Abstract

The export of glutathione from cultured human diploid fibroblasts into the surrounding medium was found by isotopic labeling experiments using [35S]cystine and by enzymatic measurements. The major part of the glutathione exported from the cells was found in normal culture medium as mixed disulfide of glutathione and cysteine. Radioactivity of 35S, mostly derived from cellular glutathione, was mainly located in glutathione moiety, not in cysteine moiety, of the mixed disulfide. Export of free glutathione was found when cystine-free medium was used. It was, therefore, concluded that mixed disulfide of glutathione and cysteine was formed in the medium by exported glutathione and medium cystine via sulfhydryl-disulfide exchange reaction. Amount of total glutathione exported from the cells was measured by enzymatic method and it was found that more than 10% of normal cellular glutathione was exported within 2 h. Apparent concentration of glutathione in the medium after a day of culture reached 3 to 4 micrometer, which was comparable to that observed in normal plasma by the same enzymatic method.

Highlights

  • The export of glutathione from cultured human diploid fibroblasts into the surrounding medium was found by isotopic labeling experiments using [3SS]cystine and by enzymatic measurements

  • Amount of total glutathione exported from the cells was measured by enzymatic method and it was found that more than 10% of normal cellular glutathione was exported within 2 h

  • No radioactivity was detected in the region of cystine, glutathione disulfide, and GSSCy. These results indicate that cystine in the culture medium is reduced within the cells and the resulting cysteine is rapidly incorporated into cellular glutathione

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Summary

PROCEDURES

Materials-Glutathione, its disulfide, L-cysteine, L-cystine, and dithiothreitol were obtained from Sigma Chemical Co. Carboxypeptidase A and yeast glutathione reductase were products of Boehringer. Taurine was obtained from Wako Chemical Co. L-Cystinyl-bisglycine was obtained from Vega Fox. L-y-Glutamyl-L-cysteine disulflde was obtained by treatment of glutathione disulfide with carboxypeptidase. L-y-Glutamyl-L-cystine was prepared by treating L-y-glutamylr,-cysteine disulfide with dithiothreitol and cysteine [4]. The unsymmetrical disulfide of glutathione and cysteine was prepared from the mixture of cysteine and glutathione disulfide by chromatography on a Dowex 1 resin column [5]. The cells were routinely grown in BME’ supplemented with 10% newborn calf serum. The cells between 18th and 28th passage were used. For each experiment cells were seeded at the required cell density in plastic dishes and were incubated for a few days at 37°C in a humid atmosphere of 5% Cog-air mixture. Cystine-free BME was prepared from Earle’s balanced salt solution, BME vitamin solution, and amino acid mixture that was lacking in cystine

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RESULTS
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Findings
DISCUSSION
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