n.h cyt c q iii ivmeetochondrie.ibgc.cnrs.fr/ateliers/atelier3/coursros_fb.pdf · 3 1 n=5 ros!...
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e -
Succinate
I III IV
n.H+
Respiratory chain complexes I, III & IV
1/2O2 H2O
Cyt C
2e-
2e-
Fumarate
II SDH
Q
n.H+ n.H+
e -
n.H+
Respiratory chain redox proton pump
2e- 2e-
e -
n.H+
Respiratory chain redox proton pump
2e- 2e-
1e- +O2 = Superoxide
e -
n.H+
Respiratory chain redox proton pump
2e- 2e-
1e- +O2 = Superoxide
2e- 2e- 2e- 2e-
e - I
III IV
n.H+
1/2O2 H2O
Cyt C
2e-
2e-
II SDH
Q
n.H+ n.H+
Rotenone
Antimycine
Cyanure
Cyt C
e -
n.H+
2e- 2e-
1e- +O2 = Superoxide
2e- 2e- 2e- 2e-
milliVolts
170 +
- - - -
+ + +
Membrane potential
e -
Respiratory chain
2e- 2e-
Superoxide
H2O2 … Antioxidant defenses
ROS related damages
ROS probe Evaluation of…
F109 F113 F115 F116
0
5
10
15
20
25
30
0:00 0:30 1:00 1:30 2:00 2:30 3:00Time
Supe
roxi
de (D
HE
red
Fluo
)
S. Raimbault, F. Bouillaud (2001)
COS cells Superoxide production, flow cytometry, DHE probe
+ Oligomycin State 4 (hyperpolarized)
Basal (DMEM medium)
+ CCCP (uncoupling)
ROS ROS
0.0 0.5 1.0 1.5 2.0
Resp. Chain
ATP
ATPase
ATP
ATPase
Resp. Chain
Resp. Chain
ATPase
Resp. Chain
ATPase
ΔΨ
Reduced
Oxidized
O2-.
Reduced
Oxidized
O2-.
Reduced
O2-.
Reduced
O2-.
Ox
Inhibition Resp. Chain
Uncoupling
Inhibition ATPsynthase
Basal Oxidation Dihydroethidin
0.0 0.5 1.0 1.5 2.0 2.5 3.0
fccp 1µM +Oligomycin
Antimycin +Oligomycin
Cyanure
Antimycin
rotenone
fccp 10µM
fccp 1µM
fccp 0.5
fccp 0.2
Oligomycin
basal
Mouse macrophages, DHE 1µM : relative slope of linear increase in fluorescence
Hyperpolarized
Uncoupled Depolarized
Inhibition Resp. Chain
Depolarized
151 142
80 98
173
100
0
50
100
150
200
5 3 3 2 4 2
Superoxide (DHE staining) and mitochondrial state CHO cells
Mozo et al (2005) Biochem. J.
DHE précautions
Effet des inhibiteurs Contrôle Ethidium
DHE
Eth DHE
Eth
Eth
Superoxide
Superoxide
cis-aconitate
Fumarate
Malate
Isocitrate
C=C
C6
C5
C4 Krebs Cycle
Aconitase
Fumarase
C=C
ROS
C=C UV: 240nm
Evaluation of mitochondrial ROS damage Use of the Aconitase/Fumarase ratio
Exemple 1: mitochondries isolées soumises à un stress oxydant externe
Exemple 2: mitochondries de tissu congelé ���(cœur après expérience Ischémie / Reperfusion)���
Exemple 3: mitochondries de levure
Adaptation au format microplaque. P r o t ocole combiné 1ère phase lecture aconitase, deuxième phase lecture fumarase. T e c a n Infinite M200 deux injecteurs. M icroplaque UV Greiner 96 puits (demi section). Séquence d’additions (pour être compatible avec le format multicanaux) disponible. Solubilisant (TritonX100 0,5%) 2 0 µ l (multicanaux) Mitochondries (10-50µg) 5µ l (P20) Tampon (TrisHCl pH 7.4 : MnCl2 1.2mM) 50µ l (multicanaux) Introduction dans le lecteur. Progamme AcoFum Lecture DO 242nm BP 5nm* Injection 2 5 µ l de citrate 120mM V f = 100µl soit 30mM final Agitation Lectures cinétiques DO 242nm BP 5nm 1 toute les minutes 10x Injection 2 5µ l de L-Malate 250mM Vf = 125µl soit 50mM final. Agitation Lectures cinétiques DO 242nm BP 5nm 1 toute les minutes 10x
En cours de validation !
0
0,5
1
1,5
2
2,5
3
3,5
4
0 200 400 600 800 1000 1200 1400
D6
E6
F6
H6
+ Isocitrate + Malate
y = 0,0014x + 1,3693
y = 0,0026x + 0,5891
y = 0,0004x + 0,5773
y = 0,0026x - 0,5875
0
0,5
1
1,5
2
2,5
3
3,5
4
0 200 400 600 800 1000 1200 1400
14 26 - 14
4 26 - 4
1,16
0,18
=
=
Rapport Aconitase/Fumarase #
ATP
JO2
ADP+Pi + ΔΨ -
H+
ATPase
Respiratory Chain
Exercise
ATP
JO2
ADP+Pi + ΔΨ -
H+
ATPase
Respiratory Chain
Rest
Subs. Subs.
2e-
∆µH+
2e-
Mild uncoupling Electron entry ctrl
Substrates
NADH ���FADH2
0.0 0.5 1.0 1.5 2.0
Resp. Chain
ATP
ATPase
ATP
ATPase
Resp. Chain
Resp. Chain
ATPase
Reduced
Oxidized
O2-.
Reduced
Oxidized
O2-.
Reduced
O2-.
Inhibition Resp. Chain
Uncoupling
State 3
State 4 « UCP » Resp. Chain
ATPase
Reduced
Oxidized
O2-.
Living cells Oxidation Dihydroethidin #superoxide production
ROS
F109 F113 F115 F116
0
5
10
15
20
25
30
0:00 0:30 1:00 1:30 2:00 2:30 3:00 Time
Supe
roxi
de (D
HE
red
Fluo
)
S. Raimbault, F. Bouillaud (2001)
Control (non transfected COS cells)
+ Oligomycin !!!
ROS
0.0 0.1 0.2 0.3 0.4 0.5 0.6
Ratio Aconitase / Fumarase
pYedp avUCP avUCP-GDP
Isolated mitochondria
State 4 KCN xxo-KCN
*
** Aconitase
Superoxide
Avian UCP expressed in yeast Criscuolo et al (2005)
ROS
Relative superoxide production rate Basal state
0 50 100 150 UCP1 clone B5-4
UCP3 clone20 UCP3 clone19 UCP3 clone13 UCP3 clone30 UCP3 clone10
clone34 clone21 clone17 clone 9 clone2
D A
AANAT 15 3 6 8 8 8 3 6 7
15 6 6 7 4
A
0
50
100
150 P = 0.076
N=8
Basal
N=5 B
Ctrl
UC
P3
UC
P1
ROS
Relative superoxide production rate Basal state
0 50 100 150 UCP1 clone B5-4
UCP3 clone20 UCP3 clone19 UCP3 clone13 UCP3 clone30 UCP3 clone10
clone34 clone21 clone17 clone 9 clone2
D A
AANAT 15 3 6 8 8 8 3 6 7
15 6 6 7 4
A
0
50
100
150 P = 0.076
N=8
Basal
N=5 B
Ctrl
UC
P3
UC
P1
0,5!
0
50
100
150
0,5!
ns + Oligomycin ?!
N=15 C
Ctrl
UC
P3
UC
P1 N=5
ROS