Demonstration of a novel type of ATP-diphosphohydrolase (EC 3.6.1.5) in the bovine lung.
A novel type of ATP-diphosphohydrolase (ATPDase) is demonstrated in bovine lung. The enzyme has an optimum pH of 7.5 and catalyzes the hydrolysis of the beta- and gamma-phosphate residues from diphospho- and triphosphonucleosides. It requires Ca2+ or Mg2+ and is insensitive to ouabain, an inhibitor of Na+/K(+)-ATPase, P1,P5-di(adenosine 5')-pentaphosphate, an inhibitor of adenylate kinase, and tetramisole, an inhibitor of alkaline phosphatase. In contrast, sodium azide (10 mM), a known inhibitor of ATPDases and mitochondrial ATPases, as well as mercuric chloride (10 microM) and gossypol (2,2'-bis[8-formyl-1,6,7-trihydroxy-5-isopropyl-3-methylnaphthalene]) (35 microM) are powerful inhibitors of this enzyme. The same inhibition profile is obtained with ATP or ADP as substrate, thereby supporting the concept of a common catalytic site for these substrates. This is further confirmed by enzyme localization after polyacrylamide gel electrophoresis under nondenaturing conditions and by kinetic properties, namely pH dependence profiles, heat inactivation, and 60Co irradiation-inactivation curves. The native molecular mass of the enzyme calculated from 60Co gamma-irradiation-inactivation curves is estimated at 70 +/- 3 kDa, whereas Km,app and Vmax,app of the ATPDase are evaluated at 7 +/- 2 microM and 1.1 +/- 0.3 mumol of Pi/min/mg protein, respectively. A comparison of the kinetic properties of this ATPDase with those of pig pancreas (Type I) and bovine aorta (Type II) lead us to believe that this enzyme is an hitherto undescribed type of ATPDase. By reference to the previously described ATPDase, we propose to identify this enzyme as ATPDase Type III (EC 3.6.1.5).
Picher
M
M
Département de Biologie, Faculté des Sciences, Université de Sherbrooke, Québec, Canada.
Côté
Y P
YP
Béliveau
R
R
Potier
M
M
Beaudoin
A R
AR
D000818:Animals; D001081:Apyrase / Q000032:analysis* / Q000037:antagonists & inhibitors; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D008861:Microsomes / Q000201:enzymology; D013379:Substrate Specificity
D001081:Apyrase