Proc. Nati. Acad. Scd. USA Vol. 77, No. 9, pp. 5149-5152, September 1980 Biochemistry Enzymatic reduction of alloxan by thioredoxin and NADPH- thioredoxin reductase (diabetes/cytotoxic action/thiol-dependent reduction/dialuric acid) ARNE HOLMGREN AND CATHARINA LYCKEBORG Department of Chemistry, Karolinska Institutet, S-104 01 Stockholm 60, Sweden Communicated by Rolf Luft, June 10, 1980 ABSTRACT Alloxan reacts with certain sulfhydryl groups Thioredoxin-S2 + NADPH + H+ either by chemical modification or reduction to dialuric acid. The effects of the drug on NADPH-thioredoxin reductase thioredoxin [thioredoxin reductase (NADPH) NADPH.oxidized-thioredoxin - thioredoxin-(SH)2 + NADP+ oxidoreductase, EC 1.6.4.5] and thioredoxin(SH)2, a ubiquitous reductase thiol-dependent disulfide reductase system, are described. Al- Thioredoxin-(SH)2 + protein-S2 loxan was a direct substrate for a nearly homogeneous prepa- ration of calf thymus NADPH-thioredoxin reductase with an ¢ thioredoxin-S2 + protein-(SH)2. apparent Km of 330 pM and a Rest of 1000 min1 at pH 7.0 and 250C. Alloxan was not a substrate for the corresponding Esch- The ubiquitous tissue and subcellular distribution of the bovine erichia coli NADPH-thioredoxin reductase. However, E. coil thioredoxin system (9) indicates that it is of major importance and calf thymus thioredoxin(SH)t both efficiently reduced al- for regulation of the protein thiol-disulfide status of cells. A role loxan. Thus, alloxan showed an apparent Km of 70 pM in the of thioredoxin in the degradation of insulin and its mechanism presence of 3.4 pM E. coil thioredoxin, 0.2 pM thioredoxin re- of action has been suggested (10, 11). ductase, and 0.4 mM NADPH. The insulin disulfide reductase activity of the complete calf thymus thioredoxin system was Because the thioredoxin system is dependent on functional inhibited by alloxan, as predicted from the reaction of the drug SH groups, we have studied the effects of alloxan on the pure with both thioredoxin4SH)k and thioredoxin reductase. The toxic enzymes from Escherichia colh and calf thymus. The results action of alloxan on animal cells, particularly the beta cells of show that alloxan is rapidly reduced to dialuric acid by the pancreas, may be caused by rapid oxidation of cellular NADPH thymus NADPH-thioredoxin reductase or by thioredoxin-(SH)2 and generation of cytotoxic dialuric acid catalyzed by the and, thus, is an efficient nondisulfide substrate for the thiore- thioredoxin system. doxin system. Since the discovery that injection of alloxan into animals causes pancreatic beta-cell necrosis, insulin deficiency, and diabetes MATERIALS AND METHODS mellitus (1), this substance has been widely used to produce NADPH, dithiothreitol, alloxan monohydrate, barbituric acid, experimental diabetes. The reason for the particular sensitivity streptozotocin, methylglyoxal, and 5,5'-dithiobis(2-nitrobenzoic of pancreatic beta cells to the cytotoxic action of alloxan is not acid) were from Sigma. 2',5'-ADP-Sepharose 4B was from known despite an intensive search for the primary molecular Pharmacia. w-Aminohexyl agarose was bought from Miles- actions of this drug (2). Yeda (Rehovot, Israel). Glutathione reductase (yeast, 120 Alloxan (2,4,5,6-tetraoxypyrimidine) is a mild oxidizing agent units/mg) was from Boehringer Mannheim. Bovine insulin and it is chemically reduced to dialuric acid (5-OH-barbituric (26.1 units/mg) was from Vitrum (Stockholm, Sweden). acid) by a thiol such as cysteine with formation of cystine (3). Thioredoxin (12) and thioredoxin reductase (13) from E. col In the presence of glutathione or certain protein thiols it forms were homogeneous preparations. Thioredoxin from calf thymus an addition product of unknown structure with an absorption was isolated as described (14). Thioredoxin reductase from calf maximum at 305 nm (3). It has been suggested that such thymus (1.5 kg) was prepared by chromatography on DEAE- modification of critical SH groups in proteins is the cause of cellulose (15), dialysis against 50 mM Tris-HCl/l mM EDTA, alloxan toxicity (4). An alternative explanation is that dialuric pH 7.5 (buffer A), and adsorption to a 15-ml column of 2',5'- acid, generated by unknown mechanisms in vvo from alloxan, 1 causes the formation of cytotoxic free radicals on reoxidation ADP-Sepharose 4B followed by elution with mM NADPH to alloxan (5-7). Dialuric acid is rapidly reoxidized to alloxan in buffer A. After dialysis against buffer A, the enzyme was by 02(2). applied to a 14-ml column of w-aminohexyl agarose and eluted Thioredoxin and NADPH-thioredoxin reductase [thioredoxin with a linear gradient of 100 ml each of buffer A and buffer reductase (NADPH); NADPH:oxidized-thioredoxin oxidore- A/1.0 