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Effect of metallothionein I on mitochondrial oxygen consumption
Life Sciences, Vol. 55, No. 3, pp. 221-226, 1994 Copyright © 1994 Elsevier Science Ltd
Pergamon
Printed in the USA. All rights reserved 0024-3205/94 $6.00 + .00 0024-3205(94) 00107-3
EFFECT
OF
Cuthbert
METALLOTHIONEIN
O.
I ON
S i m p k i n s +*, Zhao
MITOCHONDRIAL
OXYGEN
H a o - L i a o +°, and C y n t h i a
CONSUMPTION
A. T o r r e n c e +
+ U n i v e r s i t y of M a r y l a n d S c h o o l of M e d i c i n e , D e p a r t m e n t of S u r g e r y and *The S h o c k T r a u m a Institute, Baltimore, Maryland, °Jiangxi M e d i c a l C o l l e g e , N a n c h a n g , P.R. C h i n a (Received in final form May 9, 1994)
Summary The e f f e c t of the s t r e s s - i n d u c e d , c y s t e i n e rich protein, metallothionein I (MT), on o x y g e n c o n s u m p t i o n by rat liver m i t o c h o n d r i a was studied. Using a Clark-type oxygen electrode we f o u n d t h a t e l e c t r o n transport f r o m s u c c i n a t e to o x y g e n was e n h a n c e d by MT whereas ADP-initiated o x y g e n c o n s u m p t i o n was inhibited by MT. The MT effect was concentration dependent. No e v i d e n c e was f o u n d for the p r o v i s i o n by MT of an a l t e r n a t e p a t h w a y to oxygen.
Key Words: metallothionein I, mitochondrial oxygen consumption, liver Metallothioneins were discovered in 1957 by M a r g o s h e s and V a l l e e (i). T h e y h a v e b e e n f o u n d in n e a r l y all v e r t e b r a t e t i s s u e s (2). T h e s e low m o l e c u l a r w e i g h t (6-TkDa) p r o t e i n s are d e v o i d of h i s t i d i n e or a r g i n i n e and c o n t a i n few h y d r o p h o b i c residues (3). A p p r o x i m a t e l y o n e - t h i r d of the r e s i d u e s of m e t a l l o t h i o n e i n I (MT) are c y s t e i n e s t h a t are w e l l - c o n s e r v e d t h r o u g h o u t the p h y l a (3). T h e s e c y s t e i n e s are m a i n t a i n e d in the r e d u c e d s t a t e by b i n d i n g to h e a v y m e t a l s of v a l e n c e +2, s u c h as Zn, Cd, Pb or Cu (2). N M R has r e v e a l e d t h a t t h e s e m e t a l s are in constant flux, u n d e r g o i n g exchange between intramolecular thiolate binding sites (4). Because the cysteines are m a i n t a i n e d in the r e d u c e d s t a t e no d i s u l f i d e b o n d s are f o u n d in MT (2). Metallothionein is a p r o d u c t of the a c u t e r e s p o n s e to s t r e s s (5). O n e of the m a n y stressors which has b e e n studied is lipopolysaccharide (LPS) (6). The a d m i n i s t r a t i o n of LPS to rats c a u s e s i n c r e a s e d e x p r e s s i o n of the M T g e n e in n u m e r o u s t i s s u e s , w i t h r e l a t i v e e f f i c a c y in liver > k i d n e y > o v a r y > lung > b r a i n > heart > intestine. T h i s r e s p o n s e to LPS is m e d i a t e d by a c t i v a t e d macrophages (7) w h i c h r e l e a s e , a m o n g o t h e r c y t o k i n e s , IL-I~, IL-6 and TNF~. I L - l a and T N F ~ h a v e b e e n s h o w n to i n d u c e MT g e n e e x p r e s s i o n in m a n y t i s s u e s i n c l u d i n g the liver (8). IL-6 on the o t h e r h a n d i n c r e a s e s M T g e n e e x p r e s s i o n o n l y in the liver, a c t i n g via s p e c i f i c IL-6 h e p a t o c y t e b i n d i n g sites (9).
