TY - JOUR
T1 - Assembly of monomeric acetylcholinesterase into tetrameric and asymetric forms
AU - Brockman, S. K.
AU - Usiak, M. F.
AU - Younkin, S. G.
N1 - Copyright:
Copyright 2004 Elsevier B.V., All rights reserved.
PY - 1986
Y1 - 1986
N2 - 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.
AB - 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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M3 - Article
C2 - 3944084
AN - SCOPUS:0022648241
SN - 0021-9258
VL - 261
SP - 1201
EP - 1207
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 3
ER -