Single-step immunoaffinity purification and characterization of dodecylmaltoside-solubilized human neutrophil flavocytochrome b.
Single-step immunoaffinity purification and characterization of dodecylmaltoside-solubilized human neutrophil flavocytochrome b.
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十二烷基麦芽糖苷溶解的人中性粒细胞黄素细胞色素 b 的一步免疫亲和纯化和表征。
DOI：
10.1016/s0005-2736(03)00086-5
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Jesaitis,AlgirdasJ
中科院分区：
文献类型：
--
作者：
Taylor,RossM;Burritt,JamesB;Foubert,ThomasR;Snodgrass,MeaganA;Stone,KimC;Baniulis,Danas;Gripentrog,JeannieM;Lord,Connie;Jesaitis,AlgirdasJ
黄细胞色素B（Cyt B）是一种异源二聚体的膜蛋白，是吞噬细胞清除细菌和真菌病原体的电子转移系统的核心成分。本报告描述了一种快速，有效的一步纯化细胞色素B从人中性粒细胞质膜溶解在非离子去污剂十二烷基麦芽糖苷（DDM）和免疫亲和层析。一个类似的程序，直接从完整的中性粒细胞分离的细胞色素B的肝素和免疫亲和层析的组合。Cyt B在DDM中的稳定性相对于先前使用的增溶剂得到增强，这通过监测粗膜提取物中的血红素光谱和测定纯化后对蛋白水解降解的抗性来确定。凝胶过滤色谱和动态光散射表明，DDM保持一个主要的单分散人口的细胞色素B免疫亲和纯化后。用这种分离方法获得的高纯度允许直接测定血红素与蛋白质的2：1化学计量比，证实了以前的结构模型。通过基质辅助激光解吸/电离（MALDI）质谱法分析分离的异源二聚体，可以准确测定基因序列所示的p22 phoxas的质量。亲和纯化的Cyt B功能性地重建成人工双层，并证明在整个纯化过程中有效地保留了蛋白质的催化活性。
Flavocytochrome b (Cyt b) is a heterodimeric, integral membrane protein that serves as the central component of an electron transferase system employed by phagocytes for elimination of bacterial and fungal pathogens. This report describes a rapid and efficient single-step purification of Cyt b from human neutrophil plasma membranes by solubilization in the nonionic detergent dodecylmaltoside (DDM) and immunoaffinity chromatography. A similar procedure for isolation of Cyt b directly from intact neutrophils by a combination of heparin and immunoaffinity chromatography is also presented. The stability of Cyt b was enhanced in DDM relative to previously employed solubilizing agents as determined by both monitoring the heme spectrum in crude membrane extracts and assaying resistance to proteolytic degradation following purification. Gel filtration chromatography and dynamic light scattering indicated that DDM maintains a predominantly monodisperse population of Cyt b following immunoaffinity purification. The high degree of purity obtained with this isolation procedure allowed for direct determination of a 2:1 heme to protein stoichiometry, confirming previous structural models. Analysis of the isolated heterodimer by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry allowed for accurate mass determination of p22phoxas indicated by the gene sequence. Affinity-purified Cyt b was functionally reconstituted into artificial bilayers and demonstrated that catalytic activity of the protein was efficiently retained throughout the purification procedure.
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DOI：
10.1016/0005-2728(88)90140-5
发表时间：
1988
期刊：
Biochimica et biophysica acta
影响因子：
--
作者：
Charles A. Parkos;Rodger A. Allen;Charles G. Cochrane;A. J. Jesaitis
通讯作者：
Charles A. Parkos;Rodger A. Allen;Charles G. Cochrane;A. J. Jesaitis
正辛基 β-D-吡喃葡萄糖苷溶解的晶状体纤维质膜主要多肽 (MP26) 的高效液相色谱分析
DOI：
--
发表时间：
1988
期刊：
影响因子：
--
作者：
S. Manenti;I. Dunia;M. le Maire;E. Benedetti
通讯作者：
E. Benedetti
白细胞氧化损伤的分子基础
DOI：
--
发表时间：
1992
期刊：
影响因子：
--
作者：
E. Dratz;A. J. Jesaitis
通讯作者：
A. J. Jesaitis
慢性肉芽肿病的分子基础[见评论]
影响因子：
20.3
作者：
R. Smith;J. Curnutte
通讯作者：
J. Curnutte
p22phox 的磷酸化是由磷脂酶 D 依赖性和非依赖性机制介导的。
DOI：
10.1074/jbc.m004703200
发表时间：
2000
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Regier,DS;Greene,DG;Sergeant,S;Jesaitis,AJ;McPhail,LC
通讯作者：
McPhail,LC