Purification of ATP diphosphohydrolase from bovine aorta microsomes.
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.
Yagi
K
K
Department of Biochemical Engineering, Faculty of Pharmaceutical Sciences, Osaka University, Japan.
Arai
Y
Y
Kato
N
N
Hirota
K
K
Miura
Y
Y
D000818:Animals; D001011:Aorta / Q000201:enzymology*; D001081:Apyrase / Q000302:isolation & purification*; D002417:Cattle; D002845:Chromatography / Q000379:methods; D006868:Hydrolysis; D008861:Microsomes / Q000201:enzymology*; D017830:Octoxynol; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification*; D011092:Polyethylene Glycols; D012995:Solubility; D013379:Substrate Specificity
D011092:Polyethylene Glycols; D017830:Octoxynol; D010744:Phosphoric Monoester Hydrolases; D001081:Apyrase