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.     

Regulation of crystalline lens aldose reductase activity. Nonhyperbolic oxidation kinetics of NADPH by glucose

A homogeneous aldose reductase was isolated from bovine eye lens tissue by using affinity chromatography on blue agarose. A kinetic analysis of the initial rates of NADPH oxidation at 0.5-100 mM glucose and at 1.2-10 microM NADPH was carried out. The Line-weaver-Burk plots for glucose concentration were nonlinear at fixed concentrations of NADPH and linear at fixed concentrations of glucose. It was shown that the experimental plots reflect the mechanisms, in which substrate regulation of enzyme activity is effectuated by glucose binding to the regulatory site or is due to the shift of the equilibrium between the isomeric forms of aldose reductase.     

Kinetic and structural effects of activation of bovine kidney aldose reductase

Aldose reductase, purified to homogeneity from bovine kidney, is converted in a temperature-dependent process from a low-Km/low-Vmax form to a high-Km/high-Vmax form of the enzyme. Activation, which results in significant changes in the protein secondary structure, as detected by fluorescence spectroscopy, circular dichroism, and thiol modification with 5,5'-dithiobis(2-nitrobenzoic acid), has no effect on the apparent Mr, pI, or homogeneity of the enzyme, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and agarose isoelectric focusing. Vmax, which varied less than 3-fold for a series of aldehyde substrates with either activation state of the enzyme, increased an average of (17 +/- 4)-fold upon activation of the enzyme. V/Kaldehyde increased or decreased up to 4-fold, depending on the substrate. Activation desensitized the enzyme to inhibition by aldose reductase inhibitors, with the apparent Ki value increasing from 2-fold for Epalrestat [ONO-2235, (E)-3-(carboxymethyl)-(E)-5-[2-methyl-3-phenylpropenylidene]-rhoda nine] to 200-fold for AL-1576 (spiro [2,7-difluorofluorene-9,4'-imidazolidine]-2',5'-dione). Biphasic double-reciprocal plots for the aldehyde substrates and biphasic Dixon plots for inhibition by AL-1576 and Statil [ICI-128,436; 3-[(4-bromo-2-fluorobenzyl)-4-oxo-3H-phthalazin-l-ylacetic acid], observed during the course of activation, are quantitatively accounted for by the individual contributions of the two enzyme forms. On the basis of an analysis of the kinetic data, a mechanism is proposed in which isomerization of the free enzyme limits the rate of the forward reaction for the unactivated enzyme and is the primary step affected by activation.     

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.     

Aldose reductase from human psoas muscle. Affinity labeling of an active site lysine by pyridoxal 5'-phosphate and pyridoxal 5'-diphospho-5'-adenosine

The reaction of aldose reductase from human psoas muscle with either pyridoxal 5'-phosphate (PLP) or pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP) results in a pseudo first-order 2-fold activation of the enzyme with the stoichiometric incorporation of 1 mol of either reagent per mol of enzyme. However, in addition to an increase in Vmax there was also an increase in Km for both substrate, DL-glyceraldehyde, and coenzyme, NADPH. This resulted in an overall decrease in catalytic efficiency (kcat/Km). Spectral analysis indicated that activation by both PLP and PLP-AMP was accompanied by Schiff's base formation and epsilon-pyridoxyllysine was identified in hydrolysates of the reduced enzyme PLP-complex. Digestion of either PLP-modified or PLP-AMP-modified aldose reductase with endoproteinase Lys-C followed by high performance liquid chromatography purification and amino acid sequencing of the pyridoxyllated peptide revealed that PLP and PLP-AMP had modified the same lysine residue. A 32-residue peptide containing the essential lysine was found to be highly homologous with a segment of the sequence of both human liver aldehyde reductase and rat lens aldose reductase. A tetrapeptide (Ile-Pro-Lys-Ser) containing the essential lysine was identical in all three enzymes. These results highlight the close structural similarity between members of the aldehyde reductase family.     

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.     

The crystal structure of the aldose reductase.NADPH binary complex.

