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Biochimica el Biophysica Acta, 464 (1977 ) 287--294
© Elsevier/North-Holland Biomedical Press
BBA 77575 EFFECT OF INHIBITORS ON THE SIGMOIDICITY OF THE CALCIUM ION TRANSPORT KINETICS IN RAT LIVER MITOCHONDRIA
KARL E.O. ~KERMAN, MARTEN K.F. WIKSTROM and NILS-ERIK SARIS Department of Medical Chemistry, University o f Iielsinki, Siltavuorenpenger 10 A, SF001 70 Ifelsinki 1 7 (Finland)
(Received June 2nd, 1976) (Revised manuscript received September 6th, 1976)
Summary T h e kinetic p l o t (initial rate o f Ca 2÷ t r a n s p o r t versus c o n c e n t r a t i o n ) of m i t o c h o n d r i a l Ca z÷ t r a n s p o r t is h y p e r b o l i c in a sucrose m e d i u m . The p l o t b e c o m e s sigmoidal in the p r e s e n c e o f c o m p e t i t i v e inhibitors o f Ca 2÷ binding to low affinity sites o f t h e m e m b r a n e surface such as Mg 2÷ and K +. T h e p l o t also b e c o m e s sigmoidal in the p r e s e n c e o f Ba 2÷. Ba 2+ is a c o m p e t i t i v e i n h i b i t o r of b o t h Ca :÷ t r a n s p o r t and Ca 2÷ binding to the l o w a f f i n i t y sites. T h e Ki f o r the i n h i b i t i o n of Ca 2÷ t r a n s p o r t b y Ba 2÷ increases in the p r e s e n c e o f K ÷ and Mg :÷, which suggests a c o m p e t i t i o n f o r the low a f f i n i t y sites b e t w e e n the cations. T h e p l o t is still h y p e r b o l i c in the p r e s e n c e o f L a 3+, which inhibits Ca 2+ t r a n s p o r t c o m p e t i t i v e l y . R u t h e n i u m red which is a p u r e n o n - c o m p e t i t i v e i n h i b i t o r o f m i t o c h o n d r i a l Ca 2+ t r a n s p o r t , does n o t a f f e c t the s h a p e o f t h e kinetic plot. These results indicate t h a t the surface p o t e n t i a l , which d e p e n d s on the ions b o u n d to the low a f f i n i t y sites, d e t e r m i n e s w h e t h e r the kinetics o f Ca 2+ u p t a k e in m i t o c h o n d r i a is sigrnoidal or h y p e r b o l i c .
Introduction A s i g m o i d ~ p l o t is o b t a i n e d w h e n the initial rate o f m i t o c h o n d r i a l Ca 2~ t r a n s p o r t is p l o t t e d against the free Ca ~+ c o n c e n t r a t i o n [ 1 - - 3 ] . T h e s e results have b e e n i n t e r p r e t e d to be due to e o o p e r a t i v i t y in the Ca 2+ t r a n s p o r t s y s t e m . T h e sigmoidal kinetics i m p l y t h a t m i t o e h o n d r i a l Ca 2+ t r a n s l o e a t i o n w o u l d be very slow at the l o w (10 -6 M) Ca 2+ c o n c e n t r a t i o n s in the c y t o s o l . This clearly w o u l d i n f l u e n c e t h e i n t e r p r e t a t i o n o f the role o f m i t o c h o n d r i a in the r e g u l a t i o n o f c y t o s o l i c Ca 2+. Ca 2÷ is b o u n d to e n e r g y - i n d e p e n d e n t binding sites with low Abbreviations: PPO. 2,5-diphenyloxazole; POPOP. 1,4-bis-(5-phenyloxazolyl-2)-benzene; EGTA, cthylencglycol bis(ct-aminocthylether)-N,N'-tetraacetic acid.
288 affinity at the outer surface of the mitochondrial membrane [4--6]. The a m o u n t o f these sites is about 20 nmol/mg protein [5] and they are competitively inhibited by Mg2+> K+> H* [7--9] and are mainly phospholipid in nature [7--10]. These binding sites have in some models been considered to be involved in the transport of Ca 2. [8,11,12]. A n u m b e r of inhibitors of Ca 2~ transport have been used in order to gain more insight into the mechanism ot transport. La 3÷ and related rare earth cations inhibit mitochondrial Ca 2÷ transport in a competitive manner [13--17], while Ruthenium red is a c o m m o n l y used noncompetitive inhibitor [16,18,19]. Their K i is very low (about 0.03 pM), compared to the Ca 2÷ concentrations c o m m o n l y used for Ca :+ transport measurements. Ba 2÷ is taken up by m i t oc ho ndri a in a similar way to Ca 2÷ [17, 20] and thus would be a competitive inhibitor of Ca 2+ transport. The aim of this work was to study the effects of these inhibitors of Ca e+ binding and transport on the transport kinetics. Methods and Materials Rat-liver mito c hondr i a were prepared from young male rats (Sprague-Dawley) by a conventional procedure [21]. Ca 2+ uptake was measured by using the EG T A quenching m e t h o d essentially as described by Reed and Bygrave [16], in order to be able to distinguish between external binding and transport of Ca 2÷. No Ca 2+ buffers were used because the agents studied might affect the buffering. The buffers might also bind the inhibitors and thus decrease their active concentration. The mitochondria were removed from the medium either by Millipore filtration (pore size 0.6 pro) or rapid centrifugation. Energy-independent Ca 2+ binding was measured as described before [22 ]. The 4SCa was c ount ed in a Packard Tricarb liquid scintillation spectrometer in Bray's solution (PPO, POPOP, naphthalene, ethylenglycol, dioxan and methanol). Three different reaction media were used: 0.25 M sucrose/20 mM Tris • CI, pH 7.4 (sucrose medium). The same medium with 2 mM MgC12 (sucrose/Mg medium), and 130 mM KC1/20 mM Tris . C1/2 mM MgC12, pH 7.4 (KC1/Mg medium). Reagents: FCCP (carbonylcyanide p - t r i f l u o r o m e t h o x y p h e n y l h y d r a z o n e ) was obtained from Dr. P.G. Heytler and R ut heni um red from BDtt Chemicals Ltd, Poole, England. The Ruthenium red was recrystallized accoring to Luft [23] before use. Results E f f e c t o f Mg 2÷ and K+ on the Ca 2÷ transport kinetics
A hyperbolic plot is obtained when mitochondrial C a 2+ uptake is measured in the sucrose medium at +5°C and the initial rate is plotted against the Ca 2* concentration. In the presence of Mg 2+ the kinetic plot becomes sigmoidal. In the KCI/Mg medium the sigmoidicity is still more p r o n o u n c e d (Fig. 1). This effect is of interest because neither Mg 2÷ nor K* is translocated by rat-live," mitochondria under these conditions, but both are competitive inhibitors of the binding o f Ca 2. to the low affinity sites [5,7--9].
