Abstract
Four aldose reductases, designated here as aldose reductases Ia, Ib, IIa and IIb, have been purified to homogeneity from rabbit lens by a combination of several procedures such as ammonium sulfate precipitation, gel filtration, dye-affinity chromatography and chromatofocusing. The molecular weights of the four aldose reductases were estimated to be 33000 by Sephadex G-100 gel filtration, and to be 37000 by SDS-polyacrylamide gel electrophoresis. These enzymes had an identical pH optimum of 5.6. Substrate specificity studies showed that the four enzymes were capable of reducing various aldehydes and aldoses. However, D-hexoses (D-glucose and Dgalactose) were poor substrates for aldose reductases IIa and IIb. Aldose reductases Ia and Ib used both reduced nicotinamide adenine dinucleotide (NADH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) as coenzymes, but aldose reductases IIa and IIb used NADPH specifically. Very high substrate concentrations were required to demonstrate the reaction in the reverse direction with these aldose reductases. Aldose reductases Ia and Ib were activated by sulfate ion, but aldose reductases IIa and IIb were not, and they were all inhibited remarkably by phenobarbital (1 mM). On the basis of the above results, aldose reductases Ia and Ib could be classified as aldose reductase (alditol : NADP+ oxidoreductase, EC 1.1.1.21).
