Neurotoxin Activity Of Phospholipase A₂ From Gloydius Ussuriensis Snake Venom

Phospholipase A₂ homogue with Gln at 49 sites, named Gln49-PLA₂, is isolated and purified from Gloydius ussurensis snake venom by Hitrap SP cation exchange and Superdex 75 gel filtration chromatography. It is the single component by SDS-PAGE. The molecular mass of Gln49-PLA₂ is 13880.79, which shows myotoxicity, neurotoxicity and cytotoxic activity, but lacks the phospholipase A₂ activity.The in vitro effects of Gln49-PLA₂ were examined using the chick biventer cervicis nerve muscle (BCp) preparation. Gln49-PLA₂ caused a dosedependent blockade of neuromuscular transmission. The time required to cause 50% blockade was about 150 min at the highest concentration 100μg/ml. This inhibition appeared to be presynaptic in origin as evidenced by the lack of effect of toxin on responses to exogenous acetylcholine in the non-stimulated BCp.The effect of Gln49-PLA₂ on compound action potentials and conduction velocity was remarkable to significant changes in the amplitude of the potentials, The toxin 100μg/ml completely decreased the amplitude of mouse sciatic-nerve compound action potentials and conduction velocity at 40min.Though lack the PLA₂ enzyme activity, the Gln49-PLA₂ also appears to be myotoxic at high concentration (≥50μg/ml), as indicated by increasing the release of creatine kinase, and induced the appearance of degenerating muscle fibers about 30.7%. Patch clamp tests proved that Gln49-PLA₂ remarkably worked on decrease neural excitability by the excessive activation of sodium channels and the blockade of nerve terminal potassium channels, potassium currents elicited in fresh hippocampal pyramidal neuron blocked to 45.75% and sodium currents increased 158.99% in the presence of 20μg/ml Gln49-PLA₂.These findings demonstrate that Gln49-PLA₂ showed 'in vitro' a potent neuromuscular pre-synaptically blocking activity in chick nerve-muscle preparations. In fact, this toxin may mainly function at voltage-dependent ion channel to blocked neuron signal transmission. In conclusion, Gln49-PLA₂ is a new presynaptic phospholipase A₂.