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

Abstract

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 m M NADH [12–29 nmol (mg protein) −1 min −1 ] was 66% inhibited by 1 m M KCN and ca 40% by 1 m M EDTA. It was unaffected by rotenone, antimycin A, carbonyl cyanide trifluoromethoxyphenylhydrazone (FCCP), mersalyl, chlorotetracycline + Ca 2+ , 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 m M NAD(P)H with an apparent K m (SHAM) of ca 40 μ M (with NADH). The dependence of O 2 consumption on NADH concentration in the presence of SHAM (2 m M ) was sigmoidal, possibly due to endogenous catalase activity, and half‐maximal rate was obtained at 1.5 m M . 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 H 2 O 2 was produced for every 1 mol O 2 consumed. Endogenous catalase converted this H 2 O 2 to O 2 and after complete conversion the stoichiometry was 2 mol NADH consumed for every mol O 3 . SHAM was not consumed in the reaction. The possible involvement of a cytochrome P‐450/420 system is discussed.