Regulation of the Embden-Meyerhof Pathway in a...

[CANCER RESEARCH 32, 2793-2798, December 1972]

Regulation of the Embden-Meyerhof Pathway in a Transplantable

Rat Thyroid Tumor

M. F. Meldolesi and V. Macchia

Centro di Endocrinologia e di Oncologia Sperimentale del C. N. R., Istituto di Patologia Generale, UniversitÃ di Napoli, Naples, Italy

SUMMARY

The amount of lactate, pyruvate, and glycerol 1-phosphateformed from each of the available intermediates of theEmbden-Meyerhof pathway has been measured (by thesequence method) in extracts of a transplantable thyroidtumor and of normal rat thyroid.

The regulation of the rate-limiting step catalyzed byphosphofructokinase was modified in the tumor with respectto the thyroid as evidenced by the concurrent enhancement ofphosphofructokinase activity, by the increase in lactateproduction from fructose 6-phosphate, and by the lack of theinhibition of lactate production from glucose 6-phosphate byadenosine triphosphate up to 6 mM. Moreover the partiallypurified phosphofructokinase of the tumor was less inhibitedby 4 mM citrate and less sensitive to the reversal of citrateinhibition by cyclic 3',5'-adenosine monophosphate than was

the thyroid enzyme.Therefore it seems possible that, besides the enhancement

of the phosphofructokinase activity, a modification in thecontrol by allosteric effectors of such an enzyme may modifythe control of the glycolytic pathway in the thyroid tumor.

INTRODUCTION

Although several studies on the glycolytic enzyme activitiesin various tumors have been reported (14), it is not yet clearhow these modifications may influence, in vivo or in vitro, theoverall rate and hence the regulation of the Embden-Meyerhofpathway (13, 27). In normal tissues this pathway is controlledby at least 4 rate-limiting steps catalyzed by hexokinase,P-fructokinase, glyceraldehyde 3-P dehydrogenase, andpyruvate kinase (21). Therefore it seems possible that in thetumors the modification of the glycolytic pathway may berelated either to a different amount of 1 or more of these keyenzymes or to a variation in the control mechanism of suchenzymes by allosteric effectors.

For clarification of this possibility, it was consideredinteresting to study sequentially (18, 21) the activities of thekey enzymes of the glycolytic pathway in a transplantablethyroid tumor as compared to those of normal thyroid.

The rat thyroid tumor used throughout these studies wasone of a series of tumors developed in Fischer rats by Wollman(25) and designated at line 1-8. This tumor resembles the

Received February 14, 1972; accepted September 13, 1972.

thyroid in that it elaborates a periodic acid-Schiff-positivecolloid material and is capable of trapping and organifyingiodide (5), although its growth is thyroid-stimulating hormoneindependent (25) since it does not respond to the in vitroaddition of thyroid-stimulating hormone (16).

MATERIALS AND METHODS

The auxiliary enzymes, the coenzymes, and the substrates,including DL-glyceraldehyde 3-P diethylacetal, barium salt(converted to the sodium salt by utilizing Dowex 50-H+), were

from Boehringer/Mannheim, Mannheim, Germany; bovineserum albumin and EDTA were from Sigma Chemical Co., St.Louis, Mo.; and dithiothreitol was from Calbiochem, LosAngeles, Calif., DEAE-cellulose (Whatman DE-52) had anexchange capacity of 1.0 mEq/g. Male Fischer rats, weighingapproximately 200 g, were from Charles River (Laboratories,Chicago, 111.).The thyroid tumor, 1-8, kindly supplied by Dr.S. H. Wollman, NIH, Bethesda, Md., was transplanted s.c. inFischer rats and was excised 2 months after implantation. Thethyroid glands were removed from normal animals. Thethyroids and the tumors were rapidly weighed, minced, andhomogenized for 3 min in a Potter-Elvehjem homogenizer in 4volumes of sucrose (250 mM) and Tris-HCl buffer (50 mM),pH 7.4. The homogenate was then centrifuged at 22,000 X gfor 60 min, and the supernatant was collected and diluted withanother 2 volumes of the homogenization medium immediately before the assay or centrifuged at 105,OOOX# for 60min before dilution. All operations were performed at 4Â°.

