PHOSPHOCREATINE-DEPENDENT PROTEIN-PHOSPHORYLATION IN RAT SKELETAL-MUSCLE

Phosphocreatine (PCr) was found to alter the phosphorylation state of two proteins of apparent molecular masses 18 and 29 kDa in dialysed cell-free extracts of rat skeletal muscle in the presence of [gamma-P-32]ATP. The 29 kDa protein was identified as phosphoglycerate mutase (PGM), phosphorylated at the active-site histidine residue by 2,3-bisphosphoglycerate (2,3-biPG). 2,3-biPG labelling from [gamma-P-32]ATP occurred through the concerted action of phosphoglycerate kinase and 2,3-bisphosphoglycerate mutase. PCr-dependent labelling, which required creatine kinase, resulted from a shift in the phosphoglycerate kinase equilibrium towards 1,3-bisphosphoglycerate (1,3-biPG) synthesis, ultimately resulting in an increase in available [2-P-32]2,3-biPG. The maximal catalytic activity of PGM was unaffected by PCr. The 18 kDa protein was transiently phosphorylated at a histidine residue, probably by 1,3-biPG. No proteins of this monomeric molecular mass are known to bind 1,3-biPG, suggesting that the 18 kDa protein is an undescribed phosphoenzyme intermediate. Previous observations of 2- and 3-phosphoglycerate-dependent protein phosphorylation in cytosolic extracts [Ueda & Plagens (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1229-1233; Pek, Usami, Bilir, Fischer-Bovenkerk & Ueda (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4294-4298], attributed to the action of novel kinases, are likely to represent phosphoenzyme intermediates labelled by bisphosphorylated metabolites in a similar manner.