Schumacher R J, Hurst R, Sullivan W P, McMahon N J, Toft D O, Matts R L
Department of Biochemistry and Molecular Biology, Mayo Graduate School, Rochester, Minnesota 55905.
J Biol Chem. 1994 Apr 1;269(13):9493-9.
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.
我们开发了一种利用热变性的萤火虫荧光素酶进行伴侣介导的蛋白质复性分析方法。将变性的荧光素酶（活性损失>99%）稀释到网织红细胞裂解物中，通常会使活性恢复5%-15%。添加ATP再生系统可将产量提高到>60%，而热休克或添加变性蛋白会抑制伴侣活性。网织红细胞裂解物含有大量的热休克蛋白hsp90和hsp70，以及一种与酵母应激蛋白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%的荧光素酶活性恢复，表明这些蛋白或相关蛋白参与了荧光素酶的复性。此外，我们观察到荧光素酶复性活性与网织红细胞裂解物制剂中hsp70和hsp90的水平之间存在相关性。纯化的hsp90和hsp70，连同ATP再生系统，能够使荧光素酶复性至其原始活性的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物中的浓度相似时，这种复性最为有效。这项研究为网织红细胞裂解物中涉及hsp70和hsp90协同作用的ATP依赖伴侣活性提供了证据。
