MECHANISM OF INHIBITION OF ANAEROBIC GLYCOLYSIS OF BRAIN BY SODIUM IONS*

BY M. F. UTTER

(From the Department of Biochemistry, School of Medicine, Western Reserve University, Cleveland)

(Received for publication, February 25, 1959)

During the course of an investigation of the anaerobic glycolysis of nervous tissue of cotton-rats, it was observed that sodium ions had a con- siderable inhibitory effect under certain conditions. These results are in accord with an earlier report by Racker and Krimsky (1) who found that Na+ inhibited the glycolysis of mouse brain homogenates. LePage (2) has reported that the glycolysis of tumor homogenates is much reduced when sodium salts of buffers and substrates are employed as compared with glycolysis in the presence of potassium salts. On the basis of rather general observations, Na+ has been excluded from systems with homogenates of liver (3, 4) and chick embryo (5) for the study of glycolytic and oxidative reactions of carbohydrate metabolism. In view of the lack of specific in- formation concerning the nature of the inhibition caused by Na+, an at- tempt has been made to investigate the extent, specificity, and mechanism of the inhibition on preparations obtained from nervous tissues. The re- sults are presented in this communication.

The report of Racker and Krimsky was one of the first to emphasize the inhibitory nature of Na+ upon metabolic processes, although the phenome- non might have been predicted from earlier studies by various workers on the stimulatory effect of K+. In the most definitive studies of the stimu- latory effect of K+ upon carbohydrate metabolism, Boyer et al. (6, 7) concluded that K+ stimulated the transfer of phosphate from phospho- pyruvic acid to adenylic acid.’ These workers also mentioned that a high concentration of Naf was inhibitory to the same reaction. There have been numerous other studies showing the stimulatory effect of K+ upon glycogen deposition by liver slices (8), fermentation by yeast extracts (9, lo), and aerobic glycolysis of brain (11). In some of these studies, it seems likely that the effect under study could have been termed an Na+ inhibi- tion as well as a K+ stimulation if the experiments had been performed in a different fashion.

* 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

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500 INHIBITION OF GLYCOLYSIS BY NA+

Materials and Methods

Sodium and potassium salts of hexose diphosphate were prepared from the commercial barium salt. The solutions contained a small amount of an unknown inhibitor which was removed by treating briefly with charcoal.

Sodium and potassium phosphopyruvate were prepared from the silver barium salt of a sample synthesized by the method of Schmidt.2

ATP was isolated from rabbit muscle by a method closely resembling that recently described by Dounce et al. (12).

ADP was prepared from ATP by an unpublished method which utilized an ATPase obtained by isoelectric precipitation at pH 6 of a water extract of rabbit muscle, as described by Kalckar (13) for the hydrolysis of the terminal phosphate. The ADP was isolated as the barium salt after puri- fication as the mercury salt. Enzymatic analyses with hexokinase and AMP deaminase showed an ATP and AMP content, respectively, of less than 2 per cent for each component.

AMP was prepared by neutralization of a sample of the commercial acid.3 DPN was prepared by the method of Williamson and Green (14). The various preparations used in these experiments had a purity of 40 to 60 per cent.

Cotton-rats weighing 90 to 150 gm., obtained from Tumblebrook Farms, were used in this study. The animals were decapitated while under light ether anesthesia and the brain and cord removed. The medulla was de- tached and pooled with the cord. The remaining brain tissue was homoge- nized thoroughly with 10 volumes of ice-cold 1.6 X 1O-4 M ammonium phos- phate buffer (pH 7.4) in a Potter-Elvehjem homogenizer. The cord-me- dulla mixture was homogenized with 8 volumes of cold water. Extracts were prepared from the homogenates by centrifuging for 15 minutes at 1800 r.p.m. in a Servall angle centrifuge and carefully removing the super- natant.

The manometric experiments were carried out at 38” in Warburg vessels with a total volume of 7 to 9 ml. by the following experimental procedure. The main chamber of the vessel contained the following substances, with molarity expressed on the basis of the final volume of reactants: 1.4 X 1O-2 M glucose, 1 X 1O-3 M ATP, 4 X 1O-3 M MgC12, 2.4 or 4.8 X 1O-2 M KHC03, 4.7 X 10e4 M reduced glutathione, 5 X lo4 M DPN, and 1.3 X 10-s M nicotinamide. In some experiments sodium or potassium HDP was added to give a final concentration of about 2.5 X 10s3 M; in others NaHC03 has been substituted for KHCO, and in many experiments NaCl or KC1 has

* Schmidt, G., private communication to Dr. H. G. Wood. * The author wishes to acknowledge a generous gift of AMP from the Ernst Bis-

chaff Company, Inc.

