Identification of a soluble auxin-binding protein as a glutathione-dependent formaldehyde dehydrogenase

Abstract

Antibodies against the putative auxin-binding site of an ER auxin-binding protein from maize coleoptiles were raised by injecting rabbits with a synthetic oligopeptide (Ile-His-Arg-His-Ser-Cys-Glu) conjugated to hemocyanin from the Keyhole limpet. Soluble auxin-binding proteins were isolated from an extract of etiolated mung bean hypocotyls by affinity chromatography on 2,4-dichlorophenoxyacetic acid-linked (2,4-D-linked) Sepharose 4B. The antiserum against the oligopeptide recognized a specific 40 kDa polypeptide among auxin-binding proteins from mung bean hypocotyls. An auxin-binding protein of 40 kDa was then purified by several column-chromatographic steps. The apparent molecular mass of the protein was estimated to be 80 kDa by gel-filtration and 40 kDa by SDS-PAGE. We designated this protein ABP40. The dissociation constant of purified ABP40 for [ 14C]-2,4-D was calculated to be 1 × 10 −5 M. Binding of [ 14C]-2,4-D was completely inhibited by p-chlorophenoxyisobutyric acid (PCIB) and weakly inhibited by indole-3-acetic acid (IAA) and naphthalene-1-acetic acid (NAA). Benzoic acid and tryptophan had no effect on the binding. The partial amino acid sequences of ABP40, obtained after chemical cleavage by CNBr, revealed high homology to glutathione-dependent formaldehyde dehydrogenase (FDH; EC 1. 2. 1. 1). Moreover, the purified ABP40 had FDH activity.