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.

PERILLO MA; DUTTO J; CLOP EM; TURINA AV

La Plata

Congreso; XLVII Reunión Anual de la Sociedad Argentina de Biofísica; 2018

Sociedad Argentina de Biofísica

BEA is a GPI-anchoredenzyme that hydrolyzes seric acetylcholine. The ?anionic? subsite in the activesite determines the specificitywith respect to the choline moiety through electrostatic interactions. Since a) changes on the molecular environment ofGPI-anchored enzymes affect their kinetic parameters and b) monoterpenes (MT) affectbiomembranes order and electrostatics according to their dipole moment modulusand orientation, here we tested the effects of MTs (1-8 cineol, CIN and camphor,CAM) on the hydrolysis of acethylthiocholine (ATC, Ellman's method) catalyzed byBEA present in BEM. The affinity of the BEA-ATC complex in the absence of MTs(KM=0.1) was significantly affected by CIN which resulted a strongerinhibitor (KM= 0.81) than CAM (KM=0.11) (both at 0.3 mM).Moreover, CIN exhibited an IC50=0.3mMwhereas the IC50 of CAM was >> 0.6 mM. Measurements of the fluorescenceanisotropy (A) of DPH and TMA-DPH in BEM, demonstrated that both MTs affectedthe organization of the inner regions of the bilayer (both MTs reduced about a10% the ADPH) but not the polar head group region (ATMA-DPHwas almost unaffected). The effect of MTs on the lateral pressure (π) and surface potential (DV) vs Area compression isotherms in Langmuir filmswere also studied. In the presence of CIN, the transition found in the control π-A isotherm become less cooperative and the πcollapse decreased. At low π, the slopesof both isotherms (π-A andDV-A) changed; e.g. we found a DDV~20mV with respect to the control without CIN. At high π, CIN and control isotherms converged suggestingthe CIN molecules expulsion from the film upon compression. CAM did not producesignificant effects on DV, but expanded slightly the whole π-A isotherm upto the collapse point. Concluding,the inhibitory activity of CIN on BEA may be related with its effect on themembrane order and electrostatics which may be interfering unspecifically with theBEA-ATC electrostatic interaction at the active site