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.

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: https://theses.gla.ac.uk/id/eprint/73818