Creatine Kinase of Rat Heart Mitochondria

Abstract Intact mitochondria from human, beef, and rat heart, as well as rat brain and rat skeletal and smooth muscle, contain considerable creatine kinase activity; those from rat or rabbit liver, kidney, and testis contain essentially none. In rat heart mitochondria creatine kinase activity is far higher than the activity of adenylate kinase and nucleoside diphosphokinase. The heart mitochondrial creatine kinase is specific for ATP and ADP as phosphate donor and acceptor. The optimum pH is 6.7 in the direction of ATP formation and 8.0 in the direction of phosphocreatine formation. The mitochondrial isoenzyme of creatine kinase is not present in the same compartment as the intermembrane marker enzyme adenylate kinase, but may be released in soluble form by exposure to sodium acetate or sodium phosphate medium at pH 7.4. The phosphorylation of creatine by intact mitochondria takes place outside the atractyloside-sensitive ADP-ATP carrier system of the inner membrane and thus requires external ATP. When creatine in the concentration range known to be present in intact heart is added to rat heart mitochondria neither the initial State 4 or State 3 rate of respiration (succinate) is affected; however, there is a large increase in the State 4 rate following addition of ADP, whose magnitude increases as the creatine concentration is increased. In the presence of physiological concentrations of creatine and ADP rat heart mitochondria respire at a constant high rate equal to 75 to 85% of their maximal State 3 rate; under these conditions phosphocreatine is the end product of oxidative phosphorylation. The Km values for rat heart mitochondria are 35 µm for ADP, 0.72 mm for phosphocreatine, 100 µm for ATP, and 6 mm for creatine. These and other properties suggest that in addition to the ∼P storage function usually postulated for it, the phosphocreatine-creatine kinase system serves to smooth out or buffer the rate of respiration of heart mitochondria at a high and constant rate, as well as to channel ∼P to the contractile system via the mitochondrial and sarcoplasmic isoenzymes of creatine kinase.