RESUMO
Venom samples of Russell's viper (Vipera russelli) from three localities in India were analysed for their composition and toxicity. Column chromatographic fractionation on CM-Sephadex C-25 showed the absence of three fractions in the venom samples of southern India compared with the samples from northern and western India. The SDS-PAGE pattern of southern Indian venom samples also showed lack of three protein bands corresponding to molecular weights of 66,000, 39,000 and 9000. Venom samples from northern and western India possessed high acidic phospholipase activity while acidic phospholipase activity was absent in the samples from southern India, which in contrast showed large basic fractions with phospholipase activity. Proteolytic activity was present in all the venom samples; however, this activity, as well as trypsin inhibitor activity, was very low in the southern Indian samples. The ratio of proteolytic activity to inhibitor activity remained constant in most of the venom samples studied. LD50 values for most of the venom samples from northern and western India were twice as high as that of the samples from southern India. High phospholipase activity correlated with high lethal potency in the venom samples studied.
Assuntos
Venenos de Víboras/análise , Animais , Eletroforese em Gel de Poliacrilamida , Esterases/análise , Geografia , Índia , Dose Letal Mediana , Camundongos , Peptídeo Hidrolases/análise , Fosfolipases/análise , Dodecilsulfato de Sódio , Tripsina/análise , Venenos de Víboras/toxicidadeRESUMO
An acidic proteolytic enzyme, RVVX, was purified from Vipera russelli venom by successive chromatography on CM-Sephadex C-25, DEAE-cellulose and Sephadex G-100 columns. RVVX is a glycoprotein with a mol. wt of 79,000. It exhibited caseinolytic and factor X activating properties. Two trypsin inhibitors, TI-I and TI-II, were purified from V. russelli venom in a single step by CM-Sephadex C-25 column chromatography. The trypsin inhibitors interacted with the proteolytic enzyme RVVX. TI-I inhibited only the factor X activating property of RVVX while TI-II inhibited both, the caseinolytic and also factor X activating properties of RVVX. The edema inducing activity of RVVX increased markedly in the presence of non-edema inducing doses of TI-I and TI-II. RVVX, TI-I and TI-II were non-lethal in mice. The combination of RVVX and TI-II demonstrated enhanced toxicity.
Assuntos
Peptídeo Hidrolases/toxicidade , Inibidores de Proteases/toxicidade , Venenos de Víboras/toxicidade , Animais , Cromatografia DEAE-Celulose , Cromatografia em Gel , Sinergismo Farmacológico , Edema/induzido quimicamente , Eletroforese em Gel de Poliacrilamida , Fator X/metabolismo , Dose Letal Mediana , Camundongos , Peptídeo Hidrolases/análise , Peptídeo Hidrolases/isolamento & purificação , Inibidores de Proteases/análise , Inibidores de Proteases/isolamento & purificação , Espectrometria de Fluorescência , Inibidores da Tripsina/isolamento & purificação , Venenos de Víboras/análiseRESUMO
A neurotoxic phospholipase A2, VRV PL-V was purified from Vipera russelli venom in a single step by CM-Sephadex C-25 column chromatography. VRV PL-V is a basic PLA2 with a mol. wt of approximately 10,000. The lethal potency of VRV PL-V was greater than that of the crude V. russelli venom. VRV PL-V showed anticoagulant activity and induced edema in the foot pad of the mouse. VRV PL-V undergoes aggregation at pH 4.8. The size of the aggregate increased as the temperature at which the enzyme was incubated was raised. A highly aggregated form with a mol. wt of 53,100 was formed at 96 degrees C. This aggregate showed a two-fold increase in its catalytic activity, while its neurotoxic activity disappeared. The aggregate also showed a significant increase in its anticoagulant activity when compared to the monomeric form. Edema-inducing activity decreased upon association to higher molecular form.
Assuntos
Neurotoxinas , Fosfolipases A/toxicidade , Fosfolipases/toxicidade , Venenos de Víboras/toxicidade , Anticoagulantes , Catálise , Edema/induzido quimicamente , Eletroforese em Gel de Poliacrilamida , Hemólise/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Focalização Isoelétrica , Dose Letal Mediana , Peso Molecular , Fosfolipases A/isolamento & purificação , Fosfolipases A2 , Espectrometria de Fluorescência , Temperatura , Venenos de Víboras/análiseRESUMO
Rat liver ribonuclease P was isolated from a cytosolic fraction and shown to have optimal activity in the presence of 1 mM MgCl2 and 150-200 mM KCl using Escherchia coli pre-tRNA(Tyr) as substrate. In cesium sulfate isopycnic density gradients, the enzyme had a buoyant density of 1.36 g/ml, indicating that it is a ribonucleoprotein complex. Analysis of the RNAs in the enzyme sample purified through two successive Cs2SO4 density gradient steps revealed the copurification of two major species of RNA (RRP1 and RRP2) along with several less abundant RNAs. Rat liver ribonuclease P activity was insensitive to micrococcal nuclease pretreatment. However, the nuclease-treated preparations contained several incompletely degraded RNA species that may have been sufficient to support the ribonuclease P activity. When RNase A was substituted for micrococcal nuclease, the ribonuclease P activity was diminished by greater than 90%, suggesting the requirement for an RNA subunit for activity.