Glutaredoxin (Grx) contains a redox-active disulfide and catalyzes thiol-disulfide interchange reactions with specificity for GSH. The dithiol form of Grx reduces mixed disulfides involving GSH or protein disulfides. During oxidative refolding of 8 µM reduced and denatured ribonuclease RNase-(SH) in a redox buffer of 1 mM GSH and 0.2 mM GSSG to yield native RNase-(S), a large number of GSH-mixed disulfide species are formed. A lag phase that precedes formation of folded active RNase at a steady-state rate was shortened or eliminated by the presence of a catalytic concentration (0.5 µM) of Escherichia coli Grx together with protein disulfide-isomerase (PDI), its procaryotic equivalent E. coli DsbA, or the PDI analogue the E. coli thioredoxin mutant protein P34H. A mutant Grx in which one of the active site cysteine residues (Cys-11 and Cys-14) had been replaced by serine, C14S Grx, had similar effect compared with its wild-type counterpart. This demonstrated that Grx acted by a monothiol mechanism involving only Cys-11 and that RNase-S-SG-mixed disulfides were the substrates. Grx displayed synergistic activity together with PDI only in GSH/GSSG redox buffers with sufficiently low redox potential (E` of -208 or -181 mV) to allow reduction of the active site of Grx. In refolding systems that do not depend on glutathione, like cystamine/cysteamine or in the presence of selenite (SeO), no synergistic activity of Grx was observed with PDI. We conclude that Grx acts by reducing mixed disulfides between GSH and RNase that are rate-limiting in enzyme-catalyzed refolding.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				S. Arredondo, L. Segatori, H. F. Gilbert, and G. Georgiou De Novo Design and Evolution of Artificial Disulfide Isomerase Enzymes Analogous to the Bacterial DsbC J. Biol. Chem., November 14, 2008; 283(46): 31469 - 31476. [Abstract] [Full Text] [PDF]

				C. Berndt, C. H. Lillig, and A. Holmgren Thiol-based mechanisms of the thioredoxin and glutaredoxin systems: implications for diseases in the cardiovascular system Am J Physiol Heart Circ Physiol, March 1, 2007; 292(3): H1227 - H1236. [Abstract] [Full Text] [PDF]

				R. Ivarsson, R. Quintens, S. Dejonghe, K. Tsukamoto, P. in 't Veld, E. Renstrom, and F. C. Schuit Redox Control of Exocytosis: Regulatory Role of NADPH, Thioredoxin, and Glutaredoxin Diabetes, July 1, 2005; 54(7): 2132 - 2142. [Abstract] [Full Text] [PDF]

				R. Xiao, J. Lundstrom-Ljung, A. Holmgren, and H. F. Gilbert Catalysis of Thiol/Disulfide Exchange: GLUTAREDOXIN 1 AND PROTEIN-DISULFIDE ISOMERASE USE DIFFERENT MECHANISMS TO ENHANCE OXIDASE AND REDUCTASE ACTIVITIES J. Biol. Chem., June 3, 2005; 280(22): 21099 - 21106. [Abstract] [Full Text] [PDF]

				J. B. Park and M. Levine Cloning, Sequencing, and Characterization of Alternatively Spliced Glutaredoxin 1 cDNA and Its Genomic Gene: CHROMOSOMAL LOCALIZATION, mRNA STABILITY, AND ORIGIN OF PSEUDOGENES J. Biol. Chem., March 18, 2005; 280(11): 10427 - 10434. [Abstract] [Full Text] [PDF]

				P. Christmas, B. M. Weber, M. McKee, D. Brown, and R. J. Soberman Membrane Localization and Topology of Leukotriene C4 Synthase J. Biol. Chem., August 2, 2002; 277(32): 28902 - 28908. [Abstract] [Full Text] [PDF]

				D. Goldstone, P. W. Haebel, F. Katzen, M. W. Bader, J. C. A. Bardwell, J. Beckwith, and P. Metcalf DsbC activation by the N-terminal domain of DsbD PNAS, August 1, 2001; (2001) 171315498. [Abstract] [Full Text] [PDF]

				A. Puig, T. P. Primm, R. Surendran, J. C. Lee, K. D. Ballard, R. S. Orkiszewski, V. Makarov, and H. F. Gilbert A 21-kDa C-terminal Fragment of Protein-disulfide Isomerase has Isomerase, Chaperone, and Anti-chaperone Activities J. Biol. Chem., December 26, 1997; 272(52): 32988 - 32994. [Abstract] [Full Text] [PDF]

				H. F. Gilbert Protein Disulfide Isomerase and Assisted Protein Folding J. Biol. Chem., November 21, 1997; 272(47): 29399 - 29402. [Full Text] [PDF]

				D. A. Davis, F. M. Newcomb, D. W. Starke, D. E. Ott, J. J. Mieyal, and R. Yarchoan Thioltransferase (Glutaredoxin) Is Detected Within HIV-1 and Can Regulate the Activity of Glutathionylated HIV-1 Protease in Vitro J. Biol. Chem., October 10, 1997; 272(41): 25935 - 25940. [Abstract] [Full Text] [PDF]

				D Tremousaygue, C Bardet, P Dabos, F Regad, F Pelese, R Nazer, E Gander, and B Lescure Genome DNA sequencing around the EF-1 alpha multigene locus of Arabidopsis thaliana indicates a high gene density and a shuffling of noncoding regions. Genome Res., March 1, 1997; 7(3): 198 - 209. [Abstract] [PDF]

				D. Goldstone, P. W. Haebel, F. Katzen, M. W. Bader, J. C. A. Bardwell, J. Beckwith, and P. Metcalf DsbC activation by the N-terminal domain of DsbD PNAS, August 14, 2001; 98(17): 9551 - 9556. [Abstract] [Full Text] [PDF]