Bovine spleen, a convenient source for purifying a type I interferon receptor.
Bovine spleen, a convenient source for purifying a type I interferon receptor.
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牛脾，是纯化 I 型干扰素受体的便捷来源。
DOI：
10.1089/jir.1987.7.77
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发表时间：
1987
期刊：
影响因子：
--
通讯作者：
Branca,AA
中科院分区：
文献类型：
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作者：
Branca,AA
研究了牛脾脏中是否存在放射性碘标记人干扰素-α2（125 I标记HuIFN-α2）的受体。通过均质化和差速离心制备膜，并用最近开发的组织膜测定法分析标记的IFN结合。IFN与牛脾膜的亲和常数（Ka）为3.1 ± 0.99 ± 10 ~（10）M ~（-1），受体含量为8.4 ± 0.74 fmol/mg（湿重）。将这些膜上的标记的IFN-受体复合物与1.0 m亚甲基二醇双（琥珀酰亚胺基琥珀酸酯（EGS））化学交联，并进行SDS-PAGE和放射自显影。未标记的HuIFN-α2以剂量依赖性方式抑制放射自显影上观察到的137 kD复合物的形成。在抑制标记的IFN与膜结合的浓度下。在SDS存在下，通过在Ultragel AcA 34柱上的凝胶过滤纯化交联反应的产物，并通过SDS-PAGE和放射自显影检查。除137-kD复合物外，在放射性柱剖面中观察到几种低分子量物质，其迁移至10%聚丙烯酰胺凝胶底部。合并含有137-kD复合物的级分，浓缩，并用作内切糖苷酶消化测定的底物。内切糖苷酶H（EndoH）对137-kD复合物的迁移没有影响，而肽：N-糖苷酶F（PNGase F）增加了137-kD带迁移到105 kD的位置。这些研究首次提供了牛I型IFN受体的结构表征，并证明牛脾是纯化受体糖蛋白的起始材料的方便来源。
Bovine spleen was investigated for the presence of receptors for radioiodinated human interferon-α2(125I-labeled HuIFN-α2). Membranes were prepared by homogenization and differential centrifugation and analyzed for labeled IFN binding with recently developed tissue membrane assays. The characteristics of IFN binding included an affinity constant (Ka) of 3.1 ± 0.99 ± 1010M-1and a receptor content of 8.4 ± 0.74 fmoles/mg (wet weight) of bovine spleen membranes. The labeled IFN-receptor complex on these membranes was chemically cross-linked with 1.0 mMethylene glycol bis(succinimidyl succinate (EGS), and subjected to SDS-PAGE and autoradiography. The formation of a 137-kD complex observed on autoradiographs was inhibited in a dose-dependent manner by unlabeled HuIFN-α2. at concentrations that inhibited the binding of labeled IFN to the membranes. The products of the cross-linking reaction were purified by gel filtration on a column of Ultragel AcA34 in the presence of SDS and examined by SDS-PAGE and autoradiography. In addition to the 137-kD complex, several low-molecular-weight species were observed in the column profile of radioactivity which migrated to the bottom of 10% polyacrylamide gels. The fractions containing the 137-kD complex were pooled, concentrated, and utilized as a substrate for endoglycosidase digestion assays. Endoglycosidase H (EndoH) had no affect on the migration of the 137-kD complex while peptide:N-glycosidase F (PNGase F) increased the migration of the 137-kD band to a position with anMrof 105 kD. These studies provide for the first time the structural characterization of the bovine Type I IFN receptor and demonstrate that the bovine spleen is a convenient source of starting material for purification of the receptor glycoprotein.