394
BIOCHIMICA ET BIOPHYSICA ACTA
VOL.'23 (1957)
T H E I N F L U E N C E O F SOME C A T I O N S ON AN
ADENOSINE TRIPHOSPHATASE
FROM PERIPHERAL
NERVES
JENS CHR. SKOU
Institute o] Physiology, University of Aarhus (Denmark)
Stimulation of a nerve leads to an influx of sodium ions into the fibre and hence to an
increase in the intra-axonal sodium concentration 1. Normal conditions are restored
by an outward transport of the sodium ions, and this process requires energy because
the etttux takes place against an electrochemical gradient. The mechanism of this
transport is not known.
In experiments with giant axons from Sepia o~cinalis and from Loligo/orbesi,
HODGKIN AND KEYNES2 found that dinitrophenol, azide and cyanide inhibit the active
transport of sodium ions out of the nerve ; this inhibition is reversible. In the concentrations used all three substances also inhibit the oxydative phosphorylation which
takes place in mitochondria; dinitrophenol and azide do so through an uncoupling of
the phosphorylation 3, ~, and cyanide through an inhibition of the oxydation ~. CALDWELL6 observed correspondingly that addition of these substances, in the concentrations used by HODGKIN AND KEYNES, led to a reduction of the content of energy-rich
phosphate esters in the axoplasm of giant axons. This seems to indicate that energyrich phosphate esters are somehow involved in the active transport of sodium ions out
of the nerve fibres.
In this connexion it is of interest that LIBET7 and ABOODAND GERARD8 were able
to demonstrate an adenosine triphosphatase (ATPase) in the sheath of giant axons.
A further study on the ATPase in nerves and its possible role in the active outward
transport of sodium ions seems warranted.
According to LIBET, the ATPase in the sheath of giant axons is calcium-activated,
while the experiments by ABOODAND GERARD suggest that it is activated by magnesium and located in submicroscopic particles. In peripheral nerves from the rat the
latter authors found both a calcium- and a magnesium-activated ATPase. The calciumactivated enzyme was predominantly located in the mitochondria, while the magnesium-activated, as in giant axons, was mainly located in the submicroscopic particles.
Giant axons were not available to us. In preliminary experiments we found that
a homogenate of leg nerves from the shore crab (Carcinus maenas) contained both a
calcium- and a magnesium-activated ATPase, and that their localization was similar
to that of the ATPase found by ABOOD AND GERARDin rat-nerve homogenates. For
our study we have chosen the magnesium-activated enzyme, because it resembles the
magnesium-activated ATPase from the sheath of giant axons in that it is located in
submicroscopic particles.
The present investigation is concerned with the effect on the enzyme activity
exerted by the cations normally present in the tissue-sodium, potassium, magnesium
and calcium.
Re#rences p. 4oi.
VOL. 23 (1957)
INFLUENCEOF CATIONS ON AN ATPASE
395
EXPERIMENTAL
The ATPase was p r e p a r e d b y homogenization and s u b s e q u e n t differential centrifugation 8 of leg
nerves from the shore crab (Carcinus maenas).
The isolated nerves were washed and homogenized in io volumes of o.25 M ice-cold sucrose
buffered w i t h histidine, 30 mM]l, pFI 7.6. The a d m i x t u r e of alkali metal ions was avoided b y using
the histidine base, and the p H was a d j u s t e d b y addition of I N HC1. The h o m o g e n a t e was centrifuged in a Servall angle centrifuge at o ° C. F r a g m e n t s and s t r o m a were removed b y centrifugation
at 2,ooo × g for 15 minutes, m i t o c h o n d r i a and some submicroscopic particles b y centrifugation
a t io,ooo × g for 15 minutes. The s u p e r n a t a n t was centrifuged at 2o,ooo x g ( m a x i m u m available)
for 3 hours. D u r i n g this centrifugation the t e m p e r a t u r e rose from o ° to 8-1o ° C.
