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Plant Physiol, November 1999, Vol. 121, pp. 793-803
In Organello and in Vivo Evidence of the Importance of the
Regulatory Sulfhydryl/Disulfide System and Pyruvate for Alternative
Oxidase Activity in Tobacco1
Greg C.
Vanlerberghe,*
Justine Y.H.
Yip, and
Hannah L.
Parsons
Division of Life Science and Department of Botany, University of
Toronto at Scarborough, 1265 Military Trail, Scarborough, Ontario,
Canada M1C 1A4
After isolation of tobacco
(Nicotiana tabacum) leaf mitochondria, alternative
oxidase (AOX) is predominantly present as the disulfide-linked,
less-active "oxidized" form. In an in organello assay, significant
AOX activity was dependent upon both the reduction of the regulatory
disulfide bond (such as occurs by dithiothreitol) and upon the presence
of the activator pyruvate. However, AOX activity in these assays was
substantially affected when mitochondria were isolated in the presence
of pyruvate. First, pyruvate protects against the oxidation of the
regulatory sulfhydryl during isolation, such that subsequent in
organello AOX activity is not dependent upon dithiothreitol. Second,
pyruvate stabilizes AOX activity, such that mitochondria kept in the
presence of pyruvate have higher maximum rates of AOX activity than
mitochondria kept for some time in the absence of pyruvate. The ability
of 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 protein 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 significant AOX activity, it is not sufficient for activity and other factors must also be critical.
1
This work was funded by a research grant from
the Natural Sciences and Engineering Research Council of Canada to
G.C.V.
*
Corresponding author; e-mail gregv{at}scar.utoronto.ca; fax
416-287-7642.
© 1999 American Society of Plant Physiologists
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