Salmon E D, Saxton W M, Leslie R J, Karow M L, McIntosh J R
J Cell Biol. 1984 Dec;99(6):2157-64. doi: 10.1083/jcb.99.6.2157.
The diffusion coefficient of tubulin has been measured in the cytoplasm of eggs and embryos of the sea urchin Lytechinus variegatus. We have used brain tubulin, conjugated to dichlorotriazinyl-aminofluorescein, to inject eggs and embryos. The resulting distributions of fluorescence were perturbed by bleaching with a microbeam of light from the 488-nm line of an argon ion laser. Fluorescence redistribution after photobleaching was monitored with a sensitive video camera and photography of the television-generated image. With standard photometric methods, we have calibrated this recording system and measured the rates of fluorescence redistribution for tubulin, conjugated to dichlorotriazinyl-aminofluorescein, not incorporated into the mitotic spindle. The diffusion coefficient (D) was calculated from these data using Fick's second law of diffusion and a digital method for analysis of the photometric curves. We have tested our method by determining D for bovine serum albumin (BSA) under conditions where the value is already known and by measuring D for fluorescein-labeled BSA in sea urchin eggs with a standard apparatus for monitoring fluorescence redistribution after photobleaching. The values agree to within experimental error. Dcytoplasmtubulin = 5.9 +/- 2.2 X 10(-8) cm2/s; DcytoplasmBSA = 8.6 +/- 2.0 X 10(-8) cm2/s. Because DH2OBSA = 68 X 10(-8) cm2/s, these data suggest that the viscosity of sea urchin cytoplasm for protein is about eight times that of water and that most of the tubulin of the sea urchin cytoplasm exists as a dimer or small oligomer, which is unbound to structures that would impede its diffusion. Values and limitations of our method are discussed, and we draw attention to both the variations in D for single proteins in different cells and the importance of D for the upper limit to the rates of polymerization reactions.
已测定了多色紫球海胆卵和胚胎细胞质中微管蛋白的扩散系数。我们使用与二氯三嗪基 - 氨基荧光素偶联的脑微管蛋白来注射卵和胚胎。用氩离子激光488纳米线的微光束漂白会扰乱产生的荧光分布。光漂白后的荧光重新分布用灵敏的摄像机监测，并对电视生成的图像进行拍照。我们用标准光度法校准了这个记录系统，并测量了未掺入有丝分裂纺锤体的、与二氯三嗪基 - 氨基荧光素偶联的微管蛋白的荧光重新分布速率。利用菲克第二扩散定律和一种用于分析光度曲线的数字方法，从这些数据计算出扩散系数（D）。我们通过在已知值的条件下测定牛血清白蛋白（BSA）的D值，以及用用于监测光漂白后荧光重新分布的标准仪器测量海胆卵中荧光素标记的BSA的D值，来测试我们的方法。这些值在实验误差范围内相符。D细胞质微管蛋白 = 5.9 ± 2.2×10⁻⁸ 平方厘米/秒；D细胞质BSA = 8.6 ± 2.0×10⁻⁸ 平方厘米/秒。因为D水相BSA = 68×10⁻⁸ 平方厘米/秒，这些数据表明海胆细胞质中蛋白质的粘度约为水的八倍，并且海胆细胞质中的大多数微管蛋白以二聚体或小寡聚体形式存在，未与会阻碍其扩散的结构结合。讨论了我们方法的数值和局限性，我们提请注意不同细胞中单一蛋白质D值的变化以及D对聚合反应速率上限的重要性。
