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

doi:10.1016/0003-9861(85)90213-9

Archives of Biochemistry and Biophysics

Volume 238, Issue 2, 1 May 1985, Pages 670-679

https://doi.org/10.1016/0003-9861(85)90213-9 Get rights and content

Abstract

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, M_r 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.

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Among the four different substrates tested, the activity of AR was found to be markedly affected by D(+)-glyceraldehyde substrate. The enzyme has a broad substrate specificity, besides catalyzing the reduction of various aldehydes, it can utilize aldo-sugars such as glucose and galactose (Das and Srivastava, 1985). However, its affinity for glucose is low hence, the enzyme operating at low catalytic rates.
This study aimed, for the first time, to assess the purification of aldose reductase (AR) in Jaculus orientalis (Dipodidae family) kidney and to evaluate the in vitro aldose reductase inhibitory (ARI) effects of Euphorbia regis-jubae (Euphorbiaceae family) aqueous and hydroethanolic extracts. Initial screening assay of the enzymatic AR activity in different jerboa states (euthermic, prehibernating and hibernating) and tissues (brain, brown adipose tissue, liver and kidneys) was assessed. Then, AR has been purified to homogeneity from the kidneys of prehibernating jerboas by a series of chromatographic technics. Furthermore, the in vitro and in silico ARI effects of E. regis-jubae (Webb & Berth) extracts, characterized by hight performance liquid chromatography (HPLC) on the purified enzyme were evaluated. Our results showed that the highest enzyme activity was detected in the kidneys, followed by white adipose tissue and the lungs of pre-hibernating jerboa. The enzyme was purified to homogeneity from jerboa kidneys during prehibernating state with a purification factor of 53.4-fold and a yield of about 6%. AR is monomeric, active in D(+)-glyceraldehyde substrate and in disodium phosphate buffer. The pH and temperature for AR were determined to be 6.5–7.5 and 35 °C, respectively. Results of the in vitro ARI activity was strongest with both the hydroethanolic extract (IC₅₀ = 96.45 μg/mL) and aqueous extract (IC₅₀ = 140 μg/mL). Molecular docking study indicated that catechin might be the main component in both aqueous and hydroethanolic extracts to inhibited AR. This study provides new evidence on the ARI effect of E. regis-jubae (Webb & Berth), which may be related to its phenolic constituents.
Triple aldose reductase/α-glucosidase/radical scavenging high-resolution profiling combined with high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy foridentification of antidiabetic constituents in crude extract of radix scutellariae
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This is however complicated by potential auto-oxidation of NADPH [35,36], which emphasizes the importance of starting the reaction by addition of NADPH (rather than starting the reaction by adding the substrate or enzyme) to obtain the lowest possible degree of auto-oxidation of the coenzyme. Furthermore, it has been reported that rhAR activity is stimulated by lithium sulfate [37,38], and the effect of lithium sulfate in the buffer was therefore investigated (Fig. 2B). A 40% increase in enzyme activity using 0.15 M lithium sulfate (final concentrations) relative to no lithium sulfate was obtained; but no additional increase in activity at higher concentrations.
In this work, development of a new microplate-based high-resolution profiling assay using recombinant human aldose reductase is presented. Used together with high-resolution radical scavenging and high-resolution α-glucosidase assays, it provided the first report of a triple aldose reductase/α-glucosidase/radical scavenging high-resolution inhibition profile – allowing proof of concept with Radix Scutellariae crude extract as a polypharmacological herbal drug. The triple bioactivity high-resolution profiles were used to pinpoint bioactive compounds, and subsequent structure elucidation was performed with hyphenated high-performance liquid chromatography–high-resolution mass spectrometry–solid-phase extraction–nuclear magnetic resonance spectroscopy. The only α-glucosidase inhibitor was baicalein, whereas main aldose reductase inhibitors in the crude extract were baicalein and skullcapflavone II, and main radical scavengers were ganhuangemin, viscidulin III, baicalin, oroxylin A 7-O-glucuronide, wogonoside, baicalein, wogonin, and skullcapflavone II.
Structure-activity relations on [1-(3,5-difluoro-4-hydroxyphenyl)-1H- pyrrol-3-yl]phenylmethanone. the effect of methoxy substitution on aldose reductase inhibitory activity and selectivity
2011, Bioorganic and Medicinal Chemistry
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Sorbitol production was significantly inhibited by compound 5b with efficacy comparable to that of equimolar tetramethylene glutaric acid (TMGA). The latter is a specific inhibitor of ALR2, used also by other authors at the cellular level of isolated red blood cells.28–30 The structure of ALR2 active site has been extensively analyzed by both crystallographic and modeling studies.
