In organello and in vivo evidence of the importance of the regulatory sulfhydryl/disulfide system and pyruvate for alternative oxidase activity in tobacco

After isolation of tobacco (Nicotiana tabacum) leaf mitochondria, alternati ve oxidase (AOX) is predominantly present as the disulfide-linked, less-act ive "oxidized" form. In an in organello assay, significant AOX activity was dependent upon both the reduction of the regulatory disulfide bond (such a s occurs by dithiothreitol) and upon the presence of the activator pyruvate . However, AOX activity in these assays was substantially affected when mit ochondria were isolated in the presence of pyruvate. First, pyruvate protec ts against the oxidation of the regulatory sulfhydryl during isolation, suc h that subsequent in organello AOX activity is not dependent upon dithiothr eitol. Second, pyruvate stabilizes AOX activity, such that mitochondria kep t in the presence of pyruvate have higher maximum rates of AOX activity tha n mitochondria kept for some time in the absence of pyruvate. The ability o f pyruvate to protect against AOX oxidation was exploited to assess the in vivo status of the regulatory sulfhydryl/disulfide system. In both tobacco suspension cells and tobacco leaves with high levels of AOX protein, the pr otein is predominantly present as the "reduced" active form in vivo under a range of respiratory conditions. Experiments also indicate that, while the presence of reduced protein may be a necessary prerequisite for significan t AOX activity, it is not sufficient for activity and other factors must al so be critical.