Purification and properties of rat heart 5'-nucleotidase.

Abstract

59-Nucleotidase (EC 3.1.3.5) has been purified 26-fold from rat heart and obtained substantially free of nonspecific phosphatase activity. The pH optimum for the enzyme was 7.6; at this pH, 7 mM Mg++ and 2 mM Ca++ caused 40% inhibition of 59-nucleotidase activity, and 0.17-0.69 mM Mg++ was without effect on activity. The nucleoside triphosphates ATP, UTP, CTP, GTP, and ITP inhibited 59-nucleotidase in a noncompetitive manner with respect to adenosine 59-monophosphate. The AMP analogues adenosine 59-phosphorothioate (AMPS) and 2-chloroadenosine 59-monophosphate (2-chloro-AMP), which are vasodilators, and 2-methylthioadenosine 59-monophosphate (2-methylthio-AMP), which has no vasodilatory properties, were all substrates of 59-nucleotidase, with Michaelis constants similar to that of AMP. The maximum velocities of hydrolysis of AMPS, 2-chloro-AMP, and 2-methylthio-AMP were 36%, 79%, and 116%, respectively, of the maximum velocity for AMP. The rates of hydrolysis of these analogues by 59-nucleotidase are considered in relation to their vasodilatory effects.