Ballabh Das

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 pHs 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.

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 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-alpha and anti-beta antisera were purified from placenta anti-aldehyde reductase I antiserum, using immunoaffinity techniques. Anti-alpha antiserum precipitated both aldehyde reductase I and aldose reductase, whereas anti-beta 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 alpha subunits, aldehyde reductase I is a dimer of alpha and beta subunits and aldehyde reductase II is a monomer of delta subunits.

Activation of Aldose Reductase from Human Tissues

Human aorta, brain, and muscle aldose reductase, partially purified by DEAE-cellulose (DE-52) column chromatography, is activated 2–2.5-fold on incubation with 10 μM each of glucose-6-phosphate, NADPH, and glucose for 20 min at 25°C. The activation of the enzyme was established by following the NADPH oxidation as well as the sorbitol formation using glucose as substrate. The activated form of aldose reductase exhibited monophasic kinetics with glucose and glyceraldehyde, whereas the unactivated or native enzyme exhibited a biphasic kinetics with both the substrates. The activated enzyme was less susceptible to inhibition by aldose reductase inhibitors such as sorbinil, alrestatin, and quercetrin as compared with the unactivated enzyme. Similarly, the native enzyme was strongly inhibited by some of the phosphorylated intermediates of glycolytic pathway, such as 3-phosphoglycerate, 1,3-diphosphoglycerate, 2,3-diphosphoglycerate, and ADP,whereas the activated enzyme was either not inhibited or inhibition was 20–30% only. Partially purified aldose reductase from the normal human lens exhibited properties similar to the native enzyme of other tissues, whereas the enzyme from clear lens obtained from diabetic subjects with severe hyperglycemia expressed properties similar to the in vitro activated enzyme of aorta, brain, and muscle.

Hyperglycemia-induced activation of human erythrocyte aldose reductase and alterations in kinetic properties

Incubation of human erythrocytes with varying concentrations of glucose resulted in a several-fold increase in aldose reductase (alditol:NADP+ 1-oxidoreductase, EC 1.1.1.21) activity as determined by the rate of NADPH oxidation and the rate of sorbitol formation. As compared to aldose reductase from human erythrocytes not incubated with glucose (native enzyme), aldose reductase from 30 mM glucose-incubated erythrocytes (activated enzyme) exhibited altered kinetic and inhibition properties. Native enzyme showed biphasic kinetics with substrates (glucose and glyceraldehyde), was strongly inhibited by 15 microM ADP, 1,3-diphosphoglycerate, 2,3-diphosphoglycerate and 3-phosphoglycerate, and aldose reductase inhibitors such as sorbinil and alrestatin. The activated enzyme, on the other hand, exhibited monophasic kinetics, low Km for substrates, was not inhibited by the phosphorylated intermediates, and was less susceptible to inhibition by aldose reductase inhibitors. In erythrocytes of the diabetic subjects, we have found an excellent correlation between aldose reductase activity and plasma glucose levels and have observed that whenever the blood glucose level was higher than 15 mM, all of the erythrocyte aldose reductase was present in the activated form and exhibited properties similar to those observed with aldose reductase obtained from 30 mM glucose-incubated erythrocytes.

The effect of oxidants on biomembranes and cellular metabolism

During the reductive process in the tissues, the aerobes generate a number of oxidants. Unless these oxidants are reduced, oxidative damage and cell death would occur. Oxidation of plasma membrane lipids leads to autocatalytic chain reactions which eventually alter the permeability of the cell. The role of oxidative damage in the pathophysiology of diabetic complications and ischemic reperfusion injury of myocardium, especially the changes in the channel activity which may lead to arrhythmia have been studied. Hyperglycemia activates aldose reductase which could efficiently reduce glucose to sorbitol in the presence of NADPH. Since NADPH is also aldose required by glutathione reductase for reducing oxidants, its diversion would lead to membrane lipid oxidation and permeability changes which are probably responsible for diabetic complications such as cataractogenesis, retinopathy, neuropathy etc. Antioxidants such as butylated hydroxy toluene (BHT) and also reductase inhibitors prevent or delay some of these complications. By using patch-clamp technique in isolated frog myocytes, we have shown that hydroxy radicals generated by ferrous sulfate and ascorbate as well as lipid peroxides such as t-butyl hydroperoxide facilitate the entry of Na+ by oxidizing Na+-channels. Increased intracellular Na+ leads to an increase in Na+/Ca2+ exchange. The increased Na+ concentration by itself may produce electrical disturbance which would result in arrhythmia. Increased Ca2+ may affect proteases and may help in the conversion of xanthine dehydrogenase to xanthine oxidase, consequently increased production of super oxide radicals. Increased membrane lipid peroxidation and other oxygen free-radical associated membrane damage in myocytes has been demonstrated.

