PLANT PHYSIOLOGY , Vol 107, Issue 2 443-450, Copyright © 1995 by American Society of Plant Biologists
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BIOCHEMISTRY AND ENZYMOLOGY |
Purification, Characterization, and Submitochondrial Localization of a 58-Kilodalton NAD(P)H Dehydrogenase
M. H. Luethy, J. J. Thelen, A. F. Knudten and T. E. Elthon
School of Biological Sciences and the Center for Biotechnology, University of Nebraska-Lincoln, Lincoln, Nebraska 68588-0118
An NADH dehydrogenase activity from red beet (Beta vulgaris L.) root
mitochondria was purified to a 58-kD protein doublet. An immunologically
related dehydrogenase was partially purified from maize (Zea mays L. B73)
mitochondria to a 58-kD protein doublet, a 45-kD protein, and a few other
less prevalent proteins. Polyclonal antibodies prepared against the 58-kD
protein of red beet roots were found to immunoprecipitate the NAD(P)H
dehydrogenase activity. The antibodies cross-reacted to similar proteins in
mitochondria from a number of plant species but not to rat liver
mitochondrial proteins. The polyclonal antibodies were used in conjunction
with maize mitochondrial fractionation to show that the 58-kD protein was
likely part of a protein complex loosely associated with the membrane
fraction. A membrane-impermeable protein cross-linking agent was used to
further show that the majority of the 58-kD protein was located on the
outer surface of the inner mitochondrial membrane or in the intermembrane
space. Analysis of the cross-linked 58-kD NAD(P)H dehydrogenase indicated
that specific proteins of 64, 48, and 45 kD were cross-linked to the 58-kD
protein doublet. The NAD(P)H dehydrogenase activity was not affected by
ethyleneglycol-bis([beta]-aminoethyl ether)-N,N[prime] -tetraacetic acid or
CaCl2, was stimulated somewhat (21%) by flavin mononucleotide, was
inhibited by p-chloromercuribenzoic acid (49%) and mersalyl (40%), and was
inhibited by a bud scale extract of Platanus occidentalis L. containing
platanetin (61%).
