RESUMO
Acute renal failure is a serious condition of Crotalus bites, which could be treated with statins. The effects of Crotalus durissus terrificus venom (vCdt) and simvastatin on renal function, oxidative stress and representative plasma, urinary and renal aminopeptidase (AP) activities were evaluated in mice. Eighty percent LD50 of vCdt caused hyperuricemia and urinary hypoosmolality (100%) and hypercreatinemia (60%). Plasma neutral, pyroglutamyl and dipeptidyl IV and renal soluble and membrane-bound APs were susceptible to vCdt. Cortical and medullar oxidative stress (GSSG/GSH ratio) was increased by vCdt. Simvastatin (3mg/kg body wt.) altered urinary creatinine and urea, membranal protein in cortex and medulla, plasma neutral and dipeptidyl IV APs and most of renal APs in nonenvenomed, and exacerbated hypercreatinemia, but mitigated uricosuria, renal oxidative stress and protein increase in envenomed. Hyperuricemia and urinary hypoosmolality are early signs of indirect myotoxicity of vCdt with diagnostic significance. In kidney, oxidative stress and alteration of protein content and AP activities suggest membrane destruction, enzyme release and protein loss, which may be due to direct tissue damage. Plasma AP activities might be nephrotoxicity markers of C. d. terrificus envenomation. The deleterious effects of simvastatin on creatinemia and APs constitute important restrictions to its use within the antivenom therapy.
Assuntos
Injúria Renal Aguda/induzido quimicamente , Aminopeptidases/metabolismo , Venenos de Crotalídeos/toxicidade , Crotalus/fisiologia , Estresse Oxidativo , Aminopeptidases/sangue , Aminopeptidases/urina , Animais , Rim/enzimologia , Dose Letal Mediana , Masculino , CamundongosRESUMO
The effect of 2-naphthylamine, p-nitroaniline, o-phenanthroline, sodium deoxycholate and hydrocortisone succinate on the activity of human urine aminopeptidase, rat kidney methionyl and arginyl aminopeptidase, soybean and Enterolobium contortisiliquum seed aminopeptidase was studied using aminoacyl-2-naphthylamide and L-Leu-p-nitroanilide as substrates. Ki values ranged from 10 microM to 2.7 mM. On the basis of Ki and Km values, and catalytic efficiency for each enzyme, it is clear that the aminopeptidases from human urine and from soybean seed should be assayed with both substrates, whereas L-Leu-p-nitroaniline is a more appropriate substrate for the rat kidney aminopeptidases. Sodium deoxycholate is a better inhibitor than hydrocortisone succinate. Non-competitive inhibition was observed in all cases except for E. contortisiliquum seed aminopeptidase.
Assuntos
Aminopeptidases/antagonistas & inibidores , Compostos Policíclicos/farmacologia , Aminopeptidases/efeitos dos fármacos , Aminopeptidases/urina , Animais , Relação Dose-Resposta a Droga , Humanos , Rim/enzimologia , Ratos , Sementes/enzimologia , Glycine max/enzimologia , Especificidade por Substrato/efeitos dos fármacos , Árvores/enzimologiaRESUMO
The effect of 2-naphthylamine, p-nitroaniline, o-phenanthroline, sodium deoxycholate and hydrocortisone succinate on the activity of human urine aminopeptidase, rat kidney methionyl and arginyl aminopeptidase, soybean and Enterolobium contortisiliquum seed aminopeptidase was studied using aminoacyl-2-naphthylamide and L-Leu-p-nitroanilide as substrates. Ki values ranged from 10 microM to 2.7 mM. On the basis of Ki and Km values, and catalytic efficiency for each enzyme, it is clear that the aminopeptidases from human urine and from soybean seed should be assayed with both substrates, whereas L-Leu-p-nitroaniline is a more appropriate substrate for the rat kidney aminopeptidases. Sodium deoxycholate is a better inhibitor than hydrocortisone succinate. Non-competitive inhibition was observed in all cases except for E. contortisiliquum seed aminopeptidase
Assuntos
Animais , Humanos , Aminopeptidases/antagonistas & inibidores , Hidrocarbonetos Cíclicos/farmacologia , Aminopeptidases/efeitos dos fármacos , Aminopeptidases/urina , Relação Dose-Resposta a Droga , Rim/enzimologia , Ratos , Sementes/enzimologia , Glycine max/enzimologia , Especificidade por Substrato/efeitos dos fármacos , Árvores/enzimologiaRESUMO
Se desarrolló un método de punto final para la determinación de la actividad urinaria de la alanilaminopeptidasa a partir de un método cinético. Una concentración 0,5 M de HCl resultó eficaz para detener la reacción enzimática. Con un tiempo de incubación de 15 minutos se logra compensar la disminución de la sensibilidad debido a la adición del ácido. En estas condiciones los resultados entre el método cinético y el nuestro son similares. Este hecho unido a la alta precisión, avalan la introducción de este método en los laboratorios de nuestro país para la detección de daño renal de diferente causa
Assuntos
Humanos , Aminopeptidases/urina , Ensaios Enzimáticos ClínicosRESUMO
