Purification of ATP diphosphohydrolase from bovine aorta microsomes

Abstract

ATP diphosphohydrolase (EC 3.6.1.5) hydrolyzes pyrophosphate bonds of nucleoside di- and triphosphates in the presence of divalent cations. We purified the enzyme from the vessel wall of bovine aortas. The procedure gave a homogeneous preparation of ATP diphosphohydrolase for the first time from an animal source. Bovine aorta microsomes were treated with 50 mM bicarbonate buffer (pH 10.0) containing 0.025% Triton X-100. The enzyme was then solubilized from the microsomes with 0.5% Triton X-100 and purified to homogeneity by DEAE-Sepharose CL-6B chromatography and 5'AMP-Sepharose 4B affinity chromatography. The apparent molecular mass of the pure enzyme was 110 kDa. The activity recovered was 6% of that of the microsomes. The enzyme was more active with Ca2+ than Mg2+. The sensitivity of ADPase activity to divalent cations was higher than that of ATPase activity. The enzyme had broad substrate specificity to nucleoside di- and triphosphates.