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/ ) 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 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 (ADHase) and beef liver catalase of the dissociated 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 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 (transcarbamoylase) (carbamoylphosphate:iaspar- I-20- 100 , tate carbamoyltransferase, EC 2.1.3.2.); DHOase, dihydrooro- tase (i-5,6-dihydroorotate , EC 3.5.2.3.); CATase, catalase (hydrogen peroxide:hydrogen peroxide , 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. 5 mM MgPP- ribose-P 560 107 85 84 60 10 mM Pi 320 90 78 5 mM Mg++ 160 100 86 92

40 A 50% ammonium sulfate suspension of fraction E was dialyzed for 4 hr against buffer A. The dialysate was diluted to 0.24 mg 1 of protein per ml with the same buffer. Control activities were: CPSase assayed with 5 mM MgATP, 0.17 Mumol/hr per ml; 20 CPSase assayed with 25 mM MgATP, 0.95 ,umol/hr per ml; ATCase, 120 smol/hr per ml; DHOase 3.8 ,umol/hr per ml. c a . calcium phosphate gel, chromatography on Sepharose 6B, and 0 0 a centrifugation on glycerol gradients. Eluate from the Sephar- 0 2 60 L. ose 6B column was used for the experiments described in this 9 paper. The detailed purification of the complex will be de- scribed elsewhere. A typical purification is outlined in Table 1. 40 0- 1 Glycerol Gradient Centrifugation. Linear glycerol gradients, 13.5-30.5% (v/v), were prepared in 17 ml cellulose nitrate Ul 0 tubes with a Beckman gradient former. The gradients con- 0 20 C)0 0 tained 0.01 M Hepes, pH 7.4, 10 mM glutamine, 10 mM co0 sodium bicarbonate, and 1 mM dithiothreitol. Samples 93 0.5 were over each The 0 0 (approximately ml) layered gradient. 0 0 gradients were centrifuged in a SW27 rotor at 26,000 rpm in a 2 x a Beckman L2-65B ultracentrifuge maintained at 4°. After 18 hr of centrifugation, the rotor was allowed to decelerate without braking. Fractions (0.85 ml) were collected from the bottom of A40 each centrifuge tube. 1 RESULTS

f 920 Enzyme Purification. CPSase, ATCase, and DHOase exist as a complex in egg-ovary of R. catesbeiana. The ratio of the three enzymatic activities was approximately the same throughout the purification (Table 1) and the activities of all 60 . three enzymes migrated as a single peak on a Sepharose 6B column (Fig. 1) and on a glycerol gradient (Fig. 2). The activ- ity of CPSase with 100 mM NH4Cl as a substrate was also monitored and was found to be the same as that with gluta- 440 - mine at each stage of purification. 1. The molecular weight of the complex is approximately 900,000 based on the sedimentation in glycerol gradients of 220 w bovine liver catalase with a molecular weight of 248,000 and

, ' . | adex G-50 column containing buffer A, and concentrated by ultra- .. Portions (0.5 ml) of the concentrated Sephadex G-50 0 8 12 2filtration.16 20 eluate were preincubated with various effectors of CPSase activity Top for 5 hr at 4°. The samples were then centrifuged on glycerol gradients that contained the same concentration of effectors pres- FRACTION NUMBER ent during preincubation. (A) control-no effectors present. (B) FIG. 4. Effect of modiifiers of CPSase activity on the complex 2.5 mM UTP and MgCl,. (C) 10 mM ATP and MgCl2. (D) 0.5 after removal of UTP. andI Mg++. Enzyme from Fraction E was mM PP-ribose-P and 1 mM MgC12. (E) 10 mM potassium phos- concentrated, freed of UTIP and Mg++ by passage through a Seph- phate, pH 7.4. Downloaded by guest on September 29, 2021 Proc. Nat. Acad. Sci. USA 72 (1975) Pyrimidine Synthesis Complex: Reversible Dissociation 1715

