Regulation of the Embden-Meyerhof Pathway in a Transplantable Rat Thyroid Tumor

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

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 thyroid in that it elaborates a periodic acid-Schiff-positive colloid material and is capable of trapping and organifying The amount of lactate, pyruvate, and glycerol 1-phosphate iodide (5), although its growth is thyroid-stimulating hormone formed from each of the available intermediates of the independent (25) since it does not respond to the in vitro Embden-Meyerhof pathway has been measured (by the addition of thyroid-stimulating hormone (16). sequence method) in extracts of a transplantable thyroid tumor and of normal rat thyroid. The regulation of the rate-limiting step catalyzed by MATERIALS AND METHODS phosphofructokinase was modified in the tumor with respect to the thyroid as evidenced by the concurrent enhancement of The auxiliary enzymes, the coenzymes, and the substrates, phosphofructokinase activity, by the increase in lactate including DL-glyceraldehyde 3-P diethylacetal, barium salt production from fructose 6-phosphate, and by the lack of the (converted to the sodium salt by utilizing Dowex 50-H+), were inhibition of lactate production from glucose 6-phosphate by from Boehringer/Mannheim, Mannheim, Germany; bovine adenosine triphosphate up to 6 mM. Moreover the partially serum albumin and EDTA were from Sigma Chemical Co., St. purified phosphofructokinase of the tumor was less inhibited Louis, Mo.; and dithiothreitol was from Calbiochem, Los by 4 mM citrate and less sensitive to the reversal of citrate inhibition by cyclic 3',5'-adenosine monophosphate than was Angeles, Calif., DEAE-cellulose (Whatman DE-52) had an exchange capacity of 1.0 mEq/g. Male Fischer rats, weighing the thyroid enzyme. approximately 200 g, were from Charles River (Laboratories, Therefore it seems possible that, besides the enhancement Chicago, 111.).The thyroid tumor, 1-8, kindly supplied by Dr. of the phosphofructokinase activity, a modification in the S. H. Wollman, NIH, Bethesda, Md., was transplanted s.c. in control by allosteric effectors of such an enzyme may modify Fischer rats and was excised 2 months after implantation. The the control of the glycolytic pathway in the thyroid tumor. thyroid glands were removed from normal animals. The thyroids and the tumors were rapidly weighed, minced, and homogenized for 3 min in a Potter-Elvehjem homogenizer in 4 INTRODUCTION volumes of sucrose (250 mM) and Tris-HCl buffer (50 mM), Although several studies on the glycolytic enzyme activities pH 7.4. The homogenate was then centrifuged at 22,000 X g in various tumors have been reported (14), it is not yet clear for 60 min, and the supernatant was collected and diluted with how these modifications may influence, in vivo or in vitro, the another 2 volumes of the homogenization medium immedi ately before the assay or centrifuged at 105,OOOX# for 60 overall rate and hence the regulation of the Embden-Meyerhof min before dilution. All operations were performed at 4Â°. pathway (13, 27). In normal tissues this pathway is controlled by at least 4 rate-limiting steps catalyzed by hexokinase, Assay of Glycolysis. Lactate, pyruvate, and glycerol-1-P P-fructokinase, glyceraldehyde 3-P dehydrogenase, and formation was measured from all the available substrates of pyruvate kinase (21). Therefore it seems possible that in the the glycolytic pathway. Each reaction mixture contained, in a tumors the modification of the glycolytic pathway may be final volume of 1.45 ml: 150 mM KC1; 7 mM MgCl2 ; 25 mM KHCO3 buffer, pH 7.5; 40 mM potassium phosphate buffer, related either to a different amount of 1 or more of these key pH 7.5; 1.5 mM ATP or 4.0 mM ADP; 1.5 mM NAD* (unless enzymes or to a variation in the control mechanism of such enzymes by allosteric effectors. otherwise stated); one of the following substrates at saturating For clarification of this possibility, it was considered concentrations: 20 mM glucose, 10 mM glucose-6-P, 10 mM fructose-6-P, 10 mM fructose 1,6-di-P, 40 mM DL-glyceralde- interesting to study sequentially (18, 21) the activities of the hyde-3-P, 15 mM 3-P-glycerate, 15 mM 2-P-glycerate, 15 mM key enzymes of the glycolytic pathway in a transplantable P-enolpyruvate, 0.25 ml of the supernatant at 22,000 X g or thyroid tumor as compared to those of normal thyroid. The rat thyroid tumor used throughout these studies was 105,000 X g (or alternatively 0.140 or 0.040 ml with 2-P-glycerate or P-enolpyruvate as substrate, respectively). one of a series of tumors developed in Fischer rats by Wollman (25) and designated at line 1-8. This tumor resembles the ATP was used with glucose, glucose-6-P or fructose-6-P as substrate; ADP was used with fructose-1,6-di-P, glyceraldehyde-3-P, 3-P-glycerate, 2-P-glycerate or P-enolpyruvate. The Received February 14, 1972; accepted September 13, 1972. acidic solutions were previously neutralized with l N KOH and

