Localization, isolation and properties of three NADPH-dependent aldehyde reducing enzymes from dog kidney

Three kinds of NADPH-dependent aldehyde reducing enzymes were present in the dog kidney. Aldose reductase was located in the inner medulla region and aldehyde reductase in all regions of the renal cortex, outer medulla and inner medulla. In addition, a new reductase designated tentatively as high-Km aldose reductase, which was converted into an aldose reductase-like enzyme, was present in the inner medulla region of the kidney. Aldose reductase, aldehyde reductase and high-Km aldose reductase were purified to homogeneity from each region of the dog kidney. The molecular weight of aldose reductase was estimated to be 38,500 by SDS-polyacrylamide gel electrophoresis and the isoelectric point was found to be 5.7 by chromatofocusing. Aldose reductase had activity for aldo-sugars such as D-xylose, D-glucose and D-galactose as substrates and utilized both NADPH and NADH as coenzymes. Sulfate ions resulted in over 2-fold activation of aldose reductase. All aldehyde reductases from the three regions had the same properties. The molecular weights and isoelectric points of aldehyde reductases were 40,000 and 6.1, respectively. The aldehyde reductases were inactive for D-hexose, utilized only NADPH as coenzyme and were not affected by sulfate ions. High-Km aldose reductase had a molecular weight of 38,500 and an isoelectric point of 5.4. It had activity for aldo-sugars, but showed much higher Km and lower kcat/Km values than aldose reductase. Sulfate ions inhibited high-Km aldose reductase. It was converted into an aldose reductase-like enzyme by incubation in phosphate buffer at pH 7.0. The three kinds of enzymes were strongly inhibited by the known aldose reductase inhibitors. However, aldehyde reductase and high-Km aldose reductase were, in general, less susceptible than aldose reductase.     

Aldose reductase: physiological role, properties and prospects for regulating activity]

Some properties of aldose reductase isolated from various sources and possible ways of regulation of the enzyme catalytic activity are reviewed. Mammalian aldose reductases are monomeric enzymes with M(r) of 30-40 kDa and a broad substrate specificity towards aldoses. The physiological role of this enzyme consists, apparently, in providing an additional pathway for utilization of glucose and removing toxic compounds carrying an aldehyde group from the cell. Aldose reductase is thought to play a key role in various hyperglycemic states, including diabetic cataract. The kinetics of the aldose reductase reaction is hyperbolic with NADPH and nonhyperbolic with glucose. The rate of the enzyme-catalyzed reaction is determined by the effector binding in the active of inhibitory center of the enzyme. Incubation with substrates leads to the activation of the enzyme which is accompanied by a decrease of the effector binding in the enzyme inhibitory center with a sharp decrease in the sensitivity of the activated enzyme to NADPH concentration changes in the presence of glucose excess. A mechanism underlying the catalytic effect of both native and activated forms of the enzyme is proposed.     

Bovine lens aldehyde reductase (aldose reductase). Purification, kinetics and mechanism.

Aldehyde reductase (aldose reductase) was purified to homogeneity (as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis) from bovine lens by affinity chromatography on NADP+-Sepharose. The enzyme, a monomer of Mr about 40000, was active with a variety of alpha- hydroxyketones , including fructose. The minimum degree of the rate equation was 2:2 in the case of DL-glyceraldehyde, but linear kinetics were observed for glucose and NADPH over the concentration range studied. The enzyme largely followed a ternary-complex mechanism, with initial binding of NADPH before glucose and final release of NADP+.     

New inhibitors of aldose reductase: anti-oximes of aromatic aldehydes

Aldose reductase is an NADPH-dependent enzyme which catalyzes the reduction of glucose to sorbitol. Specific potent inhibitors of aldose reductase are of potential pharmacological use because elevated levels of sorbitol produced by this enzyme in lens, peripheral nerve, retina, and renal glomeruli may be responsible for the pathogenesis associated with chronic diabetes. These inhibitors could also serve as probes of the mechanism of action of aldose reductase. anti-Oximes of aromatic aldehydes (e.g., benzaldoxime and 4-fluorobenzaldoxime) have proved to be effective inhibitors of aldose reductase rivaling pharmacological agents currently used to inhibit this enzyme in vivo. The kinetic patterns of inhibition in which benzyl alcohol is used as the oxidizable substrate suggest that the inhibition is due to the formation of a stable ternary complex composed of aldose reductase, NADP+, and the anti-oxime. Analogus ternary complexes are formed at the active site of horse liver alcohol dehydrogenase which is also inhibited by anti-oximes of efficient substrates.     