horse liver alcohol dehydrogenase, 83,000 (Fig. 2). The accu- 6O0 racy of the molecular weight of the complex, calculated by the method of Martin and Ames (9), is open to question in this instance because of the wide separation between the enzyme I 1.0 complex and the markers on the glycerol gradient. 44 Effect of UTP Removal on the Complex. Passage of the en- zyme complex (Fraction E) through a Sephadex G-50 column 0.5. 24 to remove Mg++ and UTP resulted in dissociation of the complex (Fig. 3A). ATCase activity was spread over much of the gradient, possibly reflecting an equilibration between subunits. CPSase migrated as a distinct peak with a molecular weight of 260,000 + 20,000. The molecular weight of the 6C DHOase was approximately 280,000 i 20,000. Because of the slight difference in molecular weights between DHOase and 1.0 - CPSase, it is unclear whether they are separate proteins or 4CDi remain together in a smaller cpmplex. When fraction E en- zyme was passed through a Sephadex G-50 column which in 0.5 included UTP and MgCl2 the buffer, the complex remained 2C) intact with no shift in its apparent molecular weight (Fig. 3B). Effect of Modifiers of CPSase Activity on the Dissociated 0c Complex. Compounds known to modify CPSase activity in a I 6 variety of eukaryotes also affect the activity of CPSase of R. 60 catesbeiana without pronounced effects on ATCase or DHOase 0 I- (Table 2). The activation of R. catesbeiana CPSase by PP- 0 1.0 ribose-P is similar to that described earlier for the CPSases 40 from mouse spleen (10), Ehrlich ascites cells (2), and adult rat E liver (3). These compounds affected the profile of enzyme a on as shown in 4. ATP no w activities glycerol gradients Fig. had 0.5 20 discernible effect on CPSase and DHOase peaks but caused a 0 new peak of ATCase activity to appear with an apparent I- molecular weight of 110,000. There was also a shoulder of 0 0 ATCase activity with higher molecular weight (Fig. 4C). PP- a 0 0 0 0 Ribose-P (Fig. 4D) and Pi (Fig. 4E) had the same effect as E I-- ATP. On the other hand, the presence of equimolar UTP and MgCl2 during preincubation and in the gradient resulted 1.0- in partial restoration of the high-molecular-weight complex 40 (Fig. 4B). There appeared to be an excess of CPSase and DHOase over that needed to reform the complex with ATCase so that residual peaks of CPSase and DHOase remained at 0.5 20 lower-molecular-weight positions. The ratio of activities of ATCase: DHOase: CPSase in fraction 11 (Fig. 4B) is 86:7: 1, which is similar to the ratio of activities in the final stages of purification (Table 1) (73: 11: 1). The molecular weight of the complex was calculated to be 800,000 based on sedimentation 60- in a glycerol gradient with CATase and ADHase as markers. Reassociation the There remained the 1.0 of Complex by UTP. 40- possibility that after removal of Mg++ and UTP by chroma- tography on Sephadex G-0 the three enzymes of the complex continued to be loosely associated and that the readdition of 0.5 20- scribed in the legend to Fig. 4. The profile of enzyme activities was ' f - essentially the same as in Fig. 4E. Fractions 15-19 from all gradients were pooled and a 50% . . ammonium. . . .sulfate. .~ suspension was prepared. Prior to use, the sus 0 8 12 16 20 pended protein was collected by centrifugation, dissolved in buf- Top fer A, and dialyzed overnight against buffer A in a continuous flow apparatus. Preincubation conditions and centrifugation are FRACTION NUMBER described in the legend to Fig. 4. (A) no effectors present. (B) 2.5 mM UTP and 2.5 mM MgC12. (C) 2.5 mM UTP and 5 mM FIG. 5. Reassociation off the complex. Potassium phosphate MgCI2. (D) 5 mM MgC12. (E) 2.5 mM UTP, 5 mM MgC12, and 10 (10 mM) was used to dissociiate the complex by the procedure de- mM potassium phosphate, pH 7.4.

Downloaded by guest on September 29, 2021 _, 1716 Biochemistry: Kent et al. Proc. Nat. Acad. Sci. USA 72 (1976)

