Immunofluorescent visualization of specifically bound thrombin reveals cellular heterogeneity in number and density of preclustered receptors.
Immunofluorescent visualization of specifically bound thrombin reveals cellular heterogeneity in number and density of preclustered receptors.
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特异性结合的凝血酶的免疫荧光可视化揭示了预簇受体的数量和密度的细胞异质性。
DOI：
10.1002/jcp.1041170304
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发表时间：
1983
影响因子：
5.6
通讯作者：
Carney,DH
中科院分区：
文献类型：
--
作者：
Carney,DH
通过间接免疫荧光将凝血酶与甲醛固定的小鼠胚胎 (ME) 细胞的结合可视化为点状图案，直径约 500 nm 的点分布在整个细胞表面。比较平行培养物上 125 I-凝血酶的结合和点出现的实验表明，免疫荧光模式对于凝血酶与高亲和力受体的结合具有特异性。在凝血酶结合之前固定在乙醇或戊二醛中的细胞以及维持在 4°C 的细胞上观察到类似的模式。在许多已建立的细胞系中也观察到了这些模式。因此，在凝血酶与所有细胞上这些受体结合之前，凝血酶受体可以聚集。将与 CHO 细胞结合的 125 I-凝血酶的量与每个细胞的荧光点的数量进行比较表明，每个点代表超过 1000 个受体的簇。在 ME 细胞上，每个细胞的凝血酶受体簇数量从少于 50 个到超过 5,000 个不等。根据之前的研究，这表明在 ME 细胞上，每个簇平均包含约 200 个凝血酶结合位点，其中一些细胞具有很少（如果有的话）受体，而另一些细胞每个细胞具有超过一百万个受体。
Thrombin binding to formaldehyde‐fixed mouse embryo (ME) cells was visualized by indirect immunofluorescence as a dot‐like pattern with dots of approximately 500 nm diameter located over the entire cell surface. Experiments comparing the binding of125I‐thrombin and dot appearance on parallel cultures indicate that the immunofluorescent pattern is specific for thrombin‐binding to high‐affinity receptors. Similar patterns were observed on cells fixed in ethanol or glutaraldehyde prior to thrombin binding and on cells maintained at 4°C. These patterns were also observed in a number of established cell lines. Thus, thrombin receptors may be clustered prior to thrombin binding on all cells with these receptors. Comparing the amount of125I‐thrombin bound to CHO cells with the number of fluorescent dots per cell indicated that each dot represents a cluster of over 1000 receptors. On ME cells, the number of thrombin receptor clusters per cell ranged from fewer than 50 to over 5,000. Based on previous studies, this indicates that on ME cells each cluster contains an average of approximately 200 thrombin binding sites with some cells having few, if any, receptors and others having more than a million receptors per cell.