Multiple factors influence the binding of a soluble, Ca2+-independent, diacylglycerol kinase to unilamellar phosphoglyceride vesicles

We studied the influence of membrane lipids, MgCl2, and ATP on the ability of a soluble diacylglycerol kinase to bind to 100-nm lipid vesicles. The en zyme did not bind detectably to vesicles that contained phosphatidylcholine alone or to vesicles that contained 50 mol % phosphatidylcholine + 50 mol % phosphatidylethanolamine. But it did bind to vesicles that contained anio nic phosphoglycerides, and maximal binding occurred tin the presence of MgC l2) when the vesicles contained anionic phosphoglycerides alone. When incre asing amounts of phosphatidylcholine were included in phosphatidylserine-co ntaining vesicles, enzyme binding to the vesicles decreased by as much as 1 000-fold. However, when increasing amounts of phosphatidylethanolamine were included in phosphatidylserine-containing vesicles, little change in bindi ng occurred until the concentration of phosphatidylserine was reduced to be low 25 mol %. These results and results obtained with vesicles that contain ed various mixtures of anionic phosphoglycerides, phosphatidylcholine, phos phatidylethanolamine, and unesterified cholesterol provided evidence that a nionic phosphoglycerides were positive effecters of binding, phosphatidylch oline was a negative effector, and phosphatidylethanolamine and unesterifie d cholesterol were essentially neutral diluents. Other experiments showed t hat diacylglycerol and some of its structural analogues also were important , positive effectors of enzyme binding and that addition of ATP to the medi um increased their effects, The combined results of the study suggest that the enzyme may bind to vesicles via at least two types of binding sites: on e type that requires anionic phospholipids and is enhanced by Mg2+ but inhi bited by phosphatidylcholine, and one type that requires diacylglycerol and is enhanced by ATP.