Se estudió el valor diagnóstico de la alanilaminopeptidasa urinaria en pacientes con transplante renal, en dependencia del criterio de decisión indicativo de un aumento en su excreción. Los mejores resultados se obtienen cuando la excreción diaria de esta enzima es normalizada en forma de promedios de tres días consecutivos y tiene lugar en aumento igual o superior a el 20% en comparación con el dia anterior
Assuntos
Humanos , Aminopeptidases/urina , Rim/transplanteRESUMO
1. Arylamidases were isolated from rat urine using L-aminoacyl-2-naphthylamides and L-Leu-p-nitroanilide as substrates to monitor the purification. 2. Ion-exchange chromatography separated three peaks of activity (A, B and C). After gel filtration chromatography, the second and third peaks (B and C) were further purified to provide B1 and C1. Each behaved like a single active protein band on 7.5% polyacrylamide gel electrophoresis without SDS. The molecular weights of fractions B1 and C1 determined by SDS-PAGE were 440 and 270 kDa, respectively. 3. The pH optimum for arylamidase activity was 7.5 for both forms on all substrates tested. The maximum value for the Vmax/Km ratio was obtained using L-Leu-2-naphthylamide as substrate for both forms. 4. The arylamidase activity of B1 and C1 was not affected by the presence of chloride ions and was increased in the presence of CaCl2 and MnCl2 only when L-Glu-2-naphthylamide was used as substrate. EDTA (3.3-33.0 microM) and o-phenanthroline (0.1-1.0 mM) but not -SH (0.08-0.67 mM) or -S-S-(0.42-3.3 mM) group reagents inhibited the arylamidase activity. Hydrolysis of L-Leu-2-naphthylamide by fractions B1 and C1 was competitively inhibited by leucine (0.14-0.56 mM), indomethacin and puromycin (67-267 microM) and bestatin (8.3-33.3 microM). For each inhibitor, the Ki values were similar in the two fractions: 100 microM for L-leucine, 10 microM for indomethacin and puromycin and 1.0 microM for bestatin.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Aminopeptidases/isolamento & purificação , Aminopeptidases/urina , Animais , Cálcio/farmacologia , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Feminino , Masculino , Ratos , Ratos EndogâmicosRESUMO
Arylamidases were isolated from rat urine using L-aminoacyl-2-naphthylamides and L-Leu-p-nitroanilide as substrates to monitor the purification. Ion-exchange chromatography separated three peaks of activity (A, B and C). after gel filtration chromatography, the second and third peaks (B and C) were further purified to provide B1 and C1. Each behaved like a single active protein band on 7.5% polyacrylamide gel electrophoresis without SDS. he molecular weights of fractions B1 and C1 determined by SDS-PAGE were 440 and 270 kDa, respectively. The pH optimum for arylamidase activity was 7.5 for both forms on all substrate for both forms. The arylamidase activity of B1 and C1 was not affected by the presence of chloride ions and was increased in the presence of CaCl2 and MnCl2 only when L-Glu-2-naphthylamide was used as substrate. EDTA (3.3-33.0 micronM) and o-phenanthroline (0.1-1.0mM) but not -SH(0.08-0.67 mM) or -S-S-(0.42-3.3mM) group reagents inhibited the arylamidase activity. Hydrolysis of L-Leu-2-naphthylamide by fractions B1 and C1 was competitively inhibited by leucine (0.14-0.56 mM), indomethacin and puromycin (67-267 micronM) and bestatin (8.3-33.3 micronM). For each inhibitor, the Ki values were similar in the two fractions: 100 micronM for L-leucine, 10 micronM for indomethacin and puromycin and 1.0 micronM for bestatin. The enzymatic properties of fractions B1 and C1 were similar to those reported for fraction A1 by Alves et al. (Brazilian Journal of Medical and Biological...
Assuntos
Aminopeptidases/isolamento & purificação , Aminopeptidases/urina , Cálcio/fisiologia , Eletroforese em Gel de Poliacrilamida , Ratos EndogâmicosRESUMO
The aminopeptidase activity of rat urine was tested using L-aminoacyl-2-naphthylamides and L-Leu-p-nitroanilide. The enzyme preparation was purified by ion-exchange and gel filtration chromatography and showed only a single active protein band on 7.5% polyacrylamide gel electrophoresis. The enzyme was characterized by studying the effects of ions (divalent cations and chloride), chelating agents and -SH and -S-S- group reagents and by determining kinetic parameters (Km and Vmax for different substrates and Ki for amino acids, antibiotics and anti-inflammatory drugs).
Assuntos
Aminopeptidases/metabolismo , Aminopeptidases/urina , Aminopeptidases/antagonistas & inibidores , Aminopeptidases/isolamento & purificação , Animais , Cátions Bivalentes/farmacologia , Cinética , RatosRESUMO
The aminopeptidase activity of rats urine was tested using L-aminoacy1-2-naphtylamides and L-Leu-p-nitroanilide. The enzyme preparation was purified by ion-exchange and gel filtration chromatography and showed only a single active protein active protein band on 7.5% polyacrylamide gel electrophoresis. The enzyme was characterized by studying the effects of ions (divalent cations and chloride), chelating agents and -SH and -S-S- group reagents and by determining kinetic parameters (Km and Vmax for different substrates and K1 for amino acids, antibiotics and anti-inflammatory drugs)