Plasma-membrane (PM) vesicles isolated from 6-d-old corn roots by sucrose gradient centrifugation or two-phase partitioning showed an NADH-dependent nitrate reductase (NR) activity averaging at 40 nmol per milligram PM protein per hour. This membrane-associated NR activity could not be removed from two-phase-partitioned PM vesicles by salt washing, osmotic shock treatment, sonication, or freeze-thawing to reverse vesicle sidedness. Therefore, it could not be attributed to contamination of membrane vesicles by the soluble, cytosolic NR. Plasma-membrane vesicles reduced NO3-in the presence of the electron donors NADH or NADPH at an activity ratio of 2.2. The NADH- and NADPH-dependent NR activities of outside-out oriented PM vesicles differed in their sensitivity toward the detergent Brij 58, leading to a latency of 65 or 29 using NADH or NADPH as electron donor, respectively. The activities of NO3-reduction in the presence of saturating concentrations of NADH and NADPH were additive. Furthermore, both activities were characterized by a different pH dependence with a pH optimum of 7.5 for the NADH-dependent activity and of 6.8 for the NADPH-dependent activity. The membrane-associated NAD(P)H-dependent NR activities responded to different nitrogen nutrition of plants in a manner different from the soluble forms of the enzyme. The data confirm the existence of a corn PM NR and suggest that there may be two different NO3--reducing enzymes located at the PM of corn roots.
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