Purification and Immunologic Identification of Aldose Reductases

Abstract

A general method for the identification and purification of aldose reductase (alditohNADP oxidoreductase, EC 1.1.1.21) and NADP-L-hexonate dehydrogenase (L-gulonate:NADP oxidoreductase, EC 1.1.1.19) from tissues is described. Two aldose reductase isoenzymes, AR-A and AR-B, were isolated from renal papilla and were characterized. These two forms were also found in brain, retina, pancreas, lens and optic nerve. AR-A and AR-B differed primarily in glucuronate-reducing activity. All tissues, with the exception of lens and optic nerve, also contained NADP-L-hexonate dehydrogenase activity. Human red blood cells contained NADP-L-hexonate dehydrogenase activity but not aldose reductase. The aldose reductases and the two isoenzyme forms isolated from these tissues were immunologically identical as demonstrated with a specific antiserum prepared in rabbits against papillary AR-B. The antiserum inhibited AR-A and AR-B activity equally in vitro. The extent of enzyme inhibition by the antiserum was similar when glucuronate, glucuronolactone, xylose and glyceraldehyde were used as substrates. The antiserum did not react with NADP-L-hexonate dehydrogenase. Therefore, the method provides a specific means for identifying aldose reductase in crude tissue homogenates.