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MT on Mitochondrial Oxygen Consumption
Vol. 55, No. 3, 1994
We r e c e n t l y r e p o r t e d t h a t c y t o c h r o m e c is r e d u c e d by M T (i0). S i n c e t h e o u t e r m e m b r a n e of t h e m i t o c h o n d r i o n is c a p a b l e of f r e e l y a d m i t t i n g m o l e c u l e s of m o l e c u l a r w e i g h t u p to 10kDa, we e n v i s i o n e d an i n t e r a c t i o n b e t w e e n M T and c y t o c h r o m e c in t h e i n t e r m e m b r a n e space. E v i d e n c e for t h e m o d u l a t i o n of o x i d a t i v e p h o s p h o r y l a t i o n by M T is p r e s e n t e d below. Materials
and M e t h o d s
Materials Male Sprague-Dawley rats were obtained from Charles River (Wilmington, MA.). These rats weighed from 200 to 250gm. Succinate, ADP, KCN, s u c r o s e , MgCI2, KH2PO4, K2HP04, KCI, CdCI2, ZnCI2, HEPES, d i t h i o n i t e a n d Cd, Zn, m e t a l l o t h i o n e i n I (MT) f r o m r a b b i t l i v e r w e r e p u r c h a s e d f r o m SIGMA, C h e m i c a l Co. (St. Louis, MO.). MT e l u t e d as a s i n g l e p e a k d u r i n g H P L C on a C - 1 8 c o l u m n u s i n g 0.1% T F A as the ion p a i r i n g a g e n t and 0.1% T F A w i t h 80% a c e t o n i t r i l e as the organic modifier. Methods S p r a g u e - D a w l e y r a t s w e r e k i l l e d u s i n g CO 2. M i t o c h o n d r i a w e r e i s o l a t e d as p r e v i o u s l y d e s c r i b e d (Ii). Experiments were carried out in a b u f f e r (pH = 7.4) consisting of s u c r o s e (0.225M), K2HPO,/KH2PO 4 b u f f e r (10mM), MgCI2(5mM), KCI (20mM), H E P E S (20mM) and supplemented w i t h A D P (0.4mM) a n d s u c c i n a t e 4mM as noted. P r o t e i n d e t e r m i n a t i o n s w e r e m a d e u s i n g the B i o r a d a s s a y ( R o c k v i l l e C e n t r e , N.Y.). V a r i o u s c o n c e n t r a t i o n s of M T in d i s t i l l e d d e i o n i z e d H20 w e r e a d d e d to a m i t o c h o n d r i a l s u s p e n s i o n of 0 . 2 m g p r o t e i n / m l . In c o n t r o l e x p e r i m e n t s d i s t i l l e d d e i o n i z e d w a t e r a l o n e in t h e s a m e v o l u m e w a s a d d e d to t h e m i t o c h o n d r i a l suspension. The total r e a c t i o n m i x t u r e v o l u m e w a s 2ml. A d d i t i o n s of M T or o t h e r r e a g e n t s e m p l o y e d v o l u m e s w h i c h n e v e r e x c e e d e d 20ul. O x y g e n c o n s u m p t i o n (VO2) w a s m e a s u r e d p o l a r o g r a p h i c a l l y u s i n g a C l a r k - t y p e o x y g e n e l e c t r o d e (12). All e x p e r i m e n t s w e r e p e r f o r m e d at 25°C. A d d i t i o n s w e r e m a d e at i n t e r v a l s of 4 m i n u t e s . Results M T at 5 0 u M s i g n i f i c a n t l y increased the r a t e of VO2 w i t h s u c c i n a t e as s u b s t r a t e . A l s o A D P - i n i t i a t e d V O 2 w a s i n h i b i t e d in the presence of 5 0 u M M T ( f i g u r e i). No c h a n g e in V02 w a s n o t e d when MT was added to m i t o c h o n d r i a in t h e a b s e n c e of s u c c i n a t e or ADP. T a b l e o n e s h o w s t h e r e s u l t s of e i g h t e x p e r i m e n t s u s i n g M T at 50uM. T h e c o n c e n t r a t i o n d e p e n d e n c e of M T e f f e c t s on 02 c o n s u m p t i o n are s h o w n in f i g u r e 2. T h e e n h a n c e m e n t of s u c c i n a t e - i n i t i a t e d V02 and t h e i n h i b i t i o n of A D P - i n i t i a t e d V02 b o t h e x h i b i t e d a p l a t e a u b e g i n n i n g at 5 0 u M MT.
Vol. 55, No. 3, 1994
MT on Mitochondrial Oxygen Consumption
$
%
2 min.
l
l
Fig.
1
E f f e c t of M T o n m i t o c h o n d r i a l V O z. = no MT, = 5 0 u M MT. S=succinate (4mM) a n d A = A D P (0.4mM). Mitochondria, succinate, and ADP were added sequentially at intervals of 4 minutes.
