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 [32P]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 Sepharose CL-6B, and (b) comigration of the area containing [32P]phosphate and Coomassie-Blue-staining protein following SDS-polyacrylamide gel electrophoresis of homogenous PK-anoxic. The [32P]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 32P-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 32P-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.
D000332:Aerobiosis; D000469:Alkaline Phosphatase / Q000378:metabolism; D000693:Anaerobiosis; D000818:Animals; D002847:Chromatography, Agarose; D004591:Electrophoresis, Polyacrylamide Gel; D008974:Mollusca / Q000201:enzymology*; D009132:Muscles / Q000201:enzymology; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011770:Pyruvate Kinase / Q000378:metabolism*; D013379:Substrate Specificity
D011494:Protein Kinases; D011770:Pyruvate Kinase; D000469:Alkaline Phosphatase