Reversible Dissociation of a Carbamoyl Phosphate Synthase
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Proc. Nat. Acad. Si. USA Vol. 72, No. 5, pp. 1712-1716, May 1975 Reversible Dissociation of a Carbamoyl Phosphate Synthase-Aspartate Transcarbamoylase-Dihydroorotase Complex from Ovarian Eggs of Rana catesbeiana: Effect of Uridine Triphosphate and Other Modifiers (multienzyme complex/pyrimidine biosynthesis) RAYMOND J. KENT, RENG-LANG LIN, H. J. SALLACH*, AND PHILIP P. CQHENt The Department of Physiological Chemistry, University of Wisconsin Medical School, Madison, Wisc. 53706 Contributed by Philip P. Cohen, January 27, 1976 ABSTRACT Glutamine-dependent carbamoyl phos- associate under conditions which might reflect in vivo regula- phate synthase [ATP:carbamate phosphotransferase (de- phosphorylating), EC 2.7.2.91, aspartate transcarbamoylase tion. (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) and dihydroorotase (L-5,6-dihydroorotate ami- EXPERIMENTAL dohydrolase, EC 3.5.2.3), are copurified as a high-molecular- Materials. Gravid females of Rana catesbeiana were pur- weight complex from extracts of unfertilized eggs of Rana catesbeiana. UTP is required to maintain the integrity of chased from Mogul-Ed, Oshkosh, Wisc. Sodium [14C]bi- the complex during the last two purification steps. Re- carbonate (4.7 Ci/mol), obtained from New England Nuclear moval of the nucleotide results in dissociation of the com- Corp., was stored at -20° as a solution in 10 mM N-2-hy- plex. Based on sedimentation behavior in glycerol gra- droxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) buffer, dients, the dissociated carbamoyl phosphate synthase 50 was has an apparent molecular weight of 260,000 ±4 20,000 and pH 8.0, at uCi/ml. The solution diluted with unlabeled that of dihydroorotase is estimated at 280,000 At 20,000. As- sodium bicarbonate to prepare a 50 mM, 2.5 MuCi/ml stock partate transcarbamoylase is broadly distributed over the solution for the routine assay of CPSase. The stock solution gradient. The addition of ATP, 5-phosphoribosyl-1-pyro- was stored in 0.5 ml portions at -20°. Sepharose 6B was phosphate, Mg++, or inorganic phosphate to the disso- purchased from Pharmacia Fine Chemicals. ATP, UTP, PP- ciated complex results in the appearance of a peak of as- partate transcarbamoylase activity with an apparent mo- ribose-P, carbamoyl phosphate, calcium phosphate gel, horse lecular weight of 110,000 4 10,000. Incubation of a mixture liver alcohol dehydrogenase (ADHase) and beef liver catalase of the dissociated enzymes with UTP and Mg++ leads to (CATase) were obtained from Sigma Chemical Co. The proce- their reassociation into the high-molecular-weight com- dure used for the preparation of ornithine transcarbamoylase plex. and the definition of units are those described by Marshall and Recent studies have shown that, in at least two mammalian Cohen (5). Triton X-100, 2,5-diphenyloxazole (PPO), and species, the first three enzymes of pyrimidine biosynthesis, 1,4-bis[2-(5-phenyloxazole) ]-benzene (POPOP) were pur- i.e., carbamoyl phosphate synthase (CPSase), aspartate trans- chased from Research Products International Corp. All other carbamoylase (ATCase), and dihydroorotase (DHOase), exist reagents were commercial preparations of the highest purity as a macromolecular complex. Ehrlich ascites cells possess available. such a complex with a moleular weight of 800,000-850,000 Enzyme Assays. For the routine assay of CPSase, the incu- (1, 2). The extensively purified CPSase-ATCase-DHOase bation system contained in 0.2 ml: 50 mM Hepes buffer, pH complex from rat liver has a sedimentation coefficient of 27 S (approximately 900,000 daltons) (3). All of the enzymes for the de novo biosynthesis of pyrimi- dines are found in soluble extracts of egg-ovary preparations of 0 3000i Rana catesbeiana (4). Therefore, it was of interest to determine if CPSase, ATCase, and DHOase exist as a macromolecular 0- 300- S- complex in bullfrog egg-ovary preparations and whether such a complex, if it exists, could be induced to dissociate and re- 020001 OD Abbreviations: CPSase, glutamine-dependent carbamoyl phos- 4200C) phate synthase (II) [ATP:carbamate phosphotransferase (de- phosphorylating), EC 2.7.2.9]; ATCase, aspartate carbamoyl- 0o transferase (transcarbamoylase) (carbamoylphosphate:iaspar- I-20- 100 , tate carbamoyltransferase, EC 2.1.3.2.); DHOase, dihydrooro- tase (i-5,6-dihydroorotate amidohydrolase, EC 3.5.2.3.); CATase, catalase (hydrogen peroxide:hydrogen peroxide oxidoreductase, EC 1.11.1.6); ADHase, alcohol dehydrogenase (alcohol:NAD oxidoreductase EC 1.1.1.1.); Hepes, N-2-hydroxyethylpiperazine- 0 20 30 40 50 60 N'-2-ethanesulfonic acid. FRACTION NUMBER * Deceased September 14, 1974. FIG. 1. Profile of