Table
1
Effect of MT on V O 2. Data are in units of nmole O2/min.mg. Data from e a c h of t h e e i g h t rats is t h e result of 2-4 replicates. There was less than 10% variation between replicates, p < 0.05 by paired "t" test for differences between rates with and without MT after addition of s u c c i n a t e (4mM) o r A D P ( 0 . 4 m M ) . MT = 50uM. Rat
Mean
#
1 2 3 4 5 6 7 8 ± SE
Succinate without MT 28.4 32.3 36.6 28.5 18.8 29.2 17.9 26.1 2 7 . 2 ± 2.2
oxidation with MT 87.9 35.0 54.9 31.8 43.8 32.3 47.7 47.8 47.7 ± 6.5
A D P i n i t i a t e d VO2 without MT with MT 104.5 79.3 116 3 93.8 87 6 48.4 69 5 43.5 50 2 43.8 106 2 55.6 54 7 47.7 71.7 44.5 8 2 . 6 + 8.8 57.1+6.7
223
224
MT on Mitochondrial Oxygen Consumption
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80 70
O
40
o
20
2b 4b 6b ~
160 i~0 i~0 i~0 i~0 260 [MT](uM)
Fig.
2
Concentration dependence of t h e e f f e c t of M T on mitochondrial respiration. 02 consumption is in n m o l e 0 2 / m i n . m g . o = VO 2 after addition of 4mM succinate. . = VO 2 after further addition of 0.4mM ADP. O t h e r d e t a i l s w e r e as f o r Fig. i. E a c h d a t a p o i n t is t h e r e s u l t of 2 - 3 replicates. Replicates w e r e w i t h i n 10% of e a c h o t h e r . One possible mechanism f o r t h e e f f e c t of M T w a s t h e p r o v i s i o n b y M T of a n a l t e r n a t e pathway for election transfer to oxygen. In order to test for this we used 10mM KCN to block electron transfer from cytochrome oxidase to oxygen. MT did not overcome the complete inhibition of succinate or ADP initiated VO 2 by KCN (data not shown). Furthermore, when MT was added to all reactants in buffer without mitochondria, no oxygen was consumed. This ruled out the possibility that MT alone was removing oxygen from the solution. Another possibility was that the actions of M T w e r e c a u s e d solely by the metals Zn +2 o r C d +2, e x c l u s i v e of t h e protein. Therefore, we measured the effect of ZnCl 2 and CdCI 2 on 02 consumption of mitochondria. The data are summarized in T a b l e 2. This experiment demonstrated t h a t t h e e f f e c t s of t h e m e t a l s alone are very different from those of MT. At a metal concentration of luM there was complete inhibition of VO2 consumption, r e g a r d l e s s of w h e t h e r t h e i n i t i a t i n g f a c t o r w a s A D P o r succinate.
Vol. 55, No. 3, 1994
MT on Mitochondrial Oxygen Consumption
Table
225
2
E f f e c t of C d C I 2 a n d ZnCI 2 on o x y g e n c o n s u m p t i o n . Data are the r e s u l t of 2 r e p l i c a t e s . T h e r e w a s less t h a n 10% d i f f e r e n c e b e t w e e n replicates. A D P = 0.4mM. S u c c i n a t e = 4mM. R a t e s a r e in u n i t s of n m o l e O=/min.mg. [ZnCl~ 0 0.01uM 0.10uM 1.0 u M [CdCl~1 0 0.01uM 0.10uM 1.0 u M ZnCI= 0 . 0 5 u M + CdCI2 0 . 0 5 u M
Succinate oxidation 37.8 34.4 25.8 0
34.4 27.5 37.8 0 34.4
ADP
initiated 123.8 130.7 61.9 0
VO2
87.7 92.9 72.2 0 75.7
Discussion An a b n o r m a l p a t t e r n of 02 u t i l i z a t i o n and i n c r e a s e d a n a e r o b i c m e t a b o l i s m a r e c h a r a c t e r i s t i c of s e v e r e c r i t i c a l i l l n e s s e s , s u c h as s e p t i c s h o c k (13). A t t e m p t s to e x p l a i n t h e s e c h a r a c t e r i s t i c s b y f i n d i n g d e f e c t i v e f u n c t i o n in i s o l a t e d m i t o c h o n d r i a l p r e p a r a t i o n s have not been successful. O u r f i n d i n g in m i t o c h o n d r i a t h a t M T enhances succinate oxidation and inhibits ADP initiated 02 consumption provides a possible explanation. Several characteristics of M T m a k e it a g o o d c a n d i d a t e for t h i s role. O n e c h a r a c t e r i s t i c is