Ward L D, Cantrill R C, Heithier H, Peters R, Helmreich E J
Department of Physiological Chemistry, University of Würzburg, F.R.G.
Biochim Biophys Acta. 1988 Oct 7;971(3):307-16. doi: 10.1016/0167-4889(88)90146-2.
When monolayer cultured hepatocytes were incubated with 1 nM [125I]glucagon at 30 degrees C, equilibrium was reached after 10 min, whereas at 4 degrees C, equilibrium was reached after 60 min. At the higher temperature, 11.2% of the bound ligand was broken down after 60 min, at the lower temperature, the amount of degradation was negligible. At 30 degrees C, acid-washing did not remove specifically bound ligand; thus, it was assumed that the ligand was internalised at this temperature, since some of the specifically bound ligand could be washed off at lower temperatures. This was confirmed in experiments when monolayer cultures of hepatocytes were incubated with fluorescein-labelled derivatives of glucagon. The distribution of specific binding on the cell surface was studied at both 30 and 4 degrees C using video intensification microscopic techniques. In keeping with studies using radiolabelled glucagon, more fluorescence was detected following incubation at 4 degrees C than at 30 degrees C and it could be removed by washing the cells. Video intensification microscopy indicated that at the lower temperature, the bound ligand was distributed all over the cell surface. At the higher temperature, ligand-derived fluorescence could only be detected in mobile intracellular vesicles.
当单层培养的肝细胞在30℃下与1 nM [125I]胰高血糖素孵育时，10分钟后达到平衡，而在4℃下，60分钟后达到平衡。在较高温度下，60分钟后11.2%的结合配体被分解，在较低温度下，降解量可忽略不计。在30℃下，酸洗不能去除特异性结合的配体；因此，推测该配体在该温度下被内化，因为在较低温度下一些特异性结合的配体可以被洗去。当肝细胞单层培养物与荧光素标记的胰高血糖素衍生物孵育时，实验证实了这一点。使用视频增强显微镜技术在30℃和4℃下研究了细胞表面特异性结合的分布。与使用放射性标记胰高血糖素的研究一致，在4℃孵育后比在30℃孵育后检测到更多的荧光，并且可以通过洗涤细胞将其去除。视频增强显微镜显示，在较低温度下，结合的配体分布在整个细胞表面。在较高温度下，只能在移动的细胞内囊泡中检测到配体衍生的荧光。
