Isolation and characterization of acetylcholinesterase from Drosophila.

A. L. Gnagey, M. Forte, T. L. Rosenberry

Research output: Contribution to journalArticle

148 Citations (Scopus)

Abstract

The purification and characterization of acetylcholinesterase from heads of the fruit fly Drosophila are described. Sequential extraction procedures indicated that approximately 40% of the activity was soluble and 60% membrane-bound and that virtually none (less than 4%) corresponded to collagen-tailed forms. The membrane-bound enzyme was extracted with Triton X-100 and purified over 4000-fold by affinity chromatography on acridinium resin. Hydrodynamic analysis by both sucrose gradient centrifugation and chromatography on Sepharose CL-4B revealed an Mr of 165,000 similar to that observed for dimeric (G2) forms of the enzyme in mammalian tissues. In contrast, the purified enzyme gave predominant bands of about 100 kDa prior to disulfied reduction and 55 kDa after reduction on polyacrylamide gel electrophoresis in sodium dodecyl sulfate, values that are significantly lower than those reported for purified G2 enzymes from other species. However, the presence of a faint band at 70 kDa which could be labeled by [3H]diisopropyl fluorophosphate prior to denaturation suggested that the 55-kDa band as well as a 16-kDa species arose from proteolysis. This was confirmed by reductive radiomethylation and amine analysis of the 70-, 55-, and 16-kDa bands. All three contained ethanolamine and glucosamine residues that are characteristic of a C-terminal glycolipid anchor in other G2 acetylcholinesterases. The catalytic properties of the enzyme were examined by titration with a fluorogenic reagent which revealed a turnover number for acetylthiocholine that was 6-fold lower than eel and 3-fold lower than human erythrocyte acetylcholinesterase. Furthermore, the Drosophila enzyme hydrolyzed butyrylthiocholine much more efficiently than these eel or human enzymes, an indication that the fly head enzyme has a substrate specificity intermediate between mammalian acetylcholinesterases and butyrylcholinesterases.

Original languageEnglish (US)
Pages (from-to)13290-13298
Number of pages9
JournalJournal of Biological Chemistry
Volume262
Issue number27
StatePublished - Sep 25 1987
Externally publishedYes

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Acetylcholinesterase
Drosophila
Enzymes
Eels
fluorophosphate
Butyrylthiocholine
Acetylthiocholine
Head
Proteolysis
Membranes
Butyrylcholinesterase
Affinity chromatography
Denaturation
Ethanolamine
Centrifugation
Glucosamine
Glycolipids
Octoxynol
Hydrodynamics
Substrate Specificity

ASJC Scopus subject areas

  • Biochemistry

Cite this

Gnagey, A. L., Forte, M., & Rosenberry, T. L. (1987). Isolation and characterization of acetylcholinesterase from Drosophila. Journal of Biological Chemistry, 262(27), 13290-13298.

Isolation and characterization of acetylcholinesterase from Drosophila. / Gnagey, A. L.; Forte, M.; Rosenberry, T. L.

In: Journal of Biological Chemistry, Vol. 262, No. 27, 25.09.1987, p. 13290-13298.

Research output: Contribution to journalArticle

Gnagey, AL, Forte, M & Rosenberry, TL 1987, 'Isolation and characterization of acetylcholinesterase from Drosophila.', Journal of Biological Chemistry, vol. 262, no. 27, pp. 13290-13298.
Gnagey AL, Forte M, Rosenberry TL. Isolation and characterization of acetylcholinesterase from Drosophila. Journal of Biological Chemistry. 1987 Sep 25;262(27):13290-13298.
Gnagey, A. L. ; Forte, M. ; Rosenberry, T. L. / Isolation and characterization of acetylcholinesterase from Drosophila. In: Journal of Biological Chemistry. 1987 ; Vol. 262, No. 27. pp. 13290-13298.
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