Affinity purification and glucose specificity of aldose reductase from bovine lens

Abstract

Aldose reductase, a possible key enzyme of sugar-cataract formation in diabetes, has been purified from bovine lens by a five-step procedure including affinity chromatography with Mātrex gel red A. The enzyme was purified 12,600-fold and was apparently homogeneous by polyacrylamide gel electrophoresis. The glucose specificity of the purified enzyme was studied with d-glucose anomers and d-glucitol as substrates. The ratios of the reduction rate of α- d-glucose to that of β- d-glucose at 10, 13, and 20 m m were 1.90, 1.76, and 1.72, respectively. These values were in good agreement with the ratios (1.92, 1.81, and 1.66) calculated on the basis of the rate constants reported for d-glucose mutarotation equilibrium ( J. M. Los, L. B. Simpson, and K. Wiesner, 1956, J. Amer. Chem. Soc. 78, 1564–1568 ) and the assumption that aldose reductase acts on the aldehyde form of d-glucose. In addition, the composition of d-glucose produced from d-glucitol in the reverse reaction was 63% α anomer and 37% β anomer, which also agreed well with the values, 65 and 35%, respectively, calculated from the rate constants in reactions from the aldehyde form to both the α anomer and the β anomer. It was suggested from these kinetic analyses that aldose reductase acts on the aldehyde form of d-glucose ( K m = 0.66 μm) but not on either the α or the β anomer of d-glucose.