CPSase, ATCase, and DHOase activities t Author to whom reprint requests should be addressed. and of Am after chromatography of Fraction D on Sepharose 6B. 1712 Downloaded by guest on September 29, 2021 Proc. Nat. Acad. Sci. USA 72 (1975) Pyrimidine Synthesis Complex: Reversible Dissociation 1713 TABLE 1. Purification of CPSawe, ATCae, and DHOase from ovarian eggs Volume, Protein, /Amol/hr product Fold purification Purification step ml mg CPSase ATCase DHOase CPSase ATCase DHOase A First supernate 540 27,000 610 48,100 4,640 1 1 1 BCTABsupernate 525 23,600 470 39,400 4,620 0.9 0.9 1.1 C Ammonium sulfate 73 4,520 580 46,000 5,810 5.7 5.7 7.5 DCalciumphosphategel 6.9 250 340 18,200 2,670 60.2 40.9 62.1 E Sepharose 6B 24 34 130 9,700 1,620 169 160 277 F Glycerol gradient 20 8.5 68 5,000 770 354 330 527 7.4; 25 mM ATP; 30 mM MgCl2; 10 mM glutamine (or 100 DHOase activity was determined by measuring carbamoyl mM ammonium chloride); 20 mM ornithine; 10 mM sodium aspartate formation in a system (0.5 ml) containing 0.1 M ['4C]bicarbonate (0.1 IACi); 10 units of ornithine transcar- potassium phosphate buffer, pH 6.8, 2 mM L-dihydroorotate, bamoylase; and enzyme. The reaction was initiated by the and enzyme. The incubation was terminated after 30 min at addition of enzyme and terminated after 30 min at 370 by the 370 by the addition of 0.1 ml of 6 N HCl. Carbamoyl aspartate addition of 0.04 ml of 6 N HCJ. Acid-stable radioactivity was was determined by the method of Prescott and Jones (7). determined by pipetting 0.2 ml of the acidified incubation mix- Protein was determined by the method of Lowry et al. (8) ture into a scintillation vial and directing a stream of air over with bovine serum albumin as the standard. the vial for at least 30 min in a ventilated hood. The sample volume was then brought up to 1 ml with water and analyzed Enzyme Purification. The purification of the complex in- in a liquid scintillation spectrometer (Packard) after the addi- volves treatment with hexadecyltrimethylammonium bromide tion of 10 ml of a solution consisting of 5.5 g/liter of PPO and (CTAB), ammonium sulfate precipitation, adsorption on 0.1 g/liter of POPOP in toluene: Triton-X-100 (2:1, v/v). A portion of the unacidified assay mixture without enzyme was measured immediately after incubation to standardize the 0.9- assay. In a modification of the CPSase assay which was used in certain experiments, 0.2 ml of the acidified reaction mixture 60- was pipetted onto 1.5 X 2.5 cm rectangles of glass fiber paper 0.6 - 9 and dried for 4 hr at 50-60° in a vented drying oven. The glass 0 fiber rectangles were then analyzed in 10 ml of a counting mix- 0 30- ture of 4 g/liter of POP and 0.05 g/liter of POPOP in toluene. 12- The assay system for ATCase contained in 0.2 ml: 75 mM 0 mM E Tris acetate buffer, pH 8.5, 10 aspartate, 10 mM carbam- 3 oyl phosphate, and enzyme. Because of its instability in solu- a w tion, carbamoyl phosphate was not dissolved in the incubation 2 buffer until just prior to assay. Incubation was terminated after 0 20 min at 370 by the addition of reagents for the colorimetric crI- 60- determination of carbamoyl aspartate according to the method 0 0.6- of Hunninghake and Grisolia (6). 30 0.3 z CATose ADHose 0 ,o0 - Top a0l. 60 x FRACTION NUMBER 1 & E Q6- FIG. 3. Effect of removal of UTP and Mg++ on the CPSase- -S ZD ATCase-DHOase complex. Enzyme from Fraction E was con- O 5F 0 0 centrated to 6 ml by ultrafiltration. One-half of the enzyme solu- cr !-) LA. I-l tion was placed on a 1.8 X 90 cm column of Sephadex G-50 equil- ibrated with 0.01 M Hepes, pH 7.4, 10 mM glutamine, 10 mM I-CQ3- 3- sodium bicarbonate, 1 mM dithiothreitol, and 10% glycerol (v/v) ) 2 (Buffer A). The other half was chromatographed on an identical o column except that 0.5 mM UTP and MgCl2 were included in the , buffer (Buffer B). Material in the void volume from each column was concentrated to 2-3 ml by ultrafiltration, and 0.5 ml portions 0 8 12 16 20 were centrifuged on glycerol gradients. The gradient buffers were Bottom FRACTION NUMBER Top the same as the buffers for the Sephadex G-50 columns. (A) UTP FIG. 2. Profile of activities of CPSase, ATCase, and DHOase and Mg++ absent during chromatography and centrifugation. after centrifugation of Fraction E on a preparative glycerol (B) 0.5 mM UTP and MgC12 present during chromatography and gradient. centrifugation. Downloaded by guest on September 29, 2021 1714 Biochemistry: Kent et al. Proc. Nat. Acad. Sci. USA 72 (1975) TABLE 2. Effect of modifiers of CPSase activty on CPSase, 60 ATMase, and DHOase activities % of control activity 40 CPSase CPSase Addition to (5 mM (25 mM 1 assay mixture MgATP) MgATP) ATCase DHOase 20 None (control) 100 100 100 100 10 mM MgATP - 81 92 2.5 mM MgUTP 15 74 100 89 0.