β-Glucuronidase of Bovine Liver

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Abstract
β-Glucuronidase [EC 3.2.1.31] was purified from bovine liver by ammonium sulfate precipitation, ethanol fractionation, organic solvent fractionation, gel filtration, and isoelectric focusing without the need for incubation of the crude liver homogenate at 37° for 11 days, as done by Plapp and Cole (3). The last step resulted in the separation of two enzyme forms, isoelectric at pH values of 5.1 and 5.9. The enzyme isoelectric at pH 5.1 was homogeneous by analytical ultracentrifugation and polyacrylamide gel electrophoresis. This bovine liver β-glucuronidase was purified 10,700-fold, to the highest specific activity so far reported. The enzyme has a molecular weight of approximately 290,000 and a sedimentation coefficient of 14S. The Km with p-nitrophenyl β-D-glucosiduronic acid as a substrate was about 0.33 nM A broad pH activity curve with a single optimum at pH 4.4 was observed. The amino acid and carbohydrate compositions were determined. The enzyme has a very low content of sulphur-containing amino acids and 6.3% of carbohydrate, consisting of mannose, galactose, glucose, glucosamine, and sialic acid in a ratio of 45; 8; 7; 32; 5; there are also trace amounts of fucose.

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