Biochemical characterization of the arginine degrading enzymes arginase and arginine deiminase and their effect on nitric oxide production.
BACKGROUND
Nitric oxide (NO) is a biomediator believed to be synthesized primarily from extracellular arginine. Various methodologies have been used to inhibit NO synthesis so as to elucidate its physiological and pathophysiological functions. Several investigators have utilized various argin ine degrading enzymes as a means of lowering extracellular arginine. Arginase, most commonly derived from mammalian sources, has been most often used. However, arginase has failed to inhibit NO synthesis. Therefore, a systematic biochemical characterization of arginase and arginine deiminase (ADI) derived from M. Hominus was undertaken.


METHODS
The murine macrophage cell line N-9 was treated with either arginase or arginine deiminase to determine the effect on intracellular and extracellular arginine and nitric oxide production.


RESULTS
Arginase was found to have an alkaline pH optima(approximately 9.5) with little enzyme activity at physiological pH. In contrast, the pH optima of ADI was approximately 6.5, retaining >70% of its activity at physiological pH. ADI had more than 1000 fold higher affinity for arginine (Km approximately 30 KM for ADI vs approximately 45 mM for arginase), and was able to lower arginine levels to a much greater extent than arginase. ADI, unlike arginase, was effective in lowering extracellular arginine in tissue culture media and inhibit NO production by the murine macrophage cell line N-9 in response to gamma interferon and LPS stimulation.


CONCLUSIONS
These data suggest that ADI may be useful for delineating the role of NO in a variety of biological systems as well as determining the role of extracellular arginine in its synthesis.
Department of Biology, University of Kentucky, Lexington 40506, USA.
Brian J
BJ
Dillon
Frederick W
FW
Holtsberg
C Mark
CM
Ensor
John S
JS
Bomalaski
Mike A
MA
Clark
D000818:Animals; D001119:Arginase / Q000378:metabolism*; D001120:Arginine / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000235:genetics / Q000378:metabolism*; D007371:Interferon-gamma / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008099:Liver / Q000201:enzymology; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013552:Swine
D008070:Lipopolysaccharides; D011994:Recombinant Proteins; D009569:Nitric Oxide; D007371:Interferon-gamma; D001120:Arginine; D019001:Nitric Oxide Synthase; D006867:Hydrolases; D001119:Arginase; C019723:arginine deiminase