Title

Purification of toxins from green mamba venom with distinct receptor selectivities

Date of Award

1997

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

First Committee Member

Lincoln T. Potter, Committee Chair

Abstract

The venom of the green mamba, Dendroaspis angusticeps, contains toxins that selectively recognize m1, m2 and m4 muscarinic receptors. Most of the anti-m1 activity in the venom is due to four newly isolated toxins, named m1-toxins1-4. Sequencing data indicated higher than 90% homology among m1-toxins, and it also provided residues that are presumably important for m1 selectivity. Binding studies with tritiated N-methylscopolamine ($\sp3$H-NMS) demonstrated that m1-toxins exhibit similar binding properties; they bound selectively, rapidly, irreversibly, allosterically, and with equivalent IC$\sb{50}$ values to m1 muscarinic receptors. Conjugation of m1-toxin2 with biotin yielded active derivatized toxin. Biotinylation did not affect the prolonged binding to m1 receptors or the selectivity observed with the native toxin. The exceptional specificity of m1-toxins, combined with their irreversible binding under physiological conditions, and their stability to derivatization, makes these toxins ideal tools for studies in vivo.At least three toxins contribute to the anti-m2 activity seen in the venom of the green mamba. One of these toxins was purified and was named m2-toxin. This toxin inhibits the binding of $\sp3$H-NMS to m2 receptors with a Hill coefficient close to one, suggesting competition between the two ligands for the receptor binding site. When tested in CHO cells expressing each of the five muscarinic receptor subtypes and in brain tissue, m2-toxin selectively blocked the binding of $\sp3$H-NMS to m2 receptors, while it increased the binding of the tritiated ligand to the other muscarinic receptor subtypes. This toxin presumably binds allosterically to muscarinic receptors.Besides muscarinic toxins and other toxins well characterized, mamba venoms have been shown to contain toxins that affect blood coagulation. Part of the work presented in this dissertation involved the isolation and complete amino acid sequence of the first platelet aggregation inhibitor described from green mamba venom. This toxin, which was named thrombostatin, inhibited ADP-induced platelet aggregation in human platelet-rich plasma with remarkable potency.

Keywords

Health Sciences, Toxicology; Health Sciences, Pharmacology; Chemistry, Biochemistry

Link to Full Text

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