CAJ | 학술논문

以猪脑为材料，经匀浆、差速离心、蔗糖密度梯度离心分离突触体.低渗破膜得到突触体膜.Triton X 100增溶后，经钙调蛋白亲和层析可得去脂的质膜Ca2+-ATPase.用大体积亲和柱和大体积低Ca2+淋洗液淋洗，可得产率、纯度和活性均较高的质膜Ca2+-ATPase.与大豆磷脂保温后，去脂的Ca2+-ATPase的水解活力可恢复达3.32 μmol/(mg·min).SDS-聚丙烯酰胺凝胶电泳银染显示单一蛋白质带，分子质量约为140 ku，纯度在 90％以上.不同Ca2+浓度明显影响酶的活力.
이저뇌위재료，경균장、차속리심、자당밀도제도리심분리돌촉체.저삼파막득도돌촉체막.Triton X 100증용후，경개조단백친화층석가득거지적질막Ca2+-ATPase.용대체적친화주화대체적저Ca2+림세액림세， 가득산솔、순도화활성균교고적질막Ca2+-ATPase.여대두린지보온후，거지적Ca2+-ATPase적수해활력가회복 체3.32 μmol/(mg·min).SDS-취병희선알응효전영은염현시단일단백질대，분자질량약위140 ku，순도재 90％이상.불동Ca2+농도명현영향매적활력.
Synaptosomes were isolated from pig brain by homogenization, differential centrifugation and sucrose gradient centrifugation. After synaptosome lysis in hypoosmotic buffer, the plasma membrane vesicles were collected. Following the solubilization of plasma membrane vesicles in Triton X-100, the solubilized protein was applied to calmodulin affinity chromatography colurnn, and the delipidated plasma membrane Ca2 + -ATPase was purified to nearly homogeneity. The novel feature of this purification is the use of large affinity column and heavy washing to facilitate the purified Ca2+ -ATPase with higher activity and protein yield. The specific activity of the purified Ca2+ -ATPase was recovered to a maximum of 3.32 μmol· mg-1· min-1 after incubation with asolectin. Silver staining of SDS-PAGE revealed a single protein band around Mr 140 000, showing the purity was over 90 %. Different Ca2 + concentrations dramatically affect the specific activity of Ca2 + -ATPase.