Urease from a potentially pathogenic coccoid isolate: purification, characterization, and comparison to other microbial ureases.
Strain SL100 is a gram-positive coccoid isolate prototype with an adhesin specific for gastric mucin and is representative of potentially pathogenic organisms obtained at biopsy from patients with gastric disorders. The urease of this isolate constitutes a significant fraction of the total cell protein, and the outcome of the purification strategy described herein suggests that it is associated with a cell wall fraction. The urease was purified 138-fold to apparent homogeneity, as indicated by gel electrophoresis, to a specific activity of 1,120 U/mg. The urease was unstable during purification in the absence of nickel, which is present in a metallocenter in other microbial ureases. When nickel sulfate was present during growth (5 microM) and in buffers during sonication and purification (100 microM), the urease was completely stable at room temperature during the purification procedure. The native urease was approximately 260 kDa and was composed of three subunits of 65 kDa and three subunits of 21 kDa. The purified urease was relatively stable in acid and retained most of its activity after incubation for 30 min at pH 1.3. The K(m)s for urease measured from whole cells and for the purified enzyme were 0.56 and 1.7 mM, respectively, indicating that some cell wall component(s) affects the affinity of the enzyme for urea. The V(max)s for urea hydrolysis measured from whole cells and for the purified enzyme were 8.1 and 1,120 mol/min/mg of protein, respectively. The kinetic parameters, relative abundance, and subunit composition are more similar to those of the ureases of Helicobacter than to those of the ureases of other microbial species. These similarities are consistent with an adaptation of this organism to colonization of the stomach and indicate that the urease may be a virulence factor during colonization.
Lee
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The Graduate School and University Center and Department of Chemistry, City College of New York, The City University of New York, New York 10031, USA.
Calhoun
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D004795:Enzyme Stability; D006095:Gram-Positive Cocci / Q000201:enzymology* / Q000472:pathogenicity; D016998:Helicobacter / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013045:Species Specificity; D013272:Stomach Diseases / Q000382:microbiology; D014510:Urease / Q000302:isolation & purification* / Q000378:metabolism
D014510:Urease