Analysis of a novel diacylglycerol kinase from Dictyostelium discoideum: DGKA.
Analysis of a novel diacylglycerol kinase from Dictyostelium discoideum: DGKA.
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盘基网柄菌中一种新型二酰甘油激酶的分析：DGKA。
DOI：
10.1021/bi0507276
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发表时间：
2005
期刊：
影响因子：
2.9
通讯作者：
Raben,DanielM
中科院分区：
文献类型：
--
作者：
Ostroski,Michele;Tu-Sekine,Becky;Raben,DanielM
二酰基甘油激酶（DGKs）催化atp依赖的二酰基甘油磷酸化生成磷脂酸，并已在原核和真核生物中进行了研究。最近，在indictyosteliumdisideum中发现了一种与人类DGK-θ非常相似的蛋白DGKA。当使用中链二酰基甘油，1,2-二辛醇-sn-甘油（DiC8）进行测定时，它已被证明具有DGK活性。全面了解DGK的催化和调控机制，以及生理作用，需要了解其生化和动力学性质。本报告对DGKA的这些特性进行了分析。该酶以Michaelis - Menten方式催化DiC8和另一个中链DAG DiC6（1,2-二己醇-sn-甘油）的磷酸化。有趣的是，使用生理上相关的长链dag的DGKA动力学取决于底物表面浓度和使用的洗涤剂。当表面底物浓度等于或低于3.5 mol %时，DGKA对辛基葡萄糖苷混合胶束中的散装底物浓度（1,2-二油基-sn-甘油）表现出Michaelis - Menten动力学。然而，在较高的表面浓度下，初始速度与基体浓度呈s型关系。相比之下，DGKA在Triton X-100混合胶束的所有表面浓度下都表现出相对于体积底物浓度的s型动力学。最后，我们发现磷脂酰丝氨酸（PS）和磷脂酸（PA）显著增强了DGKA的催化活性。
Diacylglycerol kinases (DGKs) catalyze the ATP-dependent phosphorylation of diacylglycerols to generate phosphatidic acid and have been investigated in prokaryotic and eukaryotic organisms. Recently, a protein that is significantly similar to human DGK-θ, DGKA, was identified inDictyosteliumdiscoideum.It has been shown to possess DGK activity when assayed using a medium-chain diacylglycerol, 1,2-dioctanoyl-sn-glycerol (DiC8). A complete understanding of DGK catalytic and regulatory mechanisms, as well as physiological roles, requires an understanding of its biochemical and kinetic properties. This report presents an analysis of these properties for DGKA. The enzyme catalyzes the phosphorylation of DiC8, and another medium-chain DAG, DiC6 (1,2-dihexanoyl-sn-glycerol), in a Michaelis−Menten manner. Interestingly, the kinetics of DGKA using physiologically relevant long-chain DAGs was dependent on substrate surface concentration and the detergent that was used. DGKA displayed Michaelis−Menten kinetics with respect to bulk substrate concentration (1,2-dioleoyl-sn-glycerol) in octyl glucoside mixed micelles when the surface substrate concentration was at or below 3.5 mol %. At higher surface concentrations, however, there was a sigmoidal relationship between the initial velocity and bulk substrate concentration. In contrast, DGKA displayed sigmoidal kinetics with respect to bulk substrate concentrations at all surface concentrations in Triton X-100 mixed micelles. Finally, we show the catalytic activity of DGKA was significantly enhanced by phosphatidylserine (PS) and phosphatidic acid (PA).