[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 SEPTEMBER1976 3189 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 37°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).