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E f f e c t o f Ba 2+ on the Ca z+ transport kinetics and on Ca 2+ binding to the low a f f i n i t y sites Ba 2+ is transloeated into m i t o e h o n d r i a in a similar w a y as Ca 2+ [ 1 7 , 2 1 ] . It also induces similar respiratory stimulation [ 2 1 ] . Thus Ba :+ m a y be e x p e c t e d to inhibit m i t o c h o n d r i a l Ca 2+ transport c o m p e t i t i v e l y . Fig. 2 s h o w s the effect of Ba 2+ on the Ca 2+ transport kinetics. In the sucrose m e d i u m the plot b e c o m e s
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B(] 2° c0ncentretl0n Fig. 3. D i × o n plots f o r the i n h i b i t i o n o f Ca 2+ b i n d i n g to the low affinity sites by Ba 2+. M i t o e h o n d r i a (5 m g p r o t e i n / m l w e r e i n c u b a t e d in t h e s u c r o s e m e d i u m c o n t a i n i n g 5 p M F C C P a n d 1 /aM R u t h e n i u m r e d f o r 1 r a i n . T h e r e a f t e r 4 5 C A C 1 2 at a c o n c e n t r a t i o n o f 2 5 p M (c~ ~), 50pM (e-e) and lOOpM (,",-~) was added and the incubation was continued for 1 min. The mitochondria were removed from the medium by centrifugation (14 000 Xg) and samples were taken from the pellets for counting a f t e r s o l u b i l i s a t i o n in 1 M f o r m i c a c i d .
sigmoidal in the p r e s e n c e o f Ba 2+ (Fig. 2a). In the KC1/Mg m e d i u m Ba 2+ increases the s i g m o i d i c i t y o f t h e p l o t (Fig. 2b). In Fig. 2 it is also seen that Ba 2. inhibits Ca 2÷ transport m o r e s t r o n g l y in the sucrose m e d i u m as c o m p a r e d to the KC1/Mg m e d i u m . D i x o n and L i n e w e a v e r - B u r k plots ( n o t s h o w n ) indicate that the K i o f Ba 2+ varies b e t w e e n less than 10 pM at l o w e r Ca 2+ c o n c e n t r a t i o n s ( 1 0 - - 3 0 pM), to a b o u t 70 pM, at higher Ca 2+ c o n c e n t r a t i o n s ( 5 0 - - 1 0 0 pM). In the KC1/Mg m e d i u m t h e Ki o f Ba 2+ is a b o u t 1 0 0 pM over t h e w h o l e Ca 2+ c o n c e n t r a t i o n range as c a l c u l a t e d f r o m D i x o n plots. D u e to the similar c h e m i s t r y o f Ba 2+ and Ca 2+ the f o r m e r m a y be e x p e c t e d to inhibit all individual steps in the Ca :+ transport process. Fig. 3 s h o w s D i x o n plots for the i n h i b i t i o n o f Ca 2+ binding to the l o w affinity sites by Ba 2+.
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F i g . 4. E f f e c t o f L a 3+ o n m i t o e h o n d r i a l Ca 2+ t r a n s p o r t k i n e t i c s . E x p e r i m e n t a l c o n d i t i o n s as in l~'ig. 1. I n c u b a t i o n s w e r e m a d e i n ( A ) t h e s u c r o s e m e d i u m (~) c,) c o n t a i n i n g 0 . 0 6 p M L a C I 3 ( e - . e ) , (B) t h e K C I / M g m e d i u m (,i >) c o n t a i n i n g 0 . 0 3 p M ( e - e) and 0.06 pM LaCl 3 (:2,). M i t o c h o n d r i a l p r o t e i n I m g / m ] i n ( A ) a n d 0 . 5 m g / m l i n (B).
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F i g . 5. E f f e c t o f R u t h e n i u m r e d o n m i t o c h o n d r i a l Ca 2+ t r a n s p o r t k i n e t i c s . E x p e r i m e n t a l c o n d i t i o n s as in Fig. 1. I n c u b a t i o n s w e r e m a d e ( A ) i n t h e K C 1 / M g m e d i u m ( A - - - - A ) containing 0.06 pM Ruthenium red (f, ~/) o r ( B ) in t h e s u c r o s e m e d i u m ( ) - - c ) c o n t a i n i n g 0 . 0 3 ~ M (z~-:',) o r 0 . 0 6 p M R u t h e nium red (e o).
