Modification of Protein by Disulfide S-Monoxide and Disulfide S-Dioxide:  Distinctive Effects on PKC

Abstract

Disulfide S-monoxide (DSMO) and disulfide S-dioxide (DSDO) have been proposed as proximal mediators for the oxidant-mediated modification of proteins. These disulfide S-oxides (DSOs) derived from glutathione (GSH) and captopril (CPSH) were synthesized by iron- or methyltrioxorhenium (VII)-catalyzed oxidation of the thiols with H2O2. Treatment of mouse hippocampal extracts with [35S]GS-DSOs revealed that a large number of proteins were susceptible to thionylation; however, only a limited number of the them were detectable by the commonly used antibody against GS-associated proteins. Using protein kinase C (PKC) as a model, we found that DSOs derived from different thiols modified this kinase with different efficacy and specificity; for example, the inhibitory potency of the kinase was glutathione disulfide S-dioxide (GS-DSDO) (IC50, approximately 30 microM) > captopril disulfide S-dioxide (CPS-DSDO) (IC50, approximately 450 microM) > glutathione disulfide S-monoxide (GS-DSMO) and captopril disulfide S-monoxide (CPS-DSMO). The stoichiometries of thionylation of PKC beta mediated by [35S]GS-DSMO and [35S]GS-DSDO were approximately 1 and 5 mol/mol, respectively, and at least four glutathionylation sites were identified in the GS-DSDO-treated kinase. Modification of PKC by GS-DSDO and CPS-DSDO rendered the kinase very susceptible to limited proteolysis; the former preferentially caused the degradation of the catalytic and the latter the regulatory domain of the kinase. Furthermore, CPS-DSDO-mediated modification of PKC increased the autonomous kinase activity; this was not the case for GS-DSDO-mediated modification. Since DSOs of different oxidative states as well as those derived from different thiols exert different effects on a target protein, these molecules could cause distinct cellular responses if derived from endogenous cellular reactions or even if they arise from exogenous sources.

Ischemia-elicited Oxidative Modulation of Ca2+/Calmodulin-dependent Protein Kinase II

“…The sedimentation of standard proteins was identified by staining the SDS-gel with Coomassie Blue. Preparation and assay of GS-DSMO and GS-DSDO were carried as previously described (33). Immunoblot analysis was carried out by resolving proteins in 8 -16% gradient gels containing 0.1% SDS, followed by transfer to nitrocellulose mem-brane.…”

“…Previously, we showed that activation of NMDA receptors or direct administration of oxidants to the brain slices could trigger thionylation of neurogranin and formation of intra-molecular disulfide of this protein (29,30). The oxidant-induced modification of protein was hypothesized as a result of oxidation by the endogenously produced glutathione disulfide S-oxides (GS-DSOs), which have a high activity to form glutathione-protein-mixed disulfide (glutathionylation) and inter-and/or intra-molecular disulfide bonds of proteins (31)(32)(33).…”

Análise de agrotóxicos em água usando extração líquido-líquido com partição em baixa temperatura por cromatografia líquida de alta eficiência

“…Esta etapa visa remover seletivamente resíduos de agrotóxicos destas amostras e pré-concentrá-los. 12 As principais técnicas existentes são a extração em fase sólida, [13][14][15] microextração em fase sólida, [16][17][18][19] microextração líquido-líquido [20][21][22][23] e microextração em gota única. 24 Apesar do elevado número de técnicas existentes, a extração líquido-líquido com partição em baixa temperatura (ELL-PBT) tem se apresentado como uma alternativa para extração de agrotóxicos em diversas matrizes.…”

Simultaneous derivatization and extraction of free cyanide in biological samples with home-made hollow fiber-protected headspace liquid-phase microextraction followed by capillary electrophoresis with UV detection

“…In order to decrease the extraction solvent loss, some groups introduced the dynamic HF-LPME [24] in which the extraction solvent was withdrawn into the syringe barrel with the automated movement of the plunger during extraction, or solvent cooling assisted dynamic HF-LPME [31] in which the syringe was mounted in a cooling system. Further studies are still in progress.…”