The binding of auxins to membrane fractions from Zea mays

Batt, Susan (1975) The binding of auxins to membrane fractions from Zea mays. PhD thesis, University of Glasgow.

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Abstract

This thesis describes the interaction of auxins with membrane fractions prepared from shoot tissue of etiolated Zea mays seedlings. The binding of radioactive 1-naphthylacetic acid (NAA) to these membrane fractions is investigated using a pelletting technique originally described by Hertel et al., (1972). Modifications of this technique lead to greatly improved binding data. Optimal conditions for the binding of NAA-14C are established, and the possibility that binding is merely transport-into membrane vesicles is eliminated. A Scatchard analysis of the binding of NAA-14C to a crude membrane pellet from coleoptiles reveals two sets of high affinity binding sites, referred to as site 1 and site 2. Estimations of dissociation constants (K) and site concentration values (n) show that for site 1, K1 = 1.8 x 10-7M and n2 = 52 pmole per g fresh weight, and for site 2, K2 = 14.5 x 10-7M and n2 = 101 pmole per g fresh weight. Two sets of binding sites are also demonstrated for 3-indolylacetio acid (IAA), and these are of somewhat lower affinity than those described for NAA. The auxin-specificity of the NAA binding sites is investigated using a technique analogous to that used for enzyme inhibitor studies. The interaction of a selected range of auxins, auxin analogues and other phytohormones with the NAA binding sites shows that auxin-specificity resides in site 2 whereas site 1 is less specific. Sucrose gradient fractionation techniques are used in an attempt to characterise further the membrane pellets. A partial separation of site 1 and site 2 binding activities is achieved, with site 1 sedimenting in the lighter regions of the gradient and site 2 in the heavier bands. A study of marker enzymes and chemical composition of the gradient bands reveals that the heavier regions are enriched in plasma membranes, whereas a distinct band of Golgi activity is observed in the lighter regions. Using a phosphotungstic acid-chromic acid staining procedure, electron microscopy also demonstrates an enrichment of plasma membrane content in the heavy bands. The binding of NAA-14C to membrane fractions from mesocotyl tissue is also investigated. Early experiments suggest only one set of binding sites, kinetically equivalent to site 2 in coleoptiles. However, analogue competition and fractionation studies indicate the presence of two sets of sites with only a two-to-three-fold difference in dissociation constants.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: M B Wilkins
Keywords: Plant sciences
Date of Award: 1975
Depositing User: Enlighten Team
Unique ID: glathesis:1975-73818
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 14 Jun 2019 08:56
Last Modified: 14 Jun 2019 08:56
URI: http://theses.gla.ac.uk/id/eprint/73818

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