Purification of aldose reductase from human placenta and stabilization of the inhibitor binding site.
Aldose reductase from human placenta was purified to homogeneity by a rapid (2 day) and efficient purification scheme involving Red Sepharose affinity chromatography, chromatofocusing and high performance liquid chromatography on a size-exclusion column. Addition of NADP+ at all steps in the purification of aldose reductase and during storage of the enzyme at -20 degrees stabilized both the enzyme active site and the major site for binding of aldose reductase inhibitors such as sorbinil and tolrestat. Aldose reductase is a monomer with a molecular mass of 38 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, apparent pI 5.9. Placenta aldose reductase exhibited no cross-reactivity with aldehyde reductase from human liver in an ELISA assay. Aldose reductase showed broad specificity for aldehydes, was specific for NADPH, and was activated by sulfate.
D000426:Alcohol Dehydrogenase / Q000276:immunology; D000449:Aldehyde Reductase / Q000037:antagonists & inhibitors / Q000276:immunology / Q000302:isolation & purification*; D001665:Binding Sites; D003920:Diabetes Mellitus / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009249:NADP / Q000494:pharmacology; D010920:Placenta / Q000201:enzymology*; D013379:Substrate Specificity; D013401:Sugar Alcohol Dehydrogenases / Q000302:isolation & purification*
D009249:NADP; D013401:Sugar Alcohol Dehydrogenases; D000426:Alcohol Dehydrogenase; D000449:Aldehyde Reductase