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Characterization of nicked myotoxin a and its effect on the sarcoplasmic reticulum calcium pump
1Department of Biochemistry, Colorado State University, Fort Collins 80523.
Abstract:
Myotoxin a, a muscle-necrotizing polypeptide isolated from Crotalus viridis viridis (prairie rattlesnake) venom, was nicked at Met-28 by cyanogen bromide. Amino acid analysis indicated that the methionine content was reduced to zero from the original 1 mol. Judging from circular dichroism, the nicked myotoxin a had a conformation similar to that of original myotoxin. Raman spectra indicated that the conformations of the three disulfide bonds are not affected in nicked myotoxin a. Like the original toxin, nicked myotoxin a was myotoxic and inhibited calcium ion loading activity, although the inhibitory action was slightly lower than that of the original myotoxin a. Both modified and unmodified myotoxin a showed myonecrotic activity as determined by examining histological slides. The modified toxin also inhibited the formation of decavanadate-induced two-dimensional crystalline arrays of the sarcoplasmic reticulum Ca2+-ATPase just as the original myotoxin a does.
Insights
Prairie rattlesnake venom
Area of Science:
- Biochemistry
- Toxicology
- Molecular Biology
Background:
- Myotoxin a is a polypeptide from prairie rattlesnake venom known for muscle necrosis.
- Its specific mechanism of action, particularly concerning calcium ion transport and enzyme inhibition, requires further elucidation.
Purpose of the Study:
- To investigate the structural and functional consequences of modifying Myotoxin a at methionine-28.
- To compare the myotoxic and inhibitory activities of native and modified Myotoxin a.
Main Methods:
- Chemical modification of Myotoxin a using cyanogen bromide to nick methionine-28.
- Amino acid analysis to confirm methionine reduction.
- Circular dichroism and Raman spectroscopy to assess conformational changes.
- In vitro assays to evaluate myotoxicity, calcium ion loading inhibition, and effects on sarcoplasmic reticulum Ca2+-ATPase.
Main Results:
- Cyanogen bromide nicking at Met-28 reduced methionine content to zero without significantly altering overall conformation or disulfide bond integrity.
- Nicked Myotoxin a retained myotoxic and calcium ion loading inhibitory activities, though slightly reduced compared to the original.
- Both forms demonstrated myonecrotic effects and inhibited decavanadate-induced crystalline arrays of Ca2+-ATPase.
Conclusions:
- Methionine-28 is not essential for Myotoxin a's myotoxicity or its ability to inhibit Ca2+-ATPase function.
- The structural integrity of Myotoxin a is largely maintained after modification, preserving key biological activities.