ATP-dependent chaperoning activity of reticulocyte lysate.
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.
Schumacher
R J
RJ
Department of Biochemistry and Molecular Biology, Mayo Graduate School, Rochester, Minnesota 55905.
Hurst
R
R
Sullivan
W P
WP
McMahon
N J
NJ
Toft
D O
DO
Matts
R L
RL
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002474:Cell-Free System; D001517:Coleoptera / Q000201:enzymology; D004789:Enzyme Activation; D006360:Heat-Shock Proteins / Q000378:metabolism*; D007700:Kinetics; D008156:Luciferases / Q000737:chemistry* / Q000378:metabolism*; D011489:Protein Denaturation; D017510:Protein Folding; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*
D006360:Heat-Shock Proteins; D000255:Adenosine Triphosphate; D008156:Luciferases