Behavior of Transaldolase (EC 2.2.1 .2) and Transketolase (EC 2.2.1.1

Home , Transaldolase, Transketolase

[CANCER RESEARCH 36, 3189-3197, September 1976] Behavior of Transaldolase (EC 2.2.1 .2) and Transketolase (EC 2.2.1 .1) Activities in Normal, Neoplastic, Differentiating, and Regenerating Liver

Peter C. Heinrich, Harold P. Morris,1 and George Weber2

Laboratory for Experimental Oncology, Indiana University School of Medicine, Indianapolis, Indiana 46202 (P. C. H., G. W.J, and the Department of Biochemistry, Howard University College of Medicine, Washington, D. C. 20001 (H. P. M.J

SUMMARY In the regenerating liver at 24 hr after partial hepatec tomy, the activity of both pentose phosphate pathway en The objective of this investigation was to throw light on zymes was in the same range as that of the sham-operated the biological behavior and metabolic regulation of hepatic controls. enzymes of the nonoxidative branch of the pentose phos In differentiation at the postnatal age of 5, 12, 23, and 32 phate pathway. The activities of transaldolase (EC 2.2.1 .2) days, hepatic transaldolase activities were 33, 44, 55, and and transketolase (EC 2.2.1 .1) were compared in biological 72%, respectively, of the activities observed in the 60-day conditions that involve modulation of gene expression such old, adult male rat. During the same period, transketolase as in starvation, in differentiation, after partial hepatectomy, activities were 18, 21, 26, and 55% of the activities observed and in a spectrum of hepatomas of different growth rates. in liver of adult rat. The enzyme activities were determined under optimal ki The demonstration of increased transaldolase activity in netic conditions by spectrophotometric methods in the hepatomas, irrespective of the degree of tumor malignancy, 100,000 x g supernatant fluids prepared from tissue ho differentiation, orgnowth rate, suggeststhatthe reprogram mogenates. ming of gene expression in malignant transformation is The kinetic properties of transaldolase and tnansketolase linked with an increase in the expression of this pentose were similar in normal liver and in rapidly growing hepa phosphate pathway enzyme. Since no similar alteration was toma 3924A. For transaldolase, apparent K,, values of 0.13 found in the differentiating or regenerating liven, the in mM (normal liven) and 0.17 mM (hepatoma) were observed creased transaldolase activity appears to be specific to the for erythrose 4-phosphate and of 0.30 to 0.35 mM for fruc neoplastic transformation. The increase in transaldolase tose 6-phosphate. The pH optima in liven and hepatoma activity in conjunction with the earlier observed increase in were at approximately 6.9 to 7.2. For the transketolase glucose-6-phosphate dehydnogenase activity should pro substrates, nibose S-phosphate and xylulose S-phosphate, vide selective advantages to the neoplastic cells. the apparent K@values were 0.3 and 0.5 mM, respectively, in both liver and hepatoma. A broad pH optimum around 7.6 INTRODUCTION was observed in both tissues. In organ distribution studies, enzyme activities were mea In neoplastic transformation and progression, there is a sured in liver, intestinal mucosa, thymus, kidney, spleen, stringent need for a supply of nibose 5-phosphate for DNA brain, adipose tissue, lung, heart, and skeletal muscle. Tak synthesis and cell proliferation. In liven, the production of ing the specific activity of liver as 100%, transaldolase activ ribose 5-phosphate may proceed through the oxidative ity was the highest in intestinal mucosa (316%) and in thy pathway, chiefly by action of glucose 6-phosphate and 6- mus (219%); it was the lowest in heart (53%) and in skeletal phosphogluconate dehydrogenases, and through the non muscle (21%). Transketolase activity was highest in kidney oxidative pathway, primarily by the action of transaldolase (155%) and lowest in heart (26%) and skeletal muscle (23%). and tnansketolase. Earlier work in this laboratory demon Starvation decreased transaldolase and tnansketolase ac strated that the activity of glucose-6-phosphate dehydro tivities in 6 days to 69 and 74%, respectively, of those of the genase was increased in all the tumors of the Morris hepa liver of the normal, fed mat.This was in the same range as toma spectrum (35). Whereas much has been learned of the the decrease in the protein concentration (66%). behavior and modulation of the glucose-6-phosphate and In the liver tumors, transaldolase activity was increased 6-phosphogluconate dehydrogenases, relatively little is 1.5- to 3.4-fold over the activities observed in normal control known of the biological chemistry of the transaldolase and rat liver. Transketolase activity showed no relationship to tnansketolase enzymes in normal and neoplastic liver (35). tumor proliferation mate. The purpose of this work was to elucidate the possible linkage of transaldolase and transketolase to metabolic

I Recipient of USPHS Grant CA-10792. transformation and progression by examining the enzyme 2Recipient of USPHS Grants CA-13526 and CA-05034. To whom requests activities in liven tumors of different malignancy. To under for reprints should be addressed, at the Laboratory for Experimental Oncol ogy, Indiana University School of Medicine, Indianapolis, Ind. 46202. stand more deeply the metabolic roles of these enzymes, Received April 6, 1976; accepted June 3, 1976 transaldolase and transketolase activities were also investi

