PURIFICATION AND CHARACTERIZATION OF NITRIC-OXIDE SYNTHASE (NOSNOC) FROM A NOCARDIA SPECIES

被引:94
作者
CHEN, YJ
ROSAZZA, JPN
机构
[1] UNIV IOWA,COLL PHARM,DIV MED & NAT PROD CHEM,IOWA CITY,IA 52242
[2] UNIV IOWA,COLL PHARM,CTR BIOCATALYSIS & BIOPROC,IOWA CITY,IA 52242
关键词
D O I
10.1128/jb.177.17.5122-5128.1995
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
We previously reported on the occurrence, partial purification, and preliminary characterization of the first reported bacterial nitric oxide synthase. The soluble Nocardia enzyme, designated NOSNoc, has now been purified 1,353-fold by a combination of 2',5'-ADP-agarose affinity chromatography and hydroxylapatite chromatography. NOSNoc runs as a band of M(r) 51,900 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass was estimated to be 110.6 +/- 0.5 kDa by gel filtration, indicating that the native enzyme exists as a homodimer in solution. An N-terminal 15-amino-acid sequence was determined for NOSNoc, showing it to be different from known mammalian NOSs. N-G-Hydroxy-L-arginine was confirmed to be an intermediate in the enzymatic reaction by stoichiometric determinations of oxygen uptake, NADPH oxidation, NO formation as measured by nitrite determinations, citrulline formation, and kinetic studies. NOSNoc was competitively inhibited by N-G-methyl- and N-G-nitro-L-arginine with either L-arginine or N-G-hydroxyl-L-arginine as the substrate. Furthermore, the stability and pH and temperature optima of NOSNoc have been established.
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收藏
页码:5122 / 5128
页数:7
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