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.     

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.     

Characterization of aldose reductase and aldehyde reductase from rat testis

Aldose reductase (alditol:NAD(P)+ 1-oxidoreductase, EC 1.1.1.21) and aldehyde reductase (alcohol:NADP+ oxidoreductase, EC 1.1.1.2) were purified to a homogeneity from rat testis. The molecular weights of aldose reductase and aldehyde reductase were estimated to be 38,000 and 41,000 by SDS-polyacrylamide gel electrophoresis, and the pI values of these enzymes were found to be 5.3 and 6.1 by chromatofocusing, respectively. Aldose reductase had activity for aldo-sugars such as xylose, glucose and galactose, whereas aldehyde reductase was virtually inactive for these aldo-sugars. The Km values of aldose reductase for aldo-sugars were relatively high. When a correction was made for the fraction of aldo-sugar present as the aldehyde form, which is the real substrate of the enzyme, the Km values were much lower. Aldose reductase utilized both NADPH and NADH as coenzyme, whereas aldehyde reductase utilized only NADPH. Aldose reductase was activated significantly by sulfate ion, while aldehyde reductase was little affected. Both enzymes were inhibited strongly by the known aldose reductase inhibitors. However, aldehyde reductase was in general less susceptible to these inhibitors when compared to aldose reductase. Both aldose reductase and aldehyde reductase treated with pyridoxal 5-phosphate have lost the susceptibility to aldose reductase inhibitor, suggesting that in these two enzymes aldose reductase inhibitor interacts with a lysine residue.     

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.     

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.     

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.     

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.     

Chromatographic separation of activated and unactivated forms of aldose reductase

Chromatography of bovine kidney aldose reductase using Matrex Orange A affinity gel results in the separation of the unactivated and activated enzyme forms. The former washes through the column, while the latter is eluted with an NADPH step-gradient. The separated enzyme forms display Vmax and Km glycolaldehyde values, and relative sensitivities to inhibition by the aldose reductase inhibitor AL-1576 (spiro[2,7-difluorofluorene-9,4'-imidazolidine]-2',5'- dione), that are similar to those reported previously for the individual forms. However, because Vmax is 17-fold lower for the unactivated enzyme, the purification of aldose reductase via NADP(H) elution from a dye-ligand affinity matrix can result in the selective purification of only the activated enzyme form. These results have direct implications for the study of potential aldose reductase inhibitors, and may explain why linear double-reciprocal plots are commonly observed for enzyme prepared in this manner, while nonlinear plots are seen in other cases.     

Interrelationships among human aldo-keto reductases: immunochemical, kinetic and structural properties

We have proposed earlier a three gene loci model to explain the expression of the aldo-keto reductases in human tissues. According to this model, aldose reductase is a monomer of alpha subunits, aldehyde reductase I is a dimer of alpha, beta subunits, and aldehyde reductase II is a monomer of delta subunits. Using immunoaffinity methods, we have isolated the subunits of aldehyde reductase I (alpha and beta) and characterized them by immunocompetition studies. It is observed that the two subunits of aldehyde reductase I are weakly held together in the holoenzyme and can be dissociated under high ionic conditions. Aldose reductase (alpha subunits) was generated from human placenta and liver aldehyde reductase I by ammonium sulfate (80% saturation). The kinetic, structural and immunological properties of the generated aldose reductase are similar to the aldose reductase obtained from the human erythrocytes and bovine lens. The main characteristic of the generated enzyme is the requirement of Li2SO4 (0.4 M) for the expression of maximum enzyme activity, and its Km for glucose is less than 50 mM, whereas the parent enzyme, aldehyde reductase I, is completely inhibited by 0.4 M Li2SO4 and its Km for glucose is more than 200 mM. The beta subunits of aldehyde reductase I did not have enzyme activity but cross-reacted with anti-aldehyde reductase I antiserum. The beta subunits hybridized with the alpha subunits of placenta aldehyde reductase I, and aldose reductase purified from human brain and bovine lens. The hybridized enzyme had the characteristic properties of placenta aldehyde reductase I.     