Assay of Glycolysis. Lactate, pyruvate, and glycerol-1-Pformation was measured from all the available substrates ofthe glycolytic pathway. Each reaction mixture contained, in afinal volume of 1.45 ml: 150 mM KC1; 7 mM MgCl2 ; 25 mMKHCO3 buffer, pH 7.5; 40 mM potassium phosphate buffer,pH 7.5; 1.5 mM ATP or 4.0 mM ADP; 1.5 mM NAD* (unless

otherwise stated); one of the following substrates at saturatingconcentrations: 20 mM glucose, 10 mM glucose-6-P, 10 mMfructose-6-P, 10 mM fructose 1,6-di-P, 40 mM DL-glyceralde-hyde-3-P, 15 mM 3-P-glycerate, 15 mM 2-P-glycerate, 15 mMP-enolpyruvate, 0.25 ml of the supernatant at 22,000 X g or105,000 X g (or alternatively 0.140 or 0.040 ml with2-P-glycerate or P-enolpyruvate as substrate, respectively).ATP was used with glucose, glucose-6-P or fructose-6-P assubstrate; ADP was used with fructose-1,6-di-P, glyceralde-hyde-3-P, 3-P-glycerate, 2-P-glycerate or P-enolpyruvate. Theacidic solutions were previously neutralized with l N KOH and

DECEMBER 1972 2793

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the pH was finally adjusted to 7.5 after addition of cofactorsand substrate. The reaction was carried out in a Dubnoffmetabolic incubator, with N2 :C02 (95:5) as the gas phase at35Â°,pH 7.5, for 5 min. With glucose as substrate incubation

was also for 15 min. Some experiments were carried out in theabsence of KHCO3 with air as the gas phase, pH 7.5, asindicated in the text. The extra enzymes added to theincubation mixture were previously dialyzed with 50 mMTris-HCl buffer, pH 7.5; when undialyzed enzymes were used,a control was run with the corresponding amount of(NH4)2S04. The incubation was stopped by adding 0.1 ml of18% (w/v) HC1O4. After centrifugation, samples of theprotein-free supernatant were used for the determination ofJÃ¡ctate,pyruvate, and glycerol-1-P by enzymatic methods (8).The amounts of lactate, pyruvate, or glycerol-1-P presentbefore incubation were measured and subtracted from theamounts present after incubation. Lactate formation frompyruvate was also measured at 36Â°,pH 7.5 (10).

Hexokinase (6), P-fructokinase (19), glyceraldehyde-3-Pdehydrogenase (2), P-glycerate kinase (4), P-glycerate mutase(3), enolase (1), pyruvate kinase (22), and cytochrome oxidase(24) activities were determined at 22Â°and expressed as nmoles

of substrate utilized by the supernatant at 22,000 X g or at105,000 X^Xmg (wet weight)"1 X min"1. The thyroid

glands from 20 to 35 animals were used for each experiment;all experiments were repeated at least 5 times and the resultsfrom a typical experiment are presented.

Partial Purification of Thyroid and Tumor P-fructokinase.The enzyme was partially purified according to the method ofLayzer and Conway (11) by centrifugation at 22,000 X g andby fractionation on a DEAE-cellulose column with a lineargradient of Tris-phosphate buffer (pH 8) formed by mixing 50ml each of 50 mM and 600 mM buffer containing 0.2 mMEDTA and 0.2 mM ATP and 0.7 mM dithiothreitol. Theenzyme was eluted with an approximately 300 mMTris-phosphate buffer.

