MET-8 OF THE BETA(1)-BUNGAROTOXIN PHOSPHOLIPASE-A(2) SUBUNIT IS ESSENTIAL FOR THE PHOSPHOLIPASE-A(2)-INDEPENDENT NEUROTOXIC EFFECT

Beta1-Bungarotoxin consists of a phospholipase A2 subunit and a non-ph ospholipase A2 subunit. The toxin was oxidized with a 100-fold molar e xcess of chloramine T with respect to the methionine content of the pr otein in 0.1 M Tris/HCI at pH 8.5 and at room temperature. Reactivitie s of the two methionine (Met-6 and Met-8 of the phospholipase A2 subun it), five histidine, 14 tyrosine and one tryptophan residues of one to xin molecule with chloramine T were assessed from the change in intrin sic fluorescence and amino acid composition of the protein. Met-8 and one tyrosine on the phospholipase A2 subunit and less than one histidi ne were oxidized, while Met-6 remained intact after 30 min of reaction . One histidine and approx. two tyrosine residues were oxidized when b oth methionine residues were oxidized after 90 min of reaction. The so le tryptophan was oxidized slightly throughout the reaction. The chlor amine T oxidation did not destroy the two Ca2+-binding domains, though it modified the toxin to become less effective at binding Ca2+. The m odified toxin obtained after 30 or 90 min reaction time retained 65 % or 40 % of the phospholipase A2 activity of the parent toxin, but both were not lethal to mice and showed a very weak ability to induce the indirectly evoked contraction of chick biventer cervicis muscle. It is suggested that Met-8 may play an important role in the phospholipase A2-independent interaction with the nerve terminal membrane during the neurotoxic effect of beta1-bungarotoxin.