AFFINITY PURIFICATION OF HELICOBACTER-PYLORI UREASE - RELEVANCE TO GASTRIC MUCIN ADHERENCE BY UREASE PROTEIN

A simple, reproducible and high yield method of Helicobacter pylori ur ease enzyme purification was developed using a heparinoid (Cellufine s ulfate) affinity gel. The purification method involved two sequential steps using the same gel that takes advantage of the differential affi nity of urease to the heparinoid at two levels of hydrogen ion concent ration, SDS-polyacrylamide gel electrophoresis analysis of affinity-pu rified urease revealed two major protein bands with about 62- and 30-k Da molecular mass. When whole cell lysates of clinical and laboratory strains of H. pylori were probed by Western blot, anti-urease hyperimm une serum produced by affinity-purified urease in rabbit recognized on ly the two bands corresponding to the urease A and B subunits. To prob e 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 co mmonality of urease receptors among gastric mucin, heparin, and hepari noid, Composite data on adherence kinetics modulated by pH, salt, incu bation time, and concentration of urease or mucin were indicative of c onformation-dependent ligand-receptor interaction.