Macromolecular characterization of muscle membranes. Endogenous membrane kinase and phosphorylated protein substrate from normal and denervated muscle

Light density membranes derived from the 'microsomal' fraction of rat skeletal muscle contained an endogenous protein kinase which catalyzed the phosphorylation of an endogenous membrane substrate. No other membrane fraction contained any significant protein kinase activity. The optimal specific activity of the enzyme in these membranes was 350 pmol/mg/min. The endogenous muscle membrane protein kinase required magnesium, was stimulated by micromolar concentrations of calcium, had a pH optimum between 7.0 and 7.5, and demonstrated a Km for ATP of 2.6 x 10^-5M. The enzyme was markedly heat labile and demonstrated a linear Arrhenius plot with an apparent energy of activation of 12,100 cal/mol. There was no stimulation by cyclic nucleotides; and neither monovalent cations nor various neurotransmitters exerted any effect. It is presently unclear where the membranes exhibiting protein phosphorylation are localized within the muscle fiber. Enzyme markers suggest that these membranes are not derived from sarcolemma or sarcoplasmic reticulum but may originate in transverse tubules. The membrane phosphorylation was largely confined to a polypeptide with an apparent mol wt of 28,000. Phosphorylation could also be detected in a lower mol wt substrate as well as 2 polypeptides with apparent mol wt of 95,000 and 56,000. The Mr 28,000 endogenous protein kinase substrate was isolated by preparative gel electrophoresis in sodium dodecyl sulfate. High voltage electrophoresis of a partial acid hydrolysate of the phosphorylated Mr 28,000 substrate identified the phosphate bond to be that of phosphoserine. The amino acid composition of the substrate was neither strongly acidic nor basic. It had a high content of glycine, glutamic acid, serine, and lysine. Hydrophobic residues constituted only 45% of the total composition. Following muscle denervation for 10 days, there was a significant decrease in the amount of the Mr 28,000 polypeptide as well as the extent of phosphorylation.