[ATP-metabolizing enzymes in suspensions of isolated coupled rat brain mitochondria].

An improved method for the isolation of rat brain mitochondria is described. The preparation exhibits a respiratory control index (RCI) of 6 or 7.3 in the presence of pyruvate and malate or glutamate and malate, respectively. RCI decreases to 2.5 in the presence of Mg++. When the phosphorylation of extramitochondrially added or formed ADP is suppressed by carboxyatractyloside (CAT) inhibition of the adenine nucleotide translocator, the remaining respiration amounts to 6 nmol O2/min X mg mitochondrial protein. These results and the ratio of 16 to 19 from the quotient of phosphorylating active-state respiration to CAT inhibited respiration refer to a high degree of mitochondrial coupling of respiration. Therefore the remaining respiration in the presence of Mg++ is due to a phosphokinase activity located outside the inner membrane of intact mitochondria or at nonphosphorylating mitochondrial fragments. The following activities were observed: Oligomycin sensitive ATPase, 47 mU/mg protein; hexokinase, 272 mU/mg protein; creatinphosphokinase, 116 mU/mg protein; and a surprisingly low activity of adenylatekinase, 57 mU/mg protein.