Escolar Documentos
Profissional Documentos
Cultura Documentos
OF INHIBITION
OF ANAEROBIC
OF BRAIN
BY SODIUM
IONS*
GLYCOLYSIS
BY M. F. UTTER
(From
the Department
of Biochemistry,
University,
(Received
School of
Cleveland)
for publication,
February
Medicine,
Western
Reserve
25, 1959)
* Aided by a grant from The National Foundation for Infantile Paralysis, Inc.,
and by support of the Elisabeth Severance Prentiss Foundation.
1 The following abbreviations
have been used: HDP, hexose diphosphate; ATP,
adenosine triphosphate;
ADP, adenosine diphosphate; AMP, adenylic acid; Apyrase,
adenylpyrophosphatase;
FMP, fructose monophosphate;
DPN, diphosphopyridine
nucleotide; ATPase, adenosinetriphosphatase.
499
500
INHIBITION
OF
GLYCOLYSIS
BY
NA+
M.
F.
UTTER
501
* In order to obtain full activity it was found necessary to observe carefully the
precautions of buffering the tissue in the side arm, gassing at room temperature, and
mixing the tissue with the other components at the time the vessel was placed on
the bath. When the common procedure of gassing vessels at 38, with shaking, with
the tissue unprotected by buffers is followed, most of the glycolytic activity of brain
homogenates is lost. It has been shown previously (15) that phosphohexokinase
is
inactivated by the slight acidification produced by such a procedure.
been added. The side arm of the vessel contained 0.05 ml. of 0.08 M
KHC03, 0.05 ml. of 0.08 M ammonium phosphate buffer (pH 7.4), and
0.15 ml. of a 1: 10 homogenate of the tissue.
The total volume of the cup
contents was 1.0 to 1.2 ml. The tissue was added as the last step in the
procedure before the vessels were gassed at room temperature for 7 minutes with a mixture of 95 per cent Nz-5 per cent COZ, and the contents of
the vessel were mixed with the tissue as the vessel was placed on the bath>
After 7 to 9 minutes equilibration,
the first reading was taken,
In some
cases, the gas evolution for the first 15 minute period following equilibration was not a reliable indication of lactic acid production, since hydrolysis of ATP and other factors may give anomalous gas changes during this
period.
The 15 to 60 minute period gave a fairly constant rate of acid
production in most experiments and has been chosen as representative
of
the glycolytic rate. In some cases chemical analyses were made upon the
cements of the vessel at the end of 60 or 90 minutes.
In these experiments, the manometric values were extrapolated to coincide with the incubation time in order that the chemical and manometric values might be
compared.
All manometric experiments were carried out in duplicate or
triplicate.
In the measurement of individual enzymes of the glycolytic system it has
been convenient to carry out the reactions in Thunberg tubes. The conditions closely followed those used in the manometric studies with the exception of certain substrates or coenzymes which were added or omitted according to the reaction under study.
The homogenate was placed in the
side arm with the protecting buffers as described above and the Thunberg
tubes filled with 5 per cent CO*-95 per cent Nz by three cycles of evacuation and filling. The reaction was stopped at the indicated time by the
addition of the appropriate deproteinizing
agent.
Analyses of inorganic phosphate were run on trichloroacetic
acid filtrates
by a modification of the Fiske and Subbarow method (16). In case preliminary hydrolysis
of organic phosphate compounds was necessary, the
methods are described in conjunction with the experiments.
Glucose and lactic acid were determined on aliquots of a Somogyi filtrate
(17) by the methods of Nelson (18) and Barker and Summerson (19)
respectively.
502
INEIIBITION
OF GLYCOLYSIS BY NA+
The effect of sodium and potassium ions upon the production of acid
from glucose by brain homogenates is shown in Fig. 1. It will be noted
that a high rate of glycolysis obtains when all additions were made as potassium and ammonium salts. The addition of 0.07 M NaCi to this reaction mixture caused a striking reduction in the rate, as shown by the middle
curve, although t,he activity is higher than that of an experiment in which
only sodium salts were used. The effect of the same seriesof ion combinations on glycolysis of a glucose-HDP substrate is also shown in Fig. 1. In
the presence of HDP, the addition of 0.07 M NaCl to a K+-NHJ+ medium
has little inhibitory effect. Even with all sodium salts, a reasonable rate
TIME
IN MINUTES
FIG. 1. Inhibition
of brain glycolysis by N&l and its reversal by HDP.
