Co-purification of 130 kD nitric oxide synthase and a 22 kD link protein from human neutrophils.
The synthesis of nitric oxide (NO) from L-arginine has been demonstrated in several cell types. Both constitutive and inducible forms of NO synthase have been described in different cells. We purified the constitutive form of NO synthase enzyme in human neutrophils using a two-column procedure. Crude 100,000g supernatant of human neutrophils was passed through a 2'-5'-ADP-agarose column followed by a DEAE-Bio-Gel A anion exchange column. NO synthase enzyme migrated as a single band (MW approximately 130,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Its activity was dependent upon nicotinamide adenine dinucleotide phosphate (NADPH) and (6R)-tetrahydro-L-biopterin (BH4). In addition, flavin adenine dinucleotide (FAD) was also found to be essential for its maximal activity. A second NADPH, FAD-dependent component (MW approximately 22kD) was also found consistently on the SDS-PAGE gel. These observations suggest co-regulation between NO synthase enzyme and this NADPH, FAD-dependent component, which may be associated with the superoxide radical generating system.
D000594:Amino Acid Oxidoreductases / Q000097:blood* / Q000302:isolation & purification*; D001120:Arginine / Q000031:analogs & derivatives / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D016326:Extracellular Matrix Proteins; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009504:Neutrophils / Q000201:enzymology*; D019001:Nitric Oxide Synthase; D010084:Oxidation-Reduction; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D011509:Proteoglycans; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019323:omega-N-Methylarginine; D000099249:Proteoglycan Link Protein
D000594:Amino Acid Oxidoreductases; D001120:Arginine; D002118:Calcium; D016326:Extracellular Matrix Proteins; D009243:NAD; D019001:Nitric Oxide Synthase; D011506:Proteins; D011509:Proteoglycans; D013755:Tetradecanoylphorbol Acetate; D019323:omega-N-Methylarginine; D000099249:Proteoglycan Link Protein