Molecular and kinetic characterization and cell type location of inducible nitric oxide synthase in fish

We have found conclusive evidence for inducible nitric oxide synthase (iNOS) activity in rainbow trout (Oncorhynchus mykiss) tissue by means of biochemical, immunohistochemical, and immunoblotting analyses. This Ca2+-independent enzyme uses l-arginine to produce nitric oxide and l-citrulline. It was significantly inhibited by the l-arginine analogsN ω-monomethyl-l-arginine andN G-nitro-l-arginine methyl ester. Kinetic analyses showed typical Michaelian behavior with no evidence of cooperative effects. The specific activities of the liver and head kidney enzymes were 27 and 106 pmoles · min−1· mg protein−1, respectively, with similar values forK m (11 μM), all of which correspond well with the values for other previously characterized iNOS. Western blot analyses revealed a single band of MR = 130 kDa tested with an iNOS antiserum. At the ultrastructural level, cells with NADPH-diaphorase activity and iNOS immunoreactivity were identified as being heterophilic granulocytes in head kidney tissue and neutroph...