Tsunoo H, Sussman H H
Arch Biochem Biophys. 1983 Aug;225(1):42-54. doi: 10.1016/0003-9861(83)90005-x.
This study systematically examined the characteristics of specific binding of adult diferric transferrin to its receptor using a Triton X-100 solubilized preparation from human placentas as the receptor source. The following information was obtained. The ionic strength for maximal binding is in the range of 0.1-0.3 M NaCl. The pH optimum for specific binding extends over the range, from pH 6.0-10.0. Specific binding of diferric transferrin is not affected by 2.5 approximately 50 mM CaCl2 or by 10 mM EDTA. Triton X-100 in the concentration range of 0.02-3.0% does not affect specific binding. Specific binding is saturated within 10 min at 25 or 37 degrees C in the presence of excess amounts of diferric transferrin. The binding is reversible and the dissociation of diferric transferrin from the transferrin receptor is complete within 40 min at 25 degrees C. Apotransferrin, both adult and fetal, showed less binding than the holotransferrin species by competitive binding assay in the presence of 10 mM EDTA independent of up to 20 mM CaCl2. A 1500-fold molar excess of adult and fetal apotransferrin is required to give 40% inhibition for 125I-labeled diferric transferrin binding. Since calcium ion is not a factor, and since apotransferrin has such high binding affinity for iron (Ka = 1 X 10(24], this experiment suggests that the EDTA was necessary to prevent conversion of apotransferrin to holotransferrin from available iron in the reaction system. The specificity of the transferrin receptor for transferrin was examined by competitive binding studies in which 125I-diferric transferrin binding was measured in the presence of a series of other proteins. The proteins tested in the competitive binding studies were classified into three groups; in the first group were human serum albumin and ovalbumin; in the second group were proteins containing iron ions, such as hemoglobin, hemoglobin-haptoglobin complex, heme-hemopexin complex, ferritin, and diferric lactoferrin; in the third group were the metal-binding serum proteins, ceruloplasmin and metallothionein. None of these proteins except ferritin showed inhibition of diferric transferrin binding to the receptor. The effect of ferritin was small since a 700- to 1500-fold molar excess of ferritin is required for 50% inhibition of binding of diferric transferrin to the receptor.
本研究使用从人胎盘中用Triton X-100增溶制备的物质作为受体来源，系统地研究了成人双铁运铁蛋白与其受体特异性结合的特性。获得了以下信息。最大结合的离子强度在0.1 - 0.3M NaCl范围内。特异性结合的最适pH范围为pH 6.0 - 10.0。双铁运铁蛋白的特异性结合不受2.5至50mM CaCl2或10mM EDTA的影响。浓度范围为0.02 - 3.0%的Triton X-100不影响特异性结合。在过量双铁运铁蛋白存在下，于25或37℃时，特异性结合在10分钟内达到饱和。结合是可逆的，在25℃时，双铁运铁蛋白从转铁蛋白受体上的解离在40分钟内完成。在存在10mM EDTA且与高达20mM CaCl2无关的情况下，通过竞争性结合试验发现，成人和胎儿的脱铁运铁蛋白的结合都比全铁运铁蛋白少。需要1500倍摩尔过量的成人和胎儿脱铁运铁蛋白才能对125I标记的双铁运铁蛋白结合产生40%的抑制。由于钙离子不是一个影响因素，且脱铁运铁蛋白对铁具有如此高的结合亲和力（Ka = 1×10²⁴），该实验表明EDTA对于防止反应体系中脱铁运铁蛋白从可利用的铁转化为全铁运铁蛋白是必要的。通过竞争性结合研究检测了转铁蛋白受体对转铁蛋白的特异性，其中在一系列其他蛋白质存在的情况下测量125I-双铁运铁蛋白的结合。在竞争性结合研究中测试的蛋白质分为三组；第一组是人血清白蛋白和卵清蛋白；第二组是含铁离子的蛋白质，如血红蛋白、血红蛋白-触珠蛋白复合物、血红素-血红素结合蛋白复合物、铁蛋白和双铁乳铁蛋白；第三组是金属结合血清蛋白，铜蓝蛋白和金属硫蛋白。除铁蛋白外，这些蛋白质均未显示出对双铁运铁蛋白与受体结合的抑制作用。铁蛋白的作用很小，因为需要700至1500倍摩尔过量的铁蛋白才能对双铁运铁蛋白与受体的结合产生50%的抑制。
