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.

Name thesis:degree_name

Master of Science

Level thesis:degree_level

masters

Discipline thesis:degree_discipline

Biology

Department dc:contributor.department

Biology

Grantor dc:publisher

Virginia Tech

Year dc:date.issued

1991

Author dc:creator

• Freeman, Thomas Bernard

Chair dc:contributor.committeechair

• McNabb, F. M. Anne

Committee members dc:contributor.committeemember

• McNabb, Roger A.
• Akers, Robert Michael

Statement dc:rights

• In Copyright

Licence dc:rights.uri

• http://rightsstatements.org/vocab/InC/1.0/

Language dc:language.iso

en

Dc Identifier Other

etd-08042009-040343

OAI identifier oai:identifier

oai:vtechworks.lib.vt.edu:10919/44156