Purification and characterization of nitric oxide synthase from Staphylococcus aureus.
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.
Hong
Il-sun
IS
Department of Pharmacy, College of Pharmacy, Sungkyunkwan University, 440-746, Suwon, South Korea. hylee@skku.ac.kr
Kim
Yong Kee
YK
Choi
Wahn Soo
WS
Seo
Dong Wan
DW
Yoon
Jong Woo
JW
Han
Jeung-Whan
JW
Lee
Hoi Yong
HY
Lee
Hyang Woo
HW
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*
D004791:Enzyme Inhibitors; D019001:Nitric Oxide Synthase; D019331:NG-Nitroarginine Methyl Ester