Advertisement

Journal of Biosciences

, Volume 6, Issue 2, pp 233–248 | Cite as

Purification and regulation of aspartate transcarbamylase from germinated mung bean (Vigna radiata) seedlings

  • P. V. Prasad
  • N. Appaji Rao
Article

Abstract

Aspartate transcarbamylase (EC 2.1.3.2) was purified to homogeniety from germinated mung bean seedlings by treatment with carbamyl phosphate. The purified enzyme was a hexamer with a subunit molecular weight of 20,600. The enzyme exhibited multiple activity bands on Polyacrylamide gel electrophoresis, which could be altered by treatment with carbamyl phosphate or UMP indicating that the enzyme was probably undergoing reversible association or dissociation in the presence of these effectors. The carbamyl phosphate stabilized enzyme did not exhibit positive homotropic interactions with carbamyl phosphate and hysteresis. The enzyme which had not been exposed to carbamyl phosphate showed a decrease in specific activity with a change in the concentration of both carbamyl phosphate and protein. The carbamyl phosphate saturation and UMP inhibition patterns were complex with a maximum and a plateau region. The partially purified enzyme also exhibited hysteresis and the hysteretic response, a function of protein concentration, was abolished by preincubation with carbamyl phosphate and enhanced by preincubation with UMP. All these observations are compatible with a postulation that the enzyme activity may be regulated by slow reversible association-dissociation dependent on the interaction with allosteric ligands

Keywords

Aspartate transcarbamylase mung bean purification regulation hysteresis association-dissociation 

