THE 73-KILODALTON HEAT-SHOCK COGNATE PROTEIN PURIFIED FROM RAT-BRAIN CONTAINS NONESTERIFIED PALMITIC AND STEARIC ACIDS
THE 73-KILODALTON HEAT-SHOCK COGNATE PROTEIN PURIFIED FROM RAT-BRAIN CONTAINS NONESTERIFIED PALMITIC AND STEARIC ACIDS
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DOI：
10.1002/jcp.1041280215
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发表时间：
1986-08-01
影响因子：
5.6
通讯作者：
HIGHTOWER, LE
中科院分区：
文献类型：
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作者：
GUIDON, PT;HIGHTOWER, LE
从非热应激大鼠脑组织中纯化了一种与71千顿诱导大鼠热休克蛋白相关的蛋白，以毫克量电泳均匀。根据先前的研究和本文提出的数据，该蛋白被指定为应激同源蛋白，该蛋白与最初提出的针对果蝇70千道尔顿热休克蛋白的单克隆抗体交叉反应。经十二烷基硫酸钠聚丙烯酰胺凝胶电泳分析，纯化蛋白的表观分子质量为73千道尔顿，经非解离凝胶色谱分析，其表观分子质量为150千道尔顿，表明纯化蛋白为同型二聚体。纯化后的蛋白质在非解离条件下的等电点为5.0，暴露于蛋白质变性剂时的等电点为5.6，表明在变性过程中失去了结合的阴离子分子和/或基本残基的净暴露。对纯化蛋白进行氯仿/甲醇萃取，随后进行薄层和气液色谱分析，鉴定出与蛋白质非共价结合的棕榈酸和硬脂酸。每个二聚体大约有4个脂肪酸分子与棕榈酸和硬脂酸以1:1的比例结合。纯化后的蛋白不能与外源添加的放射性棕榈酸酯结合，表明同源蛋白的脂肪酸结合位点被完全占据，而相关脂肪酸的结合过于紧密而不易交换。讨论了与73千道尔顿应激同源蛋白相关的脂肪酸的可能意义。
A protein related to the 71 kilodalton inducible rat heat shock protein was purified to electrophoretic homogeneity in milligram amounts from brain tissue of nonheat-stressed rats. The protein has been designated as a stress cognate protein based on previous studies and data presented herein that this protein cross-reacted with a monoclonal antibody originally raised against the Drosophila 70 kilodalton heat shock protein. The purified protein had an apparent molecular mass of 73 kilodaltons when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and an apparent mass of 150 kilodaltons as determined by nondissociative gel chromatography, suggesting that the purified protein is a homodimer. The purified protein had isoelectric points of 5.0 under nondissociative conditions and 5.6 when exposed to protein denaturants, suggesting loss of bound anionic molecules and/or net exposure of basic residues upon denaturation. Chloroform/methanol extraction of the purified protein and subsequent analyses by thin layer and gas-liquid chromatography resulted in the identification of palmitic and stearic acids noncovalently bound to the protein. Approximately four molecules of fatty acids were bound per dimer with palmitic and stearic acids present in a one-to-one ratio. The purified protein did not bind exogenously added radioactive palmitate, indicating that the fatty acid-binding sites of the cognate protein were fully occupied and that the associated fatty acids were too tightly bound to exchange readily. The possible significance of the fatty acids associated with the 73 kilodalton stress cognate protein is discussed.