Purification and characterization of mouse kidney beta-glucuronidase.
Beta-Glucuronidase has been purified from mouse kidneys previously induced by gonadotrophin to a specific enzyme activity 15 times higher than the non-induced kidney. The purification procedure includes ultrasonication to solubilize the enzyme, acid and ammonium sulfate precipitations, gel filtration in Sephadex G-200, DEAE-ion exchange chromatography, and isoelectric focusing. The resulting product has a specific activity of 284,000 Fishman units/mg of protein, representing a 1,090-fold purification and is 17,000-fold higher than the level in the non-induced kidney. The purified beta-glucuronidase is apparently homogeneous by criteria of gel filtration, sodium dodecyl sulfate gel electrophoresis, and immunodiffusion. Characterization of the purified enzyme showed that it is identical with the lysosomal isoenzymic from electrophoretically, has subunit molecular weight of 74,000 (estimated by sodium dodecyl sulfate gel electrophoresis) and oligomer molecular weight of 300,000. The purified enzyme is stable at high temperature (up to 55 degrees) and at wide range of pH (from 4 to 11). It has a pH optimum for its activity at 4.7 and a Km of 1.18 times 10- minus 4 M. The purification and characterization of this enzyme from mouse kidney will have significance in the understanding of the molecular nature of the isoenzymes of beta-glucuronidase and will be useful in future studies on the mechanism of intracellular transport and distribution of this hydrolase.
D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction / Q000187:drug effects; D005966:Glucuronidase / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D006062:Gonadotropins / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007668:Kidney / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008297:Male; D051379:Mice; D008970:Molecular Weight; D011817:Rabbits / Q000276:immunology; D013696:Temperature; D014465:Ultrasonics
D006062:Gonadotropins; D007527:Isoenzymes; D046911:Macromolecular Substances; D005966:Glucuronidase