[CANCER RESEARCH 39, 3909-3914, October 1979] 0008-5472/79/0039-OOOOS02.00 Glutamine-Phosphoribosylpyrophosphate Amidotransferase (Amidophosphoribosyltransferase, EC 2.4.2.14) Activity in Normal, Differentiating, and Neoplastic Kidney

Noemi Prajda,1 Harold P. Morris,2 and George Weber3

Laboratory for Experimental Oncology, Indiana University School of Medicine, Indianapolis. Indiana 46223 [N. P., G. W.I. and the Department of Biochemistry. Howard University College of Medicine. Washington. D. C. 20001 ¡H.P. M.]

ABSTRACT transplantable hepatomas to kidney tumors and further confirm the conclusion that the behavior of amidotransferase activity is The behavior of glutamine-phosphoribosylpyrophosphate a transformation-linked alteration. The elevation in the amido amidotransferase (amidophosphoribosyltransferase, EC transferase activity should increase the capacity of the de novo 2.4.2.14), the first enzyme committed to de novo purine bio purine-biosynthetic pathway, and this enzymic imbalance synthesis, was elucidated in normal, differentiating, and neo- should confer selective advantages to the noeplastic cells. plastic rat kidneys. Enzymic activities were measured spectro- photometrically in the 100,000 x g supernatants prepared INTRODUCTION from 20% tissue homogenates containing 0.25 M sucrose and 1 HIM MgCI2. Amidotransferase4 is the rate-limiting enzyme and the first Kinetic studies were carried out on the amidotransferase in enzyme committed in the de novo purine-biosynthetic pathway. the crude supernatant from rat kidney cortex and renal cell This enzyme catalyzes an irreversible reaction between PRPP carcinoma (MK-3) so that under optimum standard assay con and glutamine to form phosphoribosylamine, L-glutamate, and ditions only the enzyme amount would be the limiting factor. P¡.Previous work in this laboratory has demonstrated that the The kinetic results indicated that certain properties of the activity of amidotransferase increased 2- to 4-fold in all the amidotransferase in kidney and in the renal tubular cell tumor chemically induced, transplantable rat hepatomas examined were similar. In normal kidney cortex and in the kidney tumor (MK-3), the pH optimum was 7.2 to 8.5; the Km's for glutamine and that the behavior of this enzyme was transformation linked and characteristic of neoplasia (13, 17). The purpose of this were 2.0 and 1.7 mw, respectively, and for MgCI2 the Km was investigation was to determine the applicability to renal neopla 1 HIM. For phosphoribosylpyrophosphate of the kidney and tumor enzyme, the Sos's were 0.9 and 0.5 HIM, respectively. sia of the pattern of reprogramming of gene expression in purine metabolism, as expressed in the behavior of the ami The amidotransferase in the kidney had sigmoid kinetics for dotransferase activity. phosphoribosylpyrophosphate, but in the renal tumor the en Previous studies in this laboratory and in other centers led to zyme exhibited a hyperbolic curve. The 50% feedback inhibi the formulation of the molecular correlation concept, which tions by adenosine 5'-monophosphate of the amidotransferase resulted in the identification of an ordered imbalance in the in normal kidney and in the tumor, studied in vitro, were 9.5 activities of the key enzymes involved in purine, pyrimidine, and 9.3 mM, respectively. A standard assay was developed for carbohydrate, ornithine, and membrane cyclic adenosine 3': kidney cortex and renal cell carcinoma in which good propor 5'-monophosphate metabolism in a spectrum of rat hepatomas tionality was achieved over a 120-min incubation period and of different growth rates (2, 3, 16, 20, 22-24, 29, 30, 34, 35). with different amounts of enzyme added. In analyzing this reprogramming of gene expression, the mo The amidotransferase specific activities in rat thymus, testes, lecular correlation concept that has been the conceptual and bone marrow, gut, kidney cortex, spleen, lung, brain, adipose experimental guide in this laboratory, grouped in Class 1 those tissue, heart, and skeletal muscle were 271, 259, 173, 167, alterations in which the discriminants were linked with the 128, 96, 70, 64, 49, 24, and <1.0%, respectively, of that of degrees in the expression of malignancy in the hepatoma the liver. spectrum (22-24). In addition to these progression-linked dis In three lines of chemically induced, transplantable renal criminants, there are alterations in gene expression that are tumors (MK-1, MK-2, and MK-3), the amidotransferase specific linked with the malignant transformation per se, since they activities were increased 2.2- to 2.7-fold over that of the kidney occurred in all liver tumors, even in the slowest growing, most cortex of normal control rats. During development, the enzyme differentiated hepatomas (22-24). These transformation-linked activities in the average kidney cell of 1-, 7-, 30-, and 40-day- alterations (Class 2) include increased activity of key enzymes old rats were 57, 71, 79, and 114% of adult activity. that channel hexoses into pentose phosphate biosynthesis These studies indicate the applicability of the pattern of through the oxidative pathw. /, glucose-6-phosphate dehydro- enzymic imbalance in purine metabolism first discovered in genase (EC 1.1.1.49), and the nonoxidative pathway, transal-

