Characterization of bothrojaracin interaction with human prothrombin.

Bothrojaracin (BJC) is a 27-kD snake venom protein from Bothrops jararaca that has been characterized as a potent thrombin inhibitor. BJC binds to exosites I and II, with a dissociation constant of 0.7 nM, and influences but does not block the proteinase catalytic site. BJC also binds prothrombin through an interaction that has not been characterized. In the present work we characterize the interaction of BJC with prothrombin quantitatively for the first time, and identify the BJC binding site on human prothrombin. Gel filtration chromatography demonstrated calcium-independent, 1:1 complex formation between fluorescein-labeled BJC ([5F]BJC) and prothrombin, whereas no interactions were observed with activation fragments 1 or 2 of prothrombin. Isothermal titration calorimetry showed that binding of BJC to prothrombin is endothermic, with a dissociation constant of 76 +/- 32 nM. The exosite I-specific ligand, hirudin(54-65) (Hir(54-65) (SO(3)(-)), displaced competitively [5F]BJC from prothrombin. Titration of the fluorescent hirudin(54-65) derivative, [5F]Hir(54-65)(SO(3)(-)), with human prothrombin showed a dissociation constant of 7.0 +/- 0.2 microM, indicating a approximately 100-fold lower binding affinity than that exhibited by BJC. Both ligands, however, displayed a similar, approximately 100-fold increase in affinity for exosite I when prothrombin was activated to thrombin. BJC efficiently displaced [5F]Hir(54-65)(SO(3)(-)) from complexes formed with thrombin or prothrombin with dissociation constants of 0.7 +/- 0.9 nM and 11 +/- 80 nM, respectively, indicating that BJC and Hir(54-65)(SO(3)(-)) compete for the same exosite on these molecules. The results indicate that BJC is a potent and specific probe of the partially exposed anion-binding exosite (proexosite I) of human prothrombin.

Myocardial oxidative stress and antioxidants in hypertension as a result of nitric oxide synthase inhibition.

Rats were made hypertensive by the administration of the nitric oxide synthase inhibitor nitro-L-arginine (LNA, 2.74 mmol/L) in drinking water for 7 d. Hearts from hemodynamically assessed animals were analyzed for lipid peroxidation (LPO), gamma-glutamylcysteine-synthetase (gamma-GCS), glutathione disulfide reductase (GR), glutathione peroxidase (GSHPx), catalase (CAT), superoxide dismutase (SOD), and total radical trapping potential (TRAP) activities. LNA treatment increased the mean arterial blood pressure by 46% and the heart rate by 22% without changing plasma renin activity. LNA treatment resulted in a 30% increase in LPO. gamma-GCS was reduced by 48% and GR by 36% in the cardiac tissue of hypertensive rats as compared to controls. The activity of nonselenium GSHPx was reduced by 27%, and selenium-dependent GSHPx activity in the heart was not affected by LNA treatment. In hypertensive rats, SOD activity was increased by 16%, and CAT was decreased by 46%. TRAP was lower (27%) in the myocardium of hypertensive rats than in that of controls. These data suggest that LNA-induced hypertension is associated with increased myocardial oxidative stress.

Exercise training in aging: hemodynamic, metabolic, and oxidative stress evaluations.

The effects of exercise training on hemodynamic and metabolic parameters as well as on responses to oxidative stress in aged individuals are controversial. The aim of the present study was to investigate changes in heart hate, mean arterial pressure, vasoreactivity, and plasma levels of insulin and glucose in male aged Wistar rats submitted to exercise training for 11 weeks (1 h/d; 5 d/wk) in a treadmill. The isolated heart was perfused by H2O2, and oxidative stress was evaluated using thiobarbituric acid reactive substances. Cardiovascular functions were recorded with a data acquisition system (CODAS, 1 kHz). Trained aged rats were bradycardic as compared with sedentary aged rats (298+/-7 versus 336+/-16 bpm) but presented similar mean arterial pressure and vasoreactivity and plasma levels of insulin and of glucose, which were quantified by radioimmunoassay and colorimetric enzymatic test. Plasma levels of insulin and of glucose ratio were increased in trained aged rats (6.9+/-0.7 versus 3.5+/-0.4 in sedentary aged rats), and the response to oxidative stress was decreased (0.4+/-0.1 versus 0.7+/-0.1 nmol/mg protein in sedentary aged rats). These results showed that exercise training produced a lower resting heart rate as well as changes in metabolic and oxidative responses. This suggests a higher myocardium protection of trained than sedentary aged rats.