Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1994 Jul 15;301(Pt 2):471–476. doi: 10.1042/bj3010471

Purification and characterization of glutathione-dependent dehydroascorbate reductase from rat liver.

E Maellaro 1, B Del Bello 1, L Sugherini 1, A Santucci 1, M Comporti 1, A F Casini 1
PMCID: PMC1137105  PMID: 8042991

Abstract

GSH-dependent enzymic reduction of dehydroascorbic acid to ascorbic acid has been studied in rat liver cytosol. After gel filtration of cytosol on Sephadex G-100 SF, dehydroascorbate reductase activity was recovered in two distinct peaks, one corresponding to glutaredoxin (an enzyme already known for its dehydroascorbate reductase activity) and another, much larger one, corresponding to a novel enzyme different from glutaredoxin. The latter was purified to apparent homogeneity. The purification process involved (NH4)2SO4 fractionation, followed by DEAE-Sepharose, Sephadex G-100 SF and Reactive Red chromatography. SDS/PAGE of the purified enzyme in either the presence or absence of 2-mercaptoethanol demonstrated a single protein band of M(r) 31,000. The M(r) determined by both Sephadex G-100 SF chromatography and h.p.l.c. was found to be approx. 48,000. H.p.l.c. of the denatured enzyme gave an M(r) value identical with that obtained by SDS/PAGE (31,000). The apparent Km for dehydroascorbate was 245 microM and the Vmax. was 1.9 mumol/min per mg of protein; for GSH they were 2.8 mM and 4.5 mumol/min per mg of protein respectively. The optimal pH range was 7.5-8.0. Microsequence analysis of the electro-transferred enzyme band showed that the N-terminus is blocked. Data on internal primary structure were obtained from CNBr-and N-chlorosuccinimide-derived fragments. No significative sequence similarity was found to any of the protein sequences contained in the Protein Identification Resource database.