E f f e c t o f La 3÷on the Ca 2÷ transport kinetics
In the sucrose m e d i u m the kinetic p l o t is still h y p e r b o l i c in the presence of La 3÷ (Fig. 4a). In the KC1/Mg m e d i u m the shape of the p l o t b e c o m e s slightly m o r e sigmoidal in the presence o f La 3÷ (Fig. 4b). Dixon plots ( n o t s h o w n ) for the inhibition of Ca z÷ t r a n s p o r t b y La 3* indicate t h a t the K i o f La 3+ in the sucrose m e d i u m does n o t differ significantly f r o m t h a t in the KC1/Mg m e d i u m . Due to the very low c o n c e n t r a t i o n used ( 0 . 0 3 - - 0 . 0 6 pM) La 3+ o u g h t n o t to a f f e c t Ca'* binding to the low affinity sites significantly. E f f e c t o f R u t h e n i u m red o17 the Ca 2÷ transport kinetics
R u t h e n i u m red does n o t a f f e c t the shape o f the kinetic plot to any significant degree. It is equally p o t e n t at all Ca 2÷ c o n c e n t r a t i o n s used. This is s h o w n in Fig. 5. D i x o n plots ( n o t s h o w n ) also indicate t h a t R u t h e n i u m red is an equally p o t e n t i n h i b i t o r during the slow phase (15 pM) and during the steep rise of the kinetic p l o t (50 /aM) and t h a t the K i in the sucrose m e d i u m does n o t differ significantly f r o m the Ki in the KC1/Mg m e d i u m . Because the Ki f o r R u t h e n i u m red is in the same range as t h a t for La 3+ [16] it is n o t e x p e c t e d to i n t e r a c t with the low affinity binding o f Ca 2+. Discussion
The results o f this w o r k show t h a t a h y p e r b o l i c kinetic p l o t (initial rate versus c o n c e n t r a t i o n ) is o b t a i n e d for Ca 2÷ t r a n s p o r t in rat-liver m i t o e h o n d r i a when s u s p e n d e d in a sucrose m e d i u m . This holds for a Ca 2÷ c o n c e n t r a t i o n range of 5 - - 1 0 0 pM. In the presence o f Mg 2+ the p l o t b e c o m e s sigmoidal. This e f f e c t is e n h a n c e d w h e n b o t h Mg 2+ and K ÷ are present. Since b o t h Mg z÷ and K* inhibit Ca 2÷ binding to the low affinity sites their effects on calcium t r a n s p o r t m a y well result f r o m an i n t e r f e r e n c e with Ca 2+ binding to these sites. K* alone does n o t change the shape o f kinetic plots significantly ( s u b m i t t e d for p u b l i c a t i o n ) ,
292 possibly b e c a u s e of the sites having a l o w e r a f f i n i t y for K + or b e c a u s e of K ~ having only o n e charge. N e i t h e r Mg 2+ n o r K + is t r a n s l o c a t e d b y the m i t o c h o n dria u n d e r the e x p e r i m e n t a l c o n d i t i o n s . S o m e w o r k e r s [1,2] have r e p o r t e d sigmoidal Ca 2+ t r a n s p o r t kinetics also in a sucrose m e d i u m . H o w e v e r , t h e y used A T P as a Ca 2+ b u f f e r a n d were thus able to m e a s u r e Ca 2÷ t r a n s p o r t at very low free Ca 2+ c o n c e n t r a t i o n s . T h e sigmoidieity of these plots was a p p a r e n t at free Ca :+ c o n c e n t r a t i o n s near 1 t~M. T h u s it a p p e a r s possible t h a t Mg 2÷ and K + do slot actually change the h y p e r b o l i c kinetics to sigmoidal b u t m a y shift the p o i n t at which the s i g m o i d i c i t y occurs to higher Ca 2+ c o n c e n t r a t i o n s . T h e use of Ca 2+ b u f f e r s ill this w o r k was n o t possible due to i n t e r a c t i o n of the used inhibitors w i t h the buffer. T h e sigmoidal kinetics have previously ( V i n o g r a d o v and Scarpa, ref. 3) been ascribed t o a c o o p e r a t i v e t r a n s p o r t m e c h a n i s m w h e r e binding of t w o Ca 2+ to a carrier w o u l d be r e q u i r e d for t r a n s l o e a t i o n . T h e y m e a s u r e d Ca 2÷ t r a n s p o r t in a KC1/Mg m e d i u m using the m u r e x i d e t e c h n i q u e and o b t a i n e d a result very similar to ours (Fig. 1). T h e y also r e p o r t e d a change f r o m sigmoidal to h y p e r b o l i c Mn 2+ t r a n s p o r t kinetics in the p r e s e n c e of Ca 2÷. H o w e v e r , m e a s u r e m e n t of cation t r a n s p o r t with the m u r e x i d e t e c h n i q u e in the p r e s e n c e of o t h e r cations t h a t also i n t e r a c t with m u r e x i d e is q u e s t i o n a b l e . T h e results o f this w o r k s h o w t h a t Ba 2., which like Mn 2+ is t r a n s l o c a t e d b y m i t o e h o n d r i a , does n o t change the kinetics o f Ca 2÷ t r a n s p o r t f r o m sigmoidal to h y p e r b o l i c . If a n y t h i n g Ba > e n h a n c e s the sigmoidicity o f the kinetic p l o t (see Fig. 2b). Ca 2+, Mn 2+ and Ba 2* could of course be t r a n s l o e a t e d b y d i f f e r e n t m e c h a n i s m s . H o w e v e r , this a p p e a r s highly u n l i k e l y in view o f the m a n y similarities in t h e i r m o d e of t r a n s p o r t [ 2 0 ] . Ba 2÷ is a c o m p e t i t i v e i n h i b i t o r of Ca 2+ t r a n s p o r t . It is of interest t h a t Ba 2+ changes the Ca 2+ t r a n s p o r t kinetics f r o m h y p e r b o l i c to sigmoidal like Mg 2÷. It is also of interest t h a t the K i of Ba 2+ f o r Ca > t r a n s p o r t increases in the KC1/Mg m e d i u m as c o m p a r e d to the sucrose m e d i u m . This w o u l d suggest a c o m p e t i t i o n b e t w e e n the cations (K +, Mg > and Ba 2+) f o r the low a f f i n i t y sites. T h e inhibition o f Ca > binding to the low a f f i n i t y sites b y Ba 2÷ c o n f i r m s this. T h e results r e p o r t e d here indicate t h a t an unspecific a l t e r a t i o n of the surface o f the m e m b r a n e m i g h t d e t e r m i n e the shape of the kinetic plot. T h e surface p o t e n t i a l is d e t e r m i n e d b y the charge d e n s i t y and h e n c e p r e s u m a b l y b y the ions b o u n d at. n o n s p e c i f i c sites. S p e r m i n e , a t e t r a v a l e n t p o l y a m i n e , k n o w n to decrease the negative surface charge of m i t o c h o n d r i a significantly [ 2 4 ] , also changes the s h a p e of kinetic plots in a similar w a y to Mg > ( s u b m i t t e d for publication), which gives f u r t h e r evidence in f a v o u r of the a b o v e i n t e r p r e t a t i o n . M o r e o v e r it has r e c e n t l y b e e n d e m o n s t r a t e d [25] t h a t t r a n s p o r t kinetics of ions m a y m i m i c the kinetics of allosterie m e c h a n i s m s (sigmoidal kinetics) w h e n the surface has the same charge as the t r a n s l o c a t e d ion. O u r results indeed suggest t h a t the surface p r o p e r t i e s o f the m i t o c h o n d r i a l m e m b r a n e have great influence on the Ca :+ t r a n s p o r t kinetics. Mg 2+ and s p e r m i n e also decrease the a c t i v a t i o n energy of Ca 2+ t r a n s p o r t significantly, whereas n e i t h e r affects t h a t o f respiration [26] suggesting t h a t n o t o n l y the charge b u t also the m o b i l i t y of the polar h e a d - g r o u p s of p h o s p h o l i p i d s at the surface of the m i t o c h o n d r i a l m e m b r a n e m i g h t be o f i m p o r t a n c e in the kinetics of C a 2+ t r a n s p o r t . La 3+ and r u t h e n i u m red, which are i n h i b i t o r y at very low c o n c e n t r a t i o n s (K~ a b o u t 0.04 t~M), d o slot a f f e c t the s h a p e of the kinetic p l o t significantly.
