Vanadate-dependent NADH oxidation in microsomal membranes of sugar beet

Microsomal membranes isolated from sugar beet (Beta vulgaris L. var. GWD-2) storage tissue were found to contain a Na3VO4-dependent system for the oxidation of NADH. The system was demonstrated to be enzymatic in nature and specific for Na3VO4. Maximal Na3VO4-dependent NADH oxidation was observed at pH 6.5, when Na3VO4 was present at 200 microM and when NADH was present at 100 microM. The oxidation activity was insensitive to rotenone and antimycin A but was inhibited by NaN3, NaCN, and quinacrine. Sodium vanadate-dependent NADH oxidation occurred with a concomitant uptake of O2 from the assay solution. Both NADH oxidation and O2 consumption were dependent upon the presence of Na3VO4, inhibited by manganese, and preferred NADH to NADPH. Catalase prevented Na3VO4-dependent O2 consumption but accelerated NADH oxidation. The effects of manganese and catalase suggest that superoxide anion and hydrogen peroxide may be involved in this process. While it is unclear as to the physiological significance of Na3VO4-dependent NADH oxidation in plant cells, the presence of this system indicates that caution must be exercised when coupled ATPase assays depending upon NADH oxidation are used with plant membranes in the presence of Na3VO4.