the f a c t t h a t M T is i n d u c e d b y c y t o k i n e s s u c h as IL-I~, T N F ~ and IL-6, in a w i d e r a n g e of o r g a n s (9). M T is a b u n d a n t in t h e c y t o p l a s m - o c c u r r i n g in u p to m M l e v e l s (14). MT theoretically could impact significantly upon mitochondrial function and other cellular processes. T h e m e c h a n i s m of t h e M T e f f e c t s on m i t o c h o n d r i a n e e d s to be explored. T h e i n c r e a s e in s t a t e IV e l e c t r o n f l o w ( s u c c i n a t e only) suggests uncoupling. T h e d e c r e a s e in s t a t e III e l e c t r o n f l o w (succinate + ADP) suggests inhibition of t h e FoF I A T P a s e or decoupling (15). It is u n l i k e l y t h a t M T a c t s as a s o u r c e of a d d i t i o n a l e l e c t r o n s v i a c y t o c h r o m e c or o t h e r c o m p o n e n t s of t h e r e s p i r a t o r y c h a i n s i n c e M T a l o n e h a d no e f f e c t on m i t o c h o n d r i a V O 2. A n o t h e r p o s s i b i l i t y is t h a t t h e c y s t e i n e s of M T c o u l d act v i a t h e m i t o c h o n d r i a l e n z y m e c o n j u g a t e B - l y a s e (16). T h e a c t i o n of M T could be mediated v i a its m a n y r e d u c e d c y s t e i n e s , or by t h e cysteines and metals acting interdependently. F u t u r e w o r k w i l l be d i r e c t e d t o w a r d u n d e r s t a n d i n g t h e r e l a t i o n s h i p of t h e s t r u c t u r e of
226
MT on Mitochondrial Oxygen Consumption
M T a n d its e f f e c t on m i t o c h o n d r i a l V O 2. We w i l l further the possible r o l e of M T as a m o d u l a t o r p h o s p h y o r y l a t i o n in s t r e s s states.
Vol. 55, No. 3, 1994
also explore of o x i d a t i v e
Acknowledgements We w o u l d like to t h a n k Dr. P a t r i c i a S o k o l o v e for u s e of t h e p o l a r i m e t e r a n d h e r h e l p f u l d i s c u s s i o n s ; Drs. T y s o n T i l d o n , M a r y McKenna and Timothy Buchman for t h e i r h e l p f u l comments. We a p p r e c i a t e t h e e d i t o r i a l a s s i s t a n c e of Mrs. R e b e c c a C o l e m a n . This w o r k w a s s u p p o r t e d in p a r t by t h e O f f i c e of N a v a l R e s e a r c h , g r a n t no. N 0 0 0 1 4 - 9 3 - I - 0 2 2 7 . References I. 2. 3. 4. 5.
6. 7. 8. 9. i0. ii. 12. 13.
14.
15. 16.
M. M A R G O S H E S , B.L VALLEE, J. Am. Chem. Soc. 79 4 8 1 3 - 4 8 1 4 (1957). M . A . DUNN, T . L . BLALOCK, R . J . COUSINS, P r o c . S o c . E x p . B i o l . Med. 185 1 0 7 - 1 1 9 (1987). J. KAY, A. CRYER, B.M. DARKE, P. K I L L I E , W.E. LESS, C.G. NOREY, J.M. STARK, Int. J. B i o c h e m 23 1-5 (1991). J . R . H . KAGI, M e t h o d s in E n z y m o l . 2 0 5 6 1 3 - 6 2 6 (1991). A. KOJ, T h e A c u t e - P h a s e R e s p o n s e to I n j u r y a n d I n f e c t i o n , A.H. G o r d o n , A. Kol (eds), 139-232, E l s e v i e r S c i e n c e P u b l i s h e r s B.V., A m s t e r d a m (1985). S. OH, J.T. DEAGAN, P.D. W H A N G E R , P.H. W E S W I G , Am. J. P h y s i o l . 234 2 8 2 - 2 8 5 (1978). T. M A I T A N I , Y. SAITO, H. F U J I M K I , K.T. SUZAKI, T o x i c o l . Lett. 30 1 8 1 - 1 8 7 (1986). M. KARIN, R.J. IMBRA, A. HEGUY, G. WONG, Mol. Cell. Biol. 2 8 6 6 - 2 8 6 9 (1985). S.K. DE, M.T. M C M A S T E R , G.K. A N D R E W S , J. Biol. Chem. 265 1 5 2 6 7 - 1 5 2 7 4 (1990). C.O. S I M P K I N S , P.M. E U D A R I C , C.A. T O R R E N C E , Z. YANG, L i f e Sci. 5_/3 1 9 7 5 - 1 9 8 0 (1993). D. J O H N S O N , H. LARDY, Mets. E n z y m o l . i0 94-96 (1967). R.W. E S T A B R O O K , Mets. E n z y m o l . I0 41-47 (1967). R. S C H L I C H I I G , T.I. T O N N E S S E N , E.M. N E M O T O , C r i t i c a l Care: S t a t e of t h e Art, D.S. Prough, R.J. T r a y s t m a n (ed.), 239-273, S o c i e t y of C r i t i c a l C a r e M e d i c i n e , A n a h e i m , Calif. (1993). R.R. B A H N S O N , A. BASU, J.S. LAZO, M o l e c u l a r and Clinical A d v a n c e s in A n t i c a n c e r D r u g R e s i s t a n c e , R.F. O z a l s (ed), 251260, K l u w e r A c a d e m i c P u b l i s h e r s B o s t o n (1991). H. R O T T E N B E R G , B i o c h i m . B i o p h y s . Acta. 1018 1-17 (1990). R.G. SCHNELLMANN, T.J. CROSS, E.A. LOCK, Toxicol. Appl. P h a r m a c o l . i00 4 9 8 - 5 0 5 (1989).