293
This also is c o n s i s t e n t with the above proposal t h a t the surface p r o p e r t i e s of the m i t o c h o n d r i a l m e m b r a n e might have great influence on the Ca 2÷ t r a n s p o r t kinetics, because n e i t h e r o f these agents is e x p e c t e d to a f f e c t the surface charge significantly at the very low c o n c e n t r a t i o n used. It is c o n c l u d e d t h a t Ca 2÷ t r a n s p o r t in m i t o c h o n d r i a involves at least two steps. First, the m o v e m e n t o f the cation to the surface o f the m e m b r a n e with or w i t h o u t s u b s e q u e n t binding to the unspecific low affinity sites, and s e c o n d l y , the actual t r a n s l o c a t i o n process. The first step is strongly d e p e n d e n t on the surface p o t e n t i a l and h e n c e on the n u m b e r and charge of ions b o u n d to the surface sites as K + and Mg 2÷. It is interesting to speculate t h a t changes in the electrical surface properties of the m i t o c h o n d r i a l m e m b r a n e (by c o n t r o l of the binding c o n s t a n t s for K ÷, Mg 2+ etc.) m a y c o n t r o l the kinetics of Ca 2+ t r a n s p o r t also in vivo. A change f r o m sigmoidal to h y p e r b o l i c Ca 2÷ u p t a k e kinetics would have p r o f o u n d effects on the translocation rate especially at the low intracellular Ca 2+ c o n c e n t r a t i o n s . R e c e n t l y it has been d e m o n s t r a t e d t h a t Ca 2÷ t r a n s p o r t in s m o o t h muscle mitoc h o n d r i a o b e y s essentially h y p e r b o l i c kinetics [ 2 7 , 2 8 , 2 9 ] even in the presence of K ÷ and Mg 2÷. Thus it appears possible t h a t the surface of these m i t o c h o n d r i a might have d i f f e r e n t properties f r o m those o f liver and heart. It has also been s h o w n t h a t muscle m i t o c h o n d r i a f r o m various aquatic a r t h r o p o d s show a large variety o f energN i n d e p e n d e n t Ca 2÷ binding characteristics, i.e. variations in binding c o n s t a n t s and n u m b e r of binding sites for Ca 2÷ [ 3 0 ] . This suggests that the surface p r o p e r t i e s of m i t o c h o n d r i a l melnbranes might vary significantly b e t w e e n d i f f e r e n t tissues and species. Acknowledgements This s t u d y was aided by a grant f r o m the Sigrid Jus61ius F o u n d a t i o n . The a u t h o r s are grateful to Mrs Kaija Viitanen for skilful technical assistance. Re ferences 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
B y g r a v e , F . L . , R e e d , K.C. a n d S p e n c e r , T. ( 1 9 7 1 ) Nat. N e w Biol. 230, 89 S p e n c e r , T. a n d B y g r a v e , F.L. ( 1 9 7 3 ) B i o e n e r g e t i c s 4, 3 4 7 - - 3 6 2 V i n o g r a d o v , A. a n d S c a r p a , A. ( 1 9 7 3 ) J. Biol. C h e m . 2 4 8 , 5 5 2 7 - - 5 5 3 1 Rossi, C., Azzi, A. a n d A z z o n e , G . F . ( 1 9 6 7 ) J. Biol. C h e m . 242, 9 5 1 - 9 5 7 R e e d , K.C. a n d B y g r a v e , F.L. ( 1 9 7 4 ) B i o c h e m . J. 142, 5 5 5 566 C h a p p e l l , J.B., C o h n , M. a n d Greville, G.D. ( 1 9 6 3 ) in E n e r g y - L i n k e d F u n c t i o n s of M i t o c h o n d r i a ( C h a n c e , B., ed.), pp. 2 1 9 - - 2 3 1 , A c a d e m i c Press, N e w Y o r k a n d L o n d o n S c a r p a , A. a n d Azzi, A. ( 1 9 6 8 ) B i o c h i m . B i o p h y s . A c t a 1 5 0 , 4 7 3 - - 4 8 1 S c a r p a , A. a n d A z z o n e , G.F. ( 1 9 6 8 ) J. Biol. C h e m . 2 4 3 , 5 1 3 2 - - 5 1 3 8 J a c n b u s , W.E. a n d B r i e r l e y , G.P. ( 1 9 6 9 ) J. Biol. C h e m . 244, 4 9 9 5 - - 5 0 0 4 S c a r p a , A. a n d A z z o n e , G . F . ( 1 9 6 9 ) B i o e h i m . B i o p h y s . A c t a 173, 7 8 - - 8 5 Saris, N.