Nitric oxide (NO) can modulate red blood cell (RBC) glycolysis by translocation of the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPD) (EC 1.2.1.12) from the cytosolic domain of the membrane protein band 3 (cdb3) in the cytosol. In this study we have investigated which NO-reactive thiols might be influencing GAPD translocation and the specific role of glutathione. Two highly reactive Cys residues were identified by transnitrosylation with nitrosoglutathione (GSNO) of cdb3 and GAPD (K2 = 73.7 and 101.5 M-1 s-1, respectively). The Cys 149 located in the catalytic site of GAPD is exclusively involved in the GSNO-induced nitrosylation. Reassociation experiments carried out at equilibrium with preparations of RBC membranes and GAPD revealed that different NO donors may form -SNO on, and decrease the affinity between, GAPD and cdb3. In intact RBC, the NO donors 3-morpholinosydnonimine (SIN-1) and peroxynitrite (ONOO-) significantly increased GAPD activity in the cytosol, glycolysis measured as lactate production, and energy charge levels. Our data suggest that ONOO- is the main NO derivative able to cross the RBC membrane, leading to GAPD translocation and -SNO formation. In cell-free experiments and intact RBC, diamide (a thiol oxidant able to inhibit GAPD activity) was observed to reverse the effect of SIN-1 on GAPD translocation. The results demonstrate that cdb3 and GAPD contain reactive thiols that can be transnitrosylated mainly by means of GSNO; these can ultimately influence GAPD translocation/activity and the glycolytic flux. © 2002 Elsevier Science (USA).

Galli, F., Rossi, R., Di Simplicio, P., Floridi, A., Canestrari, F. (2002). Protein thiols and glutathione influence the nitric oxide-dependent regulation of red blood cell metabolism. NITRIC OXIDE, 6(2), 186-199 [10.1006/niox.2001.0397].

Protein thiols and glutathione influence the nitric oxide-dependent regulation of red blood cell metabolism

ROSSI, RANIERI;
2002-01-01

Abstract

Nitric oxide (NO) can modulate red blood cell (RBC) glycolysis by translocation of the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPD) (EC 1.2.1.12) from the cytosolic domain of the membrane protein band 3 (cdb3) in the cytosol. In this study we have investigated which NO-reactive thiols might be influencing GAPD translocation and the specific role of glutathione. Two highly reactive Cys residues were identified by transnitrosylation with nitrosoglutathione (GSNO) of cdb3 and GAPD (K2 = 73.7 and 101.5 M-1 s-1, respectively). The Cys 149 located in the catalytic site of GAPD is exclusively involved in the GSNO-induced nitrosylation. Reassociation experiments carried out at equilibrium with preparations of RBC membranes and GAPD revealed that different NO donors may form -SNO on, and decrease the affinity between, GAPD and cdb3. In intact RBC, the NO donors 3-morpholinosydnonimine (SIN-1) and peroxynitrite (ONOO-) significantly increased GAPD activity in the cytosol, glycolysis measured as lactate production, and energy charge levels. Our data suggest that ONOO- is the main NO derivative able to cross the RBC membrane, leading to GAPD translocation and -SNO formation. In cell-free experiments and intact RBC, diamide (a thiol oxidant able to inhibit GAPD activity) was observed to reverse the effect of SIN-1 on GAPD translocation. The results demonstrate that cdb3 and GAPD contain reactive thiols that can be transnitrosylated mainly by means of GSNO; these can ultimately influence GAPD translocation/activity and the glycolytic flux. © 2002 Elsevier Science (USA).
2002
Galli, F., Rossi, R., Di Simplicio, P., Floridi, A., Canestrari, F. (2002). Protein thiols and glutathione influence the nitric oxide-dependent regulation of red blood cell metabolism. NITRIC OXIDE, 6(2), 186-199 [10.1006/niox.2001.0397].
File in questo prodotto:
Non ci sono file associati a questo prodotto.

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11365/42780
 Attenzione

Attenzione! I dati visualizzati non sono stati sottoposti a validazione da parte dell'ateneo