Purification and Properties of an Auxin-Binding Protein from Maize Shoot Membranes1

Abstract

An auxin-binding protein was purified from membranes of maize shoots including the coleoptiles, leaf rolls and mesocotyls. The method of Ca2+-promoted sedimentation of membrane particles was adopted for large-scale preparation. The auxin-binding protein was solubilized from the acetone-washed membranes, and purified by successive chromatographies on DEAE-Sephacel, 1-naphthylacetic acid-linked AH-Sepharose 4B, and Sephadex G-100 columns. The yield of the purified protein was about 0.2 mg from 1 kg of shoots. The binding protein exists as a dimer with a subunit molecular weight of 21,000, and possesses one auxin-binding site per dimer. The preparation also contains a minor molecular form with a subunit molecular weight of 20,000. The auxin-binding protein is not a hydrophobic protein, as judged from its amino acid composition and solubility. The circular dichroic (CD) spectrum of the binding protein resembles the spectrum anticipated from the beta-structures, and shows no alpha-helix characteristic in the secondary structure. The CD spectral changes induced on the binding of auxin and its antagonist seem to be related to the receptor function. The affinity of the binding protein for auxin is dependent on pH, with an optimum at pH 5.0, while the binding protein is unstable below pH 6. We discuss here the intracellular localization of the auxin-binding protein from the view point of the controversial pH-dependence of the binding affinity and stability.

Auxin-binding protein located in the endoplasmic reticulum of maize shoots: molecular cloning and complete primary structure.

“…A possibility is that there is a (Table 1). However, the molecular weight calculated from the deduced sequence was slightly lower than that estimated for the isolated ABP from its electrophoretic mobility on NaDod-S04/PAGE (17,18).…”

“…We previously purified an auxin-binding protein (ABP) from maize shoot membranes (17). This ABP was found in the ER (18), and its amount seemed to be correlated with auxin-induced cell elongation of the coleoptile and mesocotyl (18,19).…”

AUXIN BINDING PROTEIN1: The Outsider

“…The pH in the ER lumen is estimated to be close to 7.0, and ABP1 affinity to auxin is nearly zero at this pH (Tian et al, 1995). The highest affinity was determined at approximately pH 5.5 (Lö bler and Klä mbt, 1985;Shimomura et al, 1986;Tian et al, 1995), which is a typical pH of the extracellular matrix. Purified Zm-ABP1 was found to bind specifically and saturatably to plasma membrane vesicles, independently of auxin concentrations (Schiebl et al, 1997).…”

Similarities and Differences between the Characteristics of Gibberellin-Binding Protein and Gibberellin 2-Oxidases in Adzuki Bean (Vigna angularis) Seedlings

“…The receptors for auxin, cytokinin, ethylene, and brassinosteroid have been clarified. The auxin receptor, ABP1 was purified from corn seedlings, 2,3) and has been found in many plant species, but it took many years to be accepted as an auxin receptor. Another type of auxin receptor has been expected, because some of the auxin dependent phenomena are shown to be mediated by an ABP1 independent pathway.…”