Localization of Acetylcholinesterase via Production of Osmiophilic Polymers: New Benzenediazonium Salts With Thiolacetate Functions

Abstract

By incorporating an enzyme-susceptible thiolester group and a diazonium group into the same molecule, it has been possible to obtain an osmiophilic polymer upon enzymatic hydrolysis of the thiolester, via diazothioether formation linking the units. The thiolacetates with diazonium groups are specific substrates for cholinesterase because of the strong positive charge on the diazonium group. Following osmication, sites of acetylcholinesterase in motor end plate are notable as black deposits in light microscopy and opaque deposits in electron microscopy. This is the first cytochemical demonstration of a hydrolytic enzyme using a single agent as both substrate and capture reagent to form a polymer. The insolubility of the polymer in both water and lipid before osmication provides precision of localization needed for the high resolution of electron microscopy. However, the effectiveness of the new principle for electron microscopy depends as well upon the rate of the capture reaction (polymerization), which in turn depends upon the pK a of the thiol and pH and temperature at which the cytochemical reaction is conducted.