Lindley I, Aschauer H, Seifert J M, Lam C, Brunowsky W, Kownatzki E, Thelen M, Peveri P, Dewald B, von Tscharner V
Sandoz Forschungsinstitut Ges.m.b.H., Vienna, Austria.
Proc Natl Acad Sci U S A. 1988 Dec;85(23):9199-203. doi: 10.1073/pnas.85.23.9199.
The neutrophil-activating factor (NAF) purified from the conditioned medium of lipopolysaccharide-stimulated human monocytes was sequenced and found to consist of 72 amino acids: SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRA ENS. Purified preparations of natural NAF contained, in addition to this main form, minor amounts of three amino-terminal variants with 77 (+AVLPR), 70, and 69 residues. A gene coding for the 72-amino acid NAF was synthesized, cloned, and expressed in Escherichia coli. Western (immunologic) blot analysis of crude bacterial extracts, with an antiserum raised against natural NAF, revealed a single band that comigrated with natural NAF. Recombinant NAF purified to homogeneity had identical amino- and carboxyl-terminal sequences to the 72-amino acid natural NAF. Recombinant NAF was tested on human neutrophils and had the same activity and potency as natural NAF in inducing chemotaxis, rapidly increasing cytosolic free Ca2+, activating the respiratory burst, and releasing specific and azurophilic granular contents.
从脂多糖刺激的人单核细胞条件培养基中纯化的中性粒细胞激活因子（NAF）进行了测序，发现由72个氨基酸组成：SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRA ENS。天然NAF的纯化制剂除了这种主要形式外，还含有少量三种氨基末端变体，分别具有77个（+AVLPR）、70个和69个残基。合成了编码72个氨基酸的NAF的基因，将其克隆并在大肠杆菌中表达。用针对天然NAF产生的抗血清对粗细菌提取物进行蛋白质免疫印迹分析，显示出一条与天然NAF迁移率相同的条带。纯化至同质的重组NAF的氨基末端和羧基末端序列与72个氨基酸的天然NAF相同。重组NAF在人中性粒细胞上进行了测试，在诱导趋化性、迅速增加胞质游离Ca2+、激活呼吸爆发以及释放特异性和嗜天青颗粒内容物方面，具有与天然NAF相同的活性和效力。
