Active transport of potassium by mitochondria I. Exchange of K+ and H+

BIOCHIMICA ET BIOPHYSICA ACTA

432 BBA 45121

ACTIVE TRANSPORT OF POTASSIUM BY MITOCHONDRIA I. E X C H A N G E OF K+ AND H+ G. S. CHRISTIE*, K. AHMED, A. E. M. McLEAN, AND J. D. J U D A H

Division of Metabolic Research, The Chicago Medical School, Institute for Medical Research, Chicago, Ill. (U.S,A.)

"

(Received May 28th, I964)

SUMMARY

I. Conditions for controlled K + depletion of rat-liver mitochondria are described. These depleted mitochondria will reaccumulate K+ when supplied with an energy source and external K +. 2. Determination of mitochondrial water and K+ showed that K + was transported against a concentration gradient. 3- It was demonstrated that K + uptake was accompanied by H ÷ efflux. There appeared to be a I :I exchange of K + for H +. 4. No evidence for K+-Na ÷ exchange was obtained. No inward shift of anions was necessary for K ÷ uptake. 5. ATP and Mgz+, but not ATP alone, energised K + transport. 6. Glutamate together with Pl also energised K + transport. Other substrates tested were not effective with PI alone. In the presence of a system capable of supporting oxidative phosphorylation, all the substrates were effective.

INTRODUCTION

It is generally agreed that isolated mitochondria from a variety of tissues can maintain their internal K + against a concentration gradientl, 2. A great deal of work has been done on the conditions necessary to minimise loss of K + from isolated mitochondria a-6. The only indication of active transport against a concentration gradient has been by SHARE7, who demonstrated K+ accumulation by K+-depleted mitochondria; the particles were incubated in a medium which supported oxidative phosphorylation. No indication was given of the extent of passive inclusion of K ÷. In the present work, we have defined conditions for the loss and reaccumulation of K + by mitochondria from rat liver. We have shown that ATP is capable of driving the system, that K+ exchanges with H+ and that K+ uptake is not associated with any major movement of anions. * Present address: Department of Pathology, University of Queensland, Brisbane, Australia.

Biochim. Biophys. Acta, 94 (1965) 432-44o

MITOCHONDRIAL POTASSIUM TRANSPORT

433

METHODS

Animals Male Sprague-Dawley rats of 200-300 g body weight, were used for this work. They were fed a normal laboratory pellet diet, supplemented with vitamin E (2o mg a-tocopherol thrice weekly b y mouth) 8

Preparation of mitochondria These were isolated from IO % (w/v) homogenates made in 0.25 M sucrose. The "nuclear" debris was sedimented at 700 × g (5 rain). Mitochondria were then centrifuged down at least 3 times (80o0 × g; IO min). The loosely packed pink sediment which appears after the second mitochondrial centrifugation was discarded.

Dry weight and water content For determination of ion and water content, mitochondrial pellets were obtained b y centrifugation after diluting the flask contents 7-fold with 0.25 M sucrose containing 25 mM Tris and 5 mM MgCI~. Tared stainless-steel tubes (5° ml) were used for separation, the Sorvall RC-2 centrifuge was equipped with a rapid acceleration device. This enabled the machine to attain a force of 19000 × g within 45 sec. The centrifugation was carried on for 3 min. The tubes were drained carefully, wiped inside with absorbent paper, capped with Parafilm and inverted; they warmed rapidly to room temperature, and were wiped and capped again. The tubes were then weighed, dried to constant weight (overnight at IO5 o) and weighed again for estimation of dry weight. For experiments where water content was not needed, dry weights were obtained b y addition of trichloroacetic acid (5 % (w/v) final concentration) to the mitochondrial pellet. The precipitate was washed once with water (IO ml) and then dried at lO5 ° overnight. Both methods were highly reproducible. The results from each are distinguished in the tables b y calling the first "mitochondrial pellet" and the second "mitochondrial dry weight".

