Destabilization and denaturation of cellular protein by glutathione depletion.
This investigation tested the hypothesis that depletion of intracellular glutathione, in contrast to its oxidation could lead to non-native oxidation of protein thiols, thereby trapping proteins in an unstable conformation. Chinese hamster cells were exposed to the alpha, beta-unsaturated dicarboxylic acid diethylmaleate in order to produce rapid glutathione (GSH) depletion without oxidation. Measurement of the fluorescence of oxidized 2',7'-dichlorofluorescein diacetate indicated that reactive oxygen species accumulated in GSH depleted cells. Glutathione depletion was found to alter protein thiol/disulfide exchange ratios such that 17 to 23 nmol of protein SH/mg protein underwent oxidation. Differential scanning calorimetry (DSC) of glutathione depleted cells yielded a profile of specific heat capacity versus temperature that was characteristic of cells containing destabilized and denatured protein. In addition, cells depleted of glutathione exhibited a two-fold increase in NP-40 insoluble protein. These results indicate that depletion of intracellular glutathione caused oxidation of protein thiols, protein denaturation and aggregation and provide a mechanism to explain how GSH depletion can initiate stress responses.
Freeman
M L
ML
Vanderbilt Center for Radiation Oncology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA. Freemaml@ctrvax.vanderbilt.edu
Huntley
S A
SA
Meredith
M J
MJ
Senisterra
G A
GA
Lepock
J
J
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D005978:Glutathione / Q000378:metabolism*; D007424:Intracellular Fluid / Q000378:metabolism; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D011506:Proteins / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism
D011506:Proteins; D017382:Reactive Oxygen Species; D013438:Sulfhydryl Compounds; D005978:Glutathione