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1976 American Association for Cancer Research. P. Heinrich et a!. gated in conditions that involve regulation of gene expres to enythnose 4-phosphate. The formation of glycenaldehyde sion such as in differentiation, in regeneration, and during 3-phosphate was followed by measuring the decrease in long-term starvation in rat liven. absorbance of NADH at 340 nm in the presence of tniose The results showed that, in a spectrum of hepatomas of phosphate isomerase and glycerophosphate dehydnogen vastly different growth rates, transketolase activity showed ase (4). The standard assay mixture had the following com no pattern, but transaldolase activity was increased in all position: 60 mM tniethanolamine buffer, pH 7.4; 6 mM EDTA; the tumors studied. Both transaldolase and transketolase 15 mM fructose 6-phosphate; 1.0 mM enythrose 4-phos responded with a marked decrease in activity to starvation, phate; 0.24 mM f3-NADH; 3.6 @gglycerophosphate dehydno exhibited a characteristic rising pattern in differentiation, genase; and 0.4 @gtniosephosphate isomerase. The final and showed no change in the regenerating liven. It was volume was 1.0 ml. concluded that the increased transaldolase activity in the Transketolase Assay. The principle of the method was hepatomas is a transformation-linked alteration in gene the measurement of the formation of glycenaldehyde 3- expression that appears to be specific to neoplastic prolifer phosphate from xylulose S-phosphate and nibose 5-phos ation. phate as receptor aldehyde by following the oxidation of NADH at 340 nm in the presence of tniosephosphate isomer ase and glycerophosphate dehydnogenase (7). Mg2@and MATERIALS AND METHODS thiamine diphosphate were added, although they did not influence the enzymatic activity. The standard assay mix Experimental animals were kept in individual cages and tune was as follows: SOmM Tnis-HCI, pH 7.5; 5 mM MgCI2; Purina Laboratory chow and water were available ad libi 0.06 mM thiamine diphosphate; 0.24 mM NADH; 3.6 @g tum , except in starvation experiments where only water was glycerophosphate dehydrogenase, 0.4 p.g tniosephosphate provided (20). isomenase; 5.0 mM nibose 5-phosphate; and 5.0 mM xylulose Tumor-bearing and Control Animals. The hepatomas 5-phosphate. The final volume was 1.0 ml. were transplanted s.c. bilaterally in inbred strains of male Protein determinations were made by the procedure de Buffalo on ACI/N rats. Normal rats of the same strain, sex, scnibed by Lownyetal. (17). The cell counts were carried out age, and weight were killed with the tumor-beaning rats as described previously (27). under the same experimental conditions. To have a wide Expression and Evaluation of Results. Transaldolase spectrum of malignancy, we examined a number of tumor and transketolase activities were calculated as j@molesof lines, including the slowly growing 66, 47C, 8999, and 44; substrate metabolized per hr at 37Â°andwere expressed pen the intermediate growth rate tumors 9633 and 7794A, and g wet weight of tissue, pen mg of protein, on pen average the rapidly growing hepatomas 7777, 3924A, 3683F, and cell. Cell counts were expressed as cellularity calculated in 9618A2. The neoplasrns were harvested when they had millions of nuclei per g wet weight oftissue (27). The expeni reached a diameter of about 1.5 cm (20). mental results were subjected to statistical evaluation by Regenerating Liver. Partial hepatectomy was carried out means of the t test for small samples. Differences between by the standard procedure of Higgins and Anderson (13). means giving a probability of less than 5% were considered The remaining liven lobes were examined at 24 hr after to be significant. operation. Sham-operated animals were used as controls, as described elsewhere (20). Differentiating Liver. Pregnant Wistan rats were pun RESULTS chased from Harlan Industries, Cumbenland, Ind., and the litters were allowed to remain in the same cage with the In order to establish that linear kinetics operate in the mother for 18 days after birth; then each rat was placed in crude supernatant system used in the transaldolase and an individual cage. transketolase assays, the properties and behavior of these Effects of Starvation. Male albino Wistan rats (Harlan enzymes were compared in extracts from normal liven and Industries),190to 200 g, were used. hepatomas. Preparation of Liver and Tumor Samples. The rats were Comparison of Kinetic Properties of Transaldolase in stunned, decapitated, and exsanguinated. Livers and tu Liver and Hepatoma 3924A. Chart 1 shows the effect of moms were rapidly removed, and preparation of homoge enythrose 4-phosphate on transaldolase activity in rat liven nates and supennatant fluids was carried out as described (ACI/N strain) and in rapidly growing hepatoma 3924A (can previously (20, 33). ned in ACI/N rats). The enzyme activity in the liven and Chemicals. D-Xylulose 5-phosphate, sodium salt (Grade hepatoma was saturated at an erythnose 4-phosphate con Ill), D-erythrose 4-phosphate (disodium salt), D-nibose 5- centration of 0.5 to 1 mM. Enzyme activity was not inhibited phosphate (disodiurn salt), and thiamine diphosphate chlo by substrate levels up to 2 mM. Studies on affinity of transal ride were obtained from Sigma Chemical Co. (St. Louis, dolase to enythrose 4-phosphate from liver and hepatoma Mo.). Fructose 6-phosphate (disodium salt), f3-NADH, tn gave apparent Kfl)values of 0.13 and 0.17 mM, respectively. osephosphate isomerase, and glycerophosphate dehydro The effect of fructose 6-phosphate on transaldolase activ genase were purchased from Boehningen Mannheim (New ity in liver and hepatoma is compared in Chart 2. In both York, N. Y.). All other reagents were of the highest available tissues, transaldolase was saturated at a fructose 6-phos analyticalgrade. phate concentration of approximately 5.0 mM. The apparent Transaldolase Assay. Transaldolase catalyzes the trans K,@valueswere 0.3 to 0.35 mM for both tissues. The enzyme fer of the dihydroxyacetone moiety of fructose 6-phosphate activities were not inhibited by fructose 6-phosphate levels

3190 CANCER RESEARCH VOL. 36

400@

Chart 1. Effectof erythrose4-phosphateconcentration C) on the activity of transaldolase in liver and hepatoma 3924A. The standard assay concentration of erythrose 4- phosphate is 1 mM. app. , apparent.

