[Essential carboxylic groups of active sites of creatine kinase M- and M'-subunits in rabbit skeletal muscles].
The influence of carboxylic group modification with N-cyclohexyl-N'-beta-(4-methyl-morpholine)ethylcarbodiimide on the activity of creatine kinase was examined. The modification rate for M- and M'-subunits of the enzyme depends on the reagent concentration and the presence of Mg2+ ions. The process is described by linear dependences of logarithms of activity vs. time, indicating a pseudofirst order of reactions. The reagent inactivates M- and M'-subunits of the enzyme at approximately the same rate, modification being characterized by rate constants of 0.17 min-1 and K1 of 0.17 M. Mild alkaline hydrolysis (pH 9.2) of the modified enzyme leads to partial (30-60%) restoration of its activity. The addition of [14C]glycine methyl ester results in the irreversible incorporation of radioactivity into the protein. AMP and ATP gamma-(p-azido-anilide), the inhibitors acting competitively to nucleotide substrates, protect the enzyme against inactivation with the reagent, whereas creatine and creatine phosphate exert no influence on the modification rate of the enzyme. It is suggested that modification affects the aspartate or glutamate carboxyls at or near the ATP-binding sites in the M- and M'-subunits of the enzyme.
D000818:Animals; D001665:Binding Sites; D003028:CME-Carbodiimide / Q000494:pharmacology; D002264:Carboxylic Acids; D055598:Chemical Phenomena; D002621:Chemistry; D003402:Creatine Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007202:Indicators and Reagents; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D011817:Rabbits
D002264:Carboxylic Acids; D007202:Indicators and Reagents; D003028:CME-Carbodiimide; D003402:Creatine Kinase
Sushchestvennye dlia aktivnosti karboksil'nye gruppy aktivnykh tsentrov M- i M'-sub''edinits kreatinkinazy iz skeletnykh myshts krolika.