Rai S S, Wolff J
Laboratory of Biochemical Pharmacology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Eur J Biochem. 1997 Dec 1;250(2):425-31. doi: 10.1111/j.1432-1033.1997.0425a.x.
Vinblastine promotes tubulin polymerization as measured by turbidity at 400 nm. Unlike microtubule assembly, this mode of polymerization does not require GTP and, in fact, GTP inhibits polymerization, as do other G nucleotides in the potency order: GtetraP > or = GTP > GDP > GMP > no nucleotide. Inhibition is not nucleoside-specific as ATP, ADP, and CTP also inhibit, and inorganic oligophosphates are as inhibitory as nucleotides in the order tetraphosphate approximately triphosphate > pyrophosphate >> phosphate. Inhibition of polymerization is a rough function of the number of anionic charges and can be mimicked by suramin or tartrate. It is not due to sequestration of magnesium or to debinding of vinblastine. The anion-induced decrease in turbidity generation is reflected in the amount of tubulin that is pelletable, but even in the absence of turbidity significant pelletable tubulin persists which can be assessed by 90 degree light scattering. Formation of this polymer is less sensitive to anions. Shearing of GTP-inhibited and vinblastine-induced samples promotes turbidity and addition of seeds made from vinblastine polymers leads to rapid increases in turbidity in a concentration-dependent manner. Adjustment of the vinblastine concentration permits the demonstration of a latent period for polymerization that can be shortened by polymer seeds. Vinblastine-induced polymerization shows a critical concentration, and, in the presence of GTP, two distinct critical concentrations can be identified. We conclude that charge-charge interactions play a significant role in the formation of vinblastine-induced polymers, and that their formation is a two-step process resembling a nucleation/elongation mechanism.
通过在400nm处的吸光度测量，长春碱可促进微管蛋白聚合。与微管组装不同，这种聚合模式不需要GTP，事实上，GTP会抑制聚合，其他G核苷酸也有抑制作用，其效力顺序为：四磷酸鸟苷≥GTP＞GDP＞GMP＞无核苷酸。抑制作用并非核苷特异性的，因为ATP、ADP和CTP也有抑制作用，无机寡磷酸盐与核苷酸的抑制作用相似，顺序为：四磷酸盐≈三磷酸盐＞焦磷酸盐＞＞磷酸盐。聚合抑制是阴离子电荷数量的大致函数，可被苏拉明或酒石酸盐模拟。这不是由于镁的螯合或长春碱的解离。阴离子诱导的吸光度降低反映在可沉淀的微管蛋白量上，但即使在没有吸光度的情况下，仍有大量可沉淀的微管蛋白存在，这可通过90度光散射评估。这种聚合物的形成对阴离子不太敏感。剪切受GTP抑制和长春碱诱导的样品会促进吸光度增加，添加由长春碱聚合物制成的种子会导致吸光度以浓度依赖的方式迅速增加。调整长春碱浓度可证明聚合存在潜伏期，聚合物种子可缩短潜伏期。长春碱诱导的聚合表现出临界浓度，在GTP存在的情况下，可确定两个不同的临界浓度。我们得出结论，电荷-电荷相互作用在长春碱诱导的聚合物形成中起重要作用，其形成是一个类似于成核/延伸机制的两步过程。
