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

在激动剂刺激的细胞中，二酰基甘油（DG）激酶在第二信使DG的代谢中起核心作用，DG在受体激活后从磷酸肌苷中释放出来。我们打算用纯化的酶和兔抗80 kDa DG激酶的抗体来表征这种脂质激酶。主要研究成果如下：1)猪不同组织中DG激酶的免疫化学研究采用免疫印迹法和免疫沉淀法对猪不同组织中DG激酶进行检测。在检测的组织中，我们先前从脑中纯化的免疫反应性80 kDa激酶(J。医学杂志。化学，258,1767,-83)仅在胸腺中发现，在脾脏中发现的程度要小得多。其他组织，如血小板、心脏、肾脏和肝脏，即使有，也只含有很少的免疫反应酶。凝胶过滤的胞质酶有三个主要的活性峰，分别对应280、120和80 kDa。更有趣的是，血小板激酶几乎完全由非免疫反应性的120 kDa组成，而明显缺乏免疫反应性的80 kDa激酶。非免疫反应性280 kDa激酶在淋巴组织中大量存在，与80 kDa酶相比，其热稳定性要高得多。需要纯化不同形式的激酶来阐明它们在每个组织中的生理意义。DG激酶和PKC （Protein Kinase C， PKC）从猪胸腺中高纯度分离得到。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抗体免疫沉淀来证实DGK的磷酸化。我们发现DGK作为PKC的良好底物，DGK几乎实现了化学计量磷酸化。PKA对DGK的磷酸化作用非常有限。磷酸化的DGK在pI 6.5附近呈现宽频带。两种蛋白激酶磷酸化DGK分子的不同位点。部分磷酸化DGK的Vmax增加约2倍。DGK和PKC均与磷脂酰丝氨酸结合。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