ATP-dependent chaperoning activity of reticulocyte lysate

Robert J Schumacher, Robin Hurst, William P. Sullivan, Nancy J. McMahon, David O. Toft, Robert L. Matts

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)9493-9499
Number of pages7
JournalJournal of Biological Chemistry
Volume269
Issue number13
StatePublished - Apr 1 1994

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Schumacher, R. J., Hurst, R., Sullivan, W. P., McMahon, N. J., Toft, D. O., & Matts, R. L. (1994). ATP-dependent chaperoning activity of reticulocyte lysate. Journal of Biological Chemistry, 269(13), 9493-9499.