In the
experiments represented by the right-hand
half of the graph 2.5 X lo+ M potassium or sodium HDP was added. Other experimental
conditions ss described in
the text.
. Na*
M.
F.
503
UTTFR
Chemical
Analysis
of Effect
I
of Glucose-HDP
Tissue
by Homogenates
Results expressed as & values (microliters per mg. of dry weight per hour). In
these experiments, the components of the reaction mixture were those described
under Methods
with the following changes. In the Na+ experiments, 0.024 M
NaHCOa was present, but K+ salts of ATP and HDP gave a K+ concentration
of
about 1 X 10-r M and ammonium phosphate buffer gave a final ammonium concentration of 8 X 10-r M. In the K+ experiments 0.024 M KHCOZ replaced the Na+
buffer. The vessels contained homogenate or extract equivalent to 2 mg., dry
weight, of the original tissue. The experiments with extracts were conducted
for 90 minutes; with homogenates for 60 minutes.
Measurement
Bufkrs
Brain
Brain
homogenste
extract
Cord
homogenate
Cord
extract
~-
COz produced
Lactic acid produced
Glucose utilized
Inorganic
phosphate utilized
Na+
K+-NH,
Na+
K+-NHd+
NC%+
K+-NH$
Na+
K+-NHd+
62.4
66.4
60.7
61.7
23.8
37.8
-0.5
23.4
40.3
44.8
38.3
43.6
22.0
28.5
24.5
32.3
29.2
29.3
23.7
31.6
9.6
16.0
-3.3
11.6
26.6
27.4
24.3
29.1
11.8
16.9
10.1
15.8
of NaCl
on Glycolysis
a.nd Extracts
of Nervous
504
INHIBITION
OF
GLYCOLYSIS
BY
NA+
of lactic acid, while the actual production was 60.2 ~1. The balance of the
lactic acid was formed from the HDP, of course.
The inorganic phosphate changes follow the same pattern as the glucose
values, although the effect of Na+ is perhaps even more striking.
With the
homogenates no net esterification of inorganic phosphate occurs in the
presence of Na +, although considerable net esterification occurs with only
K+ andNHd+ present.. It is interesting to note that the changes caused by
the Na+ are again much smaller with the extracts. It is possible to suggest
two hypotheses from the foregoing observations: (a) that Na+ acts in some
fashion to decrease the phosphorylative efficiency of the glycolytic system,
thereby creating an organic phosphate deficit which must be rectified by
the addition of an organic phosphate donor such as HDP; (b) that, since
the effect is considerably reduced in extracts as compared with homogenates, one or more of the enzymes affected by Na+ is removed by
centrifugation.
Because of the inhibition of esterification processes, it seemed likely that
Na+ is affecting one or more of the following reactions: (1) hexokinase, (2)
phosphohexokinase, (3) the phosphorylation
coupled with the oxidation of
glyceraldehyde phosphate, (4) the transfer of phosphate from phosphopyruvate to the adenylic acid system, and (5) Apyrase.
An inhibition by Na+ of one or more of the first four reactions would decrease the glycolytic rate by lowering the rate of ATP formation or utilization. A stimulatory effect upon the fifth enzyme, Apyrase, would produce
the same effect by lowering the concentration of ATP.
In a later section the effect of Naf upon each of these reactions is described. Before proceeding to the testing of the individual enzymes, we
attempted to obtain more information
concerning the inhibition of the
entire glycolytic system, particularly in so far as concentration effects and
specificity were concerned.
Concentration of Sodium Ions-In
Fig. 2 various concentrations of NaCl
were superimposed upon the usual K+-NHd+ buffer system which was described earlier. The results varied somewhat from preparation to preparation, but the experiments are typical.
NaCl was markedly inhibitory at
concentrations as low as 0.035 M in most experiments and the inhibition
increased rapidly with increasing NaCl concentration. .
