Klausner R D, Van Renswoude J, Ashwell G, Kempf C, Schechter A N, Dean A, Bridges K R
J Biol Chem. 1983 Apr 25;258(8):4715-24.
Human diferric transferrin binds to the surface of K562 cells, a human leukemic cell line. There are about 1.6 X 10(5) binding sites per cell surface, exhibiting a KD of about 10(-9) M. Upon warming cells to 37 degrees C there is a rapid increase in uptake to a steady state level of twice that obtained at 0 degree C. This is accounted for by internalization of the ligand as shown by the development of resistance to either acid wash or protease treatment of the ligand-cell association. After a minimum residency time of 4-5 min, undegraded transferrin is released from the cell. Internalization is rapid but is dependent upon cell surface occupancy; at occupancies of 20% or greater the rate coefficient is maximal at about 0.1-0.2 min-1. In the absence of externally added ligand only 50% of the internalized transferrin completes the cycle and is released to the medium with a rate coefficient of 0.05 min-1. The remaining transferrin can be released from the cell only by the addition of ligand, suggesting a tight coupling between cell surface binding, internalization, and release of internalized ligand. There is a loss of cell surface-binding capacity that accompanies transferrin internalization. At low (less than 50%) occupancy this loss is monotonic with the extent of internalization. Even at saturating levels of transferrin, the loss of surface receptors upon internalization never exceeds 60-70% of the initial binding capacity. This suggests that receptors enter the cell with ligand but are replaced so as to maintain a constant, albeit reduced, receptor number on the cell surface. In the absence of ligand, the cell surface receptor number returns at 37 degrees C. Neither sodium azide nor NH4Cl blocks internalization of ligand. However, they both prevent the release of transferrin from the cell thus halting the transferrin cycle. Excess ligand can overcome the block due to NH4Cl but not azide although the cycle is markedly slower. Iron is delivered to these cells by transferrin at 37 degrees C with a rate coefficient of 0.15 to 0.2 min-1. The iron is released from the transferrin and the majority is found in intracellular ferritin. There is a large internal receptor pool comprising 70 to 80% of the total cell receptors and this may be involved in maintaining the steady state iron uptake.
人去铁转铁蛋白可与人类白血病细胞系K562细胞的表面结合。每个细胞表面约有1.6×10⁵个结合位点，解离常数约为10⁻⁹M。将细胞升温至37℃时，摄取量迅速增加至稳态水平，该稳态水平是0℃时的两倍。这是由于配体的内化作用，酸洗或蛋白酶处理配体 - 细胞结合物后产生的抗性证明了这一点。经过4 - 5分钟的最短停留时间后，未降解的转铁蛋白从细胞中释放出来。内化过程很快，但取决于细胞表面的占有率；占有率达到20%或更高时，速率系数在约0.1 - 0.2分钟⁻¹时达到最大值。在没有外部添加配体的情况下，只有50%内化的转铁蛋白完成循环并以0.05分钟⁻¹的速率系数释放到培养基中。其余的转铁蛋白只有通过添加配体才能从细胞中释放出来，这表明细胞表面结合、内化和内化配体的释放之间存在紧密的耦合。转铁蛋白内化会伴随细胞表面结合能力的丧失。在低占有率（低于50%）时，这种丧失与内化程度呈单调关系。即使在转铁蛋白饱和水平下，内化时表面受体的丧失也不会超过初始结合能力的60 - 70%。这表明受体与配体一起进入细胞，但会被替换，从而在细胞表面维持一个恒定但减少的受体数量。在没有配体的情况下，细胞表面受体数量在37℃时会恢复。叠氮化钠和氯化铵都不会阻断配体的内化。然而，它们都能阻止转铁蛋白从细胞中释放，从而使转铁蛋白循环停止。过量的配体可以克服氯化铵造成的阻断，但不能克服叠氮化钠造成的阻断，尽管循环明显变慢。在37℃时，转铁蛋白将铁传递给这些细胞，速率系数为0.15至0.2分钟⁻¹。铁从转铁蛋白中释放出来，大部分存在于细胞内铁蛋白中。存在一个庞大的内部受体池，占细胞总受体的70%至80%，这可能参与维持铁摄取的稳态。
