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Biochemical Screening of Pyrimidine Antimetabolites I

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Biochemical Screening of Pyrimidine Antimetabolites I Biochemical Screening of Pyrimidine Antimetabolites I. Systems with Oxidative Energy Source* JOSEPHE. STONEANDVANR. POTTER (McArdle Memorial Laboratory, Medical School, University of Wisconsin, Madison, Wis.} Orotic acid (uracil-4-carboxylic acid) has been the livers were quickly excised and placed in a chilled bath of isotonic saline solution. A 20 per cent homogenate in chilled shown under both in vivo and in vitro conditions, 0.25 M sucrose was made with the use of an all-glass Potter- in both microorganisms and mammals, to be a pre Elvehjem homogenizer, and Ihis homogenate was centrifuged cursor of the mono-, di-, and triphosphate pyrim- at approximately 600 g for 10 minutes to remove nuclei and idine nucleotides, the pyrimidine coenzymes, and whole cells. also of the pyrimidine moieties in both ribo- and Portions of 0.8 ml. of the cytoplasmic liver fraction were deoxyribonucleic acid (5, 6, 11, 12, 13, 15-17). placed in 25-ml. Erlenmeyer flasks, each of which contained 2.20 ml. of a reaction mixture which had the following com This study was prompted by the current position: progress in the study of nucleic acid metabolism Potassium glutamate 15.0 Amóles and its relationship to tumor growth and metabo Potassium fumarate 6.0 /«moles lism. The purposes of this research are the Potassium pyruvate 15.0 /iiiiole-s selection of agents as possible components of KHjPO, 15.0 /¿moles sequential (9) or concurrent blocks (4), the clari MgCl2 9.0 /uñóles fication of biochemical pathways, and the develop Ribose-5-phosphate (RSP) 6.0 /¿moles Uridine-5-monophosphate (UMP-5') 1.0 /imoles ment of concepts and technics in a biochemical Orotic acid-6-C" 0.3 /tmoles approach to pharmacological research. Adenosine triphosphate (ATP) 3.0 /uñóles The data presented here represent the results of A sufficient quantity of sucrose to make an isotonicity equiva a screening program with a biochemical system lent to 0.25 Msucrose. which converts orotic acid to the uridine nucleo A sufficient quantity of water to make a volume of 2.20 ml. tides. Normal rat liver cytoplasm furnished the The pH was adjusted to 7.2-7.4 with 0.2 N KOH. All flasks enzyme source, and oxidative substrates furnished were in duplicate. Drugs were added to the system in a drug: orotic acid ratio of 10:1 (3.0 /IMof the drug to 0.3 /IMof the the source of energy for the reactions. A primary orotic acid).2 Whenever possible, the drugs were added in objective of the study was to ascertain which aqueous solution; however, when necessary, acetate solutions drugs could be shown to act upon one or more or propylene glycol solutions were utilized. In these cases, ap steps in the conversion of orotic acid to the uridine propriate control flasks were added. The reaction mixtures were incubated with constant agita nucleotides. tion for 20 minutes at 30°C.The reactions were terminated by the addition of 1.50 ml. of 1.5 N perchloric acid to each flask, MATERIALS AND METHODS which resulted in a final perchloric acid concentration of 0.5 N. It has been demonstrated that under in vitro The resulting mixture was centrifuged at 600 g for 10 minutes conditions orotic acid is converted to uridine and the pellet discarded. The acid-soluble supernatant liquid was neutralized with 2.0 NKOH with phenol red as an internal nucleotides by preparations of normal rat liver indicator, and the neutral solution was then allowed to stand (6, 10). The latter observation is the basis of the for 10 minutes at —¿10°C.for the precipitation of residual test system used in this study. The drugs were potassium perchlorate. After recentrifugation, the supernatant added to a standardized system, and the con fluid was placed upon 5-cni. Dowex l (X10) aniónexchange resin columns (14). version of orotic acid to the uridine nucleotides The Chromatographie columns were submitted to the elu- was measured and compared with the conversion tion scheme which is summarized in Table 1. in appropriate controls. Fractions 1, 2, and 3 were not examined and were discarded White rats of either sex,1weighing 150-450 gm., were used (see below). Fractions 4, 5, 6, and 7 were read at 260 and 275 in this study. The animals were sacrificed by decapitation, and 1Obtained from the Holtzman-Rolfsmeyer Rat Company, * This work was supported in part by a grant (No. C2409) Madison, Wis. from the National Cancer Institute, National Institutes of 2The orotic acid used in this study was labeled in carbon 6. Health, United States Public Health Service. Its specific activity was 4.1 X 10* counts/min/mg (5.75 /imoles). Approximately 213,000 counts/min were added in Received for publication July 10, 1956. 0.3 /imoles added to each flask. 1033 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1956 American Association for Cancer Research. 