GLUCURONIDATION OF ALL-TRANS-RETINOIC ACID AND 5,6-EPOXY-ALL-TRANSRETINOIC 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 glucuronidatio n of all-trans-retinoic acid (atRA) and 5,6-epoxy-atRA have been inves tigated using liver microsomes from Sprague-Dawley and Fischer 344 rat s, Conditions for enzymatic glucuronidation were optimized for substra te concentration, protein, and time by using atRA and Sprague-Dawley m icrosomes. With detergent-activated Sprague-Dawley microsomes, 5,6-epo xy-atRA was shown to be a significantly better substrate than atRA for microsomal glucuronidation (263 vs. 116 pmol/mg/min for 5,6-epoxy-atR A and atRA, respectively). The product of incubation of microsomes wit h 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 a nd 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 micros omes from Fischer 344 rats treated with phenobarbital and 3-methylchol anthrene, compared with untreated controls, The two compounds were equ ally effective inducers of activity, although 5,6-epoxy-atRA was again the better substrate. The same control and induced Fischer rat micros omes were photolabeled with [P-32]5-azido-UDP-glucuronic acid in the a bsence or presence of detergent, two concentrations of alamethicin, an d a 10-fold molar excess of unlabeled UDP-glucuronic acid. Photoincorp oration into microsomal proteins from detergent-disrupted induced micr osomes was 2-3 times greater than that of controls. Alamethicin increa sed photoincorporation of the probe into UDP-glucuronosyltransferase p roteins an additional 1.5-2-fold in control and induced microsomes, co mpared with the respective detergent-activated samples.