ISOLATION AND CHARACTERIZATION OF A SOLUBLE ACTIVE FRAGMENT OF HYDROGENASE ISOENZYME-2 FROM THE MEMBRANES OF ANAEROBICALLY GROWN ESCHERICHIA-COLI
ISOLATION AND CHARACTERIZATION OF A SOLUBLE ACTIVE FRAGMENT OF HYDROGENASE ISOENZYME-2 FROM THE MEMBRANES OF ANAEROBICALLY GROWN ESCHERICHIA-COLI
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DOI：
10.1111/j.1432-1033.1986.tb09578.x
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发表时间：
1986-04-15
期刊：
影响因子：
--
通讯作者：
BOXER, DH
中科院分区：
文献类型：
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作者：
BALLANTINE, SP;BOXER, DH
从厌氧培养的大肠杆菌中纯化了膜结合氢化酶同工酶2的活性胰蛋白酶片段。可溶性酶衍生物通过胰蛋白酶裂解从膜组分中释放出来。提纯过程包括离子交换、羟基磷灰石和凝胶渗透色谱。该酶衍生物经100倍纯化，最终产物的比活性为320 .mu。mol紫苄基还原min-1 mg蛋白-1 （H2：紫苄基氧化还原酶）。天然酶衍生物的Mr为180,000，由Mr为61,000和30,000的等摩尔多肽组成。它含有12.5 mol Fe， 12.8 mol酸不稳定的S2-和3.1 mol Ni/180,000 g酶。纯化后的产物与氢化酶同工酶2交叉反应，而与洗涤剂分散制剂中的同工酶1不交叉反应。Western免疫印迹分析显示，未暴露于胰蛋白酶的同工酶2含有Mr 61,000和35,000的交叉反应多肽。胰蛋白酶处理膜结合酶以形成同工酶2的可溶性衍生物，因此，切割35000 Mr的多肽以产生30000 Mr的片段。胰蛋白酶处理洗涤剂分散同工酶2产生相同的酶碎片。该酶的两个亚基与氢化酶同工酶1的亚基在免疫学上没有任何相同之处。
An active tryptic fragment of membrane-bound hydrogenase isoenzyme 2 from anaerobically grown E. coli has been purified. The soluble enzyme derivative was released from the membrane fraction by trypsin cleavage. The purification procedure involved ion-exchange, hydroxyapatite and gel permeation chromatogrpahy. The enzyme derivative was purified 100-fold from the membrane fraction and the specific activity of the final preparation was 320 .mu.mol benzyl viologen reduced min-1 mg protein-1 (H2:benzyl viologen oxidoreductase). The native enzyme derivative had an Mr of 180,000 and was composed of equimolar amounts of polypeptides of Mr 61,000 and 30,000. It possessed 12.5 mol Fe, 12.8 mol acid-labile S2- and 3.1 mol Ni/180,000 g enzyme. Antibodies were raised to the purified preparation which cross-reacted with hydrogenase isoenzyme 2 but not with isoenzyme 1 in detergent-dispersed preparations. Western immunoblot analysis revealed that isoenzyme 2 which had not been exposed to trypsin contained cross-reacting polypeptides of Mr 61,000 and 35,000. Trypsin treatment of the membrane-bound enzyme to form the soluble derivative of isoenzyme 2, therefore, cleaves a polypeptide of Mr 35,000 to produce the 30,000-Mr fragment. Trypsin treatment of the detergent-dispersed isoenzyme 2 produces the same fragmentation of the enzyme. Neither of the subunits of the enzyme revealed any immunological identity with those of hydrogenase isoenzyme 1.