3-PHOSPHOGLYCERATE-DEPENDENT PROTEIN PHOSPHORYLATION

总的目标是了解糖酵解中间体的性质 3-磷酸甘油酸依赖性蛋白激酶，一类新的蛋白 激酶，并提供探针，这将有助于阐明 3-磷酸甘油酸依赖性蛋白磷酸化 系统和神经功能，这是严重依赖于 葡萄糖代谢。 为了实现这一目标，我建议（1） 纯化和表征这些新型蛋白激酶， 脑内主要内源性底物，（2）研究其 分布（种属、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 3-磷酸甘油酸依赖性蛋白磷酸化 内源底物 激酶和底物将从 通过（a）硫酸铵分级， (b)凝胶过滤，（c）使用离子交换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 色谱聚焦柱。 蛋白质磷酸化将进行 用（γ-32 P）ATP和SDS-聚丙烯酰胺凝胶电泳分析 和放射自显影。 这项研究预计将开辟一条新的途径， 蛋白质磷酸化，并可能提供新的见解的机制 一些糖酵解调节的神经功能和其他 代谢和细胞过程，并可能进入某些方面， 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.