CHARACTERIZATION OF NEUROTROPHIN DIMERS AND MONOMERS
CHARACTERIZATION OF NEUROTROPHIN DIMERS AND MONOMERS
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DOI：
10.1111/j.1432-1033.1994.0995b.x
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发表时间：
1994-11-01
期刊：
影响因子：
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通讯作者：
BARDE, YA
中科院分区：
文献类型：
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作者：
KOLBECK, R;JUNGBLUTH, S;BARDE, YA
当重组脑源性神经营养因子（BDNF）和神经营养素-3（NT-3）通过反相色谱纯化时，这些神经营养素呈现两个不同的峰。当从脑组织中纯化天然存在的BDNF时也是如此。如凝胶过滤实验所示，具有较短保留时间的峰对应于神经营养因子二聚体，具有较长保留时间的峰对应于单体。相反，在所有神经营养因子前体共同的基本加工位点具有单个氨基酸置换（Arg-1->Lys）的BDNF突变体洗脱为单峰。凝胶过滤色谱显示该峰由分子量几乎为野生型二聚体两倍的二聚体组成。N-末端测序表明延伸了19个氨基酸，包括糖基化天冬酰胺残基。当在神经元存活测定中测试时，BDNF突变体（[R-1 K]BDNF）的生物活性与野生型BDNF的生物活性相同。使用该测定，发现盐酸胍变性的神经营养素单体的生物活性远低于二聚体的生物活性，并且用NT-3单体和表达trkC的NIH 3 T3细胞的实验表明，这样的单体以防止与神经营养素受体有效相互作用的构象存在于溶液中。
When recombinant brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are purified by reverse-phase chromatography, these neurotrophins elute as two distinct peaks. This is also the case when naturally occurring BDNF is purified from brain tissue. As indicated by gel filtration experiments, the peaks with shorter retention times correspond to neurotrophin dimers, those with longer retention times to monomers. In contrast, a BDNF mutant with a single aminoacid replacement (Arg-1-->Lys) in the basic processing site common to all neurotrophin precursors elutes as a single peak. This peak is shown by gel-filtration chromatography to consist of dimers with a molecular mass almost twice that of wild-type dimers. N-terminal sequencing indicates an extension of 19 amino acids, including a glycosylated asparagine residue. The biological activity of the BDNF mutant ([R-1K]BDNF) is identical with that of wild-type BDNF when tested in a neuron survival assay. Using this assay, the biological activities of guanidine-hydrochloride-denatured neurotrophin monomers were found to be much lower than that of the dimers, and experiments with NT-3 monomers and NIH3T3 cells expressing trkC suggest that such monomers exist in solution in a conformation that prevents efficient interactions with neurotrophin receptors.