GLUCURONIDATION OF THYROXINE AND 3,5,3'-TRIIODOTHYRONINE BY HEPATIC MICROSOMES IN RAINBOW-TROUT, ONCORHYNCHUS-MYKISS

We studied the glucuronidation of thyroxine (T-4) and 3,5,3'-triiodoth yronine (T-3) in the hepatic microsomal fraction of rainbow trout, Onc orhynchus mykiss. Uridine diphosphoglucuronosyl transferase (UDPGT) ac tivity toward T-4 depended on both protein concentration (linear up to about 0.75 mg/ml) and time (linear up to 60 min). The optimal pH for glucuronidation was 6.8-7.8 units for T-4 and greater than or equal to 8.5 units for T-3. At a common pH of 7.8, the apparent K-m and V-max values were, respectively, 6.0 mu M and 42 nmol/mg protein/hr for T-4, and 1.1 mu M and 4.3 nmol/mg protein/hr for T-3. At concentrations of 100 mu M, T-4 inhibited T-3-glucuronidation, but T-3 did not inhibit T-4-glucuronidation. T-4-glucuronidation was inhibited by 3,3', 5'-tri iodothyronine (rT(3)) and tetraiodothyroacetic acid (Tetrac); T-3-gluc uronidation was inhibited by rT(3), T-4, Tetrac, and triiodothyroaceti c acid. Therefore, analogues with two outer-ring iodines were the most effective inhibitors, showing that outer-ring iodine substitution inf luences UDPGT substrate specificity. Following a 15-min pre-incubation at 24 degrees C, UDPGT thermal stability was higher for T-4 than T-3. Polyoxyethylene 10 cetyl ether (Brij 56) maximally stimulated UDPGT a ctivity for both T-4 and T-3 about two-fold at 0.0125% (w/v) detergent , suggesting that the UDPGTs are transmembrane proteins with the activ e site facing the lumen of the microsomal vesicles. Clofibrate did not affect either T-4- or T-3-UDPGT activity. On a per mg microsomal prot ein basis, T-4-glucuronidation in the rat liver exceeded that in the t rout 3-fold. We conclude that (a) trout liver microsomes have more tha n one form of UDPGT for the glucuronidation of T-4 and T-3 and (b) the se trout UDPGTs share several properties of with those of the rat. (C) 1997 Elsevier Science Inc.