Purification and characterization of human colony-stimulating factor 1 from human pancreatic carcinoma (MIA PaCa-2) cells.
Purification and characterization of human colony-stimulating factor 1 from human pancreatic carcinoma (MIA PaCa-2) cells.
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从人胰腺癌 (MIA PaCa-2) 细胞中纯化和表征人集落刺激因子 1。
DOI：
10.1016/0003-9861(87)90653-9
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发表时间：
1987
影响因子：
3.9
通讯作者：
Yunis,AA
中科院分区：
文献类型：
--
作者：
Shieh,JH;Cini,JK;Wu,MC;Yunis,AA
采用常规层析和高效液相层析相结合的方法，从人胰腺癌细胞系（MIA PaCa-2）的无血清条件培养基中纯化集落刺激因子1（CSF-1）。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和谷氨酸残基的N-末端氨基酸分析，证明了人CSF-1的纯度，其具有Mr 42，000- 50，000的弥散性单一条带。CSF-1在50 °C下稳定30分钟。它对胰蛋白酶、胰凝乳蛋白酶和枯草杆菌蛋白酶处理敏感，但对木瓜蛋白酶消化不太敏感。用不同的糖苷酶处理CSF-1不影响生物活性。巯基试剂如二硫苏糖醇（DTT）、碘乙酸和N-乙基马来酰亚胺在1 mM浓度下对CSF-1的生物活性无影响，而10 mmDTT和1 mmSDS联合使用则完全抑制CSF-1的活性。在变性和还原条件下，CSF-1在SDS-PAGE上显示为Mr 21，000- 25，000的单一蛋白条带，同时失去其活性，表明人CSF-1可能由两个相似的亚基组成，完整的四级结构是生物活性所必需的。当用神经氨酸酶和内切-β-d-N-乙酰氨基葡萄糖苷酶D处理时，CSF-1的分子量降低至36，000 - 40，000，并且在巯基乙醇存在下降低至18，000 - 20，000。由于内切-β-d-N-乙酰氨基葡萄糖苷酶D的特异性，建议糖部分是Asn连接的“复合型”单元。
Colony-stimulating factor 1 (CSF-1) was purified from the serum-free conditioned medium of a human pancreatic carcinoma cell line (MIA PaCa-2) by a combination of conventional chromatography and high-performance liquid chromatography. The purity of human CSF-1 was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a diffuse single band ofMr42,000–50,000 and by N-terminal amino acid analysis of glutamate residue. The CSF-1 was stable at 50 °C for 30 min. It is sensitive to treatment with trypsin, chymotrypsin, and subtilisin but less sensitive to papain digestion. Treatment of CSF-1 with different glycosidases did not affect the biological activity. Sulfhydryl reagents such as dithiothreitol (DTT), iodoacetic acid, andN-ethylmaleimide did not affect the biological activity at the concentration of 1 mm. However, CSF-1 activity was inhibited totally by the combination of 10 mmDTT and 1 mmSDS. Under denaturing and reducing conditions, CSF-1 appeared on SDS-PAGE as a single protein band ofMr21,000–25,000 and concurrently lost its activity, indicating that human CSF-1 possibly consists of two similar subunits and that the intact quaternary structure is essential for the biological activity. When treated with neuraminidase and endo-β-d-N-acetylglucosaminidase D, the molecular weight of CSF-1 was reduced to 36,000–40,000, and to 18,000–20,000 in the presence of mercaptoethanol. Because of the specificity of endo-β-d-N-acetylglucosaminidase D, it is suggested that the carbohydrate moieties are Asn-linked “complex-type” units.