Purification, Characterization, and Submitochondrial Localization of a 58-Kilodalton NAD(P)H Dehydrogenase

Abstract

An NADH dehydrogenase activity from red beet (Befa vulgaris 1.) root mitochondria was purified to a 58-kD protein doublet. An immunologically related dehydrogenase was partially purified from maize (Zea mays 1. 873) 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 crosslinking 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(P-aminoethyl ether)-N,N'-tetraacetic acid or CaCI,, was stimulated somewhat (21 %) by flavin mononucleotide, was inhibited by pchloromercuribenzoic acid (49%) and mersalyl (40%), and was inhibited by a bud scale extract of Platanus occidentalis L. containhg plataneth (61 %).In contrast to their mammalian counterparts, plant mitochondria are capable of oxidizing cytoplasmic NAD(P)H directly, coupling this oxidation to the electron transport chain (Moller and Lin, 1986). This occurs via exogenous NAD(P)H DHs located on the cytosolic face of the inner mitochondrial membrane (Palmer and Moller, 1982;Moller, 1986;Moller and Lin, 1986; Douce and Neuberger, 1989). The oxidation of endogenous mitochondrial matrix substrates (NADH and succinate) has been shown to take precedence over the oxidation of exogenous NAD(P)H (Dry et al., 1983;Day et al., 1985). Thus, it has been suggested that the exogenous NAD(P)H DHs may function in balancing the redox levels between NAD(P)H pools in the cytosol and the mitochondrion (Moller and Lin, 1986; Douce and Neuberger, 1989). As a result, the exogenous NAD(P)H DH could coordinate glycolytic flwc with flow through the Krebs cycle. If this is true, then regulation of exogenous NAD(P)H DH activity could greatly influence plant metabolism. Consistent with this hypothesis, Kromer and Heldt (1991) have shown that the oxidation by mitochondria of reducing equivalents generated during photosynthesis is vital for obtaining maximum photosynthetic rates. Exogenous NAD(P)H DH activity can be distinguished from the other mitochondrial NAD(P)H DH activities by its insensitivity to rotenone (Wilson and Hanson, 1969), stimulation by Ca2' and inhibition by EGTA (Coleman and Palmer, 1971), and sensitivity to platanetin (Ravanel et al., 1986). Although these characteristics ca...

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