Purification of the blood vessel ATP diphosphohydrolase, identification and localisation by immunological techniques.
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.
Sévigny
J
J
Département de biologie, Faculté des sciences, Université de Sherbrooke, Québec, Canada.
Levesque
F P
FP
Grondin
G
G
Beaudoin
A R
AR
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
D000345:Affinity Labels; D006023:Glycoproteins; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D001081:Apyrase; D000241:Adenosine