Modulation of erythrocyte acetylcholinesterase activity and its association with G protein-band 3 interactions.
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 Galpha(i1/2) and G(beta) with band 3 protein. Galpha(i1/2) and G(beta) may be linked to the band 3 C-terminal site. Galpha(i1/2) is associated with the band 3 N-terminal domain, except for the control and ACh aliquots. G(beta) is associated with both phosphorylated and dephosphorylated band 3 in the presence of velnacrine. We conclude that an erythrocyte G protein with subunits Galpha(i1/2) and G(beta) is associated with band 3. AChE depends on the degree of band 3 phosphorylation and its association with Galpha(i1/2) and G(beta).
Carvalho
F A
FA
Institute of Biochemistry, University of Lisbon Medical School, Lisbon, Portugal.
de Almeida
J P Lopes
JP
Freitas-Santos
T
T
Saldanha
C
C
D000110:Acetylcholinesterase / Q000378:metabolism*; D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism*; D015153:Blotting, Western; D004151:Dipeptides / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004912:Erythrocytes / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism*; D044385:GTP-Binding Protein alpha Subunits / Q000378:metabolism; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D008297:Male; D011485:Protein Binding; D013619:Tacrine / Q000031:analogs & derivatives / Q000494:pharmacology
D001457:Anion Exchange Protein 1, Erythrocyte; D004151:Dipeptides; D004791:Enzyme Inhibitors; D044385:GTP-Binding Protein alpha Subunits; D044387:GTP-Binding Protein beta Subunits; C071482:calpeptin; D013619:Tacrine; D000110:Acetylcholinesterase; D019204:GTP-Binding Proteins; C056424:velnacrine