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-

,
, ,,
, ,,
,
NADPH-
NADPH-,
, .
.

,
, (,
( ,,
,


A,
A,C,
C,E,
E,,
,N-
N-


,),
, ),
x
x




-
-

AO
AO










( Cu/Zn Mn):
2- + 2- + 2+ 22 + 2
( ):
222 22 + 2
( ):
2GSH + 22 GSSG + 22O
( FAD):
GSSG + 2 2GSH + 2

(. )
(. )
(. )

N-
,



-

/
0,2
1,6 ()
/

1,95

Knock-out
Glu-peroxidase -/-
202 202

+ BSO (
-) + 3-

+/- 3- ( +/- S
)


GSH
-L-glutamyl-L-cysteinylglycine
1 - 10
~ 1 - 5 ,

(
)
,

GSH
(
GSH )
GluCys

(GSSG),
(NADPH), GSH/GSSG
1000/1

( 40-50% ,
, )
GSH
-
(Saer et al.,1990; Winterbourn, 1994);
,
(Chance et al., 1979)

(Cotdrave and
Gudes, 1997)
,
, (Meister
and Anderson, 1983; Cooper, 1997)
(Pool et al,. 1995)
(Chibelli et al., 1998; Hall, 1999)
NEW! (

; SH
NMDA ) (Janaky et al., 1999)
NEW!
(Paolicchi et al., 2002)

in vitro

- GSH +GSH
CDNB (chloro-2,4- et-GSH (
dinitrobenzene)
+
DEM (diethyl maleimide)

GSH )
,
-S-

DEM 1 mM, 1h
GSH CDNB - 1 mM, 30 min
( ) Et-GSH 5 mM, 30 min

CMFD (chloromethyl fluorescein ) N=5
GSH
CDNB ,
,
Na,K-, , ,

200

150


DHRfluorescencerelatedtocontrol, %

100

50
+CDNB 70
60

protein
50
40

ATP, nM/mg
0 30
Control CDNB DEM et-GSH 20
10
123(DHR) dihydrorhodamine, 0
0 5 10 15 20 25
rhodamine 123,
Incubation time, min


()

, , 1932 .,
De Vigneaud

- ,
, [Welch G.,
1998].

(1%),
(70%).

,
[ .., .., 2002].

,
15 /.

1530 / ,
30 100 / ,
100 500 / [Welch G., Loscalo J., 1998; Warren C., 2002].


-




-
-


( (
)

) 1975 . McCully


(
)
-

( )



( -
)


,



---------------------

In vitro

in vitro



:
PI ( ) ex=485 , em=610 (
)

DCFH-DA (2,7 ) exc=485 , em=530


( )
Fluo3 exc=488 , em=530 (
)
V FITC exc=488 , em=530 (
)

in vitro

130
125
120
115

DCF, %
110
105
100
95
MK-801 90 130
85 125
80 120
115

DCF, %
110
105
100
95

AIDA 90
85
80
I

MSOP III
In vivo
Experimental protocol

Methionine with drinking water Cytometric test


1 g/kg dayly
2 weeks

Pregnant rat Pups


4 weeks

Behavioral test

Treatment with possible protector


18


()
(
10 )


1 6 14 2
()
23,3 0,4



( 4
10,01 /
2
(6 2)
() ) 71 18,9 0,5

B

3 +
0,10,01 /
( + 6 11 2 22,9 0,9
)
Content of HC in the blood of rats
under methionine
over-loading

Control 8-13 mkM


Methionine
overload 48-52 mkM



NMDA,
HCA HC

500 30

1. , ,
.
2.
NMDA, HC HCA .
HC HCA
-NMDA

AIDA -

mGlu 1

MSOP

mGlu3

R .G. M. Morris et al. 1982. Nature, 297, 681-683.

,

:

1)

( );
2) ( );
3) ( /c);
4)
,
( %
);
5)
( )
( ),

2 - 4
2 - 4 .
, .

:

4 -5

1 2 3

( () () ()

)
20 7 140 18 45 6
,

, 52 20 5 8 2
, 0,24 0,02 0,18 0 02 0,25 0,04
/c


20 7 75 35 5
, %




( Cu/Zn Mn):
2- + 2- + 2+ 22 + 2
( ):
222 22 + 2
( ):
2GSH + 22 GSSG + 22O
( FAD):
GSSG + 2 2GSH + 2
(. )
(. )
(. )

N-
,


GSH

GSH
1) GSH+ Cu2+/Fe3+ Cu+/Fe2+ + GS + H+

(2) Cu+/Fe2+ + O2 Cu2+/Fe3++ O2-

(3) GS + GSH GSSG- + H+

(4) GSSG- + O2 O2- + GSSG

(5) 2O2- + 2H+ H2O2 + O2

(6) H2O2 + Cu+/Fe2+ OH- + OH-


.

2 - 2 - -
22 - - - + 2+ 2
+ NO +2+
+ 2 - + Fe2+
ONOO- 22 + 2
2 + - + - + - + Fe3+
( )
( -)
DCFH2 DCF

-
DCF
,

100 M
PI versus DCF coordinates

PI

ROS signal

Boldyrev et al, Ann NY Acad. Sci,


2003, 814, 613-618. DCF

NMDA-
I(3-
HPG) III(ACPD)
350

300
Fluorescence, % to Control
3-HPG
250
NMDA

200
ACPD

150

100

50

0
0 100 250 500
Concentration of Ligands, mkM
Effect of
Effect of Glutamate
Glutamate Ligands
Ligands
on Na/K-ATPase
on Na/K-ATPase

160
3-HPG
140
Na/K-ATPase, % to Control

120

100
NMDA
80

60
ACPD
40

20

0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 1.0
Ligand Concentration, mM

SH-SY5Y

1


(2+3)
D Aktivated PkB, %

-
14-3-3
Kulikov et al,
Biochemistry, submitted
Na-

+D-AP5



NMDA-

MAPK
NMDA-


(* p<0.05 )

-801, D-AP5, NAC, BAPTA,


No
onditions additions 10 10 1 m 50
Ouabain 0.1
1.24 1.12 0.99 1.05 0.87
180 min

Ouabain 1 1.53 1.40 1.16 0.76 0.89


m
180 min 0.03 0.02 (*) (0.04)* (0,10)* 0.08 (8)


Na/K-


Na/K-AT

2+ Na+
- Na+

2+3 1

NMDA K+
K+

Ca2+ MAP
MAPK
K
IP3K
Ca2+ NADPH

Bcl2

kC
kC 14-3-3 +

k
k p53P -








.
OH
Fe2+
.-
02 202 Glu-SH NAD

20 GSSG NADH2







.
OH
Fe2+
.-
02 202 Glu-SH NAD

20 GSSG NADH2





GS-SG NAD


*
GluSH

-Tocoferyl*
NADH2

*
-Tocoferol Ascorbat

Asc*