Expression and immunoaffinity purification of human inducible nitric-oxide synthase. Inhibition studies with 2-amino-5,6-dihydro-4H-1,3-thiazine.
Recombinant human inducible nitric-oxide synthase (rH-iNOS) was expressed in the baculovirus system and purified by a novel immunoaffinity column. rH-iNOS and its native counterpart from cytokine-stimulated primary hepatocytes exhibited similar molecular mass of 130 kDa on SDS-polyacrylamide gel electrophoresis, recognition by antipeptide antibodies, specific activities, and IC50 values for inhibitors. The active dimeric form exhibited a specific activity range of 114-260 nmol/min/mg at 37 degrees C and contained 1.15 +/- 0.04 mol of calmodulin/monomer. The enzyme exhibited a Soret lambdamax at 396 nm with a shoulder at 460 nm and contained 0. 28-0.64 mol of heme/monomer. Dithionite reduction under CO yielded an absorbance maximum at 446 nm, indicating a P450-type heme. Imidazole induced a type II difference spectrum, reversible by L-Arg. 2-Amino-5,6-dihydro-4H-1,3-thiazine (ADT) was competitive versus L-Arg (Ki = 22.6 +/- 1.9 nM), reversed the type II difference spectrum induced by imidazole (Kd = 17.7 nM), and altered the CO-ferrous absorbance of rH-iNOS. L-Arg did not perturb the CO-ferrous adduct directly, but it partially reversed the ADT-induced absorbance shift, indicating that both bind similarly to the protein but interact differently with the heme.
Department of Molecular Design, Merck Research Laboratories, P.O. Box 2000, Rahway, New Jersey 07065, USA. jimmy_calaycay@merck.com
J R
JR
Calaycay
T M
TM
Kelly
K L
KL
MacNaul
E D
ED
McCauley
H
H
Qi
S K
SK
Grant
P R
PR
Griffin
T
T
Klatt
S M
SM
Raju
A K
AK
Nussler
S
S
Shah
J R
JR
Weidner
H R
HR
Williams
G C
GC
Wolfe
D A
DA
Geller
T R
TR
Billiar
M
M
MacCoss
R A
RA
Mumford
M J
MJ
Tocci
J A
JA
Schmidt
K K
KK
Wong
N I
NI
Hutchinson
D002851:Chromatography, High Pressure Liquid; D004790:Enzyme Induction; D006801:Humans; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D011837:Radiation-Protective Agents / Q000494:pharmacology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013843:Thiazines / Q000494:pharmacology*
D011837:Radiation-Protective Agents; D013843:Thiazines; C018450:2-amino-5,6-dihydro-4H-1,3-thiazine; D009249:NADP; D019001:Nitric Oxide Synthase