Preparation from mammallian brain and kidney of the enzyme system involved in active transport of Na+ and K+

314

BIOCHIMICA ET BIOPHYSICAACTA

PREPARATION

FROM MAMMALLIAN BRAIN AND KIDNEY OF T H E E N Z Y M E SYSTEM

I N V O L V E D I N A C T I V E T R A N S P O R T O F N a + A N D K+ JENS CHR, SKOU Institute o/ Physiology, ~Tnivevsity o/ Aarhus (Denmark)

(Received July 27th, 1961)

SUMMARY A procedure is described tor the preparation from mammalian brain and kidney of an enzyme system which hydrolyses ATP to ADP and PI, and which requires both Mg~+, lqa + and K + for full activity. The enzyme system prepared behaves qualitatively in its relations to cations and to g-strophanthin as the Mg 2+ + Na + + K+-activated, ATP-hydrolysing enzyme system previously isolated from peripheral crab nerves and red blood cells. The finding of this enzyme system in tissues from brain and kidney in which there is an active, linked transport of Na + and K + lends support to the assumption made on basis of the experiments with the enzyme system from peripheral nerves and from red blood-cell membranes that this enzyme system is involved in the active, linked transport of Na+ and K + across the cell membrane.

INTRODUCTION In previous papers 1, ~ it was shown that there can be isolated an enzyme or enzyme system from peripheral crab nerves which hydrolyses ATP to ADP and Pt, and which requires both Mg~+, Na + and K + for full activity. This enzyme system fulfils a number of the requirements to a system involved in the active linked transport of Na + and K + across the cell membrane. The same enzyme system was later found in membrane fragments from red blood ceils 3-s and in tissues from brain, kidney and frog skin s. The main difficulty in preparing the Mg~+ + Na + + K+-activated enzyme system is to get rid of a Mg~+-activated ATP-hydrolysing enzyme which does not require Na + and K + for activity. From the peripheral crab nerves this can be done by differential centrifugation of a nerve homogenate. The tissue was homogenised in 0.25 M sucrose; 0.03 M histidine, p H 7.2. By this procedure it is possible to obtain a preparation in which the Mg ~+ + Na+ + K+]MgZ+ activity ratio is 4-1o, and in the best preparation up to 20 (see ref. I). From brain, kidney and frog skin the procedure used for the isolation of the enzyme system from the nerve tissue gave preparations with a much lower activity ratio. For kidney and frog skin it was less than 2 and for the brain less than 3. It is Abbreviation: DOC, deoxycholate. Biochim. Biophys. Acta, 58 (1962) 314-325

ENZYME SYSTEM INVOLVED IN ACTIVE TRANSPORT

315

the same low ratio as was found in the preparation from the red blood-cell membranes in which the best obtained ratio was 2 (see ref. 3). By trial and error it was found, however, that b y addition of DOC and Versene to the solution in which the tissue was homogenised it was possible b y differential centrifugation to obtain preparations from kidney and brain in which the Mg 2+ + Na + + K+/Mg 2+ activity ratio is of the same size as in the preparations from the peripheral crab nerves. The procedure for the isolation of the enzyme system from these two tissues is given in this paper. EXPERIMENTAL

