NADH oxidase activity present on both the external and internal surfaces of soybean plasma membranes

Abstract

Soybean plasma membranes exhibit NADH oxidase activities accessible to NADH supplied to either the external or internal membrane surfaces. Activity at the external surface was demonstrated using plasma membranes of right side-out orientation prepared by aqueous two-phase partition and using intact soybean cells grown in culture. Activity at the internal membrane surface was demonstrated using vesicles of inside-out orientation obtained by preparative free-flow electrophoresis or by aqueous two-phase partition following freeze–thaw or Brij detergent treatment to invert some of the right side-out vesicles. The NADH oxidase activity of both membrane surfaces appeared to be stimulated by the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D). Following the freeze–thaw treatment utilized to evert the vesicles, however, the NADH oxidase activity of right side-out vesicles was greatly reduced. A 2,4-D-stimulated activity of these vesicles could be restored by treatment first with reduced glutathione and then with hydrogen peroxide or oxidized glutathione. Upon treatment with 0.1% Triton X-100, both right side-out and inside-out vesicles exhibited approximately the same specific activity and the same 2,4-D-stimulated component of activity equal to the sum of the activities of the right side-out and inside-out vesicles assayed separately. These findings suggest that a significant NADH oxidase activity of the soybean plasma membrane responsive to 2,4-D resides on the external plasma membrane surface. This latter activity is unlikely to function under physiological conditions in the direct oxidation of NADH and alternative functions, such as in protein disulfide–thiol interchange, would be more appropriate.