Pyrimidine biosynthesis and its regulation in embryos of the sea urchin, Arbacia punctulata

Comp. Biochem. Physiol.,

1976,Vol. 55B,pp. 571 to 581.Pergamon Press. Printed in Great Britain

PYRIMIDINE BIOSYNTHESIS AND ITS REGULATION IN EMBRYOS OF THE SEA URCHIN, ARBACIA P U N C T U L A T A * DAVID E. CRANDALL ~ AND GEORGE C. "I~MBLAY2 1Graduate School of Oceanography, University of Rhode Island, Kingston, RI 02881, U.S.A. and 2Department of Biochemistry and Biophysics, University of Rhode Island, Kingston, RI 02881, U.S.A. (Received 10 March 1976)

Abstraet--l. Evidence for the occurrence of the orotate pathway for the de novo biosynthesis of pyrimidines in the developing sea urchin embryo was provided by the incorporation of (14C) NaHCO3, (t4C) carbamoylaspartate, and (6J4C) orotic acid into the pyrimidines of RNA, in vivo. 2. This interpretation was supported by the observation that the radio-label from (~4C) NaHCO 3 appeared in the C-2 position of the uracil ring and by the demonstration of the activity of all of the enzymes of the orotate pathway in cell-free extracts of sea urchin eggs. 3. Evidence was obtained for the regulation of the de novo pathway by end-product inhibition. INTRODUCTION

The de novo biosynthesis of pyrimidines proceeds via the 6 enzymes of the orotate pathway (Fig. 1); this sequence of reactions appears to be universal, having been demonstrated in detail in bacteria, fungi, and in vertebrates. Recent'reviews have considered pyrimidine biosynthesis in microorganisms (O'Donovan & Neuhard, 1970) and in eucaryotic organisms (Jones, 1972). In invertebrates, the study of pyrimidine biosynthesis has received less attention. Demonstrations of the incorporation of (14C) bicarbonate into pyrimidine nucleotides in the snail, Helix pomatia, the moth Celerio euphorbiae (Porembska et al., 1966), the fiatworm Mesocestoides (Heath & Hart, 1970), and the silk worm, Bombyx mori (Moriuchi et al., 1972), suggest the synthesis of pyrimidines via the orotate pathway; verification of this interpretation by demonstrating the incorporation of the radio-labeled precursor into the pyrimidine ring, and specifically into the C-2 position, remains to be achieved. In addition to the tracer studies, recent reports have demonstrated the activity of the first two enzymes of the orotate pathway in several invertebrates. Carbamoylphosphate synthetase activity has been found in several invertebrates (see Table 5); aspartate carbamoyltransferase activity has been demonstrated in Lumbricus terrestris (Bishop & Campbell, 1965), the flatworms Fasciola hepatica, Moneizia benedeni, and Paramphistomum cervi (Kurelec, 1972), Strophocheilus obloru2us (Tramell & Campbell, 1970), Aldrichina grahami (Abe & Miura, 1972), and Drosophila melanogaster (Norby, 1973). To date, no study has demonstrated the operation of the entire orotate pathway in an invertebrate.

In sea urchin eggs, fertilization initiates a period of rapid cell division that continues to the blastula stage before slowing. During this cleavage period, which requires 7-8hr at room temperature, the number of cells increases from 1 to > 1000, and a central metabolic activity is the biosynthesis of DNA, which can be detected within 15-20 min after fertilization (Anderson, 1969; Longo & Plunkett, 1973). RNA synthesis begins almost immediately after fertilization (Rinaldi & Monroy, 1969) and continues throughout cleavage (for recent reviews see Giudice, 1973; and Stearns, 1974). In order to maintain this

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* This investigation was supported in part by Public Health Research Grant No. CA 17131 from the National Cancer Institute and by the facilities of the Graduate School of Oceanography, University of Rhode Island, Kingston, RI 02881, U.S.A. The material presented here is excerpted from a dissertation submitted in partial fulfillment of the requirements for the Ph.D. in Oceanography.

