Studies on the ability of the centrosome to nucleate microtubules

(1)从海胆卵中分离有丝分裂器（MAs），通过冷却和匀浆MA悬浮液分离中心体。加入从猪脑中纯化的微管蛋白后，形成了许多紫菀，GTP、GTP- gammas或GMP-PNP促进了从分离的中心体开始的紫菀的重建。相反，发现GDP抑制中心体成核微管的活性。(2)利用与G蛋白偶联的固定抗G蛋白抗体，通过亲和层析新分离的45K/30K复合物，提出了G蛋白中结合GTP*GDP相互转化的调控机制。发现该复合物增强了G蛋白对GTP的亲和力。(3)在精子轴突中加入G蛋白后，整个轴突表面被抗G蛋白抗体染色，表明α -微管蛋白的c端从微管壁突出。当G蛋白片段与精子轴素或微管蛋白二聚体片段孵育，并与EDAC（零长度交联剂）交联，然后进行SDS-PAGE处理时，发现α -微管蛋白优先与G蛋白交联。纺锤体微管的极性是这样的，负端向近端。因此，G蛋白将与中心体上的α -微管蛋白结合。(4)利用乳胶珠（聚苯乙烯珠）、G蛋白和微管蛋白，新建立了中心体模型体系。在GTP存在的情况下，用G蛋白部分包覆乳胶珠，然后加入脑微管蛋白。乳胶珠的聚集有利于形成紫苑。

(1) The mitotic apparatuses (MAs) were isolated from sea urchin eggs and the centrosomes were separated by chilling and homogenizing the MA suspension. Upon addition of tubulin purified from porcine brain, many asters were formed, GTP,GTP-gammaS,or GMP-PNP promoted the reconstruction of asters initiated from the isolated centrosomes. In contrast, GDP was found to inhibit the activity of the centrosome to nucleate microtubules.(2) The regulatory mechanism of bound GTP*GDP interconversion in the G protein was suggested by the action of 45K/30K complex which was newly isolated by affinity chromatograpy using fixed anti-G protein antibody conjugated with the G protein. The complex was found to enhance the affinity of the G protein to GTP.(3) Upon addition of the G protein to sperm axonemes, the entire surface of the axonemes was stained with anti-G protein antibody, suggesting that the C-terminal of alpha-tubulin is protruded from the microtubule wall. When the G protein fraction was incubated with the spem axoneme or tubulin dimer fraction and cross-linked with EDAC (a zero lenghth cross linker) followed by processing for SDS-PAGE,alpha-tubulin was found to be preferentially cross-linked with the G protein. The polarity of the spindle microtubules is such that the minus end is toward proximal. Therefore, the G protein will bind to alpha-tubulin at the centrosome.(4) A model system fo the centrosome has newly established using latex beads (polystirene beads), G protein and tubulin. In the presence of GTP,latex beads were coated with the G protein fraction, followed by additon of brain tubulin. An aggregate of latex beads favored the formation of asters.