' Visiting Assistant Professor. Permanent address: National Institute of Oncol dolase (EC 2.2.1.2) (32). These enzymic indications are in line ogy, Budapest, Hungary. with isotopie evidence for an increased potential for pentose 2 Recipient of USPHS Grant CA-10729. phosphate formation in the hepatomas (19). Ribose 5-phos- 3 Recipient of USPHS Grants CA-13526 and CA-05034. To whom requests for reprints should be addressed, at the Laboratory for Experimental Oncology, * The abbreviations used are: amidotransferase, glutamine-phosphoribosyl- Indiana University School of Medicine, Indianapolis, Ind. 46223. pyrophosphate amidotransferase (amidophosphoribosyltransferase. EC Received April 5. 1979; accepted June 26, 1979. 2.4.2.14): PRPP. phosphoribosylpyrophosphate.

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phate ¡schanneled into purine biosynthesis by PRPP synthe- assay system was worked out which was an adaptation of the tase (EC 2.7.6.1), the activity of which was increased in liver technique of Prajda eíal. (17) to the kinetic conditions of the tumors (6). The transformation-linked increase in the activity of rat kidney cortex and renal tumor systems. The reaction mix amidotransferase in all rat hepatomas examined was an impor ture contained, in final concentrations: PRPP (5 HIM); glutamine tant observation because it suggested the operation of an (20 HIM); MgCI2 (1 5 HIM); Tris-HCI buffer (50 HIM, pH 7.2); and increased capacity for purine biosynthesis in the presence of KF (1.0 mw). The determinations were carried out at 37°and a decrease in the activity of the rate-limiting enzyme of purine were stopped at 0, 30, and 45 min of incubation by boiling for catabolism, xanthine oxidase (18). exactly 5 min. The tubes were centrifuged, and in the clear The present work reports that, in a spectrum of chemically supernatant the concentration of glutamic acid that had formed induced, transplantable rat kidney tumors, the amidotransfer in the presence of PRPP was determined. This was carried out ase activity increased 2- to 3-fold. This investigation indicates by coupling to the reduction of NAD+ in the presence of added the applicability of the enzymic imbalance observed in purine excess glutamate dehydrogenase in a Gilford 2400 recording metabolism in hepatomas to renal tumors. spectrophotometer. The blanks contained the identical reaction mixture without PRPP, and the activity was recorded simulta MATERIALS AND METHODS neously. The enzyme activity was provided by the difference between the rate observed in the full reaction mixture and that All rats were housed in individual cages in air-conditioned of the blank which contained no added PRPP. Under these rooms that were illuminated daily from 6 a.m. to 7 p.m. Purina experimental conditions, no PRPP-independent ammonia was laboratory chow and water were available ad libitum. Rats were liberated from glutamine, indicating that glutaminase activity killed between 9 and 10 a.m. Examination of stomach contents did not interfere with this assay. Through careful studies, the at autopsy confirmed that all rats were well fed during the night various kinetic constants were established for the crude en before death. zyme in kidney cortex and renal tumors, and a standard assay Tumor-bearing and Control Animals. Renal cell tumors (MK- was designed where the enzyme activity was proportionate 1, MK-2, and MK-3) were implanted bilaterally in a s.c. position with the amount of tissue equivalent added and the time in male Buffalo rats. These rat kidney tumors were induced in elapsed. The relevant kinetic studies will be described below. Buffalo rats that were fed a diet containing 4'-fluoro-4-biphen- The PRPP was purchased as sodium salt, approximately 88% ylacetamide. Histologically, they