Abbreviations Used

Pi

Orthophosphate

DEAE

diethylaminoethyl

GdmCl

guanidinium chloride

Buffer A

50 mM sodium acetate-acetic acid buffer, pH 4.7 containing 6 M GdmCl

Mr

molecular weight

Tris

Tris-(hydroxymethyl) aminomethane

SDS

sodium dodecyl sulphate

PALA

N-phosphonoacetyl-L-aspartate

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Achar, B. S., Savithri, H. S., Vaidyanathan, C. S. and Appaji Rao, N. (1974)Eur. J. Biochem.,47, 15.PubMedCrossRefGoogle Scholar
  2. Adler, S. P., Purich, D. and Stadtman, E. R. (1975)J. Biol. Chem.,250, 6264.PubMedGoogle Scholar
  3. Bethel, M. R. and Jones, M. E. (1969)Arch. Biochem. Biophys.,134, 252.CrossRefGoogle Scholar
  4. Boivin, P., Galand, C. and De Martial., M. C. (1972)Pathol. Biol.,20, 583.Chem. Abst.,77, 1490742.PubMedGoogle Scholar
  5. Barbson, J. S. and Switzer, R. L. (1975)J. Biol. Chem.,250, 8664.Google Scholar
  6. Calhoun, D. H., Rimerman, R. A. and Hatfield, G. W. (1973)J. Biol. Chem.,248, 3511.PubMedGoogle Scholar
  7. Collins, K. D. and Stark, G. R. (1971)J. Biol. Chem.,246, 6599.PubMedGoogle Scholar
  8. Datta, P., Gest, H. and Segal., H. L. (1964)Proc. Natl. Acad. Sci. USA,51, 125.PubMedCrossRefGoogle Scholar
  9. Davis, B. J. (1964)Ann. N. Y. Acad. Sci.,121, 404.PubMedCrossRefGoogle Scholar
  10. Feldberg, R. S. and Datta, S. P. (1971)Eur. J. Biochem.,21, 438.PubMedCrossRefGoogle Scholar
  11. Freiden, C. (1971)Ann. Rev. Biochem.,40, 653.CrossRefGoogle Scholar
  12. Freiden, G (1979)Ann. Rev. Biochem.,48, 471.CrossRefGoogle Scholar
  13. Freiden, C. (1981) inProtein-Protein interactions, eds. Freiden, C. and Nichol, L. W., (New York: John Wiley and Sons) p. 289.Google Scholar
  14. Gerhart, J. C. and Pardee, A. B. (1962)J. Biol. Chem.,237, 891.PubMedGoogle Scholar
  15. Graf, E., Verma, A. K., Gorski, J. P., Lopaschuk, G., Niggli, V., Zurini, M., Calafoli, E. and Penniston, J. T. (1982)Biochemistry,21, 4511.PubMedCrossRefGoogle Scholar
  16. Grayson, J. E. and Yon, R. J. (1978)Biochem. Soc. Trans.,6, 197.PubMedGoogle Scholar
  17. Hatfield, G. W. (1971)Biochem. Biophys. Res. Commun.,44, 464.PubMedCrossRefGoogle Scholar
  18. Kagan, Z. S., Dorozkko, A. I, Kovaleva, S. V. and Yakovelera, L. I. (1975)Biochim. Biophys. Acta.,403, 208.PubMedGoogle Scholar
  19. Kelley, J. H. and Plant, G. W. E. (1981)J. Biol. Chem.,256, 330.Google Scholar
  20. Koistinen, V. U. (1980)J. Virol.,35, 20.PubMedGoogle Scholar
  21. Kurganov, B. I. (1977)J. Theor. Biol.,68, 521.PubMedCrossRefGoogle Scholar
  22. Kurganov, B. L, Dorozkko, A. I., Kagan, Z. S. and Yakovlev, V. A. (1976)J. Theor. Biol.,60, 247, 271, 287.PubMedCrossRefGoogle Scholar
  23. Lowry, O. H., Rosenbrough, N. J., Farr, A. L. and Randall, R. J. (1951)J. Biol. Chem.,193, 265.PubMedGoogle Scholar
  24. Maddy, A. H. (1976)J. Theor. Biol.,62, 315.PubMedCrossRefGoogle Scholar
  25. Mann, K. G. and Fish, W. W. (1972)Methods Enzymol.,26, 28.PubMedCrossRefGoogle Scholar
  26. March, C. S., Parikh, I. and Cuatrecasas, P. (1974)Anal. Biochem.,60, 149.PubMedCrossRefGoogle Scholar
  27. Neumann, J. and Jones, M. E. (1964)Arch. Biochem. Biophys.,104, 438.PubMedCrossRefGoogle Scholar
  28. Ong, B. L. and Jackson, J. F. (1972)Biochem. J.,129, 571.PubMedGoogle Scholar
  29. Prescott, L. M. and Jones, M. E. (1969)Anal. Biochem.,32, 408.PubMedCrossRefGoogle Scholar
  30. Rao, G. S. J., Savithri, H. S., Seethalakshmi, S. and Appaji Rao, N. (1979)Annal. Biochem.,95, 401.CrossRefGoogle Scholar
  31. Rao, G. S. J., Prasad, P. V. and Appaji Rao, N. (1982)Biochem. International.,4, 503.Google Scholar
  32. Reddy, A. R. V., Sobhanaditya, J. and Appaji Rao, N. (1980)J. Indian Inst. Sci.,62, 39.Google Scholar
  33. Savithri, H. S., Vaidyanathan, C. S. and Appaji Rao, N. (1978a)Proc. Indian Acad. Sci.,87B, 67.Google Scholar
  34. Savithri, H. S., Vaidyanathan, C. S. and Appaji Rao, N. (1978b)Proc. Indian Acad. Sci.,87B, 81.Google Scholar
  35. Shaltiel, S. and Er-El, Z. (1973)Proc. Natl. Acad. Sci. USA,70, 778.PubMedCrossRefGoogle Scholar
  36. Teipel, J. and Koshland, D. E. Jr. (1969)Biochemistry,8, 4656.PubMedCrossRefGoogle Scholar
  37. Vickers, L. P. (1981)Trends. Biochem. Sci.,6, 11.Google Scholar
  38. Weber, K. (1968)Nature (London),218, 1116.CrossRefGoogle Scholar
  39. Yon, R. J. (1981)Anal. Biochem.,113, 219.PubMedCrossRefGoogle Scholar
  40. Yon, R. J., Grayson, J. E., Chawda, A. and Butterworth, P. J. (1982)Biochem. J.,203, 413.PubMedGoogle Scholar

Copyright information

© Indian Academy of Sciences 1984

Authors and Affiliations

  • P. V. Prasad
    • 1
  • N. Appaji Rao
    • 1
  1. 1.Department of BiochemistryIndian Institute of ScienceBangaloreIndia

Personalised recommendations