3-PHOSPHOGLYCERATE-DEPENDENT PROTEIN PHOSPHORYLATION

总体目标是了解糖酵解中间体的性质。 3-磷酸甘油依赖的蛋白激酶--一类新的蛋白质 并提供有助于阐明该蛋白的 一种依赖3-磷酸甘油酸的蛋白质磷酸化关系 系统和神经功能，它严重依赖于 葡萄糖的代谢。为了实现这一目标，我建议(1) 提纯和鉴定这些新型蛋白激酶及其两种 大脑中的主要内源性底物，(2)研究它们的 分布(物种、组织、脑区域、细胞和亚细胞) 和个体发育，以及(3)产生那些特异性抑制 这些蛋白的3-磷酸甘油酸依赖的蛋白磷酸化 内源底物。这些酶和底物将从 通过(A)硫酸铵分级得到牛突触胞质组分， (B)凝胶过滤；(C)使用离子的快速蛋白质液相色谱(FPLC) 交换、羟基磷灰石、疏水相互作用、凝胶过滤和 色谱柱。将进行蛋白质的磷酸化 用(伽玛-32P)-三磷酸腺苷和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法分析 和放射自显影。这项研究有望开辟一条新的途径 蛋白质磷酸化，并可能为机制提供新的见解 对于一些糖酵解调节的神经功能和其他 代谢和细胞过程，并潜在地进入到 与低血糖和糖尿病相关的病理生理学。

The overall goal is to understand the nature of the glycolytic intermediate 3-phosphoglycerate-dependent protein kinases, a novel class of protein kinase, and to provide probes which will be helpful in elucidating the relationship between a 3-phosphoglycerate-dependent protein phosphorylation system and a neural function which is critically dependent upon the metabolism of glucose. In order to achieve this goal, I propose to (1) purify and characterize these new types of protein kinases and the two major endogenous substrates in the brain, (2) investigate their distributions (species, tissue, brain regional, cellular and subcellular) and ontogeny, and (3) produce those antibodies which specifically inhibit the 3-phosphoglycerate-dependent protein phosphorylation of these endogenous substrates. The kinases and substrates will be purified from the bovine synaptic cytosol fraction by (a) ammonium sulfate fractionation, (b) gel filtration, (c) fast protein liquid chromatography (FPLC) using ion exchange, hydroxylapatite, hydrophobic interaction, gel filtration, and chromatofocussing columns. The protein phosphorylation will be carried out with (Gamma-32P)ATP and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. This research is expected to open a new avenue for protein phosphorylation, and may provide fresh insight into the mechanism for some of the glycolytically-regulated neural functions and other metabolic and cellular processes, and potentially into some aspect of the pathophysiology associated with hypoglycemia and diabetes.