Institute of Agricultural Biotechnology, 12 Pskovskaya st., 27253, Moscow, Russia.
Planta. 1993 Mar;189(3):301-5. doi: 10.1007/BF00194425.
Plasma membranes have been purified from roots of maize (Zea mays L.) using a two-phase aqueous polymer system, dextran-polyethylene glycol. The plant material was homogenized in the presence of a mixture of natural protease inhibitors from potato (Solanum tuberosum L.); these inhibitors have been shown to be more effective than phenylmethylsulfonyl fluoride in suppressing the endogenous proteases in maize roots. Inhibition of proteolysis in the homogenization medium markedly increased (about tenfold) the number of lowaffinity binding sites for fusicoccin (FC). In addition, storage of plasma membranes at -20° C decreased both the number of the low-affinity sites and their dissociation constant (KD); this effect was in all probability caused by lipid peroxidation. The presence of EDTA throughout isolation and storage of the plasma membranes stabilized the parameters of FC binding to the membranes. The kinetics of binding of [(3)H]dihydroFC and the competition between [(3)H]dihydroFC and FCs A, C, J, and H were determined for the low-affinity sites. It was found that (i) the rate constant of association between FC and the low-affinity binding sites is about two orders of magnitude lower than that for the high-affinity sites; (ii) different FCs can be arranged in the order of decreasing avidity for the low-affinity FCbinding site: FC A>FC C>FC J>FC H.
使用双水相聚合物系统（葡聚糖-聚乙二醇）从玉米（Zea mays L.）根部分离纯化质膜。在存在来自马铃薯（Solanum tuberosum L.）的天然蛋白酶抑制剂混合物的情况下匀浆植物材料；这些抑制剂在抑制玉米根内源蛋白酶方面比苯甲基磺酰氟更有效。在匀浆介质中抑制蛋白水解作用显著增加（约十倍）腐胺（FC）的低亲和力结合位点的数量。此外，质膜在-20°C 下储存会降低低亲和力位点的数量及其解离常数（KD）；这种效应很可能是由脂质过氧化引起的。在质膜的分离和储存过程中始终存在 EDTA 稳定了 FC 与膜结合的参数。确定了低亲和力位点对[(3)H]二氢 FC 的结合动力学和[(3)H]二氢 FC 与 FCs A、C、J 和 H 之间的竞争。结果发现：（i）FC 与低亲和力结合位点之间的缔合速率常数比高亲和力位点低约两个数量级；（ii）不同的 FC 可以按对低亲和力 FC 结合位点的亲和力递减的顺序排列：FC A>FC C>FC J>FC H。
