Caplow M, Shanks J
Department of Biochemistry, University of North Carolina, Chapel Hill 27599-7260, USA.
Biochemistry. 1995 Dec 5;34(48):15732-41. doi: 10.1021/bi00048a018.
The recent discovery that GTP linked to latex beads binds to microtubule ends suggested that nucleotide interactions at this site may play a role in regulating microtubule (MT) dynamics. Evidence for this was sought using DIC microscopy to analyze effects of the free GTP and GDP concentration on the rates of MT elongation and phase transition to rapid shortening (catastrophe, kc). That nucleotide can dissociate and thereby destabilize the plus end by forming nucleotide-free (apotubulin) subunits was indicated by an increase in kc from 0.001 to 0.05 s-1, when the free GTP concentration was reduced from 100 to 0.5 microM, during assembly with 15 microM tubulin--GTP subunits (TuT). That nucleotide can bind to the minus end was indicated by a nearly 5-fold decrease in the rate of elongation when the free GDP concentration was increased from 1.6 to 175 microM, during assembly with a mixture of 36 microM TuT and 54 microM TuD. Further evidence that nucleotide can bind to both ends was provided by the observation that with a mixture of 36 microM TuT and 54 microM TuD, kc was increased from 0.0036 to 0.05 s-1 at the plus end, and from 0.0005 to 0.005 s-1 at the minus end, when the free GDP concentration was increased from 1.6 to 175 microM. Our evidence for destabilization of microtubules by formation of apotubulin and by nucleotide exchange to form terminal TuD subunits suggests that microtubule dynamics can be regulated in cells by an exchange factor that generates apotubulin subunits, or by a GTPase activating protein that forms TuD subunits at microtubule ends.
近期发现与乳胶珠相连的GTP可与微管末端结合，这表明该位点的核苷酸相互作用可能在调节微管（MT）动力学中发挥作用。为此，利用微分干涉差显微镜（DIC显微镜）来分析游离GTP和GDP浓度对MT伸长速率以及向快速缩短（灾变，kc）的相变的影响，以寻找相关证据。当在与15μM微管蛋白 - GTP亚基（TuT）组装过程中，游离GTP浓度从100μM降至0.5μM时，kc从0.001增至0.05 s-1，这表明核苷酸可解离，从而通过形成无核苷酸（脱辅基微管蛋白）亚基使正端不稳定。当在与36μM TuT和54μM TuD混合物组装过程中，游离GDP浓度从1.6μM增至175μM时，伸长速率降低近5倍，这表明核苷酸可与负端结合。当使用36μM TuT和54μM TuD混合物时，进一步证明核苷酸可与两端结合的证据是，当游离GDP浓度从1.6μM增至175μM时，正端的kc从0.0036增至0.05 s-1，负端的kc从0.0005增至0.005 s-1。我们关于通过形成脱辅基微管蛋白以及通过核苷酸交换形成末端TuD亚基使微管不稳定的证据表明，微管动力学在细胞中可通过产生脱辅基微管蛋白亚基的交换因子，或通过在微管末端形成TuD亚基的GTP酶激活蛋白来调节。