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M. F. UTTER 501

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 min- utes 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 equilibra- tion was not a reliable indication of lactic acid production, since hydroly- sis 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 experi- ments, the manometric values were extrapolated to coincide with the in- cubation 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 condi- tions closely followed those used in the manometric studies with the excep- tion of certain substrates or coenzymes which were added or omitted ac- cording 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 evacua- tion 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 pre- liminary 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.

* 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.

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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 po- tassium and ammonium salts. The addition of 0.07 M NaCi to this reac- tion 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 series of ion combina- tions 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

. Na*

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 potas- sium or sodium HDP was added. Other experimental conditions ss described in the text.

of glycolysis is obtained in the presence of HDP, demonstrating the reversal of the Na+ effect by the ester.

These experiments are in general agreement with the results of Racker and Krimsky (l), although the experimental procedure, as well as the spe- cies of experimental animal, was different in the two series of experiments.

Balance Studies on Sodium Inhibition-In the presence of HDP, the gly- colytic activity of brain homogenates, as measured by acid production, is essentially unaffected by the addition of Na+ to a medium which already contains potassium and ammonium salts, as shown in Fig. 1. When a chemical balance of the glycolytic process is carried out, however, Na+ causes certain alterations in the reactions, even though the over-all activity is not changed. Such an experiment is presented in Table I. Also, exten- sion of the Naf inhibition to cord-medulla preparations is demonstrated in this experiment.

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M. F. UTTFR 503

In Table I, in addition to the total acidity changes as measured by COZ production, lactic acid,’ glucose, and inorganic phosphate were determined chemically. Measurements of the latter two substances give a picture of the intermediate phosphorylation reactions. In the presence of a sufficient phosphate reservoir such as HDP, some of the phosphorylation reactions may be inhibited substantially without slowing the over-all production of lactic acid, but the change in the rate of the phosphorylation reactions will be shown by a decreased esterification of inorganic phosphate and usually by an accompanying decrease in glucose utilization.

TABLE I Chemical Analysis of Effect of NaCl on Glycolysis of Glucose-HDP by Homogenates

a.nd Extracts of Nervous Tissue

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 concen- tration 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

COz produced Na+ K+-NH,’

Lactic acid produced Na+ K+-NHd+

Glucose utilized NC%+ K+-NH$

Inorganic phosphate utilized Na+ K+-NHd+

Brain Brain Cord Cord homogenste extract homogenate extract

~-

62.4 40.3 29.2 26.6 66.4 44.8 29.3 27.4 60.7 38.3 23.7 24.3 61.7 43.6 31.6 29.1 23.8 22.0 9.6 11.8 37.8 28.5 16.0 16.9

-0.5 24.5 -3.3 10.1 23.4 32.3 11.6 15.8

For sake of comparison the values are expressed as Q values. In general, in accord with the results of Fig. 1, Naf caused no appreciable decrease in lactic acid production or total acid production in any of the four different preparations tested. It is also apparent that the chemical and manometric values are in quite good agreement in the various cases. In contrast to the acid values, the utilization of glucose was inhibited by the addition of NaCl in all four cases, although the effect was somewhat smaller with the extracts than with the corresponding homogenates. In the case of the two homoge- nates, the glucose utilization was not large enough to account for the lactic acid production. For example, with the brain homogenate, the glucose utilized amounted to 23.8 ~1. per mg., which could account-for only 47.6 ~1.

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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 homog- enates, 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 phospho- pyruvate to the adenylic acid system, and (5) Apyrase.

An inhibition by Na+ of one or more of the first four reactions would de- crease the glycolytic rate by lowering the rate of ATP formation or utiliza- tion. 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 de- scribed. 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 de- scribed earlier. The results varied somewhat from preparation to prepara- tion, 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 glyco- lytic system containing all Naf was employed and KC1 was added in increasing amounts. Added.KCI at the lowest level tested, 0,014 M, more

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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

HL. BRAIN HOMOGENATE

FIG. 2 FIG. 3

FIG. 2. Effect of NaCl and KC1 concentration on glycolysis of brain homog- enates. The experimental conditions were those described in the text with a K+- N&C medium to which was added NaCl (Curve A) or KC1 (Curve B) added to an Na+ medium.

