Mirbod F, Schaller RA, Hausinger RP, Cole GT; American Society for Microbiology. General Meeting.
Abstr Gen Meet Am Soc Microbiol. 1999 May 30-Jun 3; 99: 300 (abstract no. F-23).
Medical College of Ohio, Toledo.
Urease of Coccidioides immitis has been shown to be an important immunoprotective antigen and may represent a virulence factor of this human respiratory fungal pathogen. Ammonia/ammonium generated by hydrolysis of urea and secreted by the fungus may compromise host defenses during the process of infection. Growth of the mycelial phase of C. immitis isolate 735 in glucose-yeast extract liquid media resulted in an increase in pH from 6.7 to 7.8 after incubation for 8 days at 30 degrees C. Urease was purified to homogeneity (1048-fold) from the mycelial cytosol by con- secutive chromatographic separations which included use of DEAE-Sepharose, Phenyl-Sepharose, Mono Q ion exchange, and Superose 6 gel filtration columns. The native molecular mass of the active, purified enzyme is 450 kDa. The urease of C. immitis shares significant amino acid sequence homology with the reported urease of Cryptococcus neoformans and jack bean (66% and 58% identity, respectively). The C. immitis urease exhibited a specific activity of 1750 micromolars min-1 per mg protein, with a Km, for urea of 4.1 mM and a pH optimum of 8.0. Acetohydroxamic acid, hydroxyurea and boric acid were strong inhibitors of activity of the purified enzyme, while thiourea acted as a weak inhibitor. Activity of the C. immitis urease was enhanced by the presence of Mg2+ and Mn2+ at 5 to 10 mM. Urease activity was not affected by Na+, K+, Ca2+, or Na2EDTA, but was significantly decreased in the presence of Li+, Ni2+, CU2+, or Zn2+ at 5 to 10 mM. The enzyme was shown to be heat stable. Urease activity decreased by only 28% after 30 min at 65 degrees C. Characterization of the native urease of C. immitis contributes to our understanding of the role of this enzyme in the survival of the pathogen in vivo.
Publication Types:
Keywords:
- Ammonia
- Chromatography, Gel
- Coccidioides
- Humans
- Hydrogen-Ion Concentration
- Hydroxamic Acids
- Molecular Weight
- Sequence Homology, Amino Acid
- Urea
- Urease
- acetohydroxamic acid
- genetics
- isolation & purification
Other ID:
UI: 102195650
From Meeting Abstracts