Auxin-binding protein from coleoptile membranes of corn (Zea mays L.). I. Purification by immunological methods and characterization.
The purification of a putative auxin receptor is one possibility to elucidate the first event in the mechanism of auxin action. By affinity chromatography of membrane proteins on 2-OH-3,5-diiodobenzoic acid-Sepharose and gel filtration on Ultrogel a fraction enriched in auxin-binding protein (ABP) was obtained and used for rabbit immunization. From the immunoglobulin G (IgG) fraction of the antisera IgGs against proteins not binding auxin (nonABP) could be obtained which were used to eliminate the nonABP from the eluates of the 2-OH-3,5-diiodobenzoic acid-Sepharose. The remainder fraction was further purified and concentrated on IgG-Sepharose which retained the ABP that could be eluted without loss of binding activity. A 600-fold purification with a yield of 42% was achieved. The ABP could be identified as the site I "receptor" described by Dohrmann et al. (Dohrmann, U., Hertel, R., and Kowalik, H. (1978) Planta (Berl.) 140, 97-106). It is shown that the competitors tested reduce [14C]1-naphthylacetic acid-(NAA) binding in the following order of effectiveness: NAA greater than 2-naphthylacetic acid greater than 1-phenylacetic acid greater than 2,3,5-triiodobenzoic acid greater than 3-indolylacetic acid greater than 2,4-dichlorophenoxyacetic acid. The ABP has a sharp binding optimum at pH 5.5, and the KD was calculated to be 5.7 X 10(-8) M to [14C]NAA. The binding activity of the ABP linearly decreased with increasing temperature but could partially be restored upon chilling in the presence of auxin. The ABP seems to be a 40-kDa dimer in its native form without disulfide bonds between its monomers.
Löbler
M
M
Klämbt
D
D
D000818:Animals; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008566:Membranes / Q000032:analysis; D010937:Plant Growth Regulators; D010940:Plant Proteins / Q000302:isolation & purification*; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000276:immunology / Q000302:isolation & purification*; D013696:Temperature; D003313:Zea mays
D010937:Plant Growth Regulators; D010940:Plant Proteins; D011956:Receptors, Cell Surface; C046131:auxin receptor, plant