Gossuin A, Maloteaux J M, Trouet A, Laduron P
Biochim Biophys Acta. 1984 May 22;804(1):100-6. doi: 10.1016/0167-4889(84)90103-4.
Binding properties of [3H] dexetimide , L-quinuclidinyl[phenyl-4-3H] benzilate and [3H]methylscopolamine were compared with intact 108 CC 15 cells and membrane preparations of those. The ability of the three ligands to label specifically muscarinic receptors on membrane fractions was quite similar. By contrast, when performed with intact cells, [3H] dexetimide and L-quinuclidinyl [phenyl-4-3H]benzilate revealed higher nonspecific binding which was prevented by methylamine, suggesting a trapping of the ligands within the cells presumably in the lysosomes. To the contrary, such nonspecific 'binding' or trapping was not detectable when [3H]methylscopolamine was used as ligand, a fact which makes this ligand particularly appropriate for labelling cell surface muscarinic receptors. It is concluded that more caution is needed in binding studies when performed with intact cells; indeed, besides specific binding on receptor sites, [3H]ligand can be entrapped within the cell and can even sometimes give the illusion of specific binding. The use of lysosomal agents which do not interfere with specific receptors on membrane preparations should allow one, in most cases, to discard the possibility of a trapping phenomenon in intact cells.
将[3H]右苄替米特、L-喹核醇基[苯基-4-3H]苯甲酸酯和[3H]甲基东莨菪碱的结合特性与完整的108 CC 15细胞及其膜制剂进行了比较。这三种配体特异性标记膜组分上毒蕈碱受体的能力相当相似。相比之下，当用完整细胞进行实验时，[3H]右苄替米特和L-喹核醇基[苯基-4-3H]苯甲酸酯显示出较高的非特异性结合，甲胺可阻止这种结合，这表明配体可能在溶酶体中被困在细胞内。相反，当使用[3H]甲基东莨菪碱作为配体时，这种非特异性“结合”或捕获无法检测到，这一事实使得该配体特别适合标记细胞表面的毒蕈碱受体。得出的结论是，在用完整细胞进行结合研究时需要更加谨慎；实际上，除了在受体位点上的特异性结合外，[3H]配体可能被困在细胞内，甚至有时会产生特异性结合的假象。使用不干扰膜制剂上特异性受体的溶酶体剂，在大多数情况下，应该可以排除完整细胞中存在捕获现象的可能性。
