In situ structural analysis of microsomal UDP-glucuronyltransferases by radiation inactivation.
The structure of the UDP-glucuronyltransferases in microsomes from guinea pig and rat liver was examined in situ by radiation inactivation analysis. The p-nitrophenol conjugating activity of guinea pig microsomes increased at lower doses of radiation; at higher doses (greater than or equal to 36 megarads), activity showed a first order decline yielding a target size of 71 +/- 9 kDa. Treating microsomes with Triton X-100 eliminated the activation seen at lower doses of radiation and yielded a simple exponential decrease in activity which gave a larger target size (95 +/- 18 kDa). A monoexponential decrease in activity was seen in sonicated microsomes, at greater than or equal to 36 megarads. The same response was obtained when the reaction was assayed in the reverse direction. The estrone conjugating activity of guinea pig microsomes was similarly activated at lower doses of radiation and declined at higher doses (greater than or equal to 36 megarads), with a target size of 57 +/- 11 kDa. Allosteric activation of the enzyme by UDP-N-acetylglucosamine was eliminated by lower doses of radiation. Thus, activation of the enzyme by radiation, detergent, sonication, and UDP-N-acetylglucosamine appear to be interdependent. These activations are postulated to be due to the existence of the enzyme in an oligomeric form which can be dissociated into monomers with higher activity. The same biphasic activation-inactivation curves were obtained for p-nitrophenol conjugation in rat liver microsomes. The target sizes were 54 +/- 8 kDa (p-nitrophenol in the forward direction) and 66 +/- 10 kDa (p-nitrophenol in the reverse direction). Thus, the enzyme appears to be smaller in rat liver as compared with guinea pig liver. Lithocholate glucuronidating activity in rat liver microsomes (at greater than 36 megarads) gave a target size of 74 +/- 1 kDa.
Vessey
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Liver Study Unit, Veterans Administration Medical Center, San Francisco 94121.
Kempner
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D000818:Animals; D004970:Estrone / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000528:radiation effects*; D006168:Guinea Pigs; D066298:In Vitro Techniques; D008862:Microsomes, Liver / Q000201:enzymology*; D009596:Nitrophenols / Q000378:metabolism; D010315:Particle Accelerators; D011487:Protein Conformation; D051381:Rats
D009596:Nitrophenols; D004970:Estrone; D014453:Glucuronosyltransferase; C024836:4-nitrophenol