Skip to main content
Biochemical Journal logoLink to Biochemical Journal
. 1996 May 1;315(Pt 3):931–938. doi: 10.1042/bj3150931

Purification, cloning and expression of dehydroascorbic acid-reducing activity from human neutrophils: identification as glutaredoxin.

J B Park 1, M Levine 1
PMCID: PMC1217296  PMID: 8645179

Abstract

Dehydroascorbic acid-reducing activity in normal human neutrophil lysates was characterized and identified by activity-based purification and measurement of newly synthesized ascorbate by HPLC. The initial reducing activity was non-dialysable and could not be accounted for by the activity of glutathione as a reducing agent. The reducing activity was purified to homogeneity as an 11 kDa protein. The protein had a specific activity of 3 mumol/min per mg of protein and was glutathione dependent. Kinetic experiments showed that the protein had a K(m) for glutathione of 2.0 mM and a K(m) for dehydroascorbic acid of 250 microM. Dehydroascorbic acid reduction by the purified protein was pH dependent and was maximal at pH 7.5. Peptide fragments from the purified protein were analysed for amino acid sequence and the protein was identified as glutaredoxin. By using degenerate oligonucleotides based on the amino acid sequence, glutaredoxin was cloned from a human neutrophil library. Expressed purified glutaredoxin displayed reducing activity and kinetics that were indistinguishable from those of native purified enzyme. Several approaches indicated that glutaredoxin was responsible for the most of the protein-mediated dehydroascorbic acid reduction in lysates. From protein purification data, glutaredoxin was responsible for at least 47% of the initial reducing activity. Dehydroascorbic acid reduction was at least 5-fold greater in neutrophil lysates than in myeloid tumour cell lysates, and glutaredoxin was detected in normal neutrophil lysates but not in myeloid tumour cell lysates by Western blotting. Glutaredoxin inhibitors inhibited dehydroascorbic acid reduction in neutrophil lysates as much as 80%. These findings indicate that glutaredoxin plays a major role in dehydroascorbic acid reduction in normal human neutrophil lysates, and represent the first identification of dehydroascorbic acid reductase in human tissue by activity-based purification.

Full Text

The Full Text of this article is available as a PDF (588.7 KB).

