Studies on a testosterone glucuronyltransferase from the cytosol fraction of human liver.
An enzyme that conjugates the 17beta-hydroxyl group of testosterone was found in the cytosol fraction of human liver. The same enzyme preparation also conjugates the 16alpha-hydroxyl group of oestriol. The enzymic activity could not be sedimented by centrifuging the cytosol fraction at 158000g(av.) for 120min. The testosterone-conjugating as well as the oestriol-conjugating activities were found in the precipitate obtained after 30% saturation of the cytosol fraction with ammonium sulphate. Filtration of the precipitate through Sephadex G-200 enriched the testosterone-conjugating enzyme 50-fold and the oestriol-conjugating enzyme 100-fold. No separation of the two activities was achieved. With labelled testosterone the product of the reaction, testosterone 17beta-glucuronide, was identified by paper chromatography and by crystallization to constant specific radioactivity. Testosterone 17beta-glucuronyltransferase was active between pH7.0 and 8.6 in tris-HCl and tris-maleate buffers. The apparent K(m) values for testosterone and UDP-glucuronic acid were 6.4 and 25mum respectively. The enzyme was active between 37 and 45 degrees C; the activation energy was calculated to be 5kcal/mol. Oestriol did not influence the glucuronidation of testosterone. Controlled heating as well as alternate freezing and thawing of the purified enzyme preparation led to an inactivation of both testosterone-conjugating and oestriol-conjugating activities at similar rates. Testosterone and oestriol, when incubated together, gave a reaction rate that was approximately equal to the sum of the rates when the two substrates were incubated separately. The present findings suggest that testosterone and oestriol are conjugated by two separate enzymes.
D000293:Adolescent; D002247:Carbon Isotopes; D002845:Chromatography; D002850:Chromatography, Gel; D003545:Cysteine; D004964:Estriol / Q000378:metabolism; D005965:Glucuronates / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008274:Magnesium; D008297:Male; D013696:Temperature; D013739:Testosterone / Q000378:metabolism; D014166:Transferases / Q000302:isolation & purification*; D014500:Uracil Nucleotides / Q000378:metabolism
D002247:Carbon Isotopes; D005965:Glucuronates; D014500:Uracil Nucleotides; D013739:Testosterone; D014166:Transferases; D004964:Estriol; D008274:Magnesium; D003545:Cysteine