Douglas G C, King B F
Biochem J. 1985 Apr 15;227(2):639-50. doi: 10.1042/bj2270639.
We have developed conditions for studying the binding, uptake, degradation and transport of 125I-labelled IgG by yolk sac in vitro. Specific binding to tissue at 4 degrees C and to paraformaldehyde-treated tissue at 37 degrees C was time- and temperature-dependent and showed saturation kinetics (Kd,4 degrees C = 2.9 X 10(-6) M, Kd,37 degrees C = 5.3 X 10(-6) M). Uptake was studied at 37 degrees C using untreated tissue (K uptake = 13.3 X 10(-6) M) and was inhibited by preincubation with metabolic poisons but not with cycloheximide. Tissue that had been incubated with 125I-labelled IgG at 37 degrees C released radiolabelled degradation products and intact 125I-labelled IgG into the medium. Experiments with paraformaldehyde-treated and untreated tissue showed that release of intact 125I-labelled IgG was mostly the result of ligand dissociation from surface binding sites. However, more 125I-labelled IgG was released from untreated tissue than could be accounted for solely by loss of surface-bound ligand and the difference was presumed to reflect uptake, transport and exocytosis of 125I-labelled IgG. Degradation of 125I-labelled IgG was inhibited by leupeptin and lysosomotropic amines. These drugs had no detectable effect on 125I-labelled IgG release. The results suggest that degradation and transport of IgG are not intimately related and are consistent with a previously proposed model for IgG transport via coated vesicles which do not fuse with lysosomes and for non-selective uptake into another class of vesicle which does fuse with lysosomes.
我们已经建立了体外研究卵黄囊对125I标记的IgG的结合、摄取、降解和转运的条件。在4℃下与组织以及在37℃下与经多聚甲醛处理的组织的特异性结合具有时间和温度依赖性，并呈现饱和动力学（4℃时的解离常数Kd = 2.9×10⁻⁶ M，37℃时的Kd = 5.3×10⁻⁶ M）。在37℃下使用未处理的组织研究摄取情况（摄取常数K = 13.3×10⁻⁶ M），并且摄取受到与代谢毒物预孵育的抑制，但不受环己酰亚胺的抑制。在37℃下用125I标记的IgG孵育过的组织将放射性标记的降解产物和完整的125I标记的IgG释放到培养基中。对经多聚甲醛处理和未处理的组织进行的实验表明，完整的125I标记的IgG的释放主要是配体从表面结合位点解离的结果。然而，从未处理的组织中释放的125I标记的IgG比仅由表面结合配体的丢失所解释的要多，这种差异被推测反映了125I标记的IgG的摄取、转运和胞吐作用。亮抑酶肽和溶酶体促渗胺抑制125I标记的IgG的降解。这些药物对125I标记的IgG的释放没有可检测到的影响。结果表明，IgG的降解和转运并非密切相关，这与先前提出的通过不与溶酶体融合的包被小泡进行IgG转运以及通过与溶酶体融合的另一类小泡进行非选择性摄取的模型一致。
