Assay validation and characterization of hepatic 5'-deiodinase activity in ring doves using reverse-T3 as substrate.
Adult ring dove hepatic 5'-deiodinase (5'D) activity was studied in vitro using reverse T3 (rT3) as substrate. A previous study of ring dove hepatic deiodinase in our laboratory did not include characterization studies and was unsuccessful in relating the pattern of 5'D development to that of plasma T3. In the present study we established valid assay conditions and characterized the hepatic 5'D in doves to provide a comparison with other avian species and 5'D activity is proportional to enzyme concentration (as postmitochondrial fraction (PMF) protein) over the range measured (0-0.163 mg PMF protein/ml, representing 0-2.19 mg original tissue/ml). Activity was linear with time from 5 to 30 min of incubation at 0.163 mg PMF protein/ml. Our 5'D assays used 20 mM DTT; activity was maximal from 5 to 30 mM DTT. Using the validated conditions the following characteristics were found: the apparent Km was 0.44 microM rT3, Vmax was 255 pM rT3 degraded/min-mg PMF protein, and activity was completely inhibited by 1 mM PTU. Activity was maximal at pH 8.04 and at 37.5 degrees (although this did not differ from activity at 41.5 degrees, the body temperature of doves). In summary, this study demonstrates conditions that measure 5'D at initial velocities in dove liver and demonstrates that the hepatic 5'D enzyme in ring doves is similar to the deiodinase activity in liver of galliform birds and mammals.
Rieman
J D
JD
Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg 24061.
McNabb
F M
FM
D000818:Animals; D010856:Columbidae / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D007453:Iodide Peroxidase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D011441:Propylthiouracil / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature; D014284:Triiodothyronine / Q000097:blood; D014285:Triiodothyronine, Reverse / Q000378:metabolism*
D014284:Triiodothyronine; D014285:Triiodothyronine, Reverse; D011441:Propylthiouracil; D007453:Iodide Peroxidase; D004229:Dithiothreitol