Phytotropin-binding sites and auxin transport in Cucurbita pepo: evidence for two recognition sites

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Abstract
Two properties of phytotropins, their ability to bind to 1-N-naphthylphthalamic acid (NPA) receptors located on microsomal vesicles isolated from Cucurbita pepo L. hypocotyls, and to stimulate auxin (indol-3-yl acetic acid, IAA) accumulation into such vesicles by blocking its efflux from them, were assessed in double labelling experiments using [2,3,4,5-3H]1-N-naphthylphthalamic acid and 3-indolyl-[2-14C]acetic acid. Two sites of differing affinities and activities on IAA accumulation were found. 1-N-Naphthylphthalamic acid was found to have high affinity (KD at 10-8mol·l-1) for one site and low affinity (KD at 10-6 mol·l-1) for the other, whereas 2-(1-pyrenoyl)benzoic acid displaced NPA with high efficiency (KD below 10-8 mol·l-1) from both sites. Other phytotropins had intermediate affinities for either site. Occupation of the site with low affinity for NPA stimulated auxin accumulation, while occupation of the high-affinity site with a phytotropin did not interfere with auxin accumulation into vesicles.