Aldose Reductase from Rhodotorula: I. PURIFICATION AND PROPERTIES

Abstract

Abstract A TPN-specific aldose reductase has been purified 1310-fold from yeast (Rhodotorula sp.). Cell growth and preparation of a crude extract are described. Acidification of the extract (pH 5.0) resulted in precipitation of approximately 60% of the inert protein. Further treatment with protamine sulfate, ammonium sulfate fractionation, and negative absorption to calcium phosphate gel produced a 24-fold purification over the crude extract. Electrofocusing (pH 4 to 6 range) gave greater than a 1000-fold purification and subsequent column chromatography on Sephadex G-100 removed the Ampholine and remaining proteins from the reductase enzyme. Recovery of aldose reductase units averaged about 57% of those present in the initial extract. Homogeneity of the purified enzyme was indicated by both disc gel electrophoretical and immunological criteria. In the presence of 10-3m mercaptoethanol, the enzyme was dissociated into two protein bands by disc gel electrophoresis. The enzyme has an average molecular weight of 61,000 as estimated by gel filtration, and an isoelectric point of 5.05 (electrofocusing). When TPNH was the varied substrate, competitive inhibition was given by both TPN+ (Ki = 2.0 x 10-5 m) and 2'-AMP (Ki = 5.0 x 10-4 m).