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PLANT PHYSIOLOGY , Vol 103, Issue 2 449-456, Copyright © 1993 by American Society of Plant Biologists

METABOLISM AND ENZYMOLOGY

Evidence for a Single Naphthylphthalamic Acid Binding Site on the Zucchini Plasma Membrane

G. K. Muday, S. A. Brunn, P. Haworth and M. Subramanian
Wake Forest University, Department of Biology, Box 7325, Winston-Salem, North Carolina 27109-7325 (G.K.M.);

The binding of [2,3,4,5,(n)-3H]N-1-napthylphthalamicacid ([3H]-NPA) to zucchini (Cucurbita pepo L.) plasma membranes was examined in detail using two different filtration assays and the results were rigorously analyzed by saturation curves, double-reciprocal plots, Scatchard plots, Hill plots, and the computer program Ligand (P.J. Munson, D. Rodbard [1980] Anal Biochem 107: 220-239). To facilitate these analyses, a new assay that allows rapid and quantitative analysis of [3H]NPA binding with high reproducibility and ease of manipulation has been developed. These detailed kinetic analyses indicate that only one binding site for [3H]NPA (Kd = 16 nM) was associated with the zucchini plasma membrane. Analysis of [3H]NPA dissociation by several auxin transport inhibitors revealed similar dissociation constants with both plasma and microsomal membrane. Collectively, these data indicate the presence of only one binding site for NPA associated with the zucchini plasma membrane.


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