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.     

Structural and functional properties of aldose xylose reductase from the D-xylose-metabolizing yeast Candida tenuis

The primary structure of the aldose xylose reductase from Candida tenuis (CtAR) is shown to be 39% identical to that of human aldose reductase (hAR). The catalytic tetrad of hAR is completely conserved in CtAR (Tyr51, Lys80, Asp46, His113). The amino acid residues involved in binding of NADPH by hAR (D.K. Wilson, et al., Science 257 (1992) 81-84) are 64% identical in CtAR. Like hAR the yeast enzyme is specific for transferring the 4-pro-R hydrogen of the coenzyme. These properties suggest that CtAR is a member of the aldo/keto reductase superfamily. Unlike hAR the enzyme from C. tenuis has a dual coenzyme specificity and shows similar specificity constants for NADPH and NADH. It binds NADP(+) approximately 250 times less tightly than hAR. Typical turnover numbers for aldehyde reduction by CtAR (15-20 s(-1)) are up to 100-fold higher than corresponding values for hAR, probably reflecting an overall faster dissociation of NAD(P)(+) in the reaction catalyzed by the yeast enzyme.     

Crystal structure of CHO reductase, a member of the aldo-keto reductase superfamily

Chinese hamster ovary (CHO) reductase is an enzyme belonging to the aldo-keto reductase (AKR) superfamily that is induced by the aldehyde-containing protease inhibitor ALLN (Inoue, Sharma, Schimke, et al., J Biol Chem 1993;268: 5894). It shows 70% sequence identity to human aldose reductase (Hyndman, Takenoshita, Vera, et al., J Biol Chem 1997;272:13286), which is a target for drug design because of its implication in diabetic complications. We have determined the crystal structure of CHO reductase complexed with nicotinamide adenine dinucleotide phosphate (NADP)+ to 2.4 A resolution. Similar to aldose reductase and other AKRs, CHO reductase is an alpha/beta TIM barrel enzyme with cofactor bound in an extended conformation. All key residues involved in cofactor binding are conserved with respect to other AKR members. CHO reductase shows a high degree of sequence identity (91%) with another AKR member, FR-1 (mouse fibroblast growth factor-regulated protein), especially around the variable C-terminal end of the protein and has a similar substrate binding pocket that is larger than that of aldose reductase. However, there are distinct differences that can account for differences in substrate specificity. Trp111, which lies horizontal to the substrate pocket in all other AKR members is perpendicular in CHO reductase and is accompanied by movement of Leu300. This coupled with movement of loops A, B, and C away from the active site region accounts for the ability of CHO reductase to bind larger substrates. The position of Trp219 is significantly altered with respect to aldose reductase and appears to release Cys298 from steric constraints. These studies show that AKRs such as CHO reductase are excellent models for examining the effects of subtle changes in amino acid sequence and alignment on binding and catalysis.     

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。     

Aldose reductase from human skeletal and heart muscle. Interconvertible forms related by thiol-disulfide exchange

Aldose reductase was purified from human skeletal and heart muscle by a rapid and efficient scheme involving Red Sepharose chromatography, chromatofocusing on Pharmacia PBE 94, and hydroxylapatite high pressure liquid chromatography. The scheme afforded homogeneous enzyme, 65% recovery, in 2 days. All muscle samples express aldose reductase but not the closely related aldehyde reductase. Aldose reductase is isolated in one of two forms that are distinguishable by their kinetic patterns with glyceraldehyde as substrate and which are interconvertible by treatment with dithiothreitol. Both forms are capable of catalyzing the reduction of glucose (Km = 68 mM), and both are highly sensitive to inhibition by aldose reductase inhibitors. The reduction of glucose was shown to be nearly stoichiometric with production of sorbitol (92 +/- 2%). Dialysis of aldose reductase in the absence of thiols or NADP converts it into a form that shows markedly different kinetic properties, including very weak catalytic activity toward glucose and insensitivity to aldose reductase inhibitors. This modified form can be converted back into the native form by dithiothreitol. Thiol titration of the two forms of aldose reductase with Ellman's reagent indicated that two thiol groups were lost when the enzyme was dialyzed in the absence of dithiothreitol or NADP.     

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.     