Interrelationships among human aldo-keto reductase: immunochemical, kinetic and structural properties

We have propsed earlier a three gene loci model to explain the expression of the aldo-keto reductases in human tissues. According to this model, aldose reductase is a monomer of α subunits, aldehyde reductase I is a dimer of α, β subunits, and aldehyde reductase II is a monomer of δ subunits. Using immunoaffinity methods, we have isolated the subunits of aldehyde reductase I (α and β) and characterized them by immunocompetition studies. It is observed that the two subunits of aldehyde reductase I are weakly held together in the holoenzyme and can be dissociated under high ionic conditions. Aldose reductase (α subunits) was generated from human placenta and liver aldehyde reductase I by ammonium sulfate (80% saturation). The kinetic, structural and immunological properties of the generated aldose reductase are similar to the aldose reductase obtained from the human erythrocytes and bovine lens. The main characteristic of the generated enzyme is the requirement of Li₂SO₄(0.4 M) for the expression of maximum enzyme activity, and its K_m for glucose is less than 50 mM, whereas the parent enzyme, aldehyde reductase I, is completely inhibited by 0.4 M Li₂SO₄ and its K_m for glucose is more than 200 mM. The β subunits of aldehyde reductase I did not have enzyme activity but cross-reacted with anti-aldehyde reductase I antiserum. The β subunits hybridized with the α subunits of placenta aldehyde I, and aldose reductase purified from human brain and bovine lens. The hybridized enzyme had the characteristics properties of placenta aldehyde reductase I.     

Expression of human aldose and aldehyde reductases. Site-directed mutagenesis of a critical lysine 262.

Human aldose reductase (EC 1.1.1.21) and aldehyde reductase (EC 1.1.1.2) are implicated in the development of diabetic complications by a variety of mechanisms, and a number of drugs to inhibit these enzymes have been proposed for the therapy and prevention of these complications. To probe the structure and function of these two enzymes, we used site-directed mutagenesis in the cDNAs of both enzymes to replace lysine 262 with methionine. Wild-type and mutant enzymes were overexpressed in Escherichia coli and purified by anion exchange and affinity chromatography. N-terminal sequence analysis, Western blots, and kinetic studies confirmed the identity of the recombinant wild-type enzymes with the native human placental and liver enzymes. Recombinant aldose reductase (hAR) and aldehyde reductase (hGR) have apparent kinetic constants virtually identical to their respective native enzymes. The mutant aldose reductase (hARK262 greater than M) shows a 66-fold increase in Km for NADPH with respect to the wild type (1.9 +/- 0.4 microM versus 125 +/- 14 microM), whereas the Km for DL-glyceraldehyde increased 35-fold (20 +/- 2 versus 693 +/- 41 microM). The same constants for the mutant aldehyde reductase (hGRK262 greater than M) increased 97- and 86-fold, respectively (from 2.0 +/- 0.4 to 194 +/- 16 microM and from 1.6 +/- 0.4 to 137 +/- 3 mM). These results indicate that lysine 262 in aldose reductase and aldehyde reductase is crucial to their catalytic activity by affecting co-factor binding.     

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.     

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.     

Spectral properties of human placental aldose reductase and aldehyde reductase II

Circular dichroism and fluorescence spectra of aldose reductase (E.C.1.1.1.21) and aldehyde reductase II (E.C.1.1.1.19) purified to homogeneity from human placenta have been studied. The alpha helical content of aldose reductase and aldehyde reductase II was 51% and 56%, respectively, whereas no beta helical structure was found in either case. In the case of aldose reductase, the secondary structure was unaffected at alkaline pH (9.5), whereas a drastic alteration in the structure was observed at 58 degrees C. The secondary structure of aldehyde reductase II, on the other hand, remained unaffected at higher pH and temperature.     

