El plaguicida glifosato incrementa la Vmax de la Ca2 +-ATPasa de membrana plasmática de eritrocito humano sin afectar su afinidad (Km) por el substrato
The pesticide glyphosate increases the Vmax of the Ca2 +-ATPase from plasma membrane of human erythrocyte without affecting its affinity (Km) for the substrate
Resumen
Los plaguicidas son un amplio grupo de sustancias heterogéneas que producen un beneficio mediante la disminución de vectores que trasmiten enfermedades, y en el control de plagas que afectan la producción agrícola. En el presente trabajo estudiamos el efecto del glifosato (GF) sobre la actividad hidrolítica de la ATPasa dependiente de Ca2+ de membrana (PMCA) de eritrocito humano en su forma nativa (sin calmodulina [CaM]) o en el complejo activo CaM-PMCA. Nuestros resultados evidenciaron que el GF produjo un efecto dual, estimulatorio e inhibitorio, y el efecto fue dosis-dependiente: concentraciones bajas de GF (0.05-0.1 mM) estimularon la hidrólisis de ATP por PMCA en su conformación nativa en un 62.5%, y en el complejo activo por CaM el incremento fue de tan solo del 20% (basados en sus respectivos controles sin GF). Ambas formas conformaciones de PMCA fueron parcialmente inhibidas con GF 0.3-0.5 y 0.4-0.5 mM. El análisis sobre el efecto de GF en la afinidad de la enzima por el substrato (ATP), mostró que GF fue capaz de incrementar significativamente el consumo de ATP (Vmax), pero solo para la conformación nativa de PMCA. Sin embargo, la afinidad de la enzima por el sustrato (Km) no fue afectada en ninguna de las dos conformaciones de PMCA. En conclusión, GF afecta la velocidad del ciclo catalítico de PMCA sin afectar su afinidad por el sustrato, ello puede deberse a una interacción del plaguicida con el C-terminal de PMCA. Este efecto, pudiera ocasionar disturbios sobre la homeostasis celular del calcio desencadenando la activación de procesos celulares tales como la apoptosis.
Abstract
Pesticides are a broad group of heterogeneous substances with great benefit to the human population, as they are used for decreasing or killing a broad number of pests involved in transmission of diseases or involved in decreasing the levels of food production. In this work we studied the effect of glyphosate (GF) over the hydrolytic activity of plasma membrane Ca2+-ATPase (PMCA) from human erythrocytes, either in their native conformational state (without calmodulin [CaM]) or in the active complex CaM-PMCA. Our results showed that GF produced a dual effect, activation and inhibition, and the effect was a dose-response effect: at low-concentrations of GF (0.05-0.1 mM), ATP hydrolysis mediated by PMCA was stimulated up to 62.5%, however with CaM the increase was only 20% (with respect to the controls without GF). Both conformational forms of PMCA were partially inhibited with GF 0.3-0.5 and 0.4- 0.5 mM respectively. An analysis performed on PMCA affinity for the substrate (ATP) showed that GF was significantly increasing the ATP consumption (Vmax) and it was only significant for PMCA in their native conformation. However, for the two conformations studied, GF was unable to significantly affect the affinity of PMCA for the substrate ATP (Km). In conclusion, GF affects the velocity of the PMCA’s catalytic cycle without affecting its affinity for ATP, and this may be due to an interaction between GF and the C-terminus of PMCA. This effect may disturb calcium homeostasis in the cell, leading to the activation of several cellular processes such as apoptosis.
Keywords: Ca2+-ATPase, plasma membrane, glyphosate, calmodulin, human erythrocyte.
Citas
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