DECEMBER 1972 2793

the pH was finally adjusted to 7.5 after addition of cofactors production by the 22,000 X g supernatant of the thyroid and and substrate. The reaction was carried out in a Dubnoff of the thyroid tumor was measured in the presence of metabolic incubator, with N2 :C02 (95:5) as the gas phase at nonlimiting amounts of cofactors and substrates. In the 35Â°,pH 7.5, for 5 min. With glucose as substrate incubation presence of glucose, lactate production is linear up to 30 min was also for 15 min. Some experiments were carried out in the of incubation. With the other substrates, lactate, pyruvate, and absence of KHCO3 with air as the gas phase, pH 7.5, as glycerol-1-P production is linear up to at least 10 min. The indicated in the text. The extra enzymes added to the NADH required in vitro for lactate formation from pyruvate is incubation mixture were previously dialyzed with 50 mM generated mostly at the glyceraldehyde-3-P dehydrogenase Tris-HCl buffer, pH 7.5; when undialyzed enzymes were used, step. For this reason the principal end product formed from a control was run with the corresponding amount of substrates such as 3-P-glycerate, 2-P-glycerate, and P-enolpyru- (NH4)2S04. The incubation was stopped by adding 0.1 ml of vate is pyruvate. Therefore, in thyroid extracts, lactate 18% (w/v) HC1O4. After centrifugation, samples of the production from the various intermediates of the Embden- protein-free supernatant were used for the determination of Meyerhof pathway increases progressively from glucose to JÃ¡ctate,pyruvate, and glycerol-1-P by enzymatic methods (8). glyceraldehyde-3-P, whereas it diminishes when 3-P-glycerate The amounts of lactate, pyruvate, or glycerol-1-P present or other substrates such as 2-P-glycerate or P-enolpyruvate are before incubation were measured and subtracted from the added to the medium (Table 1). On the other hand, pyruvate amounts present after incubation. Lactate formation from accumulation, which is very low in the 1st part of the pyruvate was also measured at 36Â°,pH 7.5 (10). sequence, greatly increases when the in vitro generation of Hexokinase (6), P-fructokinase (19), glyceraldehyde-3-P NADH becomes too low, i.e., after the glyceraldehyde-3-P dehydrogenase (2), P-glycerate kinase (4), P-glycerate mutase dehydrogenase step (Table 1). The total amount of (3), enolase (1), pyruvate kinase (22), and cytochrome oxidase pyruvate + lactate formed from the various substrates of the (24) activities were determined at 22Â°and expressed as nmoles glycolytic pathway shows significant increases at 5 points of of substrate utilized by the supernatant at 22,000 X g or at the sequence, i.e., between glucose and glucose-6-P; between 105,000 X^Xmg (wet weight)"1 X min"1. The thyroid fructose-6-P and fructose-1,6-di-P; between glyceraldehyde-3-P glands from 20 to 35 animals were used for each experiment; and 3-P-glycerate;between 3-P-glycerate and 2-P-glycerate, and all experiments were repeated at least 5 times and the results finally between 2-P-glycerate and P-enolpyruvate (Table 1). from a typical experiment are presented. These steps are catalyzed, respectively, by hexokinase, Partial Purification of Thyroid and Tumor P-fructokinase. P-fructokinase, glyceraldehyde-3-P dehydrogenase + P-glycer The enzyme was partially purified according to the method of ate kinase, P-glycerate mutase, and enolase. The last reaction, Layzer and Conway (11) by centrifugation at 22,000 X g and catalyzed by enolase, could be also considered rate-limiting in by fractionation on a DEAE-cellulose column with a linear the Embden-Meyerhof pathway (12). Further evidence that gradient of Tris-phosphate buffer (pH 8) formed by mixing 50 hexokinase and P-fructokinase are rate-limiting steps in ml each of 50 mM and 600 mM buffer containing 0.2 mM thyroid extracts was obtained by adding 3.6 fig of yeast EDTA and 0.2 mM ATP and 0.7 mM dithiothreitol. The hexokinase (final concentration, 0.34 unit/ml) to glucose or 9 to 18 fig of rabbit muscle P-fructokinase (final concentration, enzyme was eluted with an approximately 300 mM Tris-phosphate buffer. 0.34 to 0.7 units/ml) to fructose-6-P; this addition resulted in Citrate inhibition of P-fructokinase was measured at pH 7.4 the increase of the rates obtained with saturating concentra tions of glucose or fructose-6-P to those obtained with in a reaction mixture containing (final concentrations): 35 mM glucose-6-P or fructose-1,6-di-P as substrate, respectively triethanolamine 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; (Table 1). aldolase (35 jug/ml); triose-P isomerase (2.0 /Â¿g/ml); In the thyroid tumor lactate production from glucose glycerol-1-P dehydrogenase (10 Mg/ml); bovine serum albumin, (Table 1) as well as hexokinase activity (Table 2) are both 0.01%. Rates of decrease in absorbance at 340 nm were higher than those of normal thyroid. Moreover the addition of determined 3 to 5 min after the reaction was started by yeast hexokinase to glucose enhances lactate production to the addition of the P-fructokinase preparation in the absence or in levels obtained from glucose-6-P. the presence of citrate. Cyclic 3',5'-AMP (0.6 mM final In the thyroid tumor extracts, lactate formation from glucose-6-P and from fructose-6-P is as high as that obtained concentration), pH 7.4, was added 4.5 min after the reaction from fructose-1,6-di-P. The addition of rabbit muscle was started, and the rate of decrease in absorbance was P-fructokinase enhances only slightly lactate production from determined 1.5 to 2.5 min after its addition. NADH oxidase fructose-6-P (Table 1). It seems therefore possible, as shown activity was measured in the absence of ATP and subtracted from P-fructokinase activity. by the sequence method, that at least in vitro P-fructokinase of thyroid tumor has lost its rate-limiting function and that the control may be transferred to another site in the glycolytic RESULTS AND DISCUSSION chain, as shown in other tissues and in other experimental conditions (18, 23). For clarification of this hypothesis a series Thyroid tumor extracts (supernatant at 22,000 X g) contain of experiments has been done. Some of them have been an amount of endogenous lactate slightly but significantly performed in the presence of various concentrations of higher (p < 0.05) than does normal thyroid (10.8+1.0 and fructose-1,6-di-P, ADP, and NAD+ (Table 3), in order to show 8.0 Â±0.5 nmoles X mg (wet weight)"1, respectively). Lactate that a maximal lactate production from fructose-1,6-di-P was