Analytical methods Inorganic orthophosphate was estimated either b y the method of BERENBLUM that of F I S K E AND S U B B A R o w 1°. Chloride was determined b y the iodometric method of SENDROY11. Na + and K + were determined b y flame photometry of the trichloroacetic acid extract of the mitochondrial pellets. ATP was determined after adsorption onto charcoal as previously described 1~ Calculations of concentrations of ions were always done on the basis of pellet water. No corrections have been applied for extramitochondrial space or for sucrose content. The contribution of the extramitochondrial fluid to the ion content of the pellet is small since the incubation fluid is diluted before the mitochondria are sedimented. AND CHAIN ~ o r

RESULTS

Depletion of mitochondrial K + Mitochondria suspended in K+-free sucrose, buffered at p H 7.4 with 5o mM Tris, and held at 38°, lose K + rapidly. Table I shows ATP, together with Mg ~÷, Biochim. Biophys. Acta, 94 (1965) 432-44 o

G.S. CHRISTIEet al.

434 TABLE

I

K + EFFLUX

FROM

MITOCHONDRIA

SUSPENDED

IN

K+-FREE MEDIUM

A

M i t o c h o n d r i a (9.5o m g d r y w t . ) w e r e i n c u b a t e d i n o.21 M s u c r o s e , 5 ° m M T r i s b u f f e r ( p H 7.4o), 6 m M A T P , 5.o m M M g z+, a n d i . o m M E D T A w h e r e s h o w n . T e m p e r a t u r e 38°. I n c u b a t i o n t i m e , 20 r a i n . M i t o c h o n d r i a w e r e c e n t r i f u g e d a t 1 9 0 0 0 × g a s d e s c r i b e d i n t h e METHODS s e c t i o n . T h e pellets were s u s p e n d e d in 5 % (w/v) trichloroacetic acid, a n d t h e p r e c i p i t a t e c e n t r i f u g e d down, to be w a s h e d a n d t h e n dried for weighing. T h e clear trichloroacetic acid s u p e r n a t a n t w a s used for K+ analysis.

Sample

Additions

mequiv K + per kg mitochondrial dry wt.

AK +

Initial Incubated Incubated Incubated Incubated Incubated

-Nil A T P p l u s M g ~+ ATP M g 2+ EDTA

142 21 90 69 48 50

-- - 12 i - - 52 - - 73 - - 94 - - 92

TABLE

II

EFFECT OF A T P AND M g 2+ ON K + UPTAKE M i t o c h o n d r i a ( I 8 m g d r y w t . p e r r a i n ) w e r e d e p l e t e d of K + b y i n c u b a t i o n i n o.21 M s u c r o s e , 5o m M T r i s b u f f e r ( p H 7.4), I.O m M E D T A f o r 2o m i n a t 38°. o . 5 - m l s a m p l e s w e r e t h e n t r a n s f e r r e d t o v e s s e l s c o n t a i n i n g o.21 M s u c r o s e , 50 m M T r i s b u f f e r , I.O m M E D T A a n d 20 m M KCI, t o g e t h e r w i t h a d d i t i o n s of N a + - A T P a n d MgCI~ a s s h o w n i n t h e t a b l e . A f t e r i o r a i n , a t 380 t h e m i t o c h o n d r i a w e r e s e p a r a t e d f o r K + a n a l y s i s a s d e s c r i b e d i n T a b l e I.

Mitoehondria

Initial K + depleted P l u s 2o m M P l u s 2o m M P l u s 20 m M P l u s 20 m M

Time (rain)

K+ K+ K+ K+

2o io IO io io

6 mM A TP

5 m2l/I MgCl 2

--+ + ---

--+ -+ --

mequiv K + per kg mitochondrial dry wt.

AK +

149 55 io 5 74 72 72

--+5 ° +19 +17 +17

decreases the loss but does not prevent it; addition of EDTA (I mM) has even less effect. Measurements of absorbancy and of mitochondrial water showed that swelling of the mitochondria was prevented by either ATP or Mg~+, and also by EDTA. It was thought that mitochondria depleted of K + by incubation in sucrose-EDTA at 380 would provide a useful system for the study of K + accumulation, since they lost some 60-7 ° % of their K+ within 20 min with no alteration of water content.