In no experiment did we detect appreciable inhibition with concentrations
of NaCl below 0.01 M. From a practical standpoint, this observation
demonstrates that it is not necessary to exclude rigidly all Na+ from the
reaction mixture, but merely to hold the concentration at a low level.
The converse experiment is shown in Curve B of Fig. 2. Here a glycolytic system containing all Naf was employed and KC1 was added in
increasing amounts.
Added.KCI at the lowest level tested, 0,014 M, more
M.
F.
U!lTER
50.5
than doubled the activity of the control, but it was impossible to increase
the-activity further by adding more KCl, and high concentrations actually
proved inhibitory.
From the results of this experiment it would appear
that, although the Naf-Kf effect is reciprocal in nature, it is not probable
that the effect upon glycolysis can be expressed as a simple ratio of the two
FIG.
HOMOGENATE
ions. If this were so, the activity in Curve B should have increased proportionately to the KC1 concentration.
Specificity of NaCl in Inhibition of Glycolysis-In view of the inhibition
of higher concentrations of KC1 it was desirable to learn whether the NaCl
effect was a non-specific one. In Table II the effect of the addition of
various concentrations of other salts to the usual K+-NH4+ salt-glycolytic
system is presented. The results are reported in terms of per cent inhibition of glycolysis. In contrast to the significant inhibition by Naf at a
HL. BRAIN
FIG.
506
INHIBITION
OF
GLYCOLYSIS
BY
NA+
TABLE
II
SpecijEcity of Inhibition
of Glycolysis by NaCl
In this experiment, the glycolytic medium contains the K+-NH,+ salt mixture
previously described, to which were added the various concentrations of the salts as
indicated.
The manometric readings for the 15 to 60 minute period were used for
the calculations.
COIl~lra~~
Inhibition
of
produced
by
NaCl
KC1
LiCl
)rn rcnl
fie? ten:
per Cd
0.035
0.07
0.14
47.3
78.0
85.2
-16.4
3.3
48.9
5.6
53.8
since they observed that the production of lactic acid was influenced far less by Na+ with HDP and FMP than it was with glucose.
Recently Wiebelhaus and Lardy (20) reported that Na+ in high concentrations was inhibitory to partially purified hexokinase obtained from beef
brain.
The effect of NaCl upon the hexokinase reaction in brain homogenate of
the cotton-rat is shown in Fig. 3. The reaction was followed by chemical
determination
of glucose disappearance in a filtrate free from the various
phosphate esters which interfere with glucose determinations.
Since the
hexokinase reaction is essentially irreversible, it is not appreciably influenced by subsequent reactions as long as an adequate supply of ATP is
available.
Other methods of hexokinase assay, such as the manometric
method of Colowick and Kalckar (21) in which the activity is followed by
acid production, were found inapplicable to homogenates in which phos-
reaction,
M.
F.
UTIER
507
phatase activity and other phosphate transfer reactions may influence the
acid production also. Likewise, with a complicated system, the disappearance of ATP may be influenced by reactions other than hexokinase.
Glucose disappearance, appears, however, to be equivalent to hexokinase
activity,
even in complicated systems.
The effect of 0.07 M NaCl upon the hexokinase reaction in brain homogenates of the cotton-rat at different levels of tissue is shown in Fig. 3 in
Curves A and B, where glucose disappearance has been plotted against the
concentration
of tissue.
The relationship
is linear, indicating that the
assay is valid and that ATP is not a limiting factor.
It will be noted that
the NaCl had no appreciable effect despite the fact that similar concentrations of NaCl cause a 75 per cent inhibition of the glycolysis.
This experiment, in which each concentration of brain was run in triplicate, is entirely
representative
of experiments with some half dozen different brain homogenates.
The conditions were essentially those of the glycolytic experiment with the exception of the shorter incubation period.
This alteration
was necessary because it was not feasible to maintain the level of ATP required for maximum activity over longer periods of time.
The effect of varying concentrations
of NaCl upon the same reaction is
plotted in Curve C of Fig. 3. This experiment also shows that the NaCl
had no influence with the exception of the highest concentration,
0.14 M,
at which a slight inhibition was observed.
The foregoing results do not support the hypothesis that the inhibition
of glycolysis by Na+ operates through an effect upon hexokinase.