were well-differentiated ade- pure, from Sigma Chemical Co., St. Louis. Mo. The glutamine nocarcinomas composed of generally uniform cells resembling and glutamate dehydrogenase were also obtained from Sigma. kidney tubular cells, and they appeared to deviate only slightly Protein concentration was assayed by the biuret reaction (5) from normal kidney tissue. The induction, transplantation, and with a standard curve, using crystalline bovine serum albumin. some biological and histological characteristics of these tumors Expression and Evaluation of Results. Amidotransferase were reported previously (9, 15). All three are classified as activity was calculated in jumol of glutamate formed per hr per slow-growing tumors, and they would relate to the slow-grow mg of protein (specific activity), per g (wet weight) of tissue, or ing part of the spectrum of hepatomas of different growth rates per cell. The cell counts were made as described previously (14, 15). Tumors MK-1 and MK-2 had a very slow growth rate and were expressed as cellularity calculated in millions of (10 to 11 months old when assayed); Tumor MK-3 was some nuclei per g, wet weight, of tissue (25). what faster (4 to 5 months old). Kidney cortex from normal The results were subjected to statistical evaluation by means animals of the same strain, sex, age, and weight was compared of the f test for small samples. Differences between means with the tumors. giving a probability of less than 5% were considered to be Amidotransferase Activity in Different Rat Organs. For significant. investigation of organ distribution of the amidotransferase ac tivity, male ACI/N rats (180 to 220 g; HaríanIndustries, Cum RESULTS berland, Ind.) were used. Differentiating Kidney. Pregnant Wistar rats were pur Comparison of Kinetic Conditions of Amidotransferase in chased from HaríanIndustries, and the litters were allowed to Normal Kidney Cortex and Renal Tumor. In order to establish remain in the same cage with the mother for 18 days after that linear kinetics operates in the crude system used in the birth; then each rat was placed in an individual cage. amidotransferase measurements and that under the assay con Preparation of Kidney and Tumor Extracts. The rats were ditions the enzyme activity measured reflected the enzyme stunned, decapitated, and exsanguinated. Tumors and kidney concentration, the properties and behavior of the amidotrans cortex were quickly excised and placed in beakers imbedded ferase were compared in extracts from normal rat kidney cortex in crushed ice. The neoplasms were dissected free of hemor- (Buffalo rats) and Renal Tumor MK-3 (carried in Buffalo rats). rhagic, necrotic, and nontumorous material. The careful selec Effect of Glutamine Concentration. Chart 1 shows the effect tion of the viable tumor tissue parts is important in ensuring the of glutamine concentration on amidotransferase activity in nor comparability of neoplasms of different age groups and tumor mal rat kidney and in Renal Tumor MK-3. The enzyme activity lines. For the enzyme assay, 20% homogenates were prepared in both normal and neoplastic tissue was saturated at a gluta from normal kidney cortex and tumor tissues in 0.25 M sucrose mine concentration of approximately 10 mw, and it was not containing 1 mvi MgCI2 (17). The homogenate was centrifuged inhibited by excess levels of glutamine up to concentrations of at 100,000 x g for 30 min at 2°in a Beckman Model L3-50 44 mW. The affinity of amidotransferase to glutamine for kidney and Tumor MK-3 yielded apparent Km's of 2.0 and 1.7 rriM, preparative centrifuge. The resulting clear supernatant was used for the assays. respectively. Assay of Amidotransferase Activity. A standard enzyme Effect of Magnesium Concentration. The enzyme activity of

