Purification and Properties of a 5'-Nucleotidase from Rat Renal Membranes

Abstract

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

The Catalytic Mechanisms of Binuclear Metallohydrolases

“…The selective inhibition by ATP and ADP of di- but not monoesterase activity in E. coli 5‘-NT has been interpreted in terms of two substrate binding sites, a catalytic site with high affinity and an inhibitory site with low affinity for substrate . The presence of two distinct substrate binding sites may also account for the observed deviation of E. coli 5‘-NT from common Michaelis−Menten behavior, suggesting uncompetitive substrate inhibition. ,,,, This deviation is not observed in enzymes from other sources. ,, …”

“…For di- and triphosphates, the optimum pH range is broad, ranging between pH 5.5 and 8.5. The ecto-5‘-NTs derived from chicken gizzard and rat liver exhibit a pH optimum of 7.5, , while other enzymes exhibit broader pH optima: pH 6.8−8.0 for the rat renal enzyme and pH 8.0−9.0 for guinea pig skeletal muscle enzyme. , …”