| Autor: |
R J, Schumacher, R, Hurst, W P, Sullivan, N J, McMahon, D O, Toft, R L, Matts |
| Rok vydání: |
1994 |
| Předmět: |
|
| Zdroj: |
The Journal of biological chemistry. 269(13) |
| ISSN: |
0021-9258 |
| Popis: |
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 to60%, 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. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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