Citrate inhibition of P-fructokinase was measured at pH 7.4in a reaction mixture containing (final concentrations): 35 mMtriethanolamine buffer, pH 7.4; 1.5 mM MgCl2; 7 mM(NH4)2SO4;0.3 mM fructose-6-P; 1 mM ATP; 0.1 mM NADH;aldolase (35 jug/ml); triose-P isomerase (2.0 /Â¿g/ml);glycerol-1-P dehydrogenase (10 Mg/ml); bovine serum albumin,0.01%. Rates of decrease in absorbance at 340 nm weredetermined 3 to 5 min after the reaction was started byaddition of the P-fructokinase preparation in the absence or inthe presence of citrate. Cyclic 3',5'-AMP (0.6 mM final

concentration), pH 7.4, was added 4.5 min after the reactionwas started, and the rate of decrease in absorbance wasdetermined 1.5 to 2.5 min after its addition. NADH oxidaseactivity was measured in the absence of ATP and subtractedfrom P-fructokinase activity.

RESULTS AND DISCUSSION

Thyroid tumor extracts (supernatant at 22,000 X g) containan amount of endogenous lactate slightly but significantlyhigher (p < 0.05) than does normal thyroid (10.8+1.0 and8.0 Â±0.5 nmoles X mg (wet weight)"1, respectively). Lactate

production by the 22,000 X g supernatant of the thyroid andof the thyroid tumor was measured in the presence ofnonlimiting amounts of cofactors and substrates. In thepresence of glucose, lactate production is linear up to 30 minof incubation. With the other substrates, lactate, pyruvate, andglycerol-1-P production is linear up to at least 10 min. TheNADH required in vitro for lactate formation from pyruvate isgenerated mostly at the glyceraldehyde-3-P dehydrogenasestep. For this reason the principal end product formed fromsubstrates such as 3-P-glycerate, 2-P-glycerate, and P-enolpyru-vate is pyruvate. Therefore, in thyroid extracts, lactateproduction from the various intermediates of the Embden-Meyerhof pathway increases progressively from glucose toglyceraldehyde-3-P, whereas it diminishes when 3-P-glycerateor other substrates such as 2-P-glycerate or P-enolpyruvate areadded to the medium (Table 1). On the other hand, pyruvateaccumulation, which is very low in the 1st part of thesequence, greatly increases when the in vitro generation ofNADH becomes too low, i.e., after the glyceraldehyde-3-Pdehydrogenase step (Table 1). The total amount ofpyruvate + lactate formed from the various substrates of theglycolytic pathway shows significant increases at 5 points ofthe sequence, i.e., between glucose and glucose-6-P; betweenfructose-6-P and fructose-1,6-di-P; between glyceraldehyde-3-Pand 3-P-glycerate;between 3-P-glycerate and 2-P-glycerate, andfinally between 2-P-glycerate and P-enolpyruvate (Table 1).These steps are catalyzed, respectively, by hexokinase,P-fructokinase, glyceraldehyde-3-P dehydrogenase + P-glycerate kinase, P-glycerate mutase, and enolase. The last reaction,catalyzed by enolase, could be also considered rate-limiting inthe Embden-Meyerhof pathway (12). Further evidence thathexokinase and P-fructokinase are rate-limiting steps inthyroid extracts was obtained by adding 3.6 fig of yeasthexokinase (final concentration, 0.34 unit/ml) to glucose or 9to 18 fig of rabbit muscle P-fructokinase (final concentration,0.34 to 0.7 units/ml) to fructose-6-P; this addition resulted inthe increase of the rates obtained with saturating concentrations of glucose or fructose-6-P to those obtained withglucose-6-P or fructose-1,6-di-P as substrate, respectively(Table 1).

In the thyroid tumor lactate production from glucose(Table 1) as well as hexokinase activity (Table 2) are bothhigher than those of normal thyroid. Moreover the addition ofyeast hexokinase to glucose enhances lactate production to thelevels obtained from glucose-6-P.

