Minor thiols cysteine and cysteinylglycine regulate the competition between glutathione and protein SH groups in human platelets subjected to oxidative stress.
Changes in the concentrations of protein-mixed disulfides (XS-SP) of glutathione (GSH), cysteine (CSH), and cysteinylglycine (CGSH) were studied in human platelets treated with diamide and t-BOOH in timecourse experiments (time range, 1-30 min) in order to understand the contribution of minor thiols CSH and CGSH to the regulation of glutathione-protein mixed disulfides (GS-SP). Diamide was much more potent than t-BOOH in altering the platelet thiol composition of XS-SP (threshold dose: diamide, 0.03 mM; t-BOOH, 0.5 mM) and caused reversible XS-SP peaks whose magnitude was related to the concentration of free thiols in untreated cells. Thus maximum levels of GS-SP (8 min after 0.4 mM diamide) were about 16-fold higher than those of controls (untreated platelets, GS-SP = 0.374 nmol/10(9) platelets), whereas those of CS-SP and CGS-SP were only 4-fold increased (untreated platelets, CS-SP = 0.112 nmol/10(9) platelets; CGS-SP = 0.024 nmol/10(9) platelets). The greater effects of diamide with respect to t-BOOH were explained on the basis of the activities of fast reactive protein SH groups for diamide and glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G-6-PDH) for t-BOOH. The addition of cysteine (0.3 mM, at 4 min) after treatment of platelets with 0.4 mM diamide increased the rate of reversal of GS-SP peaks to normal values, but also caused a relevant change in CGS-SP with respect to that of platelets treated with diamide alone. An increased gamma-glutamyltranspeptidase activity was found in platelets treated with diamide. Moreover, untreated platelets were found to release and hydrolyze GSH to CGSH and CSH. Ratios of thiols/disulfides (XSH/XSSX) and activities of GR and G-6PDH were also related to a high reducing potential exerted by GSH but not by minor thiols. The lower mass and charge of minor thiols is a likely requisite of the regulation of GS-SP levels in platelets.
Giustarini
D
D
Department of Neuroscience-Pharmacology Section, University of Siena, Via A. Moro 4, Siena, 53100, Italy.
Campoccia
G
G
Fanetti
G
G
Rossi
R
R
Giannerini
F
F
Lusini
L
L
Di Simplicio
P
P
D000975:Antioxidants / Q000378:metabolism; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism*; D003958:Diamide / Q000494:pharmacology; D004151:Dipeptides / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004912:Erythrocytes / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D018384:Oxidative Stress; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds / Q000502:physiology*; D013997:Time Factors; D018909:src Homology Domains; D020122:tert-Butylhydroperoxide / Q000494:pharmacology
D000975:Antioxidants; D004151:Dipeptides; D004220:Disulfides; D013438:Sulfhydryl Compounds; D003958:Diamide; C028505:cysteinylglycine; D020122:tert-Butylhydroperoxide; D004228:Dithionitrobenzoic Acid; D005978:Glutathione; D003545:Cysteine