FIG. 3. Effect of NaCl on the hexokinase activity of brain homogenates. The experiments were conducted in Thunberg tubes at 38” for 4 minutes under 5 per cent COP-95 per cent N2. The tubes contained 2 X 10-* M ATP, 1.4 X 10-* M glu- cose, 2 X 10-* M KF, 4 X 10-a M MgCL, and K+-N&+ buffers in a total volume of 1.0 ml. In Curve A (a), 0.07 M NaCl was added, while in Curve B (0), no Nat was present. In Curve C the indicated amounts of NaCl were added. 0.1 ml. of brain homogenate was used in the experiments of Curve C.

ions. If this were so, the activity in Curve B should have increased pro- portionately 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 inhibi- tion of glycolysis. In contrast to the significant inhibition by Naf at a

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506 INHIBITION OF GLYCOLYSIS BY NA+

level of 0.035 M, a similar concentration of KC1 actually appeared to be stimulatory and a significant inhibition appeared only at a concentration of 0.14 M. LiCl resembled KC1 rather than NaCl in its effect. The fact that KC1 and LiCl were not inhibitory except in high concentration demon- strates that the inhibition by lower concentrations of NaCl is due neither to an increased tonicity of the reaction medium nor to the chloride ions.

In other experiments not shown here, calcium ions have been found to be inhibitory in concentrations as low as 0.02 M and sulfate ions caused marked inhibition in concentrations as low as 0.04 M. The mechanisms of the inhibition of glycolysis by calcium and sulfate ions have not been investi- gated further.

Effect of Sodium Ions on Hexolcinase-Racker and Krimsky (1) suggested that the inhibition of brain glycolysis by Na+ was exerted on the hexokinase

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~~ of

Y

0.035 0.07 0.14

Inhibition produced by

NaCl KC1 LiCl

)rn rcnl fie? ten: per Cd

47.3 -16.4 78.0 3.3 5.6 85.2 48.9 53.8

reaction, since they observed that the production of lactic acid was influ- enced far less by Na+ with HDP and FMP than it was with glucose. Recently Wiebelhaus and Lardy (20) reported that Na+ in high concentra- tions 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 influ- enced 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-

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M. F. UTI’ER 507

phatase activity and other phosphate transfer reactions may influence the acid production also. Likewise, with a complicated system, the disappear- ance 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 homo- genates 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 concentra- tions of NaCl cause a 75 per cent inhibition of the glycolysis. This experi- ment, in which each concentration of brain was run in triplicate, is entirely representative of experiments with some half dozen different brain homo- genates. The conditions were essentially those of the glycolytic experi- ment with the exception of the shorter incubation period. This alteration was necessary because it was not feasible to maintain the level of ATP re- quired 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 alter- native 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 homo- genate, and the absence of other proteins may have influenced the proper- ties 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 con- siderably 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,

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508 INHIBITION OF GLYCOLYSIS BY N-4+

an experimental period of 30 minutes was employed and no appreciable inhibition by Na+ was observed.

E$ect of iVa+ on Phosphohexokinase and Phosphorylation Coupled with Oxidation of Phosphoglyceraldehyde-The effect of 0.07 M NaCl upon the second phosphorylative step of glycolysis was also investigated. The phos- phohexokinase activity of cotton-rat brain homogenates was measured in Thunberg tubes after incubation with FMP, ATP, MgC&, and the usual buffers together with KF to prevent Apyrase activity and potassium iodo- acetate to prevent further oxidation of phosphoglyceraldehyde. The end- products of the reaction were HDP and dihydroxyacetone phosphate and phosphoglyceraldehyde in an equilibrium mixture. After determination of the triose phosphates by alkaline hydrolysis (22) the HDP could be calculated from the equilibrium constant.5 The sum of HDP-P and triose phosphate P divided by 2 gives the phosphate transferred from ATP to FMP and can be used as a measure of phosphohexokinase activity.

The results of a typical experiment are shown in Table III. The addi- tion of the Na+ seemed to cause a slight increase in the rate of transfer but this difference is neither constant nor significant. The Q value is given to show that the reaction rate is reasonably proportional to enzyme concen- tration and is also rapid enough to be consistent with the over-all glyco- lytic rate. It should be noted that the conversion of 1 molecule of FMP to HDP will be equivalent to the production of 2 molecules of lactic acid at a later stage in the reaction.

