Isolation and characterization of aldose reductase from calf brain

Abstract

Aldose reductase activity (alditol: NADP + 1-oxidoreductase, EC 1.1.1.21) from calf brain was separated into two protein fractions by DEAE chromatography. Further purification by molecular sieve chromatography and electrofocusing yielded two distinctive enzymes, which were designated AR I and AR II. AR I was purified 646-fold and found to have an isoelectric point of 6.18. AR I was most active as a monomer with a molecular weight of 29 000 and appeared to be in equilibrium with a less active dimer. AR II was purified 425fold and found to have an isoelectric point of 4.88. The molecular weight of this enzyme was 30 000. Although both enzymes had specificity for aldoses as substrates, AR I had two to three 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 n ot have antigenic similarity as tested by Ouchterlony immunodiffusion and counter immunoelectrophoresis. An immunochemical cross-reaction was observed between AR II and lens aldose reductase.