Poster: Temperature responses

Abs # 125: Characterization of maize acetylcholinesterase

Presenter:	Momonoki, Yoshie S, y-momono@bioindustry.nodai.ac.jp
Authors	Momonoki, Yoshie S (A)   Sagane, Yoshimasa  (A)   Nakagawa, Tomoyuki  (A)   Michikawa, Soichi  (A)   Yamamoto, Kosuke  (A)
Affiliations:	(A): Tokyo University Of Agriculture

We previously purified the 44 kDa-AChE, which respond to environmental stimuli,such as gravity, heat and salt stresses, from etiolated 5-d-old maize seedlings and determined its cDNA sequence. The present study was clarified kinetic properties, substrate specificity of the maize AChE compared to eel AChE (commercial product). The localization, posttranslational modifications and protein family were predicted by the computer-assisted motif analysis. The AChE activity was determined by DTNB method. Neostigmine was used as an AChE inhibitor. Both maize- and eel AChE hydrolyzed acetyl- and propionylthiocholine but not butyrylthiocholine. The Km values for acetyl- and propionylthiocholine were 8.34μM and 4.28μM to maize AChE and 0.25 mM and 0.52 mM to eel AChE, respectively. The sorting server TargetP predicts that the maize AChE precursor sequence contains a signal sequence consists of 29 residues, which routes the protein to secretory pathway. This agrees with observation that 29 amino acid residues are removed from the mature AChE. The apparent molecular mass of the purified AChE was estimated as approximately 44 kDa by SDS-PAGE, which differs from calculated value (39722.3 Da). Six consensus sequences for the N-glycosilation site were identified in the deduced sequence. Further, 20 residues were predicted as probable phosphorylation sites. The discrepancy observed between estimated and calculated molecular masses might appear due to post-translational modifications. Based on comparison with protein family database, maize AChE was predicted to belong to GDSL lipase family, and predicted to possess catalytic Ser/Asp/His triad.