DEMONSTRATION OF AN ECTOATP-DIPHOSPHOHYDROLASE (EC-3.6.1.5.) IN NONVASCULAR SMOOTH MUSCLES OF THE BOVINE TRACHEA

An ectoATP-diphosphohydrolase (ATPDase) is put in evidence in non-vasc ular smooth muscles of the bovine trachea. The enzyme has an optimum p H 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 Ap(5)A. Sodium azide (20 mM), mercuric chloride (10 mu M) and gos sypol (35 mu M) inhibit the enzyme activity by more than 45%. Polyacry lamide gel electrophoresis under non-denaturing conditions and kinetic properties, namely pH dependency profiles, heat inactivation and Co-6 0 gamma-irradiation-inactivation curves, support the view that the sam e catalytic site is responsible for the hydrolysis of ATP and ADP to A MP. Accordingly, when both ATP and ADP were combined, reaction rates w ere not additive. With ATP, K-m,K-app and V-max,V-app were estimated a t 15 +/- 2 mu M and 1.9 +/- 0.1 mu mol inorganic phosphate/min per mg of protein, respectively. From Co gamma-irradiation-inactivation curve s, the molecular mass of the enzyme was estimated at 71 +/- 5 kDa. Enz yme markers indicate that the ATPDase is associated with the plasma me mbrane. Enzyme assays on trachea smooth muscle cells in suspension con firm that the catalytic site of this ATPDase is localized on the outer surface of the plasma membrane. Analysis of the biochemical propertie s shows many points of similarity between the tracheal ATPDase and the ATPDase recently described in the bovine lung.