NAD(P)H oxidase and peroxidase activities in purified plasma membranes from cauliflower inflorescences

Abstract

An NAD(P)H oxidase activity stimulated by phenolic compounds has been investigated in purified plasma membranes (pm) and in an intracellular membrane (icm) fraction depleted in plasma membranes, both obtained from a microsomal fraction from cauliflower inflorescences (Brassica oleracea L.). The phenolic compounds salicylhydroxamic acid (SHAM), ferulic acid, coniferyl alcohol, n‐propyl gallate, naringenin, kaempferol and caffeic acid all strongly stimulated the activity. Peroxidase (EC 1.11.1.7), or a peroxidase‐like enzyme, was responsible for the NAD(P)H oxidase activity, which proceeded through a free‐radical chain reaction and was inhibited by catalase (EC 1.11.1.6), superoxide dismutase (EC 1.15.1.1) and KCN. Most of the total activity was soluble; however, the membrane‐bound activity was highly enriched in the pm compared to the icm. The catalase activity was 6 times higher in the icm‐fraction than in the pm‐fraction, but this was not the reason for the much lower phenol‐stimulated NADH oxidase activity in the icm. Peroxidase activity measured with o‐dianisidine and H2O2 had about the same specific activities in the pm‐and icm‐fractions.Neither the phenol‐stimulated NADH oxidase nor the peroxidase activity could be washed away from the pm even by 0.7 M NaCl, indicating that these activities are truly membrane‐bound. SHAM as well as the other phenolic compounds capable of stimulating the NADH oxidase reaction were potent inhibitors of blue light‐induced cytochrome b‐reduction in the pm fraction.