Quantitative assay of esterases in end plates of mouse diaphragm by electron microscope autoradiography

Abstract

The inhibitor diisopropyl fluorophosphate (DFP) reacts covalently with a number of enzymes including acetylcholinesterase (AChE). The compound pyridine-2-aldoxime methiodide reactivates phosphorylated AChE faster than the other DFP-sensitive sites. Mouse diaphragm was incubated with 3H-DFP, and the radioactivity in the motor end plates was assessed by electron microscope autoradiography. Both the sites which are rapidly reactivated and those which are slowly reactivated by pyridine-2-aldoxime methiodide were evaluated. The distribution of silver grains was compatible with several alternative hypotheses, i.e., that both AChE and other DFP-sensitive enzymes are located on or around the postsynaptic membrane alone, are distributed uniformly throughout the synaptic cleft and are associated equally with both pre- and postsynaptic membranes. This distribution of developed grains was identical with that found in the larger end plates of mouse sternomastoid. The values for the number of molecules of 3H-DFP, whether expressed in terms of square micra of junctional membrane or of cubic micra of synaptic cleft, were also practically identical with those found for sternomastoid. These similarities between the diaphragm, a red muscle, and the sternomastoid, which is predominantly white, suggest that there may be a constant relationship between the sites that bind DFP and the unit dimensions of the subneural compartments concerned.