[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. OCTOBER 1979 3909 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1979 American Association for Cancer Research. N. Prä/daef al. 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