Abstract
The first cytochemical demonstration of a hydrolytic enzyme using a single agent as both substrate and capture reagent to form an osmiophilic polymer was reported earlier. The first results were obtained with substrates containing an enzyme-susceptible thiolester group and a diazonium group in the same molecule, which yielded osmiophilic polymers upon enzymatic hydrolysis via diazothioether formation linking the units. An enzyme-susceptible ester group has also been incorporated in the same indole molecule with a diazonium group to yield an osmiophilic polymer upon esterolysis via coupling to azo dye formation linking the units. The schemas of these two systems are given. The thiol substrate is α-acetylthiol-m-toluenediazonium ion (ATTD, compound II) and the indoxyl substrate is 3-acetoxy-5-indolediazonium ion (AID, compound VII). The preparation of AID is given here. Because of the strongly positive charges on the diazonium ion, neither ATTD nor AID is hydrolyzed by the aliesterases of rat kidney. The results with ATTD and AID in the ultrastructural demonstration of cholinesterases in the motor end plate of the rat are compared, as well as the influence of varying the method of fixation, substrate concentration, duration and temperature of incubation and the use of selected inhibitors with ATTD. Evidence was provided with ATTD that both acetylcholinesterase and pseudocholinesterase are present on the inner surface of the synaptic clefts of the motor end plate and that the deposits are discrete and not linear as has been reported with some other methods. On the other hand, deposits with AID are more finely granular and are also limited to the inner aspect of the synaptic clefts. The reasons for the differences in appearance of the deposits in ultrastructural preparations are discussed.