Inhibition of smooth muscle 5′-nucleotidase by imidazole and its reversal by magnesium

Imidazole, commonly used as an effective pH-buffering reagent in aqueous media maintained at pH 7-8, was found to depress the 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) activity of microsomal membrane fraction isolated from rat vas deferens smooth muscle in a dose-dependent manner in the absence of added Mg2+. Such an inhibitory effect of imidazole on the smooth muscle 5'-nucleotidase was not dependent upon the purity or integrity of the membrane fractions used and could be fully reversed by the inclusion of 5-10 mM Mg2+ in the assay medium. Of the five different pH-buffering reagents tested, imidazole was specific in exerting inhibitory effect on the 5'-nucleotidase in the absence of Mg2+ and this inhibition could not be accounted for by the impurities present in the imidazole. Differential effects of chelating reagents and other divalent metal ions on the 5'-nucleotidase activity were also observed in imidazole and Tris buffer solutions. The 5'-nucleotidase activity was not affected if the membranes were preincubated and washed with a large volume of 50 mM imidazole and subsequently assayed in 50 mM Tris in the absence of Mg2+. Similar findings were obtained with EDTA treated membrane. These results suggest that imidazole does not act by removal of the activating metal ion but rather interacts directly with 5'-nucleotidase and alters the metal-enzyme interactions.