ERYTHROSE .4.PHOSPHATE mM)

.1J-_ . . . . - . .C

0 0 400 â€” S F- F- F- C.) F- C.) C,) 200 [LIv@j 0 -C 0

Cl) z C,, z F- S F- 0 â€” 0 5 10 15 @- I V I I I 0 6 7 8 9 FRUCTOSE -6- PHOSPHATE (mM) pH Chart 2. Effect of fructose 6-phosphate concentration on the activity of Chart 3. Effect of pH on transaldolase activity in liver and hepatoma transaldolase in liver and hepatoma 3924A. The standard assay concentra 3924A. Assays were performed as described in â€œMaterialsandMethods.' The tion of fructose 6-phosphate is 15 mu., app., apparent. pH of the reaction mixture was adjusted prior to the addition of enzyme. up to 13 mM. The addition of nibose S-phosphate up to a toma extracts, thiamine diphosphate and Mg2' ions were final concentration of 1.0 mrvi did not influence the meas added to the assay mixture as specified in â€˜â€˜Materialsand urement of tnansaldolase activity in the assay with fructose Methods.â€• 6-phosphate and enythrose 4-phosphate. A broad pH optimum around 7.6 was observed for liver The pH optima for transaldolase in liven and hepatoma and hepatoma tnansketolase, as shown in Chart 7. 3924A were at approximately 6.9 to 7.2 (Chart 3). Through such kinetic studies, a standard assay was es Through such kinetic studies, a standard assay was es tablished for measurement of liver and tumor tnansketolase tablished for determination of liven and hepatoma transal activities. The assays were carried out at pH 7.6 and at 37Â° dolase activities. The assays were carried out at pH 7.2 and with the reaction mixture described in â€œMaterialsand Meth 37Â°under the reaction conditions given in â€œMaterialsand ods.â€•With this procedure, proportionality with amount of Methods.â€• With this procedure, proportionality with enzyme added (Chart 8) and length of reaction time oven a amount of enzyme added (Chart 4) and length of reaction 30-mm incubation period was achieved for both liver and time over a 30-mm incubation period was achieved. hepatoma. Comparison of Kinetic Properties of Transketolase in Comparison of Activities of Transaldolase and Transke Liver and Hepatoma 3924A. The effect of xylulose 5-phos tolase in Various Rat Organs. The activities of transaldo phate on transketolase activity is shown in Chart 5. The lase and tnansketolase were of the same order of magnitude enzyme activity in hepatoma and in liver was saturated at a in the liver, 4.3 and 3.1 @moles/hn/mgprotein, respectively xylulose 5-phosphate concentration of approximately 1.0 to (Table 1). A comparison of the specific activities in the 3.0 mM and the apparent K,) values for the liven and hepa various rat organs indicated that the highest tnansaldolase toma enzyme were 0.3 and 0.1 mM, respectively. activities were in thymus and intestinal mucosa, and the The effects of nibose 5-phosphate on transketolase activ lowest activities were in heart and skeletal muscle. The ity in liver and hepatoma are compared in Chart 6. The transketolase activities in intestinal mucosa and thymus enzyme activity in the 2 tissues was saturated at a ribose 5- were in the same range as in the liver, and lowest activities phosphate concentration of about 2 to 4 mM. The apparent were observed in heart and muscle. K,@'sfor liver and hepatoma were 0.3 and 0.5 mM, respec Effect of Starvation on Hepatic Transaldolase and tively. The enzyme activities were not inhibited by substrate Transketolase Activities. In 6-day starvation, the hepatic levels as high as 10 mM. Although no cofacton requirement cellularity nearly doubled, whereas the protein content of could be demonstrated for tmansketolase in liver and hepa the supemnatant fluid in the average cell decreased to 66%

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of the values of the liver of normal, fed mats(Table 2). These results agree with earlier observations (24). The transaldo lase and tnansketolase activities, on a wet weight basis, appeared to be increased; however, when alterations in hepatic cellularity were taken into consideration, the en zyrne activities per cell were decreased to about the same [email protected] a, level (69 and 74%) as the protein (66%). E0 Behavior of Hepatic Transaldolase and Transketolase Activity In Differentiation. During differentiation , marked I- changes occurred in the cellularity in the developing rat

