Metabolic Control of Glycolysis in Normal and Tumor Permeabilized Cells1

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1978;38:142-148. Cancer Res Mario Gosalvez, Socorro Garcia-Suarez and Luisa Lopez-Alarcon Permeabilized CellsMetabolic Control of Glycolysis in Normal and Tumor

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(CANCER RESEARCH 38. 142-148, January 1978]

Metabolic Control of Glycolysis in Normal and Tumor PermeabilizedCells1

Mario Gosalvez, Socorro Garcia-Suarez, and Luisa Lopez-Alarcon

BioquÃmica Experimental, ClÃnica Puerta de Hierro, Facultad de Medicina, Universidad AutÃ³noma, Madrid-35, Spain

ABSTRACT

Our previous reports have presented evidence suggesting the existence in tumor cells of a second control site ofglycolysis at pyruvate kinase and a competition foradenosine diphosphate between this enzyme and mitochondria, which is responsible for the Crabtree effect.Now, by using cells partially permeabilized to nucleotidesand phosphorylated substrates, we provide evidence supporting the existence in hepatocytes of a partial control byadenosine triphosphate at phosphofructokinase, which isfollowed by the total control by adenosine triphosphate atpyruvate kinase. The partial or nonoperation of this second site in Ehrlich ascites tumor cells appears to be thecause for the characteristic aerobic glycolysis, Crabtreeeffect, and low Pasteur effect of these cells.

INTRODUCTION

The metabolic control of glycolysis and the nature of thePasteur effect in normal and tumor cells are far from beingdefinitively established. We have recently reported (5, 6)evidence suggesting the existence of a second control siteof glycolysis at pyruvate kinase. At the same time weproposed that the characteristic Crabtree and Pasteur effects of tumor cells could be due to a competition for ADPbetween mitochondria and pyruvate kinase. Using normaland tumor cells that are partially permeable to nucleotidesand phosphorylated substrates, we have now collectedevidence supporting the hypothesis that the control ofglycolysis in rat liver cells consists of a partial control byATP at phosphofructokinase and a second total control byATP at pyruvate kinase. The second pyruvate kinase controldoes not operate or is only partially operative in Ehrlichascites tumor cells due to the lack of sensitivity of theenzyme to allosteric inhibition by ATP. The lack of sensitivity of pyruvate kinase to ATP in Ehrlich ascites cells closelycorrelates with the Pasteur and Crabtree effects of thesecells.

MATERIALS AND METHODS

The isolation of rat hepatocytes was done essentially bythe method of Howard and Pesch (8) as modified by Wagle(17). Male Wistar rats weighing 250 g were starved for 24 hrand killed by decapitation; the liver was extracted rapidlythrough the vena cava for perfusion. The liver was perfusedat 4Â°with oxygenated buffer (140 rriM NaCI, 5 ITIMKCI, 0.8

'This work was supported by Grants 12-201-75 and 12-241-76 from the

Instituto Nacional de PrevisiÃ³n, Spain.Received January 26, 1977; accepted October 18. 1977.

mM MgS04, 25 rriM NaHC03, and 1% albumin, pH 7) untilblood free. The liver was minced with scissors and homogenized in a Servali Omnimixer 230 with the small blades at1000 rpm 2 times for 30 sec each. The homogenate wasthen filtered through gauze and centrifugea at 500 rpm for5 min in a clinical centrifuge. With this procedure a cellpellet of 80 to 100 million cells with 100% viability wasroutinely obtained. The cells had a mean gluconeogenesisfrom lactic acid of 0.6 /umole/min/106 cells and an activeendogenous respiration completely inhibited by oligomy-cin.

Two strains of Ehrlich ascites tumors were used, a hyper-triploid tumor originally received from the Institute GustavRoussy (Paris strain) and a hypertriploid tumor receivedfrom the UniversitÃ Cattolica Sacro Cuore (Rome strain).The Ehrlich ascites cells from the 2 different strains showeddifferent values for glycolysis, respiration, Crabtree effect,Pasteur effect, and cell growth (Table 1). Ascites tumorcells were collected 8 days after transplantation, washed inisotonic phosphate saline (6.16 mM KCI, 9.35 mM Na2HPO4,1.65 mM NaH2PO4,and 0.9% NaCI, pH 7) at 22Â°,and freedof blood by osmotic shock for 15 sec in 10 HIMTris-HCI, pH6.5. All centrifugations were done at 900 x g for 5 min.

