PLANT PHYSIOLOGY , Vol 106, Issue 3 1115-1122, Copyright © 1994 by American Society of Plant Biologists
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METABOLISM AND ENZYMOLOGY |
Purification, Characterization, and Submitochondrial Localization of the 32-Kilodalton NADH Dehydrogenase from Maize
A. F. Knudten, J. J. Thelen, M. H. Luethy and T. E. Elthon
School of Biological Sciences and the Center for Biotechnology, University of Nebraska-Lincoln, Lincoln, Nebraska 68588-0118
Plant mitochondria have the unique ability to directly oxidize exogenous
NAD(P)H. We recently separated two NAD(P)H dehydrogenase activities from
maize (Zea mays L.) mitochondria using anion-exchange (Mono Q)
chromatography. The first peak of activity oxidized only NADH, whereas the
second oxidized both NADH and NADPH. In this paper we describe the
purification of the first peak of activity to a 32-kD protein. Polyclonal
antibodies to the 32-kD protein were used to show that it was present in
mitochondria from several plant species. Two-dimensional gel analysis of
the 32-kD NADH dehydrogenase indicated that it consisted of two major and
one minor isoelectric forms. Immunoblot analysis of submitochondrial
fractions indicated that the 32-kD protein was enriched in the soluble
protein fraction after mitochondrial disruption and fractionation; however,
some association with the membrane fraction was observed. The
membrane-impermeable protein cross-linking agent 3,3[prime]
-dithiobis-(sulfosuccinimidylpropionate) was used to further investigate
the submitochondrial location of the 32-kD NADH dehydrogenase. The 32-kD
protein was localized to the outer surface of the inner mitochondrial
membrane or to the intermembrane space. The pH optimum for the enzyme was
7.0. The activity was found to be severely inhibited by
p-chloromercuribenzoic acid, mersalyl, and dicumarol, and stimulated
somewhat by flavin mononucleotide.
