Characterization of aldose reductases Ia and Ib from rabbit lens.

Abstract

The properties of two aldose reductases (Ia and Ib) 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 second-order rate constant (kcat/Km) values, both enzymes had the highest reductive efficiency toward aromatic aldehydes such as pnitrobenzaldehyde. Among the aldoses tested, the aldose reductases exhibited a high affinity for DL-glyceraldehyde (Km of 31μM for Ia and 32μM for Ib) and a low affinity for D-glucose (Km of 92mM for Ia and 126mM for Ib). Aldose reductase I's could utilize both reduced nicotinamide adenine dinucleotide phosphate (NADPH) and reduced nicotinamide adenine dinucleotide (NADH) as coenzymes, but NADH was less effective than NADPH. The Km values for NADPH (1.4μM for Ia and 1.3μM for Ib) were much smaller than those for NADH (420μM for Ia and 270μM for Ib). Aldose reductase I's 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 0.3μM quercitrin, 65% by 1.6μM quercetin and about 70% by 8.0μM 3, 3-tetramethyleneglutaric acid. NADP+ and adenosine 2', 5'-diphosphate (2', 5'-ADP) were strong competitive inhibitors of both aldose reductase I's with respect to the coenzyme. The K1 values for 2', 5'-ADP were about 30μM, and those for NADP+ were about 70μM.