Sandvig K, Olsnes S, Pihl A
J Biol Chem. 1976 Jul 10;251(13):3977-84.
Kinetic parameters of the interaction of the toxic lectins abrin and ricin with human erythrocytes and HeLa cells have been measured. The binding of 125I-labeled abrin and ricin to human erythrocytes and to HeLa cells at 37 degrees was maximal around pH 7, whereas at 0 degrees the binding was similar over a broad pH range. The binding occurred at similar rates at 0 degrees and 37 degrees with rate constants in the range 0.9 to 3.0 X 10(5) M-1 s-1. The dissociation was strongly temperature-dependent with rate constants in the range 3.4 to 45 X 10(-4) s-1 at 0 degrees and 3.9 to 18 X 10(-3) s-1 at 37 degrees. The presence of unlabeled lectins as well as lactose increased the rate of dissociation. The association constants measured at equilibrium or calculated from the rate constants were between 0.64 X 10(8) M-1 and 8.2 X 10(8) M-1 for abrus lectins, and between 8.0 X 10(6) M-1 and 4.2 X 10(8) M-1 for ricinus lectins. The association constants for the toxins were lower at 37 degrees than at 0 degrees. Isolated ricin B chain appeared to bind with similar affinity as intact ricin. The number of binding sites was estimated to be 2 to 3 X 10(6) per erythrocyte and 1 to 3 X 10(7) per HeLa cell. The binding sites of HeLa cells all displayed a uniform affinity towards abrin and ricin, both at 0 degrees and at 37 degrees. The same was the case with the binding sites of erythrocytes at 0 degrees. However, the data indicated that at 20 degrees erythrocytes possessed binding sites with two different affinities. Only a fraction of the cell-bound toxin appeared to be irreversibly bound and could not be removed by washing with 0.1 M lactose. The fraction of the total amount of bound toxin which became irreversibly bound to HeLa cells was for both toxins about 2 X 10(-3)/min at 37 degrees, whereas no toxin was irreversibly bound at 0 degrees. In the case of erythrocytes no toxin became irreversibly bound, either at 0 degrees or 37 degrees, indicating that the toxins are unable to penetrate into these cells.
已测定了有毒凝集素相思子毒素和蓖麻毒素与人红细胞及HeLa细胞相互作用的动力学参数。125I标记的相思子毒素和蓖麻毒素在37℃时与人类红细胞及HeLa细胞的结合在pH 7左右达到最大值，而在0℃时，在较宽的pH范围内结合情况相似。在0℃和37℃时结合速率相似，速率常数在0.9至3.0×10⁵ M⁻¹ s⁻¹范围内。解离强烈依赖温度，0℃时速率常数在3.4至45×10⁻⁴ s⁻¹范围内，37℃时在3.9至18×10⁻³ s⁻¹范围内。未标记的凝集素以及乳糖的存在会增加解离速率。在平衡时测定或根据速率常数计算得到的结合常数，相思子凝集素在0.64×10⁸ M⁻¹至8.2×10⁸ M⁻¹之间，蓖麻凝集素在8.0×10⁶ M⁻¹至4.2×10⁸ M⁻¹之间。毒素的结合常数在37℃时低于0℃时。分离出的蓖麻毒素B链似乎与完整蓖麻毒素具有相似的亲和力。估计每个红细胞的结合位点数为2至3×10⁶个，每个HeLa细胞为1至3×10⁷个。HeLa细胞的结合位点在0℃和37℃时对相思子毒素和蓖麻毒素均表现出均匀的亲和力。红细胞在0℃时的结合位点情况也是如此。然而，数据表明在20℃时红细胞具有两种不同亲和力的结合位点。只有一部分细胞结合的毒素似乎是不可逆结合的，用0.1 M乳糖洗涤无法去除。在37℃时，与HeLa细胞不可逆结合的毒素占总结合毒素量的比例，两种毒素均约为2×10⁻³/分钟，而在0℃时没有毒素不可逆结合。对于红细胞，在0℃或37℃时均没有毒素不可逆结合，这表明毒素无法穿透这些细胞。