The
observation that Na+ inhibits the fermentation of glucose but not of HDP
and FMP could be explained by an inhibition of hexokinase, but an alternative explanation may be offered.
The molar requirements of phosphate
(as ATP) required to initiate fermentation on glucose, fructose-6-phosphate,
and HDP are 2: 1: 0 respectively.
Therefore, an inhibition of any reaction
influencing t,he formation or dest,ruction of ATP will be noted much more
readily on a glucose substrate, since the ATP requirement is higher.
Although our results are in apparent disagreement with the inhibition of
hexokinase observed by Wiebelhaus and Lardy, it should be noted that the
species from which the brain was obtained was different.
Also the latter
authors were using a partially purified hexokinase rather than a homogenate, and the absence of other proteins may have influenced the properties of the enzyme.
The inhibition fouhd by Wiebelhaus and Lardy, 18.5
per cent at a level of 0.06 M NaCl and 37.2 per cent at 0.1 M NaCI, is considerably smaller than that observed on the entire glycolytic system.
The experimental period used in these experiments was short, but in
experiments on the phosphorylation
of glucose by hexokinase coupled with
the oxidation of phosphoglyceraldehyde,
as described in the next section,
508
INHIBITION
OF
GLYCOLYSIS
BY
N-4+
(1)
HDP * glyceraldehyde
(2) Glyceraldehyde
phosphate
phosphate
+ inorganic
+ dihydroxyacetone
phosphate
phosphate
DPN
+ pyruvate (-+
phosphoglycerate
(3)
(4)
1,3-di+ lactate
1,3 Diphosphoglycerate
+ ADP FI 3-phosphoglycerate
+ ATP
hexokinase
Glucose + ATP ------+
glucose-6-phosphate
+ ADP
Id.
F.
III
TABLB
Naf
E$ect
509
UTTER
on Phosphohexokinase
Reaction
in Brain
These experiments were run in Thunberg tubes for 4 minutes at 38 under 5 per
cent COe-95 per cent Nt. The tubes contained in a volume of 1.15 ml. the following:
2 X 10e3 M ATP, 2.5 X lo-5 M FMP, 4 X 10-3 M K iodoacetate, 2 X lo- M KF and
the usual I(+-NH*+ buffers, and 4 X 10e5 M MgCle.
0.07
0.07
-
-2.0
-0.3
1.3
2.3
3.5
2.5
10.0
10.0
14.1
14.9
19.4
21.6
0.8
* FructoseS-phosphate
microliters.
.-
_-
converted
transferred
fkom ATP to
fructose-6-P
(calculated)
Q* values
--
0.8
2.3
2.5
4.3
5.2
5.4
5.4
8.2
3.7
11.9
13.4
65.0
65.0
49.3
52.4
47.3
54.0
TABLE
IV
of NaCl on Coupled Phosphorylation
These experiments were carried out in Warburg vessels at 38 for 30 minutes under
5 per cent CO,-95 per cent Nr with a total volume of 1.15 ml. The reaction mixture
contained 5 X 10-4 M ATP, 5 X 10-s M HDP, 2 X 10-P M K pyruvate, 1.4 X lo-* M
glucose, 4 X 10-S M MgCl*, 2 X 10-o r.r KF, 4.6 X lo- M reduced glutathione, and
the usual buffers.
Effect
Lactate
production
981.
0.10
0.07
0.15
0.10
0.15
Ratio,
P to lactic acid
0.07
0.07
0.07
66.1
65.4
65.9
67.0
51.9
50.7
58.9
58.9
loo.4
93.7
110.9
99.5
96.8
loo.5
96.9
97.1
0.66
0.59
0.70
0.67
0.54
0.50
0.61
0.61
-!-
0.07
(calculated)
_Y
In;crps..in
Change in
NaCl (final
concentration 1 inorganic P
--
510
INHIBITION
OF
CtLYCOLYSIS
BY
N-4+
It will be noted that the rate of lactate production is high, yielding a Qz&,
of 94 to 100 compared with values of 65 to 80 in glycolysis experiments.
Since the over-all rate of this system is greater than that of the glycolytic
system as a whole, it seems unlikely that the inhibition of glycolysis by
Na+ occurs through an effect upon this particular segment of the system.