Hepatocytes and Ehrlich ascites tumor cells were permeabilized by different methods, as indicated in the "Results." Glycolytic rates were measured in permeabilized

cells by diluting the cell suspensions with incubation medium immediately after permeabilization to a concentrationof 8 x 10" cells. The hepatocytes were incubated for 10min at 30Â°in the medium of perfusion, and the Ehrlichascites cells were incubated at 30Â°in phosphate saline for

30 min. The medium was supplemented with nucleotides,cofactors, or substrates as indicated in the charts. Allexperimental points are from duplicate determinations, andthe results of at least 5 equal experiments were compared.The ATP used in all the incubations was the sodium form.At the end of the incubation period, the reaction wasstopped by adding cold 12% perchloric acid, and aftercentrifugation of the precipitated protein the supernatantwas neutralized with 6 N K2CO3and again centrifuged. Foreach experiment, basal samples were made, which werestopped at zero time of incubation. Lactic acid was determined enzymatically (1) in the final supernatant of allsamples.

The assay of pyruvate kinase was as previously described(4). Washed cells were suspended in 10 mM Tris-HCI, pH7.4, and 0.5 mM dithioerythritol; placed in a propylenetube; and frozen and thawed twice in liquid nitrogen. Theresulting homogenate was centrifuged at 30,000 x g for 15min at 2-4Â°.Samples of the supernatant were used for

kinetic studies, generally within 1 or 2 hr after its prepara-

142 CANCER RESEARCH VOL. 38

Metabolie Control of Glycolysis

Table 1Metabolic parameters

No difference in glycolytic rate was found in hepatocytes when 3 mw glucose was used. The cells were incubatedexactly as described in "Materials and Methods." Glucose concentration was 10 HIM; cyanide concentration was 2

rriM. Pyruvate kinase was isolated and assayed as previously described. The Pasteur effect, besides being shown inrelative percentage, is also computed in absolute terms (extent of glycolysis inhibited by oxygen) as recommendedby Weinhouse (18).

O2 uptake"Glycolysis''Relative

PasteurAbsoluteSystemEAC

(Rome)EAC(Paris)RathepatocytesEAC-pyruvatekinase(Rome)EAC-pyruvate

kinase(Paris)Rat

hepatocytes-pyruvatekinaseEndogenoussubstrates0.921.271.50Glucose0.210.601.50Crabtreeeffect

(%)77.252.8002+CNr3.204.802.45effectO.,

(%)2.40

252.80420.10

96Pasteureffect''0.802.02.35Inhibitionby

2mMATP(%)43996Cell

growthrate

(lifespan)(days)1440

" Nanoatoms of oxygen per min and per million cells.h Nanomoles of lactic acid produced per min and per million cells from glucose (10 mM).c CN, cyanide; EAC, Ehrlich ascites cells.

tion, and the supernatant was kept on ice until added to theassay mixture.

The measurement of K+ and Na+ content in sonicated

Ehrlich ascites cells was made as previously reported (16),separating the cells from the medium by centrifugingthrough a layer of silicone oil. The release of LDH2 from

permeabilized cells was determined by assaying the enzymein the supernatant (1) after centrifugation of the cells at1000 rpm for 5 min in a clinical centrifuge. The releasedLDH is expressed as the percentage of the total enzymecontent of the cells. The total LDH was estimated as thetotal enzyme activity released by cells completely fragmented through a treatment with ultrasounds, which doesnot cause a loss in enzyme activity (30 sec of sonic treatment with an MSE 150-watt ultrasonic desintegrator withthe 43301 titanium probe, the power set at high, and theamplitude set at 2 Mm)- Cell viability was determined in aNebauer chamber after incubation for 10 min with 0.2%nigrosin, 1% erythrosin, or 0.2% trypan blue in phosphatesaline. Nucleotides, substrates, cofactors, and enzymeswere purchased from Boehringer Mannheim. Salts andchemicals were purchased from Merck Laboratories.