Full text

PDF
471

Images in this article

474Figure 4
on p.474
475Figure 6
on p.475

Selected References

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

  1. Basu S., Som S., Deb S., Mukherjee D., Chatterjee I. B. Dehydroascorbic acid reduction in human erythrocytes. Biochem Biophys Res Commun. 1979 Oct 29;90(4):1335–1340. doi: 10.1016/0006-291x(79)91182-3. [DOI] [PubMed] [Google Scholar]
  2. Bianchi J., Rose R. C. Dehydroascorbic acid and cell membranes: possible disruptive effects. Toxicology. 1986 Jul;40(1):75–82. doi: 10.1016/0300-483x(86)90047-8. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Bodannes R. S., Chan P. C. Ascorbic acid as a scavenger of singlet oxygen. FEBS Lett. 1979 Sep 15;105(2):195–196. doi: 10.1016/0014-5793(79)80609-2. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Comporti M. Three models of free radical-induced cell injury. Chem Biol Interact. 1989;72(1-2):1–56. doi: 10.1016/0009-2797(89)90016-1. [DOI] [PubMed] [Google Scholar]
  7. Diliberto E. J., Jr, Dean G., Carter C., Allen P. L. Tissue, subcellular, and submitochondrial distributions of semidehydroascorbate reductase: possible role of semidehydroascorbate reductase in cofactor regeneration. J Neurochem. 1982 Aug;39(2):563–568. doi: 10.1111/j.1471-4159.1982.tb03982.x. [DOI] [PubMed] [Google Scholar]
  8. Frei B., England L., Ames B. N. Ascorbate is an outstanding antioxidant in human blood plasma. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6377–6381. doi: 10.1073/pnas.86.16.6377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. GIVOL D., GOLDBERGER R. F., ANFINSEN C. B. OXIDATION AND DISULFIDE INTERCHANGE IN THE REACTIVATION OF REDUCED RIBONUCLEASE. J Biol Chem. 1964 Sep;239:PC3114–PC3116. [PubMed] [Google Scholar]
  10. HUGHES R. E. REDUCTION OF DEHYDROASORBIC ACID BY ANIMAL TISSUES. Nature. 1964 Sep 5;203:1068–1069. doi: 10.1038/2031068a0. [DOI] [PubMed] [Google Scholar]
  11. Halliwell B., Wasil M., Grootveld M. Biologically significant scavenging of the myeloperoxidase-derived oxidant hypochlorous acid by ascorbic acid. Implications for antioxidant protection in the inflamed rheumatoid joint. FEBS Lett. 1987 Mar 9;213(1):15–17. doi: 10.1016/0014-5793(87)81456-4. [DOI] [PubMed] [Google Scholar]
  12. Hillson D. A., Lambert N., Freedman R. B. Formation and isomerization of disulfide bonds in proteins: protein disulfide-isomerase. Methods Enzymol. 1984;107:281–294. doi: 10.1016/0076-6879(84)07018-x. [DOI] [PubMed] [Google Scholar]
  13. Holmgren A. Glutaredoxin from Escherichia coli and calf thymus. Methods Enzymol. 1985;113:525–540. doi: 10.1016/s0076-6879(85)13071-5. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Lambert N., Freedman R. B. Kinetics and specificity of homogeneous protein disulphide-isomerase in protein disulphide isomerization and in thiol-protein-disulphide oxidoreduction. Biochem J. 1983 Jul 1;213(1):235–243. doi: 10.1042/bj2130235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lambert N., Freedman R. B. Structural properties of homogeneous protein disulphide-isomerase from bovine liver purified by a rapid high-yielding procedure. Biochem J. 1983 Jul 1;213(1):225–234. doi: 10.1042/bj2130225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Maellaro E., Casini A. F., Del Bello B., Comporti M. Lipid peroxidation and antioxidant systems in the liver injury produced by glutathione depleting agents. Biochem Pharmacol. 1990 May 15;39(10):1513–1521. doi: 10.1016/0006-2952(90)90515-m. [DOI] [PubMed] [Google Scholar]
  18. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem. 1987 Jul 25;262(21):10035–10038. [PubMed] [Google Scholar]
  19. Mãrtensson J., Meister A., Mrtensson J. Glutathione deficiency decreases tissue ascorbate levels in newborn rats: ascorbate spares glutathione and protects. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4656–4660. doi: 10.1073/pnas.88.11.4656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Nishikimi M. Oxidation of ascorbic acid with superoxide anion generated by the xanthine-xanthine oxidase system. Biochem Biophys Res Commun. 1975 Mar 17;63(2):463–468. doi: 10.1016/0006-291x(75)90710-x. [DOI] [PubMed] [Google Scholar]
  21. PATTERSON J. W. Course of diabetes and development of cataracts after injecting dehydroascorbic acid and related substances. Am J Physiol. 1951 Apr 1;165(1):61–65. doi: 10.1152/ajplegacy.1951.165.1.61. [DOI] [PubMed] [Google Scholar]
  22. Packer J. E., Slater T. F., Willson R. L. Direct observation of a free radical interaction between vitamin E and vitamin C. Nature. 1979 Apr 19;278(5706):737–738. doi: 10.1038/278737a0. [DOI] [PubMed] [Google Scholar]
  23. Pillsbury S., Watkins D., Cooperstein S. J. Effect of dehydroascorbic acid on permeability of pancreatic islet tissue in vitro. J Pharmacol Exp Ther. 1973 Jun;185(3):713–718. [PubMed] [Google Scholar]
  24. Rose R. C. Renal metabolism of the oxidized form of ascorbic acid (dehydro-L-ascorbic acid). Am J Physiol. 1989 Jan;256(1 Pt 2):F52–F56. doi: 10.1152/ajprenal.1989.256.1.F52. [DOI] [PubMed] [Google Scholar]
  25. Scarpa M., Rigo A., Maiorino M., Ursini F., Gregolin C. Formation of alpha-tocopherol radical and recycling of alpha-tocopherol by ascorbate during peroxidation of phosphatidylcholine liposomes. An electron paramagnetic resonance study. Biochim Biophys Acta. 1984 Sep 28;801(2):215–219. doi: 10.1016/0304-4165(84)90070-9. [DOI] [PubMed] [Google Scholar]
  26. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  27. Sies H. Oxidative stress: from basic research to clinical application. Am J Med. 1991 Sep 30;91(3C):31S–38S. doi: 10.1016/0002-9343(91)90281-2. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Sun I., Morré D. J., Crane F. L., Safranski K., Croze E. M. Monodehydroascorbate as an electron acceptor for NADH reduction by coated vesicle and Golgi apparatus fractions of rat liver. Biochim Biophys Acta. 1984 Feb 14;797(2):266–275. doi: 10.1016/0304-4165(84)90130-2. [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. Wolff S. P., Wang G. M., Spector A. Pro-oxidant activation of ocular reductants. 1. Copper and riboflavin stimulate ascorbate oxidation causing lens epithelial cytotoxicity in vitro. Exp Eye Res. 1987 Dec;45(6):777–789. doi: 10.1016/s0014-4835(87)80095-7. [DOI] [PubMed] [Google Scholar]

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

RESOURCES