Kanoh H, Kondoh H, Ono T
J Biol Chem. 1983 Feb 10;258(3):1767-74.
Diacylglycerol kinase (EC 2.7.1.-) was purified 1,650-fold from pig brain cytosol. The purified enzyme showed a single protein band on polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The molecular weight of the kinase was estimated to be 78,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A similar value (76,000) was obtained by Sephadex G-150 gel filtration. The activity of the purified enzyme was markedly enhanced by either deoxycholate or phospholipids. The extent of activation by phospholipids was in the order of phosphatidylcholine greater than lysophosphatidylcholine greater than phosphatidylethanolamine approximately equal to phosphatidylserine greater than sphingomyelin. Other phospholipids and unsaturated fatty acids were ineffective. Phosphatidylcholines from egg yolk and pig brain, and dioleoyl phosphatidylcholine were similarly effective. Saturated phosphatidylcholines with acyl chain lengths shorter than palmitate also gave a considerable activation. The activity with phosphatidylcholine was from 1.5- to 2.5-fold higher than that measured with deoxycholate. A very small amount of phosphatidylinositol or phosphatidylglycerol potently inhibited the phosphatidylcholine-dependent (but not deoxycholate-dependent) kinase activity. The inhibition by phosphatidylinositol was varied according to its molar ratio to phosphatidylcholine. As little as about 2.5 mol per cent of phosphatidylinositol resulted in 50% inhibition of the phosphatidylcholine-dependent kinase activity. The deoxycholate- and phosphatidylcholine-dependent kinase activities showed almost the same Km values for the substrates. In both cases, the apparent Km values for ATP and diacylglycerol were 300 microM and about 60 microM, respectively. The kinase required Mg2+ for its activity. When compared to deoxycholate, phosphatidylcholine was more effective at higher Mg2+ concentrations. The deoxycholate-dependent activity showed a broad pH optimum at around 8.0, whereas the phosphatidylcholine-dependent activity formed a clear peak at pH 7.4.
二酰基甘油激酶（EC 2.7.1.-）从猪脑细胞质中纯化了1650倍。纯化后的酶在有无十二烷基硫酸钠的情况下，在聚丙烯酰胺凝胶电泳上均显示出一条单一的蛋白带。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳估计该激酶的分子量为78,000。通过葡聚糖G-150凝胶过滤获得了类似的值（76,000）。脱氧胆酸盐或磷脂均可显著增强纯化酶的活性。磷脂的激活程度依次为：磷脂酰胆碱＞溶血磷脂酰胆碱＞磷脂酰乙醇胺≈磷脂酰丝氨酸＞鞘磷脂。其他磷脂和不饱和脂肪酸无效。来自蛋黄和猪脑的磷脂酰胆碱以及二油酰磷脂酰胆碱同样有效。酰基链长度短于棕榈酸酯的饱和磷脂酰胆碱也能产生相当程度的激活作用。磷脂酰胆碱的激活活性比脱氧胆酸盐高1.5至2.5倍。极少量的磷脂酰肌醇或磷脂酰甘油可有效抑制磷脂酰胆碱依赖性（而非脱氧胆酸盐依赖性）激酶活性。磷脂酰肌醇的抑制作用因其与磷脂酰胆碱的摩尔比而异。低至约2.5摩尔百分比的磷脂酰肌醇即可导致磷脂酰胆碱依赖性激酶活性50%的抑制。脱氧胆酸盐依赖性和磷脂酰胆碱依赖性激酶活性对底物的Km值几乎相同。在这两种情况下，ATP和二酰基甘油的表观Km值分别为300μM和约60μM。该激酶的活性需要Mg2+。与脱氧胆酸盐相比，磷脂酰胆碱在较高Mg2+浓度下更有效。脱氧胆酸盐依赖性活性在pH约8.0时呈现较宽的最佳pH值，而磷脂酰胆碱依赖性活性在pH 7.4时形成一个明显的峰值。
