Diadenosine tetraphosphatase from human leukemia cells. Purification to homogeneity and partial characterization.

Diadenosine tetraphosphatase, an enzyme splitting diadenosine tetraphosphate to AMP and ATP, has been purified to apparent homogeneity from a permanent cell line derived from a leukemic child. The purification procedure consisted of fractionation by ammonium sulfate precipitation, followed by Sephacryl 200 and DEAE-cellulose chromatography, and finally a differential membrane filtration. The enzyme is a single polypeptide chain of Mr = 17,500 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. The apparent molecular weight of the native enzyme was calculated as 20,000 from gel filtration data. The apparent Km for Ap4A was 0.5 microM as determined by two independent kinetic assays. None of the following compounds were substrates of the enzyme: diadenosine triphosphate, NAD, nucleoside 5'-phosphates (AMP, ATP, GDP, GTP, and UTP). The enzyme had optimal activity in the presence of 1 mM Mg2+, showing no activity in the presence of EDTA.