Acutolysin D, isolated from the venom of Agkistrodon acutus, possesses marked haemorrhagic and proteolytic activities. The molecular weight and the absorption coefficients (A 1% 280) of acutolyisn D have been determined to be 47,850 ± 8 amu and 9.3 by mass spectrometer and UV spectrum, respectively. The effects of metal ions on the conformation and activity of acutolysin D have been studied by following fluorescence, circular dichroism and biological activity measurements. Acutolysin D contains two Ca2+-binding sites and two Zn2+-binding sites determined by atomic absorption spectrophotometer. Zn2+ is essential for the enzyme activities of acutolysin D, however, the presence of 1 mM Zn2+ significantly decreases its caseinolytic activity and intrinsic fluorescence intensity at pH 9.0 due to Zn(OH)2 precipitate formation. Ca2+ is important for the structural integrity of acutolysin D, and the presence of 1 mM Ca2+ markedly enhances its caseinolytic activity. Interestingly, the caseinolytic activity which is inhibited partly by Cu2+, Co2+, Mn2+ or Tb3+ and inhibited completely by Cd2+, is enhanced by Mg2+. The fluorescence intensity of the protein decreases in the presence of Cu2+, Co2+, Cd2+ or Mn2+, but neither for Ca2+, Mg2+ nor for Tb3+. Zn2+, Ca2+, Mg2+, Cu2+, Mn2+, Co2+ and Tb3+ have slight effects on its secondary structure contents. In addition, Cd2+ causes a marked increase of antiparallel β-sheet content from 45.5% to 60.2%.
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Abbreviations
- MMP:
-
matrix metalloproteinases
- holo-acutolysin D:
-
native Ca2+- and Zn2+-containing acutolysin D
- apo-acutolysin D:
-
Ca2+-free and Zn2+-free acutolysin D
- CA:
-
caseinolytic activity
- EDTA:
-
ethylenediamine tetraacetic acid
- UV:
-
ultraviolet
- AU:
-
absorbance unit
- PAGE:
-
polyacrylamide gel electrophoresis
- AAS:
-
atomic absorption spectrophotometer
References
Andersson I.., Maret W., Zeppezauer M., Brown R. D. 3rd., Koenig S. H (1981) Biochemistry 20:3433–3438
Bjarnason J. B., Fox J. W (1994) Phar. Ther. 62:325–372
Bjarnason J. B., Fox J. W (1995) Meth. Enzymol. 248:345–368
Bjarnason J. B., Fox J. W (1983) Biochemistry 22:3770–3778
Blundell T. L. (1994) Nat. Struct. Biol. 1:73–75
Brustein E. A., Vedenkina N. S., Irkova M. N. (1973) Photochem. Photobiol. 18:263–279
Chen Z. L., Liu Q. L., Wang S. Y., Xu X. L., Yu H. M. (1999) Spectrochim. Acta A 55:1909–1914
Dockal M., Carter D.C., Rüker F. (2000) J. Biol. Chem. 275:3042–3050
Gomis-Rüth F. X., Kress L. F., Kellermann J., Mayr I., Lee X., Huber R., Bode W. (1994) J. Mol. Biol. 239:513–544
Gong W. M., Zhu X. Y., Liu S. J., Teng M. K., Niu L. W. (1998) J. Mol. Biol. 283:657–668
Hakansson K., Wehnert A., Liljas A. (1994) Acta Cryst. D50:93–100
Kamiguti A. S., Hay C. R. M., Theakston R. D. G., Zuzel M. (1996) Toxicon 34:627–642
Kondo H., Kondo S., Ikezawa H., Murata R., Ohsaka A. (1960) Jpn. J. Med. Sci. Biol. 13:43–51
Krepkiy D., Forsterling F. H., Petering D. H. (2004) Chem. Res. Toxicol. 17:863–870
Liu Q. D., Xu W. H., Cheng X., Jin G., Shen X., Lou H. B., Liu J. (1999) Toxicon 37:1539–1548
Liu Q. L., Xu X. L., Wang C., Xie B. P. (1993) J. Rare Earth 11(1):15–18
Perczel A., Park K., Fasman G D. (1992) Anal. Biochem. 203:83–89
Van der Walt S. J., Joubert F. J. (1971) Toxicon 9:153–161
Weber G., Rosenheck K. (1964) Biopolymers Symp. 1:333–344
Xu X. L., Liu Q. L., Xie Y. S. (2002a) Biochemistry 41:3546–3554
Xu X. L., Liu Q. L., Yu H. M., Xie Y. S. (2002b) Protein Sci. 11:944–956
Xu X., Wang C., Liu J., Lu Z. (1981) Toxicon 19:633–644
Yamniuk A. P., Nguyen L. T., Hoang T. T., Vogel H. J. (2004) Biochemistry 43:2558–2568
Zhu Z., Gong W., Niu L., Teng M., He H. (1996) Acta Crystallogr. D 52:407–408
Zhu X. Y., Teng M. K., Niu L. W. (1999) Acta Crystallogr. D 55:1834–1841
Acknowledgments
We thank Dr. Zhenlong Chen for assistance in preparation of the manuscript. This research was supported by grants from the National Natural Science Foundation of China (Grant No. 20571069, 20171041, X. L. Xu).
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Xu, X., Liu, X., Zhang, L. et al. Effects of Metal Ions on the Conformation and Activity of Acutolysin D from Agkistrodon Acutus Venom. Protein J 25, 423–430 (2006). https://doi.org/10.1007/s10930-006-9036-1
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DOI: https://doi.org/10.1007/s10930-006-9036-1