Pergamon
Printed in the USA. All rights reserved 0024-3205/94 $6.00 + .00 0024-3205(94) 00107-3
EFFECT
OF
Cuthbert
METALLOTHIONEIN
O.
I ON
S i m p k i n s +*, Zhao
MITOCHONDRIAL
OXYGEN
H a o - L i a o +°, and C y n t h i a
CONSUMPTION
A. T o r r e n c e +
+ U n i v e r s i t y of M a r y l a n d S c h o o l of M e d i c i n e , D e p a r t m e n t of S u r g e r y and *The S h o c k T r a u m a Institute, Baltimore, Maryland, °Jiangxi M e d i c a l C o l l e g e , N a n c h a n g , P.R. C h i n a (Received in final form May 9, 1994)
Summary The e f f e c t of the s t r e s s - i n d u c e d , c y s t e i n e rich protein, metallothionein I (MT), on o x y g e n c o n s u m p t i o n by rat liver m i t o c h o n d r i a was studied. Using a Clark-type oxygen electrode we f o u n d t h a t e l e c t r o n transport f r o m s u c c i n a t e to o x y g e n was e n h a n c e d by MT whereas ADP-initiated o x y g e n c o n s u m p t i o n was inhibited by MT. The MT effect was concentration dependent. No e v i d e n c e was f o u n d for the p r o v i s i o n by MT of an a l t e r n a t e p a t h w a y to oxygen.
Key Words: metallothionein I, mitochondrial oxygen consumption, liver Metallothioneins were discovered in 1957 by M a r g o s h e s and V a l l e e (i). T h e y h a v e b e e n f o u n d in n e a r l y all v e r t e b r a t e t i s s u e s (2). T h e s e low m o l e c u l a r w e i g h t (6-TkDa) p r o t e i n s are d e v o i d of h i s t i d i n e or a r g i n i n e and c o n t a i n few h y d r o p h o b i c residues (3). A p p r o x i m a t e l y o n e - t h i r d of the r e s i d u e s of m e t a l l o t h i o n e i n I (MT) are c y s t e i n e s t h a t are w e l l - c o n s e r v e d t h r o u g h o u t the p h y l a (3). T h e s e c y s t e i n e s are m a i n t a i n e d in the r e d u c e d s t a t e by b i n d i n g to h e a v y m e t a l s of v a l e n c e +2, s u c h as Zn, Cd, Pb or Cu (2). N M R has r e v e a l e d t h a t t h e s e m e t a l s are in constant flux, u n d e r g o i n g exchange between intramolecular thiolate binding sites (4). Because the cysteines are m a i n t a i n e d in the r e d u c e d s t a t e no d i s u l f i d e b o n d s are f o u n d in MT (2). Metallothionein is a p r o d u c t of the a c u t e r e s p o n s e to s t r e s s (5). O n e of the m a n y stressors which has b e e n studied is lipopolysaccharide (LPS) (6). The a d m i n i s t r a t i o n of LPS to rats c a u s e s i n c r e a s e d e x p r e s s i o n of the M T g e n e in n u m e r o u s t i s s u e s , w i t h r e l a t i v e e f f i c a c y in liver > k i d n e y > o v a r y > lung > b r a i n > heart > intestine. T h i s r e s p o n s e to LPS is m e d i a t e d by a c t i v a t e d macrophages (7) w h i c h r e l e a s e , a m o n g o t h e r c y t o k i n e s , IL-I~, IL-6 and TNF~. I L - l a and T N F ~ h a v e b e e n s h o w n to i n d u c e MT g e n e e x p r e s s i o n in m a n y t i s s u e s i n c l u d i n g the liver (8). IL-6 on the o t h e r h a n d i n c r e a s e s M T g e n e e x p r e s s i o n o n l y in the liver, a c t i n g via s p e c i f i c IL-6 h e p a t o c y t e b i n d i n g sites (9).