2794 CANCER RESEARCH VOL. 32

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1972 American Association for Cancer Research. Regulation ofGlycolysis in a Thyroid Tumor obtained in our experimental conditions and that lactate 3',5'-AMP was not included in the elution mixture to avoid production in the tumor extracts from fructose-6-P reached interference with citrate inhibition. The thyroid enzyme was the same optimal levels obtained from fructose-1,6-di-P. strongly inhibited by 4 mM citrate, whereas the tumor enzyme Moreover some differences between thyroid and tumor, as far activity was only slightly depressed under the same as the effects produced by ATP on lactate production from experimental conditions (Table 4). The percentage of glucose and glucose-6-P are concerned, have been shown. In inhibition by citrate is decreased slightly by increasing enzyme fact, when increasing the ATP concentration in the presence of concentration. However, the differences between thyroid and 1 of these 2 substrates, a progressive inhibition of lactate tumor P-fructokinase are quite evident. The reversal of citrate production from thyroid extracts has been observed (Chart 1). inhibition by cyclic 3',5'-AMP addition was less effective on This inhibition is probably due to the effect produced by ATP the tumor than on the thyroid enzyme (Table 4). Therefore, it at the step catalyzed by P-fructokinase which is sensitive to seems possible that the allosteric regulation of P-fructokinase the allosteric control by ATP. In fact the limiting effect is by such substances is less effective in the tumor than in the partially overcome when fructose-6-P is added as substrate thyroid. These modifications may influence the control of the (Chart 1). These data are in agreement with observations glycolytic pathway. In fact, like in yeast extracts, conforma- previously reported in other tissues (17). On the contrary, in tional changes may be an effective controlling factor in tumor extracts, there is a progressive increase in lactate P-fructokinase regulation more than the total amount of the production with increasing ATP concentrations. Such increase enzyme (7). is evident with ATP up to 6 mM, when glucose is used as substrate (Chart 2). The P-fructokinase activity is also slightly higher in the tumor than in the thyroid extracts (Table 2). Table 2 In order to investigate whether the tumor enzyme shows a Enzymatic activities of the glycolytic pathway in the supernatant at different control by allosteric effectors such as citrate and 22,000 X g of rat thyroid and thyroid tumor homogenates cyclic 3',5'-AMP (9, 11, 17, 20, 26), P-fructokinase from Activity [nmoles substrate utilized tumor and from thyroid extracts was partially purified (about X mg (wet wt) "' X min "' ] 18-fold and 11-fold, respectively) on DEAE-cellulose column with a linear gradient of Tris-phosphate buffer (11). The Rat thyroid Thyroid tumor degree of purification was low because the enzyme loses some HexokinaseP-fructokinaseGlyceraldehyde-3-P Â±0.14.55 +0.36.75 activity during purification, even in the presence of ATP and +0.421.201 i0.437.50Â± dithiothreitol. Moreover the thyroid P-fructokinase was not dehydrogenaseP-glycerate 1.578.00 2.090.00 completely separated from thyroglobulin, which behaves as a kinaseP-glycerate Â±3.028.80 Â±6.546.80 broad peak. The different degree of purification between mutaseEnolasePyruvate Â±2.011.50+ +3.914.40 1.028.50+ +1.026.901 thyroid and tumor enzyme may be related to the lowest kinase1.98 1.53.35 1.5 amount of thyroglobulin present in the tumor (5). Cyclic

Table 1 Lactate 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, NAD*ATP, Â±2"23.5 t0.25.310.55.5 t2115.0+ +0.21.1 NAD*ATP, +230.5 9123.01 tO.21.5 NAD*ADP, Â±350.0 t0.620.5 10132.0+ +0.11.7 6-di-PGlyceraldehyde-3-P3-P-glycerate2-P-glycerateP-enolpyruvateGlucoseNAD*ADP, +453.0+ i2.022.5 10138.0 i0.21.5 NAD*ADP, 520.5 +3.0480.01 Â±1538.8 i0.2755.0t NAD*ADP, Â±421.51622.5 15.01200.0+ +442.0 20.01250.0+ NAD*ADP, 22.04400.0 Â±645.0 30.04400.0 NAD*ATP, i722.3 +60.03.1 i7143.0 i100.01.1

unit/ml)Fructose-6-P+ hexokinase (0.34 NAD*ATP, +246.5 i0.323.2 Â±9170.01 Â±0.21.3+ unit/ml)Fructose-1+ P-fructokinase (0.7 NAD*ADP, Â±4140.0 Â±2.0IS.Oi 11160.01 0.22.0 glyceraldehyde-3-Pdehydrogenase, 6-di-P + NAD*6.0 i 70.5 0.427.9 120.7 i 0.4 (6.1 units/ml)ATP, "Substrate concentration: 20 mM glucose; 10 mM glucose-6-P, fructose-6-P, fruciÃ³se-1,6-di-P2; 40 mM glyceraldehyde-3-P; 15 mM 3-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 gas phase. c Mean Â±S.E.

