ADENOSINE TRIPHOSPHATE-CREATINE PHOSPHOTRANSFERASE FROM OX BRAIN. 2. PROPERTIES AND FUNCTION

Abstract

The properties of adenosine triphosphate-creatine phosphotransferase prepared from ox brain were investigated and compared with those of the muscle enzyme. Inhibition was produced by p-chloromercuribenzoate and "aged" sodium thioglycollate, and fresh sodium thioglycollate could give rise to activation. On storage of partially pure preparations at 5[degree], the activity assayed in the absence of thioglycollate gradually declined, although the activity assayed with thioglycollate present showed little change. It is suggested that thiol groups essential for activity are sensitive to aerial oxidation. The enzyme was unable to catalyse a reaction with adenosine monophosphate as the phosphoryl-acceptor. It contained similar amounts of tyrosine and tryptophan to the muscle enzyme but had a considerably greater electrophoretic mobility at pH 8[center dot]0 and 8[center dot]6. The optimum pH for the "forward reaction" was 9[center dot]0 and for the "reverse reaction" was 7[center dot]9. The enzyme was stable at 0[degree] over the range pH 5[center dot]5-11[center dot]8. Reaction rates in both directions were measured at 20[degree], 30[degree] and 37[degree], and the activation energies that were calculated were very close to those for the muscle enzyme. Maximal velocities for the "forward" and "reverse reactions" and Michaelis constants for each of the four Substrates creatine, phosphocreatine, adenosine diphosphate and adenosine triphosphate were determined. The effect of the magnesium concentration on the reaction was also investigated. The apparent equilibrium constant of the reaction at pH 7[center dot]0 was determined at 30[degree] and at 37[degree]. From the data obtained, maximal rates of phosphocreatine breakdown and resynthesis in guinea-pig cerebral tissue under conditions postulated to exist in vivo were calculated and compared with the observed values. Observed concentrations of phosphorylated intermediates in vivo are discussed in the light of the measured equilibrium constant of the phosphotransferase reaction and the function of the enzyme.