- Email: [email protected]
The reaction of aldehydic lipid peroxidation products with xanthine oxidase
Antioxidant Enzymes
14.9
14.11
129
INCREASE OF LIFE SPAN OF F R O G S A F T E R I N D U C T I O N OF SOD, GR, G S H A N D A S C O R B A T E G. B a r j a , M. L G p e z and R. P e r e z . Dept. of Animal Biology-ll. Complutense U n i v e r s i t y . M a d r i d 28040. Spain.
SCAVENGING OF HYDROGEN PEROXIDE BY ISOLATED PERFUSED RABBIT LUNGS Michele L. Bamard and Sadis Matalon Department of Anesthesiology, The University of Alabama at Birmingham, Birmingham, AL 35233, U.S.A.
An a~in~ experiment is performed t h r o u g h o u t the life span of R a n a p e r e z i . Two ~rouos (young and old) of ii0 animals each were established. They were d i v i d e d into two s u b ~ r o u D s t r e a t e d e v e r y 15 days w i t h s a l i n e ( c o n t r o l s ) or a m i n o tr'i0zole ( a c a t a l a s e - t A T i n h i b i t o r ) at i mK/~. After 2.5 months liver SOD and GSH-reduc:ta~e (GR) i n c r e a s e d (to 250 a n d ]90% oi controls) in CAT deDleted animals. GSH did not vary. After- 14.5 months CA']7 r e m a i n e d depleted. Idver induration of ~ O D was a ~ a i n 260% but GR now increased to i 0(]0%, GSH was now i n c r e a s e d (250%) and a s c o r b a f e i n c r e a s e d to 250%. GR was a l s o i n d u c e d in k i d n e y (700%), b r a i n (165%) and ]un~ (147%). Kidney ascorbate (230%) and G S H (260%) a l s o i n c r e a s e d . Se and n o n Se G,%'H-PX, in vivo or in vitro TBA-RS, GSSG, cyt o c h r o m e oxidase, 02 c o n s u m p t i o n or MDA (HPLC) did not change. Mortality rate was 4 (in young animals) to 8 (in old) times s m a l l e r in t r e a t e d a n i m a l s . These results show that at least the mean life span of a v e r t e b r a t e can be increased through induction of e n d o g e nous a n t i o x i d a n t s . SupDor'ted by F I S t s g0/C)074 and 90/0013.
We investigated the scavenging ability of isolated rabbit lungs perfused with buffer at a flow rate of 108 ml/min. Lungs were extremely effective at removing an exogenous challenge of hydrogen peroxide. Peroxide was added in a bolus to produce an original calculated concentration of 0.25 raM, and was removed by the lungs with a halflife of 0.32 _+ 0.03 min. Inhibition of catalase with 0.5 mM azide or inhibition of the giutathione redox cycle with 0.2 mM 1-chloro2,4-dinitrobenzene (CDNB) did not affect the half life of hydrogen peroxide in the lungs (0.34 ± 0.01 and 0.33 _+ 0.02 min. respectively), lf, however, both azide and CDNB were added to the lungs, the halflife was increased to 0.72 _+ 0.09 min. (p < 0.05). These data indicate that either enzymatic scavenging system is able to rapidly and efficiently remove low concentrations of hydrogen peroxide if one is inhibited, but that scavenging is significantly diminished if both systems are inhibited. The rapid removal of the peroxide that remains after inhibition of catalase and the glutathione redox cycle also indicates that non-enzymatic scavenging is also very important in removal of hydrogen peroxide from the pulmonary circulation.
THE REACTION OF ALDEHYDIC LIPID PEROXIDATION PRODUCTS WITH XANTHINE OXIDASE Patrieia L. Bounds and Gary W. Winston Department of Biochemistry and Biodynamics Institute, Louisiana State University, Baton Rouge, LA 70803, U.S.A. A series of ~,fl-unsaturated aldehydes, 2-butenal through 2-nonenal and 4-hydroxy-2-nonenal, were recently identified as substrates for xanthine oxidase. The reaction proceeds with conversion of the aldehyde to the corresponding carboxylie acid, and reduction of dioxygen to 0~-. Kinetic parameters k=a t and KM and the hydrophobicity constant log k~ were determined for each aldehyde. A linear relationship was found between log k~ and pK~, and log k=at/K~, suggesting that substrate binding and the efficiency of the reaction of xanthine oxidase with unsaturated aldehydes depends on the hydrophobieity of the substrate, and that the binding site for these substrates is very lipophilic. These aldehydes, which are alternative substrates for xanthine oxidase, were studied as inhibitors in the conversion of xanthine to urate. We found noncompetitive inhibition of urate production, indicating that aldehydes and purines bind at distinct sites on the enzyme. As ~,~-unsaturated aldehydes are products of lipid peroxidation, and xanthine oxidase has been implicated in the initiation of lipid peroxidation, it is suggested that the reaction of xanthine oxidase with unsaturated aldehydes could be an important route of amplification of oxidative damage. Supported by NIAAA Grant AA06752-02.