DECEMBER 1972 2795

Table 3 presence of thyroid or tumor extracts, resulted also in the LÃ¡clateproduction by thyroid tumor supernatant at 22,000 X g in the increase of the rates of pyruvate production obtained from presence of various concentrations ofcofactors and substrate 2-P-glycerate to those obtained with P-enolpyruvate as Incubation was carried out for 5 min as described in Table 1. substrate (Tables 5 and 6, respectively). Moreover lactate production from pyruvate is higher in the tumor than in the thyroid (14.8 Â±0.5 and 4.9 Â±0.3 /uniÃ³lesof substrate utilized LÃ¡clateproduction Jnmoles X mg (wet wt)'1 X hr'1 ] X mg (wet weight)"1 X hr"1, respectively). Thyroid extracts contain an amount of endogenous Concentration glycerol-1-P (2.1 Â±0.3 nmoles X mg (wet weight)'1) quite 1,6-di-Pc50 (mM)0.51.01.52.03.04.08.010.0NAD*089 similar to that of the tumor (2.3 Â±0.3 nmoles X mg (wet weight)"'). In normal thyroid glycerol-1-P production Â±795 Â±470 Â±465 Â±898 Â±597 Â±696 increases, like lactate formation, when the control points of Â±898 the pathway are overcome by the addition of the substrate +999 Â±892 Â±898 further down in the sequence (Table 5). NADH required for Â±8101 glycerol-1-P formation at the step catalyzed by glycerol-1-P Â±9103 Â±870 Â±897 Â±898 dehydrogenase is probably generated mostly at the glyceralde- Â±9ADPb45 Â±6Fructose- Â±7 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 "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 1.5 mMNAD*.

cn E

IO 4- o (O

10 QJ 3.0 6.0 9.0 O ATP (mM) C Chart 2. Lactate production by thyroid tumor supernalanl at 3.0 6.0 9.0 22,000 X g in the presence of various concentralions of ATP wilh the following subslrales: â€¢¿â€”â€¢,20mM glucose; â€¢¿-â€¢,10mM glucose-6-P; ATP (mM) oâ€”o, 10 mM fruclose-6-P. Incubalion was carried oui as described in Chart 1. LÃ¡clateproduction by thyroid supernatant at 22,000 X g in Charl 1. Ihe presence of various concenlralions of ATP wilh Ihe following subslrales: â€¢¿â€”Â»,20mM glucose; â€¢¿Â»,10mM glucose-6-P; o o, IO mM fructose-6-P. Incubation was carried out at 35Â°,pH 7.5, in Ihe Table 4 Effects of citrate and of cyclic 3',5'-AMP on P-fructokinase activity0 presence of 150 mM KC1, 7 mM MgCl2, 40 mM potassium phosphate buffer (pH 7.5), and 1.5 mM NAD*, wilh air as Ihe gas phase. fcontrol =1001 Addition In normal thyroid extracts, pyruvate production from (4 mM) 3-P-glycerate is efficiently increased by addition of P-glycer- 3' ,5'-AMP and cyclic 3',5'- ate mutase (12.5 units/ml) (Table 5). The same results are TissueRat (0.6mM)102 (4 mM)20 AMP(0.6mM)10382 obtained in tumor extracts (Table 6), where lactate and pyruvate production from 3-P-glycerate and P-glycerate thyroid mutase activity are enhanced with respect to normal thyroid Thyroid tumorCyclic 101Citrate 66Citrale (Table 2). a P-fructokinase activity was measured at pH 7.4 as previously The addition of enolase (10 units/ml) to 2-P-glycerate in the described under "Materials and Melhods."

