Glucuronidation of thyroxine and 3,5,3'-triiodothyronine by hepatic microsomes in rainbow trout, Oncorhynchus mykiss.
We studied the glucuronidation of thyroxine (T4) and 3,5,3'-triiodothyronine (T3) in the hepatic microsomal fraction of rainbow trout, Oncorhynchus mykiss. Uridine diphosphoglucuronosyl transferase (UDPGT) activity toward T4 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 T4 and > or = 8.5 units for T3. At a common pH of 7.8, the apparent Km and Vmax values were, respectively, 6.0 muM and 42 nmol/mg protein/hr for T4, and 1.1 muM and 4.3 nmol/mg protein/hr for T3. At concentrations of 100 muM, T4 inhibited T3-glucuronidation, but T3 did not inhibit T4-glucuronidation. T4-glucuronidation was inhibited by 3,3', 5'-triiodothyronine (rT3) and tetraiodothyroacetic acid (Tetrac); T3-glucuronidation was inhibited by rT3, T4, Tetrac, and triiodothyroacetic acid. Therefore, analogues with two outer-ring iodines were the most effective inhibitors, showing that outer-ring iodine substitution influences UDPGT substrate specificity. Following a 15-min pre-incubation at 24 degrees C, UDPGT thermal stability was higher for T4 than T3. Polyoxyethylene 10 cetyl ether (Brij 56) maximally stimulated UDPGT activity for both T4 and T3 about two-fold at 0.0125% (w/v) detergent, suggesting that the UDPGTs are transmembrane proteins with the active site facing the lumen of the microsomal vesicles. Clofibrate did not affect either T4- or T3-UDPGT activity. On a per mg microsomal protein basis, T4-glucuronidation in the rat liver exceeded that in the trout 3-fold. We conclude that (a) trout liver microsomes have more than one form of UDPGT for the glucuronidation of T4 and T3 and (b) these trout UDPGTs share several properties of with those of the rat.
Finnson
K W
KW
Department of Zoology, University of Manitoba Winnipeg, Canada.
Eales
J G
JG
D000818:Animals; D005965:Glucuronates / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism*; D017686:Oncorhynchus mykiss / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013974:Thyroxine / Q000378:metabolism*; D014284:Triiodothyronine / Q000378:metabolism*
D005965:Glucuronates; D014284:Triiodothyronine; D014453:Glucuronosyltransferase; D013974:Thyroxine