K+-accumulation by depleted mitochondria Fig. I shows the effect of ATP (6 mM) together with MgCI~ (5 raM) in the presence of 20 mM KC1 upon mitochondrial K +. There is a brisk K + uptake which is not seen upon addition of KC1 alone. Table I I shows that the system requires both ATP B i o c h i m . Biophys. Acta, 94 (1965) 4 3 2 - 4 4 o

MITOCHONDRIAL TABLE

POTASSIUM

435

TRANSPORT

III

K + CONCENTRATION REACCUMULATION

OF

AND WATER

CONTENT

OF MITOCHONDRIAL

PELLETS

AFTER

DEPLETION

AND

I~ +

Mitochondrial K + depletion and transfer as described in Table II. At the times shown they were transferred to tared stainless-steel tubes and centrifuged at 19o00 × g for wet wt. and dry wt. determinations as described.

Mitochondric~

Time (rain)

l water per kg dry wt.

mequiv K +per I pellet water

Initial K + depleted P l u s 20 m M KC1 P l u s 20 m M KC1 P l u s 20 m M KC1

-20 io 20 4°

2. i 2.2 2.2 2.15 2.20

55.0 23.0 43.0 50.o 65.o

AK +

-

-

-

-+20.0 +27.o +42.0

)50

2

,so~

/t

//

I/

120

t/~J2

g,oo I10

// 40

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$

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I0

20

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Time (min)

4

40

$0

60

t

i

I

i

I

I

I0

~

~0

40

50

60

Time (min)

F i g . i. K + a c c u m u l a t i o n b y d e p l e t e d m i t o c h o n d r i a . C u r v e I : d e p l e t i o n of m i t o c h o n d r i a l K +. C u r v e 2: a t t h e a r r o w , 2 0 m M KC1, 6 . 0 m M A T P a n d 5 . o m M MgC12 w e r e a d d e d . C u r v e 3: a t t h e a r r o w , o n l y 2 0 m M KC1 w a s a d d e d . C u r v e 4 : N a + c o n t e n t t h r o u g h o u t t h e e x p e r i m e n t . T h e e x p e r i mental details are exactly as described in Table II. F i g . 2. T h e e f f e c t of K + c o n c e n t r a t i o n o n K + u p t a k e b y d e p l e t e d m i t o c h o n d r i a . C u r v e I : d e p l e t i o n o f m i t o c h o n d r i a l K +. C u r v e 2 : K + u p t a k e i n p r e s e n c e o f 6 . 0 m M A T P , 5 . 0 m M M g CI~ a n d 4 0 m M K +. C u r v e 3 : a s c u r v e 2, b u t K + a t 2 0 m M . C u r v e 4 : a s c u r v e 2, b u t K + a t i o m M . C u r v e 5 : a s c u r v e 2, b u t K + a t 5 r a M . A T P , MgCI~ a n d K + w e r e a d d e d a t t h e a r r o w . O t h e r d e t a i l s a s i n T a b l e I I .

and Mg 2+ i: neither alone gives rise to a K + uptake b e y o n d that observed in the presence of K + alone. Table I I I shows the K ÷ concentrations in the mitochondrial pellets before and after K + loss, and also after reaccumulation of K +. It is clear that no water shifts have taken place and that K + uptake is against a concentration gradient. Fig. 2 s h o w s the K+ uptake at different K + concentrations. The rates of K + uptake are nearly m a x i m a l in 20 mM KC1. It is to be noted that uptake in the presence of I0 mM Bioehim. Biophys. Acta, 9 4 (1965) 4 3 2 - 4 4 °

G.S. CHRISTIEet al.

436

K + is at about 5o To of that in the presence of 2o mM K+; the results shown in the figure support the conclusion that K + uptake is against a gradient.

Nature of K+ uptake It has been suggested 1 that K + uptake into mitochondria is associated with an equivalent uptake of an anion. Other possibilities are (a) exchange of K ÷ for Na ÷ or (b) exchange of K + for H + (refs. 13, 14). Table IV shows that K + uptake will proceed in the total absence of sodium salts. Fig. I (bottom curve) shows that no appreciable movement of Na + occurs during loss of mitochondrial K+ or during the subsequent uptake. TABLE

IV

UPTAKE

OF

K +

IN

ABSENCE

OF

Na+

IN

THE

MEDIUM

Mitochondria were treated as described in Table II, except that ATP was the Tris salt and not t h e N a + s a l t . T h e N a + c o n t e n t o f t h e i n c u b a t i o n m e d i u m w a s less t h a n o.5 r n M .

Mitochondria

Time (rain)

mequiv I4+ per kg mitochondrial dry wt.

AK +

mequiv Na + per kg mitochondrial dry wt.