I-, liver (32), and the relevant analysis should take these altera 1.) tions into consideration. The protein content in the super I,) natant fluid of the average hepatic cell was 26% of the adult values at 5 days of age. In the subsequent time intervals, at 12, 23, and 32 days, the protein concentration increased to 34, 45, and 68%, reaching the adult values in the 50-day-old 200-g rat. During the same time periods, the transaldolase I- activity was 33, 44, 55, and 72% of the activities of the adult liver. The relative activities of transketolase were percent age-wise about one-half of those observed for transaldolase. Thus, the transketolase activity during differentiation was 18, 21, 26, and 55% of the values observed in the liven of the adult rat. Comparison of Transaldolase and Transketolase Activi WET WEIGHT (mg) ties in Normal and Regenerating Liver and in Hepatomas Chart 4. Proportionality of transaldolase activity with amount of liver su of Different Growth Rates (Table 3). Investigation was car pernatant added . The standard assay described in â€œMaterialsandMethodsâ€• ned out to establish the relationship of transaldolase and was used , varying only the amount of supernatant fluid . Units on the abscissa are equivalent weights of fresh tissue. The standard enzyme assay used 1.0 transketolase activities to hepatoma growth rate. Table 3 mg. gives the activities of transaldolase and tnansketolase in

@ 200 0 S â€˜3

Chart5. Effect of xylulose 5-phosphateconcentration I- on the activity of transketolasein liver and hepatoma C.) 3924A. The standard assay concentration of xylulose 5- C,) phosphateis5.0mM. app.,apparent. -C 0 C,)F- z S F-

XYLULOSE-5-PHOSPHA'FE (mM)

-.--

[email protected]

200 0 S 1- F-

F- Chart6. Effectof ribose5-phosphateconcentrationon C.) C,) the activity of transketolase in liver and hepatoma 3924A. 100@ The standard assay concentration of ribose 5-phosphate is -C 5.0 [email protected]., apparent. 0 F-

3 6 R IBOSE â€˜5-PHOSPHATE (mM)

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hepatomas of different growth rates in comparison with the Similar behavior was observed when the activities were activities observed in the liven of control normal rats of the expressed on a per g wet weight basis or pen average cell same strain, sex, and age killed concurrently with the tu (not shown). The existence of noncovalently bound inhibi mon-bearing animals. The values of the regenerating liven tons on activators in the liven or tumor supemnatant fluid was were related to those observed in the sham-operated rats. excluded by estimation of the enzyme activity in combined Enzyme activities were calculated in @molesof substrate supemnatants of liver and tumor; only the sum of the sepa metabolized at 37Â°per hr per mg protein. Activities were rate activities was found. In the 24-hr regenerating liver, also expressed as percentages of activities observed for the transaldolase and tnansketolase activities were not altered appropriate controls and are given in parentheses. The transaldolase specific activity was increased 1.5- to 3.4-fold in all hepatomas examined. In contrast, transketo lase activity did not correlate with tumor proliferation rate.

S .3

F-

F- I 200 C-) C,) F- -C F- 0 C.) F- C,) 100 z -C S 0 F- F-

z S F-

WET WEIGHT (mgI 0 6 i a 9 Chart 8. Proportionality of transketolase activity with amount of liver su PH pernatant added. The standard assay described in â€œMaterialsandMethodsâ€• Chart 7. Effect of pH on transketolase activity in liver and hepatoma was used , varying only the amount of supernatant fluid . Units on the abscissa 3924A. Assays were performed as described in â€œMaterialsandMethods.â€•The are equivalent weights of fresh tissue. The standard enzyme assay uses 1.0 pH of the reaction mixtures was adjusted prior to the addition of enzyme. mg.

Table1 activitiesTenTissue distribution of transaldolase and transketolase theliver% homogenateswere preparedand enzymeassayscarried out as outlined for organarein â€œMaterialsandMethods.â€•Theresults of 4 or more pooled tissuesfor each (@.tmoles/hr/mgprotein)tabulated. Enzymeactivities were calculated as specific activities and also expressed as percentagesliver.Transaldolase of the values observed in the activity Transketolaseactivity Protein @moles/Tissues (mg/g wet @zmoles/ %Liver wt) hr/mg % hr/mg 100Intestinal 92 4.3 100 3.1 116Thymusmucosa 38 13.6 316 3.6 90Kidney 54 9.4 219 2.8 155Spleen 64 8.1 188 4.8 84Brain 81 7.8 181 2.6 71Adipose 28 4.3 100 2.2 94Lung 14 4.1 95 2.9 52Heart 68 4.0 93 1.6 26Muscle 52 2.3 53 0.8 53 0.9 21 0.7 23

Table2 Effect of starvation on hepatic transa!do!ase and transketolase activities MeansÂ±SE. of 4 animalsin eachgroup are given with percentagesof valuesof liver of fed rats in parentheses.Theactivities per cell should be multiplied by the exponentialgiven to arrive at the actual values.

Experimentalcondi (mil content activity (@.tmoles/activity (@moles/ 10@)NormaltionsCellularity Iions/g)Protein (mg/cell x 10@)Transaldolasehr/cell x 10w)Transketolasehr/cell x (100)Starvedfed250 Â±8(100)2.9 Â±0.2(100)12 Â±1(100)11 Â±0.2 for 6 days451 Â±9(180)Â°1 .9 Â±0.1(66)â€•8.3 Â±0.2(69y'8.1 Â±0.4 (74) a Statistically significantly different from values of normal, fed rats (p < 0.05).