The procedure for the determination of the enzyme activity was the same as described previously 1. The activity was measured at 37 °. The nitrogen was determined b y the micro-Kjeldahl method on an appropriate amount of the enzyme solution after dialysis against a IO m M phosphate buffer, p H 7.o. The brain and kidney were in most cases from rabbits, in a few cases from rats and guinea pigs. The brain and the kidney were removed from the animal inlmediately after it was killed, put into an ice-cold solution of o.25 M sucrose, o.o3 M histidine, o.oo5 M Versene, o.I ~/o DOC, p H 6.8. After removal of connective tissue, the tissue was cut to small pieces and thereafter homogenised in IO volumes of the abovementioned solution. The homogenate was centrifuged at ioooo × g for 15 min. The supernatant herefrom was centrifuged at 2o ooo × g for 6o min, see I in Tables I and II. After the centrifugation at 2o0oo × g the sediment from brain and kidney is resuspended in half the original volume in a solution of o.25 M sucrose, o.o3 M histidine, o.ooi M Versene, p H 6.8, which contain o.o5% DOC for the sediment from brain and o . 1 % DOC for the sediment from kidney. This resuspended sediment is thereafter centrifuged as shown under I I in Tables I and II. All the centrifugations were performed at o ° in a Christ ultra-centrifuge, model Omega, and lasted for 3o rain. After this centrifugation the sediments were all resuspended in i/4 of the original volume in a solution of o.25 M sucrose, o.o3 M histidine, o.ooi M Versene, p H 6.8, and stored at - - 2 o ° until use. RESULTS

As mentioned above, the main difficulty in preparing the Mg z+ + Na + + K+-acti rated, ATP-hydrolysing enzyme is to separate it from a Mg~+-activated, ATP-hydrolysing enzyme which is not stimulated b y Na + + K +. In the preparative procedure we have therefore used both the Mg ~+ + Na + + K+/Mg ~+ activity ratio and the activity with Mg z4 + Na + + K+/mg N oE the enzyme as a measure of the purification. Brain When the tissue was homogenised in 0.25 M sucrose, 0.03 M histidine, p H 6.8, without the addition of DOC and Versene, the best obtained Mg 2+ + Na + + K+/Mg 9+ activity ratio was 2-3. With o.I ~/o DOC and o.oo5 M Versene added to the solution in which the tissue is homogenised the resuspended sediment after 2000o × g of the first centrifugation has a ratio of 3-4 (Table I). When this suspension is recentrifuged Biochira. Biophys. Acta, 58 CI962) 314-32.~

316

j.C. SKOU TABLE

I

THE M g 2+ + N a + + K + / M g ~+ ACTIVITY RATIO FOR DIFFERENT FRACTIONS OF THE ENZYME PREPARED BY DIFFERF.NTIAL CEN~RIFUGATION OF A HOMOGENATE FROM BRAIN TISSUE The test solutions contained

M g 2+ 6, N a + i o o , K + 2 0 a n d M g ~+ 3 m m o l e s / 1 , r e s p e c t i v e l y . T h e ATP concentration was 3 mmoles[1. Activity ratio Mg =+ + N a + + K+IMg =+

Enzyme #action

Sedzo ooo Sed2o ooo S u p 2 o ooo II

z

2

3

4

5

6

7

3.0 4.1 2. 4

2.7 3.6 2.1

2.5 3.4 2. 3

3.1 2. 7

4-5 2. 7

3.5 3.2

1.7 4.7 3.o

2.6

5.2

2.I

3.1

5.9

8.1

6. 9

7.6

3-9

4.9

4 -1

4.7

S e d t o ooo S e d l o ooo S u p 2 o ooo Seds6 ooo S u p s 5 ooo Sed~5o ooo Supz~o ooo

8

9

zo

2.I 3.5 1.6 3.8 5 .8 3.9

3.7 6.4 8.1

6.8

3.9 2.9

5.4 5.0 4.6 5.3 4.4 6.8

the best ratio will increase to 6-8. In most of the experiments this ratio was found in the suspended 20000 × g sediment (Expts. 4-7, Table I). In a few experiments (8-1o, Table I) the best ratio is found in the supernatant after 20o00 × g. When this supernatant was further centrifuged, the best ratio is in some experiments found in the supernatant after 85o00 × g (8 and IO, Table II) and if this supernatant was further centrifuged it was still in the supernatant even after 25000o x g (Expt. io, Table II). In other experiments the best ratio was found in the sediment after 85000 × g (Expt. 9, Table II). In Table I I is shown the activity ratio and the activity per mg N of the enzyme solution ior Expt. IO, Table I. The activity per mg N is expressed both as the activity of the enzyme with Mg ~+, Na + and K + in the solution and as the activity with Mg 2+, Na + and K + in the solution minus the activity with Mg z+ alone. I t is seen t h a t the preparation with the highest activity ratio, the sediment after 85 ooo × g, also has the highest activity per mg N. TABLE

