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[Properties of creatine kinase from skeletal muscle mitochondria]
Abstract:
Creatine kinase from pigeon breast muscle was obtained in a homogeneous (as evidenced from polyacrylamide gel SDS electrophoresis) state. The molecular mass of the enzyme monomer is 43,000. Ultracentrifugation in a sucrose density gradient and gel filtration revealed that the enzyme is present in solution as a mixture of two major forms, i.e., octamer and dimer, which differ in their activity. The decrease of ionic strength from 0.25 to 0.02 results in reversible dissociation of the octameric form. A temperature rise from 5 degrees to 20 degrees C or the nature of monovalent anions (e.g., Cl-, CH3COO-, NO3-) and cations (K+, Na+) present in the medium do not influence the distribution of oligomeric forms. At pH 6.0 the major form is represented by the octamer; its dissociation is caused by an increase of pH. The octamer dissociation occurs in a mixture of substrates of the creatine kinase reaction in the presence of Mg2+; no such dissociation is observed in the absence of Mg2+ and in the presence of each of the reaction substrates. The non-interacting pair of substrates--ADP and creatine--causes the dissociation of the octamer in the presence of nitrate ions but not acetate. It is concluded that the dissociating effect of substrates is due to the conformational changes of subunits during catalysis. At physiological concentrations of nucleotide substrates the degree of octamer dissociation depends on the ratio of creatine phosphate and creatine concentrations, as well as on the presence of chlorine and phosphate ions. A qualitative estimation of the rate of pH- and substrate-dependent dissociation of creatine kinase octamer revealed that under the given experimental conditions the pH-dependent dissociation is completed within hours, whereas the substrate-dependent one--within seconds or minutes. According to its properties, mitochondrial creatine kinase from pigeon breast muscle is close to its bovine heart counterpart; the observed differences were found to be quantitative.