Caplow M, Shanks J, Ruhlen R L
Department of Biochemistry, University of North Carolina, Chapel Hill 27599-7260.
J Biol Chem. 1988 Jul 25;263(21):10344-52.
Evidence for a slowly dissociating tubulin-GTP cap at microtubule ends was derived from observation of a delay for attaining a maximum disassembly rate, after the temperature of steady state microtubules was rapidly decreased from 36 to 34 degrees C. The possibility that the microtubules were capped by a single tubulin-GTP subunit on each subhelix was ruled out, by comparison of the disassembly kinetics following a temperature decrease and dilution. The existence of a subpopulation of microtubules that underwent irreversible or near irreversible disassembly was demonstrated by a 30-s lag for attainment of a maximum assembly rate, after steady state microtubules were shifted from 34 to 36 degrees C. A dynamic instability model predicts that a maximum assembly rate will be delayed until disappearance of a subpopulation of microtubules that disassemble before being recapped. Analysis indicates that the 30-s lag resulted because approximately 2% of the mass in the steady state microtubule population was uncapped and disassembling and not readily recapped. The half-time for recapping of disassembling microtubules, by addition of tubulin-GTP subunits to ends, was equal to or greater than 20 s. Since tubulin-GDP dissociated from microtubules at a rate of about 4500 s-1, slow recapping resulted in dramatic shortening of disassembling microtubules.
微管末端存在缓慢解离的微管蛋白 - GTP帽的证据来自于以下观察结果：在稳态微管的温度从36℃迅速降至34℃后，达到最大解聚速率存在延迟。通过比较温度降低和稀释后的解聚动力学，排除了微管在每个亚螺旋上由单个微管蛋白 - GTP亚基加帽的可能性。在稳态微管从34℃转变为36℃后，达到最大组装速率存在30秒的延迟，这证明了存在一部分经历不可逆或近乎不可逆解聚的微管亚群。动态不稳定性模型预测，最大组装速率将延迟，直到在重新加帽之前解聚的微管亚群消失。分析表明，30秒的延迟是由于稳态微管群体中约2%的质量未加帽且正在解聚，并且不易重新加帽。通过向末端添加微管蛋白 - GTP亚基来重新加帽正在解聚的微管的半衰期等于或大于20秒。由于微管蛋白 - GDP以约4500 s-1的速率从微管上解离，缓慢的重新加帽导致正在解聚的微管显著缩短。
