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Properties of specific binding site of myotoxin a, a powerful convulsant, in brain microsomes
C Katagiri1, H H Ishikawa, M Ohkura
1Department of Pharmaceutical Molecular Biology, Faculty of Pharmaceutical Sciences, Tohoku University, Aoba-ku, Japan.
Abstract:
Myotoxin a, a small basic polypeptide from prairie rattlesnakes (Crotalus viridis viridis), induces myonecrosis and binds to a single class of binding sites in skeletal muscle sarcoplasmic reticulum. In the present study, [125I]myotoxin a with a high specific activity was prepared and it was shown to bind mainly to microsomes in rat whole brain. [125I]Myotoxin a was further shown to bind to microsomes prepared from all regions tested in brain. Its specific binding to whole brain microsomes was of approximately 1.9 times lower affinity (KD = 0.76 microM; Bmax = 13.1 nmol/mg) than that to skeletal muscle sarcoplasmic reticulum. [125I]Myotoxin a binding to brain microsomes was displaced by unlabeled myotoxin a with an IC50 value of 4.5 microM. [125I]Myotoxin a binding was markedly reduced by treatment of microsomes with trypsin, suggesting that the binding site of [125I]myotoxin a is partially proteins. The binding was significantly inhibited by Mg2+ at concentrations above 1 mM. Having looked at several drugs, we noted that [125I]myotoxin a binding was noncompetitively inhibited by spermine, whereas it was enhanced by heparin. On the other hand, the i.c.v. injection of myotoxin a in mice induced potent convulsive effects at 0.05 nmol/mouse or more. This paper is the first to show that the specific binding site of myotoxin a is present in mouse brain and that myotoxin a is a novel peptidic convulsant in mice.
Insights
Prairie rattlesnake venom component, myotoxin a, binds to sites in the brain and acts as a novel peptidic convulsant in mice, demonstrating a new neurotoxic effect.
Area of Science:
- Neuroscience
- Toxicology
- Biochemistry
Background:
- Myotoxin a, a polypeptide from prairie rattlesnakes (Crotalus viridis viridis), is known to cause muscle damage and bind to skeletal muscle sarcoplasmic reticulum.
- The presence and function of myotoxin a binding sites in the central nervous system were previously uncharacterized.
Purpose of the Study:
- To investigate the binding characteristics of myotoxin a in the rat brain.
- To determine if myotoxin a exhibits neurotoxic or convulsive effects in mice.
Main Methods:
- Preparation of radioiodinated myotoxin a ([125I]myotoxin a) for binding studies.
- Incubation of [125I]myotoxin a with rat brain and skeletal muscle microsomes to assess binding affinity and characteristics.
- Intracerebroventricular (i.c.v.) injection of myotoxin a in mice to evaluate convulsive effects.
Main Results:
- [125I]myotoxin a binds to microsomes in all tested regions of the rat brain with lower affinity compared to skeletal muscle.
- Binding to brain microsomes is protein-dependent and influenced by magnesium, spermine, and heparin.
- Intracerebroventricular injection of myotoxin a induces potent convulsive effects in mice at low doses.
Conclusions:
- This study identifies specific binding sites for myotoxin a in the mouse brain.
- Myotoxin a is demonstrated to be a novel peptidic convulsant, indicating a previously unrecognized neurotoxic mechanism of action.