The Preparation and Properties of p - Glucuronidase 6 . ACTIVITY IN RAT - LIVER PREPARATIONS

Previous papers in this series have dealt with the fractionation and activity of ,-glucuronidase in mouse-liver suspensions ('homogenates'). Identical fractionation of the enzyme was achieved by centrifuging at high speeds or by buffering to slightly acid pH. The sedimentable fraction was associated with subcellular particles of all sizes, and these were agglutinated in acid buffers. In water suspensions (Kerr & Levvy, 1951; Walker & Levvy, 1951), the sedimentable fraction was fully active at the usual tissue concentrations employed for ,B-glucuronidase assay with phenolphthalein glucuronide, and accounted for slightly less than half the total enzyme activity in preparations of normal adult liver. In suspensions ofmouse liver prepared in isotonic media (Walker, 1952), nearly all the enzyme was in the sedimentable fraction. These preparations, however, did not display full glucuronidase activity until some measure of disruption of the subeellular particles had occurred, with release of part of their enzyme content to the solution. This process of activation was irreversible, andwas considered to be due to readier access of the substrate to the enzyme remaining in the particles. Soluble enzyme in mouse-liver suspensions was fully active under all conditions of assay. In this connexion, soluble enzyme was defined as that fraction which was not sedimented after 15 min. at 25 000 g nor precipitated by acetate buffer, and which gave optically clear preparations under the phase-contrast microscope (Walker & Levvy, 1951). A similar approach has been made to the study of ,-glucuronidase in rat liver, in which part of the enzyme is known to be precipitable at acid pH (Becker & Friedenwald, 1949). A complication was encountered at an early stage due to reversible inhibition of soluble and insoluble enzyme by material present in the tissue.