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

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Regulation of the Embden-Meyerhof Pathway in a Transplantable Rat Thyroid Tumor [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, Università 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 4°. 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, glyceralde- hyde-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 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1972 American Association for Cancer Research. M. F. Meldolesi and V. Macchia 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 35°,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 Jáctate,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 36°,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 22°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).
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