Fanani Maria Laura, Topham Matthew K, Walsh James P, Epand Richard M
Department of Biochemistry and Biomedical Sciences, McMaster University Health Science Center, Hamilton, Ontario L8N 3Z5, Canada.
Biochemistry. 2004 Nov 23;43(46):14767-77. doi: 10.1021/bi049145z.
Diacylglycerol kinase (DGK) isoforms alpha and zeta were extracted from transfected cells that overexpressed these enzymes. We determined the lipid dependence of the binding of these isoforms to liposomes. The modulation by lipid of the rate of phosphorylation of diacylglycerol by these enzymes was also measured. Incorporation of phosphatidylethanolamine into the liposomes resulted in an increased partitioning of both isoforms of DGK to the membrane as well as an increased catalytic rate. We demonstrate that the increased catalytic rate is a consequence of both increased portioning of the enzyme to the membrane and increased catalytic activity of the membrane-bound form. DGKalpha, a calcium-dependent isoform, can be activated in a calcium-independent fashion in the presence of phosphatidylethanolamine. Similar effects are observed with cholesterol. In contrast, sphingomyelin inhibits the activity of both isoforms of DGK. Our results demonstrate that the translocation to membranes and activity of DGKalpha and DGKzeta are modulated by the composition and properties of the membrane. The enzymes are activated by the presence of lipids that promote the formation of inverted phases. However, the promotion of negative curvature is not the sole factor contributing to the lipid effects on enzyme binding and activity. A truncated form of DGKalphalacking both the E-F hand and the recoverin homology domain is constitutively active and is not further activated by any of the lipids tested or by calcium. However, a truncated form lacking only the recoverin homology domain is partially activated by either calcium or certain lipids.
从过表达二酰甘油激酶（DGK）α和ζ亚型的转染细胞中提取这两种亚型。我们测定了这些亚型与脂质体结合的脂质依赖性。还测量了脂质对这些酶催化二酰甘油磷酸化速率的调节作用。将磷脂酰乙醇胺掺入脂质体导致DGK的两种亚型在膜中的分配增加以及催化速率提高。我们证明，催化速率的提高是酶在膜中分配增加和膜结合形式催化活性增加共同作用的结果。DGKα是一种钙依赖性亚型，在磷脂酰乙醇胺存在下可通过非钙依赖方式被激活。胆固醇也观察到类似的效果。相反，鞘磷脂抑制DGK的两种亚型的活性。我们的结果表明，DGKα和DGKζ向膜的转位及其活性受膜的组成和性质调节。这些酶被促进反相形成的脂质激活。然而，促进负曲率并不是脂质对酶结合和活性产生影响的唯一因素。一种缺失E-F手和恢复蛋白同源结构域的截短形式的DGKα具有组成型活性，并且不会被任何测试的脂质或钙进一步激活。然而，仅缺失恢复蛋白同源结构域的截短形式可被钙或某些脂质部分激活。