Mg++ and UTP merely stabilized the complex during sub- DHOase, we expected ATP and PP-ribose-P, both positive sequent ultracentrifugation. Therefore, CPSase, ATCase, and effectors, to stabilize a similar complex. The failure to detect DHOase were isolated after having undergone dissociation, such a complex and the unexpected dissociating effect of to determine whether UTP and Mg++ could promote their PP-ribose-P, ATP, Mg++, and Pi on ATCase could be due in reassociation into a complex. Incubation of the dissociated part to the nature of the solvent we have chosen. Glycerol and enzymes with equimolar UTP and MgCl2 again resulted in other cryoprotectants alter the stability and kinetics of a reassociation of the complex (Fig. 5B). The molecular weight number of enzymes (13, 14) and have mimicked the qualita- of the complex was approximately 700,000. When Mg++ con- tive effects of a natural allosteric activator (15). Also, the centration was twice that of UTP, the ratio of both CPSase composition of the solvent governs the stability of the CPS- and DHOase activities in the complex to the respective un- ase-ATCase-DHOase complex from Ehrlich ascites cells complexed activities increased slightly and the apparent (1, 2). Thus, it will be necessary to know how the nature and molecular weight of the complex was 900,000 (Fig. 5C). The composition of the solvent affects the response of both the ratio of enzymatic activities in fraction 12 (Fig. 5C) was CPSase-ATCase-DHOase complex and the individual 88:13: 1 (ATCase: DHOase: CPSase). Preincubation with 5 component enzymes to effectors before we can conclude any- mM Mg++ alone produced no reassociation and resulted in the thing regarding the role of the complex in pyrimidine bio- appearance of the 110,000 dalton ATCase peak (Fig. 5D). synthesis in the bullfrog egg. Potassium phosphate at 10 mM partially inhibited the reas- The technical assistance of Mr. Edward Kmiotek is gratefully sociation of the complex (Fig. 5E). acknowledged. These studies were supported in part by research Contract no. AT(11-1)-1631 from the U.S. Atomic Energy DISCUSSION Commission, and by Grant NS 10287 from the National Insti- The CPSase-ATCase-DHOase complexes from Ehrlich as- tutes of Health. cites cells, adult rat liver, and bullfrog egg-ovary can all be 1. Shoaf, W. T. & Jones, M. E. (1971) Biochem. Biophys. Res. made to dissociate, but the bullfrog egg complex is the only Commun. 45, 796-802. one demonstrated to dissociate reversibly. The Ehrlich ascites 2. Shoaf, W. T. & Jones, M. E. (1973) Biochemistry 12, 4039- cell complex dissociated to a highly unstable CPSase and a 4051. 3. Mori, M. & Tatibana, M. (1973) Biochem. Biophys. Res. smaller complex of ATCase and DHOase when the buffer con- Commun. 54, 1525-1531. centration of dimethylsulfoxide was reduced from 30 to 10% 4. Lan, S. J., Sallach, H. J. & Cohen, P. P. (1969) Biochemistry (1, 2). After digestion with pancreatic elastase, the 27S 8, 3673-3680. complex from rat liver underwent dissociation to the compo- 5. Marshall, M. & Cohen, P. P. (1972) J. Biol. Chem. 247, nent the of 1641-1653. CPSase, ATCase, and DHOase, activities which 6. Hunninghake, D. & Grisolia, S. (1966) Anal. Biochem. 16, had sedimentation coefficients of 11, 7, and 13 S (3). 200-205. UTP and Mg++, agents which may function as in vivo 7. Prescott, L. M. & Jones, M. E. (1969) Anal. Biochem. 32, regulators, stabilize the CPSase-ATCase-DHOase complex 408-419. from bullfrog egg-ovary. Since UTP is an inhibitor of 8. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, CPSase, R. J. (1951) J. Biol. Chem. 193, 265-275. the advantage of this sort of regulation by UTP is not ap- 9. Martin, R. G. & Ames, B. N. (1961) J. Biot. Chem. 236, parent if the function of the complex is to increase the effi- 1372-1379. ciency of the initial steps of pyrimidine biosynthesis. In yeast, 10. Tatibana, M. & Shigesada, K. (1972) Biochem. Biophys. Res. UTP stabilizes a high-molecular-weight complex of CPSase Commun. 46,491-497. 11. Lue, P. F. & Kaplan, J. G. (1971) Can. J. Biochem. 49, 403- and ATCase, and its removal results in a decreased sedimenta- 411. tion rate for these enzymes (11). In Neurospora, UTP causes a 12. Williams, L. G., Bernhardt, S. & Davis, R. H. (1970) Bio- decrease in the sedimentation rate of a CPSase-ATCase chemistry 9, 4329-4335. complex (12). However, in both yeast and Neurospora, in 13. Bradbury, S. L. & Jakoby, W. B. (1972) Proc. Nat. Acad. Sci. contrast to the ATCase and CPSase remained asso- USA 69,2373-2376. bullfrog, 14. Myers, J. S. & Jakoby, W. B. (1973) Biochem. Biophys. Res. ^iated regardless of the effect of UTP on the ultimate size of Commun. 51, 631-636. the complex. 15. Ruwart, M. J. & Suelter, C. H. (1971) J. Biol. Chem. 246, Because UTP stabilizes a complex of CPSase-ATCase- 5990-5993. Downloaded by guest on September 29, 2021