In the thyroid tumor extracts, lactate formation fromglucose-6-P and from fructose-6-P is as high as that obtainedfrom fructose-1,6-di-P. The addition of rabbit muscleP-fructokinase enhances only slightly lactate production fromfructose-6-P (Table 1). It seems therefore possible, as shownby the sequence method, that at least in vitro P-fructokinaseof thyroid tumor has lost its rate-limiting function and thatthe control may be transferred to another site in the glycolyticchain, as shown in other tissues and in other experimentalconditions (18, 23). For clarification of this hypothesis a seriesof experiments has been done. Some of them have beenperformed in the presence of various concentrations offructose-1,6-di-P, ADP, and NAD+ (Table 3), in order to show

that a maximal lactate production from fructose-1,6-di-P was

2794 CANCER RESEARCH VOL. 32



Regulation ofGlycolysis in a Thyroid Tumor

obtained in our experimental conditions and that lactateproduction in the tumor extracts from fructose-6-P reachedthe same optimal levels obtained from fructose-1,6-di-P.Moreover some differences between thyroid and tumor, as faras the effects produced by ATP on lactate production fromglucose and glucose-6-P are concerned, have been shown. Infact, when increasing the ATP concentration in the presence of1 of these 2 substrates, a progressive inhibition of lactateproduction from thyroid extracts has been observed (Chart 1).This inhibition is probably due to the effect produced by ATPat the step catalyzed by P-fructokinase which is sensitive tothe allosteric control by ATP. In fact the limiting effect ispartially overcome when fructose-6-P is added as substrate(Chart 1). These data are in agreement with observationspreviously reported in other tissues (17). On the contrary, intumor extracts, there is a progressive increase in lactateproduction with increasing ATP concentrations. Such increaseis evident with ATP up to 6 mM, when glucose is used assubstrate (Chart 2). The P-fructokinase activity is also slightlyhigher in the tumor than in the thyroid extracts (Table 2).

In order to investigate whether the tumor enzyme shows adifferent control by allosteric effectors such as citrate andcyclic 3',5'-AMP (9, 11, 17, 20, 26), P-fructokinase from

tumor and from thyroid extracts was partially purified (about18-fold and 11-fold, respectively) on DEAE-cellulose columnwith a linear gradient of Tris-phosphate buffer (11). Thedegree of purification was low because the enzyme loses someactivity during purification, even in the presence of ATP anddithiothreitol. Moreover the thyroid P-fructokinase was notcompletely separated from thyroglobulin, which behaves as abroad peak. The different degree of purification betweenthyroid and tumor enzyme may be related to the lowestamount of thyroglobulin present in the tumor (5). Cyclic

3',5'-AMP was not included in the elution mixture to avoid

interference with citrate inhibition. The thyroid enzyme wasstrongly inhibited by 4 mM citrate, whereas the tumor enzymeactivity was only slightly depressed under the sameexperimental conditions (Table 4). The percentage ofinhibition by citrate is decreased slightly by increasing enzymeconcentration. However, the differences between thyroid andtumor P-fructokinase are quite evident. The reversal of citrateinhibition by cyclic 3',5'-AMP addition was less effective on

the tumor than on the thyroid enzyme (Table 4). Therefore, itseems possible that the allosteric regulation of P-fructokinaseby such substances is less effective in the tumor than in thethyroid. These modifications may influence the control of theglycolytic pathway. In fact, like in yeast extracts, conforma-tional changes may be an effective controlling factor inP-fructokinase regulation more than the total amount of theenzyme (7).

Table 2Enzymatic activities of the glycolytic pathway in the supernatant at

22,000 X g of rat thyroid and thyroid tumor homogenates

Activity [nmoles substrate utilizedX mg (wet wt) "' X min "' ]

Rat thyroid Thyroid tumor

HexokinaseP-fructokinaseGlyceraldehyde-3-P

dehydrogenaseP-glyceratekinaseP-glyceratemutaseEnolasePyruvate

kinase1.98

Â±0.14.55+0.421.201

1.578.00Â±3.028.80Â±2.011.50+

1.028.50+1.53.35

+0.36.75i0.437.50Â±

2.090.00Â±6.546.80+3.914.40+1.026.901

1.5

Table 1Lactate and pyruvate formation in the 22,000 g supernatant of rat thyroid and thyroid tumor.

