Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1971 Aug;124(1):25–30. doi: 10.1042/bj1240025

Effects of zinc and other metal ions on the stability and activity of Escherichia coli alkaline phosphatase

C N A Trotman 1,*, C Greenwood 1
PMCID: PMC1177109  PMID: 4942389

Abstract

Measurement of the ultraviolet circular dichroism of apo-(alkaline phosphatase) in urea solutions showed substantial denaturation in 3m-urea. A zinc-deficient mutant alkaline phosphatase behaved similarly. The stability of the enzyme in 6m-urea was followed as a function of its zinc content and was found to be dependent on the first two of the four zinc atoms bound by apoenzyme. Phosphatase activity was mostly dependent on a second pair of zinc atoms. Mn2+, Co2+, Cu2+ or Cd2+ also restored structural stability. Sedimentation-velocity and -equilibrium experiments revealed that dissociation of the dimer accompanied apoenzyme denaturation in urea concentrations of 1m or higher, without treatment with disulphide-reducing agent.

Full text

PDF

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Applebury M. L., Coleman J. E. Escherichia coli alkaline phosphatase. Metal binding, protein conformation, and quaternary structure. J Biol Chem. 1969 Jan 25;244(2):308–318. [PubMed] [Google Scholar]
  2. Beychok S. Circular dichroism of biological macromolecules. Science. 1966 Dec 9;154(3754):1288–1299. doi: 10.1126/science.154.3754.1288. [DOI] [PubMed] [Google Scholar]
  3. Cohen S. R., Wilson I. B. Measurement of the zinc dissociation constants of alkaline phosphatase from Escherichia coli by equilibration with zinc ion buffers. Biochemistry. 1966 Mar;5(3):904–909. doi: 10.1021/bi00867a014. [DOI] [PubMed] [Google Scholar]
  4. Csopak H., Falk K. E. The specific binding of copper(II) to alkaline phosphatase of E. coli. FEBS Lett. 1970 Apr 2;7(2):147–150. doi: 10.1016/0014-5793(70)80142-9. [DOI] [PubMed] [Google Scholar]
  5. Csopak H. The specific binding of zinc(II) to alkaline phosphatase of Escherichia coli. Eur J Biochem. 1969 Jan;7(2):186–192. doi: 10.1111/j.1432-1033.1969.tb19590.x. [DOI] [PubMed] [Google Scholar]
  6. Gratzer W. B., Cowburn D. A. Optical activity of biopolymers. Nature. 1969 May 3;222(5192):426–431. doi: 10.1038/222426a0. [DOI] [PubMed] [Google Scholar]
  7. HOLZWARTH G., DOTY P. THE ULTRAVIOLET CIRCULAR DICHROISM OF POLYPEPTIDES. J Am Chem Soc. 1965 Jan 20;87:218–228. doi: 10.1021/ja01080a015. [DOI] [PubMed] [Google Scholar]
  8. Harris M. I., Coleman J. E. The biosynthesis of apo- and metalloalkaline phosphatases of Escherichia coli. J Biol Chem. 1968 Oct 10;243(19):5063–5073. [PubMed] [Google Scholar]
  9. Jirgensons B. Circular dichroism of proteins of known and unknown conformations. Biochim Biophys Acta. 1970 Jan 20;200(1):9–17. doi: 10.1016/0005-2795(70)90037-1. [DOI] [PubMed] [Google Scholar]
  10. LEVINTHAL C., SIGNER E. R., FETHEROLF K. Reactivation and hybridization of reduced alkaline phosphatase. Proc Natl Acad Sci U S A. 1962 Jul 15;48:1230–1237. doi: 10.1073/pnas.48.7.1230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lazdunski C., Chappelet D., Petitclerc C., Leterrier F., Douzou P., Lazdunski M. The Cu2 plus-alkaline phosphatase of Escherichia coli. Eur J Biochem. 1970 Dec;17(2):239–245. doi: 10.1111/j.1432-1033.1970.tb01159.x. [DOI] [PubMed] [Google Scholar]