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1979 American Association for Cancer Research. Amidotransferase Activity in Rat Kidney both normal and neoplastic tissue was saturated at a MgCI2 were large enough to be reliable and readily repeatable. The concentration of about 10 rriM. The enzyme activity in normal results indicated that under such initial velocity-recording con kidney and in the tumor was not inhibited by excess levels of ditions the pattern of affinity curves was similar to the one given MgCI2 up to concentrations of 20 mM. The affinity of amido- in Chart 2. The affinity of amidotransferase to PRPP for kidney and tumor yielded Sos's of 0.9 and 0.5 mM, respectively. transferase to MgCI2 for both tissues yielded a Km of 1.0 mM. Effect of PRPP Concentration. The effects of PRPP on Effect of pH. Both kidney and tumor amidotransferase ex kidney and tumor amidotransferase activities are compared in hibited a pH optimum at approximately 7.2 to 8.5 (Chart 3). Chart 2. The enzymic activity of both normal control and The shape of the pH curve was similar in the 2 tissues. neoplastic tissue was saturated at a PRPP concentration of Proportionality of Amidotransferase Activity with Incuba approximately 2 mM. The substrate curve for the kidney yielded tion Time and Enzyme Amount. Through systematic kinetic sigmoid kinetics, whereas the one in the tumor exhibited only studies, a standard assay was established for determination of slight, if any, sigmoidicity. These observations were made at kidney cortex and renal tumor amidotransferase activities. In optimum concentrations of all other reactants with variations the standard assay, good proportionality was achieved with only of the PRPP levels and of stopping the enzyme assay at length of reaction time over a 120-min incubation period and 0, 30, or 45 min of incubation. In order to ascertain the behavior with amount of enzyme added (Chart 4). The standard assays of the saturation curves at initial enzyme velocity concentra of kidney and tumor were carried out at pH 7.2 at 37°with the optimum reaction mixture conditions given in "Materials and tions, these experiments were also carried out in enzyme assay systems in which the reaction was stopped at 15-min incuba Methods." Further information is provided in the legends for tion, this being the shortest incubation period when the ab- Charts 1 to 4. sorbance differences between blank and experimental systems Comparison of Activities of Amidotransferase in Different

28.

H" TUMOR MK-3 (_>x < e Chart 1. Comparison of the effect of glutamine concentration on 16 — activity of amidotransferase in kidney cortex and Renal Tumor MK- K — i 3. The standard assay described in "Materials and Methods" was U =i used for both tissues, varying only the glutamine concentration. For 12- the normal kidney and tumor, the assay was carried out on super natant fluid of 20% homogenate (0.4 and 0.2 ml. respectively). The standard assay concentration of glutamine is 20 mM. KIDNEY 4 — K •2.0mM

\ ! I I I \ 12 16 20 24 28 32 36 40 44 GLUTAMINE (m M)

TUMOR MK-3 S0 5 = 0.5 mM

Chart 2. Comparison of the effect of PRPP concentration on activity of amidotransferase in kidney cortex and Renal Tumor MK-3. The assay conditions were as described in "Materials and Methods," varying only the PRPP concentra tion. Enzyme concentrations used were as in Chart 1. The standard assay concentration of PRPP is 5 mM.