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MT on Mitochondrial Oxygen Consumption
Vol. 55, No. 3, 1994
We r e c e n t l y r e p o r t e d t h a t c y t o c h r o m e c is r e d u c e d by M T (i0). S i n c e t h e o u t e r m e m b r a n e of t h e m i t o c h o n d r i o n is c a p a b l e of f r e e l y a d m i t t i n g m o l e c u l e s of m o l e c u l a r w e i g h t u p to 10kDa, we e n v i s i o n e d an i n t e r a c t i o n b e t w e e n M T and c y t o c h r o m e c in t h e i n t e r m e m b r a n e space. E v i d e n c e for t h e m o d u l a t i o n of o x i d a t i v e p h o s p h o r y l a t i o n by M T is p r e s e n t e d below. Materials
and M e t h o d s
Materials Male Sprague-Dawley rats were obtained from Charles River (Wilmington, MA.). These rats weighed from 200 to 250gm. Succinate, ADP, KCN, s u c r o s e , MgCI2, KH2PO4, K2HP04, KCI, CdCI2, ZnCI2, HEPES, d i t h i o n i t e a n d Cd, Zn, m e t a l l o t h i o n e i n I (MT) f r o m r a b b i t l i v e r w e r e p u r c h a s e d f r o m SIGMA, C h e m i c a l Co. (St. Louis, MO.). MT e l u t e d as a s i n g l e p e a k d u r i n g H P L C on a C - 1 8 c o l u m n u s i n g 0.1% T F A as the ion p a i r i n g a g e n t and 0.1% T F A w i t h 80% a c e t o n i t r i l e as the organic modifier. Methods S p r a g u e - D a w l e y r a t s w e r e k i l l e d u s i n g CO 2. M i t o c h o n d r i a w e r e i s o l a t e d as p r e v i o u s l y d e s c r i b e d (Ii). Experiments were carried out in a b u f f e r (pH = 7.4) consisting of s u c r o s e (0.225M), K2HPO,/KH2PO 4 b u f f e r (10mM), MgCI2(5mM), KCI (20mM), H E P E S (20mM) and supplemented w i t h A D P (0.4mM) a n d s u c c i n a t e 4mM as noted. P r o t e i n d e t e r m i n a t i o n s w e r e m a d e u s i n g the B i o r a d a s s a y ( R o c k v i l l e C e n t r e , N.Y.). V a r i o u s c o n c e n t r a t i o n s of M T in d i s t i l l e d d e i o n i z e d H20 w e r e a d d e d to a m i t o c h o n d r i a l s u s p e n s i o n of 0 . 2 m g p r o t e i n / m l . In c o n t r o l e x p e r i m e n t s d i s t i l l e d d e i o n i z e d w a t e r a l o n e in t h e s a m e v o l u m e w a s a d d e d to t h e m i t o c h o n d r i a l suspension. The total r e a c t i o n m i x t u r e v o l u m e w a s 2ml. A d d i t i o n s of M T or o t h e r r e a g e n t s e m p l o y e d v o l u m e s w h i c h n e v e r e x c e e d e d 20ul. O x y g e n c o n s u m p t i o n (VO2) w a s m e a s u r e d p o l a r o g r a p h i c a l l y u s i n g a C l a r k - t y p e o x y g e n e l e c t r o d e (12). All e x p e r i m e n t s w e r e p e r f o r m e d at 25°C. A d d i t i o n s w e r e m a d e at i n t e r v a l s of 4 m i n u t e s . Results M T at 5 0 u M s i g n i f i c a n t l y increased the r a t e of VO2 w i t h s u c c i n a t e as s u b s t r a t e . A l s o A D P - i n i t i a t e d V O 2 w a s i n h i b i t e d in the presence of 5 0 u M M T ( f i g u r e i). No c h a n g e in V02 w a s n o t e d when MT was added to m i t o c h o n d r i a in t h e a b s e n c e of s u c c i n a t e or ADP. T a b l e o n e s h o w s t h e r e s u l t s of e i g h t e x p e r i m e n t s u s i n g M T at 50uM. T h e c o n c e n t r a t i o n d e p e n d e n c e of M T e f f e c t s on 02 c o n s u m p t i o n are s h o w n in f i g u r e 2. T h e e n h a n c e m e n t of s u c c i n a t e - i n i t i a t e d V02 and t h e i n h i b i t i o n of A D P - i n i t i a t e d V02 b o t h e x h i b i t e d a p l a t e a u b e g i n n i n g at 5 0 u M MT.
