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.
Département de Biologie, Faculté des Sciences, Université de Sherbrooke, Québec, Canada.
J
J
Sévigny
Y P
YP
Côté
A R
AR
Beaudoin
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