The Hormone-responsive NADH Oxidase of the Plant Plasma Membrane Has Properties of a NADH:Protein Disulfide Reductase

doi:10.1074/jbc.272.17.11221

The Hormone-responsive NADH Oxidase of the Plant Plasma Membrane Has Properties of a NADH:Protein Disulfide Reductase*

From the Departments of ^‡ Foods and Nutrition and
^¶ Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907 and
^§ University of Geneva, Geneva, Switzerland

^∥ To whom correspondence should be addressed:
Dept. of Medicinal Chemistry and Molecular Pharmacology, Hansen Life Sciences Research Bldg., Purdue University, West Lafayette, IN 47907.
Tel.: 317-494-1388; Fax: 317-494-4007.

Abstract

Plasma membranes of plant cells are characterized by a plant hormone (auxin)-responsive oxidation of NADH. The latter proceeds under argon. Also, when NADH oxidation is stimulated 50% by auxin addition, oxygen consumption is reduced by 40%. These findings are reconciled by direct assays using 5,5′-dithiobis-(2nitrobenzoic acid) (DTNB) (Ellman's reagent) that show protein disulfides to be electron acceptors for auxin-stimulated NADH oxidation. In the presence of an external reducing agent such as NADH, cysteine, or dithiothreitol, protein disulfides of the membrane are reduced with a concomitant stoichiometric increase in free thiols. In the absence of an external reducing agent, or in the presence of oxidized glutathione, DTNB-reactive thiols of the plasma membrane are decreased in the presence of auxins. Several auxin-reductant combinations were effective, but the same reductants plus chemically related and growth-inactive auxin analogs were not. A cell surface location of the affected thiols demonstrated with detergents and impermeant thiol reagents suggests that the protein may have a different physiological role than oxidation of NADH. For example, it may carry out some other role more closely related to the function of the auxin hormones in cell enlargement such as protein disulfide-thiol interchange.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

2,4-D

2,4-dichlorophenoxyacetic acid

2,3-D

2,3-dichlorophenoxyacetic acid

α-NAA

α-naphthaleneacetic acid

β-NAA

β-naphthaleneacetic acid

DTNB

5,5′-dithiobis-(2-nitrobenzoic acid)

DTT

dithiothreitol

IAA

indole-3-acetic acid

NTSB

2-nitro-5-thiosulfobenzoate

Mes

4-morpholineethanesulfonic acid.
- Received December 10, 1996.
- Revision received February 11, 1997.