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 characterization of a novel erythrose reductase from Candida magnoliae

Erythritol biosynthesis is catalyzed by erythrose reductase, which converts erythrose to erythritol. Erythrose reductase, however, has never been characterized in terms of amino acid sequence and kinetics. In this study, NAD(P)H-dependent erythrose reductase was purified to homogeneity from Candida magnoliae KFCC 11023 by ion exchange, gel filtration, affinity chromatography, and preparative electrophoresis. The molecular weights of erythrose reductase determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography were 38,800 and 79,000, respectively, suggesting that the enzyme is homodimeric. Partial amino acid sequence analysis indicates that the enzyme is closely related to other yeast aldose reductases. C. magnoliae erythrose reductase catalyzes the reduction of various aldehydes. Among aldoses, erythrose was the preferred substrate (K(m) = 7.9 mM; k(cat)/K(m) = 0.73 mM(-1) s(-1)). This enzyme had a dual coenzyme specificity with greater catalytic efficiency with NADH (k(cat)/K(m) = 450 mM(-1) s(-1)) than with NADPH (k(cat)/K(m) = 5.5 mM(-1) s(-1)), unlike previously characterized aldose reductases, and is specific for transferring the 4-pro-R hydrogen of NADH, which is typical of members of the aldo/keto reductase superfamily. Initial velocity and product inhibition studies are consistent with the hypothesis that the reduction proceeds via a sequential ordered mechanism. The enzyme required sulfhydryl compounds for optimal activity and was strongly inhibited by Cu(2+) and quercetin, a strong aldose reductase inhibitor, but was not inhibited by aldehyde reductase inhibitors and did not catalyze the reduction of the substrates for carbonyl reductase. These data indicate that the C. magnoliae erythrose reductase is an NAD(P)H-dependent homodimeric aldose reductase with an unusual dual coenzyme specificity.     

Immunohistochemical distribution of aldose reductase

Summary Aldose reductase (AR) has been purified from canine kidneys., and a monospecific antibody against the enzyme prepared. These antibodies were used in an immunohistochemical test to detect tissue sites of aldose reductase in the dog, a species known to develop diabetic lesions morphologically identical to those seen in diabetic patients. Using this method, the enzyme has been demonstrated in numerous cell types, including lens, epithelium, aortic endothelium and smooth muscle, Schwann cells of peripheral nerves, and, in the kidney, interstitial cells and cells of Henlés loop and the collecting tubules. Many other cells and tissues, including capillaries throughout the body, lack immunoreactive aldose reductase. The distribution of the immunoreactive enzyme is compatible with a potential role of the enzyme in the aetiology of some complications of diabetes, namely cataract, neuropathy, macroangiopathy and renal papillary necrosis, but not the microvascular complications.     

Characterization and purification of a mammalian osmoregulatory protein, aldose reductase, induced in renal medullary cells by high extracellular NaCl

GRB-PAP1 is a continuous line of epithelial cells derived from a rabbit renal inner medulla. Elevation of the NaCl concentration in the medium bathing these cells strongly induced the expression of a soluble protein with an apparent molecular mass of 39 kDa. The protein, purified by affinity chromatography with Amicon Matrex Gel Orange A, had enzyme activity characteristic of aldose reductase (alditol:NADPH+ oxidoreductase, EC 1.1.1.21). Goat antiserum against this purified aldose reductase selected the 39-kDa band from immunoblots of cells grown in a medium containing high NaCl. When the osmolality of the medium was increased by adding NaCl, the amount of aldose reductase protein and the aldose reductase activity increased together from very low to sustained high levels over several days. The aldose reductase protein was more than 10% of the soluble cell protein when cells were propagated in medium made hyperosmotic by adding NaCl to increase medium osmolality to 600 mosm.kg-1.     

Pig brain aldose reductase: purification, significance of the amino acid composition, substrate specificity and mechanism

1. Pig brain aldose reductase (ALR2, EC 1.1.1.21) has been purified from fresh tissue with a approximately 60% improvement in specific activity over an acetone-powder preparation. 2. Dead-end inhibition and alternate substrate studies rule out an iso Theorell-Chance mechanism but are compatible with an ordered bi bi mechanism where NADPH and NADP+ function as the outside reactants in the direction of xylitol formation. 3. Subtle but significant differences are shown to exist in the distribution of apolar and mixed amino acid residues between aldose and aldehyde reductases when the mean fractional area loss [Rose et al., 1985] is used as the measure of compositional relatedness.     

