Isozymes of phosphorylase kinase in rabbit skeletal muscle. Functional implications of differences in phosphorylase kinase and phosphorylase activities in individual muscle fibers.
Immunological and microanalytical methods were used to investigate the two isozymes of phosphorylase kinase, enzyme w and enzyme r, in psoas major and tibialis anterior muscles. Peptide mapping experiments indicated that the alpha subunit of enzyme w and alpha' subunit of enzyme r were structurally very similar. Both subunits were completely immunoprecipitated from muscle extracts with an antibody specific for the beta subunit of the kinase, indicating that alpha and alpha' subunits are completely assembled with beta subunits in adult muscle fibers. The relative amounts of enzymes w and r in single fibers were determined from amounts of alpha and alpha' subunits, which were detected by immunoblotting. Phosphorylase kinase and phosphorylase activities were measured in the same fibers, as well as in individual fibers from diaphragm and soleus muscles. Slow oxidative fibers were found to contain low levels of enzyme r, but almost no enzyme w. Considerably more enzyme r was present in fast oxidative-glycolytic fibers. Fast glycolytic fibers contained the most enzyme w, and the highest levels of enzyme r were found in a subgroup of such fibers. Interestingly, more than half of the fast glycolytic fibers analyzed contained both isozymes. In these fibers phosphorylase was positively correlated with enzyme w, but negatively correlated with enzyme r. Total kinase activity ranged 30-fold from the highest in one of the psoas fibers to the lowest in one of the soleus fibers and was closely correlated with the phosphorylase levels. In psoas and soleus fibers, calculated absolute maximal rates for phosphorylase b to a conversion varied almost 2,500-fold.
D000818:Animals; D000918:Antibody Specificity; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007163:Immunosorbent Techniques; D007527:Isoenzymes / Q000032:analysis*; D046911:Macromolecular Substances; D008291:Malate Dehydrogenase / Q000032:analysis; D008832:Microchemistry; D009132:Muscles / Q000201:enzymology*; D010764:Phosphorylase Kinase / Q000032:analysis*; D011817:Rabbits
D007527:Isoenzymes; D046911:Macromolecular Substances; D008291:Malate Dehydrogenase; D010764:Phosphorylase Kinase