The majority of stem cell factor exists as monomer under physiological conditions
The majority of stem cell factor exists as monomer under physiological conditions
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DOI：
10.1074/jbc.272.10.6406
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发表时间：
1997-03-07
影响因子：
4.8
通讯作者：
Langley, KE
中科院分区：
文献类型：
--
作者：
Hsu, YR;Wu, GM;Langley, KE
可溶性大肠杆菌来源的重组人干细胞因子(RhSCF)形成一种非共价结合二聚体。我们用沉淀平衡和尺寸排斥层析测定了二聚体缔合常数(K-a)为2-4×10(8)M(-1)。先前已经证明，SCF在人血清中的浓度约为3.3 ng/ml，根据二聚化K-a，循环中90%以上的SCF将以单体形式存在。在人血清中加入I-125-rhSCF，用体积排阻层析法分析，当总SCF浓度在10-100 ng/ml范围内，72-49%的rhSCF为单体，与K-a测定结果一致。将SCF(F63C)、SCF(V49L、F63L)和SCF(A165C)三个突变体在大肠杆菌中进行重组表达、纯化和鉴定。研究了这些突变体的二聚体K-a值、生物物理性质和生物活性。二聚缺陷突变体SCF(F63C)S-CH2CONH2和SCF(V49L，F63L)的促分裂活性显著降低，而Cys(165)-Cys(165)二硫键连接的SCF(A165C)二聚体的活性是野生型的10倍。结果表明，二聚化亲和力与生物活性之间存在相关性，这与SCF二聚化介导其受体Kit的二聚化以及随后的信号转导的模型一致。
Soluble Escherichia coli-derived recombinant human stem cell factor (rhSCF) forms a non-covalently associated dimer. We have determined a dimer association constant (K-a) of 2-4 x 10(8) M(-1), using sedimentation equilibrium and size exclusion chromatography. SCF has been shown previously to be present at concentrations of approximately 3.3 ng/ml in human serum, Based on the dimerization K-a, greater than 90% of the circulating SCF would be in the monomeric form. When I-125-rhSCF was added to human serum and the serum analyzed by size exclusion chromatography, 72-49% of rhSCF was monomer when the total SCF concentration was in the range of 10-100 ng/ml, consistent with the K-a determination. Three SCF variants, SCF(F63C), SCF (V49L,F63L), and SCF(A165C), were recombinantly expressed in Escherichia coli, purified, and characterized. The dimer K-a values, biophysical properties, and biological activities of these variants were studied. Dimerization-defective variants SCF(F63C)S-CH2CONH2 and SCF(V49L,F63L) showed substantially reduced mitogenic activity, while the activity of the Cys(165)-Cys(165) disulfide-linked SCF(A165C) dimer was 10-fold higher than that of wild type rhSCF. The results suggest a correlation between dimerization affinity and biological activity, consistent with a model in which SCF dimerization mediates dimerization of its receptor, Kit, and subsequent signal transduction.