Selected References

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

  1. Bigley R., Riddle M., Layman D., Stankova L. Human cell dehydroascorbate reductase. Kinetic and functional properties. Biochim Biophys Acta. 1981 May 14;659(1):15–22. doi: 10.1016/0005-2744(81)90266-7. [DOI] [PubMed] [Google Scholar]
  2. Bigley R., Wirth M., Layman D., Riddle M., Stankova L. Interaction between glucose and dehydroascorbate transport in human neutrophils and fibroblasts. Diabetes. 1983 Jun;32(6):545–548. doi: 10.2337/diab.32.6.545. [DOI] [PubMed] [Google Scholar]
  3. Bode A. M., Green E., Yavarow C. R., Wheeldon S. L., Bolken S., Gomez Y., Rose R. C. Ascorbic acid regeneration by bovine iris-ciliary body. Curr Eye Res. 1993 Jul;12(7):593–601. doi: 10.3109/02713689309001838. [DOI] [PubMed] [Google Scholar]
  4. Bode A. M., Vanderpool S. S., Carlson E. C., Meyer D. A., Rose R. C. Ascorbic acid uptake and metabolism by corneal endothelium. Invest Ophthalmol Vis Sci. 1991 Jul;32(8):2266–2271. [PubMed] [Google Scholar]
  5. Bode A. M., Yavarow C. R., Fry D. A., Vargas T. Enzymatic basis for altered ascorbic acid and dehydroascorbic acid levels in diabetes. Biochem Biophys Res Commun. 1993 Mar 31;191(3):1347–1353. doi: 10.1006/bbrc.1993.1365. [DOI] [PubMed] [Google Scholar]
  6. Böyum A. Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest Suppl. 1968;97:77–89. [PubMed] [Google Scholar]
  7. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  8. Coassin M., Tomasi A., Vannini V., Ursini F. Enzymatic recycling of oxidized ascorbate in pig heart: one-electron vs two-electron pathway. Arch Biochem Biophys. 1991 Nov 1;290(2):458–462. doi: 10.1016/0003-9861(91)90566-2. [DOI] [PubMed] [Google Scholar]
  9. Dyer D. L., Kanai Y., Hediger M. A., Rubin S. A., Said H. M. Expression of a rabbit renal ascorbic acid transporter in Xenopus laevis oocytes. Am J Physiol. 1994 Jul;267(1 Pt 1):C301–C306. doi: 10.1152/ajpcell.1994.267.1.C301. [DOI] [PubMed] [Google Scholar]
  10. Fernando M. R., Sumimoto H., Nanri H., Kawabata S., Iwanaga S., Minakami S., Fukumaki Y., Takeshige K. Cloning and sequencing of the cDNA encoding human glutaredoxin. Biochim Biophys Acta. 1994 Jun 21;1218(2):229–231. doi: 10.1016/0167-4781(94)90019-1. [DOI] [PubMed] [Google Scholar]
  11. Gan Z. R., Wells W. W. Identification and reactivity of the catalytic site of pig liver thioltransferase. J Biol Chem. 1987 May 15;262(14):6704–6707. [PubMed] [Google Scholar]
  12. HENDRY J. M., EASSON L. H., OWEN J. A. THE UPTAKE AND REDUCTION OF DEHYDROASCORBIC ACID BY HUMAN LEUCOCYTES. Clin Chim Acta. 1964 May;9:498–499. doi: 10.1016/0009-8981(64)90089-0. [DOI] [PubMed] [Google Scholar]
  13. Holmgren A. Thioredoxin and glutaredoxin systems. J Biol Chem. 1989 Aug 25;264(24):13963–13966. [PubMed] [Google Scholar]
  14. Hsu S. M., Raine L., Fanger H. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem. 1981 Apr;29(4):577–580. doi: 10.1177/29.4.6166661. [DOI] [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Lam K. W., Yu H. S., Glickman R. D., Lin T. Sodium-dependent ascorbic and dehydroascorbic acid uptake by SV-40-transformed retinal pigment epithelial cells. Ophthalmic Res. 1993;25(2):100–107. doi: 10.1159/000267272. [DOI] [PubMed] [Google Scholar]
  17. Maellaro E., Del Bello B., Sugherini L., Santucci A., Comporti M., Casini A. F. Purification and characterization of glutathione-dependent dehydroascorbate reductase from rat liver. Biochem J. 1994 Jul 15;301(Pt 2):471–476. doi: 10.1042/bj3010471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Mehlhorn R. J. Ascorbate- and dehydroascorbic acid-mediated reduction of free radicals in the human erythrocyte. J Biol Chem. 1991 Feb 15;266(5):2724–2731. [PubMed] [Google Scholar]
  19. Mizoguchi T., Nishnaka T., Uchida G., Mizuta J., Uchida H., Terada T., Toya H. Inhibition of bovine leukocyte thioltransferase by anti-inflammatory drugs and anti-histaminic drugs. Biol Pharm Bull. 1993 Sep;16(9):840–842. doi: 10.1248/bpb.16.840. [DOI] [PubMed] [Google Scholar]
  20. Papov V. V., Gravina S. A., Mieyal J. J., Biemann K. The primary structure and properties of thioltransferase (glutaredoxin) from human red blood cells. Protein Sci. 1994 Mar;3(3):428–434. doi: 10.1002/pro.5560030307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
  22. Stahl R. L., Liebes L. F., Silber R. A reappraisal of leukocyte dehydroascorbate reductase. Biochim Biophys Acta. 1985 Mar 29;839(1):119–121. doi: 10.1016/0304-4165(85)90189-8. [DOI] [PubMed] [Google Scholar]
  23. Stankova L., Bigley R., Ingermann R. L. The effect of cyanide on vitamin C uptake by human polymorphonuclear leukocytes. Gen Pharmacol. 1991;22(5):903–905. doi: 10.1016/0306-3623(91)90228-x. [DOI] [PubMed] [Google Scholar]
  24. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Washko P. W., Hartzell W. O., Levine M. Ascorbic acid analysis using high-performance liquid chromatography with coulometric electrochemical detection. Anal Biochem. 1989 Sep;181(2):276–282. doi: 10.1016/0003-2697(89)90243-1. [DOI] [PubMed] [Google Scholar]
  26. Washko P. W., Wang Y., Levine M. Ascorbic acid recycling in human neutrophils. J Biol Chem. 1993 Jul 25;268(21):15531–15535. [PubMed] [Google Scholar]
  27. Washko P., Rotrosen D., Levine M. Ascorbic acid transport and accumulation in human neutrophils. J Biol Chem. 1989 Nov 15;264(32):18996–19002. [PubMed] [Google Scholar]
  28. Welch R. W., Bergsten P., Butler J. D., Levine M. Ascorbic acid accumulation and transport in human fibroblasts. Biochem J. 1993 Sep 1;294(Pt 2):505–510. doi: 10.1042/bj2940505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Welch R. W., Wang Y., Crossman A., Jr, Park J. B., Kirk K. L., Levine M. Accumulation of vitamin C (ascorbate) and its oxidized metabolite dehydroascorbic acid occurs by separate mechanisms. J Biol Chem. 1995 May 26;270(21):12584–12592. doi: 10.1074/jbc.270.21.12584. [DOI] [PubMed] [Google Scholar]
  30. Wells W. W., Xu D. P., Yang Y. F., Rocque P. A. Mammalian thioltransferase (glutaredoxin) and protein disulfide isomerase have dehydroascorbate reductase activity. J Biol Chem. 1990 Sep 15;265(26):15361–15364. [PubMed] [Google Scholar]
  31. Wells W. W., Yang Y., Deits T. L., Gan Z. R. Thioltransferases. Adv Enzymol Relat Areas Mol Biol. 1993;66:149–201. doi: 10.1002/9780470123126.ch4. [DOI] [PubMed] [Google Scholar]
  32. Winkler B. S., Orselli S. M., Rex T. S. The redox couple between glutathione and ascorbic acid: a chemical and physiological perspective. Free Radic Biol Med. 1994 Oct;17(4):333–349. doi: 10.1016/0891-5849(94)90019-1. [DOI] [PubMed] [Google Scholar]
  33. Winkler B. S. Unequivocal evidence in support of the nonenzymatic redox coupling between glutathione/glutathione disulfide and ascorbic acid/dehydroascorbic acid. Biochim Biophys Acta. 1992 Oct 27;1117(3):287–290. doi: 10.1016/0304-4165(92)90026-q. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES