NOS in action

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NOS in action
Laura Castro
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NOS catalyzed reaction
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Nitric Oxide Synthases isoenzymes
Classical mammalian NOS 51-57
homology

• Neuronal NOS nNOS, Type I, NOS 1
• Inducible NOS iNOS, Type II, NOS 2
• Endothelial NOS eNOS, Type III, NOS 3

Other mammalian NOS

• Mitochondrial NOS mtNOS

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Role of nNOS in controlling synaptic plasticity
from Beckman J, 1996
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iNOS induction
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(No Transcript)
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NOS domains
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Catalytic site of human iNOS
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Domain structure of NOS
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mtNOS

• 1997-1998 NO production from mitochondrial
  preparations
• (Ghafourifar, Richter and Guilivis groups)
• purified mtNOS MW 130 kDa
• sequence has 100 homology with nNOS
• RT-PCR transcripts nNOS?
• myristoylation and phosphorylation sites
• kinetic parameters similar to nNOS
• bound to IMM
• modulates oxygen consumption and free radical
  generation by
• mitochondria

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Electron flow in NOS
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Properties of mammalian NOS
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NOS monooxygenation I reaction
Fe
3
ArgOH
Arginine
Arginine
S
O
2.
-
O
2.
OH
.
3
Fe
Fe
3
O
.-
Fe
4
P

2
.
Fe
4
P

4
Fe
P
S
S
S
S
S
FMNH
H
O
2
2
.
FMNH

H
-
OOH
R
R
R

Fe
3
Fe
2
NH
NH
NH
S
S



.
NH
NH
NH
NH
NH
2
NH
2
2
O
2
2
O
O
2
.-
2
OH
BH
BH
.

2
4
Fe
3
Fe
3
S
S
.
FMNH
FMN
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NOS monooxygenation II reaction
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NOS uncoupling




Arg O

2

-
-

e
e
3
NADPH FAD,FMN
Heme-Fe
.
NO
.-
O
O
O

2
2
2
-
ONOO
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NOS uncoupling

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.NO
high .NO (iNOS, overstimulation of nNOS)
low .NO (eNOS)
Direct Effects
Inderect Effects
Metal Reactions Radical Reactions Associated
with regulatory effects
Nitrosative Stress Oxidative Stress Associated
with pathophysiological effects and immune
responses to pathogens
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PDE II stimulated
Ion Channels (CNG)
Protein Kinases (cGK)
Phosphodieterases (PDE)
PDE III inhibited
Protein Subtrate
Phosphorylated Protein
ATP ADP
Protein Phosphatase
Biological Effects
Dephosphorylated Protein
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(No Transcript)
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NOS INHIBITORS
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Nitric oxide synthase inhibitors
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Nitric Oxide Synthase Inhibitors
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Nitric Oxide Synthase Inhibitors
 
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Lessons from knocks outs
nNOS (-/-) mice -inappropriate sexual behavior
-increased aggression -abnormalities in
balance and motor coordination at night -reduced
stroke area and neuronal apoptosis following
middle cerebral artery occlusion iNOS (-/-)
mice -macrophages exhibit reduced degree of
target cell cytostasis activity -animals are
more resistant to endotoxemic shock eNOS (-/-)
mice -mimics metabolic syndrome X of humans
(hypertension and insulin resistance) -increased
brain and miocardic damage after ischemia in
different models -increased blood presurre and
blood pressure variability -impaired oocytic
meiotic ovulation
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Some references to start with -Miranda K. et
al. 2000, Ch4 pp 41-56. In Nitric Oxide Biology
and Pathobiology. Ignarro L., ed. Academic Press,
San Diego CA. -Sue Masters B. 2000, Ch6 pp
105-128. In Nitric Oxide Biology and
Pathobiology. Ignarro L., ed. Academic Press, San
Diego CA. -Kleinert H. et al 2000, Ch7 pp
129-156. In Nitric Oxide Biology and
Pathobiology. Ignarro L., ed. Academic Press, San
Diego CA. -Alderton W. et al 2001, Biochem J.
357 593-615. -Stuehr D. 1999, Biochim Biophys
Acta 1411217-230. -Muscara M. and Wallace J.
1999 Am J Physiol 276G1313-1316 -Giulivi C.
2003 Free Rad Biol Med 34397-408. -Vazquez-Vivar
J. et al 1998 Proc Natl Acad. Sci 95
9220-9225.