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Table 3 Transaldolase and transketolase activities in hepatomas of different growth rates The data are means Â±SE. of 4 or more experiments with percentages of corresponding control liver values in parentheses.Protein activityTissues concen- Transaldolase activity Transketolase protein)Normal tration (mg/g) (.tmoles/hr/mg protein) (@moIes/hr/mg livers(Buffalo)Control 0.1Controlfor 66 78 Â±0.6 4.4 Â±0.2 3.9 Â± 0.1Controlfor 47C 87 Â±2 3.6 Â±0.1 3.2 Â± 0.2Controlfor 8999 82 Â±3 4.0 Â±0.2 3.8 Â± 0.2Controlfor 44 82 Â±1 3.1 Â±0.2 3.6 Â± 0.2Controlfor9633 81 Â±2 3.8Â±0.2 4.1Â± 0.1Controlfor 7794A 75 Â±1 5.5 Â±0.2 3.0 Â± 0.2Controlfor 7777 83 Â±2 2.8 Â±0.1 3.2 Â± 0.3Normal for 9618A2 77 Â±1 3.9 Â±0.2 3.5 Â± liver(ACI/N)Control 0.2Controlfor 3924A 71 Â±0.3 4.0 Â±0.3 3.3 Â± 0.1Sham-operatedfor 3683F 78 Â±0.5 4.2 Â±0.1 3.0 Â± 0.124-hr (Wistar) 80 Â±2 5.0 Â±0.2 3.6 Â± (97)(Wistar)Hepatomas66regeneratingliver 73 Â±1(91) 5.3 Â±0.2(106) 3.5 Â±0.1

(167)â€•47C 62 Â±2 (79)â€• 12.5Â±0.5(284)â€• 6.5Â±0.3 (169)â€•8999 64 Â±2(74)â€• 8.2 Â±0.4(228)â€• 5.4 Â±0.03 (102)44 68 Â±1 (83) 5.8Â±0.2(145)Â° 3.9Â±0.1 (153)9633 66 Â±3 (80) 6.8Â±0.6(283)â€• 5.5Â±0.5 (110)7794A 62 Â±1 (77)â€• 7.8Â±0.2(205)â€• 4.5Â±0.4 (220)â€•7777 63 Â±1 (84) 11.4 Â±0.7 (215)â€• 6.6 Â±0.4 (138)3924A 64 Â±1 (77)â€• 8.4 Â±0.4 (300)â€• 4.4 Â±0.2 (94)3683F 47 Â±1(66)â€• 9.6 Â±0.4(240)â€• 3.1 Â±0.2 (277)â€•9618A2 52 Â±1 (67)â€• 10.7 Â±0.2 (255)â€• 8.3 Â±0.3 (266)â€•â€œ 51 Â±2(64)â€• 13.2 Â±0.4(338)â€• 9.3 Â±0.3 Statistically significantly different from the respective controls (p < 0.05). as compared to values observed in the livers of sham conditions requires the study of metabolite pools and the operated rats. use of isotopes (5, 6). The present determinations of en zyme activities point out the behavior of the enzymes as DISCUSSION potential indicators of the reprogramming of gene expres sion. A major objective of this study was to throw light on the Comparison of Kinetic Properties and Organ Distribu biological behavior of transaldolase and tnansketolase by tion of Transaldolase and Transketolase. The results pre examining the organ distribution and kinetic properties of sented appear to be the 1st systematic survey of transaldo these enzymes in alterations of gene expression, such as in lase and tnansketolase activities in different rat tissues and starvation and differentiation, in induced normal prolifena the first set of kinetic curves analyzing and comparing these tion (regenerating liver), and in different degrees of malig enzyme activities in normal liver and in rapidly growing nancy. These experiments were also based on considena hepatoma. Among the 10 tissues examined, the highest tion of the role of pentose phosphate pathways in mamma transaldolase activities were found in thymus and intestinal han liver (1, 10, 12, 18, 35). In the hepatic cell, glucose 6- mucosa, tissues that carry out the most active nucleic acid phosphate might be routed from glycolysis into the oxida biosynthesis among those examined. The lowest activities tive pathway to pentose phosphates. In this process, were observed in skeletal muscle and in heart, where need NADPH is generated by the action of the glucose 6-phos for nibose 5-phosphate production is low. These obsenva phate and 6-phosphogluconate dehydnogenases. The pen tions are in line with the postulated role of transaldolase in tose phosphate may be recycled into glycolysis chiefly by channeling fructose 6-phosphate into pentose phosphate the action of transaldolase and transketolase. This pathway production (3, 35). In contrast, the transketolase activities in provides NADPH and a pool of pentose phosphates. If ni thymus and intestinal mucosa were not higher than in the bose 5-phosphate is utilized for punine biosynthesis, both liver. oxidative and nonoxidative branches of the pentose phos Kinetic studies on liver transaldolase indicated K,,'s for phate pathway might channel primarily into nibose 5-phos fructose 6-phosphate and erythnose 4-phosphate of 0.35 phate production (Chart 9). It has been suggested (3, 11, 14) and 0.13 mM, respectively. From the hepatic concentrations that in the mammalian cells both the oxidative and nonoxi of these substrates (9), one may estimate from the substrate dative pathways might proceed in the direction of nibose 5- curves that, at a hepatic tissue concentration of fructose 6- phosphate synthesis. When the utilization of nibose 5-phos phosphate of 0.060 mM and erythnose 4-phosphate, 0.004 phate declines, the pentose phosphates could be recycled mM, the extrapolated activities of transaldolase would be through transketolase and transaldolase. The precise deter about 42 or 11 @moIe/hr/g,respectively. At full substrate rnination of the functioning of these pathways under various saturation, under the optimal conditions of our assay sys

3194 CANCER RESEARCH VOL. 36

I- TRANS C,. KETOLASE 0. C,)

x F- F- 0. 0 1< F- 0 1 0 0. 0 I 0. I 0 I F- z 0.

