Properties of a Solubilized Microsomal Auxin-binding Protein from Coleoptiles and Primary Leaves of <i>Zea mays</i>

Cross, John W.; Briggs, Winslow R.

doi:10.1104/pp.62.1.152

Cited by 49 publications

(18 citation statements)

References 17 publications

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“…The NAA-binding system of maize coleoptile was chosen for the assay because it has been well characterized by Ray et al (22). We show here that 4-, 5-, and 6-N3IAA bind reversibly in the dark and that, on photolysis, 5 binding is a modification of the method of Ray et al (22) and is similar to the assay used to measure IAA uptake in zucchini vesicles (10). The modifications shorten the time, increase the number of samples that can be processed at once, and allow the use of a high speed centrifuge in place of an ultracentrifuge.…”

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confidence: 66%

Azido Auxins

Jones¹,

Melhado²,

Ho³

et al. 1984

Plant Physiol.

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The binding constants of three auxin analogs, 4-, 5-, and 6-azidoindole-3-acetic acid (4-, 5-, and 6-N3IAA), and of the photoproducts of 5-N3IAAto the naphthalene-l-acetic acid (NAA) binding sites of Zea mays L. WF9 x BR38 were determined to evaluate the potential of these analogs as photoaffinity labeling agents. We have found that 4-and 5-N3IAA bind to these sites with affinities similar to that of IAA, while 6-N3IAA and the photoproducts of 5-N3IAA bind less tightly. This 3School of Chemical Sciences. 4Abbreviations: NAA, naphthalene-l-acetic acid; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; NAA, naphthalene-1-acetic acid; N3IAA, azidoindole-3-acetic acid; 4-N3IAA, 4-azidoindole-3-acetic acid; 5-N3IAA, 5-azidoindole-3-acetic acid; 6-N3IAA, 6-azidoindole-3-acetic acid; RAC, ratio of association constants.that avoids the requirement that conditions for equilibrium binding be maintained while searching among the membrane proteins for specific auxin-binding sites. Once binding proteins are labeled, they can be isolated using standard techniques without the need to maintain equilibrium binding conditions. Therefore, the advantage of photoaffinity labeling is that binding proteins can still be isolated after they have lost the capacity for binding. Photoaffinity analogs bind reversibly to a site in the dark but can be caused to bind irreversibly by irradiation with light. Light induces the formation of reactive intermediates that attack nearby groups to form covalent bonds. The effectiveness of a photoaffinity agent, therefore, depends upon its affinity for the binding site in the dark and upon the half-lives of these photogenerated intermediates (4,24). The half-life of the ligandbinding site complex must be considerably longer than the halflives of the intermediates to achieve specific labeling.The only previous attempt to label the auxin site by affinity labeling was by Venis (26). He tested the usefulness of the diazonium salts of two auxin analogs, 2-chloro-4-aminophenoxyacetic acid and 2,5-dichloro-3-aminobenzoic acid, and reached tentative conclusions about the involvement of several amino acids in the auxin active site. The potential photoaffinity labeling agents 4-azido-2-chlorophenoxyacetic acid and 3-azido-5-chlorophenoxyacetic acid were found to have only moderate auxin activity (15), unlike the azidoindole-3-acetic acids here described. Spring and Hager (25) have recently demonstrated that sesquiterpene lactones specifically label important growth sites only when an auxin is also bound. The experiments provide an example of a nonequilibrium binding technique useful in auxin receptor research and are important because the results support a molecular model for auxin binding. The authors propose that auxin induces the receptor to undergo a conformational change, important for cell growth. Most significantly, the technique offers an independent assay for identifying auxin receptors and could be useful in conjunction with an independent approach such as we propose in this report.We ...

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confidence: 66%

Azido Auxins

Jones¹,

Melhado²,

Ho³

et al. 1984

Plant Physiol.

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“…4). This result suggests that the auxin efflux carrier was not being labeled, since TIBA has been shown to specifically inhibit auxin association with this site in vivo (4) and in vitro (5,28 (29), 40 kDa (30), and 20 kDa (31-33) have been isolated, some of which (31,32) appear to be of PM origin. It is possible that these polypeptides are related to one another as monomers of a multimeric protein, and the sizes might suggest that they are related to our polypeptides as well.…”

Section: Resultsmentioning

confidence: 99%

Specific photoaffinity labeling of two plasma membrane polypeptides with an azido auxin

Hicks

Rayle

Jones

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

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Plasma membrane vesicles were isolated from zucchini (Cucurbita pepo) hypocotyl tissue by aqueous phase partitioning and assessed for homogeneity by the use of membrane-specific enzyme assays. The highly pure (ca. 95%) plasma membrane vesicles maintained a pH differential across the membrane and accumulated a tritiated azido analogue of Exposure of N3IAA to UV light (300 nm) results in photolysis of the aryl azide (azido group). The products of this reaction are N2 gas and a highly reactive nitrene, which, via a nucleophilic reaction, would be expected to covalently label specific binding proteins. Using this compound, Jones et al. (16,17) were able to demonstrate quantitative differences in labeling of maize microsomal proteins in the presence of competing auxin or auxin analogues, although much background polypeptide labeling was evident (17). Ripp et al. (18) recently reported that a photoaffinity analogue of sucrose labeled a 62-kDa membrane protein associated with sucrose transport in soybean. They found that lowering the temperature of the reaction mixture to -1960C (liquid N2) resulted in reduced background labeling. Here we report that nonspecific [3H]N3IAA labeling of membrane proteins can also be largely eliminated by conducting the photolysis at -196C. Under these conditions, photolysis of zucchini (Cucurbita pepo L.) PM proteins with [3H]N3IAA results in the highspecific-activity labeling of a low-abundance PM polypeptide doublet that displays properties consistent with those expected for a PM auxin receptor. MATERIALS AND METHODS

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“…The proteic nature of the sites has been demonstrated (6,19) and previous attempts have been made to determine some of the amino acid residues, essential for auxin binding (6,19,29). It has been reported that at least one cysteinyl residue is likely to play a prominent role in the binding site (6,29). Other data (10, 19) suggest the presence ofan essential reducible group, possibly a disulfide.…”

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“…Since the specificity of the sites is in rather good agreement with the specificity of the coleoptile straight growth test, it has been suggested that these binding sites might represent receptors involved in the in vivo auxin action (20). The proteic nature of the sites has been demonstrated (6,19) and previous attempts have been made to determine some of the amino acid residues, essential for auxin binding (6,19,29). It has been reported that at least one cysteinyl residue is likely to play a prominent role in the binding site (6,29).…”

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confidence: 99%