Purification of pancreas type-I ATP diphosphohydrolase and identification by affinity labelling with the 5'-p-fluorosulphonylbenzoyladenosine ATP analogue.
The enzyme recently identified as type-I ATP diphosphohydrolase (ATPDase; EC 3.6.1.5) has been purified from the zymogen granule membrane of pig pancreas. After solubilization with Triton X-100 and chromatographies on ion-exchange and Affi-Gel Blue columns an approximate 3500-fold purification was obtained. The enzyme preparation with a specific activity of 45 units/mg of protein was much further purified by PAGE under non-denaturing conditions. The active band localized on the gel contained two proteins after SDS/PAGE and silver staining, corresponding to apparent molecular masses of 56 and 54 kDa. The identity of the ATPDase was confirmed by an affinity labelling technique with 5'-p-fluorosulphonylbenzoyladenosine (FSBA) as an ATP analogue. The latter was detected by a Western blot technique. A strong reaction was observed with the band corresponding to 54 kDa. N-terminal sequence analysis revealed that the 56 kDa protein has significant similarities (50-72%) with lipases, whereas the 54 kDa enzyme has no significant similarity with any known proteins. N-glycosidase F treatment confirmed the glycoprotein nature of the enzyme and suggested that the enzyme bears several N-glycosylation sites. Comparisons of molecular masses and biochemical properties show that this ATPDase is different from other reported mammalian ATPDases.
Sévigny
J
J
Département de Biologie, Faculté des Sciences, Université de Sherbrooke, Québec, Canada.
Côté
Y P
YP
Beaudoin
A R
AR
D000241:Adenosine / Q000031:analogs & derivatives* / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D001081:Apyrase / Q000302:isolation & purification* / Q000378:metabolism*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007425:Intracellular Membranes / Q000201:enzymology; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D017830:Octoxynol; D015388:Organelles / Q000201:enzymology; D010179:Pancreas / Q000201:enzymology*; D012995:Solubility; D013552:Swine
D000345:Affinity Labels; D007527:Isoenzymes; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D017830:Octoxynol; D001081:Apyrase; D000241:Adenosine