The isolation and partial characterization of the plasma membrane from Trypanosoma brucei.
The isolation and partial characterization of the plasma membrane from Trypanosoma brucei.
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布氏锥虫质膜的分离和部分表征。
DOI：
10.1042/bj1800011
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发表时间：
1979
期刊：
影响因子：
--
通讯作者：
W. Edwards
中科院分区：
文献类型：
--
作者：
H. Voorheis;J. Gale;M. Owen;W. Edwards
从布氏锥虫中提纯了完整的质膜，每个质膜上都有附着的鞭毛。为分离它们而设计的方法包括一种新的细胞破碎技术，该技术使用渗透应力和机械剪切相结合的方式，避免了极端的囊泡形成和细胞器被困在细胞内的问题。纯化后的膜均含有膜微管阵列。在分离过程中，抗原表面涂层完全从质膜上释放出来。该膜具有很高的胆固醇/磷脂比(1.54)。很大比例(42%)的细胞DNA被回收到质膜部分，除非在第二次蔗糖密度梯度离心之前包括脱氧核糖核酸酶处理的步骤，该步骤将DNA含量降低到13%以下。这一步骤也有助于将质膜与其他细胞成分分离。哇巴因敏感的Na++K+刺激的腺苷三磷酸酶和腺苷环化酶与质膜共纯化。虽然5‘-核苷酸酶被认为是质膜成分，但它很容易从膜上分离出来。纯化后的膜基本不含L-丙氨酸-α-氧代戊二酸氨基转移酶、L-天冬氨酸-α-氧代戊二酸氨基转移酶、苹果酸脱氢酶、寡霉素敏感的三磷酸腺苷酶、葡萄糖6-磷酸酶、镁离子刺激的对硝基苯基磷酸酶和过氧化氢酶。
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.