Ii

THE ACTIVITY IN # m o l e s P t HYDROLYSED FROM A T P PER HOUR PER m g N OF THE DIFFERENT FRACTIONS OF THE ENZYME SHOWN IN EXFT. IO, TABLE I I n c o l u m n 3 is s h o w n t h e a c t i v i t y w i t h M g z+ 6, N a + I o o , K + 2 o m m o l e s / 1 i n t h e m e d i u m a n d i n c o l u m n 4 t h e a c t i v i t y w i t h M g 2+ 6, N a + IOO, K + 2 o m m o l e s / 1 m i n u s t h e a c t i v i t y w i t h M g e+ 3 m m o l e s ] l . T h e A T P c o n c e n t r a t i o n w a s 3 m m o l e s ] l . T e m p e r a t u r e 37 °, p H 7.2. E m y m e fraction

Activity ratio

Mgt+ + Na + + K+IMg t+

Specific activ4~y Mg I+ + Na + + K + (Mgi+ + Na + + K ÷) - - M g a+ #moles Pilhlmg N

I II

#moles Pilhlmg N

Sed~o ooo Sup=o ooo

2. I 3.5

296 155

16I III

Sed2o ooc S u p z o o~o Sedss ooo Sed25o ooo Sup25o ooo

3.7 6.4 8.z 3.9 2.9

751 5o8

55 I 427

zz3o

992

303 131

Biochim.

224 89

Biophys.

A c t a , 58 (1962) 3 1 4 - 3 2 5

ENZYME SYSTEM INVOLVED IN ACTIVE TRANSPORT

317

TABLE III THE M g ~+ + N a + + K+/Mg 2+ ACTIVITY RATIO FOR DIFFERENT FRACTIONS OF ENZYME PREPARED BY DIFFERENTIAL

CENTRIFUGATION

OF A HOMOGENATE

FROM KIDNEY

TISSUE

The test solutions contained Mg 2+ 6, Na + ioo, K + 20 and Mg 2+ 3 mmoles/1, respectively. The ATP concentration was 3 mmoles/1. Activity ratio M g s÷ + N a + + K + / M g *+ E n z y m e fraction

Sedxo ooo Sed2o ooo SuP~o oco II

1:

z

3

i.o 2. i 1.4

2.o 1.4

2. i 1.6

Sedlo ooo Sed~o ooo SuP2o ooo Secls~ ooo Sups5 ooo Sed2~o ooo Sup~o 00o

1.2 2.0 3.5 1.9

4

5

6

7

8

2.0

1.7 1.8 4 .2

4.4

1.5 3 .6 1.8

4-9 1.8 5.5

4 .6

5.4

2. 3

6.9

6.2

T A B L E IV THE ACTIVITY IN /*moles Pt HYDROLYSED FROM ATP/h/mg N OF THE DIFFERENT FRACTIONS OF THE ENZYME SHOWN IN EXPT. 8, TABLE III. In column 3 is shown the activity with Mg 2+ 6, Na + Ioo, K + 2o mmolesfl in t h e medium a n d in column 4 the activity with Mg 2+ 6, Na + ioo, K + 20 mmoles/l minus t h e activity with Mg 2+ 3 mmoles/l. The ATP concentration was 3 mmoles/l. Temperature, 37% p H 7.2. Enoame fracti°n

Activity ratio Mg t+ + Na+ + K+/Mg t+

Specific aaivi*y Mg~+ + Na + + K + (Mg t+ + Na + + K+) --Mg *+ F,moles Pi/hlmg N

~r~

Pdhl~g N

I

Sed2o ooo Sup~o ¢oo

2.0 I. 7

375 329

187 1.38

II

Sed~o~ Supzo ooo Sed~5 ooo Sed2~o ooo Sup2~o ooo

1.5 3.6 1.8 2.3

502

163 314

6.2"