Aldose reductase is an NADPH-dependent oxidoreductase that catalyzes the reduction of a broad range of aldehydes, including glucose. Since aldose reductase has been strongly implicated in the development of the chronic complications of diabetes mellitus, much effort has been devoted to understanding the structure and mechanism of this enzyme, and many aldose reductase inhibitors have been developed as potential drugs for the treatment of these complications. We describe here the 2.75 A crystal structure of recombinant human aldose reductase (Cys-298 to Ser mutant) complexed with NADPH. This mutant displays unusual kinetic behavior characterized by high Km/high Vmax substrate kinetics and reduced sensitivity to certain aldose reductase inhibitors. The crystal structure revealed that the enzyme is a beta/alpha-barrel with the coenzyme-binding domain located at the carboxyl-terminal end of the parallel strands of the barrel. The enzyme undergoes a large conformational change upon binding NADPH which involves the reorientation of loop 7 to a position which appears to lock the coenzyme into place. NADPH is bound to aldose reductase in an unusual manner, more similar to FAD- rather than NAD(P)-dependent oxidoreductases. No disulfide bridges were observed in the crystal structure.     

Purification and partial characterization of an aldo-keto reductase from Saccharomyces cerevisiae.

A cytosolic aldo-keto reductase was purified from Saccharomyces cerevisiae ATCC 26602 to homogeneity by affinity chromatography, chromatofocusing, and hydroxylapatite chromatography. The relative molecular weights of the aldo-keto reductase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size exclusion chromatography were 36,800 and 35,000, respectively, indicating that the enzyme is monomeric. Amino acid composition and N-terminal sequence analysis revealed that the enzyme is closely related to the aldose reductases of xylose-fermenting yeasts and mammalian tissues. The enzyme was apparently immunologically unrelated to the aldose reductases of other xylose-fermenting yeasts. The aldo-keto reductase is NADPH specific and catalyzes the reduction of a variety of aldehydes. The best substrate for the enzyme is the aromatic aldehyde p-nitrobenzaldehyde (Km = 46 microM; kcat/Km = 52,100 s-1 M-1), whereas among the aldoses, DL-glyceraldehyde was the preferred substrate (Km = 1.44 mM; kcat/Km = 1,790 s-1 M-1). The enzyme failed to catalyze the reduction of menadione and p-benzoquinone, substrates for carbonyl reductase. The enzyme was inhibited only slightly by 2 mM sodium valproate and was activated by pyridoxal 5'-phosphate. The optimum pH of the enzyme is 5. These data indicate that the S. cerevisiae aldo-keto reductase is a monomeric NADPH-specific reductase with strong similarities to the aldose reductases.     

Regulation of aldose reductase activity. Mechanism of action of an activated form of the enzyme]

Activation of bovine eye lens aldose reductase during its incubation with NADPH and glucose was studied. The activated form of the enzyme was isolated, and the rate of glucose reduction measured within a broad range of substrate concentrations. Spectrophotometric titration and equilibrium gel-filtration were used to study the interaction of the enzyme active center with substrates. It was found that the reaction kinetics obeys the mechanism of a quasi-equilibrium binding of substrates with isomerization of the enzyme complexes with nicotinamide dinucleotide phosphates. This activation is accompanied by a transition from non-ordered to highly ordered binding of the substrates. The effect of ligands in the catalytic and inhibitory centers of the activated enzyme on the catalytic reaction was examined. It was found that the activated form of aldose reductase is characterized by a lower affinity of the inhibitory center for the flavonoid, morin. Morin binding not only inhibits the reaction but also prevents the activation of the enzyme.     

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.     

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 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.     

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.     

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 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.     

Bovine lens aldose reductase: identification of two enzyme forms

Two structurally different forms of bovine lens aldose reductase have been identified. Freshly prepared lens extracts contain an unactivated "b form" (ARb) which is sensitive to inhibition by Sorbinil. Upon incubation of the extracts with oxygen radical generating systems, ARb is converted to a more active "a form" (ARa), which is not inhibited by Sorbinil. ARa and ARb were purified to electrophoretic homogeneity.     

Preparation of homogenous NADPH cytochrome c (P-450) reductase from house flies using affinity chromatography techniques.

NADPH-cytochrome c (P-450) reductase (EC 1.6.2.4) was purified to apparent homogeneity from microsomes of house flies, Musca domestica L. The purification procedure involves column chromatography on three different resins. The key step in the purification scheme is the chromatography of the enzyme mixture on an affinity column of agarose-hexane-nicotinamide adenine dinucleotide phosphate. The enzyme has an estimated molecular weight of 83,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contains 1 mol each of FAD and FMN per mol of enzyme. The enzyme exhibited a Bi Bi ping-pong kinetic mechanism with NADPH and cytochrome c. The Vmax and Km for cytochrome c were 42.3 mumol min-1 mg-1 and 12.7 muM, respectively. Turnover numbers based on micromoles of enzyme were 2,600 min-1. NADP+ and 2'-AMP both inhibited the reductases with apparent Ki values of 6.9 and 187 muM, respectively. These preparations of NADPH-cytochrome c reductase were found to reduce purified house fly cytochrome P-450 in the presence of NADPH.     

