The Formation of Cytidine Nucleotides and RNA Cytosine from Orotic Acid by the Novikoff Tumor in Vitro*
Total Page：16
File Type：pdf, Size：1020Kb
The Formation of Cytidine Nucleotides and RNA Cytosine from Orotic Acid by the Novikoff Tumor in Vitro* HAROLD O. KAMMEN t AND ROBERT B. HURLBERT (Biochemistry Department, The University of Texas M. D. Anderson Hospital and Tumor Institute, Houston f~5, Tezas) The utilization of orotic acid for the formation the amino group of cytosine compounds in ma- of RNA ~uracil has been extensively studied in in- malian tissues may be derived from glutamine, in- tact animals, tissue slices, and cell suspensions (re- stead of ammonia. Eidinoff et al. (9) have shown viewed in Refs. 5, 31). The mechanism of the the glutamine antagonist, DON, to depress sig- utilization, via the initial conversion of orotic acid nificantly the incorporation of orotic acid into the to uridine nucleotides and the incorporation of uri- nucleic acid cytosine of rat tissues, both in vivo dine nucleotides into polynucleotides, has been and in tissue slices. Salzman et al. (33) have found partially described in intact animals (13, 17) and the amide group of glutamine to be the source of studied in detail in enzyme systems of microbial, the amino group of the polynucleotide cytosine avian, and mammalian origin (18, 36) (reviewed in HeLa cell cultures. In a preliminary report (31) in Refs. 33, 38, 39). The concomitant conversion we have described a soluble enzyme system, of orotic acid to RNA cytosine has usually been present in both the Novikoff rat tumor and rat observed in intact cell systems, in vivo and in liver, capable of the conversion of orotic acid to vitro, but is smaller, more variable, and apparent- uridine nucleotides and of the amination of the ly of secondary nature as compared with the con- uridine nucleotides to cytidine nucleotides. ATP version to RNA uracil (1, 7, 13, 14, 16, 18, 33, 34). and glutamine were required for the amination Lieberman (35) has demonstrated the direct step, guanosine nucleotides appeared to serve as amination of uridine triphosphate to form cytidine cofactors, and DON was inhibitory. triphosphate; the enzyme system, purified from In the work reported here, the objective was to Escherichia coli, required ammonia and ATP. The establish whether the enzymatic steps observed in existence of this reaction suggests strongly that the soluble system were part of the normal path- the uridine nucleotides and cytidine nucleotides way for the conversion of orotic acid to RNA cyto- are involved as intermediates in the conversion of sine in surviving Novikoff tumor tissue. The ef- orotic acid to RNA cytosine. fects of glutamine, other amino donors, glutamine Evidence has more recently been offered that antagonists, and uridine were examined in intact * Aided by grants from the American Cancer Society cell preparations of the tumor incubated with (P-146) and from the Anna Fuller Fund. With the assistance orotic acid-6-C TM. The labeling of the cytidine of Joyce Danielson Miller, Alice F. Wheeler, and Audrey F. nucleotides and RNA cytosine was compared with Posey. This work forms part of the dissertation submitted by the labeling of the uridine nucleotides and RNA Harold O. Kammen to the Department of Chemistry and uracil. To provide further perspective, the inter- Chemical Engineering, Stanford University, for the degree of Doctor of Philosophy, 1959. relationships among all the major "acid-soluble" pyrimidine metabolites of orotic acid were also I" Present address: Department of Pharmacology, Yale University, New Haven, Connecticut. examined. These metabolites (cytidine nucleo- ' The abbreviations used are: RNA (ribonucleieacid), DNA tides, cytidine, cytosine, uridine nucleotides, uri- (deoxyribonucleic acid), ATP (adenosine-5'-triphosphate), dine, and uracil) were separated from cold per- UMP (uridine-5'-phosphate), CMP (cytidine-5'-phosphate), chloric acid extracts of the tissue by ion-exchange DON (6-diazo-5-oxo-L-norleucine), and azaserine (0-diazo- and paper chromatographic methods. A brief re- acetyl-~serine). "Uridylic acid" and "cytidylic acid" represent the mixed $'- and 3'-monophosphates of uridine and cytidine, port has appeared (30). respectively, derived by alkaline hydrolysis of RNA. "Uridine nucleotides" and "cytidine nucleotides" represent the groups MATERIALS AND METHODS of nucleoside-5'-phosphate and -5'-pyrophosphate monomeric Materials.