Cu,Zn-superoxide dismutase-driven free radical modifications: copper- and carbonate radical anion-initiated protein radical chemistry

The understanding of the mechanism, oxidant(s) involved and how and what protein radicals are produced during the reaction of wild-type SOD1 (Cu,Zn-superoxide dismutase) with H2O2 and their fate is incomplete, but a better understanding of the role of this reaction is needed. We have used immuno-spin trapping and MS analysis to study the protein oxidations driven by human (h) and bovine (b) SOD1 when reacting with H2O2 using HSA (human serum albumin) and mBH (mouse brain homogenate) as target models. In order to gain mechanistic information about this reaction, we considered both copper- and CO3(*-) (carbonate radical anion)-initiated protein oxidation. We chose experimental conditions that clearly separated SOD1-driven oxidation via CO(*-) from that initiated by copper released from the SOD1 active site. In the absence of (bi)carbonate, site-specific radical-mediated fragmentation is produced by SOD1 active-site copper. In the presence of (bi)carbonate and DTPA (diethylenetriaminepenta-acetic acid) (to suppress copper chemistry), CO(*-) produced distinct radical sites in both SOD1 and HSA, which caused protein aggregation without causing protein fragmentation. The CO(*-) produced by the reaction of hSOD1 with H2O2 also produced distinctive DMPO (5,5-dimethylpyrroline-N-oxide) nitrone adduct-positive protein bands in the mBH. Finally, we propose a biochemical mechanism to explain CO(*-) production from CO2, enhanced protein radical formation and protection by (bi)carbonate against H2O2-induced fragmentation of the SOD1 active site. Our present study is important for establishing experimental conditions for studying the molecular mechanism and targets of oxidation during the reverse reaction of SOD1 with H2O2; these results are the first step in analysing the critical targets of SOD1-driven oxidation during pathological processes such as neuroinflammation.     

Osmotic regulation of aldose reductase protein synthesis in renal medullary cells

Renal medullary cells are normally exposed to high extracellular NaCl as part of the urinary concentrating mechanism. They react to this stress by accumulating sorbitol and other organic osmolytes. PAP-HT25, a line of epithelial cells derived from rabbit renal inner medulla, expresses this response. In hypertonic medium, these cells accumulate large amounts of sorbitol. There is a large increase in the amount of aldose reductase, which catalyzes production of sorbitol from glucose. The purpose of the present study was to investigate whether the aldose reductase protein increases because of faster synthesis or slower degradation. We measured the rate of synthesis and degradation of aldose reductase protein by pulse-chase with [35S]methionine, followed by immunoprecipitation with specific antiserum and autoradiography. The protein synthesis rate was 6 times greater in cells grown in hypertonic (500 mosmol/kg) medium, than in those grown in normal (300 mosmol/kg) medium. When control cells were switched to hypertonic medium, the synthesis rate increased 15-fold by 24 h, then decreased to 11-fold after 48 h. In contrast, synthesis rate continued to increase past 24 h when accumulation of sorbitol was prevented by inhibiting aldose reductase activity with Tolrestat. Thus, there is a feedback mechanism by which cellular sorbitol accumulation inhibits aldose reductase protein synthesis. Degradation of aldose reductase protein was slow (only about 25% in 3 days) and was not affected by osmolality. Thus, the osmoregulatory increase in aldose reductase protein is due to an increase in its synthesis rate and not to any change in its degradation.     

Aldose reductase from human psoas muscle. Purification, substrate specificity, immunological characterization, and effect of drugs and inhibitors

Aldose reductase (ALR2) has been purified to homogeneity from human psoas muscle. From sodium dodecyl sulfate-polyacrylamide electrophoresis the enzyme is monomeric and has a molecular weight of 37,000. ALR2 catalyzes the primarily NADPH-dependent reduction of a wide variety of aldehydes, although the enzyme can also utilize NADH. The best substrates for ALR2 are aromatic aldehydes (e.g. pyridine-3-aldehyde; Km = 9 microM; kcat/Km = 150,000 s-1 M-1), while among aldoses DL-glyceraldehyde is the preferred substrate (Km = 72 microM; kcat/Km = 17,250). Low (100 microM) concentrations of CaCl2 and CaSO4 cause a marked inhibition (90%) of ALR2 as do higher concentrations (0.2 M) of MgCl2. (NH4)2SO4 caused a 2-fold activation of ALR2. The enzyme is also inhibited by quercetin and the commercially developed aldose reductase inhibitors alrestatin and sorbinil. ALR2 is inhibited only very slightly by sodium valproate and barbiturates. ALR2 cross-reacts immunologically with human brain and human placental aldose reductase and with ALR2 from monkey tissue. There is no precipitin cross-reaction of ALR2 with aldose reductases from other species nor with human aldehyde reductase 1 (ALR1) or with ALR1 from other species. The data show that human muscle is a new and relatively rich source of a monomeric NADPH/NADH reductase which is clearly identifiable as aldose reductase.     

