Quantitative studies on enzymes in structures in striated muscles by labeled inhibitor methods. II. Confirmation of radioautographic measurement by liquid-scintillation counting.
Fragments of mouse diaphragm and sternomastoid muscles were incubated in diisopropyl-fluorophosphate (DFP)-(3)H in conditions known to saturate all the available DFP-sensitive reaction sites. After being extensively washed, the enzyme acetylcholinesterase (AChase) was specifically reactivated by treatment with pyridine-2-aldoxime methiodide (2-PAM). The radioactive DP-groups released into solution by 2-PAM were measured by liquid scintillation counting, and related to the known number of motor endplates present. Considerable difficulty was encountered in reducing the excess, adsorbed radioactivity to acceptable levels: long washing routines, extraction with organic solvents, and removing excess muscle fiber by microdissection were necessary. Six experiments gave a mean value of 2.4 x 10(7)molecules AChase per sternomastoid endplate, in reasonable agreement with the previously reported measurements by radioautography.
D000110:Acetylcholinesterase / Q000032:analysis*; D000818:Animals; D001345:Autoradiography; D002800:Cholinesterase Inhibitors; D003964:Diaphragm; D007531:Isoflurophate / Q000378:metabolism*; D051379:Mice; D009132:Muscles / Q000201:enzymology*; D009469:Neuromuscular Junction / Q000201:enzymology*; D010085:Oxidative Phosphorylation / Q000187:drug effects; D011220:Pralidoxime Compounds / Q000494:pharmacology; D014316:Tritium
D002800:Cholinesterase Inhibitors; D011220:Pralidoxime Compounds; D014316:Tritium; D007531:Isoflurophate; D000110:Acetylcholinesterase