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

Authors

• Michael H. Luethy, University of Nebraska-Lincoln
• Jay J. Thelen, University of Nebraska-Lincoln
• Andrew F. Knudten, University of Nebraska-Lincoln
• Thomas Elthon, University of Nebraska - LincolnFollow

Document Type

Article

Date of this Version

1995

Citation

Plant Physiol. (1 995) 107: 443-450

Comments

Copyright American Society of Plant Biologists. Used by permission.

Abstract

An NADH dehydrogenase activity from red beet (Beta vulgaris 1.) root mitochondria was purified to a 58-kD protein doublet. An immunologically related dehydrogenase was partially purified from maize (Zea mays L. 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(β-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. containing platanetin (61%).