Olivari C, Meanti C, De Michelis M I, Rasi-Caldogno F
Dipartimento di Biologia, Università di Milano, Italy.
Plant Physiol. 1998 Feb;116(2):529-37. doi: 10.1104/pp.116.2.529.
Different approaches were utilized to investigate the mechanism by which fusicoccin (FC) induces the activation of the H(+)-ATPase in plasma membrane (PM) isolated from radish (Raphanus sativus L.) seedlings treated in vivo with (FC-PM) or without (C-PM) FC. Treatment of FC-PM with different detergents indicated that PM H(+)-ATPase and the FC-FC-binding-protein (FCBP) complex were solubilized to a similar extent. Fractionation of solubilized FC-PM proteins by a linear sucrose-density gradient showed that the two proteins comigrated and that PM H(+)-ATPase retained the activated state induced by FC. Solubilized PM proteins were also fractionated by a fast-protein liquid chromatography anion-exchange column. Comparison between C-PM and FC-PM indicated that in vivo treatment of the seedlings with FC caused different elution profiles; PM H(+)-ATPase from FC-PM was only partially separated from the FC-FCBP complex and eluted at a higher NaCl concentration than did PM H(+)-ATPase from C-PM. Western analysis of fast-protein liquid chromatography fractions probed with an anti-N terminus PM H(+)-ATPase antiserum and with an anti-14-3-3 antiserum indicated an FC-induced association of FCBP with the PM H(+)-ATPase. Analysis of the activation state of PM H(+)-ATPase in fractions in which the enzyme was partially separated from FCBP suggested that the establishment of an association between the two proteins was necessary to maintain the FC-induced activation of the enzyme.
采用不同方法研究了藤霉素（FC）诱导从体内经FC处理（FC-PM）或未经FC处理（C-PM）的萝卜（Raphanus sativus L.）幼苗分离的质膜（PM）中H(+)-ATP酶激活的机制。用不同去污剂处理FC-PM表明，PM H(+)-ATP酶和FC-FC结合蛋白（FCBP）复合物的溶解程度相似。通过线性蔗糖密度梯度对溶解的FC-PM蛋白进行分级分离，结果显示这两种蛋白迁移在一起，并且PM H(+)-ATP酶保持了由FC诱导的激活状态。溶解的PM蛋白也通过快速蛋白质液相色谱阴离子交换柱进行分级分离。C-PM和FC-PM之间的比较表明，幼苗在体内用FC处理会导致不同的洗脱图谱；来自FC-PM的PM H(+)-ATP酶仅部分与FC-FCBP复合物分离，并且比来自C-PM的PM H(+)-ATP酶在更高的NaCl浓度下洗脱。用抗N端PM H(+)-ATP酶抗血清和抗14-3-3抗血清对快速蛋白质液相色谱级分进行蛋白质免疫印迹分析表明，FC诱导FCBP与PM H(+)-ATP酶缔合。对酶与FCBP部分分离的级分中PM H(+)-ATP酶激活状态的分析表明，两种蛋白之间缔合的建立对于维持FC诱导的酶激活是必要的。