Polyol pathway in tissues of spontaneously diabetic Chinese hamsters (Cricetulus griseus) and the effect of an aldose reductase inhibitor, ONO-2235

1. Sorbitol and fructose levels were significantly elevated in the lens, the sciatic nerve, the retina and the kidney of diabetic Chinese hamsters and inositol level was significantly decreased in the lens and sciatic nerve of diabetics. 2. The activity of an aldose reductase in the kidney was not different between normal and diabetic Chinese hamsters. 3. An aldose reductase inhibitor (ONO-2235) had no effect in sorbitol, fructose and inositol contents of all these tissues from diabetic Chinese hamsters. 4. These results suggest that diabetic Chinese hamsters produce polyol accumulation in tissues but that there is a clear species-specific difference to inhibition of aldose reductase.     

Cloning and expression of human aldose reductase

The complete amino acid sequence of human retina and muscle aldose reductase was determined by nucleotide analysis of cDNA clones isolated using synthetic oligonucleotide probes based on partial amino acid sequences of purified human psoas muscle aldose reductase. The cDNA sequence differs substantially in the noncoding and coding regions of recently published sequences of this enzyme. The mRNA for aldose reductase was abundantly expressed in HeLa cells, but only scarcely in a neuroblastoma cell line. Recombinant baculovirus containing one of the muscle cDNA clones was constructed and used to infect Spodoptera frugiperda (SF9) cells. A prominent protein with an apparent molecular size of 36 kDa was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the culture medium as well as in the homogenate of SF9 cells after 2 days of infection. Culture medium or the supernatant fraction of cell homogenates containing this protein had high aldose reductase activity which showed characteristics of the reported human enzyme. These findings indicate that the amino acid sequence reported in this paper represents human retina and muscle aldose reductase and that functional human aldose reductase can be expressed in large amounts in a baculovirus expression system. The result should facilitate refined structural analysis and the development of new specific aldose reductase inhibitors for the treatment of diabetic complications.     

Aldose reductase expression contributes in sorbitol accumulation and 4-hydroxynon-2-enal detoxification in two foxtail millet (<Emphasis Type="Italic">Setaria italic</Emphasis>a L.) cultivars with different salt stress tolerance

Salt stress is a major environmental factor in arid and semi-arid regions and influences many aspects of plant development. Salinity results in generation of various free radicals that can potentially damage the cellular constituents in plants. Plants were able to effectively reduce the damage caused by these free radicals by a way of enzymatic and non enzymatic defenses for better survival. Enhanced efficacy of antioxidative enzyme systems such as superoxide dismutase, catalase and ascarbate peroxidase was well documented in several plants subjected to salinity stress. Aldose reductase, an important enzyme is also known to detoxify free toxic aldehydes like HNE (4-hydroxynon-2-enal, a hydroxyalkenal) generated during oxidative damage of cellular components. However, the role of aldose reductase to impart tolerance to the plants under salt stress has not been studied in any detail. Therefore, we were interested to study the aldose reductase activity and its expression to gain an insight into the role of aldose reductase in imparting tolerance to foxtail millet cultivars (viz., Cv. Prasad and Lepakshi) subjected to NaCl stress. We observed that subjecting foxtail millets to increasing levels of stress significantly increased aldose reductase activity and in a way that correlated positively with elevated levels of sorbitol, an osmotic solute involved in osmotic balance. This suggests the involvement of aldose reductase in sorbitol biosynthesis in foxtail millet. Additionally, we observed higher levels of 4-hydroxynon-2-enal, a major lipid peroxidation product, in the susceptible than the tolerant cultivar indicating a higher proportion of cellular damage in former than in the latter. This high content of 4-hydroxynon-2-enal in the susceptible cultivar was negatively correlated with its aldose reductase activity, indicating the involvement of aldose reductase in detoxification of 4-hydroxynon-2-enal. 4-hydroxynon-2-enal is also known to be a catalyzed by glutathione-S-transferase. Glutathione-S-transferase activity was found higher in the tolerant foxtail millet than the sensitive cultivar: the tolerant cultivar showed a low 4-hydroxynon-2-enal content compared to the susceptible cultivar, demonstrating a possible mechanism for detoxification of 4-hydroxynon-2-enal by two enzymes, glutathione-S-transferase and aldose reductase in plants under stressful conditions.     

