Affinity purification of Helicobacter pylori urease. Relevance to gastric mucin adherence by urease protein.
A simple, reproducible and high yield method of Helicobacter pylori urease enzyme purification was developed using a heparinoid (Cellufine sulfate) affinity gel. The purification method involved two sequential steps using the same gel that takes advantage of the differential affinity of urease to the heparinoid at two levels of hydrogen ion concentration. SDS-polyacrylamide gel electrophoresis analysis of affinity-purified urease revealed two major protein bands with about 62- and 30-kDa molecular mass. When whole cell lysates of clinical and laboratory strains of H. pylori were probed by Western blot, anti-urease hyperimmune serum produced by affinity-purified urease in rabbit recognized only the two bands corresponding to the urease A and B subunits. To probe the molecular relevance of affinity gel adherence to mucin adherence, the purified urease was derivatized with N-hydroxysuccinimidobiotin and used in adherence assays. Competitive inhibition tests revealed commonality of urease receptors among gastric mucin, heparin, and heparinoid. Composite data on adherence kinetics modulated by pH, salt, incubation time, and concentration of urease or mucin were indicative of conformation-dependent ligand-receptor interaction.
Icatlo
F C
FC
Immunology Research Institute, Ghen Corporation, 839-1 Sano, Gifu City 501-11, Japan.
Kuroki
M
M
Kobayashi
C
C
Yokoyama
H
H
Ikemori
Y
Y
Hashi
T
T
Kodama
Y
Y
D000818:Animals; D001667:Binding, Competitive; D019921:Biotinylation; D015153:Blotting, Western; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D005752:Gastric Mucins / Q000378:metabolism*; D016480:Helicobacter pylori / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D011485:Protein Binding; D011817:Rabbits; D013552:Swine; D014510:Urease / Q000302:isolation & purification* / Q000378:metabolism
D005752:Gastric Mucins; D014510:Urease