Burgess A W, Metcalf D, Russell S H, Nicola N A
Biochem J. 1980 Feb 1;185(2):301-14. doi: 10.1042/bj1850301.
The formation of mature haemopoietic cells is controlled by hormones that specifically stimulate the progenitor cells of the granulocyte/macrophage, eosinophil, megakaryocyte and erythroid pathways. PWMSC medium (pokeweed-mitogen-stimulated spleen-cell-conditioned medium) is known to contain the biological activities that control the clonal proliferation of these four progenitor cells in vitro in semi-solid agar cultures. In this study the molecular properties of these biological activities were characterized, and all four colony-stimulating factors appear to be associated with glycoproteins. These factors were precipitated between 50 and 80%-satd. (NH(4))(2)SO(4) and could be concentrated by ultrafiltration over a 10000-mol.wt.-cut-off hollow-fibre membrane. Megakaryocyte- and erythroid-colony-stimulating factors were lost when the conditioned medium was dialysed at low ionic strength (<0.03m). Neither asialo- nor sialo-erythropoietin was detectable in concentrated PWMSC medium or in the fractions purified from it by gel filtration on Sephadex G-150. The factors bound to concanavalin A-Sepharose were eluted with alpha-methyl-d-glucopyranoside (0.10m). Analysis by gel filtration on Sephadex G-150 indicated that the apparent molecular-weight distributions of all colony-stimulating factors were identical (37000). Treatment with neuraminidase did not alter the biological activities of any of these factors, but when the molecular weights were analysed, after neuraminidase treatment, on Sepharose CL-6B in the presence of guanidine hydrochloride (6m) all were eluted with a mol.wt. of 24000. Although the apparent molecular weights of the different factors were identical, charge differences were detectable by isoelectric focusing on thin-layer granulated gels. There appeared to be considerable charge heterogeneity associated with each factor, as all were focused over 2-4 pH units. The maximum activity of the granulocyte/macrophage-colony-stimulating factor on isoelectric focusing was at pH4.8, whereas the maximum activity for the eosinophil-colony-stimulating factor was at pH5.8. The erythroid- and megakaryocyte-colony-stimulating activities were detected in the pH ranges 4.8-5.8 and 4.6-7.1 respectively. Chromatographic differences between the granulocyte/macrophage- and eosinophil-colony-stimulating factors were also detected by hydrophobic chromatography at low ionic strength (0.15m-NaCl) on Cibacron Blue-Sepharose and at high ionic strength [2m-(NH(4))(2)SO(4)] on phenyl-Sepharose. Eosinophil-colony-stimulating factor bound more strongly than the other factors to both matrices. The megakaryocyte- and erythroid-colony-stimulating activities were always associated with those for granulocytes/macrophages and eosinophils. Preparations highly enriched for eosinophil-colony-stimulating factor were also obtained by DEAE-cellulose chromatography. An overall purification of 100-fold for all of the factors was achieved with the present techniques, and, although differences were observed, only granulocyte/macrophage-stimulating factors and a small proportion of the eosinophil-stimulating factors could be completely separated from the others. Our results are consistent with the existence of separable factors for granulocyte/macrophage and eosinophil stimulation, but the megakaryocyte- and erythroid-stimulating activities were always associated with the granulocyte/macrophage- and eosinophil-stimulating activities. Thus there may be one molecule that is able to stimulate all four colony types or four very similar molecules that are difficult to separate.
成熟造血细胞的形成受激素控制，这些激素特异性刺激粒细胞/巨噬细胞、嗜酸性粒细胞、巨核细胞和红细胞生成途径的祖细胞。已知商陆有丝分裂原刺激的脾细胞条件培养基（PWMSC培养基）含有在半固体琼脂培养中体外控制这四种祖细胞克隆增殖的生物活性物质。在本研究中，对这些生物活性物质的分子特性进行了表征，所有四种集落刺激因子似乎都与糖蛋白有关。这些因子在50%至80%饱和度的硫酸铵中沉淀，可通过截留分子量为10000的中空纤维膜超滤进行浓缩。当条件培养基在低离子强度（<0.03m）下透析时，巨核细胞和红细胞集落刺激因子会丢失。在浓缩的PWMSC培养基或通过Sephadex G-150凝胶过滤从中纯化的组分中均未检测到去唾液酸或唾液酸促红细胞生成素。与伴刀豆球蛋白A-琼脂糖结合的因子用α-甲基-D-吡喃葡萄糖苷（0.10m）洗脱。通过Sephadex G-150凝胶过滤分析表明，所有集落刺激因子的表观分子量分布相同（37000）。用神经氨酸酶处理不会改变这些因子中任何一个的生物活性，但在神经氨酸酶处理后，在含有盐酸胍（6m）的Sepharose CL-6B上分析分子量时，所有因子均以24000的分子量被洗脱。尽管不同因子的表观分子量相同，但通过在薄层颗粒凝胶上进行等电聚焦可检测到电荷差异。似乎每个因子都存在相当大的电荷异质性，因为所有因子都聚焦在2 - 4个pH单位范围内。粒细胞/巨噬细胞集落刺激因子在等电聚焦时的最大活性在pH4.8，而嗜酸性粒细胞集落刺激因子的最大活性在pH5.8。红细胞和巨核细胞集落刺激活性分别在pH范围4.8 - 5.8和4.6 - 7.1中检测到。在低离子强度（0.15m氯化钠）下通过Cibacron Blue-琼脂糖和在高离子强度[2m硫酸铵]下通过苯基-琼脂糖进行疏水色谱分析时，也检测到粒细胞/巨噬细胞和嗜酸性粒细胞集落刺激因子之间的色谱差异。嗜酸性粒细胞集落刺激因子比其他因子与两种基质的结合更强。巨核细胞和红细胞集落刺激活性总是与粒细胞/巨噬细胞和嗜酸性粒细胞的活性相关。通过DEAE-纤维素色谱法也获得了高度富集嗜酸性粒细胞集落刺激因子的制剂。采用当前技术对所有因子实现了100倍的总体纯化，尽管观察到了差异，但只有粒细胞/巨噬细胞刺激因子和一小部分嗜酸性粒细胞刺激因子能够与其他因子完全分离。我们的结果与存在可分离的粒细胞/巨噬细胞和嗜酸性粒细胞刺激因子一致，但巨核细胞和红细胞刺激活性总是与粒细胞/巨噬细胞和嗜酸性粒细胞刺激活性相关。因此，可能存在一种能够刺激所有四种集落类型的分子，或者是四种非常相似且难以分离的分子。
