摘要
采用经蔗糖密度梯度法纯化的大豆 (GlycinemaxL .)下胚轴质膜微囊为材料 ,分析了胰蛋白酶处理对质膜H+ ATPase钒酸钠抑制效应的影响。实验结果显示 ,温和胰蛋白酶处理显著提高H+ ATPase的ATP水解活力。并且发现酶切处理降低了钒酸钠对ATPase的抑制效应 ,当钒酸钠浓度为 2mmol/L时 ,ATPase活力仅被抑制 5 3.49% ,而未经酶切的对照组则被抑制 6 4.13%。ATP水解动力学分析表明 ,胰蛋白酶酶切处理既不影响ATP水解的Km 值也不影响钒酸钠的抑制类型 ,酶切前后的Km 值都等于 0 .34mmol/L ,并且都属于反竞争抑制。以上结果显示胰蛋白酶酶切处理可能改变了磷酸酶结构域的结构而影响了钒酸钠的抑制效应 。
The changes in inhibitory effect of vanadate on the plasma membrane H + ATPase were studied with mild trypsin treatment using plasma membrane vesicles purified from soybean ( Glycine max L.)hypocotyles by sucrose gradient centrifugation. Results showed that under mild trypsin treatment the ATPase ATP hydrolysis activity was increased significantly. It was also found that the inhibitory effect of vandate was reduced after proteolysis. In the presence of 2 mmol/L vanadate, the ATP hydrolysis activity of the cleaved ATPase was inhibited by only 53.49%,while that of the un_cleaved ATPase was inhibited by 64.13%. Kinetic studies indicated that both the K m values and the inhibition type of vanadate were not affected by trypsin treatment. Upon proteolysis, K m remained as 0.34 mmol/L,while vanadate was still an uncompetitive inhibitor. Taking together, the structure and activity of the ATPase phosphatase domain were affected by trypsin treatment, implying that this domain might be regulated by the C_terminal end of the plasma membrane H + ATPase.
基金
国家重点基础研究发展规划项目!(G19990 1170 5 )
国家自然科学基金项目!( 3 9770 0 77)
教育部科学技术重点项目!( 9913 3 )资助项
