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.
