ON THE SUBSTRATE-SPECIFICITY OF NITRIC-OXIDE SYNTHASE

Nitric oxide (.NO) synthase (NOS) activity in subcellular fractions from cultured endothelial cells (EC) and lipopolysaccharide-activated J774.2 monocyte/macrophages was investigated by monitoring the .NO-mediated increase in intracellular cyclic GMP in LLC-PK1 pig kidney epithelial cells. The constitutive NOS in EC (NOS(c)) was largely membrane-bound, whereas the inducible NOS in J774.2 cells (NOS(i)) was equally distributed among cytosol and membrane(s). Both the cytosolic NOS(c) in EC and the membrane-bound NOS(i) in J774.2 cells were strictly Ca2+-dependent, whereas the membrane-bound NOS(c) in EC and the cytosolic NOS(i) in J774.2 cells were not. L-Homoarginine and L-arginine-containing small peptides, such as L-arginyl-L-phenylalanine, replaced L-arginine as a substrate for the NOS(c) in EC and the Ca2+-independent NOS(i) in J774.2 cells, but not the Ca2+-dependent NOS(i). Thus, irrespective of their intracellular localisation, at least three isoforms of NOS exist, which can be differentiated by their substrate specificity and Ca2+-dependency.