Speaker
Stanislav Pavelka
(1Department of Radiometry, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague and 2Central-European Technology Institute, Masaryk University, Brno, Czech Republic)
Description
Details of newly elaborated radiometric methods for extremely sensitive determination of enzyme activities of iodothyronine deiodinases (IDs) of types 1, 2 and 3 are described. IDs catalyze selective 5’- (outer ring) and 5- (inner ring) monodeiodinations of iodothyronines and play crucial roles in the biotransformations of thyroid hormones (TH).
Our novel radiometric assays for IDs are based on the use of appropriate high-specific-radioactivity 125I-labeled iodothyronines as substrates; optimized TLC separation of radioactive products from the unconsumed substrates; film-less autoradiography of radio-chromatograms using storage phosphor screens; and quantification of the separated compounds with a BAS-5000 (Fujifilm Life Science Co.) laser scanner. During the developmental process, we found out optimum assay conditions, including concentrations of the respective radioactively labeled substrates; appropriate concentrations of thiol cofactor; the amount of total protein and enzyme concentration in the incubation mixtures; and suitable incubation times for the proper measurement of the individual IDs activities.
This methodology enabled us to determine IDs enzyme activities as low as 10 exp -18 katals, in microsomal fractions of different rat and human tissues, as well as in homogenates of cultured mammalian cells. We demonstrated the applicability of our sophisticated radiometric methods by following the alterations of IDs activities induced in cultured rat astroglial cells by a series of purinergic agonists, retinoic acid, and their combination. In the case of ATP as a representative of purinergic agonists, we determined also time-course and dose-response curves to characterize in more details the induction of each type of deiodinase by purines.
This work was supported by the Academy of Sciences of the Czech Rep. (Research project No. AV0Z50110509), by the Ministry of Education of the Czech Rep. (Research project No. MSM0021622413), and by the Czech Science Foundation (Grant No. 304/08/0256).
Author
Stanislav Pavelka
(1Department of Radiometry, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague and 2Central-European Technology Institute, Masaryk University, Brno, Czech Republic)