Lactate and pyruvate formation [nmoles X mg (wet wt)"1 X hr"1 ]

Normal thyroid Thyroid tumor

Substrate" Cofactors6 Lactate Pyruvate Lactate Pyruvate

GlucoseGlucose-6-PFructose-6-PFructose-1,

6-di-PGlyceraldehyde-3-P3-P-glycerate2-P-glycerateP-enolpyruvateGlucose

+ hexokinase (0.34unit/ml)Fructose-6-P+ P-fructokinase (0.7unit/ml)Fructose-1

, 6-di-P +glyceraldehyde-3-Pdehydrogenase(6.1 units/ml)ATP,

NAD*ATP,NAD*ATP,NAD*ADP,NAD*ADP,NAD*ADP,NAD*ADP,NAD*ADP,NAD*ATP,

NAD*ATP,NAD*ADP,

NAD*6.0

Â±2"23.5+230.5Â±350.0+453.0+

520.5Â±421.51622.5

i722.3

+246.5Â±4140.0i 70.5

t0.25.310.55.5

t0.620.5i2.022.5+3.0480.01

15.01200.0+22.04400.0

+60.03.1

i0.323.2Â±2.0IS.Oi

0.427.9

t2115.0+9123.0110132.0+10138.0

Â±1538.8+442.0Â±645.0i7143.0

Â±9170.0111160.01

120.7

+0.21.1tO.21.5

+0.11.7i0.21.5i0.2755.0t

20.01250.0+30.04400.0

i100.01.1

Â±0.21.3+0.22.0

i 0.4

"Substrate concentration: 20 mM glucose; 10 mM glucose-6-P, fructose-6-P, fruciÃ³se-1,6-di-P2; 40 mM glyceraldehyde-3-P; 15 mM3-P-glycerate, 2-P-glycerate and P-enolpyruvate.

Cofactor concentration: 1.5 mM NAD*; 1.5 mM ATP; 4 mM ADP. Incubation was carried out at 35Â°for 5 min, final pH 7.5, in the presence

of 150 mM KC1,7 mM MgCl,, 25 mM KHCO3 buffer (pH 7.5) and 40 mM potassium phosphate buffer (pH 7.5) with 95% N, + 5% CO, as the gasphase.

c Mean Â±S.E.

DECEMBER 1972 2795




Table 3LÃ¡clateproduction by thyroid tumor supernatant at 22,000 X g in the

presence of various concentrations ofcofactors and substrate

Incubation was carried out for 5 min as described in Table 1.

LÃ¡clateproduction Jnmoles Xmg (wet wt)'1 X hr'1 ]

Concentration(mM)0.51.01.52.03.04.08.010.0NAD*089

Â±795Â±898Â±898+999Â±8101Â±9103

Â±9ADPb45

Â±470Â±597

Â±892

Â±870

Â±6Fructose-

1,6-di-Pc50

Â±465Â±696

Â±898

Â±897Â±898Â±7

"Incubation was carried out in the presence of 10 mM fructose-1,6-di-Pand 4 mM ADP.

6 Incubation was carried out in the presence of 10 mM fructose-1,6-di-Pand 1.5 mMNAD*.

c Incubation was carried out in the presence of 4.0 mM ADP and 1.5mMNAD*.

.?<u

<U

cnE

IO4-o(O

QJ

O

C

10

3.0 6.0

ATP (mM)

9.0

Chart 1. LÃ¡clateproduction by thyroid supernatant at 22,000 X g inIhe presence of various concenlralions of ATP wilh Ihe followingsubslrales: â€¢¿�â€”Â»,20 mM glucose; â€¢¿�Â»,10 mM glucose-6-P; o o, IOmM fructose-6-P. Incubation was carried out at 35Â°,pH 7.5, in Ihe

presence of 150 mM KC1, 7 mM MgCl2, 40 mM potassium phosphatebuffer (pH 7.5), and 1.5 mM NAD*, wilh air as Ihe gas phase.

