Enolase exists in the fluid phase of cytoplasm in 3T3 cells.
Enolase exists in the fluid phase of cytoplasm in 3T3 cells.
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DOI：
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发表时间：
1989-10
影响因子：
4
通讯作者：
L. Pagliaro;K. Kerr;D. Taylor
中科院分区：
文献类型：
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作者：
L. Pagliaro;K. Kerr;D. Taylor
我们研究了糖酵解酶烯醇化酶的细胞内分布和流动性，使用功能性荧光类似物标记的琥珀酰亚胺酯的羧基荧光素（F1-烯醇化酶）和羧基四甲基罗丹明（Rh-烯醇化酶）在对比醛缩酶，无论是天然烯醇化酶，也没有标记烯醇化酶凝胶化丝状肌动蛋白（F-肌动蛋白），作为测量的落球粘度，表明烯醇化酶和F-肌动蛋白之间缺乏相互作用。F1-烯醇化酶在水溶液中的扩散系数（D）的光漂白后荧光再分布（FRAP）测量值为D37，aq = 6.08 x 10（-7）cm 2 s-1，无固定部分，与90，000的天然分子量一致。这些值与Rh-烯醇化酶，或在F-肌动蛋白，2-磷酸甘油酸或F-肌动蛋白-醛缩酶凝胶的存在下没有显著差异，表明F1-烯醇化酶和Rh-烯醇化酶在体外都不与F-肌动蛋白或醛缩酶结合。FRAP测量F1-和Rh-烯醇化酶显微注射到生活瑞士3 T3细胞显示扩散系数的空间差异，但不是移动的部分。在核周细胞质中，我们测得的表观扩散系数为1.1 x 10（-7）cm 2 s-1，而在外周细胞质中为7.1 x 10（-8）cm 2 s-1，两个区域中F1-或Rh-烯醇化酶的迁移率约为100%。用Rh-烯醇化酶和大小分级的FITC-葡聚糖（FD-90）共注射的细胞的成像显示Rh-烯醇化酶进入细胞核，而FD-90被排除。比率成像显示相对高的核比率的Rh-烯醇化酶/FD-90，和一个统一的细胞质比率，没有迹象表明周围的应力纤维的烯醇化酶的浓度增加。这些数据表明Rh-和F1-烯醇化酶在体外不与F-肌动蛋白结合，并且在体内是100%移动的。再加上我们最近的发现，一个显着的分数的醛缩酶结合到F-肌动蛋白在体外和体内是固定的，这些数据表明肌动蛋白结合活性和糖酵解酶的细胞质流动性之间的相关性。
We have investigated the intracellular distribution and mobility of the glycolytic enzyme enolase, using functional fluorescent analogs labeled with the succinimidyl esters of carboxyfluorescein (F1-enolase) and carboxytetramethylrhodamine (Rh-enolase) In contrast to aldolase, neither native enolase nor labeled enolase gelled filamentous actin (F-actin), as measured by falling-ball viscometry, indicating a lack of interaction between enolase and F-actin. Fluorescence redistribution after photo-bleaching (FRAP) measurements of the diffusion coefficient (D) of F1-enolase in aqueous solutions gave a value of D37,aq = 6.08 x 10(-7) cm2s-1, and no immobile fraction, consistent with a native molecular weight of 90,000. These values were not significantly different with Rh-enolase, or in the presence of F-actin, 2-phosphoglycerate or F-actin-aldolase gels, demonstrating that neither F1-enolase nor Rh-enolase binds to F-actin or aldolase in vitro. FRAP measurements of F1- and Rh-enolase microinjected into living Swiss 3T3 cells revealed spatial differences in the diffusion coefficient, but not the mobile fraction. In the perinuclear cytoplasm, we measured an apparent diffusion coefficient of 1.1 x 10(-7) cm2s-1, compared to 7.1 x 10(-8) cm2s-1 in the peripheral cytoplasm, with approximately 100% mobility of F1- or Rh-enolase in both regions. Imaging of cells co-injected with Rh-enolase and size-fractionated FITC-dextran (FD-90) revealed that Rh-enolase entered the nucleus, while FD-90 was excluded. Ratio imaging showed a relatively high nuclear ratio of Rh-enolase/FD-90, and a uniform cytoplasmic ratio, with no indication of increased concentration of enolase around stress fibers. These data demonstrate that Rh- and F1-enolase do not bind to F-actin in vitro, and are 100% mobile in vivo. Together with our recent finding that a significant fraction of aldolase binds to F-actin in vitro and is immobile in vivo, these data suggest a correlation between actin-binding activity and cytoplasmic mobility of glycolytic enzymes.