Purification and Characterization of Microsomal and Lysosomal β‐Glucuronidase from Rat Liver by Use of Immunoaffinity Chromatography

Abstract

Rat liver β-glucuronidase (EC 3.2.1.31), both from microsomal and lysosomal fractions, were pinked about 9500-fold over the homogenate with high yield using affinity chromatography prepared by coupling purified specific immunoglobulin G against rat preputial gland β-glucuronidase to Nepharose 2B and isoelectric focusing. The purified enzymes appeared homogeneous on electrophoresis in polyacrylamide gel and had a molecular weight of approximately 310000. In dodecylsulfate polyacrylamide gel electrophoresis, the microsomal β-glucuronidase showed a single band corresponding to a molecular weight of 79000, while the lysosomal β-glucuronidase had three distinct bands which consisted of one major and two minor bands corresponding to molecular weight of 79000, 74000, and 70000, respectively. A broad pH activity curve with a single optimum at pH 4.4 was observed in both the microsomal and the lysosomal β-glucuronidases. Immunological gel diffusion technique with rabbit antiserum against rat liver lysosomal β-glucuronidase revealed that the both enzymes had the same or quite similar antigenic determinants.