Studies on the ability of the centrosome to nucleate microtubules

(1)从海胆卵中分离出有丝分裂器(MA)，并将MA悬液冷却、匀浆，分离出中心体。加入从猪脑中提纯的微管蛋白后，可形成许多ASTs，GTP、GTP-Gamma或GMP-PNP可促进从分离的中心体开始的ASTs的重建。反之，GDP则抑制中心体对微管的成核作用。(2)通过亲和层析新分离到的45K/30K复合体对G蛋白结合GTP*GDP相互转化的调节机制。该复合体能增强G蛋白与GTP的亲和力。(3)将G蛋白加入精子轴丝后，轴丝的整个表面均被抗G蛋白抗体染色，提示α-微管蛋白的C末端从微管壁伸出。当G蛋白部分与Spem轴丝或微管蛋白二聚体部分孵育，并用EDAC(一种零长交联剂)交联后，进行SDS-PAGE，发现α-微管蛋白优先与G蛋白交联。纺锤体微管的极性是负端朝向近端。因此，G蛋白将在中心体与α-微管蛋白结合。(4)利用乳胶珠(聚苯乙烯微珠)、G蛋白和微管蛋白建立了中心体的模型体系。在GTP存在的情况下，乳胶珠包覆G蛋白部分，然后添加脑微管蛋白。乳胶珠的聚集有利于紫锥花的形成。

(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.