Two small basic proteins, Cerebratulus lacteus toxin A-III and Stoichactis helianthus toxin, cause a rapid lysis of a wide variety eukaryote cells by nonenzymatic mechanisms. It is proposed that these cytotoxins possess a high affinity for the lipid bilayer domain of the plasma membrane; upon binding at the membrane surface, specific hydrophobic regions become exposed on the surfaces of the toxins and then penetrate into the hydrocarbon interior of the lipid bilayer, disrupting membrane bilayer lipid-lipid and possibly lipid-protein interactions. This leads to a large increase in the permeability of the membrane to ions and eventually cell lysis. The general goal of this investigation will be to elucidate at the molecular level, mechanisms of toxin binding and membrane disruption causing cell lysis. The covalent structure (sequence, disulfide bonds) of each purified toxin will be determined by the automatic Edman method; secondary structure will be analyzed by CD and Raman spectroscopy. The degree of exposure and functional roles of tyrosyl and tryptophanyl residues will be assessed by chemical modification and spectroscopic methods. The importance of disulfide bonds for toxin structure, binding, and action will be analyzed. Cytotoxin protein-lipid interactions will be assessed by gel filtration binding assays, CD and fluorescence spectroscopy, and lipid monolayer surface pressure changes. The kinetics of toxin binding, increased ionic permeability, and lysis of red cells will be separately measured by radioisotope and turbidometric methods. Cytotoxin actions upon ionic conductances of nerve and lipid bilayer membranes will be studied by voltage clamp techniques. The mechanism by which calcium inhibits A-III actions upon red cell, squid axon, and artificial lipid membranes, will be investigated.
NIH ExPorter
NIH ExPorter