Solubilization and Characterization of a Membrane-Bound Auxin-Binding Protein from Cell Suspension Cultures of Nicotiana tabacum.

A membrane-bound auxin-binding protein (MABP) was solubilized by Triton X-100 from cell suspension cultures of Nicotiana tabacum L. Solubilization of MABP was dependent on the detergent concentration and more than 80% of naphthalene-1-acetic acid (NAA)-binding activity was recovered by an optimum concentration of 0.2%. The solubilized MABP was highly heat-unstable and sensitive to protease. The properties of MABP (affinity, temperature dependence, pH optimum, and analog specificity for auxin binding) did not significantly change after solubilization, e.g. the solubilized MABP showed no or very low levels of NAA-binding at 0 to 4 degrees C but showed a high-affinity binding (dissociation constant K(d) = 2.7 +/- 0.3 x 10(-7)m) at 25 degrees C at an optimum pH of 5.0. NAA-binding of the solubilized MABP proceeded very slowly, i.e. a time of half-maximum binding was at least 15 minutes, although the solubilized MABP showed higher rates of association (k(1) = 1.3 versus 0.9 x 10(5)m(-1) min(-1)) and dissociation (k(-1) = 2.2 versus 1.6 x 10(-2) min(-1)) with NAA than the bound MABP. These results show that specific, saturable, and reversible auxin binding to MABP from dicotyledonous N. tabacum differs from that from monocotyledonous Zea mays, and confirm that MABP is distinct from a soluble auxin-binding protein which also is present in N. tabacum.