Microtubule-Nucleation Sites on Nuclei in Higher Plant Cells

本文研究了高等植物细胞分离核中微管的成核和伸长。分离的完整细胞核与来自植物或动物来源的纯化微管蛋白孵育时，未能从其表面形成微管。然而，通过核的温和均质获得的冻融的核或核颗粒，有核的微管和有核的微管从核的表面或从核材料径向拉长。从核颗粒辐射的微管比从冻融的核辐射的微管短得多。核粒子的洗涤削弱了粒子成核微管的能力。通过添加核匀浆的可溶部分，洗涤后的核颗粒可以恢复成核微管的能力。用抗植物微管蛋白的单抗对由动物微管蛋白和植物微管蛋白连续聚合而成的辐射微管复合体进行染色，发现微管在其近端结合了微管蛋白。这一结果表明，在动物细胞中，微管蛋白在冻融的细胞核或核颗粒中的掺入方式不同于在中心周体中的掺入方式。部分纯化的微管核化颗粒含有一个表观分子质量为50kd的多肽作为主要成分，它与抗α-微管蛋白的单抗发生交叉反应。2D-PAGE分析显示，50kd的多肽被分离成两个多肽，其中一个被认为是磷酸化的。因此，可溶组分中的基本因子可能是蛋白激酶或磷酸蛋白磷酸酶。这种必需因子的作用可能使50kd的多肽从失活状态变为活性状态。

The nucleation and the elongation of microtubules from isolated nuclei of higher plant cells were investigated. Isolated intact nuclei failed to nucleate microtubules from their surface when they were incubated with purified tubulin from plant or animal sources. However, frozen-thawed nuclei or nuclear particles obtained by gentle homogenization of nuclei nucleated microtubules and nucleated microtubules elongated radially from the surface of nuclei or from the nuclear materials. Microtubules radiating from the nuclear particles were extremely shorter than those radiating from frozen-thawed nuclei. The washing of the nuclear particles diminished the ability of the particles to nucleate microtubules. The ability of the washed nuclear particles to nucleate microtubules was restored by the addition of the soluble fraction of a nuclear homogenate. The staining with a monoclonal antibody specific for plant tubulin of the complexes of radiating microtubules-prepared by successive polymerization of animal tubulin and plant tubulin revealed that microtubules in the complex incorporated tubulin at their proximal ends. This result indicates that the mode of incorporation of tubulin in frozen-thawed nuclei or the nuclear particles is different from that in pericentriolar bodies in animal cells. The partially purified microtubulenucleation particles contained a peptide having an apparent molecular weight of 50 kd as a main constitution, which cross-reacted specifically with monoclonal antibody against alpha-tubulin. 2-D PAGE analysis showed the 50 kd peptide to be separated into two peptides, one of which was assumed to be phosphorylated. Therefore, the essential factor in the soluble fraction is possible to be estimated to be protein kinase or phosphoprotein phosphatase. The action of such essential factor may enable the 50 kd peptide to change from inactive form to active form.