Protein thiols and glutathione influence the nitric oxide-dependent regulation of the red blood cell metabolism.
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 (K(2) = 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.
Galli
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Istituto di Chimica Biologica G. Fornaini, University of Urbino, Via Saffi 2, 61029 Urbino (Ps), Italy. galli@uniurb.it
Rossi
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Di Simplicio
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Floridi
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Canestrari
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D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D006019:Glycolysis / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007700:Kinetics; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D001457:Anion Exchange Protein 1, Erythrocyte; D020030:Nitric Oxide Donors; D013438:Sulfhydryl Compounds; D009569:Nitric Oxide; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D005978:Glutathione; D003545:Cysteine