Detailed examination of binding over the range 10(-7)-10(-6) M suggests that membrane preparations from coleoptiles of Zea mays L., cv Kelvedon 33 contain at least two sets of high affinity binding sites for 1-naphthylacetic acid (NAA), with dissociation constants of 1.8×10(-7) M (site 1) and 14.5×10(-7) M (site 2). Similar studies with 3-indolylacetic acid (IAA) also indicate two sets of binding sites, whose concentrations are closely comparable to those deduced for NAA. A substantial proportion of the total binding activity is retained in a detergent-solubilized preparation. Using [(14)C]NAA the interactions of a range of analogues with each of the binding sites have been examined with the aid of double reciprocal plots. The specificity of site 2 is compatible with that expected for an auxin receptor, in that only active auxins, antiauxin transport inhibitors are able to compete with [(14)C]NAA for the binding sites. Site 1 on the other hand is less specific, since it appears to bind all compounds tested, including physiologically inactive analogues.
Further evidence is presented for the discrete nature of the two classes of high affinity auxin binding sites in corn (Zea mays L.) coleoptile membranes, site 1 and site 2. Fractions can be obtained by differential centrifugation that exhibit binding kinetics characteristic of site 2, but not site 1. Membrane preparations containing both binding sites may be resolved on sucrose gradients into a light and a heavy band, whose binding kinetics and analogue binding specificities correspond to those deduced for site 1 and site 2 respectively in unfractionated membranes. Evidence from enzymic and chemical assays and from electron microscopy suggests that site 2, the auxin-specific binding site, is located in fractions enriched in plasma membrane, whereas site 1 is associated with Golgi membranes and/or endoplasmic reticulum.
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