TRANSDUCTION OF REDUCING POWER ACROSS THE PLASMA-MEMBRANE BY REDUCED GLUTATHIONE - A H-1-NMR SPIN-ECHO STUDY OF INTACT HUMAN ERYTHROCYTES

The NMR signal of reduced glutathione (GSH) was monitored in intact hu man erythrocytes by the H-1 spin-echo Carr-Purcell-Meiboom-Gill pulse sequence. Addition of GSH, which was unable to cross the erythrocyte m embrane, 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 enzyme s was excluded by experiments with glucose-supplied or glucose-6-phosp hate dehydrogenase deficient cells. Involvement of external thiol grou ps of the erythrocyte membrane was shown by the lack of effect in cell s treated with an impermeable thiol-blocking compound. Involvement of spectrin was indicated by the proportional loss of the effect in eryth rocytes with variable genetic deficiency of spectrin. Protein-glutathi one mixed disulfides appeared to be the source of the NMR response sin ce an increase of their content, by diamide treatment or aging procedu res, produced a higher GSH signal, while their reduction by permeable reductants gave the opposite effect. It is concluded that GSH can tran sduce its reducing power by a thiol/disulfide exchange mechanism that sequentially involves sulfur-rich proteins spanning across the erythro cyte membrane.