Action of NaCl on Phosphopyruvate Transphosphoryluse-The
fourth phosphorylation reaction of the glycolytic system involves the transfer of phosphate from phosphopyruvate
to AMP or ADP.
Boyer et al. (6, 7) reported that K+ stimulated this particular reaction in muscle homogenates.
In one experiment there waa also an indication that high concentrations of
Na+ were inhibitory to the reaction, although this point was not elaborated.
The effect of Na+ upon the transfer of phosphate from phosphopyruvate
of
-~-
Amount of brain
homogenate
Added N&l
(final concentration)
Inorganic P
increase
Af
Decrease in
phosph$pyruvatr
_-
Q values*
.- ____
?A.
0.1
0.07
0.15
0.07
0.2
0.07
* Phosphopyruvate
to AMP
P utilized,
--
Inhibition by
Na+ of phospho yruvate P
ifecrease
per cent
1.2
0.5
9.7
8.8
19.2
9.3
8.6
1.9
27.8
10.6
38.8
22.9
as microliters
51.8
11.4
111.6
42.6
116.8
68.9
77.9
61.9
41.0
TABLE
V
NaCl on Transfer
of Phosphate
from
Phosphopyruvate
to Adenylic
Acid
These experiments were run in Thunberg tubes at 38 for 4 minutes under CO*-N2.
The solution contained 1 X 10eJ M AMP, 91 y of phosphopyruvate
P, 4 X 10eS M
MgCl2,2 X l(rs 12KF, and the usual K+-NHa+ buffers in a total volume of 1.15 ml.
Effect
M. F. UTTER
511
TABLE
VI
of KC1 on Transfer of Phosphate
from Phosphopyruvate
to Adenylic
Acid
Conditions as in Table V, except that 81 y of phosphopyruvate
P were used.
Effect
Amount of brain
homogenate
Added KC1
1[final concentration)
Increasein
inorganic P
_-
ml.
0.1
II
0.07
0.15
0.07
0.2
0.07
-
I
-2.2
-0.3
-0.3
3.5
1.0
4.0
Decreasein
phosphopyruvateP
Q value
_-
Y
0
3.25
0.8
8.0
3.0
18.5
19.6
3.2
32.1
9.0
55.7
AMP step was the reaction actually stimulated by K+. Table VI shows
the stimulatory
effect of K+ in a more direct fashion. The addition of
0.07 M KC1 to a reaction mixture containing only sodium salts greatly
stimulates the reaction. Presumably, the decrease in activity in this experiment as evidenced by the lower Q values was due to the fact that the
brain homogenate was frozen overnight before use. The comparable experiments of Tables V and VI are those in which both Na+ and K+ were
present; i.e., the last member of each pair in both tables.
Effect of Sod&m Ions on Apyra.se-In
addition to the inhibitory effect of
Na+ upon the transphosphorylation
reaction between phosphopyruvate and
AMP, we have noted that Na+ has still another effect on the glycolytic
system, a stimulation of Apyrase action. Stimulation of Apyrase can cause
a decreased rate of glycolysis, since the amount of ATP available for esterification reactions is diminished.
As mentioned earlier, the inhibition by
Na+ was much more marked with homogenates than with extracts, suggest-
512
INHIBITION
OP
GLYCOLYSIS
BY
NA+
REMOVED
.&i
I& BRAIN HOMOGENATE
Fro. 4
40.ATPP
M.
F.
U!lTER
513
rather delicately balanced and small changes in the rate of a limiting reaction can be magnified when repeated cyclically..
In tissue studies not depicted here it has been found that the reaction
rate of the Apyrase falls off after about 4 minutes, probably because of substrate limitations.
The reaction mixture contained about 93 y of hydrolyzable P calculated on a basis of two phosphate groups per molecule of
ATP or 46.5 y on the basis of conversion to ADP.
At 4 minutes in Fig. 4
in the presence of Na+, 43 y of ATP-P had already been released. The
amount of ATP used in these experiments is comparable to that in the
glycolytic experiments, illustrating that Apyrase is sufficiently active under
the conditions of glycolysis to destroy a major portion of the ATP in a
very short time.
