The relative role of internal urease for acid protection of Helicobacter pylori is unknown. The aim of this study was to determine the comparative importance of internal and external urease under acidic conditions.

The pH optimum and measured Michaelis constant for urea of external urease and urease in intact bacteria at different medium pH (pH_out) were measured using ¹⁴CO₂ release from ¹⁴C-urea. The effect of urea on membrane potential and bacterial cytoplasmic pH was measured at different fixed pH_out. ³⁵S-methionine labeling and sodium dodecyl sulfate–polyacrylamide gel electrophoresis of labeled proteins in the organism and medium measured protein synthesis at different pH_out and mechanisms of urease externalization.

External urease had activity between pH 5.0 and 8.5 and internal urease between pH_out 2.5 and 6.5, and its Michaelis constant at pH_out 7.5 was 300 mmol/L but at pH_out 4.5 was 0.5 mmol/L, similar to free urease. The addition of 5 mmol/L urea to bacteria at fixed pH_out from 3.0 to 6.0 elevated potential to about −105 mV and periplasmic pH to about pH 6.2. Protein synthesis occurred mainly between pH 6.5 and 8.0, and urease activity resulted in increased protein synthesis at acidic pH. The labeling pattern of intrabacterial and released protein was similar.

Intracellular urease activity is regulated by external pH, defends against gastric acidity by increasing periplasmic pH and membrane potential, and stimulates protein synthesis at acidic pH. External urease is produced mostly by cell lysis. GASTROENTEROLOGY 1998;114:58-70