Inhibition of the specific binding of human lactotransferrin to human peripheral-blood phytohaemagglutinin-stimulated lymphocytes by fluorescein labelling and location of the binding site.
Inhibition of the specific binding of human lactotransferrin to human peripheral-blood phytohaemagglutinin-stimulated lymphocytes by fluorescein labelling and location of the binding site.
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通过荧光素标记和结合位点定位抑制人乳转铁蛋白与人外周血植物血凝素刺激的淋巴细胞的特异性结合。
DOI：
10.1042/bj2760733
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发表时间：
1991
期刊：
影响因子：
--
通讯作者：
G. Spik
中科院分区：
文献类型：
--
作者：
D. Legrand;J. Mazurier;P. Maes;E. Rochard;J. Montreuil;G. Spik
以等摩尔比用荧光素5 '-异硫氰酸酯（FITC）标记人乳转铁蛋白可抑制蛋白质与植物血凝素激活的人外周血淋巴细胞的结合。因此，可以假设FITC在受体结合位点处或附近反应。三个FITC标记的肽已从FITC标记的乳转铁蛋白的胰蛋白酶消化物中纯化。它们的氨基酸序列的测定和它们在蛋白质的一级结构上的定位允许在N-末端叶中鉴定两个FITC可接近区域，在C-末端叶中鉴定一个。事实上，只有10%的总FITC与C-末端叶的一个赖氨酸残基（Lys 579）缀合，而大部分（80%）的FITC与位于人乳转铁蛋白N-末端结构域I的β-转角结构中的三个接近的赖氨酸残基[Lys 263（总荧光的65%）、Lys 280和Lys 282（总荧光的15%）]缀合。所获得的结果表明，受体结合位点应该位于FITC可接近的Lys 263、Lys 280和Lys 282附近，并且证实了我们报道的N-末端结构域I参与人乳转铁蛋白与促分裂原刺激的淋巴细胞的结合的初步结果[Rochard，Legrand，Mazurier，蒙特勒伊和Spik（1989）FEBS Lett. 255，201-204]。在任何情况下，FITC标记是不适合研究结合的乳转铁蛋白活化淋巴细胞和它的使用可能会导致错误的解释细胞结合实验。
Labelling of human lactotransferrin with fluorescein 5'-isothiocyanate (FITC) in an equimolar ratio inhibits the binding of the protein to phytohaemagglutinin-activated human peripheral-blood lymphocytes. Therefore it can be assumed that FITC reacts at, or near, the receptor-binding site. Three FITC-labelled peptides have been purified from a tryptic digest of the FITC-labelled lactotransferrin. The determination of their amino acid sequence and their localization on the primary structure of the protein permitted the identification of two FITC-accessible areas in the N-terminal lobe and one in the C-terminal lobe. In fact, only 10% of the total FITC was conjugated to one lysine residue (Lys579) of the C-terminal lobe, whereas most (80%) of the FITC was conjugated to three close lysine residues [Lys263 (65% of total fluorescence), Lys280 and Lys282 (15% of total fluorescence)] located in beta-turn structures, of the N-terminal domain I of human lactotransferrin. The results obtained show that the receptor-binding site should be located in the vicinity of the FITC-accessible Lys263, Lys280 and Lys282, and corroborate our preliminary results reporting the involvement of the N-terminal domain I in the binding of human lactotransferrin to mitogen-stimulated lymphocytes [Rochard, Legrand, Mazurier, Montreuil & Spik (1989) FEBS Lett. 255, 201-204]. In any case, FITC labelling is not suitable for studying the binding of lactotransferrin to activated lymphocytes and its use may lead to erroneous interpretations of cell binding experiments.