Physiological function of lipid-phosphorylating enzymes and their mechanism of action.

在激动剂刺激的细胞中，二酰甘油(DG)激酶在第二信使DG的代谢中起着核心作用，DG是在受体激活时从磷脂酰肌醇释放出来的。我们打算用纯化的酶和兔抗80 kDa DG激酶的抗体来鉴定这种脂蛋白激酶。1)猪不同组织中DG激酶的免疫化学研究采用免疫印迹和免疫沉淀法对不同组织中的DG激酶进行检测。在被检测的组织中，免疫活性的80 kDa激酶是我们先前从大脑中纯化的(J.Biol.化学。258、1767、-83)仅在胸腺中发现，在脾中的程度要小得多。其他组织，如血小板、心脏、肾脏和肝脏，几乎没有免疫活性酶。细胞质酶的凝胶过滤显示出三个主要的活性峰，分别对应于280、120和80 kDa。在…中间，血小板激酶更有趣的是，几乎完全由非免疫反应性120 kDa物种组成，明显缺乏免疫反应性80 kDa激酶。非免疫反应性280 kDa酶在淋巴组织中含量丰富，与80 kDa酶相比具有更高的热稳定性。2)在体外，从猪胸腺中高纯度地提纯了DG激酶和蛋白激酶C(PKC)的磷酸化。由于两种酶在SDS-PAGE上显示相同的迁移率(80 KDa)，因此必须用抗DGK抗体的免疫沉淀来证实DGK的磷酸化。我们发现DGK是一种很好的PKC底物，并且DGK几乎实现了化学计量比的磷酸化。PKA对DGK的磷酸化程度非常有限。磷酸化的DGK在等电点6.5附近有一条宽带。这两种蛋白激酶使DGK分子的不同位点磷酸化。部分磷酸化的DGK的Vmax增加了近两倍。在该体系中，DGK和PKC均与磷脂酰丝氨酸结合。较少

In the agonist-stimulated cells, diacylglycerol(DG) kinase plays a central role in the metabolism of a second messenger, DG, which is released from phosphoinositides upon receptor activation. We intend to characterize this lipid kinase by employing purified enzymes and rabbit antibody raised against 80 kDa DG kinase. The projects and major findings are summarized as follows:1) Immunochemical Studies on DG Kinase in Different Pig Tissues DG kinase from different pig tissues was examined by immunoblot as well as immunoprecipitation. Among the tissues examined, the immunoreactive 80 kDa kinase previously purified by us from the brain(J. Biol. Chem. 258, 1767, -83) was found only in the thymus and, to a much lesser extent, in the spleen. Other tissues like platelets,heart, kidney and liver, contained little, if any, immunoreactive enzymes. A gel filtration of cytosolic enzymes showed three major activity peaks, corresponding apparently to 280, 120 and 80 kDa. The platelet kinase was, inter … More estingly, consisted almost exclusively of nonimmunoreactive 120 kDa species with an apparent lack of immunoreactive 80 kDa kinase. The nonimmunoreactive 280 kDa kinase was abundant in the lymphoid tissues, and was much more heat-stable when compared with the 80 kDa enzyme. Purification of different kinase forms is required to elucidate their physiological implication in each tissur.2) In vitro Phosphorylation of DG Kinase by Protein Kinase C DG kinase and protein kinase C(PKC) were highly purified from pig thymus. Since both enzymes showed the same mobility(80 kDa) upon SDS-PAGE, the phosphorylation of DGK had to be confirmed by immunoprecipitation with anti-DGK antibody. We found that DGK serves as a good substrate for PKC, and that almost stoichiometric phosphorylation of DGK was achieved. Phosphorylation of DGK by PKA occurred to a very limited extent. Phosphorylated DGK showed a broad band around pI 6.5. The two protein kinases phosphorylated different sites of DGK molecule. The Vmax of partially phosphorylated DGK increased approximately two-fold. Both DGK and PKC were bound to phosphatidylserine in the system. Less