KIDNEY Sg 5 = 0.9 mM

III! I 1 4567 9 10 PRPP (mM)

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liver. In contrast, the amidotransferase activities in the adipose tissue, heart, and skeletal muscle were much lower than those observed in the liver. Feedback Inhibition by AMP of Amidotransferase Activity in Normal Kidney Cortex and Neoplastic Tissue. The amido transferase activity in Renal Tumor MK-3 and normal kidney was similarly sensitive to the inhibitory action of AMP. In kidney cortex, the 50% inhibition was 9.5 mM, and in kidney tumor tissue it was 9.3 rriM. Comparison of Amidotransferase Activity in Normal Kid ney Cortex and in Tumors MK-1, MK-2, and MK-3. In order to examine the behavior of this enzyme in kidney neoplasia, the activity was studied in 3 renal tumors of different growth rates (Table 2). Tumor MK-3 has a faster growth rate (4 to 5 months) than do Tumors MK-1 and MK-2 (10 to 11 months). The protein concentration in the 100,000 x g supernatant in the normal kidney cortex of Buffalo rats was 71.3 ±1.3 (S.E.) mg protein per g, wet weight, of tissue. In the tumors, protein content was slightly increased. The amidotransferase activity in normal kid ney was 8.9 ± 0.4 /imol/hr/mg protein x 10~2, and the specific activity was significantly increased in all 3 tumors, irrespective of the growth rate of the neoplasms. The amido transferase activities were significantly elevated in all 3 kidney tumors to 2.2- to 2.7-fold over the values observed in the pH (Final) normal kidney of the control rats of corresponding strain, sex, Chart 3. Comparison of the effect of pH on amidotransferase activity in kidney age, and weight. The enzyme activities per average cell were cortex and renal tumor. The standard assay was performed as described in Chart 1. varying only the pH of the reaction mixture. Acetate buffer was used for pH to 6.0; above this value, Tris buffer was used. The final pH of the reaction Table 1 ure is given. ratsTwentyOrgan distribution of amidotransferase activity in normal % homogenates were prepared, and enzyme assays were carried out moreAMIDOTRANSFERASEoc« as outlined for the liver in "Materials and Methods." The results of 4 or MM1momo*300ooC1,1,1,1,TUMOR tissues for each organ from adult ACI/N rats are tabulated. Enzyme activities per mg protein are to be multiplied by the exponential given to arrive at the absolute values and are also expressed as percentages of those observed in liver.Amidotransferase ACTIVITY MK-3 / activityfimol/hr/mg pro- Protein concen- /// (mg/g)Thymus tein x 10~2 % of liver tration

(u,mol/hrx10~2)M 18.2 271 57.7 Testis 17.4 259 41.8 Bone marrow 11.8 173 51 .0 Gut 11.2 167 32.9 ff Liver 6.7 100 101.6 Kidney 8.6 128 78.0 Spleen 6.4 96 77.0 Lung 4.7 70 79.0 Brain 4.3 64 33.6 Adipose tissue 3.3 49 27.2 Heart 1.6 24 54.9 Skeletal muscle <1.0 46.7Table <1.0

KIDNEY1^^J®""*"

1 ' 1 ' \ ' 1 ' 1 2 Comparison of amidotransferase activity in rat kidney cortex and renal tumors 0 246 8 10 The data are means ±S.E. of 5 rats in each group with percentages of PROTEIN (mu)pooled corresponding control kidney values. The activities per mg protein are to be Chart 4. Proportionality of amidotransferase activity with amount of kidney multiplied by the exponential given to arrive at the actual values. Assay conditions cortex and renal tumor MK-3 added. The standard assay described in "Materials are described in "Materials and Methods." and Methods" was used, varying only the amount of supernatant. The standard contentTissuesKidney Protein activity/imol/hr/mg enzyme assay contained approximately 6 mg normal kidney and 3 mg tumor homogenate equivalent (0.4 and 0.2 ml. respectively). pro tein x10~28.9

control ±1.3 ±0.4 Organs of the Adult Rat. Table 1 shows that the enzyme 273a Tumor MK-1 80.0 ±1.2 112100 24.3 ±0.8 273a specific activity was highest in organs of high cell renewal, the Tumor MK-2 78.5 ±1.9 24.3 ±0.8 220a thymus, testes, and bone. The activities in kidney cortex, Tumor MK-3mg/g71.3 85.1 ±2.5%100 119Amidotransferase19.6 ±1.0%100 spleen, lung, and brain were in the same range as those of the " Significantly different from values of kidney cortex of normal rats (p < 0.05).

