The proposed research will extend initial observations with respect to alterations in muscle plasma membrane phospholipids and phosphoproteins induced by direct addition of physiological concentrations of insulin to these membranes in the presence of ATP and MG++. The phospholipids and phosphoproteins whose phosphorylation state is altered by insulin will be isolated and identified using thin layer chromatography, high pressure liquid chromatography and mass spectroscopy for th phospholipids, and polyacrylamide gel electrophoresis, isoelectric focussing, high pressure liquid chromatography and other techniques of identification and purification for the proteins. Following identification of the phospholipids and phosphoproteins, they or their dephosphorylated products will be used as substrates to identify and purify the enzymes (protein kinases, phosphoprotein phosphatases and enzymes of phospholipid metabolism) whose activity is altered by insulin. """"""""Pulse-chase"""""""" type experiments will be employed initially to determine whether insulin affects phosphate-donating or phosphate-removing enzymes. If these experiments indicate that insulin affects a protein kinase, the amino acid sequence around the phosphorylation site(s) in the """"""""insulin-specific"""""""" phosphoproteins will be determined and the data used to synthesize model oligopeptides for identification and assay of an """"""""insulin-sensitive"""""""" protein kinase. For characterization of an """"""""insulin-sensitive"""""""" P-protein phosphatase, the 32P-labeled """"""""insulin-specific"""""""" phosphoproteins (or 32P-tryptic fragments thereof) would be used as substrates. The mechanisms by which interaction of insulin with its muscle plasma receptors leads to alterations in the activities of these enzymes will then be explored utilizing both intact plasma membranes and purified preparations of the insulin receptor and the other components involved. In particular, the possible involvement of a Ca++ phospholipid-sensitive protein kinase or of the tyrosine kinase associated with the insulin receptor will be examined. Finally, attempts will be made to determine the role of the phospholipid and phosphoprotein changes in the in vivo effects of insulin on membrane transport and intracellular metabolism. These experiments will focus on the translocation of glucose carriers to the plasma membrane, and the activation of glycogen synthase through dephosphorylation.
