Abstract
A derivative of the polypeptide cardiostimulant anthopleurin-B(AP-B) labeled with the spin label 1-oxyl 2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl azide has been prepared and characterized. The product was found by mass spectrometry to be labeled at a single site, which amino acid sequencing showed to be the N-terminus. It also retained positive inotropic activity when assayed on isolated guinea pig atria. The spin-labeled (SL) product was found to exist in two distinct conformations by reversed-phase HPLC and in at least two conformations by electron spin resonance spectroscopy (ESR) over thepH range 2–9. The ESR data also show evidence for multimetric states of SL-AP-B over thepH range 2–9, with maximum aggregation at pH 4.5–5, and a slow disaggregation when thepH is adjusted to 8–9. The presence of multiple conformers of SL-AP-B and its tendency to aggregate render it unsuitable for high-resolution NMR structural studies of the isolated ligand, but the retention of activity may make it useful for studies of the sodium-channel-bound form of the molecule.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- AP-A:
-
anthopleurin-A
- AP-B:
-
anthopleurin-B
- ATX Ia:
-
toxin Ia fromAnemonia sulcata
- Sh I:
-
neurotoxin I fromStichodactyla helianthus
- TFA:
-
trifluoroacetic acid
- SL-AP-B:
-
AP-B labeled at the N-terminus with the spin label 1-oxyl 2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl azide
References
Archer, S. J., Ellana, J. F., and Cafiso, D. S. (1991).Biophys. J. 60, 109–136.
Barbanin, J., Hugues, M., Schweitz, H., Vincent, J.-P., and Lazdunski, M. (1981).J. Biol. Chem. 256, 5764–5769.
Berliner, L. J. (1976).Spin Labeling: Theory and Applications, Academic Press, New York.
Berliner, L. J. (1977).Meth. Enzymol 49G, 418–480.
Berliner, L. J. (1979).Spin Labeling II: Theory and Applications. Academic Press, New York.
Berndt, K. D., Güntert, P., Orbons, L. P. M., and Wüthrich, K. (1992).J. Mol. Biol. 227, 757–775.
Davoust, J., and Devaux, P. F. (1982).J. Magn. Reson. 48, 475–486.
Fogh, R. H., Kem, W. R., and Norton, R. S. (1990).J. Biol. Chem. 265, 13016–13028.
Gallagher, M. J., and Blumenthal, K. M. (1992).J. Biol. Chem. 267, 13958–13963.
Gooley, P. R., Blunt, J. W., and Norton, R. S. (1984).FEBS Lett. 174, 15–19.
Gooley, P. R., Blunt, J. W., Beress, L., and Norton, R. S. (1988).Biopolymers 27, 1143–1157.
Gould, A. R., and Norton, R. S. (1995).Toxicon 33, 187–199.
Hideg, K., and Hankovszky, O. H. (1989).Biol. Magn. Reson. 8, 427–488.
Kem, W. R. (1988). InThe Biology of Nematocysts (Hessinger, D., and Lenhoff, H., eds.), Academic Press, New York, pp. 375–405.
Khera, P. K., and Blumenthal, K. M. (1994).J. Biol. Chem. 269, 921–925.
Khera, P. K., Benzinger, R., Limpkind, G., Drum, C. L., Hanck, D. A., and Blumenthal, K. M. (1995).Biochemistry 34, 921–925.
Kosen, P. A. (1989).Meth. Enzymol. 177, 86–121.
Mchaourab, H. S., Hyde, J. S., and Feix, J. B. (1994).Biochemistry 33, 6691–6699.
Monks, S. A., Pallaghy, P. K., Scanlon, M. J., and Norton, R. S. (1995).Structure 3, 791–803.
Norton, R. S. (1991).Toxicon 29, 1051–1084.
Odefey, C., Mayr, L. M., and Schmid, F. X. (1995).J. Mol. Biol. 245, 69–78.
Pallaghy, P. K., Scanlon, M. J., Monks, S. A., and Norton, R. S. (1995).Biochemistry 34, 3782–3794.
Renaud, J.-F., Fosset, M., Schweitz, H., and Lazdunski, M. (1986).Eur. J. Pharmacol. 120, 161–170.
Rousseau, D. L., Jr., Guyer, C. A., Beth, A. H., Papayannopoulos, I. A., Wang, B., Wu, R., Mroczkowski, B., and Staros, J. V. (1993).Biochemistry 32, 7893–7903.
Scanlon, M. J., and Norton, R. S. (1994).Protein Sci. 3, 1121–1124.
Schweitz, H., Vincent, J.-P., Barhanin, J., Frelin, C., Linden, G., Hugues, M., and Lazdunski, M. (1981).Biochemistry 20, 5245–5252.
Torda, A. E., Mabbutt, B. C., van Gunsteren, W. F., and Norton, R. S. (1988).FEBS Lett. 239, 266–270.
Widmer, H., Billeter, M., and Wüthrich, K. (1989).Proteins 6, 357–371.
Wilcox, G. R., Fogh, R. H., and Norton, R. S. (1993).J. Biol. Chem.,268, 24707–24719.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Monks, S.A., Norton, R.S., Curtain, C.C. et al. Preparation and characterization of a biologically active spin-labeled sea anemone toxin. J Protein Chem 15, 427–434 (1996). https://doi.org/10.1007/BF01886849
Received:
Published:
Issue Date:
DOI: https://doi.org/10.1007/BF01886849