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作 者 ：Chen Jia, Wu Zhong-yi and Zhu Mei-jun

Keywords:ABA, Binding protein, Plasma membrane, Solubilization,

Abstract:Abscisic acid (ABA) is a plant hormone that influences a number of essential physiological and developmental processes, including seed development and plant adaptation to environmental stress. While the molecular mechanism of ABA action is unknown, this hormone is supposed to interact with cellular receptors, leading to the activation of a number of biochemical processes. High affinity of ABA-binding proteins (ABA-BP) in some plants, either membrane-bound (ABA-BPm) or soluble, have been previously described, but the proteins have not been further characterized. The solubilization, purification and characterization of ABA-BP are essential for the preparation of immuno and cDNA-probes of ABA receptor, and they are the initial steps to elucidate how ABA triggers the signal transduction pathway that ultimately leads to the adaptation of environmental stress. By means of differential centrifugation and two-phase aqueous system, plasma membrane with high purity was prepared from maize roots which had been grown in dark for 3 days. Solubilization of ABA-BPm was dependent on the detergent (Triton X-100) concentration, the optimum concentration being 0.2% (W/V). The solubilization efficiency with 0.2% Triton X-100 (87%) was higher than that with acetone (65%). Radioactivity ligand binding assay showed that the binding of 3H-ABA to ABA-BPm was specific, reversible, saturable and of high affinity. After solubilization with the nonionic detergent (Triton X-100),ABA-BPs (solubilized ABA-BPm) retained their high affinity-binding to 3H-ABA, while BSA did not. The specific binding capacity of ABA-BPs were 2.77 nmoL/g protein, which indicated that ABA-BP were located in the plasma membrane. Some ABA-BPm properties, like temperature and pH dependence for ABA binding, were not significantly changed after solubilization. The ABA-BPm activity of binding ABA was insensitive to temperature change (0 ℃ to 25℃ ). In the first 5 minutes the amount of ABA bound reached 50% of the maximum value, which occurred in 30 min and remained unchanged for at least another 30 min. However, ABA-BPs binding ABA was sensitive to temperature. Although the maximal binding capacity of ABA-BPs was the same as that of ABA-BPm at 25 ℃, it was very low at 0 ℃. The binding of ABA to ABA-BPs proceeded very slowly, taking a time of at least 15 min to reach half of its maximum capacity. The short duration of maximal binding activity (less than 30 min) indicated that ABA-BPs became unstable after the phospholipid bilayer acting as the protective environment was removed by solubilization.

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