L]Na +

Initial K + depleted P l u s 20 m M K + P l u s 20 m M K + P l u s 20 m M K +

-20 io 20 4°

144 58 96

--+38

IIO

+5 2

151

+93

56 48 41 41 41

----7 --7 --7

TABLE

V

ION BALANCES

OF MITOCHONDRIAL

PELLET

M i t o c h o n d r i a (5 ° m g d r y w t . ) w e r e K + d e p l e t e d f o r 3 ° m i n a t 380 i n t h e p r e s e n c e of 6 . o m M A T P a n d 5 . o m M MgC1 v A t t h e e n d of d e p l e t i o n , KC1 o r N a C 1 t o 2 o m M w e r e a d d e d a n d t h e r e a c t i o n a l l o w e d t o p r o c e e d f o r IO r a i n . M i t o c h o n d r i a w e r e t r a n s f e r r e d t o t a r e d s t a i n l e s s - s t e e l t u b e s f o r s e p a r a t i o n a n d f o r d e t e r m i n a t i o n of w e t w t . a n d d r y w t . a s d e s c r i b e d i n T a b l e I I I . E x p l a n a t i o n of s y m b o l s : s u f f i x "i" r e p r e s e n t s m i t o c h o n d r i a l p e l l e t ; s u f f i x "o" r e p r e s e n t s m e d i u m . I t s h o u l d b e n o t e d t h a t all "o" f i g u r e s r e f e r t o i n c u b a t i o n m e d i u m . T h e c o n c e n t r a t i o n s i n t h e w a s h f l u i d a r e o n e - s e v e n t h o f t h e s e "o" c o n c e n t r a t i o n s .

Mitochondria

l water per kg dry wt.

K{ + Ko+ Na{ + Nao+ Cl{- Cl o- (Pi)l (P{)o A TPi A T P o (mM) (mM) (mM) (mM) (mM) (mM) (raM) (mM) (mM) (raM)

Initial K + depleted Incubated with 2o mM K + Incubated with 20 m M N a +

2.3 2.2

53 28

-I.O

12.7 16.o

-18.o

5° 47

25 25

2.2

41

2o.o

14.o

18.o

45

45

2.2

19

1.2

14.o

38.o

47

45

7-3 9.8

-3.4

1.4 6.2

-6. 3

ii.o

6.6

6.0

3.7

io.o

5.1

6. 4

3.6

The experiment in Table V was designed to determine whether any anion may move into the mitochondria together with K +. The particles were depleted of K + in the presence of ATP and MgCI~ to exclude the use of EDTA which might be hard to Bioehim. Biophys. Acta, 9 4 (1965) 4 3 2 - 4 4 °

437

MITOCHONDRIAL POTASSIUM TRANSPORT

e s t i m a t e . T h e p r e s e n c e of A T P a n d M g 2+ d u r i n g d e p l e t i o n a l s o e x c l u d e s t h e p o s s i b i l i t y t h a t s h i f t s of t h e s e m o l e c u l e s m i g h t a c c o u n t f o r s u b s e q u e n t K + u p t a k e . A f t e r d e p l e t i o n (30 r a i n ) , K + t o 20 m M w a s a d d e d t o o n e s e t of f l a s k s , a n d N a + t o a s i m i l a r c o n c e n t r a t i o n t o a n o t h e r s e t of f l a s k s . T h e d a t a ( T a b l e V) s h o w t h a t n o a n i o n m o v e m e n t occurs in sufficient a m o u n t to a c c o u n t for t h e o b s e r v e d K + u p t a k e . T h e a b s e n c e of a n y a c c u m u l a t i o n of C1- m a k e s i t u n l i k e l y t h a t K + u p t a k e i s d u e t o s e q u e s t r a t i o n of KC1 a s s u c h . T A B L E V2 E F F E C T OF G L U T A MATE AND P i

ON K + U P T A K E

Mitochondria (2o m g dry wt. per ml) were depleted as described in Table 22. o. 5 ml was transferred to flasks containing o.21 M sucrose, 50 mM Tris buffer (pH 7.4o), i.o mM E D T A a n d 2o mlVf KC1 together with additions as shown. The samples were otherwise treated as described in Table 2I.

Time (rain)

A dditions

mequiv K +per kg mitochondrial dry wt.

