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.     

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。     

Purification and properties of reductases for aromatic aldehydes and ketones from guinea pig liver.

NADPH-dependent enzymatic reduction of aromatic aldehydes and ketones observed in the cytosol of guinea pig liver was mediated by at least three distinct reductases (AR 1, AR 2, and AR 3), which were separated by DEAE-cellulose chromatography. By several procedures AR 2 and AR 3 were purified to homogeneity, but AR 1 could be purified only 30-fold because of the small amount. These enzymes were found to have similar molecular weights of 34,000 to 36,000 and similar Stokes radii of about 2.5 nm. AR 3 was identical to aldehyde reductase [EC 1.1.1.2] in substrate specificity for aromatic aldehydes and D-glucuronate and specific inhibition by barbiturates. AR 1 and AR 2 acted on aromatic ketones and cyclohexanone as well as aromatic aldehydes at optimal pHs of 5.4 and 6.0, respectively, and were immunochemically distinguished from AR 3. AR 1 was the most sensitive to sulfhydryl reagents, and AR 2 was more stable at 50 degrees C than the other enzymes. Similar heterogeneity was observed in the kidney enzymes, but other tissues had little aldehyde reductase activity and contained only AR 3. In addition, lung contained a high molecular weight aromatic ketone reductase different from the above reductases.     

Catalytic effectiveness of human aldose reductase. Critical role of C-terminal domain.

Human aldose reductase and aldehyde reductase are members of the aldo-keto reductase superfamily that share three domains of homology and a nonhomologous COOH-terminal region. The two enzymes catalyze the NADPH-dependent reduction of a wide variety of carbonyl compounds. To probe the function of the domains and investigate the basis for substrate specificity, we interchanged cDNA fragments encoding the NH2-terminal domains of aldose and aldehyde reductase. A chimeric enzyme (CH1, 317 residues) was constructed in which the first 71 residues of aldose reductase were replaced with first 73 residues of aldehyde reductase. Catalytic effectiveness (kcat/Km) of CH1 for the reduction of various substrates remained virtually identical to wild-type aldose reductase, changing a maximal 4-fold. Deletion of the 13-residue COOH-terminal end of aldose reductase, yielded a mutant enzyme (AR delta 303-315) with markedly decreased catalytic effectiveness for uncharged substrates ranging from 80- to more than 600-fold (average 300-fold). The KmNADPH of CH1 and AR delta 303-315 were nearly identical to that of the wild-type enzyme indicating that cofactor binding is unaffected. The truncated AR delta 303-315 displayed a NADPH/D isotope effect in kcat and an increased D(kcat/Km) value for DL-glyceraldehyde, suggesting that hydride transfer has become partially rate-limiting for the overall reaction. We conclude that the COOH-terminal domain of aldose reductase is crucial to the proper orientation of substrates in the active site.     

NADPH-dependent reduction of glyceraldehyde: a unusually high activity in the lens of the camel (Camelus dromedarius)

The enzymes of the polyol pathway, namely aldose reductase and sorbitol dehydrogenase, were measured in camel lens extracts. A NADPH-dependent glyceraldehyde and erythrose reductase activity 25 times higher than that of calf lens was observed in camel lens. A preliminary comparison between this enzyme activity present in the camel and aldose reductase of calf lens is reported.     

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.     

Purification and characterization of the recombinant human aldose reductase expressed in baculovirus system

Large quantities of recombinant human aldose reductase were produced using Spodoptera frugiperda cells and properties of the enzyme were characterized. Direct purification of the recombinant aldose reductase by affinity column chromatography using Matrex gel orange A yielded a single 36 kDa band, similar in size to the purified human muscle aldose reductase, on a sodium dodecyl sulfate-polyacrylamide gel after silver staining. The isoelectric point of the recombinant enzyme was 5.85 which is identical to the human muscle aldose reductase. Following the treatment with an acylamino-acid releasing enzyme, the blocked NH2-terminal amino acid was identified to be acetylalanine. The successive NH2-terminal sequence and that of the COOH-terminal peptide concurred with the expected translated sequence. Kinetic analyses of the recombinant enzyme activity for various substrates and the cofactor, NADPH, demonstrated a good agreement with the previously reported kinetic data on the purified human aldose reductase. A high concentration of (NH4)2SO4 elicited a significant increase in both Km and Kcat for DL-glyceraldehyde as well as D-glucose. Although IC50 values for most of the aldose reductase inhibitors with recombinant enzyme were found to fall within the comparable range of those obtained with nonhuman mammalian enzymes, the IC50 value for epalrestat was more than 10-fold higher in the recombinant enzyme. These results indicate that the recombinant human aldose reductase expressed in the baculovirus system possesses structurally and enzymatically similar properties as those reported for the native human enzyme and should serve as a superior enzyme preparation to nonhuman mammalian enzymes for the screening of the efficacy and potency of newly developed aldose reductase inhibitors.     

