The effect of temperature and protein synthesis on the renaturation of firefly luciferase in intact H9c2 cells.
A mild increase in temperature that does not exert an effect on tolerance development or synthesis of heat shock proteins (Hsps) in control cells can stimulate these processes when applied to cells that have previously been heat shocked. To study the underlying mechanism of this effect, H9c2 cells were stably transfected with the gene encoding firefly luciferase (Luc). Heat-shock-induced inactivation of Luc and its subsequent reactivation is frequently used as a model for cellular protein denaturation and renaturation. Luc reactivation was determined following a damaging heat shock (43 or 44 degrees C for 30 min) in cells that were subsequently exposed to either control temperatures (37 degrees C) or various mild hyperthermic conditions (from 38.5 to 41.5 degrees C for 1 h). To prevent changes in Luc activity consequent to new synthesis of Luc, Luc reactivation was monitored in the presence of cycloheximide, an inhibitor of protein synthesis. The results showed that reactivation of Luc was inhibited when heat-treated cells were post-treated under mild hyperthermic conditions. The observed increase in Hsp synthesis under mild hyperthermic post-heat shock conditions therefore appears to be the result of an increase in the period during which denatured proteins are present. In addition, we studied Luc reactivation in the absence of protein synthesis inhibitors. This condition led to much higher Luc activity. By estimating half-life times of Luc, the contribution of new Luc synthesis in this recovery could be determined, and only partially explained the observed increase in Luc reactivation after heat shock. Thus the synthesis of other proteins must be important for the renaturation of heat-damaged proteins.
Souren
J E
JE
Department of Molecular Cell Biology, Utrecht University, The Netherlands. j.e.m.souren@bio.uu.nl
Wiegant
F A
FA
van Hof
P
P
van Aken
J M
JM
van Wijk
R
R
D000818:Animals; D002460:Cell Line; D001517:Coleoptera / Q000201:enzymology / Q000235:genetics; D003513:Cycloheximide / Q000494:pharmacology; D006360:Heat-Shock Proteins / Q000096:biosynthesis; D006358:Hot Temperature; D008156:Luciferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020673:Protein Renaturation; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D006360:Heat-Shock Proteins; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; D003513:Cycloheximide; D008156:Luciferases