Hepatic 5'deiodinase activity of Japanese quail using reverse-T2 as substrate: assay validation, characterization, and developmental studies
Using reverse-triiodothyronine, rT3, as substrate, an in vitro 5'deiodinase (5'0) assay was validated for adult Japanese quail, by defining conditions under which activity is proportional to enzyme (protein) concentration and is linear with incubation time. Activity was measured as the release of 1(125) from labeled rT3. Using validated assay conditions we found the following 5'0 characteristics: maximal activity from 10-50 mM dithiothreitol (cofactor), an apparent Km of 0.52 µM rT3, pH optimum of 7.6-8.5, complete inhibition by 1 mM propylthiouracil and by 1 mM iopanoic acid, and substrate affinities of rT3 > T 4> T3 . Based on these characteristics, the quail hepatic 5'0 activity is like the Type I 5'0 activity found in mammalian liver and kidney, and embryonic chicken liver. To determine how previous unvalidated assays, that used high tissue concentrations and relatively low substrate (T",) concentrations, influenced 5'0 studies we reevaluated 5'0 development using our assay validated for each developmental stage. We found extreme quantitative differences in the activities measured and in the proportional relationships among stages; and only limited qualitative Similarity existed in the pattern of 5'0 development when compared to unvalidated T4 assays. These data show good correspondence between whole liver 5'0 activity per unit body weight and plasma T3/T ... ratios for the developmental stages sampled.