Vol. 55, No. 3, 1994
MT on Mitochondrial Oxygen Consumption
$
%
2 min.
l
l
Fig.
1
E f f e c t of M T o n m i t o c h o n d r i a l V O z. = no MT, = 5 0 u M MT. S=succinate (4mM) a n d A = A D P (0.4mM). Mitochondria, succinate, and ADP were added sequentially at intervals of 4 minutes.
Table
1
Effect of MT on V O 2. Data are in units of nmole O2/min.mg. Data from e a c h of t h e e i g h t rats is t h e result of 2-4 replicates. There was less than 10% variation between replicates, p < 0.05 by paired "t" test for differences between rates with and without MT after addition of s u c c i n a t e (4mM) o r A D P ( 0 . 4 m M ) . MT = 50uM. Rat
Mean
#
1 2 3 4 5 6 7 8 ± SE
Succinate without MT 28.4 32.3 36.6 28.5 18.8 29.2 17.9 26.1 2 7 . 2 ± 2.2
oxidation with MT 87.9 35.0 54.9 31.8 43.8 32.3 47.7 47.8 47.7 ± 6.5
A D P i n i t i a t e d VO2 without MT with MT 104.5 79.3 116 3 93.8 87 6 48.4 69 5 43.5 50 2 43.8 106 2 55.6 54 7 47.7 71.7 44.5 8 2 . 6 + 8.8 57.1+6.7
223
224
MT on Mitochondrial Oxygen Consumption
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80 70
O
40
o
20
2b 4b 6b ~
160 i~0 i~0 i~0 i~0 260 [MT](uM)
Fig.
2
Concentration dependence of t h e e f f e c t of M T on mitochondrial respiration. 02 consumption is in n m o l e 0 2 / m i n . m g . o = VO 2 after addition of 4mM succinate. . = VO 2 after further addition of 0.4mM ADP. O t h e r d e t a i l s w e r e as f o r Fig. i. E a c h d a t a p o i n t is t h e r e s u l t of 2 - 3 replicates. Replicates w e r e w i t h i n 10% of e a c h o t h e r . One possible mechanism f o r t h e e f f e c t of M T w a s t h e p r o v i s i o n b y M T of a n a l t e r n a t e pathway for election transfer to oxygen. In order to test for this we used 10mM KCN to block electron transfer from cytochrome oxidase to oxygen. MT did not overcome the complete inhibition of succinate or ADP initiated VO 2 by KCN (data not shown). Furthermore, when MT was added to all reactants in buffer without mitochondria, no oxygen was consumed. This ruled out the possibility that MT alone was removing oxygen from the solution. Another possibility was that the actions of M T w e r e c a u s e d solely by the metals Zn +2 o r C d +2, e x c l u s i v e of t h e protein. Therefore, we measured the effect of ZnCl 2 and CdCI 2 on 02 consumption of mitochondria. The data are summarized in T a b l e 2. This experiment demonstrated t h a t t h e e f f e c t s of t h e m e t a l s alone are very different from those of MT. At a metal concentration of luM there was complete inhibition of VO2 consumption, r e g a r d l e s s of w h e t h e r t h e i n i t i a t i n g f a c t o r w a s A D P o r succinate.
