Salicylhydroxamic acid‐stimulated NADH oxidation by purified plasmalemma vesicles from wheat roots

Purified, right side‐out plasmalemma vesicles were isolated from 7‐day‐old roots of dark‐grown wheat (Triticum aestivum L. cv. Drabant) by aqueous polymer two‐phase partitioning. The oxygen consumption by these vesicles at pH 6.5 in the presence of 1 mM NADH [12–29 nmol (mg protein)−1min−1] was 66% inhibited by 1 mM KCN and ca 40% by 1 mM EDTA. It was unaffected by rotenone, antimycin A, carbonyl cyanide trifluoromethoxyphenylhydrazone (FCCP), mersalyl, chlorotetracycline + Ca2+, and EGTA. Salicylhydroxamic acid (SHAM) and its analogue, m‐chlorobenzhydroxamic acid, stimulated the rate of oxygen consumption 10–20 fold in the presence of 1 mM NAD(P)H with an apparent Km (SHAM) of ca 40 μM (with NADH). The dependence of O2 consumption on NADH concentration in the presence of SHAM (2 mM) was sigmoidal, possibly due to endogenous catalase activity, and half‐maximal rate was obtained at 1.5 mM. In the absence of SHAM the rate increased with increasing acidity and no pH optimum was detectable between pH 4.5 and 8.5. In the presence of SHAM an optimum was observed at pH 6.5 and 0.8 mol of H2O2 was produced for every 1 mol O2 consumed. Endogenous catalase converted this H2O2 to O2 and after complete conversion the stoichiometry was 2 mol NADH consumed for every mol O3. SHAM was not consumed in the reaction. The possible involvement of a cytochrome P‐450/420 system is discussed.