2796 CANCER RESEARCH VOL. 32

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1972 American Association for Cancer Research. Regulation ofGlycolysis in a Thyroid Tumor activity in the glycolytic pathway. In fact the addition of far as enzymatic activities are concerned (Table 7). Super- glyceraldehyde-3-P dehydrogenase to fructose-1,6-di-P, en natants at 22,000 X g and 105,OOOXg of both thyroid and hances the production of lactate in thyroid extracts (Table 1). tumor do not show detectable cytochrome oxidase activity In the tumor, glycerol-1-P production from the different (Table 7). substrates (Table 6) and glyceraldehyde-3-P dehydrogenase On the basis of these experimental results it seems possible activity (Table 2) are both higher than in the thyroid. to conclude that the regulation of the glycolytic pathway Moreover the addition of glyceraldehyde-3-P dehydrogenase to seems probably to be modified in thyroid tumor extracts fructose-1,6-di-P does not influence remarkably lactate particularly at the step catalyzed by P-fructokinase. The production in tumor extracts (Table 1). evidence supporting this fact is (a) the enhancement of lactate No significant differences have been shown when the production from fructose-6-P in the tumor; (b) the difference sequence data obtained with 22,000 X g supernatant were in response between tumor and thyroid when extra compared with those obtained with 105,000 X g supernatant P-fructokinase is added to fructose-6-P as substrate; (c) the (Tables 5 and 6). The same results have been obtained also as Table 6 Table 5 Â¡retate, pyruvate, and glycerol-1-P formation in the 22,000 X g and Lactate, pyruvate, and glycerol-I-P formation in the 22,000 X g and 105,000 X g supernatant of the same homogenate 105,000 X g supernatant of the same homogenate of rat thyroid of thyroid Tumor 1-8 Experimental conditions are identical to those of Table 1. Experimental conditions are identical to those of Table 1. Lactate, pyruvate, and glycerol-1-P formation Lactate, pyruvate, and glycerol-1-P formation [nmoles X mg (wet wt)~' Xhr"'] [nmoles X mg (wet wt)"1 Xhr"1]

Substrate Lactate Pyruvate Glycerol-1-P Lactate Pyruvate Glycerol-1-P gGlucoseFructose-6-PFructose-l,6-di-P3-P-glycerate2-P-glycerateP-enolpyruvate3-PglycerateSupernatant at 22,000 X gGlucoseFructose-6-PFructose-l,6-di-P3-P-glycerate2-P-glycerateP-enolpyruvate3-P-glycerateSupernatant at 22,000 X 227.5 Â±0.38.0 Â±1.539 Â±2102.0 0.20.8 Â±3188 Â±348.0 Â±0.722.5 Â±2.067 +9116.0Â± Â±0.11.5 +9480 Â±434.0 Â±1.5447.0 Â±4.08.1 1169.0 Â±0.3720.0 Â±1340 Â±434.0 Â±10.01 Â±768.0 Â±20.01280.0 +538.0 390.0 Â±23.04500.0 Â±871.0 Â±22.04500.0 +536.0 Â±65.01090.0 Â±772.0 Â±75.01080.0+

42-P-glycerateP-glycerate + + Â±18.04250.0 72-P-glycerateP-glycerate + + 20.040 mutase(12.5 mutase12.5 units/ml)4.8+ units/ml)24.0 5(10 + enolase 37.0 Â± 70.0105,000 Â± 8(10 + enolase 73.0 Â± 10.0Â±80.0,000 units/mlGlucoseFructose-6-PFructose-l,6-di-P3-P-glycerate2-P-glycerateP-enolpyruvateSupernatantunits/ml)GlucoseFructose-6-PFructose-l,6-di-P3-P-glycerate2-P-glycerateP-enolpyruvateSupernatant

at4.5 Xg1.0+0.26.2 at10519.8 X g1.5 Â±125.0 Â±1.833.2 Â±296.0 Â±0.22.0 i4195+ Â±346.0 Â±0.420.0+ Â±2.067.0 Â±8100.0 Â±0.42.0 10450 Â±435.0 1.9435.0 + 3.5 Â±1065 Â±0.3685.0+25.01 Â±14 Â±335.0 Â±11.01460.0 .0Â±568.0 Â±537.0 Â±22.04280.0 Â±867.0 260.0 Â±23.04700.0 Â±41.3 + 80.010 Â±91.0Â± + 95.042

