Purification and characterization of naturally occurring and in vitro induced multiple forms of MM creatine kinase.
MM creatine kinase purified from tissue exists as a single form but upon release into the plasma exhibits three forms which, based on increasing anodal migration on polyacrylamide gel electrophoresis, are referred to as MM1, MM2, and MM3. The three forms were isolated in pure form from plasma in sufficient quantities for biochemical and physiological studies. Chromatofocusing results show that MM1, MM2, and MM3 have distinct isoelectric points of 7.58, 7.43, and 7.30, respectively. Analysis on high pressure liquid chromatography showed MM1 and tissue creatine kinase (MMt) to have identical peptide maps, but MM2 and MM3 were different in at least one amino acid. Tissue MM creatine kinase was converted in vitro to MM2 and MM3 after incubation in plasma. Conversion was unaffected by nonspecific proteases but inhibited by benzoylarginine, epsilon-amino caproic acid, and guanidinoethylmercaptosuccinic acid, specific inhibitors of carboxypeptidase N. Digestion of purified tissue MM creatine kinase with carboxypeptidases N and B produced MM2 and MM3 which were identical with the forms produced in vivo. Hybridization experiments with MM1 plus MM3 produced all three forms, as did tissue MM creatine kinase plus MM2. MM2 was also able to produce all three forms in the experiments. The plasma half-life of tissue MM creatine kinase or MM1 was shown to be about one-half that of MM3. The results suggest that hydrolytic cleavage of a basic amino acid, presumably by carboxypeptidase N, is responsible for conversion of MMt to MM2 and MM3. COOH-terminal amino acid analysis demonstrated the COOH-terminal amino acid of canine and rabbit MM1 to be lysine. Thus, we conclude that cleavage of the COOH-terminal lysine from MMt by serum carboxypeptidase N generates two additional MM creatine kinase isoforms with prolonged plasma clearance rates.
D000818:Animals; D002268:Carboxypeptidases; D003402:Creatine Kinase / Q000097:blood* / Q000302:isolation & purification; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D006207:Half-Life; D007525:Isoelectric Focusing; D007527:Isoenzymes; D007700:Kinetics; D009203:Myocardial Infarction / Q000201:enzymology; D011817:Rabbits
D007527:Isoenzymes; D003402:Creatine Kinase; D002268:Carboxypeptidases