ATP-dependent chaperoning activity of reticulocyte lysate.
ATP-dependent chaperoning activity of reticulocyte lysate.
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DOI：
10.1016/s0021-9258(17)36908-9
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发表时间：
1994-04
期刊：
影响因子：
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通讯作者：
R. Schumacher;R. Hurst;W. Sullivan;N. McMahon;D. Toft;R. Matts
中科院分区：
文献类型：
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作者：
R. Schumacher;R. Hurst;W. Sullivan;N. McMahon;D. Toft;R. Matts
我们已经开发了一种使用热变性萤火虫荧光素酶对伴侣介导的蛋白质变性进行检测。将变性荧光素酶（失去99%的活性）稀释成网状细胞裂解物，通常可恢复5-15%的活性。添加atp再生系统可将产量提高至60%，而热休克或添加变性蛋白可抑制伴随活性。网织红细胞裂解液含有大量的热休克蛋白hsp90和hsp70，以及与酵母应激蛋白STI1同源的60 kda蛋白。这三种蛋白的免疫分离样品以atp依赖的方式支持高达35%的荧光素酶活性的恢复，这表明这些或相关蛋白参与了荧光素酶的再生。此外，我们观察到网状细胞裂解液中荧光素酶再生活性与hsp70和hsp90水平之间的相关性。纯化的hsp90和hsp70，以及atp再生系统，能够将荧光素酶再生到其原始活性的20%以上。当hsp90和hsp70的比例约为2:1，且浓度与网织红细胞裂解液中的浓度相似时，这种恢复是最有效的。这项研究为网状细胞裂解液中atp依赖性的伴随活性提供了证据，该活性涉及hsp70和hsp90的协同作用。
We have developed an assay for chaperone-mediated protein renaturation using thermally denatured Firefly luciferase. Dilution of denatured luciferase (> 99% loss of activity) into reticulocyte lysate typically results in recovery of 5-15% activity. Addition of an ATP-regenerating system increases yields to > 60%, while heat shock or the addition of denatured proteins inhibits the chaperoning activity. Reticulocyte lysate contains abundant quantities of the heat shock proteins, hsp90 and hsp70, and a 60-kDa protein homologous to the yeast stress protein, STI1. Immune isolated samples of these three proteins support recovery of up to 35% of luciferase activity in an ATP-dependent manner, suggesting that these or associated proteins are involved in the renaturation of luciferase. Furthermore, we observed a correlation between luciferase renaturation activity and the levels of hsp70 and hsp90 in reticulocyte lysate preparations. Purified hsp90 and hsp70, along with an ATP-regenerating system, are able to renature luciferase to greater than 20% of its original activity. This renaturation is most efficient when hsp90 and hsp70 are at about a 2:1 ratio and at concentrations similar to those found in reticulocyte lysate. This study provides evidence for an ATP-dependent chaperoning activity in reticulocyte lysate that involves a cooperative action of hsp70 and hsp90.