Enzymes in pancreatic islets that use NADP(H) as a cofactor including evidence for a plasma membrane aldehyde reductase.

Recent evidence of a pyruvate malate shuttle capable of transporting a large amount of NADPH equivalents out of mitochondria in pancreatic islets suggests that cytosolic NADP(H) plays a role in beta cell metabolism. To obtain clues about these processes the activities of several NADPH-utilizing enzymes were estimated in pancreatic islets. Low levels of pyrroquinolone quinone (PQQ) and low levels of enzyme activity that reduce PQQ were found in islets. Low activities of palmitoyl-CoA and stearoyl-CoA desaturases were also detected. Significant activities of glutathione reductase, aldose reductase (EC.1.1.1.21) and aldehyde reductase (EC.1.1.1.2) were present in islets. Potent inhibitors of aldehyde and aldose reductases inhibited neither glucose-induced insulin release nor glucose metabolism in islets indicating that these reductases are not directly involved in glucose-induced insulin reaction. Over 90% of aldose reductase plus aldehyde reductase enzyme activity was present in the cytosol. Kinetic and chromatographic studies indicated that 60-70% of this activity in cytosol was due to aldehyde reductase and the remainder due to aldose reductase. Aldehyde reductase-like enzyme activity, as well as aldose reductase immunoreactivity, was detected in rat islet plasma membrane fractions purified by a polyethylene glycol-Dextran gradient or by a sucrose gradient. This is interesting in view of the fact that voltage-gated potassium channel beta subunits that contain aldehyde and aldose reductase-like NADPH-binding motifs have been detected in plasma membrane fractions of islets [Receptors and Channels 7: 237-243, 2000] and suggests that NADPH might have a yet unknown function in regulating activity of these potassium channels. Reductases may be present in cytosol to protect the insulin cell from molecules that cause oxidative injury.     

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.     

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.     

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.     

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.     

Purification by affinity chromatography of 2,4-dienoyl-CoA reductases from bovine liver and Escherichia coli

1. Dye-ligand chromatography using immobilized Cibacron blue F3GA (blue Sepharose CL-6B) and Procion red HE3B (Matrex gel red A) as matrices and general ligand chromatography employing immobilized 2',5'-ADP (2',5'-ADP-Sepharose 4B) and immobilized 3',5'-ADP (3',5'-ADP-Agarose) were employed for purification of NADPH-dependent 2-enoyl-CoA reductase and 2,4-dienoyl-CoA reductase from bovine liver (formerly called 4-enoyl-CoA reductase [Kunau, W. H. and Dommes, P. (1978) Eur. J. Biochem. 91, 533-544], as well as 2,4-dienoyl-CoA reductase from Escherichia coli. 2. The NADPH-dependent 2-enoyl-CoA reductase from bovine liver mitochondria was separated from 2,4-dienoyl-CoA reductase by dye-ligand chromatography (Matrex gel red A/KCl gradient) as well as by general ligand affinity chromatography (2',5'-ADP-Sepharose 4B/NADP gradient). The enzyme was obtained in a highly purified form. 3. The NADPH-dependent 2,4-dienoyl-CoA reductase from bovine liver mitochondria was purified to homogeneity using blue Sepharose CL-6B, Matrex gel red A, and 2',5'-ADP-Sepharose 4B chromatography. 4. The bacterial 2,4-dienoyl-CoA reductase was completely purified by ion-exchange chromatography on DEAE-cellulose followed by a single affinity chromatography step employing 2',5'-ADP-Sepharose 4B and biospecific elution from the column with a substrate, trans,trans-2,4-decadienoyl-CoA. 5. The application of dye-ligand and general ligand affinity chromatography for purification of NADPH-dependent 2,4-dienoyl-CoA reductases taking part in the beta-oxidation of unsaturated fatty acids is discussed. It is concluded that making use of coenzyme specificity for binding and substrate specificity for elution is essential for obtaining homogeneous enzyme preparations.