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    Sensing molecular organizational changes through the catalytic activity of acetylcholinesterase from erythrocyte membranes in Langmuir-Blodgett films

    Year: 2020

    Journal: Biochim. Biophys. Acta-Biomembr., Volume 1862, MAY 1

    Authors: Felsztyna, I; Turina, AV; Perillo, MA; Clop, EM

    Organizations: Agencia Nacional de Promocion Cientifica y Tecnologica, Ministerio de Ciencia y Tecnologia (Mincyt-Cordoba); Secretaria de Ciencia y Tecnologia (SeCyT-Universidad Nacional de Cordoba)Secretaria de Ciencia y Tecnologia (SECYT); Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET) from ArgentinaConsejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET); Consejo Interuniversitario Nacional (CIN); CONICETConsejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET)

    Keywords: Erythrocyte acetylcholinesterase; Bovine erythrocyte membranes; Langmuir-Blodgett films; Monoterpenes; Biosensor proof of principle

    Langmuir films prepared from bovine erythrocyte membranes (LFBEM) were studied and transferred to alkylated glasses (Langmuir-Blodgett films, LBBEM) in order to assess the effects of membrane molecular packing on Bovine Erythrocyte Acetylcholinesterase (BEA) catalytic activity. Surface pressure (pi) vs Area isotherms showed three 2D-transitions at similar to 7, similar to 18 and similar to 44 mN/m and a collapse pressure at pi(c) = 49 mN/m. The 0-12-0 mN/m compression-decompression cycles resulted reversible while those 0-40-0 mN/m exhibited a significant hysteresis. Taken together, EFM, BAM and AFM images and the stability of the film after 3C-D cycles, we can suggest that over the air-water interface as well as over the silanized glass substrate the surface is mostly covered by a monolayer with a few particles dispersed. Acetylthiocholine hydrolysis was assayed with BEA in bovine erythrocyte membrane suspensions (S-BEM) and in LBBEM packed at 10 (LBBEM,10) and 35 mN/m (LBBEM,35), which gave the following kinetic parameters: V-max = 3.41 +/- 0.15, 0.021 +/- 0.002 and 0.030 +/- 0.003 nmol.min(-1)center dot mu g prot(-1) and K-M = 0.11 +/- 0.02, 0.047 +/- 0.017 and 0.026 +/- 0.017 mM, respectively. Although from S-BEM to LBBEM we lost active enzyme, the catalytic efficiency (V-max/K-M) increased similar to 750 times. Eugenol and 1,8-cineol inhibited BEA catalytic activity in LBBEM,35. Our results demonstrate the transmission of information between the membrane and the environment within the subphase immediately below the membrane, where anchored proteins are hosted. This was reflected by the membrane packing-induced modulation of BEA catalytic activity. Furthermore, LBBEM provides a proof of concept for the development of biosensors to screen new green pesticides acting through BEA interaction.
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