The third phosphorylation of the glycolytic system is that coupled with the oxidation of phosphoglyceraldehyde. These reactions were measured by incubating brain homogenates with HDP, ATP, pyruvate, glucose, MgCh, KF, reduced glutathione, and the usual buffers. After 30 minutes, the lactic acid production and the inorganic phosphate uptake were deter- mined. The lactate production gives information concerning the oxidative reaction (Reaction 2).

(1) HDP * glyceraldehyde phosphate + dihydroxyacetone phosphate DPN

(2) Glyceraldehyde phosphate + inorganic phosphate + pyruvate (-+ 1,3-di-

phosphoglycerate + lactate

(3) 1,3 Diphosphoglycerate + ADP FI 3-phosphoglycerate + ATP

(4) hexokinase

Glucose + ATP ------+ glucose-6-phosphate + ADP

6 The value for the equilibrium constant of zymohexase, 7.3 X 10-a at 40°, given by Meyerhof and Lohmann (23), was used in these calculations. Experimental tests of the brain homogenates with HDP showed that the rate at which equili- brium was reached was very rapid and that the equilibrium constant was in the range given by Meyerhof and Lohmann. It should be noted that, in any case, the calculation can cause only a small error, since under the conditions prevailing the equilibrium is in favor of triose phosphate.

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Id. F. UTTER 509

TABLB III Naf E$ect 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.

-

c

--

-

NaCl (final oncentration

Y

0.07

0.07

0.07

-

1

_-

-

Change in inorganic P

Y

-2.0 -0.3

1.3 2.3 3.5 2.5

-

(

-

Y

10.0 10.0 14.1 14.9 19.4 21.6

-

I

_-

-

In;crps..in

(calculated)

Y

0.8 0.8 2.3 2.5 4.3 5.2

-

f

.-

-

’ transferred kom ATP to fructose-6-P (calculated)

Y

5.4 5.4 8.2 3.7

11.9 13.4

-

--

-

Q* values

65.0 65.0 49.3 52.4 47.3 54.0

* FructoseS-phosphate converted per mg. of dry weight per hour, expressed as microliters.

TABLE IV Effect 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.

981. Y

1 0.10 66.1 0.07 65.4

1 0.15 65.9 0.07 67.0

2 0.10 51.9 0.07 50.7

2 0.15 58.9 0.07 58.9

-!-

Lactate production

Ratio, P to lactic acid

loo.4 0.66 93.7 0.59

110.9 0.70 99.5 0.67 96.8 0.54

loo.5 0.50 96.9 0.61 97.1 0.61

The disappearance of inorganic phosphate reflects not only the oxidative reaction but also Reactions 3 and 4, whereby phosphate is transferred to glucose. The results of two experiments are shown in Table IV, expressed as Q values. The final column shows the ratio of phosphate uptake to lactate production. In both experiments 0.07 M NaCl caused a slight lowering of the phosphate-lactate ratio but the decreases are very small.

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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 phos- phorylation reaction of the glycolytic system involves the transfer of phos- phate from phosphopyruvate to AMP or ADP. Boyer et al. (6, 7) re- ported 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

TABLE V Effect of 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 12 KF, and the usual K+-NHa+ buffers in a total volume of 1.15 ml.

Amount of brain homogenate

?A.

0.1

0.15

0.2

Added N&l (final concen-

tration)

Af

0.07

0.07

0.07

Inorganic P Decrease in increase phosph$pyruvatr

r Y

1.2 8.6 0.5 1.9 9.7 27.8 8.8 10.6

19.2 38.8 9.3 22.9

.-

-

-~-

Q values*

____

51.8 11.4

111.6 42.6

116.8 68.9

_-

--

-

Inhibition by Na+ of phos-

pho yruvate P if ecrease

per cent

77.9

61.9

41.0

* Phosphopyruvate P utilized, as microliters per hour per mg. of tissue.

to AMP at t,hree levels of tissue is shown in Table V. Phosphopyruvate was det,ermined as the phosphate fraction liberated by hypoiodite hydrol- ysis (24). The amount of phosphopyruvate reacting with AMP was considered to be equivalent to the amount of phosphopyruvate which disappeared. A very definite inhibition by the Na+, ranging from 77.5 to 41.0 per cent, is shown. Some increase in inorganic phosphate occurred in all cases, although the changes do not occur in any predictable manner. Presumably the inorganic phosphate arises from the hydrolysis of ADP or ATP formed by transfer of phosphate from the phosphopyruvate and need not be considered in the calculations. The presence of fluoride prevents the conversion of phosphopyruvate to phosphoglyceric acid and also in- hibits the Apyrase action to some extent.