Purification and characterization of human testis aldose and aldehyde reductase

1. Aldose reductase and aldehyde reductase were purified to homogeneity from human testis. 2. The molecular weight of aldose reductase and aldehyde reductase were estimated to be 36,000 and 38,000 by SDS-PAGE, and the pI values of these enzymes were found to be 5.9 and 5.1 by chromatofocusing, respectively. 3. Aldose reductase had activity for aldo-sugars, whereas aldehyde reductase was virtually inactive for aldo-sugars. The Km values of aldose reductase for D-glucose, D-galactose and D-xylose were 57, 49 and 6.2 mM, respectively. Aldose reductase utilized both NADPH and NADH as coenzymes, whereas aldehyde reductase only NADPH. 4. Sulfate ion caused 3-fold activation of aldose reductase, but little for that of aldehyde reductase. 5. Sodium valproate inhibited significantly aldehyde reductase, but not aldose reductase. Aldose reductase was inhibited strongly by aldose reductase inhibitors being in clinical trials at concentrations of the order of 10(-7)-10(-9) M. Aldehyde reductase was also inhibited by these inhibitors, but its susceptibility was less than aldose reductase. 6. Reaction of aldose reductase with pyridoxal 5'-phosphate (PLP) resulted ca 2.5-fold activation, but aldehyde reductase did not cause the activation. PLP-treated aldose reductase has lost the susceptibility to aldose reductase inhibitor.     

Comparative studies on aldose reductase from bovine, rat and human lens

A purification scheme for aldose reductase (alditol: NADP+ 1-oxidoreductase, EC 1.1.1.21) developed using bovine lens tissue including an affinity chromatographic step is presented which is particularly suited for small quantities of lenses. Using the affinity chromatographic method as a key step also makes it possible to obtain preparations of rat lens aldose reductase which are homogeneous. The behavior of crude preparations of aldose reductase from human lens on both ion-exchange and affinity chromatography was similar to the chromatographic behavior of the enzyme from rat and bovine lens. Comparative studies of aldose reductase obtained from the lenses of the three species demonstrate the similarity of the enzymes. These comparisons were based on molecular weights, isoelectric points, chromatographic behavior and kinetic data. Homotropic cooperativity for both NADPH and glyceraldehyde, as evidenced by a downward curvature in the Lineweaver-Burk double-reciprocal plots, had been demonstrated for aldose reductase obtained from bovine lens (Sheaff, C.M. and Doughty, C.C. (1976) J. Biol. Chem. 251, 2696-2702). Similarly, cooperativity was observed with the enzyme from both rat and human lenses and the apparent Km values at both high and low concentrations of substrate are comparable for the lens aldose reductases from all three species for both substrates.     

The kinetic mechanism of human placental aldose reductase and aldehyde reductase II

The kinetic mechanism of NADPH-dependent aldehyde reductase II and aldose reductase, purified from human placenta, has been studied using L-glucuronate and DL-glyceraldehyde as their respective substrates. For aldehyde reductase II, the initial velocity and product inhibition studies (using NADP and gulonate) indicate that the enzyme reaction sequence is ordered with NADPH binding to the free enzyme and NADP being the last product to be released. Inhibition patterns using menadione (an analog of the aldehydic substrate) and ATP-ribose (an analog of NADPH) are also consistent with a compulsory ordered reaction sequence. Isotope effects of deuterium-substituted NADPH (NADPD) also corroborate the above reaction scheme and indicate that hydride transfer is not the sole rate-limiting step in the reaction sequence. For aldose reductase, initial velocity patterns, product, and dead-end inhibition studies indicate a random binding pattern of the substrates and an ordered release of product; the coenzyme is released last. A steady-state random mechanism is also consistent with deuterium isotope effects of NADPD on the reaction sequence catalyzed by this enzyme. However, the hydride transfer step seems to be more rate determining for aldose reductase than for aldehyde reductase II.     

