Besterman J M, Pollenz R S, Booker E L, Cuatrecasas P
Proc Natl Acad Sci U S A. 1986 Dec;83(24):9378-82. doi: 10.1073/pnas.83.24.9378.
Diacylglycerol-induced translocation of diacylglycerol kinase (ATP:1,2-diacylglycerol 3-phosphotransferase, EC 2.7.1.107) from the soluble to the membrane-bound compartments was demonstrated both in crude tissue homogenates and in a reconstituted enzyme-membrane model system. In homogenates of either rat brain or liver, incubation with diacylglycerol or phospholipase C, but not phospholipase A2 or phospholipase D, resulted in the translocation of diacylglycerol kinase activity from the soluble to the particulate fraction. This observation formed the basis for the first step in a two-step purification of diacylglycerol kinase. Enzyme extracted in 1 M salt from membranes of rat brain homogenates made in the presence of phospholipase C was purified further by affinity chromatography on a column containing phosphatidylserine, diacylglycerol, and cholesterol immobilized in polyacrylamide. This step yielded an enzyme preparation (step 2 enzyme) that was 500- to 750-fold purified (relative to the tissue homogenate) and required phosphatidylserine for stability. All other lipids tested failed to stabilize the enzyme. The properties of the enzyme preparation were similar to those of mammalian diacylglycerol kinases described by others. Reconstitution experiments showed that the soluble step 2 enzyme bound to inside-out vesicles of human erythrocytes only in the presence of diacylglycerol or phospholipase C but not phospholipase A2 or D. Redistribution of the kinase from soluble to vesicle-bound forms occurred rapidly and was dependent on the concentration of phospholipase C used to treat the vesicles. Physiological concentrations of calcium (50-1000 nM) did not enhance the phospholipase C-mediated translocation of the kinase. Thus, diacylglycerol kinase can translocate from cytosol to membranes in a manner dependent on the content of membrane-bound diacylglycerol but independent of the ambient concentration of calcium.
在粗制组织匀浆和重组酶-膜模型系统中均证实，二酰基甘油可诱导二酰基甘油激酶（ATP：1,2-二酰基甘油3-磷酸转移酶，EC 2.7.1.107）从可溶性区室转位至膜结合区室。在大鼠脑或肝的匀浆中，与二酰基甘油或磷脂酶C孵育，但不与磷脂酶A2或磷脂酶D孵育，会导致二酰基甘油激酶活性从可溶性部分转位至颗粒部分。这一观察结果构成了二酰基甘油激酶两步纯化第一步的基础。在磷脂酶C存在下制备的大鼠脑匀浆膜中，用1 M盐提取的酶通过在含有固定于聚丙烯酰胺中的磷脂酰丝氨酸、二酰基甘油和胆固醇的柱上进行亲和层析进一步纯化。这一步得到了一种酶制剂（第二步酶），其纯化倍数为500至750倍（相对于组织匀浆），且需要磷脂酰丝氨酸来维持稳定性。测试的所有其他脂质均无法稳定该酶。该酶制剂的性质与其他人描述的哺乳动物二酰基甘油激酶相似。重组实验表明，可溶性第二步酶仅在存在二酰基甘油或磷脂酶C时结合到人红细胞的内翻囊泡上，而不与磷脂酶A2或D结合。激酶从可溶性形式重新分布到囊泡结合形式的过程迅速发生，且取决于用于处理囊泡的磷脂酶C的浓度。生理浓度的钙（50 - 1000 nM）不会增强磷脂酶C介导的激酶转位。因此，二酰基甘油激酶可以以依赖于膜结合二酰基甘油含量但独立于钙的环境浓度的方式从细胞质溶胶转位至膜。
