IN-SITU CHARACTERIZATION OF HELICOBACTER-PYLORI ARGINASE

The properties of Helicobacter pylori arginase activity in metabolical ly competent cells and lysates were investigated with the aim of obtai ning a better understanding of the nitrogen metabolism of the bacteriu m. One-dimensional H-1- and C-13-nuclear magnetic resonance spectrosco py, spectrophotometry, radio tracer analysis and protein purification techniques were employed to characterize in situ the first step in the utilization of L-arginine by the bacterium. Arginase activity was ass ociated with the cell-envelope fraction obtained by centrifugation of lysates. A K-m of 22 +/- 3 mM was determined for the enzyme activity, and differences of V-max were observed between strains. Divalent catio ns stimulated arginase activity, and the most potent activators were C o2+>Ni2+>Mn2+. The activity was highly specific for L-arginine and did not catabolize analogs recognized by other arginases of prokaryote an d eukaryote origin. The K-i of several inhibitors was measured and ser ved also to characterize the enzyme activity. The presence of bicarbon ate enhanced the hydrolysis of L-arginine in cell suspensions, but not in lysates or semi-purified enzyme preparations. Amino acid sequence analyses revealed important differences between the deduced structures of H. pylori arginase and those of other organisms. This finding was consistent with experimental data which showed that H. pylori arginase has unique properties. (C) 1998 Elsevier Science B.V. All rights rese rved.