-E.L. ( 1 9 7 2 ) in B i o c h e m i s t r y a n d B i o p h y s i c s o f M i t o c h o n d r i a l M e m b r a n e s , pp. 6 4 1 - 4 4 5 2 . A c a d e m i c Press, N e w Y o r k a n d L o n d o n A z z o n e , G . F . , Massari, S., Rossi, E. a n d S e a r p a , A. in M i t o c h n n d r i a S t r u c t u r e a n d F u n c t i o n ( E r n s t e r . L. a n d D r a h o t a , Z., eds.), pp. 3 0 1 - - 3 1 8 , A c a d e m i c Press, N e w Y o r k a n d L o n d o n Mcla, L. ( 1 9 6 7 ) F e d . Proc. F e d . A m e r . Sci. E x p . Biol. 2 6 , 4 5 6 Mela, L. ( 1 9 6 8 ) A r c h . B i o c h e m . B i o p h y s . 123, 286 293 Mela, L. ( 1 9 6 9 ) B i o c h e m i s t r y 8, 2 4 8 1 - - 2 4 8 6 R e e d , K.C. a n d B y g r a v e , F.L. ( 1 9 7 4 ) B i o c h e m . J. 140, 143 155 V a i n i o , It., Mela, L. a n d C h a n c e , B. ( 1 9 7 0 ) Eur. J. B i o c h e m . 12, 3 8 7 - - 3 9 1 M o n r e , C.L. ( 1 9 7 1 ) B i o e h e m . B i o p h y s . Res. C o m m . 42, 2 9 8 3 0 5
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Biochimica el Biophysica Acta, 464 (1977 ) 287--294
© Elsevier/North-Holland Biomedical Press
BBA 77575 EFFECT OF INHIBITORS ON THE SIGMOIDICITY OF THE CALCIUM ION TRANSPORT KINETICS IN RAT LIVER MITOCHONDRIA
KARL E.O. ~KERMAN, MARTEN K.F. WIKSTROM and NILS-ERIK SARIS Department of Medical Chemistry, University o f Iielsinki, Siltavuorenpenger 10 A, SF001 70 Ifelsinki 1 7 (Finland)
(Received June 2nd, 1976) (Revised manuscript received September 6th, 1976)
Summary T h e kinetic p l o t (initial rate o f Ca 2÷ t r a n s p o r t versus c o n c e n t r a t i o n ) of m i t o c h o n d r i a l Ca z÷ t r a n s p o r t is h y p e r b o l i c in a sucrose m e d i u m . The p l o t b e c o m e s sigmoidal in the p r e s e n c e o f c o m p e t i t i v e inhibitors o f Ca 2÷ binding to low affinity sites o f t h e m e m b r a n e surface such as Mg 2÷ and K +. T h e p l o t also b e c o m e s sigmoidal in the p r e s e n c e o f Ba 2÷. Ba 2+ is a c o m p e t i t i v e i n h i b i t o r of b o t h Ca :÷ t r a n s p o r t and Ca 2÷ binding to the l o w a f f i n i t y sites. T h e Ki f o r the i n h i b i t i o n of Ca 2÷ t r a n s p o r t b y Ba 2÷ increases in the p r e s e n c e o f K ÷ and Mg :÷, which suggests a c o m p e t i t i o n f o r the low a f f i n i t y sites b e t w e e n the cations. T h e p l o t is still h y p e r b o l i c in the p r e s e n c e o f L a 3+, which inhibits Ca 2+ t r a n s p o r t c o m p e t i t i v e l y . R u t h e n i u m red which is a p u r e n o n - c o m p e t i t i v e i n h i b i t o r o f m i t o c h o n d r i a l Ca 2+ t r a n s p o r t , does n o t a f f e c t the s h a p e o f t h e kinetic plot. These results indicate t h a t the surface p o t e n t i a l , which d e p e n d s on the ions b o u n d to the low a f f i n i t y sites, d e t e r m i n e s w h e t h e r the kinetics o f Ca 2+ u p t a k e in m i t o c h o n d r i a is sigrnoidal or h y p e r b o l i c .
Introduction A s i g m o i d ~ p l o t is o b t a i n e d w h e n the initial rate o f m i t o c h o n d r i a l Ca 2~ t r a n s p o r t is p l o t t e d against the free Ca ~+ c o n c e n t r a t i o n [ 1 - - 3 ] . T h e s e results have b e e n i n t e r p r e t e d to be due to e o o p e r a t i v i t y in the Ca 2+ t r a n s p o r t s y s t e m . T h e sigmoidal kinetics i m p l y t h a t m i t o e h o n d r i a l Ca 2+ t r a n s l o e a t i o n w o u l d be very slow at the l o w (10 -6 M) Ca 2+ c o n c e n t r a t i o n s in the c y t o s o l . This clearly w o u l d i n f l u e n c e t h e i n t e r p r e t a t i o n o f the role o f m i t o c h o n d r i a in the r e g u l a t i o n o f c y t o s o l i c Ca 2+. Ca 2÷ is b o u n d to e n e r g y - i n d e p e n d e n t binding sites with low Abbreviations: PPO. 2,5-diphenyloxazole; POPOP. 1,4-bis-(5-phenyloxazolyl-2)-benzene; EGTA, cthylencglycol bis(ct-aminocthylether)-N,N'-tetraacetic acid.