AK+

o 4° 4° 4° 4°

-Nil Io mM g l u t a m a t e I.o mM Pt Io mM g l u t a m a t e plus i.o mM Pl G l u t a m a t e plus Pl plus o.i mM 2,4-dinitrophenol

79.0 69.o 90.0 58.0

---IO + iz --21

4°

147

+68

60

--19

/ tI tt I0

/

/

8

E

t

t J

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t

lt / l

7

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/ / / / t

4

2

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I 20

t 25

I

I

I

30

35

40

Time (rain)

Fig. 3. H+ p:mduction b y mitochondria in response to K +. Mitochondria (IOO m g dry wt.) were K + depleted as in Table V22 at 38°, T h e y were cooled to 20 ° a n d transferred to a beaker a t 20 ° containing o.21 M sucrose a n d I.O mM EDTA, IO mM g l u t a m a t e a n d i.o mM Pl. The p H was adjusted to ?.20 with 0.i M Tris and, s t a r t i n g at t i m e zero, further additions of Tris buffer were m a d e to m a i n t a i n p H 7.2. At the arrow, 2o mM KC1 was added (curve i) or 2o mM l~aC1 was added (curve 2).

Biochim. Biophys. Acta, 94 (1965) 432-44 o

G.S. CHRISTIEet al.

438 K + and H +

exchange

Since mitochondria break down ATP liberating acid groups, the ATP-Mg 2+ induced uptake of K + is not suitable for a study of H ÷ secretion. We therefore investigated other systems. SPECTOR1 had shown that mitochondria would take up K+ in the presence of glutamate. We repeated his experiments with depleted mitochondria and found that glutamate would induce a I(÷ uptake but that this was not sustained TABLE VII STOICHIOM~TRY

OF

K + - H + EXCHANGE

M i t o c h o n d r i a (80 m g d r y wt.) were K + d e p l e t e d a t 380 for 20 ra i n as d e s c r i b e d in T a b l e I I e x c e p t t h a t Tris buffer w a s left o u t of t h e m e d i u m . T h e y were t h e n cooled to 20 ° a n d t r a n s f e r r e d t o a b e a k e r c o n t a i n i n g o.21 M sucrose a n d I mM E D T A (vol ume 20 ml), also a t 20 °. p H w a s a d j u s t e d t o 7.20 a n d t h e s u s p e n s i o n o b s e r v e d while b e i n g s t i r r e d . No p H c h a n g e t o o k place. A ft e r IO m i n , KC1 to 20 mM was a d d e d , p H fell i m m e d i a t e l y a n d w a s t i t r a t e d b a c k to p H 7.20 b y a d d i t i o n of i o o mM T r i s buffer w i t h a m i c r o b u r e t t e . A d d i t i o n s of T r i s buffer were m a d e a t 2 - m i n i n t e r v a l s t h e r e a f t e r . A f t e r 4 ° m i n the m i t o c h o n d r i a were s e p a r a t e d as d e s c r i b e d in T a b l e If for K + a n a l y s i s .

~Iitochondria

Time (min)

K+ (#moles)

K + depleted P l u s 20 mM K + A

o 4°

6.0 12.o +6.0

Tris consumed (Fmoles) o.o 7.1o +7.1o

22

140 ]

•

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18

130

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16 ~_ 12o c~

g >,

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8o

8 6

70

4

60

Z I

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I0

20

30

.

J

40

Time (rain)

0 0

3 -'~

,

"

I

I

I

I

I

,5

I0

15

20

25

30

Time

(rain)

F i g . 4. Effect of s t r o p h a n t h i n - G on K + u p t a k e b y m i t o c h o n d r i a . All d e t a i l s as in F i g. i. The figure s h o w s c o n t r o l (curve I) a n d m i t o c h o n d r i a in t h e p r e s e n c e of 0.5 mM s t r o p h a n t h i n - G (curve 2). Fig. 5. Effect of s t r o p h a n t h i n - G a n d 2 , 4 - d i n i t r o p h e n o l on H + p r o d u c t i o n b y m i t o c h o n d r i a . All d e t a i l s as in Fig. 3. The c u r v e shows t h e p r o d u c t i o n of H + a f t e r a p r e l i m i n a r y o b s e r v a t i o n p e r i o d of 15 rain i n a b s e n c e of K +. D r u g s a n d K + were a d d e d a t t h e s a m e t i m e (zero t i m e i n t h e figure). M i t o c h o n d r i a l d r y wt., 15o rag. Curve I : control. Curve 2 : 0.5 mM s t r o p h a n t h i n - G . C urve 3 : o . i mM 2,4-dinitrophenol.