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1979 American Association for Cancer Research. Amidotransferase Activity in Rat Kidney significantly increased to 2- to 4-fold, respectively, in the renal preparation exhibited Michaelis-Menten kinetics in contrast to tumors (not shown). The existence of noncovalently bound the S-shaped kinetics observed in the crude liver extract (21). inhibitors or activators in the liver or tumor supernatant fluid It is important that under the same conditions the crude kidney was excluded by estimation of the enzyme activity in combined tumor enzyme was more rapidly saturated with low concentra supernatants of liver and tumor; only the sum of the separate tions of PRPP than was the kidney enzyme (Chart 2). activities was found. Selective Biological Advantages That the Increased Ami Amidotransferase Activity of Differentiating Kidney. Since dotransferase Activity Might Confer to the Cancer Cell. The the amidotransferase activity was markedly increased in renal results presented in Table 2 indicate that the amidotransferase tumors, it was of interest to examine the enzyme activity in the activity markedly increased in all 3 kidney tumors, irrespective rapidly growing differentiating kidney. Table 3 shows that at 1 of the growth rate of the neoplasms. Since kinetic studies day after birth the kidney cellularity was 1.7-fold higher than showed that in the standard assay the amidotransferase activity that in the kidneys of the adult animals (60-day-old, 200 to 220 was proportionate to enzyme amount, it is assumed that the g). The cellularity slowly decreased to normal levels over a 2- increased enzyme activity in the kidney tumors represents month period. The protein content in the average cell in the increased enzyme concentration. Recent studies brought evi newborn rat was 36% of the adult value, and it slowly rose, dence through 2 independent methods: by measurement of the reaching normal values in 60 days. The amidotransferase spe enzyme activity under optimum enzyme conditions where linear cific activity in the newborn rat kidney was 1.4-fold the activity kinetics were obtained with the enzyme activity and amount; observed in the adult rat, and it declined to normal values and by immunotitration, in which the amidotransferase amount during 60 days. When the kidney cellularity was taken into increased in hepatoma 3924A (21 ). The elevation in the activity consideration, the amidotransferase of the newborn rat was of this key purine-synthesizing enzyme should provide an in 57%; in the 7-, 30-, and 40-day-old animals, activities were creased potential for purine biosynthesis for the cancer cells. 71, 79, and 114%, respectively, of those observed in the This enzymatic indication is in good agreement with studies of kidney of normal adult rat. Wheeler ef al. (33) on the behavior of the overall metabolic pathway by isotope assessment of the synthetic and catabolic DISCUSSION pathways of purine metabolism. Since in the renal tumors there is more amidotransferase activity, this alteration in gene Comparison of Kinetic Parameters of Amidotransferase in expression should increase the purine-synthetic potential and Kidney Cortex and Neoplastic Tissue. The kinetic studies, might confer selective advantages to the cancer cells. including those demonstrating the optimum conditions for the General Applicability of Reprogramming of Gene Expres assay in crude extracts and those on proportionality of activity sion as Manifested in Increased Amidotransferase Activity. with enzyme amount and reaction time (Charts 1 to 4), showed The experimental results reported in Table 2 reveal that ami the applicability of the kidney enzyme assay to the renal tumor dotransferase activity increased in 3 renal tumor lines. Earlier enzyme under the conditions of the present investigation. work indicated that there was an elevated amidotransferase The kinetic conditions indicated that the pH optima and the activity in all examined rat hepatomas, irrespective of their apparent Km's for glutamine and Mg2+ were similar in kidney of growth rates (13, 17). Further study is required to ascertain control normal rats and in Tumor MK-3. For glutamine, both whether an increased amidotransferase activity is an integral enzymes were half-saturated at approximately 2.0 to 1.7 mw. part of neoplastic transformation in other types of neoplasia. This glutamine level was in the range of that observed in normal Evidence from the literature thus far supports this concept, kidney in rat by freeze-clamp determination (1.28 to 1.78 mw) inasmuch as an increased amidotransferase activity was also (7). reported in the spleen of mice bearing Friend leukemia (4), in The sigmoid kinetics for PRPP in the kidney in the standard chemically induced transplantable colon mouse tumor (27), enzyme assay (45-min incubation) was also present when initial and in primary hepatomas (28) and renal cell carcinomas (26) rates were measured (15-min incubation). The S-shaped affin in humans. Since the increase in the activity of the amidotrans ity curve to PRPP of the kidney enzyme is in agreement with ferase occurred in all 3 kidney tumors irrespective of growth other reports in the literature on crude enzyme preparations rate, this observation is in line with the interpretation that this (1,8, 17, 37). On the other hand, it has recently been shown alteration in gene expression is linked with the malignant trans in this laboratory that the highly purified liver amidotransferase formation. A minor rise in amidotransferase activity was ob-