RESULTS

Preparation and Characterization of Partially Permeabilized Cells. To investigate the allosteric effect of exoge-nously added ATP in the glycolytic flux of metabolicallyintact permeabilized cells, we tested several kinds of permeabilized cells described in the literature. Ehrlich ascitescells permeabilized with an osmotic shock in water (9)exhibited an intense glycolysis when adequately supplemented with various cofactors, but the results were irre-producible. Ehrlich ascites cells permeabilized with dextransulfate (11, 14) presented, in our hands, an inhibited glycol-

2The abbreviations used are: LDH, lactic dehydrogenase; GDP, glucose1,6-diphosphate.

ysis when compared with controls and were thus notsuitable for our purpose. Hepatocytes treated with toluene(7, 12, 15) were permeable to macromolecules, lost enzymes, and became too depleted to sustain glycolysis. Wethen tried many different mild methods to obtain cell per-meabilization to exogenous nucleotides (10) and found 4procedures suitable to obtain an active glycolysis withminimal supplementation. These new methods are assumedto result in only a partial permeabilization of the cellsbecause the effects (to be described later) of exogenousnucleotides in our permeabilized cells can be obtained atlower nucleotide concentrations when the permeabilizationprocedure is intensified. That fact would suggest that, inour permeabilized cells, the exogenously added nucleotides do not equilibrate inside and outside of the cells butseem to enter by diffusion. A simple procedure for partialpermeabilization of rat hepatocytes in a mild and reproducible manner is to resuspend the cell pellet in a smallamount of distilled water for 15 sec and immediately supplement the cells with an excess of medium. This procedureyields 100% of the cells permeable to vital dyes, and thesecells sustain intense glycolysis when supplemented withNAD+. Another method yielding equal results is merely to

isolate the hepatocytes by the procedure described in"Materials and Methods," omitting the albumin in the

medium throughout. Hepatocytes permeabilized by both ofthese methods exhibit an active endogenous respiration,which is partially sensitive to oligomycin and dinitrophenol.These characteristics are 2 of the most stringent requirements for metabolic integrity, which would indicate thatthese cells manage to maintain an ionic concentrationadequate for coupled mitochondrial respiration (3).

Ehrlich ascites tumor cells can become partially permeable to nucleotides in a reproducible manner by beingsubmitted to a very mild sonication, which does not resultin inactivation of the enzyme content of the cells. The bestprocedure for us is to treat the cell suspensions in volumesof 4 ml in glass cups, adjusting the concentration to 80 to

JANUARY 1978 143

M. Gosalvez et al.

100 x 10Hcells/ml in phosphate saline at 4Â°.The power

and thÃ©amplitude of the ultrasounds and the duration ofthe ultrasonic treatment are critical. Our best results areachieved with an MSE 150-watt ultrasonic desintegratorwith the 43301 titanium probe, the power set at low, andthe amplitude set at 2 ^m. Chart 1A shows that, underthese conditions of ultrasonic treatment, 60% of the cellsbecome permeable to vital dyes in 45 sec, while 20% of thecells remain apparently unaffected. These cell preparationsvery actively produce lactic acid from glucose when supplemented with 1 HIM NADH and have intense endogenousrespiration, which is partially sensitive to dinitrophenol butis not sensitive to oligomycin. The amount of pyruvatekinase and phosphofructokinase in the homogenates ofsonicated cells is equal to that found in homogenates ofintact cells. The contribution of the intact cells to the totalvalue of respiration and glycolysis of the cell preparationspermeabilized with ultrasounds varies between 10 and 15%,indicating that permeabilized cells have, upon adequatesupplementation, a more active glycolysis and respirationthan do intact cells. The loss of respiratory sensitivity tooligomycin is probably due to the ionic imbalance of thecells. Ehrlich ascites cells permeabilized by ultrasoundscontain 90 nmoles of sodium and 90 nmoles of potassiumper mg of protein, as opposed to 30 nmoles of sodium and180 nmoles of potassium per mg of protein in intact cells.Very similar permeabilization and metabolic characteristicsare obtained in Ehrlich ascites cells by treating the cells

