guanosine-triphosphate and cysteine-sulfinic-acid

Peroxiredoxins (Prxs) are a family of peroxidases that reduce hydroperoxides. The cysteine residue in the active site of certain eukaryotic Prx enzymes undergoes reversible oxidation to sulfinic acid (Cys-SO2H) during catalysis, and sulfiredoxin (Srx) has been identified as responsible for reversal of the resulting enzyme inactivation in yeast. We have now characterized mammalian orthologs of yeast Srx with an assay based on monitoring of the reduction of sulfinic Prx by immunoblot analysis with antibodies specific for the sulfinic state. Sulfinic reduction by mammalian Srx was found to be a slow process (kcat = 0.18/min) that requires ATP hydrolysis. ATP could be efficiently replaced by GTP, dATP, or dGTP but not by CTP, UTP, dCTP, or dTTP. Both glutathione and thioredoxin are potential physiological electron donors for the Srx reaction, given that their Km values (1.8 mM and 1.2 microM, respectively) are in the range of their intracellular concentrations, and the Vmax values obtained with the two reductants were similar. Although its pKa is relatively low (approximately 7.3), the active site cysteine of Srx remained reduced even when the active site cysteine of most Prx molecules became oxidized. Finally, depletion of human Srx by RNA interference suggested that Srx is largely responsible for reduction of the Cys-SO2H of Prx in A549 human cells.

Topics: Adenosine Triphosphate; Amino Acid Sequence; Animals; Binding Sites; Cell Line, Tumor; Cloning, Molecular; Cysteine; DNA, Complementary; Dose-Response Relationship, Drug; Escherichia coli; Glutathione; Glutathione Transferase; Guanosine Triphosphate; HeLa Cells; Humans; Hydrogen-Ion Concentration; Kinetics; Mice; Models, Chemical; Molecular Sequence Data; Oxidation-Reduction; Oxidoreductases; Oxidoreductases Acting on Sulfur Group Donors; Oxygen; Peroxidases; Peroxiredoxins; Rats; Recombinant Proteins; RNA Interference; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Schizosaccharomyces; Sequence Homology, Amino Acid; Temperature; Thioredoxins; Time Factors; Tissue Distribution

The effect of forskolin on the excitatory amino acid-induced accumulation of cyclic AMP was examined in hippocampal preparations of the guinea pig. Forskolin at concentrations of 0.1-10 microM remarkably enhanced the stimulatory effects of histamine and adenosine, whereas it markedly attenuated the stimulation induced by cysteine sulfinate, an excitatory amino acid. Forskolin reduced the maximal response to cysteine sulfinate without affecting the apparent ED50. At a concentration of 1 microM, forskolin also inhibited the stimulatory effects of glutamate, N-methyl-DL-aspartate, and veratridine without affecting those of kainate and quisqualate. Pretreatment of the slices with 0.1 mM N-ethylmaleimide partially prevented the attenuation by forskolin of cysteine sulfinate-induced accumulation of cyclic AMP without affecting the stimulation induced by cysteine sulfinate. Forskolin at concentrations of less than 1 microM did not affect GTP-stimulated activity and Cl- -dependent activity of adenylate cyclase of the hippocampal membranes.

Topics: Adenosine; Amino Acids; Animals; Aspartic Acid; Chlorides; Colforsin; Cyclic AMP; Cysteine; Drug Interactions; Ethylmaleimide; Glutamates; Glutamic Acid; Guanosine Triphosphate; Guinea Pigs; Hippocampus; Histamine; Male; N-Methylaspartate; Neurotransmitter Agents; Veratridine