The sediment after this last centrifugation was suspended in a volume of 0.25 M ice-cold
buffered sucrose corresponding to one half of the v o l u m e of the original homogenate. This supension was centrifuged at io,ooo × g for io m i n u t e s in order to r e m o v e a n y remaining mitochondria.
The final s u p e r n a t a n t was used as enzyme solution in the e x p e r i m e n t s ; it contained from one half
to t w o thirds of the activity originally p r e s e n t in the homogenate.
The e n z y m e was relatively u n s t a b l e ; w h e n it was stored in a refrigerator at 4-5 ° C, its activity
fell to one half in 3-4 days.
The disodium or d i b a r i u m salts of A T P and the b a r i u m salt of A D P (Sigma products) were
converted into free acids, the Na salt b y passage t h r o u g h an Amberlite 12o (H + form) column and
the b a r i u m salts b y precipitation with equimolar a m o u n t s of sulphuric acid and s u b s e q u e n t passage t h r o u g h an Amberlite 120 column to r e m o v e residual b a r i u m ions. The free acids were neutralized to p H 7 with a i M solution of 2-amino-2-methyl-I,3-propanediol. I n the concentrations used,
this s u b s t a n c e does n o t affect the e n z y m e activity and is w i t h o u t influence on the p h o s p h a t e determination.
ATP and A D P were determined s p e c t r o p h o t o m e t r i c a l l y 9 and b y m e a s u r e m e n t of 7'P; Na
a n d K were determined with a flame s p e c t r o p h o t o m e t e r , B e c k m a n DU, flame a t t a c h m e n t No.
92oo. The p H was m e a s u r e d w i t h a glass electrode, R a d i o m e t e r P H M 3. Inorganic p h o s p h a t e was
determined b y the m e t h o d of FISKE A N D S U B B A R O W TM with amidol as the reducing agent.
The activity of the e n z y m e was determined in a volume of i.o ml containing o.I ml of the
a b o v e - m e n t i o n e d e n z y m e solution. The reaction m i x t u r e was buffered with histidine, 3 ° mM[1,
p H 7.2. Unless otherwise stated, the m i x t u r e contained 3 m M ATP/1. The cations were added as
solution of their chloride. All e x p e r i m e n t s were performed at 36° C. After a i o - m i n u t e t e m p e r a t u r e
equilibration the e x p e r i m e n t was s t a r t e d b y the addition of enzyme, and the m i x t u r e was incubated
for 15 or 3 ° minutes, according to the reaction velocity. The hydrolysis of A T P was linear within
the e x p e r i m e n t a l period. The reaction was s t o p p e d b y the addition of o. I ml of 50 % trichloroacetic
acid, and after centrifugation aliquots of 0. 4 ml were t a k e n o u t for d e t e r m i n a t i o n of inorganic phosphate.
I n the absence of added cations, the reaction m i x t u r e contained small a m o u n t s (about o.i
mM/1) of sodium and potassium, which originated f r o m the enzyme solution a n d from the ATP.
I n all the d i a g r a m s except Fig. I, the e n z y m e activity is expressed as # g of P split off from
A T P in 3o minutes.
RESULTS
When the substrate is ADP, no inorganic phosphate is liberated (data not presented).
In the presence of ATP the hydrolysis stops when inorganic phosphate corresponding
to one phosphate group has been split off (Fig. I).
Fig. 2. shows that under the experimental conditions used the pH optimum is 7.2.
The enzyme activity is zero when no cations are added or when K + or Ca ++ alone
are added to the reaction mixture. The addition of Na + gives rise to a just measurable
activity, which is independent of the sodium concentration. When Mg ++ is added, the
enzyme shows a slight activity with a m a x i m u m at 3 mM/1 (Fig. 3).
If sodium ions are added to a reaction mixture which already contains magnesium
ions, the enzyme activity increases (Fig. 4). When the concentrations of Mg ++ and of
ATP are 3 mM/1, a m a x i m u m activity is observed when the sodium concentration is
6 mM/1. At higher sodium concentrations, the activating effect of Na ÷ decreases.