IO 20 3O 4O 50 60

TIME OF SONICATION (sec)

IO 20 3O 40mm

Chart 1. A. increasing stained cells (â€¢)and decreasing remaining cells(A) with increasing time of sonication of Ehrlich ascites cells (Rome strain).See "Materials and Methods" for conditions of the treatment with ultra

sounds. B, release of LDH in permeabilized hepatocytes by osmotic shockwith increasing the incubation time to 30 min. See conditions in "Materials and Methods."

nonomolÂ«LACTATE/min/10*etili^>p__w Â»KJa>o1~â€”

~~ â€”11

l2mM GLUCOSE I2171M GLUCOSE l2mM GLUCOSE l2mM GLUCOSEI mM NAD ImMNAD I mM NAO

075mMATP OTSmMATP02 mM AMP

Â»â€¢42 n-18 n-6

9mU GLUCOSE IZmMGLUCOSE l2mMGLUCOSE >2mMGLUCOSE l2mM GLUCOSEI mM NAD I mM NAD ImMNAD I mM NAO I mM NAD

IMS ImMATP 2mMATP 4mMATP 6mMATPn-14 n-13 n-12 n-15

Chart 2. Lactic acid production from glucose in hepatocytes permeabilized by osmotic shock. A, effects of the supplementation with NAD'. NAD'plus ATP. or NAD' plus ATP and AMP; B. inhibition of glycolysis by

increasing exogenous ATP. The concentration of the additions is shownbelow the bars. Segments on the bars. S.E.; n. the number of experiments.

with the drug S-triethyl-L-cysteine, an anticancer drug (U.S. National Cancer Institute NSC 83265), which causes theloss of microvilli in tumor cells.3 Cell suspensions of 20 to30 x 106cells/ml must be incubated for 30 min at 30Â°in the

presence of 80 to 100 /ug of the drug per ml to obtain 60 to80% of the cells permeable to vital dyes and showing anintense glycolysis when supplemented with 1 mM NAD'.

Longer incubation times or higher drug concentrationsresult in a "bubbling" of the cell surface. The hepatocytes

and Ehrlich ascites cells permeabilized by the above-mentioned procedures do not lose low-molecular-weight cyto-plasmic enzymes upon prolonged incubation at 30Â°,as do

other types of permeabilized cells (7. 9, 11). Chart 16shows the release of LDH from permeabilized hepatocytesupon incubation at 30Â°.The initial release of enzyme activity

in the first 5 min of incubation and the 6% release over thenext 55 min of incubation are considered to be due to

3D. Kessell. Oral communication to the Sixth Joint Working Conferenceof the Division of Cancer Treatment of the National Cancer Institute,Annapolis, Md., September 1975.

Metabolic Control of Glycolysis

fragmentation of fragile cells. Similar results are foundwith partially permeabilized Ehrlich ascites cells.

Effects of Cofactors and Allosteric Effectors on PartiallyPermeabilized Cells. Chart 2 shows the effects of NAD ,ATP, and AMP on lactic acid production from glucose byhepatocytes permeabilized by osmotic shock. In the presence of 5 or 12 mM glucose, the hepatocytes produceabout 1 nmole of lactic acid per min per 106 cells. This rate

is stimulated 73% with the presence of 1 HIM NAD . Furtheraddition of ATP (0.75 HIM) or AMP (0.1 rriM) results in asmall stimulation. The progressive inhibition of the glyco-lytic rate sustained by glucose plus NAD' is complete at 6

mM ATP. Chart 3 represents the release of the ATP inhibition by the addition of phosphate and GDP. GDP was usedinstead of fructose 1,6-diphosphate as a counter inhibitorof pyruvate kinase. The maximal release of the inhibition.66%, is obtained with 2 rriM GDP. Phosphate, at 60 HIM,liberates the inhibition 57%. As phosphate and GDP counteract the inhibition by ATP of the enzymes phosphofructo-

kinase and pyruvate kinase. respectively (2, 13), the experiments shown in Chart 3 suggest that none of these enzymesis completely inhibited by ATP in partially permeabilizedhepatocytes. Chart 4/4 summarizes experiments with hepa-

