Title page for ETD etd-08042009-040343
|Type of Document
||Freeman, Thomas Bernard
||Hepatic 5'deiodinase activity of Japanese quail using reverse-T2 as substrate : assay validation, characterization, and developmental studies
||Master of Science
|McNabb, F. M. Anne
|Akers, Robert Michael
|McNabb, Roger A.
|Date of Defense
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.
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