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Abstract

In the present report we describe an ATP diphosphohydrolase (apyrase EC 3.6.1.5) in rat cardiac sarcolemma. It is Ca2+ dependent and is insensitive to ouabain, orthovanadate, N-ethylmaleimide (NEM), lanthanum, and oligomycin that are classical ATPase inhibitors. Sodium azide that is a mitochondrial inhibitor at low concentrations, did not affect the enzyme activity at 5.0 mM or below. In contrast, at high concentrations (> 10 mM) sodium azide inhibited the enzyme. Levamisole, a specific inhibitor of alkaline phosphatase and P1, P5-di(adenosine 5'-)pentaphosphate (Ap5A), a specific inhibitor of adenylate kinase did not inhibit the enzyme. Mercury chloride showed a parallel inhibition of the hydrolysis of both substrates of apyrase. Similar inhibition profiles are powerful evidence for a common catalytic site for the hydrolysis of both substrates. The enzyme has an optimum pH range of 7.5-8.0 and catalyzes the hydrolysis of triphospho- and diphosphonucleosides other than ATP or ADP. The apparent Km (Michaelis constant) and Vmax (maximal velocity) are 62.1 +/- 5.2 microM and 1255.7 +/- 178 micromol inorganic phosphate liberated/min/mg with ATP and 59.4 +/- 4.3 microM and 269.2 +/- 39 micromol inorganic phosphate liberated/min/mg with ADP. Enzyme markers indicated that this apyrase is associated with the plasma membrane. A deposition of lead phosphate granules on the outer surface of the sarcolemmal vesicles was observed by electron microscopy in the presence of either ATP or ADP as substrate. It is suggested that the ATP diphosphohydrolase could regulate the concentration of extracellular adenosine, and thus is important in the control of vascular tone and coronary flow.

Moderate exercise training promotes adaptations in coronary blood flow and adenosine production in normotensive rats

“…The enzyme activity was routinely determined at 37°C in the following incubation media: (a) the blood serum was incubated with 112.5 mM Tris-HCl, pH 8.0, with approximately 1.0 mg of serum protein at 37°C for 40 min in a final volume of 0.2 mL, and the reaction mixture contained ADP or ATP as a substrate19 (2.0 mM or 3.0 mM, respectively); (b) the cardiac sarcolemmal fraction was incubated with 50 mM Tris-HCl buffer, pH 7.5, 1.5 mM CaCl, and 0.8–1.0 µg of protein in a final volume of 0.2 mL, and the reaction mixture contained ADP or ATP as a substrate20 (3.0 mM or 2.0 mM, respectively).…”

Adenosine Diphosphate Ribose Dilates Bovine Coronary Small Arteries through Apyrase- and 5′-Nucleotidase-Mediated Metabolism

“…Since inhibition of 5′-nucleotidase is accompanied by the accumulation of 5′-AMP, we next determined the enzyme activity catalyzing the conversion of ADPR to 5′-AMP. Apyrase, which is also known as ecto-ATP-diphosphohydrolase, is a membrane-bound enzyme present in a large variety of tissues including brain, heart, nonvascular and vascular smooth muscle [41, 42, 43, 44, 45, 46]. It catalyzes the hydrolysis of ATP and ADP to 5′-AMP.…”