V16-\2-0.8-0.4-1

2 mM GLUCOSE l2mM GLUCOSEI mM NAD I mM NAO

6mM ATP

l2mM GLUCOSEI mM NAO6mM ATP2OmM Pi

l2mM GLUCOSE i2rr>M GLUCOSEI mM NAD I mM NADE mM ATP Â£mM ATP40mM Pi 8OmM Pi

12-mm/IO'c.Ã¹

08-t-u|0.4-EÂ¡Ã¯111

l2mM GLUCOSEI mM NAO6mM ATP

wGLUCOSEimM NAD

6 mM ATP2 mM GO P

M GLUCOSE l2mM GLUCOSEmM NAD Im M NADmM ATP 6 mM ATPmM GDP 6mM GDP

Chart 3. Lactic acid production from glucose in hepatocytes permeabilized by osmotic shock. A, inhibition of glycolysis by ATP and partial releaseof the inhibition by increasing exogenous phosphate; B, inhibition ofglycolysis by ATP and partial release of the inhibition by increasing exogenous GDP.

tocytes permeabilized by isolation with albumin and treatedwith citrate and citrate plus phosphate. Citrate, an exclusiveinhibitor of phosphofructokinase (13), inhibits 50% of theglycolytic rate at a concentration of 4 mM, and this inhibition is completely released by the addition of 40 mMphosphate. Chart 46 illustrates the effects of ATP and ATPplus GDP in the lactic acid production from phosphoenolpyruvate in these hepatocytes. Phosphoenol pyruvate sustains an active glycolysis in permeabilized hepatocytes, 1.2nmoles of lactic acid per min per 106 cells, which is totally

inhibited by 6 mM ATP. The inhibition is fully released by 4mM GDP. The experiments shown in Chart 4 stronglysupport the hypothesis that the inhibition of pyruvate kinaseby ATP can be complete in the absence of GDP. while theinhibition of phosphofructokinase by citrate is only partial.

Chart 5 demonstrates the action of ATP in the glycolysisof Ehrlich ascites cells permeabilized by osmotic shockand supplemented with NAD . ATP concentrations greaterthan 0.75 mM cause a progressive inhibition of the glycolytic rate. The maximal inhibition attainable with ATP is

i1.6-1.2-08-04-i1][I

mM NAD 1mM NAD I mM NAD 1 mM NAD4 mM CITRATE 6 mM CITRATE 6 "iM CITRATE

B108-0.4-OmM

PEP lOmM PÂ£POmM6mMATP 6mM2

mMn-6nÂ«6 Aâ€¢.PEP

OmMATP6mMG

DP 4mMâ€¢6 nPEPATPGOPA

Chart 4 Lactic acid production from glucose or phosphoenol pyruvate(PEP) in hepatocytes permeabilized by isolation in the absence of albumin.A, inhibition of glycolysis by increasing citrate and total release of theinhibition by phosphate; B. inhibition by ATP and total release of theinhibition by increasing GDP.

JANUARY 1978 145

M. Gosalvez et al.

i1,17mM

GLUCOSE 7mM GLUCOSE 7mM GLUCOSEmW NAO ImMNAD

0 T5mM ATPn.23 "-I3 n.32

7mM GLUCOSEi mM NAD

7mM GLUCOSEIfttM NAO

2mM ATPn-9

7mM GLUCOSEâ€¢'.'-.-â€¢-

4mM ATP

7mM GLUCOSEimMNAD61IMATP

Chart 5. Lactic acid production from glucose in Ehrlich ascites cells(Rome strain) permeabilized by sonication. A. influence of supplementationwith NAD- or NAD' plus ATP; B, effect of increasing ATP.

