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- Title
Radiometric enzyme assays: development of methods for extremely sensitive determination of types 1, 2 and 3 iodothyronine deiodinase enzyme activities.
- Authors
Pavelka, Stanislav
- Abstract
We elaborated novel, reliable radiometric methods for extremely sensitive determination of enzyme activities of iodothyronine deiodinases (IDs) of types 1, 2 and 3 in microsomal fractions of different rat and human tissues, as well as in homogenates of cultured mammalian cells. These radiometric enzyme assays were based on the use of high-specific-radioactivity I-labeled iodothyronines as substrates; TLC separation of radioactive products from the unconsumed substrates; film-less autoradiography of radiochromatograms using storage phosphor screens; and quantification of the separated compounds with a BAS-5000 laser scanner. This methodology enabled us to determine IDs enzyme activities as low as 10 katals. The applicability of our sophisticated methods was demonstrated by following the alterations of IDs activities induced in cultured astroglial cells by a series of purinergic agonists, retinoic acid, and their combination. In addition, in case of ATP as a representative of purinergic agonists, we determined time-course and dose-response curves to characterize in more details the induction of each type of deiodinase by purines.
- Subjects
FIRE assay; RADIATION measurements; CELLS; ENZYME kinetics; RADIOACTIVITY; AUTORADIOGRAPHY; RADIOCHROMATOGRAPHY; PURINES; THYROID hormones
- Publication
Journal of Radioanalytical & Nuclear Chemistry, 2010, Vol 286, Issue 3, p861
- ISSN
0236-5731
- Publication type
Article
- DOI
10.1007/s10967-010-0798-8