Purification and properties of adenosine triphosphate-creatine phosphotransferase from muscle of the dogfish Scylliorhinus canicula.
1. Creatine kinase occurs in high concentration in the soluble proteins of dogfish muscle. A fourfold purification gives essentially pure enzyme but with a low specific activity. This appears to be a property of the native enzyme and not a result of the isolation procedures used. 2. The amino acid composition is similar to that of other phosphagen kinases, but the enzyme differs from mammalian creatine kinases in having four thiol groups readily reactive towards 5,5'-dithiobis-(2-nitrobenzoic acid). Titration of two thiol groups is accompanied by almost complete loss of activity. The remaining two thiol groups react at different rates, suggesting that modifying the third thiol group affects the reactivity of the fourth thiol group. 3. The enzyme is markedly protected against inactivation by iodoacetamide by MgATP or MgADP. Addition of creatine to MgADP decreases protection, but the further addition of Cl(-) restores protection to the original value. The quaternary MgADP-creatine-enzyme-nitrate complex protects very strongly as is found for the rabbit enzyme. The involvement of the conformational state of the enzyme in such effects is discussed. 4. Creatine kinase from both dogfish and rabbit is equally sensitive to urea denaturation. Urea protects the dogfish enzyme by about 9% against inhibition by iodoacetamide. 5. The formation of a hybrid between the dogfish and rabbit enzymes in vitro has been demonstrated. 6. At high substrate concentrations the dogfish enzyme shows apparent ordered kinetics. The effect of temperature on V(max.) and the Michaelis constants for MgATP and creatine were determined. These and changes in the apparent activation energy suggest that limited adaptation has occurred commensurate with physiological need.
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000577:Amides; D000818:Animals; D003401:Creatine; D003402:Creatine Kinase / Q000302:isolation & purification*; D006824:Hybridization, Genetic; D007461:Iodoacetates; D007700:Kinetics; D008274:Magnesium; D009132:Muscles / Q000201:enzymology*; D012754:Sharks / Q000378:metabolism*
D000577:Amides; D007461:Iodoacetates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D003402:Creatine Kinase; D008274:Magnesium; D003401:Creatine