Parish R W
Eur J Biochem. 1975 Oct 15;58(2):523-31. doi: 10.1111/j.1432-1033.1975.tb02401.x.
The isolation of cell organelles from Dictyostelium discoideum was attempted using a variety of techniques. Cell homogenization (e.g. Potter-Elvehjem, glass beads) gave poor yields of organelles which were, in addition, exceptionally fragile and unstable in density gradients. An isolation method was developed using Triton X-100 in buffered sorbitol/Ficoll solutions at concentrations optimal for plasma membrane rupture. Immediately following cell lysis the solutions were diluted to sub-optimal Triton X-100 concentrations. Sedimentabilities of malate dehydrogenase, citrate synthetase, urate oxidase and catalase of around 55%, 40%, 35% and 55% respectively could be demonstrated using this method. The organelles were more resistant to breakage during resuspension following differential centrifugation and remained largely intact during density gradient centrifugation. The distribution of adenylate kinase activity in gradients showed that at least half the mitochondria retained an intact outer membrane. The mitochondria and peroxisomes could not be clearly separated using conventional sucrose-Ficoll density gradients. Separation was achieved by incubating the cell homogenate with succinate and a tetrazolium dye (2-p-iodophenyl-3-p-nitrophenyl-5-phenyl monotetrazolium chloride). Succinate dehydrogenase activity of mitochondria reduced the tetrazolium dye and the product (formazan) was deposited on the mitochondrial membranes ("heavy-labelling"). The mitochondria then sedimented to denser regions of the gradient while catalase distribution remained unchanged. The treatment left both organelles intact. The mitochondria (1.21 g/ml) were slightly denser than the peroxisomes (1.19 g/ml). The peroxisomes contained catalase and urate oxidase; no other hydrogen-peroxide-producing oxidases were detected. The slime mould urate oxidase resembled the mammalian enzyme. It had an apparent Km value of 12.5 muM, an optimum of activity at pH 8.5 in borate buffer and was competitively inhibited by trichloropurine.
人们尝试使用多种技术从盘基网柄菌中分离细胞器。细胞匀浆法（如波特-埃尔维耶姆匀浆器、玻璃珠法）得到的细胞器产量很低，而且这些细胞器在密度梯度中异常脆弱且不稳定。人们开发了一种分离方法，即在缓冲的山梨醇/聚蔗糖溶液中使用 Triton X-100，其浓度对于质膜破裂而言是最佳的。细胞裂解后，立即将溶液稀释至低于最佳的 Triton X-100 浓度。使用该方法可证明苹果酸脱氢酶、柠檬酸合酶、尿酸氧化酶和过氧化氢酶的沉降率分别约为 55%、40%、35%和 55%。这些细胞器在差速离心后的重悬过程中更耐破碎，并且在密度梯度离心过程中基本保持完整。腺苷酸激酶活性在梯度中的分布表明，至少一半的线粒体保留了完整的外膜。使用传统的蔗糖-聚蔗糖密度梯度无法清晰分离线粒体和过氧化物酶体。通过将细胞匀浆与琥珀酸盐和一种四唑染料（2-对碘苯基-3-对硝基苯基-5-苯基氯化三苯基四氮唑）一起孵育实现了分离。线粒体的琥珀酸脱氢酶活性使四唑染料还原，产物（甲臜）沉积在线粒体膜上（“重标记”）。然后线粒体沉降到梯度的较密区域，而过氧化氢酶的分布保持不变。该处理使两种细胞器均保持完整。线粒体（1.21 g/ml）的密度略高于过氧化物酶体（1.19 g/ml）。过氧化物酶体含有过氧化氢酶和尿酸氧化酶；未检测到其他产生过氧化氢的氧化酶。黏菌尿酸氧化酶与哺乳动物的酶相似。其表观 Km 值为 12.5 μM，在硼酸盐缓冲液中 pH 8.5 时活性最佳，并且受到三氯嘌呤的竞争性抑制。
