Purification and characterization of the urease enzymes of Helicobacter species from humans and animals

Abstract

The urease enzymes of Helicobacter pylori, H. mustelae, H. felis, and H. nemestrinae have been purified to homogeneity by affinity chromatography and characterized. The native urease enzymes of the four organisms were found to be almost identical, with a pl of 6.1 and molecular masses of 480 to 500 kDa, as determined by electrophoretic mobility in nondenaturing polyacrylamide gels. Transmission electron microscopy of the native urease showed it to be a molecule approximately 13 nm in diameter, with hexagonal symmetry. Denaturation studies indicated that each urease enzyme molecule was composed of two nonidentical subunits with molecular masses of approximately 64 and 30 kDa. The subunits were present in a 1:1 ratio, suggesting a hexameric stoichiometry for the native molecule. The predicted molecular mass of H. pylori urease, based on subunit molecular weight and stoichiometry, is 568 kDa. N-terminal amino acid sequencing of the enzyme subunits from the four species revealed high levels of homology. The large subunits (UreB) were found to be 92 to 100%o homologous, and the small subunits (UreA) were 75 to 95% homologous over the first 12 to 20 residues. The high degree of homology suggests a common ancestral origin and an important role for the urease enzymes of these organisms. 5259 on August 5, 2020 by guest http://iai.asm.org/ Downloaded from

Immunoblot assay for serodiagnosis of Helicobacter pylori infections

“…1. Thirteen prominent protein bands (15,22,25,27,29,30,33,43,52,57,60,66, and 87 kDa) and six weaker protein bands (in the range of 17 to 110/120 kDa) were stained (Fig. 1, lane 3).…”

Identification of Helicobacter pylori by immunological dot blot method based on reaction of a species-specific monoclonal antibody with a surface-exposed protein

“…1). It has been reported that the small subunit of the urease produced by H. pylori has a molecular mass of 30 kDa (18). A ureasenegative mutant that does not produce any of the subunits of this enzyme (16) was therefore included in the experiment.…”

Cloning, expression and sequencing of Helicobacter felis urease genes

“…Western blot analysis of extracts of E, coii cells harbouring plLL205 indicated the presence of two polypeptides of approximately 30 and 66 kDa which cross-reacted with poiycionai H. felis rabbit antiserum ( Fig,2A), These proteins were not produced by bacteria carrying the vector (plLL570), Native H. felis urease has been reported to be composed of repeating monomeric subunits with calculated molecular masses of 30 and 70 kDa (Turbett et al, 1992), Thus the 30 and 66 kDa proteins were thought to correspond to the ureA and ureB gene products, respectively. Interestingly an extract of E, coli cells harbouring the recombinant plasmid plLL763 (Cussac etal., 1992), containing the H. pylori ureA and ureB genes, expressed two polypeptides with approximate molecular sizes of 30 and 62 kDa which cross-reacted with the anti-H. feiis antisera ( Fig.…”