Silver R B
Proc Natl Acad Sci U S A. 1986 Jun;83(12):4302-6. doi: 10.1073/pnas.83.12.4302.
Monospecific antibodies to the calcium transport enzyme (alpha-Ca pump) inhibit mitosis when microinjected into sand dollar embryos. Immunoglobulins were raised against the calcium transport enzyme (Ca pump) of sarcoplasmic reticulum (SR) from rat skeletal muscle and guinea pig ileum smooth muscle. Specific antibodies were further isolated from IgG fractions by using electrophoretically purified SR Ca-pump protein as the immobilized ligand for immunoaffinity chromatography. ELISA demonstrated that common antigenic determinants are shared by SR, SR Ca pump (of rat skeletal and guinea pig ileum smooth muscle), and isolated membrane containing "native" mitotic apparatus (MA). Preimmune sera gave negative results in identical control assays. Triton X-100 extraction of MA removes the Ca-pump antigen. SR Ca pump and the MA Ca pump have nearly identical molecular masses as determined by NaDodSO4/PAGE. These alpha-SR Ca-pump IgGs inhibit ATP-dependent Ca2+ sequestration by purified SR and MA membranes. Indirect immunofluorescence of isolated native MA demonstrated coincident localization of the MA Ca pump, sequestered calcium, and membrane vesicles. Fluorescent foci were regionally concentrated within the volumes of the asters and spindle. Microinjection of the anti-Ca-pump IgGs into one of two sister blastomeres at second metaphase resulted in mitotic arrest of the injected cell accompanied by a rapid loss of spindle birefringence. Karyomeres formed and fused to form nuclei either at the site of the metaphase plate or at the position the chromosomes occupied during anaphase A. The cleavage furrow did not develop in the injected cell, while the sister and neighbor cells continued normal mitotic cycling. Injection later in mitosis yielded cells with two nuclei whose cleavage furrow relaxed completely. Routine control injections of boiled immune IgG, preimmune IgG, Wesson oil, buffer, or goat anti-rabbit IgG did not affect mitosis, birefringence of the MA, or cleavage furrow activity.
将针对钙转运酶（α - Ca泵）的单特异性抗体显微注射到海胆胚胎中时，会抑制有丝分裂。制备了针对大鼠骨骼肌和豚鼠回肠平滑肌肌浆网（SR）钙转运酶（Ca泵）的免疫球蛋白。通过使用经电泳纯化的SR钙泵蛋白作为免疫亲和层析的固定配体，从IgG组分中进一步分离出特异性抗体。酶联免疫吸附测定（ELISA）表明，SR、SR钙泵（大鼠骨骼肌和豚鼠回肠平滑肌的）以及含有“天然”有丝分裂器（MA）的分离膜具有共同的抗原决定簇。免疫前血清在相同的对照试验中给出阴性结果。用Triton X - 100提取MA可去除钙泵抗原。通过十二烷基硫酸钠/聚丙烯酰胺凝胶电泳（NaDodSO4/PAGE）测定，SR钙泵和MA钙泵的分子量几乎相同。这些α - SR钙泵免疫球蛋白抑制纯化的SR膜和MA膜对ATP依赖的Ca2 + 摄取。对分离的天然MA进行间接免疫荧光显示，MA钙泵、摄取的钙和膜泡共定位。荧光焦点区域集中在星体和纺锤体区域内。在第二次中期将抗钙泵免疫球蛋白显微注射到两个姐妹卵裂球之一中，导致注射细胞的有丝分裂停滞，并伴随纺锤体双折射迅速消失。核粒形成并融合，在中期板位置或后期A染色体占据的位置形成细胞核。注射细胞中没有形成分裂沟，而姐妹细胞和相邻细胞继续正常的有丝分裂循环。在有丝分裂后期注射会产生具有两个细胞核的细胞，其分裂沟完全松弛。常规对照注射煮沸的免疫IgG、免疫前IgG、维森油、缓冲液或山羊抗兔IgG均不影响有丝分裂、MA的双折射或分裂沟活性。