620

436 497 424

223

243 519

D u r i n g t h e s t o r a g e of t h e e n z y m e s o l u t i o n a t - - 2 0 ° t h e r e is a n i n c r e a s e i n t h e a c t i v i t y r a t i o w h i c h a m o u n t s t o 2 5 - 5 0 ~/o. T h i s is d u e t o a d e c r e a s e i n t h e a c t i v i t y of t h e e n z y m e w i t h M g ~+ a l o n e . T h e a c t i v i t y w i t h M g 2+ + N a + + K + is n o t a l t e r e d for several weeks. If, a f t e r t h e s e c o n d c e n t r i f u g a t i o n a t 2 0 0 0 0 × g t h e s e d i m e n t is r e s u s p e n d e d i n 0 . 0 6 M T r i s b u f f e r , p H 6.8, i n s t e a d of i n 0.25 M s u c r o s e , o.o 3 M h i s t i d i n e , o . o o I M V e r s e n e , p H 6.8, t h e a c t i v i t y r a t i o will, a f t e r s t o r a g e f o r a f e w d a y s i n c r e a s e f u r t h e r u p t o l O - 2 O ; b u t a t t h e s a m e t i m e t h e a c t i v i t y p e r m g N will d e c r e a s e a n d r e a c h a low value. Kidney

I n e x p e r i m e n t s i n w h i c h t h e t i s s u e is h o m o g e n i s e d i n 0.25 M s u c r o s e w i t h 0.03 M h i s t i d i n e a s b u f f e r , p H 6.8, w i t h o u t t h e a d d i t i o n of D O C a n d V e r s e n e , t h e b e s t o b Biochim. Biophys.

Aaa, 58 (1962) 314-325

318

J.C. SKOU

rained Mg~+ + Na + + K+/Mg *+ activity ratio by differential centrifugation is not more than 2. With the addition of o.I ~/o DOC and 0.0o5 M Versene to the solution the ratio is increased to 4-6, Table I I I . As in the experiments with the brain tissue the best Mg 2+ + Na+ + K+/Mg ~+ activity ratio after the first centrifugation is found in the resuspended sediment after 20 ooo × g. After the second centrifugation the best ratio is in the supernatant after 2o000 × g, and in the experiments in which this supernatant is further centrifuged, the best ratio is still in the supernatant, even after 25o000 × g for 30 rain. For the enzyme preparation from kidney it is also found that the preparation with the highest Mg2+ + Na + + K+/Mg *+ activity ratio is the preparation with the highest activity per mg N (Table IV).

The effect o / D O C on the enzyme In Table V is shown the effect of different concentrations of DOC on the Mg 2+ + Na + + K+/Mg *+ activity ratio and in Fig. i A the effect on the absolute activity of the enzyme with Mg 2+ + Na + + K + and with Mg~+ alone in the medium. The tissue was from kidney and was homogenised in 0.25 M sucrose, 0.03 M histidine, pH 6.8, without DOC and Versene. The enzyme preparation was the resuspended IOOOO × g sediment. DOC was added to the enzyme preparation in the concentrations shown in the table and the figure and was allowed to act for 15 rain. Thereafter o.i ml of the enzyme solution was added to 0.9 ml of the test solution for determination of the enzyme activity. The DOC concentration in the test solution was thus IO times lower than the concentrations in the enzyme solution shown in the table and in the figure. DOC in concentrations up to o . I % give a slight increase in the activity ratio (Table V). This is due to a decrease in the activity with Mg *+ alone, Fig I A, and not to an increase in the activity with Mg ~+ + Na + + K +. At concentrations above o.1% the activity with Mg 2+, Na + and K + decreases more than the activity with Mg ~+ alone, i.e. the Mg 2+ + Na + + K+/Mg 2+ activity ratio decreases. In Fig. I B is shown the effect of DOC on the activity of an enzyme prepared with o. I °/o DOC in the solution. The enzyme is prepared from brain tissue. DOC was added to the enzyme solution as in the experiment shown in Fig. I A. As in Fig. I A, the activity with Mg ~+ + Na+ + K + decreases with increasing TABLE THE EFFECT OF ADDITION OF D O C

