Purification and identification of formyl-methionyl-leucyl-phenylalanine as the major peptide neutrophil chemotactic factor produced by Escherichia coli.
Purification and identification of formyl-methionyl-leucyl-phenylalanine as the major peptide neutrophil chemotactic factor produced by Escherichia coli.
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DOI：
10.1016/s0021-9258(18)91029-x
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发表时间：
1984-05
期刊：
影响因子：
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通讯作者：
Wayne;MarascoS;S. Phan;Henry KrutzschQ;Henry;Showell;Douglas E. FeltnerS;Roderick Nairnll;E. Becker;Peter;Ward
中科院分区：
文献类型：
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作者：
Wayne;MarascoS;S. Phan;Henry KrutzschQ;Henry;Showell;Douglas E. FeltnerS;Roderick Nairnll;E. Becker;Peter;Ward
从大肠杆菌培养物中富集趋化因子的丁醇提取物通过高压液相色谱法分级分离和纯化。来自生物活性（溶酶体酶分泌）和抗原活性（与[3 H]fMet-Leu-Phe竞争结合兔抗-fMet-Leu-Phe）的单个级分的产率显示原始材料的平均50%回收率。通过酶释放活性的测定，分离出5个生物活性峰。这些峰中有三个与抗原活性峰完全一致，表明至少有3个和多达5个不同的甲酰基-甲硫氨酰肽已被分离。大部分回收的活性出现在峰3中，占回收的总生物和抗原活性的70%。随后通过二肽基羧肽酶气相色谱-质谱法（DCP/GC-MS）分析5个峰级分，以确定氨基酸序列。消化后，仅在五个峰级分之一（峰3）中证明了甲酰基-Met肽。此外，GC保留时间和质谱均表明峰3含有甲酰基-甲硫氨酰基-亮氨酰基-苯丙氨酸。DCP/GC和MS数据通过对真实的fMet-Leu-Phe进行的测试得到证实。E.将合成的fMet-Leu-Phe加入到大肠杆菌中，导致峰3的精确高压液相色谱级分中的生物活性和抗原活性增加，其中由E.最后，对回收的生物活性和抗原活性的分析表明，在培养物中发现了纳摩尔浓度的甲酰基肽。这些结果表明，E.大肠杆菌中的甲酰基-甲硫氨酰基-亮氨酰基-苯丙氨酸似乎是其主要成分，是负责细菌培养物提取物中白细胞趋化活性的肽介质。
Chemotactic factor-enriched butanol extracts from Escherichia coli culture filtrates were fractionated and purified by high pressure liquid chromatography. The yield from individual fractions of biological activity (lysosomal enzyme secretion) and antigenic activity (competition with [3H]fMet-Leu-Phe for binding to rabbit anti-fMet-Leu-Phe) revealed an average 50% recovery of original material. Five peaks of biological activity were separated as demonstrated by enzyme-releasing activity. Three of these peaks coincided exactly with peaks of antigenic activity, suggesting that at least 3 and as many as 5 distinct formyl-methionyl peptides had been separated. The majority of recovered activity appeared in peak 3 and represented 70% of the total biological and antigenic activities recovered. The five peak fractions were subsequently analyzed by dipeptidyl carboxypeptidase gas chromatography-mass spectrometry (DCP/GC-MS) to determine amino acid sequences. After digestion, the formyl-Met peptide was demonstrated in only one of the five peak fractions (peak 3). Furthermore, both the GC retention times and mass spectra indicated that peak 3 contained formyl-methionyl-leucyl-phenylalanine. The DCP/GC and MS data were confirmed with tests made on authentic fMet-Leu-Phe. Butanol extracts from E. coli filtrates to which were added synthetic fMet-Leu-Phe resulted in increased biological and antigenic activity in the precise high pressure liquid chromatography fractions of peak 3 where the fMet-Leu-Phe produced by E. coli was found. Finally, the analysis of recovered biological and antigenic activities indicated that the formyl peptides were found in nanomolar concentrations in culture filtrates. These results demonstrate that the NH2-terminal formyl peptides produced by E. coli, of which formyl-methionyl-leucyl-phenylalanine appears to be the major component, are the peptide mediators responsible for leukocyte chemotactic activity in the bacterial culture extracts.