Table 7 Enzymatic 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 mg fwetwt)'1 X min'1 ]

Activity

Rat thyroid Thyroid tumor

Xgsupernatant1.563.8520.80 Xgsupernatant2.655.5032.00

HexokinaseP-fructokinaseGlyceraldehyde-3-P dehydrogenase P-glycerate mutase 26.30 26.00 40.80 41.70 Cytochrome oxidase22,000 N.D.b105,OOOXÂ£supernatant1.383.8520.30N.D.22,000 N.D.105,OOOXÂ£supernatant2.305.4530.80N.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 is 12. Lea, M. A., and Walker, D. G. Factors Affecting Hepatic Glycolysis used as substrate in tumor extracts, and (d) the altered and Some Changes That Occur during Development. Biochem. J., response to citrate and to cyclic 3',5'-AMP of the partially 94:655-665,1965. purified tumor P-fructokinase with respect to the thyroid 13. Lee, I. Y., Strunk, R. C., and Coe, E. L. Coordination among Rate-limiting Steps of Glycolysis and Respiration in Intact Ascites enzyme. Tumor Cells. J. Biol. Chem., 242: 2021-2028, 1967. 14. Lo, C. H., Farina, F., Morris, H. P., and Weinhouse, S. Glycolytic Regulation in Rat Liver and Hepatomas. Advan. Enzyme ACKNOWLEDGMENTS Regulation., 6: 453-464, 1968. 15. Lorenson, M. Y., and Mansour, T. E. Studies on Heart The authors are indebted to Dr. S. H. Wollman and to Dr. F. Phosphofructokinase. Binding Properties of Native Enzyme and of Eisenberg, NIH, Bethesda, Md., for helpful editorial suggestions. Enzyme Desensitized to AUosteric Control. J. Biol. Chem., 244: 6420-6431,1969. 16. Macchia, V., Meldolesi, M. F., and Chiariello, M. Effect of TSH and TSH-like Substances on Some Properties of a Transplantable REFERENCES Thyroid Tumor of the Rat. In: K. Fellinger and R. HÃ¶fer(eds.), Further Advances in Thyroid Research, pp. 1205-1213. Wien: 1. Bergmeyer, H. U., Klotzsch, H., MÃ¶llering, H., NelbÃ¶ck-Hoch- Verlag der Wiener Medizinischen Akademie, 1971. stctter, M., and Beaucamp, K. Enolase from skeletal Muscle. In: H. 17. Mansour, T. E. Studies on Heart Phosphofructokinase: Purifica U. Bergmeycr (ed.), Methods of Enzymatic Analysis, pp. 973-974. tion, Inhibition and Activation. J. Biol. Chem., 238: 2285-2292, New York: Academic Press, Inc., 1965. 1963. 2. Bergmeyer, H. U., Klotzsch, H., MÃ¶llering, H., NelbÃ¶ck-Hoch- 18. Meldolesi, M. F., Effects of 3,3',5-Triiodo-L-Thyronine Administra stetter, M., and Beaucamp, K. Glyceraldehyde-3-phosphate Dehy- tion on the Embden-Meyerohof Pathway in the Kidney Cortex of drogenase from Skeletal Muscle. In: H. U. Bergmeyer (ed.), the Rat. European J. Biochem., 22: 27-30, 1971. Methods of Enzymatic Analysis, pp. 979-980. New York: 19. Parmeggiani, A., Luft, J. H., Love, D. S., and Krebs, E. G. Academic Press, Inc., 1965. Crystallization and Properties of Rabbit Skeletal Muscle Phospho 3. Bergmeyer, H. U., Klotzsch, H., MÃ¶llering,H., Nclbock-Hoch- fructokinase. J. Biol. Chem., 241: 4625-4637, 1966. stetter, M., and Beaucamp, K. 3-Phosphoglycerate Mutase from 20. Pogson, C. I., and RÃ¤ndle, P. J. The Control of Rat-Heart Skeletal Muscle, In: H. U. Bergmeyer (ed.), Methods of Enzymatic Phosphofructokinase by Citrate and Other Regulators. Biochem. J., Analysis, pp. 995-996. New York: Academic Press, Inc., 1965. 