Investigations on the Tetrodotoxin Binding Component from Electrically Excitable Tissue

Citation

Benzer, Theodore I. (1974) Investigations on the Tetrodotoxin Binding Component from Electrically Excitable Tissue. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/3B26-2G75. https://resolver.caltech.edu/CaltechETD:etd-09282005-082018

Abstract

NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document. Tetrodotoxin from the Japanese puffer fish was labelled with tritium and purified from the crude mixture obtained. The interaction between the purified [[...]]-tetrodotoxin and membrane suspensions from the olfactory nerve of the long-nosed garfish was investigated using equilibrium dialysis. Tetrodotoxin was shown to bind to membrane suspensions with a dissociation constant [...]. The nerve preparation binds 43 picomoles of [[...]]-tetrodotoxin per gm of wet tissue at saturating toxin concentrations. Using various hydrolytic enzymes, the binding component was shown to be a protein embedded in a phospholipid environment. The binding is inhibited below pH 4.0 and is not stable towards heat. Tetrodotoxin binding is not inhibited by the local anaesthetic procaine. The tetrodotoxin binding component from garfish olfactory nerve membranes was solubilized using the nonionic detergent Triton X-100. Tetrodotoxin binds to the solubilized component with a dissociation constant [...] and under saturating conditions [...] moles of tetrodotoxin are bound per milligram of solubilized protein. Upon solubilization the toxin binding component becomes much less stable towards heat, chemical modification and enzymatic degradation. Sucrose gradient velocity sedimentation yields an S value of 9.2 for the extracted binding component and from gel filtration data the binding component appears to be slightly larger than [...]. Tetrodotoxin binding to membrane fragments of the electric organ of Electrophorus electricus was measured and found to be [...] moles of the toxin per gram of wet tissue at saturating conditions with a dissociation constant of [...]. Calcium ions at millimolar concentrations were found to inhibit toxin binding to the membrane fragments. The tetrodotoxin binding component was solubilized with the nonionic detergent Lubrol-PX and a convenient assay was developed for measuring the toxin binding to detergent extracts using gel filtration in the centrifuge to separate bound toxin from free toxin. This assay was used to investigate the problem of the stability of the tetrodotoxin binding component in the detergent extract. Two derivatives of tetrodotoxin were covalently linked to Sepharose-2B in an attempt to synthesize an affinity resin for purification of the tetrodotoxin binding component. It was found that the columns did not display properties which would make them useful since they effected only a minor purification of the binding component with low yields of activity.

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