Purification and properties of aldose reductase and aldehyde reductase II from human erythrocyte

Aldose reductase (EC 1.1.1.21) and aldehyde reductase II (L-hexonate dehydrogenase, EC 1.1.1.2) have been purified to homogeneity from human erythrocytes by using ion-exchange chromatography, chromatofocusing, affinity chromatography, and Sephadex gel filtration. Both enzymes are monomeric, Mr 32,500, by the criteria of the Sephadex gel filtration and polyacrylamide slab gel electrophoresis under denaturing conditions. The isoelectric pH's for aldose reductase and aldehyde reductase II were determined to be 5.47 and 5.06, respectively. Substrate specificity studies showed that aldose reductase, besides catalyzing the reduction of various aldehydes such as propionaldehyde, pyridine-3-aldehyde and glyceraldehyde, utilizes aldo-sugars such as glucose and galactose. Aldehyde reductase II, however, did not use aldo-sugars as substrate. Aldose reductase activity is expressed with either NADH or NADPH as cofactors, whereas aldehyde reductase II can utilize only NADPH. The pH optima for aldose reductase and aldehyde reductase II are 6.2 and 7.0, respectively. Both enzymes are susceptible to the inhibition by p-hydroxymercuribenzoate and N-ethylmaleimide. They are also inhibited to varying degrees by aldose reductase inhibitors such as sorbinil, alrestatin, quercetrin, tetramethylene glutaric acid, and sodium phenobarbital. The presence of 0.4 M lithium sulfate in the assay mixture is essential for the full expression of aldose reductase activity whereas it completely inhibits aldehyde reductase II. Amino acid compositions and immunological studies further show that erythrocyte aldose reductase is similar to human and bovine lens aldose reductase, and that aldehyde reductase II is similar to human liver and brain aldehyde reductase II.     

The aldo-keto reductase superfamily. cDNAs and deduced amino acid sequences of human aldehyde and aldose reductases

Aldehyde reductase [EC 1.1.1.2] and aldose reductase [EC 1.1.1.21] are monomeric NADPH-dependent oxidoreductases having wide substrate specificities for carbonyl compounds. These enzymes are implicated in the development of diabetic complications by catalyzing the reduction of glucose to sorbitol. Enzyme inhibition as a direct pharmacokinetic approach to the prevention of diabetic complications resulting from the hyperglycemia of diabetes has not been effective because of nonspecificity of the inhibitors and some appreciable side effects. To understand the structural and evolutionary relationship of these enzymes, we cloned and sequenced cDNAs coding for aldose and aldehyde reductases from human liver and placental cDNA libraries. Human placental aldose reductase (open reading frame of 316 amino acids) has a 65% identity (identical plus conservative substitutions) to human liver and placental aldehyde reductase (open reading frame of 325 amino acids). The two sequences have significant identity to 2,5-diketogluconic acid reductase from corynebacterium, frog rho-crystallin, and bovine lung prostaglandin F synthase (reductase). Southern hybridization analysis of human genomic DNA indicates a multigene system for aldose reductase, suggesting the existence of additional proteins. Thus, the aldo-keto reductase superfamily of proteins may have a more significant and hitherto not fully appreciated role in general cellular metabolism.     

Reduction of trioses by NADPH-dependent aldo-keto reductases. Aldose reductase, methylglyoxal, and diabetic complications.

The substrate specificities of human aldose reductase and aldehyde reductase toward trioses, triose phosphates, and related three-carbon aldehydes and ketones were evaluated. Both enzymes are able to catalyze the NADPH-dependent reduction of all of the substrates used. Aldose reductase shows more discrimination among substrates than does aldehyde reductase and is generally the more efficient catalyst. The best substrate for aldose reductase is methylglyoxal (kcat = 142 min-1, kcat/Km = 1.8 x 10(7) M-1 min-1), a toxic 2-oxo-aldehyde that is produced nonenzymatically from triose phosphates and enzymatically from acetone/acetol metabolism. D- and L-glyceraldehyde and D- and L-lactaldehyde are also good substrates for aldose reductase. The aldose reductase-catalyzed reduction of methylglyoxal produces 95% acetol, 5% D-lactaldehyde. Further reduction of acetol produces only L-1,2-propanediol. Acetol and propanediol are two products that accumulate in uncontrolled diabetes. Both acetol and methylglyoxal were compared with glucose for their abilities to produce covalent modification of albumin. All three of these carbonyl compounds reacted with albumin to produce modified proteins with new absorption and emission bands that are spectrally similar. Both methylglyoxal and acetol are much more reactive than glucose. A new integrative model of diabetic complications is proposed that combines the aldose reductase/polyol pathway theory and the nonenzymatic glycation theory except that emphasis is placed both on methylglyoxal/acetol metabolism and on glucose metabolism.     

