Demonstration of an ectoATP-diphosphohydrolase (E.C.3.6.1.5.) in non-vascular smooth muscles of the bovine trachea.
An ectoATP-diphosphohydrolase (ATPDase) is put in evidence in non-vascular smooth muscles of the bovine trachea. The enzyme has an optimum pH of 7.0 and catalyzes the hydrolysis of the gamma- and beta-phosphate residues from extracellular triphospho- and diphosphonucleosides. It requires either Ca2+ or Mg2+ and is insensitive to ouabain, oligomycin and Ap5A. Sodium azide (20 mM), mercuric chloride (10 microM) and gossypol (35 microM) inhibit the enzyme activity by more than 45%. Polyacrylamide gel electrophoresis under non-denaturing conditions and kinetic properties, namely pH dependency profiles, heat inactivation and 60Co gamma-irradiation-inactivation curves, support the view that the same catalytic site is responsible for the hydrolysis of ATP and ADP to AMP. Accordingly, when both ATP and ADP were combined, reaction rates were not additive. With ATP, Km,app and Vmax,app were estimated at 15 +/- 2 microM and 1.9 +/- 0.1 mumol inorganic phosphate/min per mg of protein, respectively. From 60Co gamma-irradiation-inactivation curves, the molecular mass of the enzyme was estimated at 71 +/- 5 kDa. Enzyme markers indicate that the ATPDase is associated with the plasma membrane. Enzyme assays on trachea smooth muscle cells in suspension confirm that the catalytic site of this ATPDase is localized on the outer surface of the plasma membrane. Analysis of the biochemical properties shows many points of similarity between the tracheal ATPDase and the ATPDase recently described in the bovine lung.
Picher
M
M
Département de Biologie, Faculté des Sciences, Université de Sherbrooke, Québec, Canada.
Béliveau
R
R
Potier
M
M
Savaria
D
D
Rousseau
E
E
Beaudoin
A R
AR
D000818:Animals; D001081:Apyrase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D002417:Cattle; D009130:Muscle, Smooth / Q000201:enzymology*; D009708:Nucleotidases / Q000378:metabolism; D013379:Substrate Specificity; D014132:Trachea / Q000201:enzymology
D009708:Nucleotidases; D001081:Apyrase