Burgess A W, Metcalf D, Sparrow L G, Simpson R J, Nice E C
Biochem J. 1986 May 1;235(3):805-14. doi: 10.1042/bj2350805.
Four forms of mouse granulocyte/macrophage colony-stimulating factor (GM-CSF) were purified 100,000-fold from mouse lung conditioned medium. Each of the CSF species stimulated the formation of both granulocyte and macrophage colonies, and half-maximal stimulation in the semi-solid mouse bone-marrow colony assay occurred at 1 pm. The four GM-CSF species exhibited similar charge microheterogeneity, focusing between pH 4.2 and pH 5.2. On SDS/polyacrylamide gels two of the GM-CSF sub-species had apparent Mr values of 23,000, and the other two, 21, 000. Treatment with neuraminidase decreased the Mr values of these two sets to 21,000 and 19,000 respectively. Incubation with endoglucosidase F decreased the charge heterogeneity and the Mr of all species to 16,500. A gas-phase radioiodination procedure was used to incorporate 2-3 atoms of 125I/molecule into purified GM-CSF without any loss of biological activity. The 125I-labelled GM-CSF was analysed on a microbore reversed-phase h.p.l.c. column to determine its specific radioactivity directly. This 125I-labelled GM-CSF molecule is suitable for cell-surface receptor-binding studies.
从小鼠肺条件培养基中纯化出四种形式的小鼠粒细胞/巨噬细胞集落刺激因子（GM-CSF），纯化倍数达10万倍。每种集落刺激因子（CSF）都能刺激粒细胞集落和巨噬细胞集落的形成，在半固体小鼠骨髓集落试验中，半数最大刺激浓度在下午1点时出现。这四种GM-CSF表现出相似的电荷微不均一性，聚焦在pH 4.2至pH 5.2之间。在十二烷基硫酸钠/聚丙烯酰胺凝胶上，其中两种GM-CSF亚类的表观相对分子质量（Mr）值为23,000，另外两种为21,000。用神经氨酸酶处理后，这两组的Mr值分别降至21,000和19,000。用内切葡糖苷酶F孵育后，所有亚类的电荷不均一性和Mr值均降至16,500。采用气相放射性碘化方法，将每分子2 - 3个125I原子掺入纯化的GM-CSF中，且不丧失任何生物活性。对125I标记的GM-CSF在微径反相高效液相色谱柱上进行分析，以直接测定其比放射性。这种125I标记的GM-CSF分子适用于细胞表面受体结合研究。
