title: Purification, characterization, and localization of an ATP diphosphohydrolase in porcine kidney.
abstract: Membranes of pig kidney cortex tissue were solubilized in the presence of Triton X-100. Partial purification of ATP diphosphohydrolase (ATPDase) was achieved by successive chromatography on concanavalin A-Sepharose, Q-Sepharose Fast Flow, and 5'-AMP-Sepharose 4B. Monoclonal antibodies against ATPDase were generated. Further purification of the ATPDase was obtained by immunoaffinity chromatography with these monoclonal antibodies. NH(2)-terminal amino acid sequencing of the 78-kDa protein showed a sequence very homologous to mammalian CD39. The protein is highly glycosylated, with a nominal molecular mass of approximately 57 kDa. The purified enzyme hydrolyzed di- and triphosphates of adenosine, guanosine, cytidine, uridine, inosine, and thymidine, but AMP and diadenosine polyphosphates could not serve as substrates. All enzyme activities were dependent on divalent cations and were partially inhibited by 10 mM sodium azide. The distribution of the enzyme in pig kidney cortex was examined immunohistochemically. The enzyme was found to be present in blood vessel walls of glomerular and peritubular capillaries.
authors.affiliation: Department Medische BasisWetenschappen, Limburgs Universitair Centrum, Belgium.
authors.forename: R
authors.initials: R
authors.lastname: Lemmens
authors.forename: L
authors.initials: L
authors.lastname: Kupers
authors.forename: J
authors.initials: J
authors.lastname: Sévigny
authors.forename: A R
authors.initials: AR
authors.lastname: Beaudoin
authors.forename: G
authors.initials: G
authors.lastname: Grondin
authors.forename: A
authors.initials: A
authors.lastname: Kittel
authors.forename: E
authors.initials: E
authors.lastname: Waelkens
authors.forename: L
authors.initials: L
authors.lastname: Vanduffel
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001081:Apyrase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002417:Cattle; D006801:Humans; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D007668:Kidney / Q000201:enzymology*; D008854:Microscopy, Electron; D008970:Molecular Weight; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013552:Swine; D014018:Tissue Distribution
chemical_list: D000911:Antibodies, Monoclonal; D001081:Apyrase