Microinjected glutathione reductase crystals as indicators of the redox status in living cells

FEBS Letters

Saffrich, Rainer¹  Dandekar, Thomas¹  Keese, Michael A.²  Becker, Katja²  Schirmer, R.Heiner² 

[1] European Molecular Biology Laboratories, Meyerhofstr. 1, 69117 Heidelberg, Germany;Center of Biochemistry, Im Neuenheimer Feld 328, Heidelberg University, 69120 Heidelberg, Germany

关键词: Protein crystal;    Glutathione reductase;    Cytosol;    Biolistic crystal transfer;    Microinjection;    Redox potential;    CHO cells;    Chinese hamster ovary cells;    CT;    charge transfer;    GR;    glutathione reductase;    GSH;    reduced glutathione;    GSSG;    glutathione disulfide;    PBS;    phosphate-buffered saline of pH 7.4;   

DOI  :  10.1016/S0014-5793(99)00296-3

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The flavoenzyme glutathione reductase catalyses electron transfer reactions between two major intracellular redox buffers, namely the NADPH/NADP⁺ couple and the 2 glutathione/glutathione disulfide couple. On this account, microcrystals of the enzyme were tested as redox probes of intracellular compartments. For introducing protein crystals into human fibroblasts, different methods (microinjection, particle bombardment and optical tweezers) were explored and compared. When glutathione reductase crystals are present in a cytosolic environment, the transition of the yellow E_ox form to the orange-red 2-electron reduced charge transfer form, EH₂, is observed. Taking into account the midpoint potential of the E_ox/EH₂ couple, the redox potential of the cytosol was found to be <−270 mV at pH 7.4 and 37°C. As a general conclusion, competent proteins in crystalline – that is signal-amplifying – form are promising probes for studying intracellular events.

【 授权许可】

Unknown