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.
D000449:Aldehyde Reductase / Q000097:blood*; D000596:Amino Acids / Q000032:analysis; D002237:Carbohydrate Dehydrogenases / Q000097:blood*; D002848:Chromatography, DEAE-Cellulose; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008970:Molecular Weight; D009249:NADP / Q000494:pharmacology; D011487:Protein Conformation; D013379:Substrate Specificity; D013401:Sugar Alcohol Dehydrogenases / Q000097:blood*; D013816:Thermodynamics
D000596:Amino Acids; D009249:NADP; D002237:Carbohydrate Dehydrogenases; D013401:Sugar Alcohol Dehydrogenases; D000449:Aldehyde Reductase; C020843:gulonate dehydrogenase