Bearer E L, DeGiorgis J A, Medeiros N A, Reese T S
Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island 02912, USA.
Cell Motil Cytoskeleton. 1996;33(2):106-14. doi: 10.1002/cm.970330202.
We previously showed that axoplasmic organelles from the squid giant axon move toward the barbed ends of actin filaments and that KI-washed organelles separated from soluble proteins by sucrose density fractionation retain a 235-kDa putative myosin. Here, we examine the myosin-like activities of KI-washed organelles after sucrose density fractionation to address the question whether the myosin on these organelles is functional. By electron microscopy KI-washed organelles bound to actin filaments in the absence of ATP but not in its presence. Analysis of organelle-dependent ATPase activity over time and with varying amounts of organelles revealed a basal activity of 350 (range: 315-384) nmoles Pi/mg/min and an actin-activated activity of 774 (range: 560-988) nmoles/mg/min, a higher specific activity than for the other fractions. By video microscopy washed organelles moved in only one direction on actin filaments with a net velocity of 1.11 +/- .03 microns/s and an instantaneous velocity of 1.63 +/- 0.29 microns/s. By immunogold electronmicroscopy, 7% of KI-washed organelles were decorated with an anti-myosin antibody as compared to 0.5% with non-immune serum. Thus, some axoplasmic organelles have a tightly associated myosin-like activity.
我们之前表明，来自鱿鱼巨大轴突的轴浆细胞器向肌动蛋白丝的带刺末端移动，并且通过蔗糖密度梯度离心从可溶性蛋白质中分离出的经碘化钾洗涤的细胞器保留了一种235 kDa的假定肌球蛋白。在此，我们研究了蔗糖密度梯度离心后经碘化钾洗涤的细胞器的类肌球蛋白活性，以解决这些细胞器上的肌球蛋白是否具有功能这一问题。通过电子显微镜观察，经碘化钾洗涤的细胞器在无ATP时与肌动蛋白丝结合，但在有ATP时则不然。随着时间推移并使用不同量的细胞器分析细胞器依赖性ATP酶活性，结果显示基础活性为350（范围：315 - 384）nmol Pi/mg/分钟，肌动蛋白激活活性为774（范围：560 - 988）nmol/mg/分钟，比其他组分具有更高的比活性。通过视频显微镜观察，洗涤后的细胞器仅在一个方向上在肌动蛋白丝上移动，净速度为1.11±0.03微米/秒，瞬时速度为1.63±0.29微米/秒。通过免疫金电子显微镜观察，7%的经碘化钾洗涤的细胞器被抗肌球蛋白抗体标记，而非免疫血清标记的比例为0.5%。因此，一些轴浆细胞器具有紧密相关的类肌球蛋白活性。