I F

.@ G L U C 0 N E 0 G S N I C P A T H W A Yâ€”â€”â€”â€” Chart 9. Behavior of key enzymes of glycolysis, gluconeogenesis, and pentose phosphate pathways in hepatomas. R-5-P, ribose 5-phosphate; Ru-5-P, ribulose 5-phosphate: 6-PG OH, 6-phosphogluconate dehydrogenase; Xu-5-P, xylulose 5-phosphate; 6-PG, 6-phosphogluconate: 5-7-P. sedoheptulose-7- phosphate; E-4-P, erythrose 4-phosphate; G-6-P, glucose 6-phosphate; G-6-P OH, glucose-6-phosphate dehydrogenase: HK, hexokinase; PHI, phospho hexoisomerase; PFK, phosphofructokinase: F-6-P, fructose 6-phosphate: F-1,6-P, fructose 1,6-diphosphate; GA-3-P, glyceraldehyde 3-phosphate: PK, pyruvate kinase, OA, oxaloacetate; FOPase, fructose diphosphatase; G-6-Pase, glucose-6-phosphatase; P cxylase, pyruvate carboxylase; PEP, phosphoenol pyruvate : PEP CK, phosphoenolpyruvate carboxykinase. tem, the transaldolase activity was approximately 340 enzymes were glucokinase, phosphofructokinase, pynuvate @moles/hr/g. Thus, the extrapolated enzyme activities at kinase (34), and 2 of the enzymes of the oxidative pathway, tissue concentrations are at about 3 to 15% of those ob glucose-6-phosphate and 6-phosphogluconate dehydno served under optimal assay conditions. With the use of a genases (29). This pattern contrasts sharply with the behav similar extrapolation when the transketolase activity at sub ion of the gluconeogenic enzymes, such as glucose-6-phos strate saturation was approximately 300 zmoles/hn/g, the phatase,which instarvationincreasedon were maintained activity would be approximately 10 to 18 @moles/g/hn at the in near normal range in the average liven cell. That the hepatic pentose phosphate level of 0.060 mM (9). These enzymes transaldolase and transketolase do depend on potential in vivo enzymic capacities would be still several insulin, in part at least, is supported by observations that orders of magnitude higher than the activities of the overall their activities decreased in diabetes and were restored to synthetic pathway of DNA production (32, 35). Whereas the normal range by insulin administration (2). concentrations of these substrates in tumors of the hepa Behavior of Transaldolase and Transketolase Activities toma spectrum are not yet known, the kinetic studies in this in Differentiation. The low activities of the 2 enzymes in the paper indicate that the properties of transaldolase and 1st postnatal week and the slow increase up to the normal transketolase are similar in normal and neoplastic liver. levels during development are similar to the pattern of activ Since enzyme activity was proportional to the amount of ities of glucose-6-phosphate and 6-phosphogluconate de enzyme extract added , it may be assumed that alterations in hydrogenases and glucoki nase, phosphofructokinase , and the measured enzyme activities reflect changes in the tissue pyruvate kinase. All these enzymes depend for their biosyn concentrations of transketolase and tnansaldolase. Deter thesis, in part at least, on insulin (25), and the plasma mination of the enzyme amount by immunological means insulin level during differentiation correlates closely with should provide an independent measure of these enzyme the activities of these enzymes (32). The pattern of develop concentrations; these are not yet available. mental change in these enzyme activities is in sharp con Behavior of Transaldolase and Transketolase Activities trast to that of the gluconeogenic enzymes and of the en In Starvation. The decrease during starvation of the enzyme zymes of pynimidine synthesis and catabolism (32). activities of the nonoxidative pathway of pentose phosphate Selective Biological Advantage that the Increased Tran production parallels the decline in protein concentration in saldolase Activity Might Confer to the Cancer Cell. The the supennatant fluid, where these enzymes were measured. results in Table 3 indicated that transaldolase activity was The decline of activities of these enzymes in starvation may increased in all the liver tumors, irrespective of the growth relate to the decrease of plasma insulin level in fasting (29), mateof the neoplasms. Tnansketolase activity exhibited no since other enzymes that depend on insulin, in part at least, trend. Since the concentration of protein in most tumors for their biosynthesis also decreased in starvation. Such was decreased, the increase in transaldolase activity in the

