The preparation and properties of β-glucuronidase. 6. Activity in rat-liver preparations

Abstract

After homogenizing rat liver in isotonic sucrose soln. nearly all the glucuronidase activity was found in the subcellular particles. Homogenizing in water made a large part of the enzyme soluble and it was brought completely into soln. by the surface-active agent Triton X-100. The activity of soluble and insoluble rat-liver glucuronidase in water suspensions was greatly reduced by a non-dialysable endogenous inhibitor. Inhibition, which was overcome by Triton X-100, was also seen in rat kidney, but was very slight in mouse liver and kidney. Rat liver enzyme was powerfully inhibited by mouse liver and kidney prepns. The inhibitor in rat liver was thermostable at neutral pH. It was not completely sedimented on the high-speed centrifuge, but was completely precipitated by buffering to pH 5.2. Incubation in acetate buffer, pH 5.2, destroyed the inhibitor in adult, but not infant liver prepns. Inhibition varied directly with substrate and inhibitor concn., and the kinetics were analyzed. The term "combined inhibition" is suggested for this effect. Inhibition by heparin and chondroitin sulfate was studied. The effects differed in important respects from those of the endogenous inhibitor. The glucuronidase activity of newborn rat liver and of rat liver regenerating after partial hepatectomy was less than that of normal adult rat liver. Sources of error in the assay of beta-glucuronidase are discussed, with particular respect to the effects of the endogenous inhibitor in rat liver.