Prostaglandin-sensitive adenylate cyclase of a murine macrophage-like cell line (P388D1): II. Isolation and partial characterization of PGE2-binding proteins.
Prostaglandin-sensitive adenylate cyclase of a murine macrophage-like cell line (P388D1): II. Isolation and partial characterization of PGE2-binding proteins.
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鼠巨噬细胞样细胞系（P388D1）的前列腺素敏感腺苷酸环化酶：II。
DOI：
10.4049/jimmunol.133.5.2662
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发表时间：
1984
影响因子：
4.4
通讯作者：
T. Suzuki
中科院分区：
文献类型：
--
作者：
R. Fernandez;T. Suzuki
研究了小鼠巨噬细胞系P388 D1前列腺素E2结合位点的性质。在细胞松弛素D（10 μ g/ml）存在下，4 ℃时[3 H]-PGE 2与完整P388 D1细胞的特异性结合在浓度大于7.5 × 10（-9）M时接近饱和，并且可以被未标记的PGE 2最有效地取代，而被未标记的PGI 2较不有效地取代。结合数据的Scatchard分析清楚地表明了PGE 2结合亲和力的异质性，并显示存在约3.9 fmol/10（8）个细胞的高亲和力位点（KD = 1.1 × 10（-9）M）和约24 fmol/10（8）个细胞的低亲和力位点（KD = 2 × 10（-8）M）。通过在与PGE 2偶联的Sepharose 4 B上进行亲和层析，从放射性标记的P388 D1细胞的去污剂裂解物中分离PGE 2结合蛋白。通过连续使用Sephadex G-100凝胶过滤和在二硫苏糖醇（1 mM）和Triton X-100（0.5%）存在下的等电聚焦进一步纯化亲和分离的材料。最后一步产生约0.25%的原始放射性，其在pH值接近6.5时急剧聚焦为单峰。电聚焦的PGE 2结合蛋白迁移为单一条带，分子量为10000。在SDS-PAGE中的95，000。电聚焦的PGE 2结合蛋白特异性结合[3 H]-PGE 2，但再次显示其亲和力的异质性。
Properties of prostaglandin (PG) E2 binding sites of a murine macrophage cell line (P388D1) were investigated. The specific binding of [3H]-PGE2 to intact P388D1 cells at 4 degrees C in the presence of cytochalasin D (10 micrograms/ml) approached saturation at concentration greater than 7.5 X 10(-9) M, and could be displaced most effectively by unlabeled PGE2 and less effectively by unlabeled PGI2. The Scatchard analysis of the binding data clearly indicated the heterogeneity with respect to the PGE2 binding affinity and showed the presence of about 3.9 fmol/10(8) cells of the high affinity sites (KD = 1.1 X 10(-9) M) and about 24 fmol/10(8) cells of the low affinity sites (KD = 2 X 10(-8) M). PGE2-binding proteins were isolated from the detergent lysate of the radiolabeled P388D1 cells by affinity chromatography on Sepharose 4B coupled to PGE2. The affinity-isolated materials were further purified by successive use of Sephadex G-100 gel filtration and isoelectric focusing in the presence of dithiothreitol (1 mM) and Triton X-100 (0.5%). The final step yielded about 0.25% of the original radioactivity, which sharply focused as a single peak at pH near 6.5. The electrofocused PGE2-binding proteins migrated as a single band with a m.w. of 95,000 during SDS-PAGE. The electrofocused PGE2-binding proteins bound specifically to [3H]-PGE2 but showed again the heterogeneity with respect to their affinity.