Erythrocytic Nucleoside Diphosphokinase

An isozyme of nucleoside diphosphokinase (NDP kinase) has been purified from human erythrocytes. The best preparations were purified about 9000-fold from hemolysates to specific activities of about 1000 units per mg of protein. The isoelectric point of the partially purified sample, with an isoelectric focusing technique, was found to be 7.3, whereas a highly purified preparation of erythrocytic NDP kinase described earlier was found to consist principally of isozymes with pI values of 5.8 and 6.3. The enzyme is stable and shows a wide pH optimum (7.0 to 9.5). Michaelis constants (Km) for ATP and dTDP were 1.3 x 10-4 m and 2.7 x 10-4 m, respectively. p-Chloromercuribenzoate (PMB) inactivates the enzyme completely which could be reactivated by use of dithiothreitol, suggesting the presence of essential sulfhydryl groups. Substrates such as ATP or dTDP, in the presence or absence of Mg++, markedly protect the enzyme against inactivation by PMB. Although urea at a concentration of 2 m does not influence the enzymic activity, this concentration of urea renders the enzyme susceptible to irreversible inactivation by PMB. The enzymic reaction requires the presence of a divalent cation. Mg++, Mn++, and Co++ are equally effective. Ca++ is 50% effective, whereas Zn++ and Cu++ cannot satisfy divalent cation requirement. Studies of the effect of temperature on reaction velocity yielded a diphasic Arrhenius plot with a transition point at 31°. Energy of activation above and below 31° are 4.8 and 10.3 kcal m-1, respectively.