288 affinity at the outer surface of the mitochondrial membrane [4--6]. The a m o u n t o f these sites is about 20 nmol/mg protein [5] and they are competitively inhibited by Mg2+> K+> H* [7--9] and are mainly phospholipid in nature [7--10]. These binding sites have in some models been considered to be involved in the transport of Ca 2. [8,11,12]. A n u m b e r of inhibitors of Ca 2~ transport have been used in order to gain more insight into the mechanism ot transport. La 3÷ and related rare earth cations inhibit mitochondrial Ca 2÷ transport in a competitive manner [13--17], while Ruthenium red is a c o m m o n l y used noncompetitive inhibitor [16,18,19]. Their K i is very low (about 0.03 pM), compared to the Ca 2÷ concentrations c o m m o n l y used for Ca :+ transport measurements. Ba 2÷ is taken up by m i t oc ho ndri a in a similar way to Ca 2÷ [17, 20] and thus would be a competitive inhibitor of Ca 2+ transport. The aim of this work was to study the effects of these inhibitors of Ca e+ binding and transport on the transport kinetics. Methods and Materials Rat-liver mito c hondr i a were prepared from young male rats (Sprague-Dawley) by a conventional procedure [21]. Ca 2+ uptake was measured by using the EG T A quenching m e t h o d essentially as described by Reed and Bygrave [16], in order to be able to distinguish between external binding and transport of Ca 2÷. No Ca 2+ buffers were used because the agents studied might affect the buffering. The buffers might also bind the inhibitors and thus decrease their active concentration. The mitochondria were removed from the medium either by Millipore filtration (pore size 0.6 pro) or rapid centrifugation. Energy-independent Ca 2+ binding was measured as described before [22 ]. The 4SCa was c ount ed in a Packard Tricarb liquid scintillation spectrometer in Bray's solution (PPO, POPOP, naphthalene, ethylenglycol, dioxan and methanol). Three different reaction media were used: 0.25 M sucrose/20 mM Tris • CI, pH 7.4 (sucrose medium). The same medium with 2 mM MgC12 (sucrose/Mg medium), and 130 mM KC1/20 mM Tris . C1/2 mM MgC12, pH 7.4 (KC1/Mg medium). Reagents: FCCP (carbonylcyanide p - t r i f l u o r o m e t h o x y p h e n y l h y d r a z o n e ) was obtained from Dr. P.G. Heytler and R ut heni um red from BDtt Chemicals Ltd, Poole, England. The Ruthenium red was recrystallized accoring to Luft [23] before use. Results E f f e c t o f Mg 2÷ and K+ on the Ca 2÷ transport kinetics
A hyperbolic plot is obtained when mitochondrial C a 2+ uptake is measured in the sucrose medium at +5°C and the initial rate is plotted against the Ca 2* concentration. In the presence of Mg 2+ the kinetic plot becomes sigmoidal. In the KCI/Mg medium the sigmoidicity is still more p r o n o u n c e d (Fig. 1). This effect is of interest because neither Mg 2÷ nor K* is translocated by rat-live," mitochondria under these conditions, but both are competitive inhibitors of the binding o f Ca 2. to the low affinity sites [5,7--9].
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E f f e c t o f Ba 2+ on the Ca z+ transport kinetics and on Ca 2+ binding to the low a f f i n i t y sites Ba 2+ is transloeated into m i t o e h o n d r i a in a similar w a y as Ca 2+ [ 1 7 , 2 1 ] . It also induces similar respiratory stimulation [ 2 1 ] . Thus Ba :+ m a y be e x p e c t e d to inhibit m i t o c h o n d r i a l Ca 2+ transport c o m p e t i t i v e l y . Fig. 2 s h o w s the effect of Ba 2+ on the Ca 2+ transport kinetics. In the sucrose m e d i u m the plot b e c o m e s
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B(] 2° c0ncentretl0n Fig. 3. D i × o n plots f o r the i n h i b i t i o n o f Ca 2+ b i n d i n g to the low affinity sites by Ba 2+. M i t o e h o n d r i a (5 m g p r o t e i n / m l w e r e i n c u b a t e d in t h e s u c r o s e m e d i u m c o n t a i n i n g 5 p M F C C P a n d 1 /aM R u t h e n i u m r e d f o r 1 r a i n . T h e r e a f t e r 4 5 C A C 1 2 at a c o n c e n t r a t i o n o f 2 5 p M (c~ ~), 50pM (e-e) and lOOpM (,",-~) was added and the incubation was continued for 1 min. The mitochondria were removed from the medium by centrifugation (14 000 Xg) and samples were taken from the pellets for counting a f t e r s o l u b i l i s a t i o n in 1 M f o r m i c a c i d .
sigmoidal in the p r e s e n c e o f Ba 2+ (Fig. 2a). In the KC1/Mg m e d i u m Ba 2+ increases the s i g m o i d i c i t y o f t h e p l o t (Fig. 2b). In Fig. 2 it is also seen that Ba 2. inhibits Ca 2÷ transport m o r e s t r o n g l y in the sucrose m e d i u m as c o m p a r e d to the KC1/Mg m e d i u m . D i x o n and L i n e w e a v e r - B u r k plots ( n o t s h o w n ) indicate that the K i o f Ba 2+ varies b e t w e e n less than 10 pM at l o w e r Ca 2+ c o n c e n t r a t i o n s ( 1 0 - - 3 0 pM), to a b o u t 70 pM, at higher Ca 2+ c o n c e n t r a t i o n s ( 5 0 - - 1 0 0 pM). In the KC1/Mg m e d i u m t h e Ki o f Ba 2+ is a b o u t 1 0 0 pM over t h e w h o l e Ca 2+ c o n c e n t r a t i o n range as c a l c u l a t e d f r o m D i x o n plots. D u e to the similar c h e m i s t r y o f Ba 2+ and Ca 2+ the f o r m e r m a y be e x p e c t e d to inhibit all individual steps in the Ca :+ transport process. Fig. 3 s h o w s D i x o n plots for the i n h i b i t i o n o f Ca 2+ binding to the l o w affinity sites by Ba 2+.
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F i g . 4. E f f e c t o f L a 3+ o n m i t o e h o n d r i a l Ca 2+ t r a n s p o r t k i n e t i c s . E x p e r i m e n t a l c o n d i t i o n s as in l~'ig. 1. I n c u b a t i o n s w e r e m a d e i n ( A ) t h e s u c r o s e m e d i u m (~) c,) c o n t a i n i n g 0 . 0 6 p M L a C I 3 ( e - . e ) , (B) t h e K C I / M g m e d i u m (,i >) c o n t a i n i n g 0 . 0 3 p M ( e - e) and 0.06 pM LaCl 3 (:2,). M i t o c h o n d r i a l p r o t e i n I m g / m ] i n ( A ) a n d 0 . 5 m g / m l i n (B).
