Pharmacologic experiments on the interaction between crotoxin and the mammalian neuromuscular junction

Crotoxin and its two subunits were tested for their neuromuscular blocking activity on the phrenic nerve-hemidiaphragm preparation. Two types of experimental paradigms were used, the first of which separated the toxin binding step from subsequent events in paralysis and the second of which did not. In both paradigms the toxin produced concentration-dependent blockade of transmission. However, the results with low concentrations were variable, and in some cases complete neuromuscular blockade did not develop. The isolated acidic and basic subunits possessed little toxicity. In experiments designed to characterize binding, the intact toxin displayed the following properties: 1) the apparent half-time for tissue association was about 22 min; 2) binding was not affected by low temperature, the presence or absence of nerve stimulation and the substitution of strontium for calcium; and 3) when binding was allowed to go to completion, reversibility was negligible. Pretreatment of tissues with the isolated subunits of crotoxin did not enhance or inhibit the binding of the parent molecule. Modification of one histidine residue in the isolated basic subunit, followed by reconstitution with unmodified acidic subunit, generated a molecule that possessed only about 10% of the neurotoxicity of the native toxin. The modified toxin could not be used to antagonize binding of the native toxin. Both polyclonal and monoclonal antibodies were generated that neutralized the biologic activity of crotoxin. In experiments that separated the binding step from later events in paralysis, the polyclonal preparation continued to locate and partially neutralize tissue-bound toxin. In experiments that initiated events that follow binding, polyclonal antibodies were progressively less effective with time in neutralizing toxin.(ABSTRACT TRUNCATED AT 250 WORDS)