Diphenylene iodonium sensitivity of a solubilized membrane enzyme from rose cells

Abstract

An NADH-specific oxidation reduction enzyme has been partially purified from rose cell microsomes by aqueous two-phase partitioning, ultracentrifugation, and ion-exchange chromatography, on the basis of the enzyme's ability to activate lucigenin chemiluminescence in the presence of NADH. The enzyme showed strong similarity to a plasma membrane NADH oxidase (“superoxide synthase” as assayed by lucigenin chemiluminescence; T. M. Murphy and C.-K. Auh, Plant Physiol. 110: 621–629, 1996) in its response to substrate, to Triton X-100, and to diphenylene iodonium, an inhibitor of mammalian neutrophil NADPH oxidase and other flavoenzymes. However, its fluorescence spectrum was not characteristic of flavins and instead was similar to that of pterins. Thus inhibition of an enzyme-catalyzed reaction by diphenylene iodonium does not necessarily imply that the reaction is catalyzed by NADPH oxidase or another flavoenzyme. Superoxide synthesis catalyzed by the enzyme preparation was very low but could be increased at least twofold by the addition of a quinone, menadione. This suggests the enzyme acting in conjunction with a natural quinone could produce activated oxygen species in stressed plant cells.