title: Purification and characterization of nitric oxide synthase from Staphylococcus aureus.
abstract: We previously reported the presence of nitric oxide synthase (NOS) in Staphylococcus aureus ATCC6538P whose activity was induced by methanol. In the present study, the methanol-induced NOS was purified 900-fold from S. aureus by means of Mono Q ion exchange column, 2',5'-ADP-agarose affinity column, and Superdex 200HR gel permeation column chromatography. The purified bacterial NOS showed two protein bands with 67 and 64 kDa molecular mass on SDS-PAGE. However, the molecular mass of the NOS was 135 kDa on Superdex 200HR gel permeation column chromatography, indicating that the native enzyme exists as a heterodimer. This bacterial NOS had K(m) value of 13.4x10(-6) M for L-arginine and V(max) of 35.3 nmol min(-1) mg(-1) protein. In addition, reduced nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide, flavin mononucleotide, tetrahydrobiopterin, calmodulin and Ca(2+) were required as cofactors in the conversion of L-arginine to L-citrulline, and NOS inhibitors selectively inhibited the activity of the purified NOS.
authors.affiliation: Department of Pharmacy, College of Pharmacy, Sungkyunkwan University, 440-746, Suwon, South Korea. hylee@skku.ac.kr
authors.forename: Il-sun
authors.initials: IS
authors.lastname: Hong
authors.forename: Yong Kee
authors.initials: YK
authors.lastname: Kim
authors.forename: Wahn Soo
authors.initials: WS
authors.lastname: Choi
authors.forename: Dong Wan
authors.initials: DW
authors.lastname: Seo
authors.forename: Jong Woo
authors.initials: JW
authors.lastname: Yoon
authors.forename: Jeung-Whan
authors.initials: JW
authors.lastname: Han
authors.forename: Hoi Yong
authors.initials: HY
authors.lastname: Lee
authors.forename: Hyang Woo
authors.initials: HW
authors.lastname: Lee
mesh_terms: D004791:Enzyme Inhibitors / Q000494:pharmacology; D008970:Molecular Weight; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000201:enzymology*
chemical_list: D004791:Enzyme Inhibitors; D019001:Nitric Oxide Synthase; D019331:NG-Nitroarginine Methyl Ester