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F i g . 5. E f f e c t o f R u t h e n i u m r e d o n m i t o c h o n d r i a l Ca 2+ t r a n s p o r t k i n e t i c s . E x p e r i m e n t a l c o n d i t i o n s as in Fig. 1. I n c u b a t i o n s w e r e m a d e ( A ) i n t h e K C 1 / M g m e d i u m ( A - - - - A ) containing 0.06 pM Ruthenium red (f, ~/) o r ( B ) in t h e s u c r o s e m e d i u m ( ) - - c ) c o n t a i n i n g 0 . 0 3 ~ M (z~-:',) o r 0 . 0 6 p M R u t h e nium red (e o).
E f f e c t o f La 3÷on the Ca 2÷ transport kinetics
In the sucrose m e d i u m the kinetic p l o t is still h y p e r b o l i c in the presence of La 3÷ (Fig. 4a). In the KC1/Mg m e d i u m the shape of the p l o t b e c o m e s slightly m o r e sigmoidal in the presence o f La 3÷ (Fig. 4b). Dixon plots ( n o t s h o w n ) for the inhibition of Ca z÷ t r a n s p o r t b y La 3* indicate t h a t the K i o f La 3+ in the sucrose m e d i u m does n o t differ significantly f r o m t h a t in the KC1/Mg m e d i u m . Due to the very low c o n c e n t r a t i o n used ( 0 . 0 3 - - 0 . 0 6 pM) La 3+ o u g h t n o t to a f f e c t Ca'* binding to the low affinity sites significantly. E f f e c t o f R u t h e n i u m red o17 the Ca 2÷ transport kinetics
R u t h e n i u m red does n o t a f f e c t the shape o f the kinetic plot to any significant degree. It is equally p o t e n t at all Ca 2÷ c o n c e n t r a t i o n s used. This is s h o w n in Fig. 5. D i x o n plots ( n o t s h o w n ) also indicate t h a t R u t h e n i u m red is an equally p o t e n t i n h i b i t o r during the slow phase (15 pM) and during the steep rise of the kinetic p l o t (50 /aM) and t h a t the K i in the sucrose m e d i u m does n o t differ significantly f r o m the Ki in the KC1/Mg m e d i u m . Because the Ki f o r R u t h e n i u m red is in the same range as t h a t for La 3+ [16] it is n o t e x p e c t e d to i n t e r a c t with the low affinity binding o f Ca 2+. Discussion
The results o f this w o r k show t h a t a h y p e r b o l i c kinetic p l o t (initial rate versus c o n c e n t r a t i o n ) is o b t a i n e d for Ca 2÷ t r a n s p o r t in rat-liver m i t o e h o n d r i a when s u s p e n d e d in a sucrose m e d i u m . This holds for a Ca 2÷ c o n c e n t r a t i o n range of 5 - - 1 0 0 pM. In the presence o f Mg 2+ the p l o t b e c o m e s sigmoidal. This e f f e c t is e n h a n c e d w h e n b o t h Mg 2+ and K ÷ are present. Since b o t h Mg z÷ and K* inhibit Ca 2÷ binding to the low affinity sites their effects on calcium t r a n s p o r t m a y well result f r o m an i n t e r f e r e n c e with Ca 2+ binding to these sites. K* alone does n o t change the shape o f kinetic plots significantly ( s u b m i t t e d for p u b l i c a t i o n ) ,
292 possibly b e c a u s e of the sites having a l o w e r a f f i n i t y for K + or b e c a u s e of K ~ having only o n e charge. N e i t h e r Mg 2+ n o r K + is t r a n s l o c a t e d b y the m i t o c h o n dria u n d e r the e x p e r i m e n t a l c o n d i t i o n s . S o m e w o r k e r s [1,2] have r e p o r t e d sigmoidal Ca 2+ t r a n s p o r t kinetics also in a sucrose m e d i u m . H o w e v e r , t h e y used A T P as a Ca 2+ b u f f e r a n d were thus able to m e a s u r e Ca 2÷ t r a n s p o r t at very low free Ca 2+ c o n c e n t r a t i o n s . T h e sigmoidieity of these plots was a p p a r e n t at free Ca :+ c o n c e n t r a t i o n s near 1 t~M. T h u s it a p p e a r s possible t h a t Mg 2÷ and K + do slot actually change the h y p e r b o l i c kinetics to sigmoidal b u t m a y shift the p o i n t at which the s i g m o i d i c i t y occurs to higher Ca 2+ c o n c e n t r a t i o n s . T h e use of Ca 2+ b u f f e r s ill this w o r k was n o t possible due to i n t e r a c t i o n of the used inhibitors w i t h the buffer. T h e sigmoidal kinetics have previously ( V i n o g r a d o v and Scarpa, ref. 3) been ascribed t o a c o o p e r a t i v e t r a n s p o r t m e c h a n i s m w h e r e binding of t w o Ca 2+ to a carrier w o u l d be r e q u i r e d for t r a n s l o e a t i o n . T h e y m e a s u r e d Ca 2÷ t r a n s p o r t in a KC1/Mg m e d i u m using the m u r e x i d e t e c h n i q u e and o b t a i n e d a result very similar to ours (Fig. 1). T h e y also r e p o r t e d a change f r o m sigmoidal to h y p e r b o l i c Mn 2+ t r a n s p o r t kinetics in the p r e s e n c e of Ca 2÷. H o w e v e r , m e a s u r e m e n t of cation t r a n s p o r t with the m u r e x i d e t e c h n i q u e in the p r e s e n c e of o t h e r cations t h a t also i n t e r a c t with m u r e x i d e is q u e s t i o n a b l e . T h e results o f this w o r k s h o w t h a t Ba 2., which like Mn 2+ is t r a n s l o c a t e d b y m i t o e h o n d r i a , does n o t change the kinetics o f Ca 2÷ t r a n s p o r t f r o m sigmoidal to h y p e r b o l i c . If a n y t h i n g Ba > e n h a n c e s the sigmoidicity o f the kinetic p l o t (see Fig. 2b). Ca 2+, Mn 2+ and Ba 2* could of course be t r a n s l o e a t e d b y d i f f e r e n t m e c h a n i s m s . H o w