Effects of conversion of phenylalanine-31 to leucine on the function of human dihydrofolate reductase

Oligonucleotide-directed, site-specific mutagenesis was used to convert phenylalanine-31 of human recombinant dihydrofolate reductase (DHFR) to leucine. This substitution was of interest in view of earlier chemical modification studies (Kumar et al., 1981) and structural studies based on X-ray crystallographic data (Matthews et al., 1985a,b) which had implicated the corresponding residue in chicken liver DHFR, Tyr-31, in the binding of dihydrofolate. Furthermore, this particular substitution allowed testing of the significance of protein sequence differences between mammalian and bacterial reductases at this position with regard to the species selectivity of trimethoprim. Both wild-type (WT) and mutant (F31L) enzymes were expressed and purified by using a heterologous expression system previously described (Prendergast et al., 1988). Values of the inhibition constants (Ki values) for trimethoprim were 1.00 and 1.08 microM for WT and F31L, respectively. Thus, the presence of phenylalanine at position 31 in human dihydrofolate reductase does not contribute to the species selectivity of trimethoprim. The Km values for nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) and dihydrofolate were elevated 10.8-fold and 9.4-fold, respectively, for the mutant enzyme, whereas the Vmax increased only 1.8-fold. Equilibrium dissociation constants (KD values) were obtained for the binding of NADPH and dihydrofolate in binary complexes with each enzyme. The KD for NADPH is similar in both WT and F31L, whereas the KD for dihydrofolate is 43-fold lower in F31L. Values for dihydrofolate association rate constants (kon) with enzyme and enzyme-NADPH complexes were measured by stopped-flow techniques.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and identification of an Escherichia coli beta-keto ester reductase as 2,5-diketo-D-gluconate reductase YqhE

An NADPH-dependent enzyme that reduces ethyl 2-methylacetoacetate stereoselectively to ethyl (2R)-methyl-(3S)-hydroxybutanoate was purified 730-fold from Escherichia coli. The N-terminal amino acid sequence data obtained from the purified reductase were used to search the E. coli genome, and a single match was found at the start of the yqhE open reading frame. The YqhE protein had been identified previously by Yum et al. as a 2,5-diketo-D-gluconate reductase on the basis of sequence similarity to other bacterial homologues [Yum, D.-Y.; Lee, B.-Y.; Pan, J.-G. Appl.Environ. Microbiol. 1999, 65, 3341-3346]; however, it had not been examined for beta-keto ester reductions. Our results thus link a key enzyme in the microbial production of ascorbate with stereoselective beta-keto ester reductions, two important fields in biocatalysis. The purified YqhE reductase accepts ethyl acetoacetate and a variety of 2-substituted derivatives, and its sequence is similar to other aldose reductase superfamily members that also reduce alpha-substituted beta-keto esters to syn-(2R,3S) alcohols.     

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.     

A vertebrate aldo-keto reductase active with retinoids and ethanol

Enzymes of the short chain and medium chain dehydrogenase/reductase families have been demonstrated to participate in the oxidoreduction of ethanol and retinoids. Mammals and amphibians contain, in the upper digestive tract mucosa, alcohol dehydrogenases of the medium chain dehydrogenase/reductase family, active with ethanol and retinol. In the present work, we searched for a similar enzyme in an avian species (Gallus domesticus). We found that chicken does not contain the homologous enzyme from the medium chain dehydrogenase/reductase family but an oxidoreductase from the aldo-keto reductase family, with retinal reductase and alcohol dehydrogenase activities. The amino acid sequence shows 66-69% residue identity with the aldose reductase and aldose reductase-like enzymes. Chicken aldo-keto reductase is a monomer of M(r) 36,000 expressed in eye, tongue, and esophagus. The enzyme can oxidize aliphatic alcohols, such as ethanol, and it is very efficient in all-trans- and 9-cis-retinal reduction (k(cat)/K(m) = 5,300 and 32,000 mm(-1).min(-1), respectively). This finding represents the inclusion of the aldo-keto reductase family, with the (alpha/beta)(8) barrel structure, into the scenario of retinoid metabolism and, therefore, of the regulation of vertebrate development and tissue differentiation.     

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.     

