Abstract
Snake venom phosphodiesterase fromCrotalus adamanteus can be purified by blue sepharose chromatography followed by gel exclusion chromatography on Sephadex G-100. The enzyme is judged to be homogeneous by SDS gel electrophoresis. Atomic absorption spectrometry indicates a stoichiometry of 1.07 g-atom of zinc per mole of purified enzyme. The enzyme is inhibited by a wide variety of structurally different metal binding agents, e.g., 1,10-phenanthroline, thioglycolic acid,l-cysteine, 8-hydroxy-5-quinoline sulfonic acid, EDTA, and dipicolinic acid. The results of both the chelator inhibition and the atomic absorption analysis indicate that snake venom phosphodiesterase is a zinc metalloenzyme. Snake venom phosphodiesterase shares a number of mechanistic features in common with the nucleotidyl transferases. All of these enzymes contain zinc, are activated by magnesium, and catalyze α-β phosphoryl bond cleavage. Mechanistic studies of phosphodiesterase may therefore be helpful in understanding the mechanism of the hydrolytic step catalyzed by all of these enzymes.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Auld, D. S. (1979). The role of zinc in biochemical processes. InAdvances in Chemistry Series, No.172, pp. 112–133.
Auld, D. S., Kawaguchi, H., Livingston, D. M., and Vallee, B. L. (1974). RNA-dependent DNA polymerase (reverse transcriptase) from avian myeloblastosis virus: A zinc metalloenzyme.Proc. Natl. Acad. Sci. USA 71, 2091–2095.
Auld, D. S., Kawaguchi, H., Livingston, D. M., and Vallee, B. L. (1975). Zine in reverse transcriptase from mammalian RNA type C viruses.Biochem. Biophys. Res. Commun. 62, 296–302.
Auld, D. S., Atsuya, I., Campino, C., and Valenzuela, P. (1976). Yeast RNA polymerase I: A eukaryotic zinc metalloenzyme.Biochem. Biophys. Res. Commun. 69, 548–554.
Coleman, J. E. (1974). The role of Zn(II) in transcription by T7 RNA polymerase.Biochem. Biophys. Res. Commun. 60, 641–648.
Drum, D. E., and Vallee, B. L. (1970). Differential chemical reactivities of zinc in horse liver alcohol dehydrogenase.Biochemistry 9, 4078–4086.
Falchuk, K. H., Mazus, B., Ulpino, L., and Vallee, B. L. (1976).Euglena gracilis DNA dependent RNA polymerase II: A zinc metalloenzyme.Biochemistry 15, 4468–4475.
Iwanaga, S., and Suzuki, T. (1979). Enzymes in snake venom. In Lec, C. Y. (ed.),Snake Venoms, Springer-Verlag, New york, pp. 61–158.
Kotsiopoulos, P. S., and Mohr, S. C. (1975). Protein synthesis elongation factor 1 from rat liver; A zinc metalloenzyme.Biochem. Biophys. Res. Commun. 67, 979–987.
Laemmli, V. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227, 680–685.
Lattke, H., and Weser, U. (1976). Yeast RNA-Polymerase B: A Zinc Protein.FEBS Lett. 65, 288–291.
Mayaux, J., and Blanquet, S. (1981). Binding of zinc to Escherichia coli phenylalanyl transfer ribonucleic acid synthetase. Comparisons with other aminoacyl transfer ribonucleic acid synthetases.Biochemistry 20, 4647–4654.
Oka, J., Ueda, K., and Hayaishi, O. (1978). Snake venom phosphodiesterase. Simple purification with blue sepharose and its application to poly (ADP-ribose) study.Biochem. Biophys. Res. Commun. 80, 841–848.
Pollack, S. E., and Auld, D. S. (1980). Fluorometric assays for nucleotidyl transferases and exonucleases.Federation Proceedings 39, 2031.
Pollack, S. E., and Auld, D. S. (1982). Fluorescent nucleotide triphosphate substrates for snake venom phosphodiesterase.Analytical Biochemistry 127, 81–88.
Posorske, L. H., Cohn, M., Yanagisawa, M., and Auld, D. S. (1979). Methionyl-t RNA synthetase of Escherichia Coli. A zinc metalloprotein.Biochem. Biophys. Acta. 576, 128–133.
Philipps, G. R. (1975). Purification and characterization of phosphodiesterase from Crotalus venom. Hoppe-Selyer's Z.Physiol. Chem. 356, 1085–1096.
Poesz, B. J., Battula, N., and Loeb, L. A. (1975). Zinc in reverse transcriptase.Biochem. Biophys. Res. Commun. 56, 959–964.
Sabbioni, E. (1976). The metalloenzyme nature of calf thymus deoxynucleotidyl transferase.FEBS Lett. 71, 233–235.
Scrutton, M. C., Wu, C. W., and Goldthwait, D. A. (1971). The presence and possible role of zinc in RNA polymerase obtained fromEscherichia coli.Proc. Natl. Acad. Sci. USA 68, 2497–2501.
Slater, J. P., and Mildvan, A. S., and Loeb, L. A. (1971). Zinc in DNA polymerases.Biochem. Biophys. Res. Commun. 44, 37–43.
Springate, C. F., Mildvan, A. S., Abramson, R., Engle, J. L., and Loeb, L. A. (1973).Escherichia coli deoxyriboncleic acid polymerase I. A zinc metalloenzyme.J. Biol. Chem. 248, 5987–5993.
Sulkowski, E., and Laskowski, M., Sr. (1971). Inactivation of 5′-nucleotidase in commercial preparations of venom exonuclease (phosphodiesterase).Biochem. Biophys. Acta. 240, 443–447.
Switzer, R. C., Merrill, C. R., and Shifrin, S. (1979). A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels.Anal. Biochem. 98, 231–237.
Theirs, R. E. (1957). Separation and concentration of millimicrogram quantities of metals and contamination hazards. In Yal, J. H. (ed.),Methods of Trace Analysis, Wiley, New York.
Yarbrough, L. R., Schlegeck, J. G., and Baughman, M. (1979). Synthesis and proeprties of fluorescent nucleotide substrates for DNA-dependent RNA polymerases.J. Biol. Chem. 254, 12069–12073.
Vallee, B. L. (1960). Metal and enzyme interactions: Correlation of composition, function, and structure. In Boyer, P. D., Lardy, H., and Myrback, K. (eds.),The Enzymes III, Academic, New York, pp. 225–276.
Vallee, B. L. (1972). In Galde, K., Horecker, B. L., and Whelan, W. J. (eds.),Molecular Basis of Biological Activity, Academic, New york, pp. 145–168.
Vallee, B. L. (1980). Zinc and other active site methods as probes of local conformation and function of enzymes.Carlsberg Res. Commun. 45, 423–441.
Vallee, B. L., and Falchuk, K. H. (1981). Zinc and gene expression.Phil. Trans. R. Soc. Lond. B 294, 185–197.
Williams, E. J., Sung, S., and Laskowski, M., Sr. (1961). Action of venom phosphodiesterase on deoxyribonucleic acid.J. Biol. Chem. 236, 1130–1134.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Pollack, S.E., Uchida, T. & Auld, D.S. Snake venom phosphodiesterase: A zinc metalloenzyme. J Protein Chem 2, 1–12 (1983). https://doi.org/10.1007/BF01025165
Received:
Published:
Issue Date:
DOI: https://doi.org/10.1007/BF01025165