V

ON THE M g ~'+ + N a + + K + / M g ~+ ACTIVITY RATIO

The enzyme was prepared from kidney tissue without DOC in to the enzyme solution in the concentrations shown in the table a f t e r w h i c h o. i m l o f t h e e n z y m e s o l u t i o n w a s a d d e d t o o . 9 m l of e n z y m e a c t i v i t y . T h e t e s t s o l u t i o n s c o n t a i n e d M g 2+ 6, N a + respectively. The ATP concentration was

the solution. The DOC was added a n d w a s a l l o w e d t o a c t f o r 15 m i n , of t e s t s o l u t i o n f o r d e t e r m i n a t i o n I o o , K + 2 o a n d M g 2+ 3 m m o l e s / 1 , 3 mmoles/1.

Per cent DOC in enxyme solutio~t E~zyrne fraction

o

0.0 5

o.z

o.2

A ctivity ratio

Sed050o S e d l 0 000 Sed~0 ooo SUp~o 0o0

1.4 1.6 1.2 I.O

1-3 1. 7 1.2 i, I

1.4 2. 3 1.8 1.3

Biochim.

1.3 1.8 1. 7 1.4

Biophys.

Acta,

5 8 (1962) 314--325

ENZYME SYSTEM INVOLVED IN ACTIVE TRANSPORT

319

Ng6,NoI00,K20 mmoles/I t.,?.

12

t.O

1.0

A

0.8

o~s/l

0.8

O.G

0.6

O~

OA

0.2

0.2 MO3-

0

I

I

0.1

02

0

I

I

O~

0.2

Percent DOC

Fig. I. The effect of DOC on the a c t i v i t y of t h e enzyme w i t h Mg I+, N a + a n d K + and w i t h Mg ~+ alone in t h e t e s t solution. The e n z y m e in A was p r e p a r e d f r o m kidney w i t h o u t DOC a n d in B from b r a i n w i t h o.I ~o DOC in the solution in which the tissue was homogenised. DOC was added t o the e n z y m e solution in the c o n c e n t r a t i o n s s h o w n in the figure a n d allowed to act for 15 rain, a f t e r which o. i ml of the e n z y m e solution was added to 0.9 ml of t h e test solution for d e t e r m i n a t i o n of e n z y m e activity. The test solutions contained Mg 2+ 6, N a + zoo, K + 20 a n d Mg 2+ 3 mmoles/1, respectively. The A T P c o n c e n t r a t i o n was 3 mmoles/1.

O.6

t,2

A

B

t.0

O.S

0.8

0.4 Hg G,No t 00,K 20 mmoMs/I

/I

O.

0.6

0,3

0.4

O,2

0.2

0,1

E

Mg3 0 O

0.0 S

O.t

0

I

I

0.05

0.1

Percent DOC

Fig. 2. The effect of DOC o n the a c t i v i t y of e n z y m e p r e p a r e d w i t h o. I ~o DOC in the solution in w h i c h t h e tissue was homogenised. A, enzyme f r o m brain; B, f r o m kidney. DOC was added to the t e s t solution in t h e c o n c e n t r a t i o n s s h o w n in the figure. The t e s t solution contained Mg~+ 3 and Mg 2+ 6, N a + ioo, K + 20 mmoles/1, respectively. The A T P c o n c e n t r a t i o n was 3 mmoles/1.

Biochim. Biophys. Acta, 58 (z962) 314-325

320

J.c.

