Assembly of monomeric acetylcholinesterase into tetrameric and asymetric forms

S. K. Brockman, M. F. Usiak, Steven G Younkin

Research output: Contribution to journalArticle

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Abstract

A pulse-chase experiment was performed in embryonic rat myotube cultures to examine possible precursor-product relationships among the various molecular forms of acetylcholinesterase (AChE was labeled with paraoxon, a compound which diethylphosphorylates AChE at its active site. Diethylsphosphorylated (labeled) AChE is inactive but can be reactivated by treatment with 1-methyl-2-hydroxyiminomethyl-pyridinium. Thus labeled enzyme could be followed as AChE that regained activity following treatment with 1-methyl-2-hydroxyiminomethylpyridium. To selctively label monomeric AChE (the hypothesized precursor form), cultures were treated with methanesulfonylfluoride which irreversibly inactivated more than 97% of total cellular AChE. Mehylsulfonylfluoride was then washed from the cultures, and they were labeled with paraoxon during a 40-55-min recovery period. AChE appearing in the cultures during this recovery period is newly synthesized and consists almost entirely (92%) of the monomeric form. Immediately and 120-130 min after labeling, cultures were subjected to a sequential extraction procedure to separate globular from asymmetric forms. Individual forms were then separated by velocity sedimentation on sucrose gradients. In our first series of experiments, we observed a 55% decrease in labeled monomers during the chase, a 36% increase in labeled tetramers, and a 36% increase in labeled asymmetric forms. In a second series of experiments focused on individual asymmetric forms, we observed a 55% decrease in labeled monomers, a 58% increase in lajbeled tetramers, an overal increase of 81% in labeled asymmetric forms, and a 380% increase in labeled A12 AChE. These data provide the first uniequivocal proof that complex forms of AChE are assembled from active monomeric precursors.

Original languageEnglish (US)
Pages (from-to)1201-1207
Number of pages7
JournalJournal of Biological Chemistry
Volume261
Issue number3
StatePublished - 1986
Externally publishedYes

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Paraoxon
Acetylcholinesterase
compound A 12
Skeletal Muscle Fibers
Monomers
Sucrose
Recovery
Catalytic Domain
Experiments
Sedimentation
Labeling
Rats
Labels
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Assembly of monomeric acetylcholinesterase into tetrameric and asymetric forms. / Brockman, S. K.; Usiak, M. F.; Younkin, Steven G.

In: Journal of Biological Chemistry, Vol. 261, No. 3, 1986, p. 1201-1207.

Research output: Contribution to journalArticle

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abstract = "A pulse-chase experiment was performed in embryonic rat myotube cultures to examine possible precursor-product relationships among the various molecular forms of acetylcholinesterase (AChE was labeled with paraoxon, a compound which diethylphosphorylates AChE at its active site. Diethylsphosphorylated (labeled) AChE is inactive but can be reactivated by treatment with 1-methyl-2-hydroxyiminomethyl-pyridinium. Thus labeled enzyme could be followed as AChE that regained activity following treatment with 1-methyl-2-hydroxyiminomethylpyridium. To selctively label monomeric AChE (the hypothesized precursor form), cultures were treated with methanesulfonylfluoride which irreversibly inactivated more than 97{\%} of total cellular AChE. Mehylsulfonylfluoride was then washed from the cultures, and they were labeled with paraoxon during a 40-55-min recovery period. AChE appearing in the cultures during this recovery period is newly synthesized and consists almost entirely (92{\%}) of the monomeric form. Immediately and 120-130 min after labeling, cultures were subjected to a sequential extraction procedure to separate globular from asymmetric forms. Individual forms were then separated by velocity sedimentation on sucrose gradients. In our first series of experiments, we observed a 55{\%} decrease in labeled monomers during the chase, a 36{\%} increase in labeled tetramers, and a 36{\%} increase in labeled asymmetric forms. In a second series of experiments focused on individual asymmetric forms, we observed a 55{\%} decrease in labeled monomers, a 58{\%} increase in lajbeled tetramers, an overal increase of 81{\%} in labeled asymmetric forms, and a 380{\%} increase in labeled A12 AChE. These data provide the first uniequivocal proof that complex forms of AChE are assembled from active monomeric precursors.",
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