[Conditions for reciprocal conversion of oligomeric forms of heart mitochondrial creatine kinase].
A procedure for purifying creatine kinase from bovine heart mitochondria, including enzyme extraction from mitochondria with salt solutions, concentration on cellulose phosphate gel and gel filtration on Sephacryl S-300 has been developed. Using ultracentrifugation in a sucrose density gradient and gel filtration, it was demonstrated that mitochondrial creatine kinase is present in solution as a mixture of two main forms, i. e., an octamer and a dimer. The distribution of the oligomeric forms is not influenced by changes in the ionic strength from 0.02 to 0.25, temperature (5-20 degrees C), freezing-thawing and the nature of monovalent anions (Cl-, NO3-, CH3COO-) and cations (Na+, K+) present in the medium. At pH 6.0, the predominant form is the octamer; an increase in pH induces its dissociation. An equilibrious mixture of the creatine kinase reaction substrates in the presence of Mg2+ also causes octamer dissociation; no dissociation is observed in the absence of Mg2+ or in the presence of one of the substrates. The non-working couple of substrates, Mg-ADP and creatine, causes dissociation of the octamer in the presence of Cl-, but not of CH3COO-. It is assumed that the dissociating effect of the substrates is due to conformational changes in the subunits concomitant with the formation of the creatine kinase active center in the course of catalysis. At physiological concentrations of nucleotide substrates, the degree of octamer dissociation depends on pH, creatine phosphate/creatine ratio and Pi. It is assumed that the above factors may regulate the reversible conversion of the octamer into the dimer in vivo.
D000818:Animals; D002417:Cattle; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D003402:Creatine Kinase / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D008929:Mitochondria, Heart / Q000201:enzymology*; D008970:Molecular Weight; D013379:Substrate Specificity
D046911:Macromolecular Substances; D003402:Creatine Kinase
Usloviia vzaimnogo perekhoda oligomernykh form mitokhondrial'noĭ kreatinkinazy serdtsa.