14.10
"E"aON~--SP'CiFIC EXP~SSIO. OFCOFPER-ZINC S.PEROXIDE14 12 DISMUTASE GENE IN T R A N S G E N I C MICE: ANIMAL G E N E D O S A G E E F F E C T IN D O W N ' S S Y N D R O M E .
I. Ceballos-Pigot,
A. Niqole,
P. Briand,
MODEL
OF
G. Grimber,
F. Javoy-Agid -, M. Lafon" and P.M. Sinet. Genetic Biochemistry Laboratory, CNRS URA 1335, Necker Hospital, 75015 Paris. *Experimental Medecine Laboratory, INSERM U289, Salpetriere Hospital, 75013 Paris. It has been Suggested that copper-zinc superoxide dismutase SOD)
increment,
promote
oxidative
involved Down's
by accelerating damage
in the various syndrome
neurological developed
such
lesions.
strains
hydrogen p e r o x i d e
within
trisomy
premature
In order
to
of transgenic
test
h~man
C~Zn
neurons,
SOD
protein
r~NA
not
in
and
this
Alzheimer-type
hypothesis,
the
human
we
have
CuZn
SOD
of brain sections was
preferentially
revealed
that
expressed
in
particularly in pyramidal cells of Ammcn's horn and granule
involve
glutathione
adaptative
percxidase,
significantly
higher
suggesting
in an
increased CuZn SOD activit}"
modifications
glu~athione
t r a n s f e r a s e activities. The a m o u n t o f t h i o b a r b i t u r i c
strongly
be
found
carrying
ceils of gyros dentate. In this tissue,
did
might
(1.93 fold). Ir~munohistochemica!
analysis and
and
resulted in increased CuZn SO~
in the brain
and in situ hybridization
(c~/zn might
abnormalities
aging
mice
gone. The human transgene expression activity predominantly
21 cells
neurobiological as
formation,
acid
transgenic increased
in
reduczase
seleno-dependenz
and
(TBA)-reactive
brains
level
of
compared
q!utathione
ma=erial
to
peroxidatlon
S-
was
controls, in
rive.
These results suppor ~- the notion that CuZn SOD gone dosage effecn could play a role in the pathogenesis cf rapid aging features in the brain of Down's syndrome patients.
"
14.9
14.11
129
INCREASE OF LIFE SPAN OF F R O G S A F T E R I N D U C T I O N OF SOD, GR, G S H A N D A S C O R B A T E G. B a r j a , M. L G p e z and R. P e r e z . Dept. of Animal Biology-ll. Complutense U n i v e r s i t y . M a d r i d 28040. Spain.
SCAVENGING OF HYDROGEN PEROXIDE BY ISOLATED PERFUSED RABBIT LUNGS Michele L. Bamard and Sadis Matalon Department of Anesthesiology, The University of Alabama at Birmingham, Birmingham, AL 35233, U.S.A.
An a~in~ experiment is performed t h r o u g h o u t the life span of R a n a p e r e z i . Two ~rouos (young and old) of ii0 animals each were established. They were d i v i d e d into two s u b ~ r o u D s t r e a t e d e v e r y 15 days w i t h s a l i n e ( c o n t r o l s ) or a m i n o tr'i0zole ( a c a t a l a s e - t A T i n h i b i t o r ) at i mK/~. After 2.5 months liver SOD and GSH-reduc:ta~e (GR) i n c r e a s e d (to 250 a n d ]90% oi controls) in CAT deDleted animals. GSH did not vary. After- 14.5 months CA']7 r e m a i n e d depleted. Idver induration of ~ O D was a ~ a i n 260% but GR now increased to i 0(]0%, GSH was now i n c r e a s e d (250%) and a s c o r b a f e i n c r e a s e d to 250%. GR was a l s o i n d u c e d in k i d n e y (700%), b r a i n (165%) and ]un~ (147%). Kidney ascorbate (230%) and G S H (260%) a l s o i n c r e a s e d . Se and n o n Se G,%'H-PX, in vivo or in vitro TBA-RS, GSSG, cyt o c h r o m e oxidase, 02 c o n s u m p t i o n or MDA (HPLC) did not change. Mortality rate was 4 (in young animals) to 8 (in old) times s m a l l e r in t r e a t e d a n i m a l s . These results show that at least the mean life span of a v e r t e b r a t e can be increased through induction of e n d o g e nous a n t i o x i d a n t s . SupDor'ted by F I S t s g0/C)074 and 90/0013.
