Involvement of sulfhydryl groups in the action of insulin and radiation on thymocyte Na +-dependent amino acid transport

Abstract

1. 1. p- Chloromercuribenzene sulfonate concentrations <10 −5 M stimulate the the uptake by thymocytes of 2-aminoisobutyrate, a non-metabolized amino acid. At concentrations > 10 −5 M of this reagent, transport is impaired and cell viability is affected. In contrast, 5,5′-dithiobis-(2-nitrobenzoate) between 10 −4 and 10 −6 M produces only stimulation of 2-aminoisobutyrate uptake after treating for 10 min. 2. 2. Treatment of thymocytes with 10 −4 M 5,5′-dithiobis-(2-nitrobenzoate) reveals at least three categories of reactive SH groups. Titration of the most rapidly reacting category, 4 · 10 7−7 · 10 7/ cell , activates 2-aminoisobutyrate transport to the same extent as does p- chloromercuribenzene sulfonate. Cells treated with 10 −6 M insulin showed a 30–50% reduction in the number of sulfhydryl groups that could be titrated with 5,5′-dithiobis-(2-nitrobenzoate). In thymocytes treated with 10 −6 M p- chloro[ 203Hg] mercuribenzene sulfonate, addition of 10 −6 or 10 −9 M insulin before treatment with the sulfhydryl reagent again reduces the number of titrable SH groups by 20%. 3. 3. Insulin (10 −10−10 −6 M) also stimulates 2-aminoisobutyrate uptake, but the effects of insulin and SH blocker are not addictive. 4. 4. Insulin, but not p- chloromercuribenzene sulfonate, prevents the impairment of 2-aminoisobutyrate transport caused by γ-irradiation. Treatment of cells with p- choloromercuribenzene sulfonate prior to irradiation increases the radiation impairment of 2-aminoisobutyrate transport. 5. 5. γ-irradiation reduces the number of 5,5′-dithiobis-(2-nitrobenzoate) reactive sulfhydryl residues by 37%. 6. 6. A model for the action of insulin and irradiation on 2-aminoisobutyrate transport is presented.