Kinetic properties of type-II ATP diphosphohydrolase from the tunica media of the bovine aorta

Abstract

The kinetic properties of type-II ATP diphosphohydrolase are described in this work. The enzyme preparation from the inner layer of the bovine aorta, mostly composed of smooth muscle cells, shows an optimum at pH 7.5. It catalyzes the hydrolysis of triand diphosphonucleosides and it requires either Ca 2+ or Mg 2+ for activity. It is insensitive to ouabain (3 mM), an inhibitor of Na + K + -ATPase , to tetramisole (5 mM), an inhibitor of alkaline phosphatase, and to Ap 5A (100μM), an inhibitor of adenylate kinase. In contrast, sodium azide (10mM), a known inhibitor for ATPDases and mitochondrial ATPase, is an effective inhibitor. Mercuric chloride (10μM) and 5′- p-fluorosulfonylbenzoyl adenosine are also powerful inhibitors, both with ATP and ADP as substrates. The inhibition patterns are similar for ATP and ADP, thereby, supporting the concept of a common catalytic site for these substrates. Apparent K m and V max, obtained with ATP as the substrate, were evaluated at 23 ± 3 μM and 1.09 μmol P i/min per mg protein, respectively. The kinetic properties of this enzyme and its localization as an ectoenzyme on bovine aorta smooth muscle cells suggest that it may play a major role in regulating the relative concentrations of extracellular nucleotides in blood vessels.