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Results obt,ained in our laboratory have demonstrated that nuclear cataract formation is accompanied by a. progressive decrease in the level of glutatbione-especially within the nucleus (Truscott a.nd Augusteyn, 1977). We wondered if this might be ascribed to a decrease in lens glutathione reductase.
The Purification and Properties of Human Lens Glutathione Reductase* '
A very simple and rapid method for the purificatiqn of human lens glutathione reductase has been developed.Themethodinvolvesonlytwosteps -affinitychromatographyonZ'.Ei'-ADPSepharosc 4B and gel filtration on Sephacryl S-200. With whole lenses, the purification achieved is over 18000-fold and 80 y0 of the activity in the tissue homogenate is recovered as an enzyme with a specific activity of 218 IU/mg-*.
glutathione reductase purification
A very simple and rapid method for the purificatiqn of human lens glutathione reductase has been developed.Themethodinvolvesonlytwosteps -affinitychromatographyonZ'.Ei'-ADPSepharosc 4B and gel filtration on Sephacryl S-200. With whole lenses, the purification achieved is over 18000-fold and 80 y0 of the activity in the tissue homogenate is recovered as an enzyme with a specific activity of 218 IU/mg-*.
Mouse-Liver Glutathione Reductase. Purification, Kinetics, and Regulation
European Journal of Biochemistry, 1979
Glutathione reductase from the liver of DBA/2J mice was purified to homogeneity by means of ammonium sulfate fractionation and two subsequent affinity chromatography steps using 8-(6-aminohexyl)-amino-2'-phospho-adenosine diphosphoribose and N6-(6-aminohexyl)-adenosine 2', 5'-bisphosphate-Sepharose columns. A facile procedure for the synthesis of 8-(6-aminohexyl)amino-2'-phospho-adenosine diphosphoribose is also presented. The purified enzyme exhibits a specific activity of 158 U/mg and an A280/A460 of 6.8. It was shown to be a dimer of M , 105000 with a Stokes radius of 4.18 nm and an isoelectric point of 6.46. Amino acid composition revealed some similarity between the mouse and the human enzyme. Antibodies against mouse glutathione reductase were raised in rabbits and exhibited high specificity. The catalytic properties of mouse liver glutathione reductase have been studied under a variety of experimental conditions. As with the same enzyme from other sources, the kinetic data are consistent with a 'branched' mechanism. The enzyme was stabilized against thermal inactivation at 80°C by GSSG and less markedly by NADP' and GSH, but not by NADPH or FAD. Incubation of mouse glutathione reductase in the presence of NADPH or NADH, but not NADP' or NAD', produced an almost complete inactivation. The inactivation by NADPH was time, pH and concentration dependent. Oxidized glutathione protected the enzyme against inactivation, which could also be reversed by GSSG or other electron acceptors. The enzyme remained in the inactive state even after eliminating the excess NADPH. The inactive enzyme showed the same molecular weight as the active glutathione reductase. The spectral properties of the inactive enzyme have also been studied. It is proposed that auto-inactivation of glutathione reductase by NADPH and the protection as well as reactivation by GSSG play in vivo an important regulatory role.
Experimental Eye Research, 1992
A method for specific determination of glutathione (GSH) is described. This method utilizes the enzymatic conjugation of GSH to 1-chloro-2,4&nitrobenzene through reaction catalyzed by glutathione Stransferase. The recovery of GSH as determined by this method is comparable to that in currently used methods. The method is specific for GSH determination. Other sulihydryl (-SH) compounds including the protein-SH or p-mercaptoethanol, which are often included in tissue homogenates, do not interfere with GSH determination. Acid extraction of the tissue is not required in this method and comparatively smaller amounts of tissue samples (as little as 20 ,~l of a 10 y0 w/v tissue homogenate) are needed for the analyses. The method when applied for GSH determination in ocular tissues yielded results in agreement with the reported values in literature. Evidence for the sensitivity, accuracy, and convenience of the method is provided by analysing the sample containing GSH in the range of l-200 nmol by this method.