Addition of potassium ions to a mixture containing magnesium ions does not
affect the enzyme activity; addition of calcium ions results in an inhibition.
Re#fences p. 4ox.
VOL. 23 (I957)
J. c. SKOU
396
~(, P
~g P
~P
0
2C
0
lC
M
5
ab go 9b l b 1gor a i;8o
n
5.0
g
~
4.0
~o
s'.o 9.'0
2
4
sb
o
pH
6
c
,Co
B
10
i
rnfl/l
~do
mM/t
Fig. 2.
Fig. 3Fig. 1.
Fig. z. Liberation of inorganic phosphate with ATP as substrate. The dotted line indicates first P
of ATP. Abscissa, time in minutes; ordinate, /tg P removed from ATP. The reaction mixture contained 5 m M Mg, 8o m M Na and 12o m M K per litre. The initial concentration of ATP was 1.16
n'lM [1.
Fig. 2. Relation between enzyme activity and pH, Abscissa, pH ; ordinate,/xg P removed from ATP
in 30 minutes. The reaction mixture contained 6 m M Mg, 80 m M Na, i2o m M K and 3 m M ATP
per litre.
Fig. 3. Enzyme activity in relation to the concentration of Mg++, Ca ++, Na +, or K ÷. Abscissa, ion
concentration in mM/1; ordinate, /~g P removed from ATP in 3° minutes.
~gP
40F
p9 P
Mg6 mM/[
NaC[40 mM/I
12
L
• w
Mg3 mM/i
:
e
30
~
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tK
NaCI
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2 4 6 8 Ca
0 0
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20
40
60
80
100
KCI mM/t
120
Fig. 5Fig. 4.
Fig. 4. Enzyme activity in relation to the concentration of Na +, K +, Ca ~+ in the presence of Mg++,
3 raM/1. Abscissa, ion concentration in mM[1; ordinate,/xg P removed from ATP in 3 ° minutes.
Fig. 5, Enzyme activity in relation to the concentration of K + in the presence of Mg ++, 6 mM/1,
and different concentrations of Na +, Abscissa, potassium concentration in mM/1; ordinate, ~tg P
removed from ATP in 3° minutes.
If both magnesium and sodium ions are present in the mixture, addition of potassium ions results in a further increase in the enzyme activity (Fig. 5). When the potassium concentration is raised, the enzyme activity reaches a m a x i m u m and then decreases, and at high potassium concentrations it is smaller than it was when no potassium ions were added. The latter phenomenon is seen only from the curves representing
3 and IO mM/1, The curve for 3 mMll shows t h a t high concentrations of potassium
inhibit only that part of the activity which is due to Na +, but not the part which is due
to Mg ++,
Re]erences p. 4oi.
VOL. 23 ( 1 9 5 7 )
INFLUENCE
OF CATIONS ON AN ATPASE
397
It is further seen from Fig. 5 that the m a x i m u m activity obtained in the presence of potassium increases with the sodium concentration. The potassium concentration required for m a x i m u m enzyme activity also depends on the sodium concentration; it increases when the sodium concentration is increased. Maximum enzyme activity is obtained when the potassium concentration is roughly equal to that of sodium.
Finally, it should be observed that the inhibition due to high potassium concentrations
decreases when the concentration of sodium is increased. The effect of potassium ions
depends accordingly not only on the presence of magnesium and sodium ions, but also
on the concentration of sodium ions.
The addition of K ÷ has also an effect on the relation between enzyme activity and
sodium concentration (Fig. 6). In the absence of K +, the activity reaches a m a x i m u m
at 6 m M Na/l; when more sodium is
Hg P
added, the activity decreases. When as
mM/I
little as 3 m M K/1 is added to the
system, the activity is not only enhanced but shows a steady rise with
the sodium concentration until it
finally levels off. The sodium concentration at which this level is reached
increases with the amount of potassium
added.
