Rabbit beta-glucuronidase. Purification and properties, and the existence of multiple forms.
1. beta-Glucuronidase (EC 3.2.1.31) was purified from rabbit liver by a procedure involving autolysis, (NH(4))(2)SO(4) fractionation, chromatography on DEAE-cellulose and hydroxyapatite, gel filtration, sedimentation in a sucrose gradient, and isoelectric focusing. 2. Electron microscopy revealed ferritin as the major contaminant in later stages of purification and also showed aggregates of enzyme molecules. Particular attention was paid to the removal of ferritin. 3. The purified enzyme was homogeneous in polyacrylamide-gel electrophoresis both in non-dissociating conditions and in the presence of sodium dodecyl sulphate, and in Ouchterlony gel diffusion and immunoelectrophoresis against polyspecific antisera. 4. Sedimentation in sucrose gradients gave a molecular weight of 300000, whereas gel filtration indicated 440000. 5. Subunits of 75000 molecular weight were observed in gel electrophoresis in the presence of sodium dodecyl sulphate and in gel filtration in the presence of urea. 6. The K(m) value for p-nitrophenyl beta-d-glucuronide was 0.6mm, and the enzyme was extremely sensitive to lactone inhibitors. It was also inhibited by Hg(2+) ions. 7. Multiple forms were observed in the pure enzyme by isoelectric focusing, with pI values of 4.5-5.8. Subunits showed similar heterogeneity. The origin of the multiple forms was investigated in detail, and the possibility of artifact generation largely excluded. Some of the forms of lowest pI disappeared after neuraminidase digestion. The nature of the residual heterogeneity remains to be elucidated.
D000818:Animals; D001329:Autolysis; D002499:Centrifugation, Density Gradient; D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005293:Ferritins; D005966:Glucuronidase / Q000302:isolation & purification*; D006882:Hydroxyapatites; D005779:Immunodiffusion; D007122:Immunoelectrophoresis; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000302:isolation & purification*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008854:Microscopy, Electron; D008970:Molecular Weight; D011817:Rabbits; D012967:Sodium Dodecyl Sulfate
D006882:Hydroxyapatites; D007527:Isoenzymes; D012967:Sodium Dodecyl Sulfate; D005293:Ferritins; D005966:Glucuronidase