In normal thyroid extracts, pyruvate production from3-P-glycerate is efficiently increased by addition of P-glycer-ate mutase (12.5 units/ml) (Table 5). The same results areobtained in tumor extracts (Table 6), where lactate andpyruvate production from 3-P-glycerate and P-glyceratemutase activity are enhanced with respect to normal thyroid(Table 2).

The addition of enolase (10 units/ml) to 2-P-glycerate in the

presence of thyroid or tumor extracts, resulted also in theincrease of the rates of pyruvate production obtained from2-P-glycerate to those obtained with P-enolpyruvate assubstrate (Tables 5 and 6, respectively). Moreover lactateproduction from pyruvate is higher in the tumor than in thethyroid (14.8 Â±0.5 and 4.9 Â±0.3 /uniÃ³lesof substrate utilizedX mg (wet weight)"1 X hr"1, respectively).

Thyroid extracts contain an amount of endogenousglycerol-1-P (2.1 Â±0.3 nmoles X mg (wet weight)'1) quite

similar to that of the tumor (2.3 Â±0.3 nmoles X mg (wetweight)"'). In normal thyroid glycerol-1-P production

increases, like lactate formation, when the control points ofthe pathway are overcome by the addition of the substratefurther down in the sequence (Table 5). NADH required forglycerol-1-P formation at the step catalyzed by glycerol-1-Pdehydrogenase is probably generated mostly at the glyceralde-hyde-3-P dehydrogenase step (Table 1) which, in the thyroid,as well as in other tissues (21) and in vitro, has a rate-limiting

3.0 6.0

ATP (mM)

9.0

Chart 2. Lactate production by thyroid tumor supernalanl at22,000 X g in the presence of various concentralions of ATP wilh thefollowing subslrales: â€¢¿�â€”â€¢,20 mM glucose; â€¢¿�-â€¢,10 mM glucose-6-P;oâ€”o, 10 mM fruclose-6-P. Incubalion was carried oui as described inCharl 1.

Table 4Effects of citrate and of cyclic 3',5'-AMP on P-fructokinase activity0

fcontrol =1001

Addition

TissueRat

thyroidThyroid tumorCyclic

3' ,5'-AMP

(0.6mM)102

101Citrate

(4 mM)20

66Citrale

(4 mM)and cyclic 3',5'-

AMP(0.6mM)10382

a P-fructokinase activity was measured at pH 7.4 as previouslydescribed under "Materials and Melhods."




Regulation ofGlycolysis in a Thyroid Tumor

activity in the glycolytic pathway. In fact the addition ofglyceraldehyde-3-P dehydrogenase to fructose-1,6-di-P, enhances the production of lactate in thyroid extracts (Table 1).In the tumor, glycerol-1-P production from the differentsubstrates (Table 6) and glyceraldehyde-3-P dehydrogenaseactivity (Table 2) are both higher than in the thyroid.Moreover the addition of glyceraldehyde-3-P dehydrogenase tofructose-1,6-di-P does not influence remarkably lactateproduction in tumor extracts (Table 1).

No significant differences have been shown when thesequence data obtained with 22,000 X g supernatant werecompared with those obtained with 105,000 X g supernatant(Tables 5 and 6). The same results have been obtained also as

Table 5Lactate, pyruvate, and glycerol-I-P formation in the 22,000 X g and

105,000 X g supernatant of the same homogenate of rat thyroidExperimental conditions are identical to those of Table 1.

Lactate, pyruvate, and glycerol-1-P formation[nmoles X mg (wet wt)~' Xhr"']

Substrate Lactate Pyruvate Glycerol-1-P

Supernatant at 22,000 XgGlucoseFructose-6-PFructose-l,6-di-P3-P-glycerate2-P-glycerateP-enolpyruvate3-Pglycerate