Chicken muscle aldose reductase: purification, properties and relationship to other chicken aldo/keto reductases

An enzyme that catalyzes the NADPH-dependent reduction of a wide range of aromatic and hydroxy-aliphatic aldehydes was purified from chicken breast muscle. This enzyme shares many properties with mammalian aldose reductases including molecular weight, relative substrate specificity, Michaelis constants, an inhibitor specificity. Therefore, it seems appropriate to call this enzyme an aldose reductase (EC 1.1.1.21). Chicken muscle aldose reductase appears to be kinetically identical to an aldose reductase that has been purified from chicken kidney (Hara et al., Eur. J. Biochem. 133, 207-214) and to hen muscle L-glycol dehydrogenase (Bernado et al., Biochim. biophys. Acta 659, 189-198). The association of this aldose reductase with muscular dystrophy in the chick is discussed.     

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.     

Aldose reductase and p-crystallin belong to the same protein superfamily as aldehyde reductase

Aldose reductase (EC 1.1.1.21) has been implicated in a variety of diabetic complications. Here we present the first primary sequence data for the rat lens enzyme, obtained by amino acid and cDNA analysis. We have found structural similarities with another NADPH-dependent oxidoreductase: human liver aldehyde reductase (EC 1.1.1.2). The identity between these two enzymes is 50%. Both enzymes share approx. 40-50% homology with p-crystallin, a major lens protein present only in the frog, Rana pipiens. We propose that aldose reductase, aldehyde reductase and p-crystallin are members of a superfamily of related proteins.     

19F NMR quantitation of lens aldose reductase activity using 3-deoxy-3-fluoro-D-glucose

In the present study we have determined the kinetics of 3-deoxy-3-fluoro-D-glucose (3-FG) as a substrate for the aldose reductase reaction in vitro. In addition, we compared the 3-deoxy-3-fluoro-sorbitol (3-FS) production rates from 3-FG in the intact lens using 19F NMR with conventional aldose reductase determinations in extracts from the same lenses. The affinity of in vitro aldose reductase for 3-FG was approximately 20 times greater (9.3 mM) than that for glucose (188 mM). An excellent correlation between the rate of 3-FS production in the intact canine lens, determined with 19F NMR, and extracted aldose reductase activity was observed. The relatively high affinity of aldose reductase for 3-FG and the correlation of 3-FS production with enzyme activity in the intact lens suggests that 3-FS production from 3-FG detected by 19F NMR could provide an accurate noninvasive determination of aldose reductase activity in the eye lens.     

Identification of Pig Brain Aldehyde Reductases with the High-Km Aldehyde Reductase, the Low-Km Aldehyde Reductase and Aldose Reductase, Carbonyl Reductase, and Succinic Semialdehyde Reductase

Four NADPH-dependent aldehyde reductases (ALRs) isolated from pig brain have been characterized with respect to substrate specificity, inhibition by drugs, and immunological criteria. The major enzyme, ALR1, is identical in these respects with the high-Km aldehyde reductase, glucuronate reductase, and tissue-specific, e.g., pig kidney aldehyde reductase. A second enzyme, ALR2, is identical with the low-Km aldehyde reductase and aldose reductase. The third enzyme, ALR3, is carbonyl reductase and has several features in common with prostaglandin-9-ketoreductase and xenobiotic ketoreductase. The fourth enzyme, unlike the other three which are monomeric, is a dimeric succinic semialdehyde reductase. All four of these enzymes are capable of reducing aldehydes derived from the biogenic amines. However, from a consideration of their substrate specificities and the relevant Km and Vmax values, it is likely that it is ALR2 which plays a primary role in biogenic aldehyde metabolism. Both ALR1 and ALR2 may be involved in the reduction of isocorticosteroids. Despite its capacity to reduce ketones, ALR3 is primarily an aldehyde reductase, but clues as to its physiological role in brain cannot be discerned from its substrate specificity. The capacity of succinic semialdehyde reductase to reduce succinic semialdehyde better than any other substrate shows that this reductase is aptly named and suggests that its primary role is the maintenance in brain of physiological levels of gamma-hydroxybutyrate.     

