Transduction of reducing power across the plasma membrane by reduced glutathione. A 1H-NMR spin-echo study of intact human erythrocytes.
The NMR signal of reduced glutathione (GSH) was monitored in intact human erythrocytes by the 1H spin-echo Carr-Purcell-Meiboom-Gill pulse sequence. Addition of GSH, which was unable to cross the erythrocyte membrane, produced an approximate twofold increase of the GSH signal in glucose-depleted cells. Addition of oxidised glutathione (GSSG), did not affect the signal, and addition of GSH to hemolysates gave a much smaller increase. Reduction of internal GSSG by NADPH-dependent enzymes was excluded by experiments with glucose-supplied or glucose-6-phosphate dehydrogenase deficient cells. Involvement of external thiol groups of the erythrocyte membrane was shown by the lack of effect in cells treated with an impermeable thiol-blocking compound. Involvement of spectrin was indicated by the proportional loss of the effect in erythrocytes with variable genetic deficiency of spectrin. Protein-glutathione mixed disulfides appeared to be the source of the NMR response since an increase of their content, by diamide treatment or aging procedures, produced a higher GSH signal, while their reduction by permeable reductants gave the opposite effect. It is concluded that GSH can transduce its reducing power by a thiol/disulfide exchange mechanism that sequentially involves sulfur-rich proteins spanning across the erythrocyte membrane.
Ciriolo
M R
MR
Department of Biology, Tor Vergata, University of Rome, Italy.
Paci
M
M
Sette
M
M
De Martino
A
A
Bozzi
A
A
Rotilio
G
G
D000328:Adult; D001786:Blood Glucose / Q000378:metabolism; D002478:Cells, Cultured; D004220:Disulfides / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D008875:Middle Aged; D010084:Oxidation-Reduction; D015398:Signal Transduction; D013438:Sulfhydryl Compounds / Q000378:metabolism
D001786:Blood Glucose; D004220:Disulfides; D013438:Sulfhydryl Compounds; D005978:Glutathione