A certain, low activity in the absence of sodium is observed in Fig. 6. It
is, as previously pointed out (c/. Fig. 4),
due to the presence of magnesium, and
° o~
J]t /
~~~o - - co
m
~50
260
it is independent of the potassium conNaCI rnM/t
centration. In the presence of high conFig. 6. E n z y m e activity in relation to the concentrations of potassium, 200 and 350 centration of N a + in the presence of M g ++, 6
mM/1, the addition of small amounts of mM/1, a n d different c o n c e n t r a t i o n s of K +. A b scissa, s o d i u m c o n c e n t r a t i o n in m M / 1 ; ordinate,
sodium, 3 and 6 mM/1, leads to an
/~g P r e m o v e d f r o m A T P in 30 m i n u t e s .
inhibition of this low, magnesiumdependent activity. Higher concentrations of sodium have, as usual, an activating
effect.
It appeared from Fig. 4 that calcium ions inhibit the activity which is due to the
presence of Mg++. An inhibition is also seen when calcium ions are added to a system
containing magnesium + sodium ions or magnesium + sodium + potassium ions
(Fig. 7).
When Mg++ or Mg ++ + Na + are the only cations present, the optimum magnesium concentration is 3 mM/1. In the presence of potassium the optimum concentration of magnesium is 6 mM/1, and this value is independent of the potassium concentration. If calcium ions are also added, the optimum magnesium concentration becomes still higher, and it increases with the calcium concentration (Fig. 8).
In Figs. 9 and IO is shown the relation between enzyme activity and sodium
concentration in the absence and presence of calcium ions at various concentrations
of potassium and magnesium. It appears that the activating effect of Na + is proportional to the Mg:Ca ratio.
It was previously noted (Fig. 6) that low concentrations of sodium in the presence
401
20[ f ///°
Re#fences p. 4oz.
K~
K120mh//l
,~----"~,~
M96mM/[
398
J.C. SKOU
J.LgP
VOL. 23 (1957)
~P
2O
3
3. K8 No8M94
15
2.
1.
25|I
201
mM/l
~_
_
K 120 N080 Ca0 rnM/I
//~'-~--'-
~
Na8 Mg4 mM/t
Mg4 mM/I
10
5
~
~
0
o
~
~,
~
~
CaCI2 mH/t
~T-
~
L
-0
Fig. 7- E n z y m e activity in relation to the concentration of Ca ++ in t h e presence of Mg ++,
Mg ++ + N a +, or Mg ++ + N a + + K +. Abscissa,
calcium c o n c e n t r a t i o n in mM/1; ordinate,/~g 1O
r e m o v e d from AT1O in 3 ° minutes.
o
0
mM/t
I
(5
,
12
18
MgC[2 m H / i
I
24
Fig. 8. E n z y m e activity in relation to the concentration of Mg ++ in the presence of K +, 12o
Na+, 80
and different concentrations of Ca ++ . Abscissa, m a g n e s i u m concentration in m M / l ; ordinate, /~g 1O r e m o v e d from
ATP in 3o minutes.
mM/1,
mM]l,
Ag P
~9 P
35
30
K 350 Mg 6 m M / I
-.K 120 Ivlg6rnM/l
3O
.
/
K 350 Mg 2 0 m M / l
/
,
2 mPq/l
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10
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/ / /
.....
¢r.,-.o-
b
go
760
7go
NaCI mM/{
26o
K 350 Mg20 Co10mtV//1
f
0
KaSOMge Co-o2mH/t
o-. . . . . . . .
I
100
I
200
300
NaCI m M / I
Fig. 9-
Fig. IO.