SKOU

concentrations of DOC, but in contrast to the effect of DOC on the enzyme prepared without DOC in the solution, there is no effect of DOC in the activity with Mg z+ alone. In Fig. 2 A and B is shown the effect of DOC on the activity of an enzyme prepared with o . I % DOC in the solution. In these experiments, the DOC was added to the test solution in the concentrations shown on the figure. Apart from a very small stimulating effect of o . o 0 i % DOC, concentrations higher than o . o i % inhibit the activity with Mg 2+ + Na + + K + in the medium. In concentrations up to 0.025 % there was no effect on the activity with Mg~+ alone, while higher concentrations inhibit the activity.

The effect o/cations The substrate for the enzyme is ATP which is hydrolysed to ADP and P I . With A D P as substrate there is a slight release of Pi which is about IO °/o of the release with ATP as substrate. This effect on ADP may be due to the presence of myokinase. 0.3 ~

0~

_~ 0.2 ~

0

0.2

No

=" 0.1

~

= K

~. o.1

--. I 2

Pig3 rnmoles/I I 1 I 4 6 rnrnoles/I

8

E I t0

Fig. 3- T h e effect of N a + a n d of K + on the a c t i v i t y of e n z y m e f r o m b r a i n in the presence of Mg ~+ 3 mmoles/1. A T P c o n c e n t r a t i o n 3 mmoles/1.

Na

V" o 0

Mg3:" rnmolas/I I 2

I I t 4 6 8 mrnoles/|

I to

Fig. 4. The effect of N a + a n d of K + on the a c t i v i t y of e n z y m e f r o m k i d n e y in the presence of Mg z+ 3 mmoles]l. ATP c o n c e n t r a t i o n 3 mmoles/1.

For the hydrolysis of ATP the enzyme requires Mg z+, but the activity is very low when Mgz+ is the only cation present. Addition of Na + besides Mg~+ leads to a slight increase in activity, Figs. 3 and 4- Maximum activity with Na + is obtained at a concentration of I.O mmole/1 for brain and 2.0 mmoles/1 for kidney. The Na + concentration which give half maximum increase in activity is 0. 4 mmole/1 for brain and 0.6 mmole/1 for kidney. If K+ or any of the other monovalent cations are added instead of Na+, the enzyme shows practically no increase in activity. When the medium contains both Mg2+ and Na +, addition of K + leads to a considerable increase in the activity of the enzyme (Figs. 5 and 6). The increase in activity produced by K + when there are Mgz+ and Na + in the medium is dependent on the concentration of Na +. At low concentrations of Na + the addition of low concentration of K + leads to an increase, while the activity decreases again when the K + concentration is increased (Figs. 5 and 6). When the Na + concentration is increased, the concentrations of K + which give a decrease in activity also increase. As is seen from Figs. 5 and 6, the concentrations of K+ which give a decrease in activity at a given Na+ concentration are lower for the kidney enzyme than for the brain enzyme. Biochira. Biophys. Acta, 58 (I962) 314-325

321

ENZYME SYSTEM INVOLVED IN ACTIVE TRANSPORT

Mg6,NalO0 rnmolesA

1.2

ID

0.8

"~'-t......._~~ MgNa20B,

x-" E

0~

0.4

02

0

I

I

I

I

I

0

25

50

75

100

K mrnoles/[

Fig. 5- T h e effect of K + on t h e a c t i v i t y of enzym, e from b r a i n in the presence of Mg a+ 6, N a + zoo a n d Mg ~+ 6, N a + 20 mmoles/1, respectively, in t h e test solution. A T P c o n c e n t r a t i o n 3 mmoles]l. 0~

0.6

E

0.4

0.2

0

MgB,Na10 -

I

I

I

I

I

0

30

60

90

t 20

K mmoles/I

~Fig. 6. The effect of K + on t h e a c t i v i t y of e n z y m e from kidney in t h e presence of Mg 2+ 6, N a + i o o ; Mg 2+ 6, N a + 5 ° a n d Mg ~+ 6, N a + IO mmoles/1, respectively, in t h e test solution. A T P c o n c e n t r a t i o n 3 mmoles/l.

