Nichols J M, Cross G A
J Gen Microbiol. 1977 Apr;99(2):291-300. doi: 10.1099/00221287-99-2-291.
Two methods were used to isolate mitochondria from Crithidia fasciculata. In the first method, cells were weakened by exposure to hypotonic conditions and then disrupted by blending; mitochondria were subsequently isolated using disodium 3,5-diacetoamido-2,4,6-triiodobenzoate gradients. In the second, cells were treated with digitonin before disruption; mitochondria were purified by differential centrifugation. Both preparations were examined with the electron microscope and were also shown to possess several characteristic biochemical properties of mitochondria. Kinetoplast DNA was present in the mitochondria, uncontaminated by nuclear DNA. Analysis by polyacrylamide gel electrophoresis showed two RNA components of molecular weights of 0-47 X 10(6) and 0-22 X 10(6), in addition to cytoplasmic RNA contamination. Four mitochondrial components with sedimentation coefficients of 14-6S, 11-4S, 10-1S and 9-9S were identified on sucrose density gradients. Ethidium bromide abolished the incorporation of [5-3H]uridine into the presumed mitochondrial RNA.
采用两种方法从梨形鞭毛虫中分离线粒体。第一种方法是，将细胞置于低渗条件下使其弱化，然后通过匀浆破碎；随后使用3,5 - 二乙酰氨基 - 2,4,6 - 三碘苯甲酸二钠盐梯度分离线粒体。第二种方法是，在破碎细胞前先用洋地黄皂苷处理；通过差速离心法纯化线粒体。对两种制备物进行了电子显微镜检查，结果表明它们还具有线粒体的几种特征性生化特性。线粒体中存在动质体DNA，未被核DNA污染。聚丙烯酰胺凝胶电泳分析显示，除了细胞质RNA污染外，还有分子量为0 - 47×10⁶和0 - 22×10⁶的两种RNA成分。在蔗糖密度梯度上鉴定出了沉降系数分别为14 - 6S、11 - 4S、10 - 1S和9 - 9S的四种线粒体成分。溴化乙锭抑制了[5 - ³H]尿苷掺入假定的线粒体RNA中。
