QUANTITATIVE STUDIES ON ENZYMES IN STRUCTURES IN STRIATED MUSCLES BY LABELED INHIBITOR METHODS

Abstract

Fragments of mouse diaphragm and sternomastoid muscles were incubated in diisopropyl-fluorophosphate (DFP)-3H 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 107molecules AChase per sternomastoid endplate, in reasonable agreement with the previously reported measurements by radioautography.