Stellan Eriksson

The reduction of the L‐cysteine‐glutathione mixed disulfide in rat liver. involvement of an enzyme catalyzing thiol‐disulfide interchange

The naturally occurring mixed disulfide of cysteine and glutathione (CySSG)** can readily be synthesized in large quantities, free from the corresponding symmetrical disulfides, cysteine (CySSCy) and glutathione disulfide (GSSG) [l] . This has made it possible to study the enzymatic reduction of this compound, which, in addition, may have relevance to the biological reduction of cystine. It has been claimed that similar reductions require only GSH and glutathione reductase and that n@ catalytic effect on the thioldisulfide interchange could be demonstrated [2,3]. However, two highly purified enzymes catalyzing such exchange reactions of low molecular weight compounds have been described [4,5 J . We have reported earlier that we have evidence for an enzymatic reaction between glutathione (GSH) and CySSG [l] and have recently presented data on a partially purified enzyme [6]. The present communication demonstrates the presence of this enzyme in rat liver.

Enzymatic catalysis of the reversible sulfitolysis of glutathione disulfide and the biological reduction of thiosulfate esters

Abstract Rat liver supernatants were shown to contain an enzymatic activity catalyzing in both forward and reverse directions the reversible sulfitolysis of glutathione disulfide. The enzymatic sulfitolysis has maximal activity at pH 7. S -Sulfoglutathione, which is a product of the sulfitolysis, was isolated by passage through an ion-exchange column. Three different assays were applied to determine S -sulfoglutathione, viz., methods based on the ninhydrin reaction, the formation of a thiazoline derivative in strong acid, and the use of radioactively labeled glutathione. The reversal of the sulfitolysis, i.e., the reaction of S -sulfoglutathione with glutathione, was studied directly by determination of sulfite with radioactive N -ethylmaleimide, or indirectly by coupling to the NADPH- and glutathione reductase-linked reduction of glutathione disulfide. Chromatographic analysis of rat liver supernatants demonstrated that all fractions catalyzing the reversible sulfitolysis did also catalyze the previously studied thiol-disulfide interchange of glutathione and the mixed disulfide of cysteine and glutathione. The reduction of thiosulfate esters, such as S -sulfocysteine and trimethylammonium-ethylthiosulfate, with glutathione was also catalyzed by the enzyme active in the sulfitolysis, which indicates an important biosynthetic role of the enzyme in microorganisms synthesizing cysteine via S -sulfocysteine. The enzyme is also capable of participating in the formation of the naturally occurring S -sulfoglutathione.

The nature of the enzymatic reduction of the mixed disulfide of coenzyme A and glutathione

The biological reduction of mixed disulfides and related compounds in various organisms has been investigated, and different mechanisms of the reduction have been considered (cf. [ 1,2] ). The mixed disulfide of coenzyme A and glutathione, CoASSG, is naturally occurring [3-51 and its concentration level in rat liver has been quantitatively estimated [6]. The enzymatic reduction of this compound has been considered to involve either GSH and an enzyme catalyzing thiol-disulfide interchange [7] , or NADPH and a specific CoASSG reductase [8,9] or glutathione reductase [IO] . Recent research in our laboratory concerns the enzyme systems involved in the reduction of disulfides and thiosulfate esters [I] , and it was consequently of interest to study the alternative mechanisms proposed for the reduction of CoASSG, and attempt to evaluate their relative importance. Some of the results have been presented [ 1 ] .

The identity of enzymes reducing a thiamine disulfide derivative and cystine derivatives via thiol-disulfide exchange

Abstract In earlier work ∗ , we have studied a labile enzyme activity catalyzing an exchange between thiol and acceptor. Glutathione (GSH) was used as the thiol and a number of low molecular weight substances such as cystine and GSH-disulfide derivatives, S -sulfocysteine (CySSO 3 H), S -sulfoglutathione and 5,5′-dithiobis(2-nitrobenzoa (DTNB) were used as acceptor substrates in the thiol transfer reaction. This broad substrate specificity led us to the tentative suggestion that thiamine disulfide derivatives also were acceptor substrates to the thioltransferase † activity, which is confirmed in this study. The methods used for the resolution of enzymes and substrate specificity were: (1) isoelectric focusing, (2) CM-cellulose chromatography, (3) labelling of the thioltransferase with [ 35 S]GSH, (4) gel filtration on Bio-Gel P-150, and (5) investigation of ratios of the specific activities of GSH-linked enzymes in different tissues. Generally it was found that bovine tissue had higher specific thioltransferase activity than rat tissue. GSH S -aryltransferase (EC 2.5.1.13) had quite different activity ratios from those obtained with the enzyme involved in cystine and thiamine disulfide reduction. This result, and dissimilar Chromatographic behavior, indicate that GSH S -aryltransferase is not involved in disulfide reduction.