Tsavaler L, Stein B S, Sussman H H
J Cell Physiol. 1986 Jul;128(1):1-8. doi: 10.1002/jcp.1041280102.
Specific binding of ferric bovine transferrin to the human transferrin receptor was investigated using K562 cells propagated in serum-free medium without transferrin supplemented with 10(-5) elemental iron. Affinity chromatography of solubilized extracts of K562 cells surface-labeled with 125I was performed using bovine transferrin- and human transferrin-Sepharose 4B resins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of resin eluates reveal that bovine transferrin specifically binds a Mr = 188,000 protein which dissociates into a Mr = 94,000 protein under reducing conditions, a finding identical to what is seen with human transferrin. The Mr = 94,000 reduced protein isolated by bovine transferrin resin shows an identical one-dimensional partial proteolytic digestion map with that of the human transferrin receptor. Unlabeled bovine transferrin was shown to specifically compete 125I-labeled human transferrin from the human transferrin receptor on the surface of K562 cells at 4 degrees C in a similar manner as unlabeled human transferrin; however, approximately a 2,000-fold higher concentration of bovine ligand was required to achieve comparable competition (50% inhibition of binding). Indirect immunofluorescence cytolocalization of bovine transferrin in K562 cells grown in serum-free medium supplemented with ferric bovine transferrin reveal patterns similar to those seen for human transferrin (both focal perinuclear and diffuse cytoplasmic fluorescence). Monensin treatment results in a dramatic accumulation of bovine ligand in perinuclear aggregates, suggesting that it is recycled through the Golgi, as is human transferrin. K562 cells grown in serum-free medium supplemented with either 300 micrograms/ml of ferric human or ferric bovine transferrin were found to demonstrate superimposable growth curves.
使用在无转铁蛋白的无血清培养基中培养并补充10⁻⁵ 元素铁的K562细胞，研究了三价铁牛转铁蛋白与人转铁蛋白受体的特异性结合。使用牛转铁蛋白和人转铁蛋白-琼脂糖4B树脂对用¹²⁵I进行表面标记的K562细胞的溶解提取物进行亲和层析。树脂洗脱液的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示，牛转铁蛋白特异性结合一种分子量为188,000的蛋白质，该蛋白质在还原条件下解离为分子量为94,000的蛋白质，这一发现与用人转铁蛋白观察到的结果相同。通过牛转铁蛋白树脂分离的分子量为94,000的还原蛋白显示出与人转铁蛋白受体相同的一维部分蛋白酶解图谱。未标记的牛转铁蛋白在4℃下能以与未标记的人转铁蛋白类似的方式，特异性地从K562细胞表面的人转铁蛋白受体上竞争¹²⁵I标记的人转铁蛋白；然而，需要大约高2000倍浓度的牛配体才能实现相当的竞争（50%的结合抑制）。在补充了三价铁牛转铁蛋白的无血清培养基中生长的K562细胞中，牛转铁蛋白的间接免疫荧光细胞定位显示出与用人转铁蛋白观察到的模式相似（核周灶性和弥漫性细胞质荧光）。莫能菌素处理导致牛配体在核周聚集体中大量积累，表明它像人转铁蛋白一样通过高尔基体循环。发现在补充有300微克/毫升三价铁人转铁蛋白或三价铁牛转铁蛋白的无血清培养基中生长的K562细胞表现出重叠的生长曲线。