45% at 6 HIM and thus is never complete. Chart 6A showsthat the ATP inhibition can be completely released by 50nriMphosphate, while it can be released only minimally byGDP. These experiments, which were done with cells ofthe Paris strain (see Table 1), suggest that almost all theglycolytic inhibition by ATP is due to the inhibition ofphosphofructokinase by ATP and that there is a lack ofsensitivity of pyruvate kinase for ATP. Chart 66 confirmsthat such is the case. Lactic acid production from phosphoenol pyruvate in these cells is only partially inhibited byATP, and this inhibition is almost completely released byGDP. The same experiments performed with the Romestrain of Ehrlich ascites cells showed a complete lack ofinhibition by ATP of the phosphoenol pyruvate-supportedlactic acid production.

In the experiments shown in Chart 66, the amount oflactate produced from phosphoenol pyruvate by the sonicated tumor cells (0.5 nmole/min/mg of protein) is clearlysignificantly less than would be expected in view of thelactic acid production with the use of glucose plus NAD'

as substrate in these cells (6 nmoles/min/mg of protein,

7mM GLUCOSE 7mMGLUCOSEimM NAD iniM NAD

6mMATP

7mM GLUCOSE 7mM GLUCOSErfYiM NAO imM NAO6mM ATP 6mM ATP5OmM Pi 6mM GDP

rranomolesLoctote/min/mgproÂ»o0gC_

UW-lOmM

PEP lOmM PEP lOmM PEP6mM ATP 6mM ATP

6mM GDP

Chart 6. A, lactic acid production from glucose in Ehrlich ascites cells(Paris strain) permeabilized by treatment with NSC 83265 and supplementedwith NAD*; partial inhibition by ATP; and total and partial release of the

inhibition by added P, and GDP, respectively. B, lactic acid production fromphosphoenol pyruvate (PEP) in Ehrlich ascites cells (Paris strain) permeabilized by ultrasounds; partial inhibition with ATP and release of the inhibitionwith GDP.

Table 2Correlation of data on different metabolic parameters for the 3 cell

populations of Table 1Correlation of PK"

inhibition by ATP with

Extent of glycolysis withCNExtentof aerobicglycolysisAbsolute

Pasteureffect0,uptake (endogenoussubstrate)Relative

Pasteureffect0.uptake(glucose)Crabtree

effectPKinhibition by ATPr

Â»r=r=T=r

=r=r=r

=0.439393-0.8657800.9031170.9673880.9880220.996199-0.9974031.000000" PK, pyruvate kinase.

Chart 5A). The low rate from phosphoenol pyruvate wasdue to a lack of NADH in the cells, because the addition of3 rriM NADH produces a lactic acid production from phosphoenol pyruvate of more than 9 nmoles/min/mg of protein. Such lactate production is completely insensitive toATP and may include an excessive activity of the enzymeLDH, which masks minor effects of ATP on pyruvate kinase.We therefore present the experiment done in the absence

Metabolie Control of Glycolysis

Table 3

Lactic acid production from fructose

Cofactors

HepatocytesIntact

Partiallypermeabilized(osmotic shock)CN

(2mM),

Citrate CN (2 citrate(4 mM) mM) (4mw)0.10

1.05 0.52NAD'

mM),NAD' (1 citrate

mM) (4mM)1

.3" 0.66"NAD+

(1mM),

citrate(4 mw),PI (40mM)1.04"NAD'

mM),ATP (6mM)0.14"NAD+

(1mM),

ATP (6mM),

GDP (4mM)0.82""

n = 6.

of NADH, which (in spite of its limited rate) shows thepartial inhibition by ATP and release by GDP.