  12. Lazdunski C., Petitclerc C., Lazdunski M. Structure-function relationships for some metalloalkaline phosphatases of E. coli. Eur J Biochem. 1969 Apr;8(4):510–517. doi: 10.1111/j.1432-1033.1969.tb00556.x. [DOI] [PubMed] [Google Scholar]
  13. MALAMY M. H., HORECKER B. L. PURIFICATION AND CRYSTALLIZATION OF THE ALKALINE PHOSPHATASE OF ESCHERICHIA COLI. Biochemistry. 1964 Dec;3:1893–1897. doi: 10.1021/bi00900a018. [DOI] [PubMed] [Google Scholar]
  14. Neu H. C., Heppel L. A. The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts. J Biol Chem. 1965 Sep;240(9):3685–3692. [PubMed] [Google Scholar]
  15. PLOCKE D. J., LEVINTHAL C., VALLEE B. L. Alkaline phosphatase of Escherichia coli: a zinc metalloenzyme. Biochemistry. 1962 May 25;1:373–378. doi: 10.1021/bi00909a001. [DOI] [PubMed] [Google Scholar]
  16. PLOCKE D. J., VALLEE B. L. Interaction of alkaline phosphatase of E. coli with metal ions and chelating agents. Biochemistry. 1962 Nov;1:1039–1043. doi: 10.1021/bi00912a014. [DOI] [PubMed] [Google Scholar]
  17. Petitclerc C., Lazdunski C., Chappelet D., Moulin A., Lazdunski M. The functional properties of the Zn2(plus)-and Co2(plus)-alkaline phosphatases of Escherichia coli. Labelling of the active site with pyrophosphate, complex formation with arsenate, and reinvestigation of the role of the zinc atoms. Eur J Biochem. 1970 Jun;14(2):301–308. doi: 10.1111/j.1432-1033.1970.tb00290.x. [DOI] [PubMed] [Google Scholar]
  18. ROTHMAN F., BYRNE R. Fingerprint analysis of alkaline phosphatase of Escherichia coli K12. J Mol Biol. 1963 Apr;6:330–340. doi: 10.1016/s0022-2836(63)80092-3. [DOI] [PubMed] [Google Scholar]
  19. Reynolds J. A., Schlesinger M. J. Conformational states of the subunit of Escherichia coli alkaline phosphatase. Biochemistry. 1967 Nov;6(11):3552–3559. doi: 10.1021/bi00863a029. [DOI] [PubMed] [Google Scholar]
  20. Reynolds J. A., Schlesinger M. J. Hydrogen ion equilibria of conformational states of Escherichia coli alkaline phosphatase. Biochemistry. 1968 Jun;7(6):2080–2085. doi: 10.1021/bi00846a009. [DOI] [PubMed] [Google Scholar]
  21. Schlesinger M. J. Activation of a mutationally altered form of the Escherichia coli alkaline phosphatase by zinc. J Biol Chem. 1966 Jul 10;241(13):3181–3188. [PubMed] [Google Scholar]
  22. Schlesinger M. J., Barrett K. The reversible dissociation of the alkaline phosphatase of Escherichia coli. I. Formation and reactivation of subunits. J Biol Chem. 1965 Nov;240(11):4284–4292. [PubMed] [Google Scholar]
  23. Simpson R. T., Vallee B. L., Tait G. H. Alkaline phosphatase of Escherichia coli. Composition. Biochemistry. 1968 Dec;7(12):4336–4342. doi: 10.1021/bi00852a028. [DOI] [PubMed] [Google Scholar]
  24. Simpson R. T., Vallee B. L. Two differentiable classes of metal atoms in alkaline phosphatase of Escherichia coli. Biochemistry. 1968 Dec;7(12):4343–4350. doi: 10.1021/bi00852a029. [DOI] [PubMed] [Google Scholar]
  25. THIERS R. E. Contamination in trace element analysis and its control. Methods Biochem Anal. 1957;5:273–335. doi: 10.1002/9780470110218.ch6. [DOI] [PubMed] [Google Scholar]
  26. Trotman C. N., Greenwood C. Structural and activational zinc in Escherichia coli alkaline phosphatase. Biochem J. 1971 Jan;121(1):12P–12P. doi: 10.1042/bj1210012pa. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Trotman C. N., Greenwood C. Zinc-dependent conformational stability in the alkaline phosphatase of Escherichia coli. Biochem J. 1969 Oct;114(4):82P–83P. doi: 10.1042/bj1140082pb. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES