Radiometric enzyme assays: development of methods for extremely sensitive determination of types 1, 2 and 3 iodothyronine deiodinase enzyme activities

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 ¹²⁵I-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 10⁻¹⁸ 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.