Human jejunal glutathione reductase: purification and evaluation of the NADPH- and glutathione-induced changes in redox state.
Human proximal jejunal glutathione reductase (EC 1.6.4.2) was purified to homogeneity by affinity chromatography on 2', 5'-ADP-Sepharose 4B. In most of its molecular and kinetic properties, the enzyme resembled glutathione reductase from other sources: The subunit mass was 56 kDa; the isoelectric point and pH optimum were 6.75 and 7.25, respectively; Michaelis constants, determined at pH 7.4, 37 degrees C, fell within the range of previously reported values [Km(NADPH) = 20 microM, Km(GSSG) = 80 microM]. The response of the enzyme to reducing conditions, on the other hand, had unique features: Preincubation with 1 mM NADPH resulted in 90% loss of activity which could be partially reversed by 2 mM GSSG, but not GSH. (Treatment with GSSG regenerated 68% of the original activity.) Reduction by GSH also caused inactivation which potentially amounted to greater than 80%. This inactivation could not be reversed by GSSG. The protective effect of GSSG against inactivation by GSH was studied. Except where [GSSG] far exceeded [GSH], the presence of GSSG in the preincubation medium decreased the extent of inhibition without affecting the rate constant for approach to equilibrium activity. At [GSSG] greater than [GSH] a decrease in the rate constant for inactivation was also observed. The results were interpreted in terms of a three-step mechanism: (1) preequilibrium reduction of Eox to Ered; (2) rate-limiting change in conformation from Ered to E'red, and (3) irreversible conversion to catalytically inferior products.
Oğüs
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H
Department of Biochemistry, Faculty of Medicine, Hacettepe University, Ankara, Turkey.
Ozer
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N
D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D019803:Glutathione Disulfide; D005980:Glutathione Reductase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007526:Isoelectric Point; D007583:Jejunum / Q000201:enzymology*; D007700:Kinetics; D009249:NADP / Q000494:pharmacology; D010084:Oxidation-Reduction
D009249:NADP; D005980:Glutathione Reductase; D005978:Glutathione; D019803:Glutathione Disulfide