It was of interest to determine the effect of the concentration of Na+ on
the action of Apyrase.
The results are shown in Fig. 5 in which micrograms of ATP-P hydrolyzed
are plotted against the log of the NaCl
concentration.
Stimulation of Apyrase by Na+ can be detected at a concentration of about 0.003 M. Since glycolysis was appreciably inhibited
only at concentrations above this point, this experiment shows that Apyrase
is at least as responsive to low concentrations
of Na+ as the entire glycolytic
system.
This observation supports the thesis that stimulation of Apyrase
may constitute a major mechanism whereby Na+ inhibits glycolysis.
In all of the foregoing experiments, effects of Naf were demonstrated by
adding Naf to a reaction mixture which contained 0.004 M Mg++, as is the
case in the glycolytic system.
As shown in Fig. 6, the action of Na+ is
conditioned by the presence of Mg.
The results are plotted as Apyrase
activity against varying MgCh concentrations
in the presence of 0.07 M
NaCl and without NaCl.
Surprisingly, the Na+ effect was negligible when
Mg++ was omitted and did not reach a maximum until a concentration of
0.004 M MgClz was attained.
Thereafter as the Mg++ concentration was
increased, the activity declined in both cases in an essentially parallel
fashion.
It should be recalled that the 0.004 M MgC12 was present in the
glycolytic experiments and in all of the foregoing experiments on Apyrase
also. In a few experiments not shown here MnClz was used to replace
MgCI,.
Although Mn++ stimulated Apyrase activity to about the same
extent as Mg++, the addition of Na+ did not enhance the effect to the same
degree as it did with Mg++.
The mechanism of the combined action of Mg++ and Na++ is not clear.
Kielley and Meyerhof
(26) have recently studied an Mg++-stimulated
ATPase from muscle and there have been other reports of Mg++-stimulated
Apyrases in brain (25) and Escherichia coli extracts (27). The decreasing
activity with higher Mg++ concentrations
may be due to the removal of
ATP as the insoluble magnesium salt, since Mg ATP has a limited solu-
514
INHIBITION
OF
GLYCOLYSIS
BY
N.k+
It is possible but unlikely that the Na+ acts by affecting the solubility.
bility of ATP. It seemsmore probable that Na+ is in someway influencing
the enzyme itself, possibly by altering the physical state. Meyerhof and
Wilson (28) have recently reported that brain Apyrase exhibits different
properties toward certain inhibitors, depending on its physical state.
In Fig. 7 the effect of Na+ upon the dephosphorylation of ADP is compared with the action on ATP. It is clear that Na+ exerts a stimulatory
effect upon the removal of the second labile phosphate group as well as
the first, although the rate of action on ADP is somewhat lower. even
though the amount of hydrolyzable phosphate was the samein both experiments. The mechanism of phosphatase action upon ADP in brain is not
known. The possibilities exist that ADP is dephosphorylated via ATP
after a myokinase-like reaction, that ADP and ATP are dephosphorylated
by different phosphatases, or that a single enzyme attacks both substrates.
It is interesting to note that the activity of the brain preparation upon a
mixed substrate of ADP and ATP is almost equal to the sum of the activity
upon the two substrates separately. It should be mentioned that, with
the short periods of incubation used in these studies, dephosphorylation of
AMP does not appear to be a significant factor. Meyerhof and Wilson
(25) came to similar conclusions in their studies on brain Apyrase.
MINUTES
FIG. 7
FIG. 6. Interaction of Mg++and Na+on brain Apyrase. 0.1 ml,of brain homogenate used. Other experimental
conditions as in Fig. 4.
FIG. 7. Activity of brain Apyrase on ATP and ADP. The tubes contained 1.5
X Wa M ATP, 3.0 X 1W3 M ADP, and 0.07 M NaCl when indicated.
Other conditions as in Figs. 4 to 6.
FIG. 6
M.
F.
UTTER
515
DISCUSSION
The relative importance of ADP and AMP as acceptors in the phosphopyruvate transphosphorylase
reaction is not clearly understood.
On the
basis of indirect evidence, Boyer et al. (7) came to the conclusion that ADP
was superior to AMP as a phosphate acceptor in the phosphopyruvate
reaction in muscle extracts.