Vol. 55, No. 3, 1994
MT on Mitochondrial Oxygen Consumption
Table
225
2
E f f e c t of C d C I 2 a n d ZnCI 2 on o x y g e n c o n s u m p t i o n . Data are the r e s u l t of 2 r e p l i c a t e s . T h e r e w a s less t h a n 10% d i f f e r e n c e b e t w e e n replicates. A D P = 0.4mM. S u c c i n a t e = 4mM. R a t e s a r e in u n i t s of n m o l e O=/min.mg. [ZnCl~ 0 0.01uM 0.10uM 1.0 u M [CdCl~1 0 0.01uM 0.10uM 1.0 u M ZnCI= 0 . 0 5 u M + CdCI2 0 . 0 5 u M
Succinate oxidation 37.8 34.4 25.8 0
34.4 27.5 37.8 0 34.4
ADP
initiated 123.8 130.7 61.9 0
VO2
87.7 92.9 72.2 0 75.7
Discussion An a b n o r m a l p a t t e r n of 02 u t i l i z a t i o n and i n c r e a s e d a n a e r o b i c m e t a b o l i s m a r e c h a r a c t e r i s t i c of s e v e r e c r i t i c a l i l l n e s s e s , s u c h as s e p t i c s h o c k (13). A t t e m p t s to e x p l a i n t h e s e c h a r a c t e r i s t i c s b y f i n d i n g d e f e c t i v e f u n c t i o n in i s o l a t e d m i t o c h o n d r i a l p r e p a r a t i o n s have not been successful. O u r f i n d i n g in m i t o c h o n d r i a t h a t M T enhances succinate oxidation and inhibits ADP initiated 02 consumption provides a possible explanation. Several characteristics of M T m a k e it a g o o d c a n d i d a t e for t h i s role. O n e c h a r a c t e r i s t i c is the f a c t t h a t M T is i n d u c e d b y c y t o k i n e s s u c h as IL-I~, T N F ~ and IL-6, in a w i d e r a n g e of o r g a n s (9). M T is a b u n d a n t in t h e c y t o p l a s m - o c c u r r i n g in u p to m M l e v e l s (14). MT theoretically could impact significantly upon mitochondrial function and other cellular processes. T h e m e c h a n i s m of t h e M T e f f e c t s on m i t o c h o n d r i a n e e d s to be explored. T h e i n c r e a s e in s t a t e IV e l e c t r o n f l o w ( s u c c i n a t e only) suggests uncoupling. T h e d e c r e a s e in s t a t e III e l e c t r o n f l o w (succinate + ADP) suggests inhibition of t h e FoF I A T P a s e or decoupling (15). It is u n l i k e l y t h a t M T a c t s as a s o u r c e of a d d i t i o n a l e l e c t r o n s v i a c y t o c h r o m e c or o t h e r c o m p o n e n t s of t h e r e s p i r a t o r y c h a i n s i n c e M T a l o n e h a d no e f f e c t on m i t o c h o n d r i a V O 2. A n o t h e r p o s s i b i l i t y is t h a t t h e c y s t e i n e s of M T c o u l d act v i a t h e m i t o c h o n d r i a l e n z y m e c o n j u g a t e B - l y a s e (16). T h e a c t i o n of M T could be mediated v i a its m a n y r e d u c e d c y s t e i n e s , or by t h e cysteines and metals acting interdependently. F u t u r e w o r k w i l l be d i r e c t e d t o w a r d u n d e r s t a n d i n g t h e r e l a t i o n s h i p of t h e s t r u c t u r e of
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M T a n d its e f f e c t on m i t o c h o n d r i a l V O 2. We w i l l further the possible r o l e of M T as a m o d u l a t o r p h o s p h y o r y l a t i o n in s t r e s s states.
Vol. 55, No. 3, 1994
also explore of o x i d a t i v e
Acknowledgements We w o u l d like to t h a n k Dr. P a t r i c i a S o k o l o v e for u s e of t h e p o l a r i m e t e r a n d h e r h e l p f u l d i s c u s s i o n s ; Drs. T y s o n T i l d o n , M a r y McKenna and Timothy Buchman for t h e i r h e l p f u l comments. We a p p r e c i a t e t h e e d i t o r i a l a s s i s t a n c e of Mrs. R e b e c c a C o l e m a n . This w o r k w a s s u p p o r t e d in p a r t by t h e O f f i c e of N a v a l R e s e a r c h , g r a n t no. N 0 0 0 1 4 - 9 3 - I - 0 2 2 7 . References I. 2. 3. 4. 5.
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