Purification and properties of L cell-derived colony-stimulating factor.
Purification and properties of L cell-derived colony-stimulating factor.
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L 细胞来源的集落刺激因子的纯化和特性。
DOI：
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发表时间：
1979
期刊：
影响因子：
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通讯作者：
Shadduck Rk
中科院分区：
文献类型：
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作者：
A. Waheed;Shadduck Rk
通过L细胞在无血清培养基中生长制备CSF。该条件培养基进行六步纯化方案，包括超滤、醇沉淀和通过DEAE-纤维素、Con A-Sepharose、Sephadex G-150和蔗糖密度梯度离心的分离。将所得CSF纯化1000倍，起始活性回收率为50%至70%。粒细胞和巨噬细胞集落形成用5 × 10 - 12 M CSF检测;最大集落用3 ng/骨髓培养物获得。获得两个主要的活性峰，一个是nonadjacent到Con A，而另一个是结合和具体的α-甲基葡萄糖苷洗脱。这两个馏分含有碳水化合物残基，因为他们与PAS染色贪婪，被高碘酸盐灭活，并表现出改变电泳迁移率与神经氨酸酶治疗后。碘化后，每个纯化的馏分在SDS-丙烯酰胺凝胶中迁移为单一条带。分子量估计为65，000至70，000道尔顿。用巯基乙醇还原后，CSF组分被还原成分子量约为35，000的亚基。这些研究证实了L细胞CSF的糖蛋白性质和亚基组成。本文所述的方法可用于纯化CSF的Con A-粘附和Con A-非粘附形式。
CSF was prepared by the growth of L cells in serum-free culture medium. This conditioned medium was subjected to a six-step purification schedule which included ultrafiltration, alcohol precipitation, and separation by DEAE-cellulose, Con A-Sepharose, Sephadex G-150, and sucrose density-gradient centrifugation. The resultant CSF was 1000-fold purified with 50% to 70% recovery of the starting activity. Granulocyte and macrophage colony formation was detected with 5 x 10(-12M CSF; maximum colonies were obtained with 3 ng per marrow culture. Two major peaks of activity were obtained; one was nonadherent to Con A, whereas the other was bound and specifically eluted with alpha-methylglucoside. Both fractions contained carbohydrate residues as they stained avidly with PAS, were inactivated by periodate, and showed altered electrophoretic mobility after treatment with neuraminidase. Following iodination, each purified fraction migrated in a single band in SDS-acrylamide gels. The molecular weight was estimated as 65,000 to 70,000 daltons. Following reduction with mercaptoethanol, the CSF fractions were reduced into subunits with molecular weights of approximately 35,000. These studies confirm the glycoprotein nature and subunit composition of L cell CSF. The methods described herein are useful for the purification of both the Con A-adherent and con A-nonadherent forms of CSF.