Characterization of the Phosphorylase b to a Converting Activity in Skeletal Muscle Extracts of Mice with the Phosphorylase b Kinase Deficiency Mutation

Abstract

Abstract The phosphorylase b kinase deficiency mutation carried in I strain mice is known to result in a marked reduction of phosphorylase b to a converting activity in skeletal muscle (Lyon, J. B., Jr. (1970) Biochem. Genet. 4, 169–185). As assayed in muscle extracts I strain had 0.3 % of the phosphorylase kinase activity found in the control strains C57BL/St and Ha/ICR (a Swiss-Webster strain). Nevertheless I strain mouse skeletal muscle contained protein that cross-reacted with an antiserum to rabbit muscle phosphorylase kinase, indicating the presence of a structurally altered enzyme protein. The amount of cross-reacting material was 118% of that found in extracts of skeletal muscle from control strains of mice. The mutant activity was shown to have the same pH optimum, Km for phosphorylase b, and Mg2+ATP concentration dependence as the control strain phosphorylase b kinase activity, and both activities were inhibited by millimolar concentrations of Ca2+, F-, and ethylene glycol bis(β-amino-ethyl ether)-N,N'-tetraacetic acid (EGTA). The mutant phosphorylase b to a converting activity incorporated 32P from [γ-32P]ATP into phosphorylase and the labeled phosphorylase a was isolated. The addition of ATP (10 mm), Mg2+ (50 mm), and Ca2+ (4 mm) to a protein-glycogen complex isolated by differential centrifugation of I strain skeletal muscle extracts increased phosphorylase activity as the -AMP: +AMP ratio from 0.021 to 0.075 within 20 s, as compared to an increase in activity from 0.08 to 0.85 in the protein-glycogen complex isolated from the control strain. In addition, incubation of diaphragm muscle from phosphorylase kinase-deficient animals with 4 µm isoproterenol for 10 min produced a conversion of phosphorylase b to a. The -AMP: +AMP ratio increased from 0.043 to 0.078 in mutant diaphragms, as compared to 0.060 to 0.168 in the control strain. Thus, functional phosphorylase kinase activity exists in skeletal muscle of animals with the phosphorylase b kinase deficiency mutation. Since epinephrine has been shown to stimulate glycogen breakdown in I strain gastrocnemius muscle (Lyon J. B., Jr., and Porter, J. (1963) J. Biol. Chem. 238, 1–11), conversion of phosphorylase b to a may be a necessary step in catecholamine-stimulated glycogenolysis in I strain skeletal muscle as it is in normal skeletal muscle.