We investigated the scavenging ability of isolated rabbit lungs perfused with buffer at a flow rate of 108 ml/min. Lungs were extremely effective at removing an exogenous challenge of hydrogen peroxide. Peroxide was added in a bolus to produce an original calculated concentration of 0.25 raM, and was removed by the lungs with a halflife of 0.32 _+ 0.03 min. Inhibition of catalase with 0.5 mM azide or inhibition of the giutathione redox cycle with 0.2 mM 1-chloro2,4-dinitrobenzene (CDNB) did not affect the half life of hydrogen peroxide in the lungs (0.34 ± 0.01 and 0.33 _+ 0.02 min. respectively), lf, however, both azide and CDNB were added to the lungs, the halflife was increased to 0.72 _+ 0.09 min. (p < 0.05). These data indicate that either enzymatic scavenging system is able to rapidly and efficiently remove low concentrations of hydrogen peroxide if one is inhibited, but that scavenging is significantly diminished if both systems are inhibited. The rapid removal of the peroxide that remains after inhibition of catalase and the glutathione redox cycle also indicates that non-enzymatic scavenging is also very important in removal of hydrogen peroxide from the pulmonary circulation.
THE REACTION OF ALDEHYDIC LIPID PEROXIDATION PRODUCTS WITH XANTHINE OXIDASE Patrieia L. Bounds and Gary W. Winston Department of Biochemistry and Biodynamics Institute, Louisiana State University, Baton Rouge, LA 70803, U.S.A. A series of ~,fl-unsaturated aldehydes, 2-butenal through 2-nonenal and 4-hydroxy-2-nonenal, were recently identified as substrates for xanthine oxidase. The reaction proceeds with conversion of the aldehyde to the corresponding carboxylie acid, and reduction of dioxygen to 0~-. Kinetic parameters k=a t and KM and the hydrophobicity constant log k~ were determined for each aldehyde. A linear relationship was found between log k~ and pK~, and log k=at/K~, suggesting that substrate binding and the efficiency of the reaction of xanthine oxidase with unsaturated aldehydes depends on the hydrophobieity of the substrate, and that the binding site for these substrates is very lipophilic. These aldehydes, which are alternative substrates for xanthine oxidase, were studied as inhibitors in the conversion of xanthine to urate. We found noncompetitive inhibition of urate production, indicating that aldehydes and purines bind at distinct sites on the enzyme. As ~,~-unsaturated aldehydes are products of lipid peroxidation, and xanthine oxidase has been implicated in the initiation of lipid peroxidation, it is suggested that the reaction of xanthine oxidase with unsaturated aldehydes could be an important route of amplification of oxidative damage. Supported by NIAAA Grant AA06752-02.
14.10
"E"aON~--SP'CiFIC EXP~SSIO. OFCOFPER-ZINC S.PEROXIDE14 12 DISMUTASE GENE IN T R A N S G E N I C MICE: ANIMAL G E N E D O S A G E E F F E C T IN D O W N ' S S Y N D R O M E .
I. Ceballos-Pigot,
A. Niqole,
P. Briand,
MODEL
OF
G. Grimber,
F. Javoy-Agid -, M. Lafon" and P.M. Sinet. Genetic Biochemistry Laboratory, CNRS URA 1335, Necker Hospital, 75015 Paris. *Experimental Medecine Laboratory, INSERM U289, Salpetriere Hospital, 75013 Paris. It has been Suggested that copper-zinc superoxide dismutase SOD)
increment,
promote
oxidative
involved Down's
by accelerating damage
in the various syndrome
neurological developed
such
lesions.
strains
hydrogen p e r o x i d e
within
trisomy
premature
In order
to
of transgenic
test
h~man
C~Zn
neurons,
SOD
protein
r~NA
not
in
and
this
Alzheimer-type
hypothesis,
the
human
we
have
CuZn
SOD
of brain sections was
preferentially
revealed
that
expressed
in
particularly in pyramidal cells of Ammcn's horn and granule
involve
glutathione
adaptative
percxidase,
significantly
higher
suggesting
in an
increased CuZn SOD activit}"
modifications
glu~athione
t r a n s f e r a s e activities. The a m o u n t o f t h i o b a r b i t u r i c
strongly
be
found
carrying
ceils of gyros dentate. In this tissue,
did
might
(1.93 fold). Ir~munohistochemica!
analysis and
and
resulted in increased CuZn SO~
in the brain
and in situ hybridization
(c~/zn might
abnormalities
aging
mice
gone. The human transgene expression activity predominantly
21 cells
neurobiological as
formation,
acid
transgenic increased
in
reduczase
seleno-dependenz
and
(TBA)-reactive
brains
level
of
compared
q!utathione
ma=erial
to
peroxidatlon
S-
was
controls, in
rive.
These results suppor ~- the notion that CuZn SOD gone dosage effecn could play a role in the pathogenesis cf rapid aging features in the brain of Down's syndrome patients.
"