In Vitro Acetylcholinesterase Inhibition by Type A Botulinum Toxin

Abstract

Type A botulinum toxin was studied for its ability to inhibit the action of acetylcholinesterase. The chromogenic substrate, indophenyl acetate, was used for assay ofenzyme activity. Inhibition ofenzyme function was detected through use ofboth 6.6 X 10-6 mg (20 LDS0) and 6.6 x I010 mg (2 x 10-3 LD50) of type A botulinal toxin. Control assays were performed by use of both homologous antitoxin and heterologous antitoxins (types B and E). Enzyme inhibition was effectively prevented by use ofhomologous antitoxin only. The inhibition noted was specific and reproducible for given substrate, enzyme, and toxin concentrations.

Assay of type A botulinal toxin using acetylcholinesterase inhibition

“…(Cook, 1955a(Cook, , 1955b. The fact that inhibition of acetyl cholinesterase occurs _in vitro through the action of type A botulinum toxin was reported earlier by Marshall and Quinn (1967). The spectrophotometric methods and equipment used by these workers were also used in these studies.…”

“…Johnson et al (1965) studied serological procedures which might be applicable to the detection of botulinal toxins in foods. However, this method is rather cumbersome and has proved to be of little practical importance, Recently, Marshall and Quinn (1967) showed that type A toxin is capable of inhibiting the enzyme acetylcholinesterase _in vitro, and also that the inhibition is detectable spectrophotometrically. Reports con cerning the vitro estimation of organic phosphate inhibitors of acetylcholinesterase are many and since type A toxin does inhibit acetyl cholinesterase, spot tests based on the methods of Cook (1955aCook ( , 1955b and Sandi and Wight (1961), for example, may be used to show enzyme in hibition qualitatively.…”

“…Each dose was characterized, using 2.50 x 10"% IPA and 0.25 nM units/ml of type III acetylcholinesterase, by the methods given by Marshall and Quinn (1967). However, in these experiments, the toxin and enzyme were incubated together for 20 min prior to IPA addition and the course of each inhibition curve was charted for 100 min.…”

“…The Table 1. Final optical density values at 625 m|i, obtained at specified time intervals by use of indophenyl acetate (IPA), acetyl cholinesterase (Ac'ase, type III), types A, B, and E antitoxin, and type A botulinum toxin in enzyme inhibition studies (Marshall and Quinn, 1967) Reaction ^Difference in the OD of the cuvette without toxin and the cuvette with toxin or with toxin and antitoxin, as specified.…”

“…The data in Table 1 indicate that some system may be causing a potentiation of toxicity such that the amount of antitoxin required for neutralization is no longer sufficient to prevent enzyme inhibition. The preceding discussion was taken from Marshall and Quinn (1967).…”