Nicola N A, Cross B, Simpson R J
Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Parkville, Victoria, Australia.
Biochem Biophys Res Commun. 1993 Jan 15;190(1):20-6. doi: 10.1006/bbrc.1993.1004.
Murine leukemia inhibitory factor (LIF) (the fully active recombinant form produced in E. coli) was digested in the unreduced state with trypsin and Staphylococcal V8 protease in 0.05% sodium dodecyl sulfate. Disulfide-bonded peptides were identified by altered mobility on reverse-phase high-performance liquid chromatography in the presence or absence of dithiothreitol and subjected to amino acid sequencing. Peptides containing more than one disulfide bond were subjected to further proteolysis and disulfide-bonded subfragments identified and sequenced. The three disulfide bonds are CYS13-CYS135, CYS19-CYS132 and CYS61-164 and the first and third of these are clearly homologous to the two disulfide bonds in oncostatin M. The spatial organization of the cysteine residues in the predicted four alpha-helical bundle structure of LIF (Bazan, Neuron 7,197;1991) is compatible with these disulfide assignments.
将小鼠白血病抑制因子（LIF）（在大肠杆菌中产生的完全活性重组形式）在未还原状态下于0.05%十二烷基硫酸钠中用胰蛋白酶和葡萄球菌V8蛋白酶进行消化。通过在存在或不存在二硫苏糖醇的情况下反相高效液相色谱上迁移率的改变来鉴定二硫键连接的肽，并对其进行氨基酸测序。含有多个二硫键的肽进行进一步的蛋白水解，鉴定并测序二硫键连接的亚片段。三个二硫键为CYS13-CYS135、CYS19-CYS132和CYS61-164，其中第一个和第三个与抑瘤素M中的两个二硫键明显同源。LIF预测的四螺旋束结构（巴赞，《神经元》7,197；1991）中半胱氨酸残基的空间组织与这些二硫键分配相符。
