ISOLATION AND CHARACTERIZATION OF ALDOSE REDUCTASE FROM CALF BRAIN

Aldose reductase activity (alditol: NADP+ 1-oxidoreductase, EC 1.1.1.21) from calf brain was separated into 2 protein fractions by DEAE chromatography. Further purification by molecular sieve chromatography and electrofocusing yielded 2 distinctive enzymes, which were designated AR I and AR II. AR I was purified 646-fold and had an isoelectric point of 6.18. AR I was most active as a monomer with a MW of 29,000 and appeared to be in equilibrium with a less active dimer. AR II was purified 425-fold and had an isoelectric point of 4.88. The MW of this enzyme was 30,000. Although both enzymes had specificity for aldoses as substrates, AR I 2-3 times larger turnover numbers with aromatic aldehydes and hexonates than did AR II. AR I was activated by sulfhydryl compounds and exhibited biphasic double reciprocal plots. AR I was more sensitive to inhibition by high substrate and phenobarbital concentrations than was AR II. AR I and AR II did not have antigenic similarity as tested by Ouchterlony immunodiffusion and counter immunoelectrophoresis. An immunochemical cross-reaction was observed between AR II and lens aldose reductase.