M NaCl. The active fractions were pooled, dialyzed ductase, EC 1.6.4.5] (the thioredoxin system) from bacterial and against buffer A, and concentrated by adsorption to a 5-ml animal cells was originally isolated as a hydrogen donor system column of 2',5'-ADP-Sepharose 4B. Thioredoxin reductase was for the enzyme ribonucleotide reductase (8). The thioredoxin eluted with 200 mM potassium phosphate, pH 7.5/1 mM system also catalyzes NADPH-dependent reduction of certain EDTA. The enzyme was concentrated by ultrafiltration and exposed disulfide bonds in proteins (8) by the following dialyzed against buffer A. It had a specific activity of 48 Mmol mechanisms: of thionitrobenzoate formed per mg of protein with 5 mM 5,5'-dithiobis(2-nitrobenzoic acid) as substrate (15). The enzyme preparation was estimated to be of >95% purity by polyac- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- rylamide gel electrophoresis (14); it had a native molecular vertisement" in accordance with 18 U. S. C. §1734 solely to indicate weight of around 110,000, determined on a calibrated Sephadex this fact. G-200 column (15). 5149 Downloaded by guest on September 25, 2021 5150 Biochemistry: Holmgren and Lyckeborg Proc. Nati. Acad. Sci. USA 77 (1980) Table 1. Effect of alloxan on E. coli thioredoxin-(SH)2 some direct oxidation of NADPH, in agreement with the results Thioredoxin of Grankvist et al. (7). These results showed that thioredoxin- activity,t ,uM Oxidation of (SH)2 was oxidized to thioredoxin-S2 by alloxan (reactions 1 and disulfide NADPHt 2): Sample* reduced * min-' AA340 *min- Thioredoxin-S2 + NADPH + H+ 1. Thioredoxin-S2 (11 AM) 13.0 0 thioredoxin 2. Thioredoxin-(SH)2 (11 AM) - thioredoxin-(SH)2 + NADP+ [1] + thioredoxin reductase (0.2 yM) reductase + alloxan (0.9 mM) 13.0 0.620 Thioredoxin-(SH)2 + alloxan 3. Thioredoxin-S2 (11 AM) thioredoxin-S2 + dialuric acid. [2] + alloxan (0.9 mM) 13.0 0.090 4. Control, only alloxan (0.9 mM) 0.090 Reduction of Alloxan by E. coli Thioredoxin and Thiore- doxin Reductase. Alloxan behaved as a substrate for the E. coli * To three cuvettes (samples 1-3) containing 500 MI of 0.10 M potas- in sium phosphate, pH 7.0/1 mM EDTA/0.4 mM NADPH was added thioredoxin system. As shown Fig. 1, variation of the con- 50 Ml of thioredoxin-S2 from E. coli (130AM). Cuvette 1 served as centration of alloxan gave an apparent Km of around 70 ,M a control. To cuvette 2 was added 7.5 ,ug (10 Al) ofE. coli thioredoxin with 3.4 ,M thioredoxin and 0.2 ,M thioredoxin reductase plus reductase, which oxidized 12AM NADPH (AA 0 = 0.092) in 30 sec, excess NADPH at pH 7.0. At 0.4 mM NADPH, the direct re- consistent with complete reduction ofthioredoxin-S2. After 3 min, duction of alloxan by thioredoxin reductase or the reaction 5 Ml of 0.10 M alloxan in 1 mM HCl was added to cuvettes 2-4. between NADPH and alloxan was low (below 1.5 ,M-min-' t After 30 min, 30 Ml was withdrawn from samples 1-4 and added to at 100 MM alloxan). When alloxan (540 MM) and thioredoxin 500 Al of 0.10 M potassium phosphate (pH 7.0), 1 mM EDTA, 1 mg of insulin per ml, 0.4 mM NADPH, and 0.2AM thioredoxin reduc- reductase (0.1 MM) were kept constant and the concentration tase. The insulin reduction activity of thioredoxin was followed at of thioredoxin was varied, the reaction rate was proportional 340 nm (10). to the concentration of thioredoxin (up to 3IMM). The K,.t for The initial change in A340 nm for the four cuvettes was followed the E. coil thioredoxin system (Fig. 1) under those conditions against a blank of 0.4 mM NADPH. was around 50% of that of insulin disulfide reduction (10), showing that alloxan was a good nondisulfide substrate. Reduction of Alloxan by Bovine NADPH-Thioredoxin Spectrophotometric Measurements. A Zeiss PMQ3 spec- Reductase. Thioredoxin reductase from calf thymus (15) was trophotometer equipped with an automatic sample changer obtained in a nearly homogeneous state. When 0.25 ,M thio- and semimicrocuvettes plus a Servogor 541 recorder was used. redoxin reductase was incubated with 0.40 mM NADPH and All measurements were done at room temperature in air 500MM alloxan, the enzyme caused oxidation of NADPH with without anaerobic precautions in 0.1 M potassium phosphate/i a rate of 190 AM-min'l (AA340 = 1.2 min'). The dependence mM EDTA, pH 7.0, in a final volume of 0.5 or 0.6 ml. of the NADPH oxidation rate on alloxan concentration is shown Activation of Bovine Thioredoxin System. Thioredoxin in Fig. 2 for 33 nM thioredoxin reductase. An apparent Km of reductase (1 mg/ml) was preincubated with 2 mM di- 330,M was calculated from a Lineweaver-Burk plot. The Kent thiothreitol for 30 min followed by extensive dialysis for 16 hr for the enzyme was calculated to be 1000 min1.