title: Separation, purification, and characterization of digitoxigenin-monodigitoxoside UDP-glucuronosyltransferase activity.
abstract: Glucuronidation of digitoxigenin-monodigitoxoside was investigated in liver microsomes from spironolactone-induced male Wistar rats. Isolation of a specific digitoxigenin-monodigitoxoside UDP-glucuronosyltransferase was possible utilizing chromatofocusing chromatography with a gradient from pH 10.1 to 8.0 after solubilizing the microsomal protein with the nonionic detergent Emulgen 911. The digitoxigenin-monodigitoxoside UDP-glucuronosyltransferase was further purified using UDP-hexanolamine Sepharose 4B affinity chromatography. The highly purified (75-fold) enzyme showed activity toward digitoxigenin-monodigitoxoside and slight activity toward digitoxigenin-bisdigitoxoside, whereas digitoxin and substrates for p-nitrophenol, 17 beta-OH steroid, and 3 alpha-OH steroid UDP-glucuronosyltransferases were not glucuronidated. In addition, bilirubin, morphine, estrone, 4-hydroxybiphenyl, and aromatic amines were not glucuronidated by this protein. These results strongly confirm the presence of a form of UDP-glucuronosyltransferase, which is highly specific for the glucuronidation of digitoxigenin-monodigitoxoside.
authors.forename: L
authors.initials: L
authors.lastname: von Meyerinck
authors.forename: B L
authors.initials: BL
authors.lastname: Coffman
authors.forename: M D
authors.initials: MD
authors.lastname: Green
authors.forename: R B
authors.initials: RB
authors.lastname: Kirkpatrick
authors.forename: A
authors.initials: A
authors.lastname: Schmoldt
authors.forename: T R
authors.initials: TR
authors.lastname: Tephly
mesh_terms: D000818:Animals; D003902:Detergents / Q000494:pharmacology; D004073:Digitoxigenin / Q000031:analogs & derivatives* / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000302:isolation & purification* / Q000378:metabolism; D066298:In Vitro Techniques; D007525:Isoelectric Focusing; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D011487:Protein Conformation; D051381:Rats; D011919:Rats, Inbred Strains; D013148:Spironolactone / Q000494:pharmacology; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D003902:Detergents; D004073:Digitoxigenin; C035486:digitoxigenin monodigitoxoside; D013148:Spironolactone; D014453:Glucuronosyltransferase