Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Affinity chromatography ofd-lactate dehydrogenases fromLimulus polyphemus (horseshoe crab) andHaliotus sp. (abalone) voluntary muscle on 8-(6-aminohexyl)-amino-adenine nucleotide-sepharose

  • 25 Accesses

Abstract

An improved method of purification employing sequential isoenzyme elution on DEAE-cellulose at pH 6.5 and biologically specific elution with the reduced NAD-pyruvate adduct from 8-(6-aminohexyl)-amino-NAD⊕-Sepharose is described forLimulus (horseshoe crab) muscle D- lactate dehydrogenase. The protein is judged as being at least 98%pure by its constant specific activity in the terminal purification steps, a molar extinction coefficient (280nm) identical with that previously reported for the purified enzyme, and its protein and enzyme electrophoretic patterns on starch and polyacrylamide gel electrophoresis at three pHs. The binding of ammonium sulfate fractionated D- lactate dehydrogenase from crude cell- free Limulus andHaliotus (abalone) muscle homogenates to 8-(6-aminohexyl)-amino-AMP-and 8-(6-aminohexyl)-amino-NAD®-Sepharose columns is demonstrated. A comparison of the binding properties of these two enzymes with those of vertebrate L-lactate dehydrogenases suggests that they may be significantly different in terms of their binding sites for the adenine portion of the coenzyme.

This is a preview of subscription content, log in to check access.

References

  1. 1.

    Pesce, A., McKay, R. H., Stolzenbach, F., Cahn, R. D., andKaplan, N. O. (1964) J. Biol. Chem. 239: 1753.

  2. 2.

    Fondy, T. P., Pesce, A., Freedberg, I., Stolzenbach, F., andKaplan, N. O. (1964) Biochemistry 3: 522.

  3. 3.

    Pesce, A., Fondy, T. P., Stolzenbach, F., Castillo, F., andKaplan, N. O. (1967) J. Biol. Chem. 242: 2151.

  4. 4.

    Fondy, T. P., Everse, J., Driscoll, G. A., Castillo, F., Stolzenbach, F. E., andKaplan, N. O. (1965) J. Biol. Chem. 240: 4219.

  5. 5.

    Kaloustian, H. D., Stolzenbach, F. E., Everse, J., andKaplan, N. O. (1969) J. Biol. Chem. 244: 2891.

  6. 6.

    Kaloustian, H. D., andKaplan, N. O. (1969) J. Biol. Chem. 244: 2902.

  7. 7.

    Eichner, R. D., andKaplan, N. O. (1977)Arch. Biochem. Biophys., in press.

  8. 8.

    Long, G. L., andKaplan, N. O. (1968) Science 162: 685.

  9. 9.

    Long, G. L., andKaplan, N. O. (1973) Arch. Biochem. Biophys. 154: 696.

  10. 10.

    Long, G. L., andKaplan, N. O. (1973) Arch. Biochem. Biophys. 154: 711.

  11. 11.

    Long, G. L. (1975) “D-Lactate Dehydrogenase from the Horseshoe Crab,”In Methods in Enzymology, Vol. 41,Wood, W. A. (ed.), Academic Press, New York, pp. 313–318.

  12. 12.

    Lee, C.-Y., Lappi, D. A., Wermuth, B., Everse, J., andKaplan, N. O. (1974) Arch. Biochem. Biophys. 163: 561.

  13. 13.

    Rossmann, M. G. (1975) “Evolutionary and Structural Relationships among Dehydrogenases,”In The Enzymes, Vol. 11,Boyer, P. D. (ed.), Academic Press, New York, pp. 62–102.

  14. 14.

    Lee, C.-Y., andKaplan, N. O. (1975) Arch. Biochem. Biophys. 168: 665.

  15. 15.

    Fine, I. H., andCostello, L. (1963) “The Use of Starch Electrophoresis in Dehydrogenase Studies,”In Methods in Enzymology, Vol. 6,Colowick, S. P., andKaplan, N. O. (eds.), Academic Press, New York, pp. 958–972.

  16. 16.

    Davis, B. J. (1964) Ann. N. Y. Acad. Sci. 121: 404.

  17. 17.

    Gabriel, O. (1971) “Analytical Disc Gel Electrophoresis,”In Methods in Enzymology, Vol. 22,Jakoby, W. B. (ed.), Academic Press, New York, pp. 565–578.

  18. 18.

    Everse, J., Zoll, E.C., Kahan, L., andKaplan, N. O. (1971) Bioorg. Chem. 1: 207.

  19. 19.

    Lee, C.-Y., andKaplan, N. O. (1976) J. Macromol. Sci. Chem. A10:15.

  20. 20.

    Dixon, J. E. Personal communication (manuscript in preparation).

Download references

Author information

Correspondence to George L. Long or Elizabeth L. Moore.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Long, G.L., Moore, E.L. Affinity chromatography ofd-lactate dehydrogenases fromLimulus polyphemus (horseshoe crab) andHaliotus sp. (abalone) voluntary muscle on 8-(6-aminohexyl)-amino-adenine nucleotide-sepharose. Journal of Solid-Phase Biochemistry 1, 307–317 (1976). https://doi.org/10.1007/BF02990969

Download citation

Keywords

  • Lactate Dehydrogenase
  • Ammonium Sulfate
  • Horseshoe Crab
  • Sepharose Column
  • Disodium EDTA