title: Purification of the blood vessel ATP diphosphohydrolase, identification and localisation by immunological techniques.
abstract: ATP diphosphohydrolase (ATPDase) or apyrase (EC 3.6.1.5), an enzyme that hydrolyses the gamma and beta phosphate residues of triphospho- and diphosphonucleosides, has been purified from the bovine aorta media. A particulate fraction was isolated by differential, and sucrose cushion centrifugations, producing a 33-fold enrichment in ADPase activity. Solubilization of the enzyme from the particulate fraction with Triton X-100 caused a partial loss of activity. The solubilized enzyme was purified by DEAE-agarose, Affi-Gel blue and Concanavalin A column chromatographies yielding an additional 138-fold enrichment of the enzyme. The enzyme preparation was further purified by PAGE under non-denaturing conditions, followed by its detection on the gel. The active band was cut out and separated by SDS/PAGE. Overstaining with silver nitrate revealed a single band corresponding to a molecular mass of 78000. Presence of an ATP binding site on the latter protein was demonstrated by labelling with 5'-p-fluorosulfonylbenzoyladenosine (FSBA), an analogue of ATP, followed by its detection by a Western blot technique. Labelling specificity was demonstrated by competition experiments with Ca-ATP and Ca-ADP. An antiserum directed against the N-terminal sequence of the pig pancreas ATPDase (54 kDa) cross-reacted with the bovine aorta ATPDase at 78 kDa. Digestion of the ATPDase with N-glycosidase F caused a marked shift of the molecular mass, thereby showing multiple N-oligosaccharide chains. Immunohistochemical localisation confirmed the presence of ATPDase on both endothelial and smooth muscle cells.
authors.affiliation: Département de biologie, Faculté des sciences, Université de Sherbrooke, Québec, Canada.
authors.forename: J
authors.initials: J
authors.lastname: Sévigny
authors.forename: F P
authors.initials: FP
authors.lastname: Levesque
authors.forename: G
authors.initials: G
authors.lastname: Grondin
authors.forename: A R
authors.initials: AR
authors.lastname: Beaudoin
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta / Q000201:enzymology*; D001081:Apyrase / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D002417:Cattle; D006023:Glycoproteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D015151:Immunoblotting; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology
chemical_list: D000345:Affinity Labels; D006023:Glycoproteins; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D001081:Apyrase; D000241:Adenosine