Glucuronidation of all-trans-retinoic acid and 5,6-epoxy-all-trans-retinoic acid. Activation of rat liver microsomal UDP-glucuronosyltransferase activity by alamethicin.
The effects of detergent, alamethicin (a channel-forming peptide), and the inducers phenobarbital and 3-methylcholanthrene on glucuronidation of all-trans-retinoic acid (atRA) and 5,6-epoxy-atRA have been investigated using liver microsomes from Sprague-Dawley and Fischer 344 rats. Conditions for enzymatic glucuronidation were optimized for substrate concentration, protein, and time by using atRA and Sprague-Dawley microsomes. With detergent-activated Sprague-Dawley microsomes, 5,6-epoxy-atRA was shown to be a significantly better substrate than atRA for microsomal glucuronidation (263 vs. 116 pmol/mg/min for 5,6-epoxy-atRA and atRA, respectively). The product of incubation of microsomes with atRA and UDP-glucuronic acid was identified as a glucuronide by beta-glucuronidase hydrolysis and by HPLC analysis. Alamethicin was shown to be a highly effective activator of glucuronidation activity; atRA and 5,6-epoxy-atRA glucuronidation rates were increased 2- and 3-fold, respectively, compared with detergent activation. Alamethicin (but not detergent) significantly increased retinoid glucuronidation by microsomes from Fischer 344 rats treated with phenobarbital and 3-methylcholanthrene, compared with untreated controls. The two compounds were equally effective inducers of activity, although 5,6-epoxy-atRA was again the better substrate. The same control and induced Fischer rat microsomes were photolabeled with [32P]5-azido-UDP-glucuronic acid in the absence or presence of detergent, two concentrations of alamethicin, and a 10-fold molar excess of unlabeled UDP-glucuronic acid. Photoincorporation into microsomal proteins from detergent-disrupted induced microsomes was 2-3 times greater than that of controls. Alamethicin increased photoincorporation of the probe into UDP-glucuronosyltransferase proteins an additional 1.5-2-fold in control and induced microsomes, compared with the respective detergent-activated samples.
Little
J M
JM
Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock 72205, USA.
Lehman
P A
PA
Nowell
S
S
Samokyszyn
V
V
Radominska
A
A
D000345:Affinity Labels; D000408:Alamethicin / Q000494:pharmacology*; D000818:Animals; D004790:Enzyme Induction; D014453:Glucuronosyltransferase / Q000378:metabolism*; D008297:Male; D008748:Methylcholanthrene / Q000494:pharmacology; D008862:Microsomes, Liver / Q000187:drug effects* / Q000201:enzymology; D010634:Phenobarbital / Q000494:pharmacology; D051381:Rats; D011916:Rats, Inbred F344; D017207:Rats, Sprague-Dawley; D014212:Tretinoin / Q000031:analogs & derivatives* / Q000378:metabolism*
D000345:Affinity Labels; C018058:5,6-epoxyretinoic acid; D000408:Alamethicin; D008748:Methylcholanthrene; D014212:Tretinoin; D014453:Glucuronosyltransferase; D010634:Phenobarbital