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‐PK₁ pig kidney epithelial cells. The constitutive NOS in EC (NOS_c) was largely membrane‐bound, whereas the inducible NOS in J774.2 cells (NOS₁) was equally distributed among cytosol and membrane(s). Both the cytosolic NOS_c in EC and the membrane‐bound NOS₁ in J774.2 cells were strictly Ca²⁺‐dependent, whereas the membrane‐bound NOS_c in EC and the cytosolic NOS₁ 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 NOS₁ in J774.2 cells, but not the Ca²⁺‐dependent NOS₁. Thus, irrespective of their intracellular localisation, at least three isoforms of NOS exist, which can be differentiated by their substrate specificity and Ca²⁺‐dependency.