Characterization of aldose reductases Ia and Ib from rabbit lens.

Abstract

The properties of 2 aldose reductases (Ia and Ib) [which play a role in the etiology of diabetic cataract] from rabbit lens were investigated. Both enzymes showed similar substrate specificity and were capable of reducing various aldoses and aldehydes. On the basis of apparent Km, Vmax and 2nd-order rate constant (kcat/Km) values, both enzymes had the highest reductive efficiency toward aromatic aldehydes such as p-nitrobenzaldehyde. Among the aldoses tested, the aldose reductases exhibited a high affinity for DL-glyceraldehyde (Km of 31 .mu.M for Ia and 32 .mu.M for Ib) and a low affinity for D-glucose (Km of 92 mM for Ia and 126 mM for Ib). Aldose reductase I could utilize both NADPH and NADH as coenzymes, but NADH was less effective than NADPH. The Km values for NADPH (1.4 .mu.M for Ia and 1.3 .mu.M for Ib) were much smaller than those for NADH (420 .mu.M for Ia and 270 .mu.M for Ib). Aldose reductase I were strongly activated by sulfate ion and their Km and Vmax values for substrate and coenzyme were increased. Aldose reductase I''s were inhibited strongly by aldose reductase inhibitors: about 80% by 0.3 .mu.M quercitrin, 65% by 1.6 .mu.M quercetin and about 70% by 8.0 .mu.M 3,3-tetramethyleneglutaric acid, NADP+ and 2'',5''-ADP were strong competitive inhibitors of both aldose reductase I with respect to the coenzyme. The Ki values for 2'',5''-ADP were about 30 .mu.M, and those for NADP+ were about 70 .mu.M.