Biochim. Biophys. Acta, 94 (1965) 432-44 o

MITOCIIONDRIAL POTASSIUM TRANSPORT

439

unless Pl in small a m o u n t s was also present. T a b l e VI shows t h e effect of g l u t a m a t e , PI a n d a c o m b i n a t i o n of these. The K+ u p t a k e u n d e r these c i r c u m s t a n c e s is b l o c k e d b y 2,4-dinitrophenol (o.I mM). A s t u d y of m i t o c h o n d r i a l A T P levels shows t h a t d u r i n g K + d e p l e t i o n m i t o c h o n d r i a l A T P levels fall considerably. G l u t a m a t e in t h e presence of Pl causes a r a p i d rise in A T P levels. T h e secretion of H + b y m i t o c h o n d r i a (previously d e p l e t e d of K +) in t h e presence of g l u t a m a t e a n d Pl, b o t h in t h e presence a n d absence of K + was therefore studied. Fig. 3 shows t h a t H+ efflux occurs slowly from K + - d e p l e t e d m i t o c h o n d r i a i n c u b a t e d i~a t h e presence of g l u t a m a t e a n d PI. W h e n K + is a d d e d , there is a r a p i d rise in the f o r m a t i o n of acid groups. T a b l e V I I shows t h e s t o i c h i o m e t r y of t h e K + - H + e x c h a n g e , a p p r o x . I K + is t a k e n up to I H + formed. 2,4-Dinitrophenol (o.I mM) blocks t h e p r o d u c t i o n of H + b y 9 ° % (see Fig. 5).

Action of strophanthin-G on K + uptake Figs. 4 a n d 5 show the effect of s t r o p h a n t h i n - G on K + u p t a k e a n d H + p r o d u c t i o n b y K + - d e p l e t e d m i t o c h o n d r i a ; the two curves are v e r y similar. T h e effect of t h e glycoside is not strong, since high c o n c e n t r a t i o n s are r e q u i r e d to d e m o n s t r a t e a n y effect, b u t these are n o t higher t h a n those r e q u i r e d to block K+ u p t a k e b y slices of r a t liver 15. T h e r e is also a lag p e r i o d of some IO m i n before a n y effect is a p p a r e n t . This is a t v a r i a n c e w i t h p r e v i o u s l y recorded effects of the cardiac glycosidesTM.

Oxidative p.~osphorylation and K+ uptake T a b l e V I I I shows a c o m p a r i s o n b e t w e e n four s u b s t r a t e s c a p a b l e of being o x i d i z e d b y r a t - l i v e r m i t o c h o n d r i a . All s u p p o r t K+ u p t a k e if A M P (with a small a m o u n t of ATP) is present, a n d no difference was f o u n d b e t w e e n the effect of these s u b s t r a t e s !(n inducing K + u p t a k e a n d t h a t of a d d e d A T P w i t h Mg *+. TABLE VIII K + UPTAKE DURING OXIDATIVE SYNTHESIS O F ATP The mitochondria (2o mg dry wt. per ml) were depleted as in Table II. They were then transferred to flasks containing 2o mM KC1, Io mM substrate (except for succinate, 25 mM); IO mM Pi, 5 mM MgC12, 0.2 mM ATP, 3 mM AMP, 5° mM Tris buffer (pH 7.4o), i.o mM EDTA and o.17 M sucrose. Terrtperature, 38°. Time, Io rain. The mitochondria were separated as in Table II for K + analysis, and the supernatant was analyzed for ATP synthesis.

Substrate

I4+ uptake (mequiv/min[kg mitochondrial dry wt.)

A TP formation (mmoles[min[kg mitochondrial dry wt.)

