Phosphorylation in vivo of red‐muscle pyruvate kinase from the channelled whelk, Busycotypus canaliculatum , in response to anoxic stress

That red muscle pyruvate kinase from anoxic Busycotypus canaliculatum (PK‐anoxic) is a phosphoprotein was demonstrated by the anoxia‐dependent, in vivo , covalent incorporation of injected [ 32 P]orthophosphate into the enzyme molecule. Specificity in labelling of PK‐anoxic was strongly suggested by: (a) coincidental elution of pyruvate kinase activity and radioactivity following chromatography of purified PK‐anoxic on Sepha‐rose CL‐6B, and (b) comigration of the area containing [ 32 P]phosphate and Coomassie‐Blue‐staining protein following SDS‐polyacrylamide gel electrophoresis of homogenous PK‐anoxic. The [ 32 P]phosphate content of the enzyme was calculated to be 7.3 mol phosphate/mol enzyme (233 kDa, 180 units/mg protein). Evidence for the reversibility of this phosphorylation was provided by the consistent kinetic similarities between purified red muscle pyruvate kinase from aerobic animals (PK‐aerobic) and homogenous, unlabelled, alkaline phosphatase treated PK‐anoxic. Comparison of the electrophoretic mobilities of products derived from acid hydrolysis of purified 32 P‐labelled PK‐anoxic with authentic substances suggest the presence of an O ‐phospho‐ L ‐threonine residue in the protein. That this residue plays a probable role in an interconversion mechanism was suggested by the lack of phosphate exchange of homogenous 32 P‐labelled PK‐anoxic in the presence of all substrates. A possible role of protein phosphorylation as a mechanism for the overall control of molluscan anaerobic metabolism is suggested.