Cystine metabolism in human fibroblasts. Comparison of normal, cystinotic, and gamma-glutamylcysteine synethetase-deficient cells.

R G Oshima,W J Rhead,J G Thoene,J A Schneider

doi:10.1016/s0021-9258(17)33294-5

Abstract

Incubation of normal human skin fibroblasts or fibroblasts derived from patients with erythrocyte deficiency of gamma-glutamylcysteine synthetase (gamma-glutamylcysteine synthetase-deficient) in culture medium containing L-[35S]cystine resulted in incorporation of radioactivity into protein, cysteine, and glutathione, gamma-Glutamylcysteine synthetase-deficient fibroblasts synthesized glutathione from [35S]cystine at 30% the rate of normal cells and contained 30% the normal amount of glutathione. Cystinotic fibroblasts incorporated [35S]cystine into the large intracellular cystine pool not found in normal or gamma-glutamylcysteine synthetase-deficient cells and also appeared to synthesize glutathione more slowly than normal cells. However, the radioactivity recovered as cystine was reduced greatly and the rate of [35S]cystine incorporation into glutathione increased if cystinotic cells were first depleted of their intracellular cystine pool before incubation in [35S]cystine. This suggests that the apparent reduced rate of glutathione synthesis observed in untreated cystinotic cells was a secondary effect caused by dilution of the [35S]cystine by the large pool of nonradioactive cystine. Cystinotic cells depleted of cystine by treatment with mercaptoethylamine reaccumulate 30 to 50% of their initial cystine in 24 hours in the absence of extracellular cystine. Both normal and cystinotic cells lose more than 90% of their intracellular glutathione in 24 hours in cystine-free medium. Both cell types can reutilize cysteine from glutathione for protein synthesis.

Highlights

From the Department of Pediatrics, University of California, San Diego, School of Medicine, La Jolla, California 92093
Incubation of normal human skin fibroblasts or fibroblasts derived from patients with erythrocyte deficiency of y-glutamylcysteine synthetase (y-glutamylcysteine synthetase-deficient) in culture medium containing L- [%]cystine resulted in incorporation of radioactivity into protein, cysteine, and glutathione. y-Glutamylcysteine synthetase-deficient fibroblasts synthesized glutathione from [%]cystine at 30% the rate of normal cells and contained 30% the normal amount of glutathione
The radioactivity recovered as cystine was reduced greatly and the rate of [SsS]cystine incorporation into glutathione increased if cystinotic cells were first depleted of their intracellular cystine pool before incubation in [SsS]cystine

Summary

PROCEDURE

Materials-L-“C-amino-acid mixture (57 mCi/mmol of carbon) uniformly labeled alanine, arginine, asparatate, glutamate, glycine, leucine, isoleucine, lysine, pbenylalanine, proline, serine, threonine, tyrosine and valine, and G [a6S]cystine (100 to 300 mCi/mmol) were purchased from Amersham/Searle. When a more nearly cystine-free medium was necessary, the concentration of dialyzed serum was reduced appropriately. Cell Culture-Control and cystinotic human fibroblasts were started from skin biopsies and maintained in modified F-12 medium supplemented with 10% fetal bovine serum [17]. Fibroblast cultures from patients wlth y-glutamylcysteine synthetase deficiency were grown from biopsies provided by Dr Frederick Richards, II (S).l Al1 experiments were done on mycoplasm-free [18] cells of less than 30 passages in culture. The resultant precipitate was removed by centrifugation and redissolved in 1 N NaOH for protein [20] and radioactivity determination. The radioactive components of the acid soluble contents of the cells pulsed with [a”S]cystine were found to co-electrophorese with unlabeled glutathione-NEM, cysteine-NEM, cystine, and small amounts of unidentified material near the origin. Protein was determined colorimetrically [20] on the precipitate recovered after centrifugation

RESULTS

F Initial GSH Protein I

Findings

DISCUSSION