The preparation and properties of β-glucuronidase. 1. The fractionation of buffered water homogenates

Abstract

Addition of acetate buffer, pH 5.2, to water homogenates of mouse liver, kidney and uterus caused agglutination of granular material which could then be separated by centrifuging at low speeds. This material had glucuronidase activity, amounting with normal adult liver and kidney to slightly less than half the total in the homo-genate. Citrate buffer caused identical fractionation of the enzyme in homogenates, provided that the pH and concn. were strictly controlled. Increasing the pH beyong 5.2 or the concentration beyond 0.1 N led to extraction of the enzyme in the granules by citrate. Changes in the glucuronidase activity of a tissue in vivo were confined to that fraction of the enzyme which was soluble in acidified water homogenates. Incubation of homogenates in acetate buffer led to a progressive release of enzyme from the granules to the soln. This did not occur in citrate buffer. In concentrated homogenates, the presence of the insoluble enzyme fraction can lead to false results for the total activity. The pH-activity curves for the hydrolysis of phenol-phthalein glucuronide in acetate buffer by the soluble and insoluble fractions were identical in having peaks at pH 4.5 and 5.2. Saccharate inhibited the enzyme in the granules without affecting its release to the soln. on incubation in acetate buffer. The significance of these findings in the assay of glucuronidase was discussed, with particular reference to tissues in varying states of proliferation.