Table 3 Behavior of amidotransferase in differentiating kidney The data are means ±S.E. of 4 or more rats in the various groups with percentages of corresponding control adult kidney values in parentheses. The activities are to be multiplied by the exponential given to arrive at the actual value. Assay conditions are described in "Materials and Methods."

Protein concentration Amidotransferase activity

protein x Postnatal(days)1 age 106630 x x10~246.8 87.4 x 10 IO'210.9 10~80.8 x ±4.0(172)" ±2.1 (62)a ±0.3 (36)a ±1.0(143)" ±0.1 (57)° 7 590 ±4.0(160)" 50.9 ±0.0 (68)a 8.6 ±0.0 (42)a 11.8 ±0.3(155)" 1.0 ±0.0 (71)" 594 ±3.0(162)" 11.8 ±0.9 (58)a 30 70.0 ±3.4 (93) 9.0 ±0.4(118) 1.1 ±0.1 (79)" 40 408 ±2.4(111) 75.2 ±0.0(100) 18.4 ±0.3 (90) 8.6 ±0.3(113) 1.6 ±0.1 (114) 60 (adult control)Cellularity/g367 ±1.5(100)mg/g 75.0 ±3.1 (100)mg/cell 20.4 ±0.8(100)(imol/hr/mg 7.6 ±0.3(100)(imol/hr/cell1.4 ±0.0(100) 1Significantly different from the respective control adult kidney values (p < 0.05).

