Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1991 Apr 15;275(Pt 2):341–348. doi: 10.1042/bj2750341

Redox properties and cross-linking of the dithiol/disulphide active sites of mammalian protein disulphide-isomerase.

H C Hawkins 1, M de Nardi 1, R B Freedman 1
PMCID: PMC1150058  PMID: 2025221

Abstract

1. The redox properties of the active-site dithiol/disulphide groups of PDI were determined by equilibrating the enzyme with an excess of GSH + GSSG, rapidly alkylating the dithiol form of the enzyme to inactivate it irreversibly, and determining the proportion of the disulphide form by measuring the residual activity under standard conditions. 2. The extent of reduction varied with the applied redox potential; to a first approximation, the data fitted a model in which all the enzyme dithiol/disulphide groups are independent and equivalent and the equilibrium constant between these sites and the GSH/GSSG redox couple is 42 microM at pH 7.5. 3. The standard redox potential for PDI active-site dithiol/disulphide couples was calculated from this result and found to be -0.11 V; hence PDI is a stronger oxidant and weaker reductant than GSH, nicotinamide cofactors, thioredoxin and dithiothreitol. 4. The redox equilibrium data for PDI with the GSH/GSSG redox couple showed sigmoidal deviations from linearity. The sigmoidicity could be modelled closely by assuming a Hill coefficient of 1.5. 5. This evidence of co-operative interactions between the four active sites in a PDI dimer was extended by studying the reaction between PDI and homobifunctional alkylating agents with various lengths between the reactive groups. A species whose electrophoretic mobility suggested it contained an intrachain cross-link was observed in all cases, whereas there was no evidence for cross-linking between the chains of the PDI homodimer. Most effective cross-linking was achieved with reagents containing five or more methylene spacer groups, implying a minimum distance of 1.6 nm (16 A) between the active-site reactive groups within the two thioredoxin-like domains of the PDI polypeptide.