~g P
Fig. 9. E n z y m e activity in relation to the concent r a t i o n of N a + in the presence of Mg ++, 6
a n d different concentrations of K + and Ca ++. Abscissa, sodium concentration in raM/l; ordinate,
# g P r e m o v e d f r o m AT1O in 3o minutes,
raM/l,
12
/
~
'
~
M96 Nu6 r n M / I
Fig. I o. E n z y m e activity in relation to the concent r a t i o n of Na + in the presence of K +, 350
and different concentrations of Mg++ and Ca ++.
Abscissa, s o d i u m concentration in raM/l; ordinate # g 1O r e m o v e d f r o m A T P in 30 minutes.
raM/l,
.e
()
46
8~D
Fig. I I.
120
mMII
Fig. I I. E n z y m e activity in relation to t h e concentration of K + or Li + in the presence of Mg ++,
and Na +, 6
Abscissa, ion concent r a t i o n in
ordinate, /2g lO r e m o v e d from
A T P in 3 ° minutes.
1£~06 mM/1,
raM~i;
mM/1.
VOL. 23 (1957)
INFLUENCEOF CATIONS ON AN ATPASE
399
of high potassium concentrations inhibited the enzyme activity due to magnesium.
Figs. 9 and IO show that this is also the case in the presence of calcium.
The effect of Li ÷ was studied in a few experiments. Lithium ions do not affect the
activity due to magnesium, but if the system contains both magnesium and sodium,
the addition of Li+ results in an increase of the activity. The action of lithium is consequently similar to that of potassium, but it should be noted that at low concentrations lithium has a weaker effect than potassium, while its effect is stronger at high
concentrations (Fig. II).
DISCUSSION
In a brain-tissue homogenate, UTTER TMdemonstrated a magnesium-activated apyrase, the activity of which varied with the sodium concentration. Homogenates of rat
nerves 8 and of crab nerves contain not only a magnesium-activated ATPase, but also
an adenylic kinase, It seems possible that the apyrase activity demonstrated by
UTTER was due to the combined effects of a magnesium-sodium-activated ATPase
and an adenylic kinase.
The ATPase from crab nerve studied here is magnesium-activated and located in
submicroscopic particles. In these respects it resembles the ATPase isolated from rat
nerve and from the sheath of giant axons b y ABOOD AND GERARD8 and also the ATPase isolated from muscle b y KIELLY and MEYERHOF11. There are further points of similarity between the magnesium-activated ATPases from crab nerve and from muscle.
Their p H optima are roughly identical, 7.2 and 6.8, respectively, and they are both
strongly inhibited b y calcium ions. The effect of sodium ions on the activity of the
magnesium-activated ATPase from muscle has not been studied.
The experiments reported here show that the activity of the ATPase from crab
nerve is highly dependent on the relative concentrations of the four cations Na +, K +,
Mg ÷+ and Ca +÷. The presence of magnesium ions is an obligatory requirement for the
activity of the enzyme; sodium ions increase the activity when magnesium ions are
present; potassium ions increase the activity when the system contains both magnesium and sodium ions. In high concentrations potassium ions inhibit that part of the
activity which is due to Na ÷, while the activity due to Mg ÷+ is not affected. Calcium
ions inhibit the activity under all conditions.
In the presence of magnesium or of magnesium + sodium the optimum magnesium concentration was found to be 3 mM/1 when the ATP concentration was also
3 mM/1. Preliminary studies indicate that the optimum concentration of magnesium
is equal to the ATP concentration also at other ATP levels. From this, one might infer
that the substrate for the enzyme is magnesium-ATP.
The differences between the effects of sodium and potassium suggest that the
activating effects of sodium and potassium differ in their point of attack, and that the
inhibitory effect of potassium is due to an interference with the sodium activation. An
hypothesis to fit this would be that the substrate most readily attacked b y the enzyme
is sodium-magnesium-ATP. Addition of K+ might then lead, on the one hand, to a
direct stimulation of the enzyme in the presence of Mg++ and Na + and, on the other,
to a displacement of sodium from the substrate, resulting in an inhibition.
This assumption would explain, firstly, why potassium is activating only in the
presence of magnesium + sodium.