--The orotic acid-6-C 14 was pur- compounds extractable by cold percMoric acid and hydrolyz- chased from Tracerlab, Inc., the uniformly C 14- able to UMP and CMP, respectively. labeled cytidine from Schwarz Laboratories, Inc., Received for publication February 9, 1959. and the amino acids (L-isomers)from the Cali- 654 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1959 American Association for Cancer Research. KAMMEN AND HURLBERT--Formation of RNA Cytosine 655 fornia Corporation for Biochemical Research. The in a 13-ml. tube, and the precipitated tissue was re- azaserine bore the Parke, Davis and Company lot extracted with 0.4 N perchloric acid; the combined number 2342 X 35, and the DON s bore the Sloan- extracts (7-8 ml. total volume) constituted the Kettering Institute number SK 13,835 ab. The "cold perchloric acid extract." The precipitate was azaserine and DON materials were checked by further washed with two 8-ml. portions of cold published spectrophotometric criteria and found 0.4 ~r perchlorie acid, and 3 ml. of cold 12 per cent to be 59 per cent azaserine (3) and 87 per cent NaC1 was added. The suspension was neutralized DON (8), respectively. Other chemicals were com- carefully with a minimum volume of NaOH solu- mercial products of reagent quality. tion (phenol red indicator) and heated at 100~ C. The Novikoff transplantable hepatoma s (27) for 30 minutes to extract the sodium nucleate (12, was carried in 350-500-gm. female Holtzmann 16). The precipitate was centrifuged and re- Sprague-Dawley rats and was transplanted every extracted for a few minutes with 1 mi. of hot 10 5th day by the intraperitoneal injection of 100- per cent NaC1. The combined salt extracts were 300 mg. of fresh, minced tissue suspended in treated with 2.5 volumes of ethanol at - 10~ C. for 0.15 M NaC1. The tumor for the experimental work several hours to precipitate the sodium nucleate, was taken on the 5th day, when the rats were which was then washed twice with 5-ml. portions usually severely hemorrhagic. The large mass of of cold 80 per cent ethanol and dissolved in 2.0 ml. tumor in the greater omentum and a smaller mass of water. The sodium nucleate solution was brought adjacent to the spleen were removed, chilled, to 0.3 s NaOH and incubated overnight at 88 ~ C. cleaned of the larger blood clots, passed through The DNA was precipitated by the addition of 1 a stainless steel mesh in a large syringe, and sus- volume of 0.4 N perchloric acid (cold); the acid pended in the Krebs-Ringer phosphate-glucose solution and a 1-ml. wash with 0.1 N perchloric (KRPG) medium (see below). The free blood cells acid constituted the "RNA" fraction. The DNA were removed by suspending and sedimenting the was dissolved in 2 ml. of 0.1 s NaOH, the solution tumor tissue 2 or 3 times in the KRPG medium, was heated at 80 ~ C. for 5 minutes (I3), neutral- with centrifugation at about 100 X g for 1-2 ized, and treated with 2.5 volumes of cold ethanol. minutes at 2 ~ C. The packed tissue was diluted The precipitated sodium deoxyribonucleate was with the KRPG solution to a 30 per cent suspen, washed twice with 5-ml. portions of 80 per cent sion (v/v). The composition of the modified ethanol and dissolved in 3.0 pal. of water for KRPG solution was: 0.125 M NaCl, 0.015 M KCI, analysis. 0.003 ~ CaCb, 0.00067 M MgCl2, 0.006 M sodium Analysis and chromatography.--The RNA and DNA con- phosphate, pH 7.4, and 0.030 M D-glucose. In some tent of the sodium nucleate solution and DNA content of the cases the tissue was washed and suspended in the sodium deoxyribonucleate solution were determined. In addi- Krebs-Ringer salts solution, with separate addi- tion, the RNA and DNA remaining in the tissue residue after extraction of the nucleates were estimated by analysis of an tion of the phosphate and glucose to the incuba- extract made with hot 0.4 N perchloric acid. All data have been tion flasks. calculated to include and correct for this residual RNA and Incubation and preparation of samples.--Ali- DNA (less than 10 per cent of the total). There was no de- quots of the tissue suspension were pipetted into tectable cross-contamination of the "RNA" and the sodium the incubation flasks; the relative amount of deoxyribonucleate fractious. The analyses for RNA were made by a modified orcinol procedure (19) and for DNA by Burton's tissue in each flask was estimated by analyses for modification of the Dische diphenylamine procedure (6). DNA at the end of the incubation. The incubation (Commercial yeast RNA and sperm DNA were used as mixtures were prepared and kept at 0 ~ in 50-ml. standards.) Erlenmeyer flasks, flushed with 95 per cent Oz-5 Each "RNA" fraction was brought to 1 N perchloric acid by the addition of 11.5 N perchloric acid, heated at 100~ C. for 80 per cent CO~ before !and after the addition of the minutes to destroy the purine nucleotides, and neutralized at tissue. During the incubation the stoppered flasks 0 ~ C. with the minimum volume of concentrated KOH. The were shaken about 100 cycles per minute at 38 ~ C. KC104 precipitate was washed twice by centrifugation with in a water bath. 'The pH of the medium decreased several ml. of cold water; the solution and washes were chro- to nearly 6.8 during the course of the incubations.