Effects of ATP on Isolated Pyruvate Kinase. Table 1shows the inhibition by ATP of pyruvate kinase isolatedfrom hepatocytes and the 2 strains of Ehrlich ascites cells.ATP, at 2 mM, totally inhibits the enzyme isolated fromhepatocytes, while it does not produce a significant effecton the enzyme from the Rome strain of Ehrlich ascitescells. The cells from the Paris strain have an intermediatesensitivity. Table 1 also shows data on various metabolicparameters as measured in the intact cells and records thelife span of the 2 strains of tumors as an index of thegrowth rate of the cells. The Rome strain of Ehrlich ascitescells has a high Crabtree effect and a very low Pasteureffect. The Paris strain of tumor cells is in an intermediateposition. As shown in Table 2, there is a great correlationbetween the sensitivity to ATP in the 3 cell types and theirCrabtree and relative Pasteur effects (r = -0.997403 and r= 0.988022, respectively). Table 3 compares the effect ofNAD+, citrate, ATP, and phosphate on the lactic acid pro

duction from fructose in intact and partially permeabilizedhepatocytes. Intact hepatocytes produce lactic acid only inthe presence of cyanide and the rate is inhibited 50% bycitrate. The inhibition with citrate in permeabilized hepatocytes is only partially released by phosphate. There is also89% inhibition with ATP, of which 66% is released by GDP.These experiments with fructose are interpreted as evidence of the existence of a part of the glycolytic flux that isnot sensitive to ATP and citrate. It would correspond to theoperation of fructokinase, which glycolyzes fructose to fructose 1,6-diphosphate without requiring the hexokinase andphosphofructokinase steps.

DISCUSSION

The effects of ATP here described in partially permeabilized hepatocytes are consistent with a partial allostericinhibition of phosphofructokinase, which is followed by atotal allosteric inhibition of pyruvate kinase. Both inhibitions are counteracted independently by phosphate andGDP, respectively, which supports the specificity of ATPfor each site and the allosteric nature of its effect. The lackof total inhibition of glycolysis by ATP in partially permeabilized Ehrlich ascites cells is clearly traced to the partialor nonsensitivity of the enzyme pyruvate kinase to allosteric

inhibition by ATP. This insensitivity is demonstrated bothin the in situ enzyme and in the homogenate enzyme andclosely correlates with the Crabtree and Pasteur effects ofthe cells. Our previous results (5, 6), together with thepresent experiments, present evidence suggesting the existence of a second site of glycolytic control at pyruvatekinase, which (by not being operative or by being onlypartially operative in tumors cells) is the cause of the aerobicglycolysis, Crabtree effect, and low Pasteur effect characteristic of these cells. Our results have been gathered inpartially permeabilized cells, a new cell system, which isassumed to represent an intermediate system between thepermeabilized cell (7, 9, 11) and the intact cell. Furthercharacterization of this cell system would be desirable.

REFERENCES

1. Bergmeyer, H. U. Method of Enzymatic Analysis. Weinheim. WestGermany: Verlag-Chemie, 1965.

2. Carbonell, J., Feliu, J. E., Marco, R., and Sols, A. Pyruvate Kinase.Classes of Regulatory Isoenzymes in Mammalian Tissues. European J.Biochem., 37. 148-156, 1973.

3. Chance, B. Technique for the Assay of the Respiratory Enzymes. Methods Enzymol.. 273-329, 1965.

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10. LÃ³pez-Alarcdn, L. Mecanismos de Control GlucolÃtico en CÃ©lulasdeTumor AscÃticode Ehrlich. Ph.D. Thesis, Universidad AutÃ³noma Madrid, 1976.

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1973.13. Salas, M. L.. ViÃ±uela,E., Salas, M., and Sols, A. Citrate Inhibition of

Phosphofructokinase and the Pasteur Effect. Biochem. Biophys. Res.Commun.. 19: 371-376, 1965.

14. Scholnick, P., Lang, D., and Racker, E. Regulatory Mechanisms inCarbohydrate Metabolism. IX. Stimulation of Aerobic Glycolysis by

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Energy-linked Ion Transport and Inhibition by Dextran Sulfate. J. Biol.Chem.. 248. 5175-5182. 1973.

15. Sols, A., Reeves, R. E., and Gancedo, C. Regulation of Enzymes "insitu." In: E. H. Fischer, E. G. Krebs, H. Neurath. and E R. Stadman

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Metabolic Control of Glycolysis in Normal and Tumor Permeabilized Cells1

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