NITROGENASE SYSTEM FROM AZOTOBACTER - 2-ENZYME REQUIREMENT FOR N2 REDUCTION ATP-DEPENDENT H2 EVOLUTION AND ATP HYDROLYSIS
NITROGENASE SYSTEM FROM AZOTOBACTER - 2-ENZYME REQUIREMENT FOR N2 REDUCTION ATP-DEPENDENT H2 EVOLUTION AND ATP HYDROLYSIS
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DOI：
10.1073/pnas.56.3.979
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发表时间：
1966-01-01
影响因子：
11.1
通讯作者：
LECOMTE, JR
中科院分区：
文献类型：
--
作者：
BULEN, WA;LECOMTE, JR
材料和方法-如前所述培养和收获棕色固氮菌6，细胞糊料在收获时或在氩气氛中冷冻保存时使用。细胞(100-120-gm细胞糊)在法国压力池中破裂，制备S1441/2上清液组分(通过144,000×g的离心法获得30分钟)，包含90-95%的粗提物活性，如上所述。7由于纯化过程中产生了对氧气的敏感性，所有的缓冲液和溶液都用Ar饱和，每毫升含有0.1 mg二硫苏糖醇。鱼精蛋白硫酸盐分级：鱼精蛋白既用于去除核酸，也用于预沉淀具有酶活性的成分。将Sigma化学公司的二级试剂配制成2%的溶液于室温水中，用1N氢氧化钠调节pH至6.0，离心除去沉淀物。将上清液倒出，在氩流下机械搅拌，以去除溶解氧。S144-1/2在超速离心过程中冷却，在鱼精蛋白分离过程中保持在00℃。以每克蛋白质5毫升的速度加入硫酸鱼精蛋白溶液，通过离心法去除沉淀的核酸。用0.5N醋酸调节上清液的pH至6.5，每克S 44-I/蛋白再加入1.2ml硫酸鱼精蛋白溶液沉淀活性成分。离心后，将沉淀物悬浮在0.01M磷酸二氢钾，pH 7.0，室温下，并与纯化的7-纤维素磷酸纤维素(Sigma化学公司)搅拌。以每克S44-I/，蛋白质100毫克的速度添加。磷酸纤维素及其结合的鱼精蛋白在烧结型979上被去除
Materials and Methods.-Azotobacter vinelandii 0 was cultured and harvested as previously described, 6 and the cellpaste either used when harvested or stored frozen in an argonatmosphere. Cells (100-120-gm cell paste) were ruptured in a French pressure cell, and the S1441/2 supernatant fraction (obtained by centrifugation at 144,000 X g for 30 min), containing90-95% of the activity of crude extracts, was prepared as previously described. 7Since a sensitivity to oxygen developed during purification, all buffers and solutions were saturated with argon and contained 0.1 mg of dithiothreitol per ml.Protamine sulfate fractionation: Protamine was used both to remove nucleic acids and to pre-cipitate the enzymatically active components. A 2% solution of thereagent (Sigma Chemical Co., Grade II) was prepared in water at room temperature, the pH adjusted to 6.0 with 1 N NaOH, and the precipitate removed by centrifugation. The supernatant solution was decanted and stirredmechanically under a stream of argon to remove dissolved oxygen. The S144-1/2 frac-tion, allowed to cool during ultracentrifugation, was held at 00 during protamine fractionation. Protamine sulfate solution was added at the rate of 5 ml per gm of protein, and the precipitated nucleic acids were removed by centrifugation. The decantedsupernatant solution was adjusted to pH 6.5 with 0.5 N acetic acid, and the activecomponents were precipitated upon the further addition of 1.2 ml of protamine sulfate solution per gm of S, 44-i/, protein. After centrifugation, the precipitate was suspended in 0.01 M potassium phosphate, pH 7.0, at room temperatureand stirred with purified7 cellulose phosphate (Sigma Chemical Co.) added at the rate of 100 mg per gm of S44-i/, protein. Cellulose phosphate with its bound protamine was removed on a fritted 979