Purification and properties of microsomal UDP-glucuronosyltransferase from rat liver.
p-Nitrophenol conjugating activity associated with liver microsomal UDP-glucuronosyltransferase (EC 2.4.1.17) was purified 150- to 200-fold from cell-free homogenates. The purification scheme included solubilization with the nonionic detergent Lubrol WX, anion exchange chromatography at pH 6.0 and 7.5, and affinity chromatography with UDP-hexanolamine Sepharose 4B. The enzyme purified as a phospholipid-protein complex and was shown to consist of a single polypeptide chain of molecular weight 59,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid analysis indicated approximately 531 mol of amino acids/59,000 g of enzyme and a molar ratio of nonpolar to polar residues of 1.08. During fractionation, the enzyme displayed instability with such steps as gel filtration, dialysis, or ultrafiltration of dilute samples; however, upon adsorption to ion exchange resins or storage in concentrated form, the enzyme was reasonably stable. The active lipoprotein complex showed both size and charge heterogeneity as judged by gel filtration and electrofocusing. Three forms of the enzyme resolved by isoelectric focusing had isoelectric points which averaged pH 6.68, 6.56, and 6.31. Polypeptide compositions of these electrophoretically distinct phospholipid protein complexes were indistinguishable on the basis of sodium dodecyl sulfate-polyacryl-amide gel electrophoresis, suggesting that the charge heterogeneity may be the result of differences in the phospholipid content of the lipoprotein complex.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002846:Chromatography, Affinity; D004790:Enzyme Induction / Q000187:drug effects; D014453:Glucuronosyltransferase / Q000302:isolation & purification / Q000378:metabolism; D007525:Isoelectric Focusing; D008566:Membranes / Q000187:drug effects / Q000201:enzymology; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D008970:Molecular Weight; D010634:Phenobarbital / Q000494:pharmacology; D051381:Rats
D000596:Amino Acids; D014453:Glucuronosyltransferase; D010634:Phenobarbital