Purification and characterization of two aldose reductase isoenzymes from rabbit muscle

Using a modification of the procedure of Kormann et al. (Kormann, A. W., Hurst, R. O., and Flynn, T. G. (1972) Biochim. Biophys. Acta 258, 40-55) for the purification of glycerol dehydrogenase, two enzymes have been purified from the skeletal muscle of male rabbits. From a consideration of their properties these enzymes have been named aldose reductase 1 and aldose reductase 2, respectively. Both enzymes are monomeric by the criteria of gel filtration and polyacrylamide gel electrophoresis in sodium dodecyl sulfate and both reductases are immunologically identical as shown by double immunodiffusion and rocket immunoelectrophoresis. Aldose reductases 1 and 2 have almost identical amino acid compositions, their NH2 termini are blocked and the COOH termini of both enzymes are apparently identical. The enzymes differ, however, in molecular weight with aldose reductase 2 having Mr = 41,500 and aldose reductase 1 Mr 40,200. Both enzymes have the broad substrate specificity typical of the aldehyde reductase family of enzymes; Km values of aldose reductase 1 for aldo sugars were similar to those reported for rabbit lens aldose reductase, and both aldose reductase 1 and 2 were inhibited by the commercial aldose reductase inhibitors Alrestatin and Sorbinil. Two aldose reductases, immunologically and electrophoretically identical to the muscle enzymes, were found in rabbit lens. Two aldose reductases were also detected in the skeletal muscle of male rats and pigs and in pig and bovine lens. The presence of relatively large amounts of aldose reductase in muscle identifies a new and rich source of the enzyme.     

Efficacy of glucose, ouabain and an aldose reductase inhibitor on 2-[3H] myo-inositol uptake by human, rat and rabbit erythrocytes

Myo-inositol uptake by erythrocytes from humans, rabbits and rats was studied with an isotope technique. In human erythrocytes, the inhibitory effect on myo-inositol uptake was stronger with glucose than with ouabain. However, an aldose reductase inhibitor (ONO-2235, 100 microM) or insulin (200 microU/ml) failed to correct the decrease in myo-inositol uptake in packed RBC, produced by either 10 mM glucose or 2mM ouabain. Ten mM ouabain had an inhibitory effect on myo-inositol uptake in all species, but an inhibitory effect was not observed with 20 mM glucose in rabbit erythrocytes. The results suggest that myo-inositol uptake by erythrocytes may be dependent on the active transport system via sodium-ATPase and that erythrocytes may not be a suitable model to monitor the possible effect of an aldose reductase inhibitor on myo-inositol concentrations in other tissues concerned with diabetic complications.     

Aldose reductase regulates miR-200a-3p/141-3p to coordinate Keap1–Nrf2, Tgfβ1/2, and Zeb1/2 signaling in renal mesangial cells and the renal cortex of diabetic mice

Aberrant regulation in oxidative stress, fibrogenesis, and the epithelial–mesenchymal transition (EMT) in renal cells under hyperglycemic conditions contributes significantly to the onset and progression of diabetic nephropathy. The mechanisms underlying these hyperglycemia-induced dysregulations, however, have not been clearly elucidated. Herein, we report that aldose reductase is capable of regulating the expression of miR-200a-3p/141-3p negatively in renal mesangial cells. MiR-200a-3p/141-3p, in turn, act to target Keap1, Tgfβ2, fibronectin, and Zeb2 directly and regulate Tgfβ1 and Nrf2 indirectly under high-glucose conditions, resulting in profound dysregulations in Keap1–Nrf2, Tgfβ1/2, and Zeb1/2 signaling. In vivo in streptozotocin-induced diabetic mice, we found that aldose reductase deficiency caused significant elevations in miR-200a-3p/141-3p in the renal cortex, which were accompanied by a significant downregulation of Keap1, Tgfβ1/2, and fibronectin but significant upregulation of Nrf2. Moreover, in vivo administration of inhibitors of miR-200a-3p in diabetic animals significantly exacerbated cortical and glomerular fibrogenesis and increased urinary albumin excretion, tightly linking dysregulated miR-200a-3p with the development of diabetic nephropathy. Collectively, our results reveal a novel mechanism whereby hyperglycemia induces aldose reductase to regulate renal expression of miR-200a-3p/141-3p to coordinately control hyperglycemia-induced renal oxidative stress, fibrogenesis, and the EMT. Our novel findings also suggest that inhibition of aldose reductase and in vivo renal cortical restoration of miR-200a-3p/141-3p or their combination are very promising avenues for the development of therapeutic strategies or drugs against diabetic nephropathy.     

