidylcholines / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols / Q000378:metabolism*; D010718:Phosphatidylserines / Q000378:metabolism; D011312:Pressure; D013499:Surface Properties; D010738:Type C Phospholipases / Q000737:chemistry* / Q000378:metabolism*
D010713:Phosphatidylcholines; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D010718:Phosphatidylserines; D010738:Type C Phospholipases