Silver R B, Cole R D, Cande W Z
Cell. 1980 Feb;19(2):505-16. doi: 10.1016/0092-8674(80)90525-5.
We present the first report of isolate mitotic apparatus with vesicular calcium sequestration. Phase-contrast, differential interference contrast and polarized light microscopy as well as transmission and scanning electron microscopic examinations revealed structures comparable to mitotic apparatus in vivo. Numerous membrane-bound vesicles which retained their osmotic activity were present throughout. Microtubules, yolk, ribosomes and condensed chromatin were also present. The protein composition of mitotic apparatus was not dramatically altered by treatment with 0.5% Triton X-100, even though vesicles were destroyed and yolk was extracted. Calcium sequestration was demonstrated with ATP-dependent accumulation of 45Ca by mitotic apparatus whose vesicles were left intact. Compared with controls for which no nucleotide was added, accumulation by mitotic apparatus with intact vesicles was enhanced to 184% when it was present. When ATP was supplemented with the divalent ionophore A23187, the calcium retention level was comparable to that of the control to which no nucleotide was added. Finally, the calcium accumulation by mitotic apparatus treated with either of the nonhydrolyzable ATP analogs AMPPCP or AMPPNP resulted in calcium retention levels similar to those of controls. The solubilization of vesicles with Triton X-100 abolished calcium accumulation in the presence or absence of any of the above additives. Resolution of vesicles on sucrose step gradients after 45Ca-oxalate loading with ATP or AMPPCP indicates that a specific vesicular fraction sequesters 45Ca.
我们首次报道了分离出的具有囊泡钙隔离功能的有丝分裂装置。相差显微镜、微分干涉相差显微镜、偏光显微镜以及透射电子显微镜和扫描电子显微镜检查显示，其结构与体内的有丝分裂装置相似。整个装置中存在大量保持其渗透活性的膜结合囊泡。还存在微管、卵黄、核糖体和凝聚染色质。用0.5% Triton X-100处理后，有丝分裂装置的蛋白质组成没有显著改变，尽管囊泡被破坏且卵黄被提取。完整囊泡的有丝分裂装置通过依赖ATP的45Ca积累证明了钙隔离功能。与未添加核苷酸的对照组相比，完整囊泡的有丝分裂装置在有核苷酸存在时积累量增加到184%。当ATP与二价离子载体A23187一起添加时，钙保留水平与未添加核苷酸的对照组相当。最后，用不可水解的ATP类似物AMPPCP或AMPPNP处理的有丝分裂装置的钙积累导致钙保留水平与对照组相似。用Triton X-100溶解囊泡后，无论是否存在上述任何添加剂，钙积累均被消除。在用ATP或AMPPCP加载4５Ca-草酸盐后，通过蔗糖阶梯梯度分离囊泡表明，特定的囊泡部分隔离了45Ca。
