斑叶阿若母花序中交替氧化酶(AOX)的分离

ISOLATION OF THE ALTERNATIVE OXIDASE FROM Arum maculatum

斑叶阿若母(Arrm,maculatum)开花时花序组织中线粒体通过交替氧化呼吸途径产生很高的热量。从花序线粒体中分离的蛋白质浓度约14.0 mg/ml。氧电极测定的线粒体交替氧化呼吸耗氧量为32μmoles/min。用渗透压法破碎线粒体,分离线粒体膜和基质,再用非离子去污剂dBC溶解线粒体膜上的交替氧化酶(AOX)。线粒体、线粒体膜和线粒体膜可溶蛋白都具有AOX活性。而线粒体基质和线粒体膜不可溶蛋白则没有AOX活性。用快速蛋白液相层析(FPLC)法纯化了AOX.其活性可达70-100%,在-70℃中保持6个月以上仍具有活性。银染显示纯化的AOX有4个不同分子量(30-32kD)的蛋白质斑点。双向电泳表明这四个蛋白质的等电点为pH 6.4至pH 7.4。分离出的斑叶阿若母AOX为测量其氨基酸序列和探索其空间结构创造了条件。

There is plenty of alternate oxidase （AOX） in the inflorescences of thermogenic Arum maculatum. The isolated mitochondria exhibited a high activity, consuming oxygen on average 32umoles/min. The concentration of the isolated protein from mitoehondria was 14.0mg/ml. The isolated mitochondria were crashed by osmotic method to isolate matrix, membrane, and membrane soluble and insoluble proteins. The whole mitochondria, membrane and membrane soluble protein showed AOX activity while the matrix and membrane insoluble protein did not displayed AOX activity. The proteins were purified with FPLC by adding deoxyeholamide dBC. The purified enzyme fraction exhibited a high specific activity, which could be kept for at least 6 months when stored at -70℃. Furthermore, we used a silver stain system to identify the AOX, which showed 4 different protein bands from 30kD to 32kD. 2-dimensional eleetrophoresis showed 4 isoelectric points in the range from pH6.4 to 7.4 respectively.