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1976 American Association for Cancer Research. P. Heinrich et al. hepatornas indicates a preferential maintenance of the pro were not altered in the 24-hr regenerating liver (30). duction of this enzyme protein in the transformed cells. General Applicability of Reprogramming of Gene In examining the behavior of enzymes in the oxidative Expression as Manifested in Increased Transaldolase Ac branch of pentose phosphate synthesis, it was observed tivity. The transaldolase activity was increased in 10 differ that glucose-6-phosphate dehydrogenase was increased in ent lines of transplantable rat hepatomas (Table 3). In this all liver tumors, whereas 6-phosphogluconate dehydrogen laboratory, increased transaldolase activity was recently ob ase showed no relationship with proliferation rate (35). In served in human primary hepatomas (28), in transplantable liver, glucose-6-phosphate dehydrogenase is the rate-limit kidney tumors in rats, and in 2 primary kidney tumors in ing enzyme for the oxidative pathway. Transaldolase is not humans (G. Weber, A. C. Jackson, and F. Goulding, to be known to be rate limiting in the nonoxidative pathway, but it published). Further studies are needed to ascertain whether might function as a rate-limiting enzyme in liver when fruc an increased transaldolase activity is an integral pant of the tose 6-phosphate is the substrate, because the hepatic con neoplastic transformation in all types of neoplasms. centration of the other reactant, erythrose 4-phosphate, is Biological Significance of Malignant Transformation very low (9). In support of a possible nate-limiting role for linked Alterations in Gene Expression. Studies conducted transaldolase is the observation that the enzyme activity is with the molecular correlation concept as a conceptual and high in intestinal mucosa and in thymus, which are actively experimental tool resulted in gaining a degree of insight engaged in cell renewal and thus should have a high nibose into the biochemical strategy of the cancer cell (26, 30, 31). 5-phosphate requirement. Our demonstration of increased glucose-6-phosphate dehy Since, in the standard assay, transaldolase activity was pro drogenase and transaldolase activities present in all hepa portionate with the enzyme amount, it is assumed that the tomas, irrespective of growth rate, indicates that the repro increased enzyme activity in the hepatomas represents in gramming of gene expression in the malignant transfonma creased enzyme concentration. Such a conclusion, reached tion is linked with an increase in the expression of the by enzyme kinetic studies, should be confirmed by inde activity of these 2 key pentose phosphate-synthesizing en pendent immunological evidence. This evidence is available zymes. There is an array of key enzymes in carbohydrate, for glucose-6-phosphate dehydrogenase by the demonstra pynimidine, and DNA metabolism that in the spectrum of tion both kinetically and through immunotitration that the hepatomas correlates with the increase in growth rate and increased activity present in liver tumors was due to in the degrees of malignancy. Such enzymes include hexoki creased enzyme concentration (22). The increased potential nase, phosphofructokinase, pyruvate kinase (26), nibonu for the operation of the direct oxidative pathway has been cleotide reductase (8), thymidine kinase (8), DNA polymer corroborated by isotope investigations in these hepatomas ase (19), and others which represent reprogramming of (23); the elucidation of the precise contribution made by the gene expression that is linked with the progression in the nonoxidative pathway requires further work. degrees of neoplasia (26). In this laboratory recently there Since in all the hepatomas there is an increased activity of were discovered 10 transformation-linked alterations in key the 2 enzymes involved in the channeling of glucose 6- enzyme activities, and they all heighten the capacity for the phosphate (glucose-6-phosphate dehydnogenase) and fruc channeling of precursors to strategic biosynthetic proc tose 6-phosphate (transaldolase) into pentose phosphate esses (30). Thus, the increased glucose-6-phosphate dehy biosynthesis, these alterations in gene expression should drogenase and transaldolase activities enhance the poten increase the potential for ribose 5-phosphate biosynthesis tial for routing glycolytic metabolites into nibose 5-phos and might confer selective advantages to the cancer cell. phate biosynthesis (35). The increase in all hepatomas of Specificity to Neoplasia of the Increase in Transaldo the activity of glutamine 5'-phosphoribosyl-l-pyrophos lase Activity. The increased transaldolase activity was pres phate amidotnansferase (EC 2.4.2.14) (20) and the concur ent in all the hepatomas, but in the 24-hr regenerating liven, rent decrease in the activities of xanthine oxidase (EC which has a growth rate similar to that of the rapidly grow 1.2.3.2) (21) and unicase (EC 1.7.3.3) (30) should provide an ing hepatomas (26), there was no alteration in this enzyme increased potential for punine biosynthesis. The elevated activity. The transaldolase activity in the rapidly growing activities of adenyloscuccinate synthetase (EC 6.3.4.4) (15), differentiating liver was lower than in the liver of adult adenylosuccinase (EC 4.3.2.2), and IMP dehydnogenase (EC normal rats. Since transaldolase activity was elevated even 1.2.1.14) (16, 30) should provide a step up in the capacity for in the very slowly growing tumors, the increased activity of the utilization of punines on the biosynthesis of adenine and this enzyme across the hepatoma spectrum does not reflect guanine nucleotides (30, 31). The increased UDP kinase (EC an increase in growth rate alone, but it appears to be char 2.7.4.6) activity should yield an enhanced potential for UTP actenistic of the neoplastic transformation. and, consequently, for RNA and DNA biosynthesis (36). The lack of alteration in transaldolase activity in regener These transformation-linked increases in the activities of ating liver sharply distinguishes it from the elevations in the the key enzymes of pentose phosphate biosynthesis, purine regenerating liver for dTMP kinase, dTMP synthase, dCMP production and utilization, and UTP biosynthesis indicate deaminase, nibonucleotide reductase, DNA polymerase, an integrated reprogramming of gene expression that IMP dehydrogenase, and the increase in the incorporation should confer selective biological advantages to the neo of thymidine into DNA (for review, see Refs. 26 and 31). The plastic cells. activities of the other 4 enzymes that are increased in all the Since glucose-6-phosphate dehydrogenase and transal hepatomas (glucose-6-phosphate dehydrogenase, UDP ki dolase are at the fountainhead of directing glycolytic pre nase, adenylosuccinate synthetase and adenylosuccinase) cursors into nibose S-phosphate biosynthesis, these 2 en

