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. 1974 Apr;53(4):1003–1016. doi: 10.1172/JCI107637

Uptake and Utilization of Exogenous Cystine by Cystinotic and Normal Fibroblasts

Beatrice States 1,2,3, Dorothy Harris 1,2,3, Stanton Segal 1,2,3
PMCID: PMC333085  PMID: 4815074

Abstract

The uptake of l-[35S]cystine was studied in six cystinotic and six normal fibroblast lines grown for five days either on cover slips or in 32-oz plastic flasks. Cystinotics showed greater uptake than normals. The apparent Kt for cystine entry in both types of cells was 0.043 mM but cystinotic cells showed a higher maximum velocity of entry. A comparison of the fate of l-[35S]cystine incubated for 20 min with monolayers of cells showed 30% and 15% of the intracellular 35S to be l-cystine in cystinotic and normal cells, respectively. The 35S effluxed more slowly from cystinotic than from normal cells after a 20-min preloading with l-[35S]cystine. Identification of 35S compounds in efflux media after 3 min showed 75% of the total 35S was l-cystine with the remainder in cysteine and acidic sulfur metabolites of cystine with no essential difference between cystinotics and normals. In paired experiments, the specific activity of the effluxed l-[35S]cystine after both efflux periods was the same as that entering the cell, thus indicating that the free l-[35S]cystine had not exchanged with the pre-existing pool in the cystinotic cells. During 3 min efflux, the l-cystine pool in normal cells was depleted mainly by loss of free cystine. In cystinotic cells, a new steady state was attained after 21 min of efflux and the intracellular l-[35S]cystine had the same percentage of total radioactivity seen after the initial 20-min uptake. After the rapid efflux of l-[35S]cystine from normals, [35S]cysteine and other labeled cystine metabolites appeared in the efflux media. By the end of a 3-min efflux, cystinotic cells had incorporated more label into reduced glutathione than had normal cells. However, when the new steady state was attained in cystinotics, the amounts of 35S in glutathione were not markedly different in the two types of cells. Approximately 95% of the total label could be accounted for in free sulfur compounds.