Ribonucleotide reductase activity in intact mammalian cells: stimulation of enzyme activity by MgCl2, dithiothreitol, and several nucleotides

An intact cell assay system based on Tween-80 permeabilization was used to investigate ribonucleotide reductase activity in Chinese hamster ovary cells. Dithiothreitol, a reducing agent, is required for optimum activity. Analysis of dithiothreitol stimulation of CDP and ADP reductions indicated that in both cases the reducing agent served only to increase the reaction rate without altering the affinity of the enzyme for substrates. Magnesium chloride significantly stimulated the reduction of CDP but not ADP; this elevation in CDP reduction was due to an increase in both the affinity of the enzyme for substrate and the Vmax. In addition to ATP and dGTP, well-known activators of CDP and ADP reductase activities, it was found that dCTP and GTP were also able to activate CDP and ADP reductase activities, respectively. For the dCTP-activated reaction the Vmax was 0.158 nmol dCDP formed 5 X 10(6) cells-1 h-1 and the Km was 0.033 mM CDP, while for the GTP-activated reduction a Vmax of 0.667 nmol dADP formed 5 X 10(6) cells(-1) h-1 and Km of 0.20 mM ADP were observed. Kinetic analysis revealed that dCTP, dGTP, and GTP stimulate ribonucleotide reduction solely by increasing the affinity of the enzyme for substrate without affecting the Vmax of the respective reactions. ATP behaves in a different manner as it stimulates CDP reduction by altering both the affinity of the enzyme for substrate and the Vmax. Cellular concentrations of ribo- and deoxyribonucleoside di- and triphosphate pools were measured to help evaluate the relative physiological importance of the nucleotide activators. These determinations, along with the reaction kinetic studies, strongly imply that ATP is a much more important regulator of CDP reduction that dCTP, whereas GTP may serve as well or better than dGTP as the in vivo activator of ADP reduction.     

Stable preparation of aldose reductase isoenzymes from human placenta

An efficient, large-scale purification has been achieved for two aldose reductase isoenzymes from human placenta in stable form. The procedure included ammonium sulfate fractionation (45-75%), followed by chromatographies on Matrex Red A, DE-52 cellulose, and Matrex Orange A. The preparations were stable for at least 3 months at 3 degrees C. IC50 values toward sorbinil were similar to those reported for crude or partially purified enzymes, indicating that they retained native structures during the purification steps. The molecular weights of purified GAR1 and GAR2, named according to their order of elution with a salt gradient from a Matrex Red A column, were 36,600 and 40,300, respectively. Kinetic studies indicate that GAR1 belongs to an aldose reductase (a low-Km form) and GAR2 to an aldehyde reductase (a high-Km form). GAR2, an aldehyde reductase, was also active in the reduction of D-glucose, with an apparent Km comparable to that of GAR1 but with a Vmax only 14% that of GAR1.     

Activation of cGMP phosphodiesterase in retinal rods: mechanism of interaction with the GTP-binding protein (transducin)

The mechanism of activation of cGMP phosphodiesterase by the GTP-binding protein in the disc membrane of retinal rods has been investigated by measuring the light-induced phosphodiesterase activity in reconstituted systems where the concentration of either the GTP-binding protein or the phosphodiesterase is varied. The results are consistent with the existence of two activator sites per phosphodiesterase functional unit: binding of one G alpha GTP (alpha subunit of the G-protein with GTP bound) with high affinity (100 +/- 50 nM) partially activates the enzyme (Vmax1 approxmately 0.05 Vmax to 0.10V max to trypsin-activated phosphodiesterase); binding of a second G alpha GTP with lower affinity (600 +/- 100 nM) induces maximal activation (Vmax2 approximately Vmax of trypsin-activated phosphodiesterase). The two different states of activated phosphodiesterase have the same Km for cGMP and the same pH dependence; they differ in their sensitivity to GMP. Micromolar concentration of protamines increases the affinity of the two activator sites and slightly increases Vmax1. When G-protein is activated with GTP-gamma S instead of GTP, the affinities of the two activator sites are not significantly modified, while Vmax1 appears to be increased.