Mouse-Liver Glutathione Reductase: Inactivation by NADPH sf Two Allelic Variants

Abstract

Mouse-liver glutathione reductase has been purified to homogeneity from strain SWR/J by ammonium sulfate precipitation (40-80 %) and two additional steps of affinity chromatography in ATPR-Sepharose and 2', 5'-ADP-Sepharose from which it was specifically eluted by using NADP+ gradients. After 2032-fold purification the pure enzyme has a specific activity of 146 Uimg. The SWRiJ protein is slightly more basic than the other allelic variant from strain DBA/2J, with PI 7.0 and 6.5 respectively. Both pure proteins are immunologically identical, either by immunodiffusion or by quantitative imrnunoprecipitation. They can however be distinguished by their rate of inactivation in the presence of NADPH, their reduced cofactor. The SWR/J protein is much more resistant to that inactivation ( t x = 14 min) than the DBA/2J enzyme (t % = 5 min).

La elizlma glutation reductasa de higado de raton ha sido purificada hasta homogeneidad a partir de la estirpe SWR/J mediante su precipitadbn fraccionada con sulfato am6nico (40-80 %) y dos pasos adicionales de cromatograiia de afinidad en geles de ATPR-Sefarosa y 2',5'-ADP-Sefarosa de 10s que fue eluida especificamente mediante sendos gradientes de NADP+. Tras una purificaci6n de 2.032 veces la enzima pura muestra una actividad especifica de 146 p/mg. La proteina de la estirpe SWR/J es ligeramente mas bhsica que la procedente de la otra variante alelica DBA/2J, con PI 7,O y 6,5, respectivamente. Ambm proteinas puras son inmunologicamente identicas, sea en inmunodifusi6n o en inmunoprecipitaci6n cuantitativa. Se distinguen, sin embargo, por su diferente velocidad de inactivacibn con