Purification and characterization of urease isolated from the pathogenic fungus Coccidioides immitis

F. Mirbod, R. A. Schaller, G. T. Cole

Research output: Contribution to journalArticle

29 Scopus citations

Abstract

Coccidioides immitis, the causative agent of San Joaquin Valley fever (coccidioidomycosis), produces a urease which has been suggested to contribute to the virulence of this fungal pathogen. Urease catalyzes the hydrolysis of urea and has been proposed to at least partly account for alkalinity of the microenvironment in which C. immitis grows due to the release of ammonia and ammonium ions. The C. immitis urease was purified to homogeneity (1048-fold) from the mycelial cytosol by chromatographic fractionation. The sequence of 12 N-terminal amino-acid residues of the purified, native polypeptide was identical to that predicted by the translated urease gene sequence which has been reported. The isolated enzyme exhibited a specific activity in the presence of urea of 1750 μmol min-1 mg-1 protein, has a native molecular mass of 450 kDa, revealed a Km for urea of 4.1 mM, had a pH optimum of 8.0 and is heat stable. Hydroxyurea, acetohydroxamic acid (AHA) and boric acid each inhibited activity of the purified enzyme. Urease activity was enhanced by the presence of 5-10 mM concentrations of Mg2+ or Mn2+, but inhibited by Li+, Ni2+, Cu2+ or Zn2+. The reversible urease inhibitor, AHA, blocked enzyme activity in the crude mycelial cytosolic fraction when added at a concentration of 10 mM. On the other hand, 10 mM AHA added to 4-day-old mycelial cultures only partially decreased the amount of ammonium detected in the culture medium. It is evident, therefore, that C. immitis urease activity does not account for the total amount of ammonia secreted during in vitro growth of the pathogen. Other metabolic sources of ammonia, which may also contribute to the virulence of C. immitis, are under investigation.

Original languageEnglish (US)
Pages (from-to)35-44
Number of pages10
JournalMedical mycology
Volume40
Issue number1
DOIs
StatePublished - Jan 1 2002
Externally publishedYes

    Fingerprint

Keywords

  • Ammonium production
  • Coccidioides immitis
  • Urease purification

ASJC Scopus subject areas

  • Infectious Diseases

Cite this