Kubowitz
and Ott (29) reported that an
enzyme isolated from human muscle catalyzed the transfer to ADP.
Although the situation in brain may be similar, it must be noted that, in the
absence of Na+, the transfer to AMP by brain homogenates is more than
adequate to account for glycolysis (Table V), making it difficult to dismiss
this reaction from consideration.
In view of the above observations it is possible to postulate a mechanism
for the inhibition of glycolysis by Na+. The importance of an adequate
supply of ATP for the maintenance of glycolysis is well known.
In any
system in which the ATP concentration
is dependent upon a balance of
synthetic and phosphatase reactions, a stimulation of the latter reactions
may lead to a decreased rate or cessation of activity in the over-all system.
The brain homogenate-glycolysis
preparation seems to be rather delicately
balanced in this regard and any increase in Apyrase activity may be expected to be reflected in decreased glycolysis.
Since the dephosphorylation
of ADP as well as ATP is promoted by Naf, the production of AMP is
enhanced.
It is interesting to note that AMP and its deaminated derivative, inosinic acid, have been reported by Greenberg (30) to be inhibitory
to glycolysis of brain preparations
when present in a high concentration
(0.001 M).
Therefore,
in addition to the loss of esterified phosphate
through a heightened Apyrase activity, an inhibitory substance is formed
at a greater rate. Likewise, the removal of AMP by rephosphorylation
with phosphopyruvate
is depressed by the Na+.
Greenberg (30) has
shown that the AMP inhibition of glycolysis can be overcome by the presence of a large excess of a phosphate ester such as HDP, although an incubation period is required after the addition of HDP before the rate returns
It would appear that there may be three interlocked effects
to normal.
of Naf: a stimulation of the conversion of ATP to AMP, an inhibition of
glycolysis by the AMP thus formed, and a decreased rate of removal of
AMP by rephosphorylation.
These three effects when taken together
seem entirely adequate as an explanation for the marked inhibition of
glycolysis by Na+.
As mentioned earlier, the stimulatory
effect of K+ and the inhibitory
effects of Na+ are partially reciprocal in nature, at least as observed in the
present experiments with brain and in t.he experiments by Boyer et al. (6,
7) with muscle preparations.
It should be emphasized, however, that the
addition of. K+ never completely reversed the effect of Na+ and that, in-
516
INHIBITION
OF
GLYCOLYSIS
BY
NA+
1. Racker, E., and Krimsky, I., J. Biol. Chem., 161, 453 (1945).
2. LePage, G. A., J. Biol. Chem., 176, 1009 (1948).
3. Reiner, J. M., Arch.
Biochem.,
12, 327 (1947).
4. Potter, V. R., Methods in medical research, Chicago, 1, 330 (1948).
5. Novikoff, A. B., Potter, V. R., and LePage, G. A., J. Biol. Chem., 173,239 (1948).
fj. Boyer, P. D., Lardy, H. A.! and phillips, P. H., J. Biol. Chem., 146, 673 (1942).
versely, Na+ inhibition was never complete in the presence of even small
concentrations
of K+. The latter observation lends some support to the
hypothesis that K+ may play some r@e other than the reversal of Na+
inhibition of certain glycolytid reactions as presented here. Muntz (10)
has shown that the addition of small amounts of K+ or NH*+ has a profound effect on the alcoholic fermentation by cell-free yeast preparations,
even in the presence of relatively large concentrations
of Na+.
It would seem from data presented here that the Na+ effect plays its
most important rble in homogenates, in which phosphate ester concentrations may be of prime importance in determining the course of the reactions
and the Apyrase concentration
is much smaller.
The Na+ inhibition can
be demonstrated
in extracts (Table I) however, and, as mentioned previously, Boyer et al. (6, 7) demonstrated the K+-Na+ effects on phosphopyruvate transphosphorylase with muscle extracts.
M.
F.
UTTER
517
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ARTICLE:
MECHANISM OF INHIBITION OF
ANAEROBIC GLYCOLYSIS OF BRAIN
BY SODIUM IONS
M. F. Utter
J. Biol. Chem. 1950, 185:499-517.