Hydrolysis of 5'-AMP by 5'-nucleotidase is a possible source of adenosine in the kidney. A renal membrane-bound ecto-5'-nucleotidase has been previously described. The present study deals with the catalytic properties of a 5'-AMP phosphohydrolase partially purified from high-speed supernatants of rat kidney homogenates. It exhibits phosphatase activity toward 5'-AMP, 5'-IMP, and 5'-GMP, but not toward 2'- and 3'-AMP and corresponds therefore to a 5'-nucleotidase. The hydrolysis of 5'-AMP by the soluble 5'-nucleotidase requires divalent cations. Maximal activity is reached with 10 microM of either Mn2+ or Co2+, whereas half-maximal activity is obtained with approximately 400 microM Mg2+. The soluble 5'-nucleotidase exhibits Michaelis-Menten kinetics with a Km of 9.5 microM for 5'-AMP. In the presence of 1 mM of free Mg2+, physiological concentrations of ATP provoke an increase of the Km for 5'-AMP and a decrease of Vmax. An increase of the pH of 0.4 units in the pH range 6.4-7.4 roughly doubles the rate of hydrolysis of 5'-AMP. The effects of ATP and of the pH are compatible with a role of the renal soluble 5'-nucleotidase in the hydrolysis of 5'-AMP and in the production of adenosine during hypoxia.
5'-Nucleotidase activity was solubilized from a particulate fraction of rat renal
homogenates by Sulphobetaine 14. An 11,430-fold purification was achieved by a two-step
chromatographic procedure using concanavalin-A Sepharose and ADP-agarose. SDS-PAGE
of the purified material revealed a single polypeptide band with a M(r) of 69,000. The enyzme
exhibited absolute specificity for 5'-mononucleotides. Among 7 tested substrates, adenosine
monophosphate (AMP) showed the highest value of V/K(m). The K(m) for 5'-AMP is 5.1 μmol/1
and V is 632 pmol/min/mg. The plot of activity versus pH shows a broad plateau between pH
6.8 and 8.0. The hydrolysis of 5'-AMP was competitively inhibited by adenosine 5'-triphosphate
(ATP; K; = 1.2 μmol/1), adenosine 5'-diphosphate (ADP; K(i) = 0.032 μmol/1) and
a,ß-methyleneadenosine 5'-diphosphate (AOPCP; K(i) = 0.005 μmol/1). All of the 5 detergents
tested activated the enzyme. Sulphobetaine 14 was the most potent and resulted in a 4-fold
stimulation by increasing V without change of Km. Addition of exogenous divalent cations
was not required for activity. However, the enzyme was inhibited by EDTA. This inhibition
was overcome by the addition of Co^2+, M^n2+ and to a lesser extent of Mg^2+. Hg^2+, Zn^2+, Cu^2+
and Pb2+ inhibited in the low micromolar range. The properties of this enzyme from the rat
kidney are similar to those reported in the literature for ecto 5'-nucleotidases from other
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