+P-glyceratemutase(12.5

units/ml)4.8+

227.5Â±348.0Â±434.0Â±434.0+538.0+536.0

+42-P-glycerate

+ enolase 37.0 Â±5(10units/mlGlucoseFructose-6-PFructose-l,6-di-P3-P-glycerate2-P-glycerateP-enolpyruvateSupernatant

at4.5Â±125.0Â±346.0Â±435.0Â±335.0Â±537.0Â±41.3

Â±0.38.0Â±0.722.5Â±1.5447.0Â±10.01

390.0 Â±23.04500.0Â±65.01090.0

Â±18.04250.0

Â±70.0105,000

Xg1.0+0.26.2

Â±0.420.0+1.9435.0

Â±11.01460.0Â±22.04280.0+ 80.010

Â±1.539Â±2.067Â±4.08.1

Â±1.833.2Â±2.067.0+ 3.5

far as enzymatic activities are concerned (Table 7). Super-natants at 22,000 X g and 105,OOOXg of both thyroid andtumor do not show detectable cytochrome oxidase activity(Table 7).

On the basis of these experimental results it seems possibleto conclude that the regulation of the glycolytic pathwayseems probably to be modified in thyroid tumor extractsparticularly at the step catalyzed by P-fructokinase. Theevidence supporting this fact is (a) the enhancement of lactateproduction from fructose-6-P in the tumor; (b) the differencein response between tumor and thyroid when extraP-fructokinase is added to fructose-6-P as substrate; (c) the

Table 6Â¡retate, pyruvate, and glycerol-1-P formation in the 22,000 X g and

105,000 X g supernatant of the same homogenateof thyroid Tumor 1-8

Experimental conditions are identical to those of Table 1.

Lactate, pyruvate, and glycerol-1-P formation[nmoles X mg (wet wt)"1 Xhr"1]

Lactate Pyruvate Glycerol-1-P

Supernatant at 22,000 XgGlucoseFructose-6-PFructose-l,6-di-P3-P-glycerate2-P-glycerateP-enolpyruvate3-P-glycerate

+P-glyceratemutase12.5

units/ml)24.0

Â±2102.0+9116.0Â±

1169.0Â±768.0Â±871.0Â±772.0

+72-P-glycerate

+ enolase 73.0 Â±8(10units/ml)GlucoseFructose-6-PFructose-l,6-di-P3-P-glycerate2-P-glycerateP-enolpyruvateSupernatant

at10519.8Â±296.0Â±8100.0Â±1065

.0Â±568.0Â±867.0Â±91.0Â±

0.20.8Â±0.11.5Â±0.3720.0Â±20.01280.0

Â±22.04500.0Â±75.01080.0+

20.040

10.0Â±80.0,000

X g1.5Â±0.22.0

Â±0.42.0Â±0.3685.0+25.01

260.0 Â±23.04700.0+ 95.042

Â±3188+9480Â±1340

i4195+10450

Â±14

Table 7Enzymatic activities" in the 22,000 X g and 105,000 X g supernatant of rat thyroid and

of thyroid tumor homogena tes f nmoles of substrate utilized X mgfwetwt)'1 X min'1 ]

Activity

Rat thyroid Thyroid tumor

HexokinaseP-fructokinaseGlyceraldehyde-3-P

dehydrogenaseP-glycerate mutaseCytochrome oxidase22,000

Xgsupernatant1.563.8520.80

26.30N.D.b105,OOOXÂ£supernatant1.383.8520.30

26.00N.D.22,000

Xgsupernatant2.655.5032.00

40.80N.D.105,OOOXÂ£supernatant2.305.4530.80

41.70N.D.

a Standard errors were less than 5%.b N.D., not detectable.

DECEMBER 1972 2797




lack of inhibition by ATP up to 6 mM when glucose-6-P isused as substrate in tumor extracts, and (d) the alteredresponse to citrate and to cyclic 3',5'-AMP of the partially

purified tumor P-fructokinase with respect to the thyroidenzyme.

ACKNOWLEDGMENTS

The authors are indebted to Dr. S. H. Wollman and to Dr. F.Eisenberg, NIH, Bethesda, Md., for helpful editorial suggestions.

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1972;32:2793-2798. Cancer Res M. F. Meldolesi and V. Macchia Transplantable Rat Thyroid TumorRegulation of the Embden-Meyerhof Pathway in a

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