In this experiment, as in others not shown, the extent of the reaction as

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M. F. UTTER 511

‘measured by phosphopyruvate utilization did not show a linear relationship to the amount of tissue used, suggesting that some uncontrolled factor is involved. In all cases, however, a Na+ inhibition comparable to that of Table V could be demonstrated. The Q values are given for comparison with the other glycolytic reactions in Table V. It will be noted that the higher concentrations of tissue give a rate exceeding that of over-all glycol- ysis, suggesting that this reaction is not necessarily a side reaction.

The finding of an Na+ inhibition of this reaction agrees with the previ- ous observation of K+ stimulation of the same reaction reported by Boyer et al. (6, 7). These investigators demonstrated the K+ effect upon the reaction in an indirect manner by showing the stimulatory action of the ions upon a system which transferred phosphate from Q-phosphoglyceric acid to creatine with AMP as a carrier. By balance studies and other considerations, they reached the conclusion that the phosphopyruvate-

TABLE VI Effect 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.

Amount of brain homogenate

ml. 0.1

0.15

0.2

-

1

_-

-

Added KC1 Increase in [final concentration) inorganic P

II

0.07

0.07

0.07

‘I -2.2 -0.3 -0.3

3.5 1.0 4.0

-

_-

-

Decrease in phosphopyruvate P

Y 0 3.25 0.8 8.0 3.0

18.5

-

Q value

0 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 ex- periment as evidenced by the lower Q values was due to the fact that the brain homogenate was frozen overnight before use. The comparable ex- periments 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 esteri- fication reactions is diminished. As mentioned earlier, the inhibition by Na+ was much more marked with homogenates than with extracts, suggest-

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512 INHIBITION OP GLYCOLYSIS BY NA+

ing that one of the affected systems is removed by centrifugation. Meyer- hof and Wilson (25) have shown that the greater portion of Apyrase is absorbed on the particles of the brain homogenates which are removed by centrifugation. IJnpublished experiments from this laboratory confirm this observation.

In Fig. 4, the stimulatory effect of Na+ upon Apyrase is shown. The results have been calculated in two ways: (a) the increase in inorganic phosphate (PO) and (5) the disappearance of easily hydrolyzable phosphate (ATP-P). Since there is a small endogenous production of inorganic

40.ATPP REMOVED

.&i I’& BRAIN HOMOGENATE

Fro. 4 LOG thCI CONCENTRATION

Fro. 5

Fro. 4. Effect of NaCl on Apyrase activity of brain homogenates. Experiments run in Thunberg tubes for 4 minutes under C02-Ne. Each tube contained, in addi- tion to the brain homogenate, 1.5 X 10-8 M ATP, 4 X 10-a M MgCl,, and the usual K+-NH4+ buffers in a volume of 1.0 ml. Where indicated 0.07 M NaCl was added.

FIG. 5. Apyrase activity with varying NaCl concentration. 0.1 ml. of brain homogenate used throughout. Other experimental conditions as described in Fig. 4.

phosphate, we have found the decrease in ATP to be a more reliable estimate.

According to either method of calculation Na+ stimulates the rate of Apyrase activity at all concentrations of brain tissue employed. The amount of stimulation varied somewhat from experiment to experiment, as was the case with the inhibitory effect in glycolysis experiments, but the results given in Fig. 4 are typical. The stimulation of Apyrase by Na+ amounts to approximately 50 per cent when the three different tissue levels are averaged, but it is probable that this loss of ester phosphate is sufficient to influence the course of glycolysis markedly, since the system is

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M. F. U!lTER 513

rather delicately balanced and small changes in the rate of a limiting reac- tion 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 sub- strate limitations. The reaction mixture contained about 93 y of hydro- lyzable 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 micro- grams of ATP-P hydrolyzed are plotted against the log of the NaCl concentration. Stimulation of Apyrase by Na+ can be detected at a con- centration 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-

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514 INHIBITION OF GLYCOLYSIS BY N.k+

bility. It is possible but unlikely that the Na+ acts by affecting the solu- bility of ATP. It seems more probable that Na+ is in some way 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 com- pared 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

FIG. 6 MINUTES

FIG. 7

FIG. 6. Interaction of Mg++and Na+on brain Apyrase. 0.1 ml,of brain homog- enate 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 condi- tions as in Figs. 4 to 6.

though the amount of hydrolyzable phosphate was the same in both experi- ments. 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.