e v e r , this a p p e a r s highly u n l i k e l y in view o f the m a n y similarities in t h e i r m o d e of t r a n s p o r t [ 2 0 ] . Ba 2÷ is a c o m p e t i t i v e i n h i b i t o r of Ca 2+ t r a n s p o r t . It is of interest t h a t Ba 2+ changes the Ca 2+ t r a n s p o r t kinetics f r o m h y p e r b o l i c to sigmoidal like Mg 2÷. It is also of interest t h a t the K i of Ba 2+ f o r Ca > t r a n s p o r t increases in the KC1/Mg m e d i u m as c o m p a r e d to the sucrose m e d i u m . This w o u l d suggest a c o m p e t i t i o n b e t w e e n the cations (K +, Mg > and Ba 2+) f o r the low a f f i n i t y sites. T h e inhibition o f Ca > binding to the low a f f i n i t y sites b y Ba 2÷ c o n f i r m s this. T h e results r e p o r t e d here indicate t h a t an unspecific a l t e r a t i o n of the surface o f the m e m b r a n e m i g h t d e t e r m i n e the shape of the kinetic plot. T h e surface p o t e n t i a l is d e t e r m i n e d b y the charge d e n s i t y and h e n c e p r e s u m a b l y b y the ions b o u n d at. n o n s p e c i f i c sites. S p e r m i n e , a t e t r a v a l e n t p o l y a m i n e , k n o w n to decrease the negative surface charge of m i t o c h o n d r i a significantly [ 2 4 ] , also changes the s h a p e of kinetic plots in a similar w a y to Mg > ( s u b m i t t e d for publication), which gives f u r t h e r evidence in f a v o u r of the a b o v e i n t e r p r e t a t i o n . M o r e o v e r it has r e c e n t l y b e e n d e m o n s t r a t e d [25] t h a t t r a n s p o r t kinetics of ions m a y m i m i c the kinetics of allosterie m e c h a n i s m s (sigmoidal kinetics) w h e n the surface has the same charge as the t r a n s l o c a t e d ion. O u r results indeed suggest t h a t the surface p r o p e r t i e s o f the m i t o c h o n d r i a l m e m b r a n e have great influence on the Ca :+ t r a n s p o r t kinetics. Mg 2+ and s p e r m i n e also decrease the a c t i v a t i o n energy of Ca 2+ t r a n s p o r t significantly, whereas n e i t h e r affects t h a t o f respiration [26] suggesting t h a t n o t o n l y the charge b u t also the m o b i l i t y of the polar h e a d - g r o u p s of p h o s p h o l i p i d s at the surface of the m i t o c h o n d r i a l m e m b r a n e m i g h t be o f i m p o r t a n c e in the kinetics of C a 2+ t r a n s p o r t . La 3+ and r u t h e n i u m red, which are i n h i b i t o r y at very low c o n c e n t r a t i o n s (K~ a b o u t 0.04 t~M), d o slot a f f e c t the s h a p e of the kinetic p l o t significantly.
293
This also is c o n s i s t e n t with the above proposal t h a t the surface p r o p e r t i e s of the m i t o c h o n d r i a l m e m b r a n e might have great influence on the Ca 2÷ t r a n s p o r t kinetics, because n e i t h e r o f these agents is e x p e c t e d to a f f e c t the surface charge significantly at the very low c o n c e n t r a t i o n used. It is c o n c l u d e d t h a t Ca 2÷ t r a n s p o r t in m i t o c h o n d r i a involves at least two steps. First, the m o v e m e n t o f the cation to the surface o f the m e m b r a n e with or w i t h o u t s u b s e q u e n t binding to the unspecific low affinity sites, and s e c o n d l y , the actual t r a n s l o c a t i o n process. The first step is strongly d e p e n d e n t on the surface p o t e n t i a l and h e n c e on the n u m b e r and charge of ions b o u n d to the surface sites as K + and Mg 2÷. It is interesting to speculate t h a t changes in the electrical surface properties of the m i t o c h o n d r i a l m e m b r a n e (by c o n t r o l of the binding c o n s t a n t s for K ÷, Mg 2+ etc.) m a y c o n t r o l the kinetics of Ca 2+ t r a n s p o r t also in vivo. A change f r o m sigmoidal to h y p e r b o l i c Ca 2÷ u p t a k e kinetics would have p r o f o u n d effects on the translocation rate especially at the low intracellular Ca 2+ c o n c e n t r a t i o n s . R e c e n t l y it has been d e m o n s t r a t e d t h a t Ca 2÷ t r a n s p o r t in s m o o t h muscle mitoc h o n d r i a o b e y s essentially h y p e r b o l i c kinetics [ 2 7 , 2 8 , 2 9 ] even in the presence of K ÷ and Mg 2÷. Thus it appears possible t h a t the surface of these m i t o c h o n d r i a might have d i f f e r e n t properties f r o m those o f liver and heart. It has also been s h o w n t h a t muscle m i t o c h o n d r i a f r o m various aquatic a r t h r o p o d s show a large variety o f energN i n d e p e n d e n t Ca 2÷ binding characteristics, i.e. variations in binding c o n s t a n t s and n u m b e r of binding sites for Ca 2÷ [ 3 0 ] . This suggests that the surface p r o p e r t i e s of m i t o c h o n d r i a l melnbranes might vary significantly b e t w e e n d i f f e r e n t tissues and species. Acknowledgements This s t u d y was aided by a grant f r o m the Sigrid Jus61ius F o u n d a t i o n . The a u t h o r s are grateful to Mrs Kaija Viitanen for skilful technical assistance. Re ferences 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
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