On the substrate specificity of nitric oxide synthase

Abstract

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-PK 1 pig kidney epithelial cells. The constitutive NOS in EC (NOS c) was largely membrane-bound, whereas the inducible NOS in J774.2 cells (NOS 1) was equally distributed among cytosol and membrane(s). Both the cytosolic NOS c in EC and the membrane-bound NOS 1 in J774.2 cells were strictly Ca 2+-dependent, whereas the membrane-bound NOS c in EC and the cytosolic NOS 1 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 Ca 2+-independent NOS 1 in J774.2 cells, but not the Ca 2+-dependent NOS 1. Thus, irrespective of their intracellular localisation, at least three isoforms of NOS exist, which can be differentiated by their substrate specificity and Ca 2+-dependency.