The marine environment contains many vertebrate and invertebrate organisms that can envenomate and cause disease in humans. Marine animals that have this potential include members of the family Scorpaenidae (commonly called """"""""scorpionfish""""""""), which contains ca. 30 genera and 350 species of venomous fish. The most dangerous of the venomous fish known is a scorpionfish which belongs to the genus Synanceia and is commonly called the """"""""stonefish"""""""". Although naturally occurring envenomation by stonefish and other scorpionfish causes severe local tissue damage and neuromuscular disease in humans, and envenomation of laboratory animals mimics the human disease, very little has been published concerning the physicochemical and pharmacological properties of toxins contained in scorpionfish venoms. The primary, long-term objective of the proposed research is to purify scorpionfish toxins to homogeneity and to characterize their physicochemical and neuropharmacological properties. The knowledge obtained should aid in determining whether stonefish and/or other scorpionfish venoms contain toxins useful in the study of neuromuscular tissue, and improve our understanding of the molecular pathogenesis of disease caused by scorpionfish. More specifically, the research proposed in this application focuses on the characterization of a membrane-damaging, protein toxin which the PI recently has discovered in stonefish venom (Toxican 29:733-743, 1991). The experimental protocols are designed to: (i) develop a purification scheme for obtaining the toxin in a homogeneous state, (ii) characterize the physicochemical properties of the isolated toxin (including a combined strategy of N-terminal amino acid sequencing of the toxin and nucleotide sequencing of cDNA encoding the toxin, in order to determine the toxin's primary structure), (iii) use electrophysiological techniques and electron microscopy to characterize and compare the action of the crude venom and purified toxin on the nerve terminal membrane (presynaptic compartment) of the mouse and frog neuromuscular synapse, (iv) determine whether stonefish venom and toxin form stable, cation-selective channels spontaneously in artificial lipid bilayers, (v) use light and electron microscopy to characterize and compare the local tissue damage produced in guinea pigs by the crude venom and purified toxin, (vi) determine whether active and passive immunization against the stonefish toxin protects mice and guinea pigs against disease after challenge with the venom, and (vii) use genetic and immunologic techniques to determine whether other scorpionfish (the lionfish and the California sculpin) produce a toxin which is similar or identical to the stonefish toxin.