Re]erences p. 4oL
400
J . c . SKOU
VOL. 28 (1957)
Secondly, it would explain the relation between enzyme activity and potassium
concentration (c/. Fig. 5)- The rise of activity on addition of K + must then be due to a
direct stimulation of the enzyme by this ion. With an increase in the potassium concentration this stimulation must be counteracted by an inhibition due to displacement
of sodium from the substrate. The activity must consequently pass through a maximum
and eventually approach the level observed when no sodium was added to the system.
Thirdly, it would explain the dependence of the potassium effect on the sodium
concentration. The higher the sodium concentration, the higher must be the potassium
concentration required to give a certain displacement of sodium from the substrate.
Since a higher potassium concentration means a stronger activation of the enzyme,
it follows that the maximum enzyme activity obtained by the addition of potassium
must increase with the sodium concentration. The potassium concentration required
to give maximum activity must also increase with the sodium concentration. Fig. 5
shows that maximum activity was obtained when the concentration of potassium was
approximately equal to that of sodium. The concentration of potassium required to
reduce the enzyme activity to the level observed when no sodium was added must accordingly also increase with the sodium concentration.
Although the above-mentioned assumption could explain a majority of the data,
it should be noted that a few observations are not accounted for in this way, e.g. the
decrease in the activating effect of Na + above a certain concentration in a system
which does not contain K +, and the inhibitory effect of small amounts of sodium in
the presence of a high potassium concentration. Further studies may clarify these
points.
The effect of calcium on the enzyme is purely inhibitory. The inhibition is counteracted by the addition of extra magnesium and may therefore be due to a competition
between calcium and magnesium.
It may now be asked whether the present studies have produced any evidence
of a connexion between this enzyme and the active extrusion of sodium ions from the
axon.
The process responsible for this transport must presumably be located in or in
close proximity to the nerve membrane. In homogenates of crab nerve the enzyme
studied here is located in submicroscopic particles, and we do not know its localization
in the intact nerve, but it is suggestive that ABOOD AND GERARD w e r e able to isolate
from the sheath of giant axons an ATPase which was also magnesium-activated and
located in submicroscopic particles.
As previously pointed out, studies by HODGKIN AND KEYNES and by CALDWELL
indicate a connexion between the sodium extrusion from nerve and the metabolism
of energy-rich phosphate esters. The substrate of ATPase is an energy-rich phosphate
ester.
I'{ODGI~IN AND K E Y N E S 1~ have shown that the sodium effiux from giant axons
depends on and is directly proportional to the intra-axonal sodium concentration;
they injected sodium into the axons, and the intra-axonal sodium concentrations in
their experiments varied from the 4o mM/1 normally present to 13o mM/1.
In the crab nerve the intra-axonal potassium concentration is 342 mM/kg axoplasm14; this is calculated from determinations of the potassium and sodium concentrations in whole crab nerves on the assumption that the intra-axonal sodium concentration is the same as in giant axons, viz. 4o mM/kg axoplasm.
Re/erences p. 4oz.
VOL.
23
(1957)
INFLUENCE OF CATIONS ON AN A T P A S E
401
The present experiments showed that in the presence of 35o m M K/1 the activity
of the magnesium-activated ATPase is highly dependent on the sodium concentration,
and there is an approximate direct proportionality between the enzyme activity and
sodium concentration within the concentration range used in the experiments of
HODGKIN AND KEYNES (c/. Fig. IO). The extent of the changes in enzyme activity is
influenced by the Mg: Ca ratio in the system, but the linearity is observed at all magnesium and calcium concentrations used. If, in analogy to what happens in the nerve
after stimulation, a rise in the sodium concentration is accompanied by a decrease in
the potassium concentration, the ATPase activity is further enhanced (c/. Fig. 9).
