Parkin K L, Hultin H O
J Biochem. 1986 Jul;100(1):87-97. doi: 10.1093/oxfordjournals.jbchem.a121709.
Detergent treatments were examined for their efficacy in purifying trimethylamine-N-oxide (TMAO) demethylase activity from fish muscle microsomes. Tritons X-100 and X-45, deoxycholate, Brijs, Tweens 20, 65, and 80, and SDS were generally ineffective in solubilizing demethylase activity from this membrane fraction, at concentrations up to 10 mg detergent per mg protein. In all of these cases, specific activity became enriched in the particulate fraction obtained post-treatment. Highest fold-purification was achieved by using 10 mg SDS per mg protein in 5 mM histidine, pH 7.0 at 10-14 degrees C. Activity was relatively stable to the presence of SDS at this level, and with this treatment, TMAO demethylase activity became purified in the resultant particulate fraction 28- and 58-fold for activity stimulated by ascorbate-iron-cysteine and FMN-NADH, respectively. The presence of urea or 2-mercaptoethanol, or sonication of the SDS-microsome suspension during purification resulted in significant losses of recovered activity. This partially purified fraction represented about 1% of the original microsomal protein and SDS-PAGE revealed the presence of several protein components. The partially purified demethylase could utilize the same two cofactor systems as the native microsomes. It displayed a curvilinear dependence on iron for activity and a sigmoidal response for cysteine. Utilization of NADH, FMN, and ascorbate differed for the purified fraction as compared to the microsomes. Substrate inhibition by TMAO was observed for the partially purified preparation, whereas saturation kinetics were previously noted for microsomal activity.(ABSTRACT TRUNCATED AT 250 WORDS)
研究了去污剂处理法从鱼肌肉微粒体中纯化三甲胺 - N - 氧化物（TMAO）脱甲基酶活性的效果。在每毫克蛋白质使用高达10毫克去污剂的浓度下，Triton X - 100和X - 45、脱氧胆酸盐、Brij、吐温20、65和80以及十二烷基硫酸钠（SDS）通常无法有效溶解该膜组分中的脱甲基酶活性。在所有这些情况下，比活性在处理后获得的颗粒级分中富集。在10 - 14摄氏度下，于5 mM组氨酸（pH 7.0）中每毫克蛋白质使用10毫克SDS可实现最高的纯化倍数。在此水平下，活性对SDS的存在相对稳定，经此处理，对于分别由抗坏血酸 - 铁 - 半胱氨酸和黄素单核苷酸 - 烟酰胺腺嘌呤二核苷酸（FMN - NADH）刺激的活性，TMAO脱甲基酶活性在所得颗粒级分中分别纯化了28倍和58倍。纯化过程中存在尿素或β - 巯基乙醇，或对SDS - 微粒体悬浮液进行超声处理，均会导致回收活性显著损失。这个部分纯化的级分约占原始微粒体蛋白质的1%，十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）显示存在几种蛋白质组分。部分纯化的脱甲基酶可利用与天然微粒体相同的两种辅因子系统。其活性对铁呈曲线依赖性，对半胱氨酸呈S形响应。与微粒体相比，纯化级分对烟酰胺腺嘌呤二核苷酸（NADH）、黄素单核苷酸（FMN）和抗坏血酸的利用情况有所不同。对于部分纯化的制剂观察到了TMAO的底物抑制作用，而之前微粒体活性呈现饱和动力学。（摘要截短于250字）
