NUCLEOSIDE DIPHOSPHOKINASE FROM BEEF HEART MITOCHONDRIA . PURIFICATION AND PROPERTIES

A nucleoside diphosphokinase has been partially purified from beef heart mitochondria. The purified enzyme has a molecular weight of 103,000 ± 3,000 and an isoelectric point of the order of 9.5. The optimum pH depends upon the adenosine diphosphate concentration; it is 7.4 at low concentrations of adenosine diphosphate and ranges from 6 to 8 at high concentrations of adenosine diphosphate. Inactivation of the enzyme by an SH reagent such as p-mercuribenzoate is released by incubation with dithiothreitol. The adenosine diphosphate-adenosine triphosphate exchange catalyzed by the purified enzyme is twice as fast as the uridine diphosphate-uridine triphosphate, adenosine diphosphate-uridine triphosphate, or adenosine dipbosphate-deoxyadenosine triphosphate exchanges and four to five times faster than the adenosine diphosphate-cytidine triphosphate, cytidine diphosphate-cytidine triphosphate, or guanidine diphosphate-guanidine triphosphate exchanges. The phosphonic analog of adenosine triphosphate, namely, AOPOPCP, is totally inactive as phosphate donor. The reaction mechanism obeys the Ping-Pong kinetics with Michaelis constants of 0.10 mM for adenosine diphosphate and 1.4 mM for adenosine triphosphate at a pH of 7.4. A study of the distribution pattern of the free and magnesium-bound forms of adenosine diphosphate and adenosine triphosphate shows that free adenosine diphosphate is preferred to magnesium adenosine diphosphate as phosphate acceptor, and that magnesium adenosine triphosphate is the only phosphate donor. High concentrations of magnesium adenosine diphosphate are inhibitory. Free adenosine triphosphate competes with free adenosine diphosphate for the phosphorylated form of nucleoside diphosphokinase. AMP is a noncompetitive inhibitor with respect to adenosine diphosphate. In contrast with nucleoside diphosphokinase from liver mitochondria, nucleoside diphosphokinase prepared from beef heart mitochondria does not exhibit allosteric properties under the experimental conditions reported in this paper. © 1969, American Chemical Society. All rights reserved.