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Purification and properties of a 5'-nucleotidase from rat renal membranes.

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 Mr of 69,000. The enyzme exhibited absolute specificity for 5'-mononucleotides. Among 7 tested substrates, adenosine monophosphate (AMP) showed the highest value of V/Km. The Km for 5'-AMP is 5.1 mumol/l and V is 632 mumol/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; Ki = 1.2 mumol/l), adenosine 5'-diphosphate (ADP; Ki = 0.032 mumol/l) and alpha, beta-methyleneadenosine 5'-diphosphate (AOPCP; Ki = 0.005 mumol/l). 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 Co2+, Mn2+ and to a lesser extent of Mg2+. Hg2+, Zn2+, Cu2+ 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 sources.