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 (NOSc) was largely membrane-bound, whereas the inducible NOS in J774.2 cells (NOSi) was equally distributed among cytosol and membrane(s). Both the cytosolic NOSc in EC and the membrane-bound NOSi in J774.2 cells were strictly Ca(2+)-dependent, whereas the membrane-bound NOSc in EC and the cytosolic NOSi 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 NOSc in EC and the Ca(2+)-independent NOSi in J774.2 cells, but not the Ca(2+)-dependent NOSi. 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.
Hecker
M
M
William Harvey Research Institute, St. Bartholomew's Hospital Medical College, London, UK.
Walsh
D T
DT
Vane
J R
JR
D000594:Amino Acid Oxidoreductases / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002460:Cell Line; D006152:Cyclic GMP / Q000378:metabolism; D004727:Endothelium / Q000187:drug effects / Q000378:metabolism; D004730:Endothelium, Vascular / Q000201:enzymology; D007527:Isoenzymes / Q000378:metabolism; D007668:Kidney / Q000187:drug effects / Q000378:metabolism; D008070:Lipopolysaccharides; D008264:Macrophages / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D009000:Monocytes / Q000201:enzymology; D009569:Nitric Oxide / Q000494:pharmacology; D019001:Nitric Oxide Synthase; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine
D007527:Isoenzymes; D008070:Lipopolysaccharides; D010455:Peptides; D009569:Nitric Oxide; D001120:Arginine; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D006152:Cyclic GMP; D002118:Calcium