Abstract
The histochemical reaction for acetylcholinesterase (AChE) employing acetyl disulfide ([CH3COS]2, or AcDiS) as substrate and 0.006 M Pb(NO3)2 as capturing agent was characterized by (1) a sigmoid curve for velocity of hydrolysis versus substrate concentration and (2) a red precipitate (presumably [CH3COSS]2Pb) at sites of AChE at the motor endplates (MEP's) and (from spontaneous hydrolysis) in the supernatant solution. With the addition of 0.03 M thiolacetic acid (TA) to the incubation medium, which by itself produced little or no histochemical staining, the foregoing characteristics were changed to (1) a bell-shaped curve, with the peak at 0.003 M AcDiS, and (2) a black precipitate (presumably PbS) at the MEP's and in the supernatant solution; in addition, the velocity of hydrolysis was increased approximately 100-fold. Comparable differences were obtained with AcDiS as substrate when using ionic Au+ or Au(S2O3)2 –3 as the capturing agent. Results are explained on the basis that in the absence of free heavy metal ion (i.e., with the Pb[TA]2 or Au[S2O3]2 –3 complex), AcDiS combines with AChE at both the anionic and esteratic sites, whereas, in the presence of Pb2+ or Au+, attachment of the substrate occurs only at the esteratic site of the enzyme. Results similar to the former type were obtained when the bis-(thioacetoxy) aurate (I) complex (Au[CH3COS]2 – or Au[TA]2 – served as both substrate and capturing agent. With both the AcDiS-Au(S2O3)2 –3 and Au (TA)2 – methods, extremely fine localization of AChE was obtained at the MEP's by electron microscopy.