The above considerations show that the crab-nerve ATPase studied here seems to
fulfil a number of the conditions that must be imposed on an enzyme which is thought
to be involved in the active extrusion of sodium ions from the nerve fibre. Further
studies on the enzyme and its relation to the cations m a y serve to throw light on the
nature of this process.
SUMMARY
Leg nerves from the shore crab (Carcinus maenas) contain an adenosine t r i p h o s p h a t a s e which is
located in the submicroscopic particles. The influence of sodium, potassium, m a g n e s i u m and calc i u m ions on this e n z y m e has been investigated.
The presence of m a g n e s i u m ions is an obligatory r e q u i r e m e n t for the activity of the enzyme.
S o d i u m ions increase the activity w h e n m a g n e s i u m ions are present. P o t a s s i u m ions increase the
a c t i v i t y w h e n the s y s t e m contains b o t h m a g n e s i u m and s o d i u m ions. P o t a s s i u m ions in high conc e n t r a t i o n inhibit t h a t p a r t of the activity which is due to Na +, while the activity due to Mg ++ is
not affected. Calcium ions inhibit the enzyme u n d e r all conditions.
W h e n Mg ++ or Mg ++ + Na + are p r e s e n t in the system, the o p t i m u m m a g n e s i u m concentration is equal to the concentration of ATP. If p o t a s s i u m ions are added, the o p t i m u m m a g n e s i u m
c o n c e n t r a t i o n is doubled. If calcium ions are also added, the o p t i m u m m a g n e s i u m c o n c e n t r a t i o n
becomes still higher, a n d it increases with the calcium concentration.
A m a j o r i t y of these o b s e r v a t i o n s m a y be explained by a s s u m i n g (a) t h a t the s u b s t r a t e m o s t
readily a t t a c k e d b y the e n z y m e is s o d i u m - m a g n e s i u m - A T P , (b) t h a t p o t a s s i u m ions s t i m u l a t e
the e n z y m e directly, and (c) t h a t an increase in the c o n c e n t r a t i o n of p o t a s s i u m ions leads to
a displacement of s o d i u m ions from the s u b s t r a t e and accordingly to an inhibition of the reaction.
If the s y s t e m contains the four cations in concentrations r o u g h l y equal to those in the crabnerve axoplasm, an increase in the sodium concentration as well as a decrease in the p o t a s s i u m
c o n c e n t r a t i o n will lead to an intensification of the e n z y m e activity. This observation, as well as
some o t h e r characteristics of the system, suggest t h a t the adenosine t r i p h o s p h a t a s e studied here
m a y be involved in the active extrusion of s o d i u m from the nerve fibre.
REFERENCES
1 R. I). KEYNES, J. Physiol. (London), 114 (1951) 119.
A. L. HODGKIN AND R. D. KEYNES, J. Physiol. (London), 128 (1955) 28.
3 W. F. LOOMIS AND F. LIPMANN, Federation Proc., i2 (1953) 218,
4 j . I). JUDAH, Biochem. J., 49 (1951) 2715 D. KEILIN, Proc. Roy. Soc. (London), B 121 (1936) 165.
6 p. C. CALl)WELL, J. Physiol. (London), 132 (I956) 35P.
7 B. LIBET, Federation Proc., 7 (1948) 72.
S L . G. ABOOD AND R W . GERARD, ]. Cellular Comp. Physiol., 43 (1954) 379.
9 H . M. KALCKAR, J . Biol.
Chem., 167 (1947) 4 4 5 .
10 C. H. FISKE AND Y. SUBBAROW, J. Biol. Chem., 66 (1925) 375.
11 W. W. KIELLY AND O. MEYERHOF,J. Biol. Chem., 176 (1948) 59I.
is M. F. UTTER, J, Biol. Chem., 185 (195 o) 499.
13 A. L. HODGKIN AND R. D. KEYNES, J. Physiol. (London), 13I (I956) 592.
14 R. D. KEYNES AND R. R. LEWIS, J. Physiol. (London), 113 (1951) 73.
Received June I5th, 1956