Based on our previous work, we studied the effect of methoxy-substitution as well as the regioposition of the benzoyl-moiety of 4a [(1-(3,5-difluoro-4-hydroxyphenyl)-1H-pyrrol-3-yl)(phenyl)methanone]. On this basis, compounds 4b–c and 5a–c were synthesized and assayed for aldose and aldehyde reductase inhibitory activity. Furthermore, a 4,6-difluoro-5-hydroxyphenyl pattern (9) was studied, in order to verify the optimum position of the phenol-moiety. Compound 5b emerged as the most potent and selective inhibitor. Moreover, further assays proved 5b as a potent antioxidant and an inhibitor of sorbitol accumulation in isolated rat lenses. Combining the above attributes, 5b could serve as a lead compound targeted at long-term diabetes complications.
Effects of Stobadine and Vitamin E in Diabetes-Induced Retinal Abnormalities: Involvement of Oxidative Stress
2007, Archives of Medical Research
Citation Excerpt :
In the provided supernatant activity of the enzyme, AR and the quantity of protein was measured. AR was determined spectrophotometrically according to the method of Das and Skrivastava (35) by monitoring the decrease in NADPH absorption at 340 nm at 37°C using dl-glyceraldehyde as substrate. Unspecific NADPH dehydrogenase activity was recorded for 5 min; then dl-glyceraldehyde was added and the incubation was continued for another 5 min.
Because hyperglycemia-induced oxidative stress may be a cause of retinopathy, this study examined the hypothesis that administration of exogenous antioxidants, stobadine (ST) and vitamin E (vitE), can restore retinal abnormalities in experimental diabetes.
Normal and streptozotocin (STZ)-induced male Wistar rats received daily intraoral doses of ST (24.7 mg/kg) and vitE (α-dl-tocopherol acetate, 400–500 IU/kg) individually or in combinations for 8 months. The biochemical parameters including aldose reductase enzyme (AR) activity and lipid peroxidation (MDA), and histopathological changes such as retinal capillary basement membrane thickness (RCBMT) and vascular endothelial growth factor (VEGF) expression were evaluated.
A 37.99% increase in RCBMT was observed in rats after 8 months diabetes duration. The increase in RCBMT was 12.34% in diabetic rats treated with ST and 23.07% in diabetic rats treated with vitE. In diabetic rats treated with antioxidant combination, just a 4.38% increase was observed in RCBMT. The excess VEGF immunoreactivity and increased MDA and AR activity determined in diabetic retina were significantly attenuated by individual antioxidant treatments. Although both antioxidants decreased blood glucose, HbA1c, fructosamine and triglyceride levels in diabetic rats, poor glycemic control was maintained in all experimental groups during the treatment period. However, the antioxidant combination led to almost complete amelioration in retinal MDA and RCBMT in diabetic rats.
The ability of antioxidant combination to arrest retinal abnormalities and lipid peroxidation even in the presence of poor glycemic control might advocate the key role of direct oxidative damage and the protective action of antioxidants in retinal alterations associated with diabetic retinopathy.
Aldehyde reductase
2007, xPharm: The Comprehensive Pharmacology Reference
Aldehyde reductase, also known as aldose reductase, is an NAD(P)H-dependant enzyme that catalyses the reduction of an aldose to the corresponding sugar alcohol, in particular, D-glucose to sorbitol. Under normal conditions, the production of sorbitol constitutes in some tissues a significant fraction of the cellular osmolite.
In a further enzymatic step, sorbitol can be metabolized to fructose by the NAD-specific sorbitol dehydrogenase. The conversion of glucose to fructose by this means constitutes the polyol pathway of glucose metabolism, which plays a minor role in glucose metabolism in most tissues.
Aldehyde reductase is thought to be involved in the pathogenesis of neuropathy, retinopathy, nephropathy, and cataracts in diabetes …
Water extract of Aralia elata prevents cataractogenesis in vitro and in vivo
2005, Journal of Ethnopharmacology
The water extract of Aralia elata (Aralia extract) has been used in Korean traditional medicine to treat diabetes mellitus. Here, we investigated the aldose reductase inhibitory activity, antioxidant activity and anticataract capacity of Aralia extract using various experimental systems. To determine its aldose reductase inhibitory activity and its antioxidant effect, we used rat lens homogenates. Rat lens cultures and a rat model were used to observe anticataract activity. The resulting IC₅₀ value of Aralia extract in vitro as an aldose reductase inhibitor was 11.3 μg/ml and as an antioxidant was 25.1 μg/ml. Rat lenses in media containing 20 mM xylose developed a distinctly opaque ring in 9 h, and treatment with Aralia extract at a concentration of 1 mg/ml lowered lens opacity by 36.4 and 31.3% after 24 and 48 h, respectively. In vivo experiments were performed with streptozotocin (STZ) induced diabetic rats. The diabetic control animals developed cataracts at 11 weeks after STZ injection, while oral Aralia extract administered at 300 and 600 mg/kg body weight for 11 weeks reduced cataract formation by 15 and 12%, respectively.
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Purification and properties of aldose reductase and aldehyde reductase II from human erythrocyte

Abstract

Biochem. Pharmacol

Biochim. Biophys. Acta

Exp. Eye Res

Biochem. Biophys. Res. Commun

Biochim. Biophys. Acta

Anal. Biochem

Anal. Biochem

Anal. Biochem

J. Biol. Chem

Biochim. Biophys. Acta

Biochim. Biophys. Acta

J. Biol. Chem

J. Biol. Chem

Metabolism

Science (Washington, D. C.)