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1979 American Association for Cancer Research. N. Prä/daef al. served in the newborn kidney, but only on a protein basis; nant rat tissues. J. Biochem., 766. 1-10, 1977. when the high cellularity of the postnatal kidney was taken into 12. Jackson, R. C, Morris, H. P., and Weber. G. Enzymes of the purine ribonucleotide cycle in rat hepatomas and kidney tumors. Cancer Res., 37: consideration, it was apparent that the activity of this enzyme 3057-3065, 1977. in the average cell was less than 60% of that in the adult 13. Katunuma. N., and Weber. G. Glutamine phosphoribosylpyrophosphate kidney. In contrast, the amidotransferase activity in the kidney amidotransferase: increased activity in hepatomas. FEBS Lett., 49: 53-56, 1974. tumors increased 2- to 3-fold above the normal values on a per 14. Lea, M. A.. Morris. H. P., and Weber, G. DNA metabolism in liver and kidney cell basis. Thus, the marked increase of amidotransferase tumors of different growth rates. Cancer Res.. 28. 71-74, 1968. 15. Morris, H. P.. Wagner. 8. P., and Meranre, D. R. Transplantable adenocar- activity appears to be characteristic of neoplastic transforma cinomas of the rat kidney possessing different growth rates. Cancer Res., tion. 30: 1362-1369, 1970. Biochemical studies have revealed that in kidney tumors a 16. 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- neoplastic program is displayed that is similar to that observed 1561. 1969. in liver tumors. Thus, as in hepatomas, in the renal tumors 17. Prajda, N.. Katunuma. N., Morris, H P.. and Weber, G. Imbalance of purine examined in this work the activities of the key gluconeogenic metabolism in hepatomas of different growth rates as expressed in behavior of glutamine-phosphoribosylpyrophosphate amidotransferase (amidophos- enzymes decreased, whereas those of the glycolytic enzymes phoribosyltransferase, EC 2.4 2.14). Cancer Res.. 35 3061-3068, 1975. increased (26). The incorporation of thymidine into DMA was 18. Prajda, N., Morris, H. P., and Weber, G. Imbalance of purine metabolism in hepatomas of different growth rates as expressed in behavior of xanthine increased (14) and the level of adenine nucleotides and the oxidase (EC 1.2.3.2). Cancer Res., 36: 4639-4646, 1976. concentrations of glycolytic intermediates were altered in the 19. Sweeney. M. J., Ashmore, J.. Morris, H. P., and Weber, G Comparative same direction in both kidney and liver tumors (31, 36). Studies biochemistry of hepatomas. IV. Isotope studies of glucose and fructose metabolism in liver tumors of different growth rates Cancer Res., 23: 995- in this laboratory have indicated that the activities of CTP 1002. 1963. synthetase (34), IMP dehydrogenase (10, 11), adenylosuccin- 20. Sweeney, M. J., Parton, J. W., and Hoffman. D H. Biosynthesis of uridine- 5'-monophosphate in rat liver and Morris hepatomas. Adv. Enzyme Regul.. ate synthetase, adenylosuccinase, and adenylate deaminase 12: 385-396, 1974. (10, 12) increased in both hepatic and renal neoplasms in the 21. Tsuda, M.. Katunuma, N., Morris, H. P., and Weber, G. Purification, prop rat. Recent work has shown that xanthine oxidase, the rate- erties, and immunotitration of hepatoma glutamine phosphoribosylpyrophos phate amidotransferase (amidophosphoribosyltransferase, EC 2.4.2.14). limiting enzyme of purine degradation, was low in all examined Cancer Res., 39: 305-311, 1979. transplantable hepatomas (18) and that it also decreased in the 22. Weber, G. The molecular correlation concept: recent advances and impli kidney tumors.5 While there may be differences in organ-spe cations. In: H. Busch (ed.). The Molecular Biology of Cancer, pp. 487-521. New York: Academic Press, Inc., 1974. cific characteristics of the metabolic imbalance in the various 23. Weber, G. Enzymology of cancer cells. Part 1. N. Engl. J. Med., 296: 486- types of tumors, there appears to be present a shared program 493. 1977. of biochemical alterations in various tumors that characterizes 24. Weber. G. Enzymology of cancer cells. Part 2. N. Eng. J. Med., 296. 541- 551, 1977. the enzymic and metabolic phenotype that is stringently linked 25. Weber. G., and Cantero. A. Studies on hormonal factors influencing hepatic with the malignant transformation. glucose-6-phosphatase. Endocrinology, 67: 701-712, 1957. 26. Weber. G., Goulding, F. J., Jackson, R. C.. and Eble, J. N. Biochemistry of human renal cell carcinoma. In: W. Davis and K. R. Harrap (eds.). 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3914 CANCER RESEARCH VOL. 39

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1979 American Association for Cancer Research. Glutamine-Phosphoribosylpyrophosphate Amidotransferase (Amidophosphoribosyltransferase, EC 2.4.2.14) Activity in Normal, Differentiating, and Neoplastic Kidney

Noemi Prajda, Harold P. Morris and George Weber

Cancer Res 1979;39:3909-3914.

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