title: Constitutive nitric oxide synthase is present in normal human keratinocytes.
abstract: Normal human keratinocytes in culture exhibit a nitric oxide synthase (NOS) activity ranging from 50 to 150 pmol/min/mg of protein. The enzyme is cytosolic and requires the presence of calcium, nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), and flavin adenine dinucleotide. Calmodulin antagonists (trifluoperazine and calmidazolium) inhibit the enzyme activity. We show that NG-nitro-L-arginine inhibits NOS more potently than NG-monomethyl-L-arginine and that L-canavanine is a weak inhibitor. NOS was partially purified using a 2',5'-ADP Sepharose affinity column eluted with NADPH. A partially purified fraction was analyzed by sodium dodecyl sulfate-polyacrylamide gel electro-phoresis and Western blotting. A protein with an apparent molecular weight of 152 kDa cross-reacted with monoclonal antibodies raised against the neuronal constitutive isoform of NOS. The enzyme had a Vmax of 7.3 nmol/min/mg of protein and a Km for L-arginine of 22.3 microM. These results indicate that normal human keratinocytes contain a constitutive nitric oxide synthase related to NOS I.
authors.affiliation: L'Oréal, Centre de Recherche Charles Zviak, Sciences du Vivaut, Clichy, France.
authors.forename: J E
authors.initials: JE
authors.lastname: Baudouin
authors.forename: P
authors.initials: P
authors.lastname: Tachon
mesh_terms: D001120:Arginine / Q000031:analogs & derivatives / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002172:Canavanine / Q000494:pharmacology; D002478:Cells, Cultured; D003600:Cytosol / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D015603:Keratinocytes / Q000201:enzymology*; D007700:Kinetics; D019331:NG-Nitroarginine Methyl Ester; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019335:Nitroarginine; D019323:omega-N-Methylarginine
chemical_list: D002147:Calmodulin; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D019335:Nitroarginine; D019323:omega-N-Methylarginine; D002172:Canavanine; D001120:Arginine; D019001:Nitric Oxide Synthase; D002118:Calcium; D019331:NG-Nitroarginine Methyl Ester