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M. F. UTTER 515

DISCUSSION

The relative importance of ADP and AMP as acceptors in the phospho- pyruvate 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. Al- though 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 ex- pected 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 deriva- tive, 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 pres- ence of a large excess of a phosphate ester such as HDP, although an incu- bation period is required after the addition of HDP before the rate returns to normal. It would appear that there may be three interlocked effects 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-

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516 INHIBITION OF GLYCOLYSIS BY NA+

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 pro- found 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 concentra- tions 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 pre- viously, Boyer et al. (6, 7) demonstrated the K+-Na+ effects on phospho- pyruvate transphosphorylase with muscle extracts.

It is a pleasure to acknowledge the invaluable technical assistance of Mrs. Ellen Wolfe and the constant and stimulating interest of Dr. H. G. Wood.

SUMMARY

It has been shown that Na+ inhibits glycolysis of homogenates of nervous tissue of the cotton-rat. The effect was reversed by HDP, although an imbalance of the phosphate esters persisted. The inhibition occurred at concentrations of Na+ as low as 0.03 M and was due neither to the increased tonicity caused by the addition of NaCl nor to the chloride ions. The inhibition by Na+ was less marked in extracts.

The effect of Na+ upon various individual reactions of the glycolytic system was investigated. Hexokinase, phosphohexokinase, and the cou- pled oxidation-phosphorylation reactions were not significantly affected, but the transfer of phosphate from phosphopyruvate to AMP was inhibited and the dephosphorylation of ATP and ADP was stimulated.

The stimulation of Apyrase was shown to be operative at low levels of Na+ and to be dependent upon the presence of Mg++.

The significance of the effects of Na+ upon the individual reactions has been discussed in relation to its effect upon glycolysis.

BIBLIOGRAPHY

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., 1’73,239 (1948). fj. Boyer, P. D., Lardy, H. A.! and phillips, P. H., J. Biol. Chem., 146, 673 (1942).

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M. F. UTTER 517

7. Boyer, P. D., Lardy, H. A., and Phillips, P. H., J. Biol. Chem., 149, 529 (1943). 8. Buchanan, J. M., Hastings, A. B., and Nesbett, F. B., .I. Biol. Chem., 189, 435

(1949). 9. Farmer, S. N., and Jones, D. A., Nature, 150, 768 (1942).

10. Muntz, J. A., J. Biol. Chem., 171, 653 (1947). 11. Ashford, C. A., and Dixon, K. C., Biochem. J., 29, 157 (1935). 12. Dounce, A. L., Rothstein, A., Beyer, G. T., Meier, R., and Freer, R. M., J. Biol.

Chem., 174, 361 (1948). 13. Kalckar, H. M., J. Biol. Chem., 153, 355 (1944). 14. Williamson, S., and Green, D. E., J. Biol. Chem., 136, 345 (1940). 15. Utter, M. F., Federation PTOC., 6, 299 (1947). 16. Fiske, C. H., and Subbarow, Y., J. Biol. Chem., 66, 375 (1925). 17. Somogyi, M., J. Biol. Chem., 160, 61 (1945). 18. Nelson, N., J. BioZ. Chem., 163. 375 (1944). 19. Barker, S. B., and Summerson, W. H., J. Biol. Chem., 138, 535 (1941). 20. Wiebelhaus, V. D., and Lardy, H. A., Arch. Biochem., 21, 321 (1949). 21. Colowick. S. P., and Kalckar, H. M., .T. Biol. Chem., 148, 117 (1943). 22. Meyerhof, O., and Lohmann, K., Biochem. Z., 273, 413 (1934). 23. Meyerhof, O., and Lohmann, K., Biochem. Z., 271, 89 (1934). 24. Lohmann, K., and Meyerhof, O., Biochem. Z., 273, 60 (1934). 25. Meyerhof, O., and Wilson, J. R., Arch. Biochem., 14, 71 (1947). 26. Kielley, W. W., and Meyerhof, O., J. Biol. Chem., 176, 591 (1948). 27. Utter, M. F., and Werkman, C. H., J. Biol. Chem., 146, 289 (1942). 28. Meyerhof, O., and Wilson, J. R., Arch. Biochem., 17, 153 (1948). 29. Kubowitz, F., and Ott, P., Biochem. Z., 317, 193 (1944). 30. Greenberg, G. R., J. Biol. Chem., 181, 781 (1949).

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