Purification, characterization, and localization of an ATP diphosphohydrolase in porcine kidney

Membranes of pig kidney cortex tissue were solubilized in the presence of T riton X-100. Partial purification of ATP diphosphohydrolase (ATPDase) was a chieved by successive chromatography on concanavalin A-Sepharose, Q-Sepharo se Fast Flow, and 5'-AMP-Sepharose 4B. Monoclonal antibodies against ATPDas e were generated. Further purification of the ATPDase was obtained by immun oaffinity chromatography with these monoclonal antibodies. NH2-terminal ami no acid sequencing of the 78-kDa protein showed a sequence very homologous to mammalian CD39. The protein is highly glycosylated, with a nominal molec ular mass of similar to 57 kDa. The purified enzyme hydrolyzed di- and trip hosphates of adenosine, guanosine, cytidine, uridine, inosine, and thymidin e, but AMP and diadenosine polyphosphates could not serve as substrates. Al l enzyme activities were dependent on divalent cations and were partially i nhibited by 10 mM sodium azide. The distribution of the enzyme in pig kidne y cortex was examined immunohistochemically. The enzyme was found to be pre sent in blood vessel walls of glomerular and peritubular capillaries.