Purification and Biochemical Characterization of Glutathione S-Transferases in Bactrocera Minax (Diptera: Tephritidae)
Abstract
Glutathione S-transferases (GSTs) were purified from 3 developmental stages of Bactrocera minax through glutathione-agarose affinity chromatography, and characterized subsequently using the Michaelis-Menten kinetics toward the artificial substrates 1-chloro-2, 4-dinitrobenzene (CDNB) and reduced glutathione (GSH), respectively. Compared to the counterparts of third instar larva and adult, the highest specific activity of the purified GSTs towards CDNB was observed in the pupae. Although the specific activities of purified enzymes varied among 3 developmental stages, the purification yields were similar. SDS-PAGE revealed only one band at 23 kDa for all 3 stages. GSTs of the adults exhibited the highest Km value towards CDNB, while for GSH the pupae possessed the highest Km. The optimum temperature and pH for CDNB conjugation of the 3 stages were 37 °C and 7.5, respectively. Inhibition kinetics showed that ethacrynic acid, bromosulfalein, diethyl maleate, tetraethylthiuram disulfide and curcumin possessed excellent inhibitory effects on purified GSTs in B. minax. Moreover, the pupa showed the highest catalytic capability based on Vmax values for both the CDNB and GSH, which may suggest a potentially higher GSTs detoxification ability in the pupal stage than the other 2 developmental stages.Resumen: Se purificaron las transferasas-S de glutatión (GST) de 3 estadios de desarrollo de Bactrocera minax por medio de la afinidad de cromatografía de glutation-agarosa, y caracterizada luego usando la kinética de Michaelis-Menten hacia el sustrato artificial 1-cloro-2, 4 dinitrobenceno-(CDNB) y glutatión reducida (GSH), respectivamente. En comparación con los homólogos del tercer estadio larval y del adulto, la mayor actividad específica de las GST purificadas hacia CDNB se observó en la pupas. Aunque las actividades específicas de las enzimas purificadas varian entre los 3 estadios de desarrollo, el rendimiento de purificación fueron similares. SDS-PAGE reveló una sola banda a los 23 kDa para los 3 estadios. El GST del adulto exhibió el mayor valor de Km hacia el CDNB, mientras que la GSH de la pupa posee la más alta Km . La temperatura y el pH óptimo para la conjugación de CDNB de las 3 estadios fueron 37 °C y 7.5, respectivamente. La inhibición kinética mostró que el ácido etacrínico, bromosulfalein, maleato de dietilo, disulfuro de tetraetiltiuram y la cúrcuma posee excelentes efectos inhibidores sobre la GST purificada en B. minax. Además, la pupa presentó la mayor capacidad catalítica basado en los valores de Vmax para el CDNB y GSH, lo que puede indicar una capacidad potencialmente mayor de desintoxicación de GSTs en el estadio de la pupa que las otros 2 estadios de desarrollo.
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