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1976 American Association for Cancer Research. Increased Transaldolase Activity in Hepatomas zymes, along with the other transformation-linked enzymes, Measurement with the Folin Phenol Reagent. J. Biol. Chem., 193: 265- 275, 1951. should be of particular interest in the design of selective 18. Novello, F., and McLean, P. The Pentose Phosphate Pathway of Glucose chemotherapy. Metabolism: Measurement of the Non-Oxidative Reactions of the Cycle. Biochem. J., 107: 775-791 , 1968. 19. Ove, P., Laszlo, J., Jenkins. M. D., and Morris, H. P. Increased DNA Polymerase Activity in a Series of Rat Hepatomas. Cancer Res.,29: 1557- 1@61,1969. REFERENCES 20. Prajda, N., Katunuma, N., Morris, H. P., and Weber, G. Imbalance of Purine Metabolism in Hepatomas of Different Growth Rates as Ex pressed in Behavior of Glutamine PRPP Amidotransferase (Amidophos 1. Baquer, N. Z. , Cascales, M. , Teo, B. C., and McLean, P. The Activity of phoribosyltransferase, EC 2.4.2.14), Cancer Res., 35: 3061-3068, 1975. the Pentose Phosphate Pathway in Isolated Liver Cells. Biochem. Bio 21. Prajda, N., and Weber, G. Malignant-transformation-linked Imbalance: phys. Res. Commun., 52: 263-269, 1973. Decreased xanthine Oxidase Activity in Hepatomas. Federation Euro 2. Baquer, N. Z., Sochor, M., and McLean, P. Hormonal Control of the pean Biochem. Soc. Letters, 59: 245-249, 1975. â€˜Compartmentation'of the Enzymes of the Pentose Phosphate Pathway 22. Selmeci, L. E., and Weber, G. Increased Glucose 6-Phosphate Dehydro Associated with the Large Particle Fraction of Rat Liver. Biochem. Bio genase Concentration in Hepatoma 3924A: Enzymic and Immunological phys. Res. Commun., 47: 218â€”226,1972. Evidence. Federation European Biochem Soc. Letters, 61: 63-67, 1976. 3. Bonsignore, A., Pontremoli, S., Mangiarotti, G., De Flora, A., and Mangi 23. Sweeney, M. J., Ashmore, J., Morris, H. P., and Weber, G. Comparative arotti, M. A Direct Interconversion: o-Fructose 6-Phosphate Sedoheptu Biochemistry of Hepatomas. IV. Isotope Studies of Glucose and Fructose lose 7-Phosphate and D-xylulose 5-Phosphate Catalyzed by the Enzymes Metabolism in Liver Tumors of Different Growth Rates. Cancer Res., 23: Transketolase and Transaldolase. J. Biol. Chem. 237: 3597-3602, 1962. 995-1002, 1963. 4. Brand, K. Transaldolase. In: H. U. Bergmeyer (ed), Methoden der Enzy 24. Weber, G. Effect of Six-day Starvation on Rat Liver Lactic Dehydrogen matischen Analyse, Vol. 1, Ed. 2, pp. 674-678. Weinheim, Germany: ase Activity. J. Nutr., 71: 156â€”158,1960. Verlag Chemie, 1970. 25. Weber, G. Integrative Action of Insulin at the Molecular Level. Israel J. 5. Brand, K., and Deckner, K. Quantitative Relationship between the Pen Med . Sci., 8: 325-343, 1972. toss Phosphate Pathway and the Nucleotide Synthesis in Ascites Tumor 26. Weber, G. The Molecular Correlation Concept: Recent Advances and Cells. Z. Physiol. Chem., 351: 711-717, 1970. Implications. In: H. Busch (ed), The Molecular Biology of Cancer, pp. 6. Brand, K., Deckner, K., and Musil, J. Enzyme Pattern of the Pentose 487-521 . New York: Academic Press, Inc., 1974. Phosphate Pathway in Ascites Tumor Cells and the Effect of Nucleoside 27. Weber, G., and Cantero, A. Studies on Hormonal Factors Influencing Triphosphates on Its Enzyme Activities. Z. Physiol. Chem., 351: 213-220, Hepatic Glucose-6-Phosphatase. Endocrinology, 61: 701-712, 1957. 1970. 28. Weber, G., Malt, R. A., Glover, J. L., Williams, J. C., Prajda, N. , and 7. Dc La Haba, G., Leder, I. G., and Racker, E. Crystalline Transketolase Waggoner, C. D. Biochemical Basis of Malignancy in Man. In: Biological from Bakers' Yeast: Isolation and Properties. J. Biol. Chem., 214: 409-' Characterization of Human Tumors, pp. 60-72. Amsterdam: Excerpta 426,1955. Medica Foundation, Publishers, 1976. 8. Elford, H. L., Freese, M., Passamani, E., and Morris, H. P. Ribonucleo 29. Weber, G., Morgan, C. R., Wright, P. 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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1976 American Association for Cancer Research. Behavior of Transaldolase (EC 2.2.1.2) and Transketolase (EC 2.2.1.1) Activities in Normal, Neoplastic, Differentiating, and Regenerating Liver

Peter C. Heinrich, Harold P. Morris and George Weber

Cancer Res 1976;36:3189-3197.

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