Full text

PDF
341

Images in this article

345Fig. 4.
on p.345

Selected References

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

  1. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  2. Bulleid N. J., Freedman R. B. Defective co-translational formation of disulphide bonds in protein disulphide-isomerase-deficient microsomes. Nature. 1988 Oct 13;335(6191):649–651. doi: 10.1038/335649a0. [DOI] [PubMed] [Google Scholar]
  3. Cappel R. E., Gilbert H. F. Thiol/disulfide exchange between 3-hydroxy-3-methylglutaryl-CoA reductase and glutathione. A thermodynamically facile dithiol oxidation. J Biol Chem. 1988 Sep 5;263(25):12204–12212. [PubMed] [Google Scholar]
  4. Clancey C. J., Gilbert H. F. Thiol/disulfide exchange in the thioredoxin-catalyzed reductive activation of spinach chloroplast fructose-1,6-bisphosphatase. Kinetics and thermodynamics. J Biol Chem. 1987 Oct 5;262(28):13545–13549. [PubMed] [Google Scholar]
  5. Creighton T. E. Disulfide bonds as probes of protein folding pathways. Methods Enzymol. 1986;131:83–106. doi: 10.1016/0076-6879(86)31036-x. [DOI] [PubMed] [Google Scholar]
  6. Creighton T. E. Experimental studies of protein folding and unfolding. Prog Biophys Mol Biol. 1978;33(3):231–297. doi: 10.1016/0079-6107(79)90030-0. [DOI] [PubMed] [Google Scholar]
  7. Creighton T. E., Goldenberg D. P. Kinetic role of a meta-stable native-like two-disulphide species in the folding transition of bovine pancreatic trypsin inhibitor. J Mol Biol. 1984 Nov 5;179(3):497–526. doi: 10.1016/0022-2836(84)90077-9. [DOI] [PubMed] [Google Scholar]
  8. Creighton T. E. Kinetics of refolding of reduced ribonuclease. J Mol Biol. 1977 Jun 25;113(2):329–341. doi: 10.1016/0022-2836(77)90145-0. [DOI] [PubMed] [Google Scholar]
  9. ELLMAN G. L. Tissue sulfhydryl groups. Arch Biochem Biophys. 1959 May;82(1):70–77. doi: 10.1016/0003-9861(59)90090-6. [DOI] [PubMed] [Google Scholar]
  10. Edman J. C., Ellis L., Blacher R. W., Roth R. A., Rutter W. J. Sequence of protein disulphide isomerase and implications of its relationship to thioredoxin. Nature. 1985 Sep 19;317(6034):267–270. doi: 10.1038/317267a0. [DOI] [PubMed] [Google Scholar]
  11. Freedman R. B., Hawkins H. C., Murant S. J., Reid L. Protein disulphide-isomerase: a homologue of thioredoxin implicated in the biosynthesis of secretory proteins. Biochem Soc Trans. 1988 Apr;16(2):96–99. doi: 10.1042/bst0160096. [DOI] [PubMed] [Google Scholar]
  12. Gilbert H. F. Molecular and cellular aspects of thiol-disulfide exchange. Adv Enzymol Relat Areas Mol Biol. 1990;63:69–172. doi: 10.1002/9780470123096.ch2. [DOI] [PubMed] [Google Scholar]
  13. Hawkins H. C., Blackburn E. C., Freedman R. B. Comparison of the activities of protein disulphide-isomerase and thioredoxin in catalysing disulphide isomerization in a protein substrate. Biochem J. 1991 Apr 15;275(Pt 2):349–353. doi: 10.1042/bj2750349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hawkins H. C., Freedman R. B. The reactivities and ionization properties of the active-site dithiol groups of mammalian protein disulphide-isomerase. Biochem J. 1991 Apr 15;275(Pt 2):335–339. doi: 10.1042/bj2750335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Holmgren A. Enzymatic reduction-oxidation of protein disulfides by thioredoxin. Methods Enzymol. 1984;107:295–300. doi: 10.1016/0076-6879(84)07019-1. [DOI] [PubMed] [Google Scholar]
  16. Holmgren A., Söderberg B. O., Eklund H., Brändén C. I. Three-dimensional structure of Escherichia coli thioredoxin-S2 to 2.8 A resolution. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2305–2309. doi: 10.1073/pnas.72.6.2305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Holmgren A. Thioredoxin and glutaredoxin systems. J Biol Chem. 1989 Aug 25;264(24):13963–13966. [PubMed] [Google Scholar]
  18. Holmgren A. Thioredoxin. 6. The amino acid sequence of the protein from escherichia coli B. Eur J Biochem. 1968 Dec 5;6(4):475–484. doi: 10.1111/j.1432-1033.1968.tb00470.x. [DOI] [PubMed] [Google Scholar]
  19. Holmgren A. Thioredoxin. Annu Rev Biochem. 1985;54:237–271. doi: 10.1146/annurev.bi.54.070185.001321. [DOI] [PubMed] [Google Scholar]
  20. Isaacs J., Binkley F. Glutathione dependent control of protein disulfide-sulfhydryl content by subcellular fractions of hepatic tissue. Biochim Biophys Acta. 1977 Mar 29;497(1):192–204. doi: 10.1016/0304-4165(77)90152-0. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Lin T. Y., Kim P. S. Urea dependence of thiol-disulfide equilibria in thioredoxin: confirmation of the linkage relationship and a sensitive assay for structure. Biochemistry. 1989 Jun 13;28(12):5282–5287. doi: 10.1021/bi00438a054. [DOI] [PubMed] [Google Scholar]
  24. Ludueña R. F., Roach M. C. Interaction of tubulin with drugs and alkylating agents. 1. Alkylation of tubulin by iodo[14C]acetamide and N,N'-ethylenebis(iodoacetamide). Biochemistry. 1981 Jul 21;20(15):4437–4444. doi: 10.1021/bi00518a031. [DOI] [PubMed] [Google Scholar]
  25. Lundström J., Holmgren A. Protein disulfide-isomerase is a substrate for thioredoxin reductase and has thioredoxin-like activity. J Biol Chem. 1990 Jun 5;265(16):9114–9120. [PubMed] [Google Scholar]
  26. Lyles M. M., Gilbert H. F. Catalysis of the oxidative folding of ribonuclease A by protein disulfide isomerase: dependence of the rate on the composition of the redox buffer. Biochemistry. 1991 Jan 22;30(3):613–619. doi: 10.1021/bi00217a004. [DOI] [PubMed] [Google Scholar]
  27. Meister A. Glutathione metabolism and its selective modification. J Biol Chem. 1988 Nov 25;263(33):17205–17208. [PubMed] [Google Scholar]
  28. Meredith M. J., Reed D. J. Status of the mitochondrial pool of glutathione in the isolated hepatocyte. J Biol Chem. 1982 Apr 10;257(7):3747–3753. [PubMed] [Google Scholar]
  29. Nilsson R., Peterson E., Dallner G. Permeability of microsomal membranes isolated from rat liver. J Cell Biol. 1973 Mar;56(3):762–776. doi: 10.1083/jcb.56.3.762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Parkkonen T., Kivirikko K. I., Pihlajaniemi T. Molecular cloning of a multifunctional chicken protein acting as the prolyl 4-hydroxylase beta-subunit, protein disulphide-isomerase and a cellular thyroid-hormone-binding protein. Comparison of cDNA-deduced amino acid sequences with those in other species. Biochem J. 1988 Dec 15;256(3):1005–1011. doi: 10.1042/bj2561005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pigiet V. P., Schuster B. J. Thioredoxin-catalyzed refolding of disulfide-containing proteins. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7643–7647. doi: 10.1073/pnas.83.20.7643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. ROST J., RAPOPORT S. REDUCTION-POTENTIAL OF GLUTATHIONE. Nature. 1964 Jan 11;201:185–185. doi: 10.1038/201185a0. [DOI] [PubMed] [Google Scholar]
  33. Scheele G., Jacoby R. Conformational changes associated with proteolytic processing of presecretory proteins allow glutathione-catalyzed formation of native disulfide bonds. J Biol Chem. 1982 Oct 25;257(20):12277–12282. [PubMed] [Google Scholar]
  34. Scheele G., Jacoby R. Proteolytic processing of presecretory proteins is required for development of biological activities in pancreatic exocrine proteins. J Biol Chem. 1983 Feb 10;258(3):2005–2009. [PubMed] [Google Scholar]
  35. Sedmak J. J., Grossberg S. E. A rapid, sensitive, and versatile assay for protein using Coomassie brilliant blue G250. Anal Biochem. 1977 May 1;79(1-2):544–552. doi: 10.1016/0003-2697(77)90428-6. [DOI] [PubMed] [Google Scholar]
  36. Suttie J. W. Vitamin K-dependent carboxylase. Annu Rev Biochem. 1985;54:459–477. doi: 10.1146/annurev.bi.54.070185.002331. [DOI] [PubMed] [Google Scholar]
  37. Vermeer C. Gamma-carboxyglutamate-containing proteins and the vitamin K-dependent carboxylase. Biochem J. 1990 Mar 15;266(3):625–636. doi: 10.1042/bj2660625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Walters D. W., Gilbert H. F. Thiol/disulfide redox equilibrium and kinetic behavior of chicken liver fatty acid synthase. J Biol Chem. 1986 Oct 5;261(28):13135–13143. [PubMed] [Google Scholar]

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

RESOURCES