CONSTITUTIVE NITRIC-OXIDE SYNTHASE IS PRESENT IN NORMAL HUMAN KERATINOCYTES

Normal human keratinocytes in culture exhibit a nitric oxide synthase (NOS) activity ranging from 50 to 150 pmol/min/mg of protein. The enzy me is cytosolic and requires the presence of calcium, nicotinamide ade nine dinucleotide phosphate, reduced form (NADPH), and flavin adenine dinucleotide. Calmodulin antagonists (trifluoperazine and calmidazoliu m) inhibit the enzyme activity. We show that N-G-nitro-L-arginine inhi bits NOS more potently than N-G-monomethyl-L-arginine and that L-canav anine is a weak inhibitor, NOS was partially purified using a 2',5'-AD P Sepharose affinity column eluted with NADPH. A partially purified fr action was analyzed by sodium dodecyl sulfate-polyacrylamide gel elect rophoresis and Western blotting. A protein with an apparent molecular weight of 152 kDa cross-reacted with monoclonal antibodies raised agai nst the neuronal constitutive isoform of NOS. The enzyme had a V-max o f 7.3 nmol/min/mg of protein and a K-m for L-arginine of 22.3 mu M. Th ese results indicate that normal human keratinocytes contain a constit utive nitric oxide synthase related to NOS I.