MAMMALIAN 60-KDA STRESS PROTEIN (CHAPERONIN HOMOLOG) - IDENTIFICATION, BIOCHEMICAL-PROPERTIES, AND LOCALIZATION
MAMMALIAN 60-KDA STRESS PROTEIN (CHAPERONIN HOMOLOG) - IDENTIFICATION, BIOCHEMICAL-PROPERTIES, AND LOCALIZATION
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DOI：
10.1074/jbc.270.22.13429
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发表时间：
1995-06-02
影响因子：
4.8
通讯作者：
TASHIMA, Y
中科院分区：
文献类型：
--
作者：
ITOH, H;KOBAYASHI, R;TASHIMA, Y
使用串联ATP-琼脂糖凝胶柱以及Mono和柱层析从猪肝脏胞质溶胶中纯化哺乳动物伴侣蛋白同源物（HSP 60）。该蛋白的部分氨基酸序列（96个氨基酸残基）与人HSP 60的核苷酸序列同源性为96.9%。纯化蛋白的NH 2端序列（5个氨基酸残基）与HSP 60的信号序列一致。这些事实导致了60 kDa肝蛋白与伴侣蛋白同系物的鉴定。二氢叶酸还原酶能够与肝伴侣蛋白同系物形成稳定的复合物。在二维凝胶电泳上，通过pI = 5.6附近的至少五个斑点检测到肝伴侣蛋白同系物。使用针对伴侣蛋白同系物的抗体的免疫印迹研究表明，伴侣蛋白同系物定位于猪肝的胞质溶胶、线粒体和核组分中。在电镜水平上，伴侣蛋白同系物定位于大鼠肾脏的线粒体和细胞质中。在胞质溶胶中的伴侣蛋白同系物，但不是在其他亚细胞级分中，与抗体交叉反应，对相应于HSP 60的信号肽的合成肽，以及纯化的伴侣蛋白同系物的免疫印迹。这些结果表明，细胞质中的功能性伴侣蛋白同源物可能被转运到线粒体中，并且蛋白质可能在细胞器中被加工成线粒体HSP 60。
Mammalian chaperonin homolog (HSP60) was purified from porcine livers cytosol using a tandem ATP-Sepharose column and Mono and column chromatography. A partial amino acid sequence (96 amino acid residues) of this protein was determined and coincided with those of human HSP60 with 96.9% homology, which was deduced from the nucleotide sequence of the cDNA. The sequence of the NH2 termini of the purified protein (5 amino acid residues) coincided with the signal sequence of HSP60. These facts led to the identification of the 60-kDa liver protein with the chaperonin homolog. Dihydrofolate reductase was able to form a stable complex with the liver chaperonin homolog. The liver chaperonin homolog was detected by at least five spots around pI = 5.6 on two-dimensional gel electrophoresis. Immunoblotting studies using an antibody against chaperonin homolog showed that the chaperonin homolog was localized in the cytosol, mitochondrial, and nuclear fractions of porcine Liver. The chaperonin homolog was localized both in the mitochondria and cytoplasm of rat kidneys at the electron microscopic level. The chaperonin homolog in the cytosol, but not in the other subcellular fractions, was cross-reacted with an antibody against the synthetic peptide corresponding to the signal peptide of HSP60 as well as the purified chaperonin homolog on immunoblotting. These results suggested that the functional chaperonin homolog in the cytosol may be transported into the mitochondria and the protein may be processed to mitochondrial HSP60 in the organella.