Thomas W E, Glomset J A
Howard Hughes Medical Institute, Department of Medicine, Regional Primate Research Center, University of Washington, Seattle 98195-7370, USA.
Biochemistry. 1999 Mar 16;38(11):3310-9. doi: 10.1021/bi982566u.
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 enzyme 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 anionic phosphoglycerides, and maximal binding occurred (in the presence of MgCl2) when the vesicles contained anionic phosphoglycerides alone. When increasing amounts of phosphatidylcholine were included in phosphatidylserine-containing vesicles, enzyme binding to the vesicles decreased by as much as 1000-fold. However, when increasing amounts of phosphatidylethanolamine were included in phosphatidylserine-containing vesicles, little change in binding occurred until the concentration of phosphatidylserine was reduced to below 25 mol %. These results and results obtained with vesicles that contained various mixtures of anionic phosphoglycerides, phosphatidylcholine, phosphatidylethanolamine, and unesterified cholesterol provided evidence that anionic phosphoglycerides were positive effectors of binding, phosphatidylcholine was a negative effector, and phosphatidylethanolamine and unesterified cholesterol were essentially neutral diluents. Other experiments showed that diacylglycerol and some of its structural analogues also were important, positive effectors of enzyme binding and that addition of ATP to the medium 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: one type that requires anionic phospholipids and is enhanced by Mg2+ but inhibited by phosphatidylcholine, and one type that requires diacylglycerol and is enhanced by ATP.
我们研究了膜脂、MgCl₂和ATP对可溶性二酰基甘油激酶与100纳米脂质囊泡结合能力的影响。该酶无法检测到与仅含磷脂酰胆碱的囊泡或含50摩尔%磷脂酰胆碱 + 50摩尔%磷脂酰乙醇胺的囊泡结合。但它确实能与含阴离子磷酸甘油酯的囊泡结合，并且当囊泡仅含阴离子磷酸甘油酯时（在MgCl₂存在下）会出现最大结合。当含磷脂酰丝氨酸的囊泡中磷脂酰胆碱的量增加时，酶与囊泡的结合减少多达1000倍。然而，当含磷脂酰丝氨酸的囊泡中磷脂酰乙醇胺的量增加时，在磷脂酰丝氨酸浓度降至25摩尔%以下之前，结合几乎没有变化。这些结果以及用含阴离子磷酸甘油酯、磷脂酰胆碱、磷脂酰乙醇胺和未酯化胆固醇的各种混合物的囊泡所获得的结果表明，阴离子磷酸甘油酯是结合的正效应物，磷脂酰胆碱是负效应物，而磷脂酰乙醇胺和未酯化胆固醇基本上是中性稀释剂。其他实验表明，二酰基甘油及其一些结构类似物也是酶结合的重要正效应物，并且向培养基中添加ATP会增强它们的作用。该研究的综合结果表明，该酶可能通过至少两种类型的结合位点与囊泡结合：一种类型需要阴离子磷脂，受Mg²⁺增强但受磷脂酰胆碱抑制，另一种类型需要二酰基甘油并受ATP增强。
