Panda D, Chakrabarti G, Hudson J, Pigg K, Miller H P, Wilson L, Himes R H
Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045, USA.
Biochemistry. 2000 May 2;39(17):5075-81. doi: 10.1021/bi992217f.
Deuterium oxide (D(2)O) is known to promote the assembly of tubulin into microtubules in vitro, to increase the volume of mitotic spindles and the number and length of spindle microtubules, and to inhibit mitosis. Reasoning that its actions on cellular microtubules could be due to modulation of microtubule dynamics, we examined the effects of replacing H(2)O with D(2)O on microtubule dynamic instability, treadmilling, and steady-state GTPase activity. We found that replacing 50% or more of the H(2)O with D(2)O promoted microtubule polymerization and stabilized microtubules against dilution-induced disassembly. Using steady-state axoneme-seeded microtubules composed of pure tubulin and video microscopy, we found that 84% D(2)O decreased the catastrophe frequency by 89%, the shortening rate by 80%, the growing rate by 50%, and the dynamicity by 93%. Sixty percent D(2)O decreased the treadmilling rate of microtubules composed of tubulin and microtubule-associated proteins by 42%, and 89% D(2)O decreased the steady-state GTP hydrolysis rate by 90%. The mechanism responsible for the ability of D(2)O to stabilize microtubule dynamics may involve enhancement of hydrophobic interactions in the microtubule lattice and/or the substitution of deuterium bonds for hydrogen bonds.
已知重水（D₂O）在体外可促进微管蛋白组装成微管，增加有丝分裂纺锤体的体积以及纺锤体微管的数量和长度，并抑制有丝分裂。鉴于其对细胞微管的作用可能是由于对微管动力学的调节，我们研究了用D₂O替代H₂O对微管动态不稳定性、踏车行为和稳态GTP酶活性的影响。我们发现，用D₂O替代50%或更多的H₂O可促进微管聚合，并使微管稳定，防止因稀释而解聚。使用由纯微管蛋白组成的稳态轴丝起始微管并结合视频显微镜，我们发现84%的D₂O可使灾难频率降低89%，缩短速率降低80%，生长速率降低50%，动态性降低93%。60%的D₂O可使由微管蛋白和微管相关蛋白组成的微管的踏车速率降低42%，89%的D₂O可使稳态GTP水解速率降低90%。D₂O稳定微管动力学能力的机制可能涉及增强微管晶格中的疏水相互作用和/或用氘键取代氢键。
