Microsoft word - pavelkaabstr-vienna091.doc

Adaptation of methods for radiometric determination of enzyme activities
of the key enzymes of thyroid hormones metabolism

Stanislav Pavelka a,b*
a Institute of Physiology, Academy of Sciences of the Czech Republic, v.v.i., 14220 Prague 4, Czech Republic; b Department of Biochemistry, Faculty of Science, Masaryk University, 61137 Brno, Czech Republic * Corresponding author: [email protected] or [email protected] The aims of the present studies were: first, to establish valid high bromide intake its thyrotoxic effects prevailed and TPO assay conditions for the radiometric determination of the activity was reduced. enzyme activities of thyroid peroxidase (TPO), iodothyronine Interestingly, elevated TPO activity was found in all rats sulfotransferases (ST) and uridinediphospho-glucuronyl- administered with perchlorate, regardless of the type of diet transferase (UDPGT) in various fractions of rat tissues; and (the content of iodine in the diet, respectively). second, with the aid of such developed assays, to follow the effects of some exogenous substances - bromide and perchlorate ions as supposed goitrogenic agents, and an antidepressant drug fluoxetine (Prozac) - on the metabolism The mentioned enzymes are involved either in the biosynthesis of TH in the thyroid gland (TPO), or in peripheral biotransformations of TH (ST and UDPGT, along with the most important iodothyronine deiodinases). Br conc. (g/l)
Br conc. (g/l)
Fig. 1 Specific peroxidase (TPO) activity, determined in microsomal Methods
fractions of the thyroid glands of rats maintained for up to 56 days on the iodine-sufficient diet B or iodine-deficient diet R, in dependence The procedure of the radiometric assay for TPO in vitro was on the extent of bromide intake. (The rats permanently drank based on the ability of TPO to oxidize 131I-iodide in the solutions of bromide with the concentrations of 0, 1, 2, 3 or 5 g/l). 2O2, generated in situ by glucose oxidase, and to catalyze subsequent iodination of specific tyrosyl residues The radiometric determination of iodothyronine sulfo- in the added thyroglobulin. The measure of TPO activity in transferases (ST) did not demonstrate any significant effects microsomal fractions of the thyroids was the amount of of the application to the rats of fluoxetine alone, or together radioiodine incorporated into thyroglobulin, determined with T3, on the induction of these enzyme activities. either after TLC separation of the incubated samples, or In contrast, administration of fluoxetine alone caused a simply after precipitation of radiolabeled protein and significant (about 2-fold) increase in glucuronyl-transferase measurement of 131I radioactivity in separated fractions. (UDPGT) activity (Table 1). However, the effect of The transfer of sulfonate moiety from the "active fluoxetine was completely abolished, provided it was applied sulfate", 3'-phosphoadenosine 5'-phosphosulfate, to the in combination with supraphysiological concentrations of T3. substrate L-3,3',5'-[125I]-triiodothyronine ([125I]-rT basis for radiometric determination of ST activity in liver cytosolic fractions. The extent of conversion was determined after the separation of the sulfated product from the unmodified [125I]-rT3 by Sephadex LH-20 chromatography. The rate of conjugation of the phenolic hydroxyl group 3 with glucuronic acid, catalyzed by UDPGT in rat liver microsomes, was measured in the radiometric assay for UDPGT. Reaction mixtures, containing uridinediphospho-glucuronic acid, the substrate [125I]-rT Table 1: The influence of fluoxetine, 3,5,3’-triiodothyronine (T3) and
microsomes, were analyzed after the incubation, again by their combination on specific enzyme activities (fmol rT3-G/h/mg prot.) of UDPGT in rat liver microsomes. chromatography on microcolumns of Sephadex LH-20. Results and Discussion
Acknowledgements: This work was supported by the Academy of Sciences of the Czech Republic (Research project No. AV0Z With the use of the described radiometric assay for TPO, we 50110509), by the Ministry of Education of the Czech Republic found that the influence of exogenous bromide on the TPO (Research project No. MSM0021622413), by the GA CR (Grant No. activity in the rat thyroids was biphasic, with regard to the 304/08/0256), GA AS CR (Grant No. KJB401630701) and by the extent of bromide intake in the animals (Fig. 1). An increase grant from the EC (Project MYORES, contract No. 511978). (up to 3-fold) in TPO activity was measured in rats with a low or moderate bromide intake while in animals with very


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