Voorheis H P, Gale J S, Owen M J, Edwards W
Biochem J. 1979 Apr 15;180(1):11-24. doi: 10.1042/bj1800011.
Whole sheets of plasma membrane, each with their attached flagellum, were purified from Trypanosoma brucei. The method devised for their isolation included a new technique of cell breakage that used a combination of osmotic stress followed by mechanical sheer and avoided the problem of extreme vesiculation as well as the trapping of organelles in cell 'ghosts'. The purified membranes all contained the pellicular microtubular array. The antigenic surface coat was completely released from the plasma membrane during the isolation procedure. The membranes had a very high cholesterol/phospholipid ratio (1.54). A large proportion (42%) of the cellular DNA was recovered in the plasma-membrane fraction unless a step involving deoxyribonuclease treatment, which decreased the DNA content to less than 13%, was included before secrose-density gradient centrifugation. This step also aided the separation of plasma membranes from other cellular components. The ouabain-sensitive Na+ + K+-stimulated adenosine triphosphatase and adenylate cyclase co-purified with the plasma membranes. Although 5'-nucleotidase was thought to be a plasma-membrane component, it was easily detached from the membrane. The purified membranes were essentially free of L-alanine-alpha-oxoglutarate aminotransferase, L-asparte-alpha-oxoglutarate aminotransferase, malate dehydrogenase, oligomycin-sensitive adenosine triphosphatase, glucose 6-phosphatase, Mg2+-stimulated p-nitrophenyl phosphatase and catalase.
从布氏锥虫中纯化出了整片的质膜，每片质膜都附着有鞭毛。设计的用于分离质膜的方法包括一种新的细胞破碎技术，该技术先利用渗透压应激，再结合机械剪切力，避免了过度形成囊泡以及细胞器被困在细胞“幽灵”中的问题。纯化后的质膜均含有表膜微管阵列。在分离过程中，抗原性表面被膜从质膜上完全释放。这些质膜具有非常高的胆固醇/磷脂比率（1.54）。除非在蔗糖密度梯度离心之前加入一步涉及脱氧核糖核酸酶处理的步骤（该步骤可将DNA含量降低至13%以下），否则大部分（42%）细胞DNA会在质膜组分中被回收。这一步骤还有助于质膜与其他细胞成分的分离。哇巴因敏感的Na⁺ + K⁺ 刺激的腺苷三磷酸酶和腺苷酸环化酶与质膜共同纯化。尽管5'-核苷酸酶被认为是质膜的一种成分，但它很容易从膜上脱离。纯化后的质膜基本上不含L-丙氨酸-α-酮戊二酸转氨酶、L-天冬氨酸-α-酮戊二酸转氨酶、苹果酸脱氢酶、寡霉素敏感的腺苷三磷酸酶、葡萄糖6-磷酸酶、Mg²⁺ 刺激的对硝基苯磷酸酶和过氧化氢酶。