Biochim. Biophys. Acta, 58 (I962) 314-325

322

j . c . SKOU t.2

1.0

0.8 0-

~o~ 0.4

0.2 ~'Mg6

0

I

0 0 0 150

I

I

25 125

50 t00

I

I

75 t00 75 SO rnmole=s/I

I

I

t25 25

150 0

K Na

Fig. 7- T h e e f f e c t o f N a + + K + o n t h e a c t i v i t y of e n z y m e f r o m b r a i n a n d f r o m k i d n e y i n t h e p r e s e n c e of M g ~+ 6 m m o l e s / 1 . T h e s u m of N a + a n d K + w a s k e p t c o n s t a n t a t 15o m m o l e s / 1 . A T P concentration 3 mmoles/1.

ii

C$

1D -

i

0.8

-

~6

-

0.4

-

kl

°'I 0

I

I

I

I

0

SO

t00

t50

mm01cs/I F i g . 8. T h e e f f e c t of L i +, N H ¢ +, Rb +, Cs + a n d K + o n t h e a c t i v i t y of e n z y m e f r o m b r a i n i n t h e p r e s e n c e of M g ~+ 6, N a + IOO m m o l e s / 1 . A T P c o n c e n t r a t i o n 3 m m o l e s / 1 .

Biochim. Biophys. Acta, 58 (1962) 3 1 4 - 3 2 5

ENZYME SYSTEM INVOLVED IN ACTIVE TRANSPORT

323

In Fig. 7 are shown the results of an experiment in which the test solution besides Mg 2+ 6 mmolesfl contains Na + and K + in varying concentrations, but the sum of the concentrations of Na + + K + is kept constant at I5O mrnolefl. As in Figs. 5 and 6, it is seen t h a t the activating effect of Na + and K + is due to a combined effect of these two ions. The concentrations of K+ at half m a x i m u m activity with optimum concentrations of Mg 2+ and Na + in the medium are about I mmole/l for enzyme from brain and 0.5 mmole/1 from kidney. NH4 +, Rb +, Cs + or Li + can replace K + (Fig. 8). The activating effect of NH4+ is greater t h a n for K +, but the concentration at half m a x i m u m activity is higher for NH4+ t h a n for K +, i.e. the affinity for K + is higher than for NH4 +. The other monovalent cations have a lower activating effect, and the concentration at half m a x i m u m activity is higher t h a n for K+. The o p t i m u m Mg 2+ concentration when the medium contains Mg z+ alone or Mg 2+ and Na +, is 3 mmoles/1 when the ATP concentration is 3 mmolesfl. With Na + and K + in the medium, the optimum Mg 2+ concentration increases to 4-6 mmolesfl when the ATP concentration is 3 mmoles/l. Ca 2+ inhibits the activity due to both Mg 2+, Mg ~+ + Na +, and Mg 2+ + Na + + K +.

G-strophantin G-strophanthin inhibits the activity of the enzyme with Mg 2+ + Na + + K + in the solution and the activity with Mg ~'+ + Na +, while it has no effect on the activity with Mg 2+ alone, Figs. 9 and IO. The concentration of g-strophanthin at half m a x i m u m inhibition is 2" lO -7 M for enzyme from brain and IO-e M for enzyme from kidney.

12

t.0

0.8 w

Mg6,Na4OO,K20 mmoles/I

o~

0.4

~ ~ g -

~

3,NoI0 :

Hg3o

I 0

I 0.0t

I I 0.02 0.03 g-Strophanthin mmofe*/I

I

I

0.04

0.05

Fig. 9. T h e effect of g - s t r o p h a n t i n on t h e a c t i v i t y of e n z y m e f r o m b r a i n in t h e presence of Mg 2+ 3 ; Mg ~+ 3, Na+ io a n d Mg ~+ 6, N a + Ioo, K + 2o mmolesfl, respectively, in t h e t e s t solution. A T P c o n c e n t r a t i o n 3 mmoles/1.