Activation of human erythrocyte, brain, aorta, muscle, and ocular tissue aldose reductase

Based upon kinetic, structural, and immunologic properties, we have demonstrated that human tissues have three major forms of aldo-keto reductases: aldose reductase (AR), and aldehyde reductases I (AR I) and II (AR II). The proposed subunit compositions are AR, alpha; AR I, alpha-beta; and AR II, delta. Only AR can effectively reduce glucose to sorbitol. The beta subunits in AR I alter the substrate specificity of AR and prevent conformational changes required for the activation of alpha subunits. Partially purified AR (by DE-52) from human erythrocytes expresses biphasic kinetics with glucose and glyceraldehyde. The enzyme can be activated with glucose + glucose-6-P + NADPH and is strongly inhibited by sorbinil, alrestatin, and quercetrin, and by ADP, 2,3DPG, 1,3DPG, and 3PGA. The activated enzyme expresses monophasic kinetics with substrates (Km glucose less than 1 mmol/L) and is less susceptible to inhibition by synthetic AR inhibitors and phosphorylated intermediates. The enzyme from human brain, aorta, muscle, and ocular tissues was also activated under similar conditions. Erythrocyte enzyme was activated by incubation of blood with 30 to 50 mmol/L glucose. In diabetic subjects with blood sugar levels higher than 250 mg%, almost all the erythrocyte enzyme exists in the activated form. As demonstrated by enzyme-linked immunosorbent assay (ELISA), the increase in AR activity (in vivo and in vitro) was due to the activation of the enzyme and not to the de novo synthesis. In each case, the activation of the enzyme was confirmed by NADPH oxidation and the formation of proportionate amounts of sorbitol.

Inhibition kinetics of human kidney aldose and aldehyde reductases by aldose reductase inhibitors

Kinetic patterns of inhibition of homogenous human kidney aldose reductase (AR, EC 1.1.1.21) and aldehyde reductase II (AR II, EC 1.1.1.19) by statil, ICI 105552 [1-(3,4-dichlorobenzyl)-3-methyl-1,2-dihydro-2-oxoquinol-4-yl acetic acid], tolrestat, alrestatin, chromone carboxylic acid (CCA), quercetin, phenobarbital and sorbinil were studied. On the basis of the kinetic nature of inhibition, the inhibitors were classified into four distinct categories. For aldose reductase, sorbinil and phenobarbital were noncompetitive (NC; category I) and CCA and alrestatin were uncompetitive (UC; category II) to both the aldehyde substrate and NADPH. Quercetin and ICI 105552 were NC to the aldehyde and UC to NADPH (category III) and tolrestat and statil were UC to the aldehyde and NC to NADPH (category IV). For AR II, sorbinil and alrestatin were category I inhibitors, ICI 105552 and statil belong to category II, phenobarbital, tolrestat and CCA to category III, and quercetin to category IV. To determine the specificity of inhibition, the ratios of the inhibition constants (Kii) for AR and AR II were calculated. A lower ratio indicates greater specificity. With aldehyde as the varied substrate the specificity ratios were: statil less than ICI 105552 less than alrestatin less than tolrestat less than quercetin less than CCA less than sorbinil less than phenobarbital, and with NADPH as the varied substrate, ICI 105552 less than statil less than alrestatin less than tolrestat less than quercetin less than CCA less than sorbinil less than phenobarbital. For AR, double-inhibition plots generated for one inhibitor from each kinetic category versus sorbinil showed that AR inhibitors of categories I-III bind to the same site on the protein molecule as sorbinil. However, tolrestat seemed to bind to a site different from the sorbinil binding site. For AR II, inhibitors from all the four categories appeared to bind to the same inhibitor binding site.

Interrelationships among human aldo-keto reductase: 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.

Purification and properties of aldehyde reductases from human placenta

Aldehyde reductases (alcohol: NADP+-oxidoreductase, EC 1.1.1.2) I and II from human placenta have been purified to homogeneity. Aldehyde reductase I, molecular weight about 74 000, is a dimer of two nonidentical subunits of molecular weights of about 32 500 and 39 000, whereas aldehyde reductase II is a monomer of about 32 500. Aldehyde reductase I can be dissociated into subunits under high ionic concentrations. The isoelectric pH for aldehyde reductases I and II are 5.76 and 5.20, respectively. Amino acid compositions of the two enzymes are significantly different. Placenta aldehyde reductase I can utilize glucose with a lower affinity, whereas aldehyde reductase II is not capable of reducing aldo-sugars. Similarly, aldehyde reductase I does not catalyse the reduction of glucuronate while aldehyde reductase II has a high affinity for glucuronate. Both enzymes, however, exhibit strong affinity towards various other aldehydes such as glyceraldehyde, propionaldehyde, and pyridine-3-aldehyde. The pH optima for aldehyde reductases I and II are 6.0 and 7.0, respectively. Aldehyde reductase I can use both NADH and NADPH as cofactors, whereas aldehyde reductase II activity is dependent on NADPH only. Both enzymes are susceptible to inhibition by sulfhydryl group reagents, aldose reductase inhibitors, lithium sulfate, and sodium chloride to varying degrees.

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.