SUCROSE PROTECTABLE INHIBITION OF THE SUCROSE TRANSPORTER BY N-ETHYLMALEIMIDE

To understand contradictory data published in the literature, the sens itivity of sucrose and of valine uptake to N-ethylmaleimide (NEM) was reinvestigated in detail with plasma membrane vesicles purified by pha se partitioning from mature sugar beet (Beta vulgaris) leaves. Uptake in the vesicles was energized by an artificial proton-motive force com bining a pH gradient and an electrical gradient. Three main parameters were varied in the experiments: the presence of a reducing agent, dit hiothreitol (DTT) in the medium used to store the vesicles, the temper ature of pretreatment with NEM (12 or 23 degrees C) and the temperatur e of incubation with the labelled substrate (12 or 23 degrees C). Sens itivity of sucrose uptake to NEM only appeared with vesicles that had been stored in the presence of DTT, and if the pretreatment was run at 23 degrees C. The temperature of incubation with labelled sucrose did not affect NEM sensitivity. The NEM sensitivity of valine uptake was not affected in the same way as sucrose uptake by the temperature of p reincubation, showing that the effects observed were specific for a gi ven transporter. Under conditions which normally inhibit sucrose uptak e, addition of sucrose during NEM pretreatment protected the sucrose t ransporter against NEM inhibition.