| Abstract
| - The mechanism of interaction of phorbol esters with conventionalprotein kinase Cs wasaddressed by examining the direct binding of this class of activatorsto protein kinase C βII. Bindingmeasurements reveal that the major role of phorbol esters is toincrease the affinity of protein kinase Cfor membranes by several orders of magnitude. The relativeincrease depends linearly on the mole fractionof phorbol esters in membranes, with the potency illustrated by thefinding that 1 mol % phorbol 12-myristate 13-acetate (PMA) increases protein kinase C's membraneassociation by approximately 4 ordersof magnitude. For comparison, diacylglycerol (DG), which alsoactivates protein kinase C by increasingthe enzyme's membrane affinity, is 2 orders of magnitude lesseffective than PMA in altering proteinkinase C's membrane affinity. The remarkably high-affinityinteraction with phorbol esters allowed usto measure the direct binding of protein kinase C to PMA in neutralmembranes and, thus, to evaluate theeffect of Ca2+ on the phorbol ester interaction in theabsence of Ca2+ effects on the enzyme'sinteractionwith acidic lipids. Changing the Ca2+ concentrationover 5 orders of magnitude had no effect on thedirect interaction of protein kinase C with PMA immobilized inphosphatidylcholine membranes. Thus,the Ca2+-binding site for membrane association and thephorbol ester-binding site do not interactallosterically. Lastly, a method that does not have thelimitations of the Scatchard plot for analysis ofamphitropic proteins was used to determine the dissociation constant ofprotein kinase C from phorbolesters: expressed relative to membrane lipids, the dissociationconstant is 1.5 × 10-5 mol %. Insummary,our data reveal that (1) the direct binding of protein kinase C tophorbol esters, in the absence of interactionswith acidic lipids, provides a major contribution to the free energychange involved in the association ofprotein kinase C with membranes and (2) this interaction is notregulated by Ca2+.
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