The purification and properties of aldose reductase from rat ovary

Aldose reductase has been highly purified from rat ovary to apparent homogeneity, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme proved to be a monomeric protein with a molecular weight of about 39,900. The enzyme catalyzed the NADPH-dependent reduction of a number of aromatic and aliphatic aldehydes as well as aldo-sugars. The enzyme was potently inhibited by p-chloro-mercuribenzoate and a commercially developed aldose reductase inhibitor, M79175. The result of an immunoinhibition study, using antibody against the purified enzyme, indicated that the enzyme was responsible for more than 50% of the overall catalytic activity of D-glucose reduction in rat ovarian cytosol. Western blotting analysis revealed that immunoreactive proteins to anti-ovarian aldose reductase antibody were present in adrenal gland, various reproductive tissues, brain, lung, and heart of rats. Furthermore, ovarian tissues of various species contained immunoreactive proteins, though in small amounts. The enzyme was primarily localized in the granulosa cells and oocytes of all stages of follicular development during the estrous cycle, though it was also found in the corpora lutea cells in the pregnant rats.     

Functional studies of aldo-keto reductases in Saccharomyces cerevisiae

We utilized the budding yeast Saccharomyces cerevisiae as a model to systematically explore physiological roles for yeast and mammalian aldo-keto reductases. Six open reading frames encoding putative aldo-keto reductases were identified when the yeast genome was queried against the sequence for human aldose reductase, the prototypical mammalian aldo-keto reductase. Recombinant proteins produced from five of these yeast open reading frames demonstrated NADPH-dependent reductase activity with a variety of aldehyde and ketone substrates. A triple aldo-keto reductase null mutant strain demonstrated a glucose-dependent heat shock phenotype which could be rescued by ectopic expression of human aldose reductase. Catalytically-inactive mutants of human or yeast aldo-keto reductases failed to effect a rescue of the heat shock phenotype, suggesting that the phenotype results from either an accumulation of one or more unmetabolized aldo-keto reductase substrates or a synthetic deficiency of aldo-keto reductase products generated in response to heat shock stress. These results suggest that multiple aldo-keto reductases fulfill functionally redundant roles in the stress response in yeast.     

Mammalian wax biosynthesis. I. Identification of two fatty acyl-Coenzyme A reductases with different substrate specificities and tissue distributions

The conversion of fatty acids to fatty alcohols is required for the synthesis of wax monoesters and ether lipids. The mammalian enzymes that synthesize fatty alcohols have not been identified. Here, an in silico approach was used to discern two putative reductase enzymes designated FAR1 and FAR2. Expression studies in intact cells showed that FAR1 and FAR2 cDNAs encoded isozymes that reduced fatty acids to fatty alcohols. Fatty acyl-CoA esters were the substrate of FAR1, and the enzyme required NADPH as a cofactor. FAR1 preferred saturated and unsaturated fatty acids of 16 or 18 carbons as substrates, whereas FAR2 preferred saturated fatty acids of 16 or 18 carbons. Confocal light microscopy indicated that FAR1 and FAR2 were localized in the peroxisome. The FAR1 mRNA was detected in many mouse tissues with the highest level found in the preputial gland, a modified sebaceous gland. The FAR2 mRNA was more restricted in distribution and most abundant in the eyelid, which contains wax-laden meibomian glands. Both FAR mRNAs were present in the brain, a tissue rich in ether lipids. The data suggest that fatty alcohol synthesis in mammals is accomplished by two fatty acyl-CoA reductase isozymes that are expressed at high levels in tissues known to synthesize wax monoesters and ether lipids.     

Purification and characterization of aldose reductase and aldehyde reductase from human kidney.

Aldose reductase and aldehyde reductases have been purified to homogeneity from human kidney and have molecular weights of 32,000 and 40,000 and isoelectric pH 5.8 and 5.3, respectively. Aldose reductase, beside catalyzing the reduction of various aldehydes, reduces aldo-sugars, whereas aldehyde reductase, does not reduce aldo-sugars. Aldose reductase activity is expressed with either NADH or NADPH as cofactor, whereas aldehyde reductase utilizes only NADPH. Both enzymes are inhibited to varying degrees by aldose reductase inhibitors. Antibodies against bovine lens aldose reductase precipitated aldose reductase but not aldehyde reductase. The sequence of addition of the substrates to aldehyde reductase is ordered and to aldose reductase is random, whereas for both the enzymes the release of product is ordered with NADP released last.     

四种中草药对大鼠半乳糖性白内障相关酶活性的影响

本实验测定了中草药对大鼠半乳糖性白内障延缓及治疗中五种酶活性的影响。结果表明,在白内障晶状体中,醛糖还原酶的活性明显升高;多元醇脱氢酶,己糖激酶,6磷酸葡萄糖脱氢酶及过氧化氢酶的活性明显降低。在注射半乳糖的同时,分别用黄芩、石斛、菟絲子及玉蝴蝶四种中草药水煎剂灌胃,醛糖还原酶的活性没有明显升高,其余四种酶的活性均基本恢复到正常,表明这四种中草药对半乳糖所致的酶活性异常变化有抑制或纠正作用。