ELECTROPHORESIS, 2004
Detection of glutathione reductase after electrophoresis on native or sodium dodecyl sulfate polyacrylamide gels Commercial glutathione reductase (GR) from spinach and yeast (Saccharomyces cerevisiae) were stained on 7.5% native polyacrylamide gel electrophoresis (PAGE) gels or 15% sodium dodecyl sulfate (SDS)-PAGE gels with or without further purification by a 2',5'-ADP Sepharose 4B affinity column. For SDS-PAGE gels, the SDS was removed first by washing twice with 25% isopropanol in 10 mM Tris-HCl (pH 7.9) for 10 min. The gel was then dipped in a 50 mM Tris-HCl buffer (pH 7.9) containing 4.0 mM oxidized glutathione (GSSG), 1.5 mM NADPH, and 2 mM 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) for 20 min. The GR activity was negatively stained in the dark by a solution containing 1.2 mM 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 1.6 mM phenazine methosulfate (PMS) for 5-10 min. The contrast between the clear zone of GR activity and the purple background was found in both native and SDS-PAGE gels. This negative staining method can detect GR as little as 0.064 units and 0.0032 units, respectively, for spinach and yeast sources. Under reduced SDS-PAGE gels, the GR activity band located on 72 kDa for spinach and 51 kDa for yeast. This fast and sensitive method could be used during enzyme purification and for characterization of GR from different sources under different physiological stages or conditions.
European Journal of Biochemistry, 1997
Human glutathione reductase (GR; which catalyzes the reaction NADPH + GSSG + H' -2 GSH + NADP') is an obligatory FAD-containing homodimer of known geometry. Native human GR, a potential target of antimalarial and cytostatic agents, cannot be dissociated by dilution or by means of subunitinterface mimetics, similarly to well-studied viral dimeric proteins. However, ab initio folding andlor dimerization of human GR can be inhibited by point mutations or by peptides corresponding to subunitinterface areas, for example synthetic peptide P I 1, which represents the intersubunit-contact helix HI 1. The structure of this peptide, which might assist inhibitor design, was solved by high-resolution NMR spectroscopy. Residues 440-453, were found to be a helical in the isolated peptide. To quantitate the efficacy of inhibitors such as P I 1, we developed the following unfolding/reactivation assay. The effects of various guanidine hydrochloride (Gdn/HCl) concentrations were studied by analytical ultracentrifugation. It was shown that human GR denatured by greater than 3 M Gdn/HCl is monomeric and free of FAD. Circular-dichroism experiments at 223 nm indicated a half-life of approximately 20 s at 20°C for the unfolding process. To optimize the reactivation yield, four parameters [protein concentration (x) in the range 0.3-10 pg/ml, cofactor supplementation, temperature (y; 0-32"C), and time (0-72 h)] were varied systematically, and a reactivation score z was given to each constellation of parameters. This type of analysis might be useful to optimize refolding and activation yields for other proteins. For human GR, the highest recovery was found not to occur at one of the corners of the x,y plain, but close to its center. Consequently, the optimal assay conditions for folding and dimerization inhibitors are as follows. The enzyme (at 300 pg/ml) is denatured by 5 M guanidine hydrochloride/5 mM dithiothreitol, then reactivated by dilution to 1 pg/ml at pH 6.9 and 20°C. In the absence of inhibitors, this procedure leads to 70% of the control activity within 8 h. Peptides representing the upper subunit interface (for instance residues 436-478) of human GR were found to inhibit refolding with EC,,,, values in the micromolar range, whereas fragments from other regions of the protein had no influence on this process. For peptide P11, the EC,,,, value was 20 pM. In conclusion, hGR, enzyme with a tight intersubunit contact area of 21 nm', appears to be suitable for studying protein folding, dimerization, and prosthetic-group complexation in the absence and presence of compounds that inhibit these processes. There is a shortage, at least for oligomeric enzymes of eukaryotes, of published systematic studies on protein (re)activation.