The data show an increased uptake and decreased efflux of cystine from cystinotic cells. However, it is not possible to conclude if these differences are due to primary changes in membrane function or to the reflection of metabolic defects without further investigation.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Crawhall J. C., Segal S. The intracellular ratio of cysteine and cystine in various tissues. Biochem J. 1967 Nov;105(2):891–896. doi: 10.1042/bj1050891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. FOGH J., FOGH H. A METHOD FOR DIRECT DEMONSTRATION OF PLEUROPNEUMONIA-LIKE ORGANISMS IN CULTURED CELLS. Proc Soc Exp Biol Med. 1964 Dec;117:899–901. doi: 10.3181/00379727-117-29731. [DOI] [PubMed] [Google Scholar]
  3. Foster D. O., Pardee A. B. Transport of amino acids by confluent and nonconfluent 3T3 and polyoma virus-transformed 3T3 cells growing on glass cover slips. J Biol Chem. 1969 May 25;244(10):2675–2681. [PubMed] [Google Scholar]
  4. Groth U., Rosenberg L. E. Transport of dibasic amino acids, cystine, and tryptophan by cultured human fibroblasts: absence of a defect in cystinuria and Hartnup disease. J Clin Invest. 1972 Aug;51(8):2130–2142. doi: 10.1172/JCI107020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. HAYFLICK L., MOORHEAD P. S. The serial cultivation of human diploid cell strains. Exp Cell Res. 1961 Dec;25:585–621. doi: 10.1016/0014-4827(61)90192-6. [DOI] [PubMed] [Google Scholar]
  6. Hummeler K., Zajac B. A., Genel M., Holtzapple P. G., Segal S. Human cystinosis: intracellular deposition of cystine. Science. 1970 May 15;168(3933):859–860. doi: 10.1126/science.168.3933.859. [DOI] [PubMed] [Google Scholar]
  7. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  8. Mahoney M. J., Rosenberg L. E. Uptake of alpha-aminoisobutyric acid by cultured human fibroblasts. Biochim Biophys Acta. 1970 Dec 1;219(2):500–502. doi: 10.1016/0005-2736(70)90232-4. [DOI] [PubMed] [Google Scholar]
  9. McNamara P., Rea C., Segal S. Sugar transport: effect of temperature on concentrative uptake of alpha-methylglucoside by kidney cortex slices. Science. 1971 Jun 4;172(3987):1033–1034. doi: 10.1126/science.172.3987.1033. [DOI] [PubMed] [Google Scholar]
  10. Platter H., Martin G. M. Tryptophane transport in cultures of human fibroblasts. Proc Soc Exp Biol Med. 1966 Oct;123(1):140–143. doi: 10.3181/00379727-123-31424. [DOI] [PubMed] [Google Scholar]
  11. ROSENBERG L. E., BERMAN M., SEGAL S. Studies of the kinetics of amino acid transport, incorporation into portein and oxidation in kidney-cortex slices. Biochim Biophys Acta. 1963 Jun 4;71:664–675. doi: 10.1016/0006-3002(63)91140-5. [DOI] [PubMed] [Google Scholar]
  12. ROSENBERG L. E., DOWNING S. TRANSPORT OF NEUTRAL AND DIBASIC AMINO ACIDS BY HUMAN LEUKOCYTES: ABSENCE OF DEFECT IN CYSTINURIA. J Clin Invest. 1965 Aug;44:1382–1393. doi: 10.1172/JCI105243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. SEGAL S. DISORDERS OF AMINO ACID TRANSPORT. Ann Intern Med. 1965 Apr;62:847–851. doi: 10.7326/0003-4819-62-4-847. [DOI] [PubMed] [Google Scholar]
  14. Schneider J. A., Bradley K. H., Seegmiller J. E. Transport and intracellular fate of cysteine-35S in leukocytes from normal subjects and patients with cystinosis. Pediatr Res. 1968 Nov;2(6):441–450. doi: 10.1203/00006450-196811000-00001. [DOI] [PubMed] [Google Scholar]
  15. Schneider J. A., Rosenbloom F. M., Bradley K. H., Seegmiller J. E. Increased free-cystine content of fibroblasts cultured from patients with cystinosis. Biochem Biophys Res Commun. 1967 Nov 30;29(4):527–531. doi: 10.1016/0006-291x(67)90516-5. [DOI] [PubMed] [Google Scholar]
  16. Schulman J. D., Bradley K. H. The metabolism of amino acids, peptides, and disulfides in lysosomes of fibroblasts cultured from normal individuals and those with cystinosis. J Exp Med. 1970 Dec 1;132(6):1090–1104. doi: 10.1084/jem.132.6.1090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Schulman J. D., Schneider J. A., Bradley K. H., Seegmiller J. E. Cystine, cysteine, and glutathione metabolism in normal and cystinotic fibroblasts in vitro, and in cultured normal amniotic fluid cells. Clin Chim Acta. 1972 Mar;37:53–58. doi: 10.1016/0009-8981(72)90415-9. [DOI] [PubMed] [Google Scholar]
  18. Sedwick W. D., Wiktor T. J. Reproducible plaquing system for rabies, lymphocytic choriomeningitis,k and other ribonucleic acid viruses in BHK-21-13S agarose suspensions. J Virol. 1967 Dec;1(6):1224–1226. doi: 10.1128/jvi.1.6.1224-1226.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Segal S., Crawhall J. C. Characteristics of cystine and cysteine transport in rat kidney cortex slices. Proc Natl Acad Sci U S A. 1968 Jan;59(1):231–237. doi: 10.1073/pnas.59.1.231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. States B., Segal S. Developmental aspects of cystine transport in rat intestinal segments. Biochim Biophys Acta. 1968 Sep 17;163(2):154–162. doi: 10.1016/0005-2736(68)90093-x. [DOI] [PubMed] [Google Scholar]
  21. States B., Segal S. Quantitation of cyst(e)ine in human fibroblasts and separation of cysteines sulfinic acid, cysteic acid and taurine. Clin Chim Acta. 1973 Jan 10;43(1):49–53. doi: 10.1016/0009-8981(73)90116-2. [DOI] [PubMed] [Google Scholar]
  22. States B., Segal S. Thin-layer chromatographic separation of cystine and the N-ethylmaleimide adducts of cysteine and glutathionen. Anal Biochem. 1969 Feb;27(2):323–329. doi: 10.1016/0003-2697(69)90041-4. [DOI] [PubMed] [Google Scholar]
  23. THIER S. O., SEGAL S., FOX M., BLAIR A., ROSENBERG L. E. CYSTINURIA: DEFECTIVE INTESTINAL TRANSPORT OF DIBASIC AMINO ACIDS AND CYSTINE. J Clin Invest. 1965 Mar;44:442–448. doi: 10.1172/JCI105157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Tietze F., Bradley K. H., Schulman J. D. Enzymic reduction of cystine by subcellular fractions of cultured and peripheral leukocytes from normal and cystinotic individuals. Pediatr Res. 1972 Aug;6(8):649–658. doi: 10.1203/00006450-197208000-00002. [DOI] [PubMed] [Google Scholar]

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