Identification and characterization of a metalloprotease activity from Helicobacter pylori.
Helicobacter pylori produces a metalloprotease with a native molecular size of approximately 200 kDa, as determined by size-exclusion chromatography. Subcellular distribution studies demonstrated that the activity was associated with the outer membrane fraction of the bacterium. In addition, the protease was secreted by the bacterium when grown in liquid culture. The enzyme activity was measured by hydrolysis of azocasein and biotinylated casein and exhibited optimal caseinolytic activity at pH 8.0 (37 degrees C). The activity was inhibited by EDTA, 1,10-phenanthroline, phosphoramidon, pyridine-2,6-dicarboxylic acid, and 8-hydroxyquinoline-5-sulfonic acid (HQSA). Inhibition by HQSA was reversed by zinc, whereas inhibition due to EDTA was reversed by excess calcium, thus indicating that the enzyme was a zinc-dependent, calcium-stabilized endoproteinase. Furthermore, titration with Zn2+ of a desalted, active-site zinc-chelated preparation of the protease demonstrated that Zn2+ was essential for activity. Leupeptin, phenylmethylsulfonyl fluoride, E-64, pepstatin A, dithiothreitol, and 2-mercaptoethanol had no effect on enzymatic activity. Addition of Ca2+ or Mg2+ to the incubation medium resulted in approximately a twofold stimulation of the azocaseinolytic activity of the enzyme. The protease was stably expressed since it was active even after repeated subculture of the bacterium. Bovine serum albumin, hide powder azure, and elastin-Congo red remained intact even after prolonged exposure to the enzyme. The surface expression of this metalloprotease activity raises the possibility that this enzyme may be involved in the proteolysis of a variety of host proteins in vivo and thereby contributes to gastric pathology.
Windle
H J
HJ
Department of Clinical Medicine, Trinity College, University of Dublin, Ireland. hjwindle@tcd.ie
Kelleher
D
D
D004492:Edetic Acid / Q000494:pharmacology; D016480:Helicobacter pylori / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases / Q000032:analysis* / Q000378:metabolism; D008970:Molecular Weight; D015032:Zinc / Q000494:pharmacology
D004492:Edetic Acid; D008666:Metalloendopeptidases; D015032:Zinc