Biockim, Biophys. Aaa, 58 (I962) 314-325

324

J.C. SKOU

°.I O.6

Hg6,Na'lOO, K 20 mrnoles/I

¢L

E

o.4

0.2

,i Hg3 -

I

I

I

I

I

0.0t

0.02

0,03

0.04

0.05

g-Strophonthin

mmoles/I

Fig. IO. T h e effect of g - s t r o p h a n t h i n on t h e a c t i v i t y of e n z y m e from k i d n e y i n t h e p r e s e n c e of Mg 2+ 3 a n d Mg ~+ 3, Na+ IO a n d Mg ~+ 6, N a + Ioo, K + 2o mmoles/1, r e s p e c t i v e l y , in t h e t e s t s o l u t i o n . A T P c o n c e n t r a t i o n 3 mmoles/1.

0~8

o.2

o 0 0 0 120

30 90

60 60

I

I

90

t20

K

30

0

Na

mmoles/I

Fig. I I. T h e effect of N a + + K + on t h e a c t i v i t y of e n z y m e from r a b b i t - h e a r t m u s c l e in t h e p r e s e n c e of Mg a+ 3 mmoles/1 in t h e t e s t solution. A T P c o n c e n t r a t i o n 3 mmoles/1.

DISCIJSSION

DOC seems to have at least two effects. One is to decrease the activity of the Mg 2+activated enzyme which is not activated by Na + + K +. The other is to facilitate the separation b y differential centrifugation oi the Mg~+-activated enzyme from the Biochirn. Biophys. Acta, 58 (1962) 314-325

ENZYME SYSTEM INVOLVED IN ACTIVE TRANSPORT

325

Mg2+ + Na + + K+-activated enzyme. DOC does not increase the activity of the Mg2+ + Na + + K+-activated enzyme. The enzyme prepared b y the described preparative technique behaves both in relation to ions and to g-strophanthin qualitatively like the enzyme prepared ffrom peripheral nerves from crab1, 2 and from red blood-cell membranes s-5. However, quantitatively there are some differences. The affinities for Na + and K+ are higher than the affinities of the crab-nerve enzyme for the same ions. This is not surprising when one recalls that the concentrations of Na+ and K + both intra- and extracellularly in the crab are much higher than in the rabbit and rat. The experiments with the enzyme system isolated from peripheral nerve 1, 3,6 and red blood-cell membranes s-s have given good evidence that this enzyme system is involved in the active, linked transport of Na + and K+ across the cell membrane. As is seen from the present experiments, an enzyme system with the same properties as the enzyme system from these tissues can also be isolated from other tissues where there is an active, linked transport of Na + and K +. It m a y be added that the same enzyme system has also been isolated from frog skin and from rabbit striated and heart muscle. In Fig. I I is shown the effect of Mg 2+, Na + and K + on an enzyme system prepared from heart muscle. But for these tissues it has not yet been possible to elaborate a technique to separate in a reproducible way the Mg2+ + Na + + K÷-activated enzyme from the Mg2+-activated to obtain a Mg 2+ + Na + + K+/Mg 2÷ activity ratio which is higher than 2-3. REFERENCES 1 j . C. SKou, Biochim. Biophys. Acta, 23 (1957) 394. 2 j . C. SKOU, Biochim. Biophys. Acta, 42 (196o) 6. s R. L. PosT, C. R. MERRITT, C. R. •INSOLVING AND C. D. ALBRIGHT, J. Biol. Chem., 235 (196o) 1796. 4 D. TOSTESON, p e r s o n a l c o m m u n i c a t i o n . 5 E. T. DUNHAM AND J. M. GLYNN, J. Physiol. (London), 156 (1961) 274. 6 j . C. SKOU, Symposium on Membrane Transport and Metabolism, Prague, ~96o, p. 228.

Biochim. Biophys. Acta, 58 (1962) 3 1 4 - 3 2 5