A micromethod for the assay of aldose reductase, its application to pancreatic islets

A micromethod for the assay of aldose reductase is described. The method, which is based on the fluorometric measurement of the NADP+ formed when an aldose is converted to its corresponding polyol, was applied to lens and pancreatic islet crude homogenates, as well as semipurified lens aldose reductase. The fluorometric method has proved to be reproducible, more rapid, and more sensitive than the classical spectrophotometric procedure, and should find ready application in the screening of potential aldose reductase inhibitors.     

Involvement of cysteine residues in catalysis and inhibition of human aldose reductase. Site-directed mutagenesis of Cys-80, -298, and -303.

In order to study the potential role of cysteinyl residues in catalysis and inhibition of human aldose reductase, mutants containing cysteine to serine substitution at positions 80 (ALR2:C80S), 298 (ALR2:C298S), and 303 (ALR2:C303S) were constructed. Mutation of Cys298 resulted in the most profound changes, as ALR2:C298S displayed 4- to 5-fold elevation in K'm(NADPH), K'm(DL-glyceraldehyde), and kcat(DL-glyceraldehyde) relative to wild type aldose reductase as well as a 10-fold higher Ki for the aldose reductase inhibitor sorbinil. Wild type and mutant reductases were equally sensitive to tolrestat, a structurally different reductase inhibitor. Carboxymethylation of the wild type enzyme or the C80S and C303S mutants led to a modest decrease in kcat as well as an increase in K'm(DL-glyceraldehyde) and Ki(sorbinil). These parameters were not significantly changed when ALR2:C298S was subjected to carboxymethylation. Lithium sulfate caused activation of ALR2:WT, C80S, and C303S but did not significantly affect the activity of ALR2:C298S. The differential sensitivity of wild type and mutant reductases to inhibition by sorbinil and tolrestat, before and after carboxymethylation, indicates that these inhibitors bind at different sites. These results suggest that Cys-298 is present near the active site and constitutes a regulatory group which controls the catalytic activity and inhibitor sensitivity of the enzyme.     

人脑醛糖还原酶的纯化和一些基本性质

使用DEAE纤维素柱层析、PBE-94层析聚焦、NADP~+-Sepharose 4B亲合层析及SephadexG-100凝胶过滤分离纯化了人脑醛糖还原酶。在DEAE层析中,用咪唑-HCI缓冲液替代了磷酸缓冲液,改善了分离效果。在聚丙烯酰胺及SDS聚丙烯酰胺凝胶电泳中,纯化的人脑醛糖还原酶均呈一条区带。它的pI为5.6,最适pH为6.5,分子量为36,000,底物特异性和氨基酸组成与其它哺乳动物的醛糖还原酶有相似性。开链式醛糖是醛糖还原酶的真正底物,它在开链式和半缩醛的平衡体系中占比例极小,因而推知醛糖还原酶对此底物有很高的K_(cat)和K_(cat)/K_m值,能有效地将它们还原成相应的醇。     

Metabolism of the 2-oxoaldehyde methylglyoxal by aldose reductase and by glyoxalase-I: roles for glutathione in both enzymes and implications for diabetic complications

Numerous physiological aldehydes besides glucose are substrates of aldose reductase, the first enzyme of the polyol pathway which has been implicated in the etiology of diabetic complications. The 2-oxoaldehyde methylglyoxal is a preferred substrate of aldose reductase but is also the main physiological substrate of the glutathione-dependent glyoxalase system. Aldose reductase catalyzes the reduction of methylglyoxal efficiently (k(cat)=142 min(-1) and k(cat)/K(m)=1.8x10(7) M(-1) min(-1)). In the presence of physiological concentrations of glutathione, methylglyoxal is significantly converted into the hemithioacetal, which is the actual substrate of glyoxalase-I. However, in the presence of glutathione, the efficiency of reduction of methylglyoxal, catalyzed by aldose reductase, also increases. In addition, the site of reduction switches from the aldehyde to the ketone carbonyl. Thus, glutathione converts aldose reductase from an aldehyde reductase to a ketone reductase with methylglyoxal as substrate. The relative importance of aldose reductase and glyoxalase-I in the metabolic disposal of methylglyoxal is highly dependent upon the concentration of glutathione, owing to the non-catalytic pre-enzymatic reaction between methylglyoxal and glutathione.