Inosine triphosphate pyrophosphohydrolase from human erythrocytes was purified and characterized. The enzyme is highly specific for ITP and shows optimal activity in glycine buffer pH 9.6 and 50 mM MgCl2. The Km of the enzyme is 1.3 X 10(-4), the Vmax = 1.2 X 10(-9) and the Keq = 3.8 X 10(4). Human erythrocyte ITP pyrophosphohydrolase does not require SH compounds for activation. The enzyme is inhibited by Cd++, Co++, and Ca++ ions and by p-hydroxymercuribenzoate.
The ability to synthesize inosinetriphosphate was demonstrated in blood cells as well as in a variety of tissue extracts in spite of the presence of ITP pyrophohydrolase. At the expense of having sub-optimal conditions, an assay system was selected that completely repressed the hydrolyzing enzyme, thus permitting the accumulation of ITP. In an attempt to define the biosynthetic pathway of ITP, and since guanylate kinase has been implicated in the formation of ITP, the rate of synthesis of ITP and GTP in cell extracts was compared. The comparison of the specific activities of the [14C]-labeled hypoxanthine and guanine moieties of the inosine and guanosine phosphates formed during incubation with [8-14C]-inosine and [8-14C]-guanosine respectively, revealed striking differences in the relative rates of isotope incorporation. Tentative mechanisms are proposed to explain these differences. The data obtained thus far does not discard the possibility that ITP may be formed by stepwise phosphorylation and (or) by direct pyrophosphorylation of IMP.
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