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Modulation of Erythrocyte Acetylcholinesterase Activity and Its Association with G Protein-Band 3 Interactions

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Abstract

Circulating acetylcholine, substrate of membrane acetylcholinesterase (AChE), is known to enhance the band 3 protein degree of phosphorylation. The purpose of this study was to verify whether the band 3 phosphorylation status is associated with a G protein and whether it is an influent factor on AChE enzyme activity. From blood samples of healthy donors, erythrocyte suspensions were prepared and incubated with AChE substrate (acetylcholine) and inhibitor (velnacrine), along with protein tyrosine kinase (PTK) and tyrosine phosphatase (PTP) inhibitors. AChE activity was determined by spectrophotometry and extract samples were analyzed by western blotting using primary antibodies to different G protein subunits. Our results with phosphorylated band 3 (PTP inhibitor) show an increase in erythrocyte AChE (p < 0.0001). A dephosphorylated band 3 state (PTK inhibitor) shows a significant decrease. We identified a potential linkage of protein subunits Gαi1/2 and Gβ with band 3 protein. Gαi1/2 and Gβ may be linked to the band 3 C-terminal site. Gαi1/2 is associated with the band 3 N-terminal domain, except for the control and ACh aliquots. Gβ is associated with both phosphorylated and dephosphorylated band 3 in the presence of velnacrine. We conclude that an erythrocyte G protein with subunits Gαi1/2 and Gβ is associated with band 3. AChE depends on the degree of band 3 phosphorylation and its association with Gαi1/2 and Gβ.

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Authors and Affiliations

  1. Institute of Biochemistry, Institute of Molecular Medicine, University of Lisbon Medical School, Av. Prof. Egas Moniz, 1649-028, Lisbon, Portugal

    F. A. Carvalho, J. P. Lopes de Almeida, T. Freitas-Santos & C. Saldanha

  2. Santa Maria Hospital, University of Lisbon Medical School, Av. Prof. Egas Moniz, 1649-028, Lisbon, Portugal

    J. P. Lopes de Almeida

  3. Instituto de Bioquímica, Instituto de Medicina Molecular, Faculdade de Medicina de Lisboa, Edifício Egas Moniz, Av. Prof. Egas Moniz, 1649-028, Lisbon, Portugal

    J. P. Lopes de Almeida

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  1. F. A. Carvalho
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  2. J. P. Lopes de Almeida
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  3. T. Freitas-Santos
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  4. C. Saldanha
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Correspondence to J. P. Lopes de Almeida.

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Carvalho, F.A., de Almeida, J.P.L., Freitas-Santos, T. et al. Modulation of Erythrocyte Acetylcholinesterase Activity and Its Association with G Protein-Band 3 Interactions. J Membrane Biol 228, 89–97 (2009). https://doi.org/10.1007/s00232-009-9162-8

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  • DOI: https://doi.org/10.1007/s00232-009-9162-8

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