ACETYL DISULFIDE, (CH3COS)2, AND BIS-(THIOACETOXY) AURATE (I) COMPLEX, Au(CH3COS)2–, HISTOCHEMICAL SUBSTRATES OF UNUSUAL PROPERTIES WITH ACETYLCHOLINESTERASE

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.