100: 683-693, 1966. 4. BÃ¼cher,T., Ãœber ein PhosphatÃ¼bertragendes GÃ¤rungsferment. 21. Scrutton, M. C., and Utter, M. F. The Regulation of Glycolysis and Biochim. Biophys. Acta, 1: 292-314, 1947. Gluconeogenesis in Animal Tissues. Ann. Rev. Biochem., 37: 5. De Nayer, P., Weathers, B., and Robbins, J. Thin-layer Gel 249-302, 1968. Filtration of Thyroid lodoproteins. Studies on Rat Transplantable 22. Valentine, W. N., and Tanaka, K. R. Pyruvate Kinase:Clinical Thyroid Tumors. Endocrinology, 81: 1118-1124, 1967. Aspects. Methods Enzymol., 9: 468-473, 1966. 6. Grossbard, L., and Schimke, R. T. Multiple Hexokinases of Rat 23. Williamson, J. R. Metabolic Control in the Perfused Rat Heart. In: Tissues. Purification and Comparison of Soluble Forms. J. Biol. B. Chance, R. W. Estabrook, and J. R. Williamson, (eds.), Control Chem., 247: 3546-3560, 1966. of Energy Metabolism, pp. 333-346. New York: Academic Press, 7. Hess, B., Boiteux, A., and KrÃ¼ger,J. Cooperation of Glycolytic Inc., 1965. Enzymes. Advan. Enzyme Regulation, 7: 149-167, 1969. 24. Wharton, D. C., and Tzagoloff, A. Cytochrome Oxidase from Beef 8. Hohorst, H. J., Kreutz, F. H., and BÃ¼cher,T., "Ãœber Meta- Heart Mitochondria. Methods Enzymol., 10: 245-253, 1967. bolitgehalte und Metabolit-Konzentrationen in der Leber der 25. Wollman, S. H. Production and Properties of Transplantable Ratte. Biochem. Z., 332: 18-46, 1959. Tumors of the Thyroid Gland in the Fischer Rats. Recent Progr. 9. Kemp, R. G. Rabbit Liver Phosphofructokinase: Comparison of Hormone Res., 19: 579-618, 1963. Some Properties with Those of Muscle Phosphofructokinase. J. 26. Wu, R. Further Analysis of the Mode of Inhibition and Activation Biol. Chem., 246: 245-252, 1971. of Novikoff Ascites Tumor Phosphofructokinase. J. Biol. Chem., 10. Kornberg, A. Lactic Dehydrogenase of Muscle. Methods Enzymol., 247:4680-4685, 1966. 7:441-443, 1955. 27. Wu, R., Power, H., and Hamerman, D. Rate-limiting Factors in 11. Layzer, R. B., and Conway, M. M. Multiple Isoenzymes of Human Glycolysis and Transport of Inorganic Phosphate in DBAH, Phosphofructokinase. Biochem. Biophys. Res. Commun., 40: Tumor, DBAG Tumor, Novikoff Hepatoma and Novikoff Ascites 1259-1265, 1970. Tumor. Cancer Res., 25: 1733-1742, 1965.

2798 CANCER RESEARCH VOL. 32

M. F. Meldolesi and V. Macchia

Cancer Res 1972;32:2793-2798.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/32/12/2793

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/32/12/2793. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.