Heat induced protein denaturation in the particulate fraction of HeLa S3 cells: effect of thermotolerance.
In this study we investigated the effect of heat on the proteins of the particulate fraction (PF) of HeLa S3 cells using electron spin resonance (ESR) and thermal gel analysis (TGA). ESR detects overall conformational changes in proteins, while TGA detects denaturation (aggregation due to formation of disulfide bonds) in specific proteins. For ESR measurements the -SH groups of the proteins were labelled with a maleimido bound spin label (4-maleimido-tempo). The sample was heated inside the ESR spectrometer at a rate of 1 degree C/min. ESR spectra were made every 2-3 degrees C between 20 degrees C and 70 degrees C. In the PF of untreated cells conformational changes in proteins were observed in three temperature stretches: between 38 and 44 degrees C (transition A, TA); between 47 and 53 degrees C (transition B, TB); and above 58 degrees C (transition C, TC). With TGA, using the same heating rate, we identified three proteins (55, 70, and 90 kD) which denatured during TB. No protein denaturation was observed during TA, while during TC denaturation of all remaining proteins in the PF occurred. When the ESR and TGA measurements were done with the PF of (heat-induced) thermotolerant cells, TA was unchanged while TB and TC started at higher temperatures. The temperature shift for the onset of these transitions correlated with the degree of thermotolerance that was induced in the cells. These results suggest that protection against heat-induced denaturation of proteins in the PF is involved in heat induced thermotolerance.
Burgman
P W
PW
Department of Radiobiology, University of Groningen, The Netherlands.
Konings
A W
AW
D000222:Adaptation, Physiological; D002458:Cell Fractionation; D002470:Cell Survival; D004578:Electron Spin Resonance Spectroscopy; D006367:HeLa Cells; D006358:Hot Temperature; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry*; D011489:Protein Denaturation / Q000502:physiology*
D008565:Membrane Proteins