Effect of natural terpenes on Bovine erythrocyte acetylcholinesterase (BEA) activity from bovine erythrocyte ghost membranes (BEM). Possible unspecific mechanism that tunes the BEA catalytic activity.

DUTTO, J.; CLOP, E. M.; TURINA, A. V.; PERILLO, M. A.

Córdoba

Encuentro; VII Reunión Científica del IIByT (CONICET-UNC); 2019

Instituto de Investigaciones Bilógicas y Tecnológicas

BEA is a GPI-anchored enzyme that hydrolyzessericacetylcholine. The ?anionic? subsite in the activesite determines the specificity with respect to the choline moiety through electrostatic interactions. Sincea) changes on the molecular environment of GPI-anchored enzymes affect their kinetic parameters andb) monoterpenes (MT) affects biomembrane order and electrostatics according to their dipole momentmodulus and orientation, here we tested the effects of MTs (1-8 cineol, CIN and camphor, CAM) on thehydrolysis of acethylthiocholine (ATC, Ellman´s method)catalyzed by BEA present in BEM. Theaffinity of the BEA-ATC complex in the absence of MTs (K M =0.1) was significantly affected by CINwhich resulted a stronger inhibitor (K M = 0.81) than CAM (K M =0.11) (both at 0.3mM). Moreover, CINexhibited an IC 50 =0.3mM whereas the IC 50 of CAM was >> 0.6 mM.Measurements of the fluorescenceanisotropy (A) of DPH and TMA-DPH in BEM, demonstrated that both MTs affected the organizationof the inner regions of the bilayer (both MTs reduced about a 10% the A DPH ) but not the polar headgroup region (A TMA-DPH was almost unaffected). The effect of MTs on the lateral pressure (π) and surfacepotential (DV) vs Area compression isotherms in Langmuir films were also studied. In the presence ofCIN, the transition found in the control π-Aisotherm become less cooperative and the π collapse decreased.At low π, the slopes of both isotherms (π-A and DV-A) changed; e.g.we found a DDV~20mV withrespect to the control without CIN. At high π,CIN and control isotherms convergedsuggesting theCINmolecules expulsion from the film upon compression. CAM did not produce significant effects onDV, but expanded slightly the whole π-A isotherm up to the collapse point. Concluding, the inhibitoryactivity of CIN on BEA may be related with its effect on the membrane order and electrostatics whichmay be interfering unspecificallywith the BEA-ATC electrostatic interaction at the active site.Acknowledgements: JD holds a fellowship form EVC-CIN. Financed with grants from CONICET,SECyT, FONCyT.