Characterization of cytoplasmic T3 binding sites by adsorption to hydroxyapatite: effects of drug inhibitors of T3 and relationship to glutathione-S-transferases.
To facilitate studies of thyroid hormone (T3) binding to cytoplasmic proteins, we prepared monkey (M. fascicularis) liver cytosol (100,000g supernatant) and examined T3 binding using hydroxyapatite (HAP) separation. HAP adsorbs cytoplasmic and nuclear binding sites but not serum T4 binding proteins. Cytosol was incubated with [125I]T3 for 30 min at 4 degrees C and separated by adding an equal volume of HAP (15 g/100 mL). After a further incubation of 10 min, the HAP pellet was washed three times in buffer containing Triton X-100, 0.5%. With this method, a single class of T3 binding site was observed with Kd 15.8 +/- 1.2 nM, concentration 0.62 +/- 0.17 pmol/mg protein (n = 3, mean +/- SD). We used this assay to assess potential drug inhibitors of cytoplasmic binding and to evaluate the proposal that glutathione-S-transferases (GST) and cytoplasmic T3 binding proteins are identical. Displacement of [125I]T3 by unlabeled iodothyronines relative to T3 (100) was T4 58, Triac 7, rT3 7, Tetrac less than or equal to 1. This hierarchy indicates that this binding site is distinct from nuclear or serum binding sites. T3 binding was displaceable by nonsteroidal anti-inflammatory drugs (NSAID) and nonbile acid cholephils (NBAC). Half-inhibitory concentrations (microM, mean +/- SD, n greater than or equal to 3) were diclofenac 4.9 +/- 1.3, mefenamic acid 13.6 +/- 0.6, bromosulphthalein 45 +/- 3, iopanoic acid approximately 200. Amiodarone and furosemide were inactive up to 100 microM. No displacement was observed with cortisol or the bile acid taurocholate, up to 100 microM. Dithiothreitol, 5 mM, did not change binding affinity or capacity.(ABSTRACT TRUNCATED AT 250 WORDS)
Barlow
J W
JW
Ewen Downie Metabolic Unit, Alfred Hospital, Melbourne, Australia.
Raggatt
L E
LE
Lim
C F
CF
Topliss
D J
DJ
Stockigt
J R
JR
D000638:Amiodarone / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D001667:Binding, Competitive; D003600:Cytosol / Q000378:metabolism; D004008:Diclofenac / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D017886:Durapatite; D005665:Furosemide / Q000494:pharmacology; D005982:Glutathione Transferase / Q000502:physiology*; D006854:Hydrocortisone / Q000494:pharmacology; D006882:Hydroxyapatites / Q000378:metabolism*; D066298:In Vitro Techniques; D007480:Iopanoic Acid / Q000494:pharmacology; D008099:Liver / Q000378:metabolism; D008252:Macaca fascicularis; D008528:Mefenamic Acid / Q000494:pharmacology; D013448:Sulfobromophthalein / Q000494:pharmacology; D013656:Taurocholic Acid / Q000494:pharmacology; D014284:Triiodothyronine / Q000037:antagonists & inhibitors* / Q000378:metabolism*
D006882:Hydroxyapatites; D014284:Triiodothyronine; D013448:Sulfobromophthalein; D004008:Diclofenac; D008528:Mefenamic Acid; D013656:Taurocholic Acid; D005665:Furosemide; D017886:Durapatite; D005982:Glutathione Transferase; D007480:Iopanoic Acid; D000638:Amiodarone; D004229:Dithiothreitol; D006854:Hydrocortisone