S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase decreases the enzyme affinity to the erythrocyte membrane.
The effects of nitric oxide (NO) or related molecules on the binding of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to the red blood cell (RBC) membrane were investigated. It was demonstrated that submillimolar concentrations of the NO donor sodium nitroprusside (SNP) not only strongly inactivated GAPDH by S-nitrosylation of the enzyme thiols but also decreased the binding affinity of GAPDH for the RBC membrane. In fact, the incubation with SNP for 60 min at 30 degrees C and at a concentration > 50 microM induced the dissociation of the native GAPDH from the white unsealed membranes (standard ghosts) in a concentration-dependent manner with a partial recovery of the enzyme activity and thiols when SNP concentrations higher of 1 mM were used. Binding experiments under saturating conditions indicate a Ka value for the nitrosylated GAPDH of 3.5 +/- 0.8 x 10(6) M-1, which was more than 50% less than the Ka value of 7.6 +/- 0.6 x 10(6) M-1 observed for the native enzyme. These data were also confirmed in reassociation experiments under nonsaturating conditions. Dithiothreitol (DTT), which at concentrations of less than 1 mM catalyzed the S-nitrosylation of GAPDH and the consequent modification of the binding properties described above, the concentrations higher than 5 mM restored both the enzyme activity and the binding properties. Furthermore, the enzyme-membrane association induced before the incubation step afforded at least partial protection from the loss of titrable thiols and from the inactivation induced either spontaneously or by SNP. Taken together, these data not only confirm the key role of the active site cysteine residues in the catalytic function of GAPDH but also suggest that they may be involved in the NO-dependent regulation of GAPDH binding to the RBC membrane.
Galli
F
F
G. Fornaini Institute of Biochemistry, University of Urbino, Italy.
Rovidati
S
S
Ghibelli
L
L
Canestrari
F
F
D000818:Animals; D015153:Blotting, Western; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D018482:Muscle, Skeletal / Q000187:drug effects / Q000201:enzymology; D009599:Nitroprusside / Q000494:pharmacology*; D011817:Rabbits; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D009599:Nitroprusside; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D004229:Dithiothreitol