1034 Cancer Research nan in a Beckman model DU spectrophotometer. The radio 4) UMP-5' + ATP ^ UDP + ADP (3). activity of the samples was determined by plating a 0.2-ml. 4a) UDP + ATP ^±UTP + ADP (3). aliquot from each tube upon weighed aluminum planchets 4b) UTP-> UDPX (UDP conjugates) and which were inscribed with a circle 3.8 sq. cm. in area. Approxi mately 0.05 ml. of a 0.1 per cent solution of gelatin was used to UTPXS (3). spread the solution upon the inscribed area.8 The samples were 5) The ATP renewal is accomplished by oxida- then dried under a heat lamp, and the determination of radio tive phosphorylation with the Krebs cycle activity was done in gas-flow proportional counters.4 It should be emphasized that to insure activity of the system fortified at three points. all glassware should be washed in sulfurie-nitric acid cleaning solution and glass-distilled water. All reagents should also be In testing drugs against the standard system, prepared with glass-distilled water. the following points were taken as criteria of the activity of the system: Under control conditions, the standard sys 1. The total radioactivity in the UTP-UTPX tem5 converts an average of 7 per cent (range, 3-12 complex is the most reliable index to the effect of per cent among all preparations used) of the orotic the drug upon the conversion of orotic acid to the acid to the uridine nucleotides. The UTP-UTPX6 uridine nucleotides. In this study it has been taken complex accounts for 80-90 per cent of the orotic as the major criterion. acid converted. 2. The quantity of nucleotide in the UTP- TABLE1* SCHEDULEOFELUENTSANDFRACTIONSCOLLECTED Fraction Ml/total No. samples/ no. Eluent fraction fraction Compounds eluted 1 HjO 30 Nucleosides, bases 2 3.0 N formic acid t 150 Adenosine monophosphate (AMP), UMP-5', orotic acid 3 0.3 N ammonium formate! 30 Orotic acid and ADP 4 0.4 N ammonium formate 30 Mixed uridine coenzymes (UDPX's) 5 0.7 N ammonium formate 30 Uridine diphosphate (UDP) 6 l. 0 N ammonium formate 30 Uridine triphosphate (UTP) and UTPX 7 1.25 N ammonium formate 30 ATP * AHsubstances eluted in théChromatographiescheme presented in Table 1 were characterized by either chromatography of known samples or reference to previous research done in this laboratory (3). t All formic acid solutions were at pH 2.O. ÃŽAllammonium formate solutions were at pH 5.O. The known reactions included in the test sys UTPX fraction is an indication of the trans- tem may be summarized as follows: phosphorylase ability of the system in the presence 1) R5P + ATP -> PRPP (phosphoribosylpyro- phosphate) + AMP (7). specific activity of 1200 counts/min//*mole. However, this sys 2) Orotic acid + PRPP —¿Â»Orotidylicacid (orotic tem was unsatisfactory as a test object because of the extreme ly low percentage of orotic acid converted to the uridine acid ribotide) + PP (pyrophosphate) (8). nucleotides and because of excessive variation between dupli 3) Orotidylic acid -> UMP-5' + C02 (8). cate flasks (20-40 per cent). It was found that, with this and all subsequent systems investigated, the major uridine derivative 3The gelatin solution facilitates spreading of the plated produced was uridine triphosphate (UTP). This fraction con solutions; it also equalizes the crystallization of the ammonium tains small amounts of a uridine nucleotide (Dr. L. Hecht, formate present (M. J. Johnson, personal communication). unpublished data) whose specific activity is about 60 per cent 4After being counted, the plates were again weighed, and of that of the UTP. This compound has not been completely correction was made for self-absorption. The assistance of Dr. characterized and is designated as UTPX because it follows UTP on the chromatogram. The radioactivity of the UTP- Charles Heidelberger and his staff in radioactivity determina UTPX fraction has been adopted as the major criterion of tions is gratefully acknowledged. activity. 6The first system investigated consisted essentially of the The second system was further modified by the deletion of same ox¡dativesubstrates employed in the standard system, the fructose and the reduction of the UMP-5' pool to 1.0 including 3.0 jumólesof ATP, a relatively large amount of /¿mole.Thissmall amount of UMP-5' serves as a marking pool, UMP-5' (6 /¿moles),and a pool of 3.0 /¿molesoforotic acid and its phosphorylation to UTP-UTPX serves as a measure which contained 0.3 /¿molesofthe labeled compound. These ment of the transphosphorylative ability of the preparation. substances were incubated with 0.8 ml. of 20 per cent rat liver Amounts of UMP-5' greater than 1 /¿moledepress the con homogenate for 20 minutes at 30°C.Under these conditions, version of orotic acid into the uridine nucleotides by actual the conversion of orotic acid to the uridine nucleotides could inhibition, aside from the effects due to competition for high not be demonstrated; however, under these conditions the energy phosphate and the dilution of the radioactivity.
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