The optical interconversion of the P-450 and P-420 forms of neuronal nitric oxide synthase: effects of sodium cholate, mercury chloride and urea.
We investigated whether or not neuronal nitric oxide synthase (nNOS) (EC 1.14.13.39) was converted to the P-420 form on exposure to sodium cholate, mercury chloride or urea, and the reconversion of the P-420 to the P-450 form. Sodium cholate and mercury chloride induced the conversion of nNOS from the P-450 to the P-420 form in concentration- and incubation time-dependent manners, and the nNOS activity decreased. In the presence of glycerol, L-arginine and/or tetrahydrobiopterin, the sodium cholate-treated P-420 form could be reconverted to the P-450 form under constant experimental conditions, and the nNOS activity could also be restored. The mercury chloride-treated P-420 form of nNOS could be reconverted to the P-450 form on incubation with reduced glutathione (GSH) or L-cysteine, and the nNOS activity was recovered. However, no reconversion of the mercury chloride-treated P-420 form to the P-450 form was observed in the presence of glycerol, L-arginine, or tetrahydrobiopterin. Urea (4.0 M) dissociated nNOS into its subunits, but nNOS remained in the P-450 form. The nNOS monomer was more susceptible to sodium cholate. After removing the urea by dialysis, and supplementation of the nNOS solution with glycerol, L-arginine or BH(4), the P-420 was reconverted to the P-450 form, and the reassociation of nNOS monomers was also observed. These results suggested that nNOS was more stable as to exposure to sodium cholate, mercury chloride or urea in comparison to microsomal cytochrome P-450, which may be due to the different heme environment and protein structure.
Department of Biochemistry, Kagawa Medical University, Miki-cho, Kita-gun, 761-0793, Kagawa, Japan. hjiang@kms.ac.jp
H
H
Jiang
M
M
Ichikawa
A
A
Furukawa
S
S
Tomita
T
T
Ohnishi
Y
Y
Ichikawa
D000818:Animals; D001120:Arginine / Q000494:pharmacology; D001708:Biopterins / Q000031:analogs & derivatives* / Q000494:pharmacology; D003545:Cysteine / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D003580:Cytochromes / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D005990:Glycerol / Q000494:pharmacology; D007527:Isoenzymes / Q000737:chemistry*; D008627:Mercuric Chloride / Q000494:pharmacology*; D051379:Mice; D019001:Nitric Oxide Synthase / Q000737:chemistry*; D010957:Plasmids; D020358:Sodium Cholate / Q000494:pharmacology*; D013053:Spectrophotometry; D014162:Transfection; D014508:Urea / Q000494:pharmacology*
D003580:Cytochromes; D007527:Isoenzymes; D001708:Biopterins; D008627:Mercuric Chloride; D014508:Urea; C013464:cytochrome P420; D003577:Cytochrome P-450 Enzyme System; D001120:Arginine; D019001:Nitric Oxide Synthase; C003402:sapropterin; D003545:Cysteine; D020358:Sodium Cholate; D005990:Glycerol