Glutamate Succinate c~-Oxoglutarate jS-Hydroxybutyrate

6.5 3.2 6.o 7.0

13° I27 i4 i 135

DISCUSSION The p r e s e n t w o r k p r o v i d e s evidence t h a t m i t o c h o n d r i a u n d e r t a k e active t r a n s p o r t of K+ coupled to a t r a n s p o r t o u t w a r d s of H+. S t r o p h a n t h i n - G a n d 2,4-dinitrop h e n o l i n h i b i t b o t h K+ u p t a k e a n d H + efflux. N e t K + u p t a k e a g a i n s t a g r a d i e n t is

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G.S. CHRISTIE et al.

established, and it is shown that ion transport m a y be energised by ATP in the presence of Mg 2+. Whether there is any other relation to oxidative phosphorylation is not at present certain. The relationship between the present work and that of SARIS17 and of BRIERLEY et al. TM must be discussed. In the first place, it is at least doubtful that any of these authors have demonstrated true active transport by mitochondria. In neither case has the existence of gradients been demonstrated. An apparent requirement for energy is not sufficient evidence that movement of an ion is active; it m a y be that energy-dependent processes maintain mitochondrial structures. To study active transport it should be established that the ion moves against an electrochemical gradient. In the case of divalent cations such as Mg 2+ and Ca 2+ this is very difficult, since they m a y form unionised complexes with mitochondrial components. Furthermore, BRIERLEY et al. TM state that their Mg 2+ uptake is inhibited during the active phase of oxidative phosphorylation, and that it proceeds rapidly during the oxidation of substrate in the absence of nucleotide. This does not happen with K+. When we consider t h a t mitochondrial water uptake (which is far from active) m a y also accompany oxidation of m a n y substrates TMit m a y be questioned whether the uptakes of Mg 2+ or of Ca 2÷ are models for cellular ion-transport systems. The K + reaccumulation system of mitochondria here described is strikingly similar to the reaccumulation of K+ b y depleted liver slices which we have described previously 2°. ACKNOWLEDGEMENTS

We are indebted to Miss S. ELSEY and Mr. W. GARRETT for skilled technical assistance. This work was supported by grants from the U.S. Public Health Service (AM 7226-o2), the Life Insurance Medical Research Fund, the Otho S. A. Sprague Memorial Institute and the Burroughs-Wellcome Fund. G. S. C. was in receipt of a travel grant from the Anti Cancer Council, Victoria, Australia. A. E. M. McL. was in receipt of a U.S. Public Health Service International Fellowship (FF 492). REFERENCES i 2 3 4 5 6 7 8 9 IO ii 12 13 14 15 I6 17 18 19 2o

W. G. SPECTOR, Proc. Roy. Soc. London, Ser. B, 141 (1953) 268. C. A. PRICE, A. FONNESU AND R. E. DAVIES, Biochem. J., 64 (1956) 754J- A. AMOORE AND W. ]DARTLEY, Biochem. J., 69 (1958) 348. J. L. GAMBLE, Jr., J. Biol. Chem., 228 (1957) 955. R. L. SCOTT AND J. L. GAMBLE, Jr., J. Biol. Chem., 236 (1961) 57 o. F. ULRICH, Am. J. Physiol., 198 (196o) 847. L. SHARE, Am. J. Physiol., 194 (1958) 47. A. E. M. MCLEAN, Biochem. J., 87 (1963) 164. I. BERENBLUM AND E. B. CHAIN, Biochem. J., 32 (1938) 295C. H. FISKE AND Y. SUBBARow, J. Biol. Chem., 66 (1925) 375J. SENI)ROY, f . Biol. Chem., 13o (1939) 6o5. K. AHMED AND J. D. JUDAH, Biochim. Biophys. Acta, 57 (1962) 245. P. MITCHELL, Biochem. Soc. Syrup. Cambridge, Engl., 22 (1963) 142. S. G. SCHULTZ, W. EPSTEIN AND A. K. SOLOMON, J. Gen. Physiol., 47 (1963) 329. J. D. JUDAH AND K. AHMED, Biochim. Biophys. Acta, 71 (1963) 34. M. WEATHERALL, Ciba Found. Syrup. Enzymes and Drug Action, (1962) 115. N. E. SARIS, Comment. Phys.-Math., 28-11 (1963) i. G. P. BRIERLEY, E. BACHMANN AND D. E. GREEN, Proc. Natl. Acad. Sci. U.S., 48 (1962) 1928. A. L. LEHNINGER, Physiol. Rev., 42 (1962) 467. A. E. M. McLEAN, Biochem. J., 87 (1963) 161.

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