The preparation and properties of β-glucuronidase. 2. Centrifugal fractionation of water homogenates and the nature of the sedimentable fraction

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Abstract
About 40% of the glucuronidase activity in a water homogenate of adult mouse liver was sedimented by centrifuging at 25,000 g for 15 min. This corresponded to the removal from suspension of almost all the cytoplasmic granules visible under the phase microscope. Glucuronidase activity was distributed over granules of all sizes. Making the prepn. 0.1 [image] with respect to NaCl led to slight agglutination and much easier sedimentation of the granules and the assoicated enzyme activity. Buffering the prepn. to pH 4-6 with acetate led to pronounced agglutination of the granules, which then sedimented after 15 min. at 1500 g. Adjusting the pH to within these limits with acid alone led not only to agglutination of the granules, but to precipitation of part of the water-soluble enzyme. The latter effect was overcome by adding electrolyte, as in buffered prepns., or by warming. Incubation in acetate buffer at pH 5.2 led to extraction of the enzyme from the granules. This did not occur on incubating an untreated homogenate, or after pH adjustment with acid alone. Shaking a homogenate in a tissue disintegrator, or repeated freezing and thawing, caused disintegration of the granules and release of the associated enzyme to the soln. Homogenizing the tissue in acetone followed by resuspension in water had the same effect, but release of the enzyme was not seen when the acetone-dried powder was resuspended in acetate buffer. Treatment with surface-active agents, Teepol XL and Triton X-100, brought the granular enzyme into soln. with disappearance of particulate matter from the prepn. Glucuronidase was inhibited non-competitively by the anionic Teepol XL, but not by the non-ionic Triton X-100. Inhibition by Teepol was at first reversed by adding inactive protein, but later became irreversible.