FURTHER PURIFICATION AND CHARACTERIZATION OF SCRAPIE PRIONS
FURTHER PURIFICATION AND CHARACTERIZATION OF SCRAPIE PRIONS
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DOI：
10.1021/bi00269a050
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发表时间：
1982-01-01
期刊：
影响因子：
2.9
通讯作者：
MCKINLEY, MP
中科院分区：
文献类型：
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作者：
PRUSINER, SB;BOLTON, DC;MCKINLEY, MP
痒病病原体是一类新型小型传染性病原体（称为朊病毒）的原型。长时间的生物测定以及试剂的疏水性使其纯化变得复杂。开发了仓鼠大脑中该试剂的纯化方案，其结果是该试剂的蛋白质含量浓缩了 100 至 1000 倍。该方案包括 Triton X-100/脱氧胆酸钠提取、聚乙二醇沉淀、核酸酶和蛋白酶消化、胆酸盐/十二烷基肌氨酸钠提取、(NH4)2SO4 沉淀、Triton X-100/十二烷基硫酸钠提取和通过不连续蔗糖梯度沉淀。纯化程度最高的部分来自靠近梯度底部的 25% 和 60% 蔗糖界面。通过电镜对来自梯度界面的旋转阴影样品进行检查，该样品含有> 109.5中位感染剂量单位/ml的瘙痒病剂，结果显示由无定形材料和许多直径为25 nm、长度为100-200 nm的扁平棒组成的聚集体。界面部分的放射性碘化鉴定出一种蛋白质，该蛋白质是来自瘙痒病感染的大脑的制剂所特有的。该蛋白质的表观分子量在 27,000 至 30,000 之间。这种独特的蛋白质还用[14C]焦碳酸二乙酯进行放射性标记。尽管瘙痒病朊病毒和这种独特蛋白质的特性必须足够相似才能进行共纯化，但仍需要进一步的实验数据来确定这种蛋白质是否是感染性所必需的。与蛋白质放射性标记研究相反，用[γ-32P]ATP对纯化级分中的核酸分子进行5'-末端标记未能鉴定出独特的核酸。这些放射性标记研究的结果与早期的观察结果一致，表明朊病毒可以通过修饰蛋白质的程序灭活，但可以抵抗修饰核酸程序的灭活。
The scrapie agent is prototypic of a novel class of small infectious pathogens called prions. Prolonged bioassays as well as the hydrophobicity of the agent have complicated its purification. A purification protocol for the agent in hamster brain was developed, which results in preparations enriched for the agent between 100- and 1000-fold with respect to protein. The protocol includes Triton X-100/sodium deoxycholate extraction, poly(ethylene glycol) precipitation, nuclease and protease digestion, cholate/sodium lauryl sarcosinate extraction, (NH4)2SO4 precipitation, Triton X-100/sodium dodecyl sulfate extraction and sedimentation through a discontinuous sucrose gradient. The highest degree of purification was found in a fraction from the 25% and 60% sucrose interface near the bottom of the gradient. Examination by EM of rotary-shadowed samples from the gradient interface which contained > 109.5 median infectious dose units/ml of the scrapie agent revealed aggregates composed of amorphous material and numerous flattened rods measuring 25 nm in diameter by 100-200 nm in length. Radioiodination of the interface fraction has identified a protein that is unique to preparations from scrapie-infected brains. The apparent MW of the protein is between 27,000 and 30,000. The unique protein was also radiolabeled with [14C]diethyl pyrocarbonate. Although the properties of the scrapie prion and this unique protein must be sufficiently similar to allow co-purification, further experimental data will be needed to establish whether or not this protein is required for infectivity. In contrast to the protein radiolabeling studies, 5''-end labeling with [.gamma.-32P]ATP of nucleic acid molecules in the purified fraction failed to identify a unique nucleic acid. The results of these radiolabeling studies are consistent with earlier observations showing that prions are inactivated by procedures that modify proteins but are resistant to inactivation by procedures that modify nucleic acids.