Developmental regulation of creatine kinase isoenzymes in myogenic cell cultures from chicken. Biosynthesis of creatine kinase subunits M and B.

At various intervals after plating, cultures were exposed to 2-h pulses of tritium-labeled leucine. For determination of incorporated radioactivity, the creatine kinases were isolated by specific immunoprecipitation. Sodium dodecyl sulfate-gel electrophoresis was used to separate the two types of creatine kinase subunits MCK and B-CK from each other and their radioactivity was determined in the respective gel slices. In cells cultured in standard medium, B-CK synthesis increased sharply up to a maximal incorporation at 72 h and then it decreased. Synthesis of M-CK could not be detected in 24-h cultures, it increased dramatically during the 3rd and 4th day of culture, and its synthesis was maintained at a high level afterwards. Depletion of Ca2+ in such cultures blocked fusion and creatine kinase synthesis still took place but was reduced a-fold for B-CK and 3.5fold for M-CK. Differentiation was inhibited by growth in 5-bromo-2’-deoxyuridine-containing media and incorporation into B-CK was reduced; however, the synthesis of M-CK could not be measured even after prolonged culturing. Rates of synthesis for B-CK in fibroblasts 4 days after subculturing were comparable to the ones measured in myogenic cells at 24 h but much lower than in cells grown in 5bromo-2’-deoxyuridine for the same period. Changing rates of synthesis of the two types of creatine kinase subunits seem to govern the creatine kinase isoenzyme transition. The early increase in B-CK synthesis could be a property of differentiating myogenic cells, whereas a high rate of M-CK synthesis is characteristic for myotubes.