Parvalbumin isoforms in chicken muscle and thymus. Amino acid sequence analysis of muscle parvalbumin by tandem mass spectrometry

Parvalbumins are high-affinity Ca(2+)-binding proteins characterized by an EF-hand structure. Muscles of lower vertebrates contain up to five isoparvalbumins whereas higher vertebrates were believed to contain only one isoform per species. Recently Brewer et al. [Brewer, J.M., Wunderlich, J.K., & Ragland, W. (1990) Biochimie 72, 653-660] purified and sequenced a protein that they named avian thymic hormone, from chicken thymus. This protein, promoting immunological maturation of bone marrow cells in culture, was identified as a parvalbumin. The amino acid composition of this thymic parvalbumin was, however, considerably different from those of chicken muscle parvalbumin [Strehler, E.E., Eppenberger, H.M., & Heizman, C.W. (1977) FEBS Lett. 75, 127-133], suggesting the existence of two tissue-specific parvalbumins in chicken. We purified parvalbumin from chicken muscle, determined its complete amino acid sequence by tandem mass spectrometry, and showed that this protein is rather homologous to muscle parvalbumins from other species but different in 45 positions from the thymic parvalbumin. We discuss the possibility that a parvalbumin gene family might exist in higher vertebrates, expressed in a tissue-specific and developmentally regulated manner.     

Properties of the NAD(P)H-dependent xylose reductase from the xylose-fermenting yeast Pichia stipitis.

Xylose reductase from the xylose-fermenting yeast Pichia stipitis was purified to electrophoretic and spectral homogeneity via ion-exchange, affinity and high-performance gel chromatography. The enzyme was active with various aldose substrates, such as DL-glyceraldehyde, L-arabinose, D-xylose, D-ribose, D-galactose and D-glucose. Hence the xylose reductase of Pichia stipitis is an aldose reductase (EC 1.1.1.21). Unlike all aldose reductases characterized so far, the enzyme from this yeast was active with both NADPH and NADH as coenzyme. The activity with NADH was approx. 70% of that with NADPH for the various aldose substrates. NADP+ was a potent inhibitor of both the NADPH- and NADH-linked xylose reduction, whereas NAD+ showed strong inhibition only with the NADH-linked reaction. These results are discussed in the context of the possible use of Pichia stipitis and similar yeasts for the anaerobic conversion of xylose into ethanol.     

Change in stereospecificity of bovine lens aldose reductase modified by oxidative stress

Bovine lens aldose reductase (alditol:NADP+ oxidoreductase, EC 1.1.1.21) undergoes an oxidative modification, greatly stimulated by high ionic strength, upon incubation in the presence of oxygen radical generating systems (Del Corso, A., Camici, M., and Mura, U. (1987) Biochem. Biophys. Res. Commun. 148, 369-375). The enzyme modification is accompanied by a change in stereospecificity toward the two enantiomers of glyceraldehyde. In particular, the Km for L-glyceraldehyde of the native form increased over 150 times after the enzyme modification, with a decrease in the catalytic efficiency of over 200 times. By contrast, for the D-enantiomer the Km increased only 7 times with respect to the native form, with a concomitant decrease in the catalytic efficiency of only approximately 3 times. This dramatic change in stereospecificity may account for the reported apparent cooperative behavior exhibited also by highly purified electrophoretically homogeneous preparations of aldose reductase.     

Aldose and aldehyde reductases in human tissues

Immunochemical characterizations of aldose reductase and aldehyde reductases I and II, partially purified by DEAE-cellulose (DE-52) column chromatography from human tissues, were carried out by immunotitration, using antisera raised against the homogenous preparations of human and bovine lens aldose reductase and human placenta aldehyde reductase I and aldehyde reductase II. Anti-aldose reductase antiserum cross-reacted with aldehyde reductase I, anti-aldehyde reductase I antiserum cross-reacted with aldose reductase and anti-aldehyde reductase II antiserum precipitated aldehyde reductase II, but did not cross-react with aldose reductase or aldehyde reductase I from all the tissues examined. DE-52 elution profiles, substrate specificity and immunochemical characterization indicate that aldose reductase is present in human aorta, brain, erythrocyte and muscle; aldehyde reductase I is present in human kidney, liver and placenta; and aldehyde reductase II is present in human brain, erythrocyte, kidney, liver, lung and placenta. Monospecific anti-α and anti-β antisera were purified from placenta anti-aldehyde reductase I antiserum, using immunoaffinity techniques. Anti-α antiserum precipitated both aldehyde reductase I and aldose reductase, whereas anti-β antibodies cross-reacted with only aldehyde reductase I. Based on these studies, a three gene loci model is proposed to explain the genetic interrelationships among these enzymes. Aldose reductase is a monomer of α subunits, aldehyde reductase I is a dimer of α and β subunits and aldehyde reductase II is a monomer of δ subunits.