1887

Abstract

The urease from (serotype 8) has been purified by immuno-affinity column chromatography. Two active nickel-containing forms of the enzyme were demonstrated by non-denaturing electrophoretic analysis and a single active peak of apparent molecular mass 190 kDa was shown by FPLC. Total inactivation and denaturation of the enzyme to give three subunit polypeptides (one of 72 kDa containing nickel, one of 14 kDa and one of 11 kDa) was achieved by treatment with SDS and boiling. Densitometry suggested that the active enzyme contains equimolar ratios of the three subunits and hence is a hexamer. The enzyme displayed a pH optimum of 6·9 and pI values were determined. Storage of the purified enzyme at −70 °C followed by thawing to 20 °C caused a partial breakdown to inactive subunits. Anti-urease monoclonal antibodies bound both to the active enzyme and to the inactive 72 kDa subunit, and the antibodies cross-reacted with ureases from all of the other human serotypes. Competition assays with the antibodies revealed four distinct epitopes on the enzyme, all distinct from its active site.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-135-2-315
1989-02-01
2021-05-08
Loading full text...

Full text loading...

/deliver/fulltext/micro/135/2/mic-135-2-315.html?itemId=/content/journal/micro/10.1099/00221287-135-2-315&mimeType=html&fmt=ahah

References

  1. Axen R., Porath J., Ernback S. 1967; Chemical coupling of peptides and proteins by means of cyanogen halides. Nature, London 214:1302–1304
    [Google Scholar]
  2. Dixon N.E., Gazzola C., Blakeley R.L., Zerner B. 1975; Jack bean urease (EC 3.5.1.5). A metalloenzyme.A simple biological role for nickel?. Journal of the American Chemical Society 97:4131–4133
    [Google Scholar]
  3. Dulbecco R., Vogt M. 1954; Plaque formation and isolation of pure lines with poliomyelitis virus. Journal of Experimental Medicine 99:167
    [Google Scholar]
  4. Eng H., Robertson J.A., Stemke G.W. 1986; Properties of urease from Ureaplasma urealyticum: kinetics, molecular weight and demonstration of multiple enzyme isoelectric point forms. Canadian Journal of Microbiology 32:487–493
    [Google Scholar]
  5. Fahey J.L., Terry E.W. 1978; Ion exchange chromatography and gel filtration. In Immurtochemistry 1 pp Weir D. M. Edited by Oxford: Blackwell Scientific Publications;
    [Google Scholar]
  6. Fishbein W.N. 1969; A sensitive and non-inhibitory catalytic gel stain for urease. In 5th International Symposium on Chromatography and Electrophoresis, pp 238–242 Ann Arbor, Michigan: Ann Arbor Scientific:
    [Google Scholar]
  7. Geigy J.R. 1970 In Documenta Geigy, Scientific Tables p. 280 Diem K., Lentner C. Edited by Basle: J. R. Geigy;
    [Google Scholar]
  8. Goswami K.K.A., Russell W.C. 1983; Monoclonal antibodies against human paramyxovirus type 3 and against SV5 virus: preparation and preliminary characterization. Journal of General Virology 64:1163–1172
    [Google Scholar]
  9. Hunter W.M. 1978; Radioimmunoassay. In Immunochemistry, 1 pp 14.1–14.40 Weir D. M. Edited by Oxford: Blackwell Scientific Publications;
    [Google Scholar]
  10. Kenny G.E. 1983; Inhibition of growth of Ureaplasma urealyticum by a new urease inhibitor, fluorofamide. Yale Journal of Biology and Medicine 56:717–722
    [Google Scholar]
  11. Kenny G.E., Cartwright F.D. 1977; Effect of urea concentration on growth of Ureaplasma urealyticum (T-strain mycoplasma). Journal of Bacteriology 132:144–150
    [Google Scholar]
  12. Masover G.K., Razin S., Hayflick L. 1977a; Localisation of enzymes in Ureaplasma urealyticum (T-strain mycoplasma). Journal of Bacteriology 130:297–302
    [Google Scholar]
  13. Masover G.K., Razin S., Hayflick L. 1977; (b)Effects of carbon dioxide, urea and ammonia on growth of Ureaplasma urealyticum (T-strain mycoplasma). Journal of Bacteriology 130:292–296
    [Google Scholar]
  14. Pollack J.D. 1986; Metabolic distinctiveness of ureaplasmas. Pediatric Infectious Disease 5:S305–S307
    [Google Scholar]
  15. Precious B.L., Thirkell D., Russell W.C. 1987; Preliminary characterization of the urease and a 96kDa surface-expressed polypeptide of Ureaplasma urealyticum . Journal of General Microbiology 133:2659–2670
    [Google Scholar]
  16. Randall R.E., Young D.F., Goswami K.K.A., Russell W.C. 1987; Isolation and characterisation of monoclonal antibodies to Simian virus 5 and their use in revealing antigenic differences between human, canine and simian isolates. Journal of General Virology 68:2769–2780
    [Google Scholar]
  17. Romano N., Lalicata R., Alesi D.R. 1986; Energy production in Ureaplasma urealyticum . Pediatric Infectious Disease 5:S308–S312
    [Google Scholar]
  18. Russell W.C., Blair G.E. 1977; Polypeptide phosphorylation in adenovirus-infected cells. Journal of General Virology 34:19–35
    [Google Scholar]
  19. Stemke G.W., Robertson J.A., Nhan M. 1987; Purification of urease from Ureaplasma urealyticum . Canadian Journal of Microbiology 33:857–862
    [Google Scholar]
  20. Swanberg S.L., Masover G.K., Hayflick L. 1978; Some characteristics of Ureaplasma urealyticum Urease activity in a simple buffer: effect of metal ions and sulphydryl inhibitors. Journal of General Microbiology 108:221–225
    [Google Scholar]
  21. Todd M.J., Hausinger R.P. 1987; Purification and characterisation of the nickel-containing multicomponent urease from Klebsiella aerogenes . Journal of Biological Chemistry 262:5963–5967
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-135-2-315
Loading
/content/journal/micro/10.1099/00221287-135-2-315
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error