Assembly of monomeric acetylcholinesterase into tetrameric and asymetric forms

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

Research output: Contribution to journalArticle

38 Scopus citations

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

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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