COOH

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../C~c.

II

O,P ~-*'~'~'~',~ ,

RIBOSE-5-P OMP

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II RIBOSE-5'- P UMP

Fig. 1. The orotic acid pathway for the de novo biosynthesis of UMP.

572

DAVID E. CRANDALL AND GEORGE C. TREMBLAY

rapid rate of synthesis of nucleic acids during cleavage, a supply of nucleotide precursors of D N A and R N A must be provided and maintained at adequate levels. Because of the heavy demands for pyrimidine nucleotides during early development, the sea urchin embryo is an especially attractive candidate for a study of the orotate pathway and the possible regulatory mechanisms controlling its output. In the studies reported here we have demonstrated the operation of the complete orotate pathway in sea urchin embryos by measuring the incorporation of (~4C) bicarbonate, (~4C) carbamoylaspartate, and (~4C) orotic acid into R N A ; proof that (~4C) bicarbonate enters pyrimidines via the orotate pathway is provided by the observation that the radio-labeled carbon is incorporated into the C-2 position of the pyrimidine ring. In addition, we have demonstrated the activities of all of the component enzymes of the orotate pathway in cell-free extracts of sea urchin eggs. We also present evidence that the orotate pathway in sea urchin embryos is regulated by end-product inhibition, in which pyrimidine nucleosides or their metabolites inhibit the activity of this biosynthetic pathway. MATERIALS A N D METHODS

Chemicals All radio-labeled chemicals except (t4C) carbamoylphosphate were purchased from New England Nuclear Corp., Boston, MA; (t4C) carbamoylphosphate was synthesized from (~4C) KCNO by the method of Jones et al. (1955). All other chemicals were obtained from Sigma Chemical Co., St. Louis, MO,

Preparation of sea urchin eggs Ripe specimens of Arbacia punctulata were obtained from Mr. Glendle Noble, Panama City, FL. During the summer months, Arbacia were also obtained from the Supply Department of the Marine Biological Laboratory, Woods Hole, MA, and from Quonochontaug Pond, Charlestown, RI. Mature unfertilized eggs were obtained by electrical stimulation and the eggs were washed repeatedly in millipore-filtered (0,45/~m) seawater to remove bacteria and the jelly coats; the completeness of jelly coat removal was monitored by staining with Janus Green, which makes the jelly coat visible under the microscope. Eggs from several females were pooled for each experiment. Sperm was obtained from a single male by electrical stimulation and diluted with ten vol filtered seawater; the eggs, in 200 ml of seawater, were fertilized with I ml of sperm suspension. The completeness of fertilization was determined by observing the elevation of fertilization membranes under the microscope; only cultures showing 95~o or better fertilization were used for further work. Culture medium and conditions of incubation Incubations were carried out in 250 ml beakers. Each incubation vessel contained approx 200 mg of fertilized eggs in a vol of 30ml of millipore-filtered seawater to which the following additional components had been added: streptomycin, 50/~g/ml; penicillin, 150 units/ml, and various intermediates and inhibitors of pyrimidine biosynthesis as indicated. All chemicals were dissolved in illtered seawater and brought to pH 8.0 with KOH or HC1 before being added to the incubation vessels. The embryos * One unit catalyzes the conversion of 1 pmole orotic acid to U M P per hr at 25°C.

were cultured at room temperature and were kept in suspension on a rotating shaker. Embryonic development was allowed to proceed for 3 hr, at which time the 14C-labeled precursor was added; after 2 more hr of incubation, the incubation mixture was placed on ice. The embryos were harvested by centrifugation, the pellet of embryos was suspended in 2 ml of water, and the suspensions were frozen until analyzed.

Preparation of the acid-soluble fraction The frozen embryos were thawed and homogenized; an aliquot was removed for measurement of protein content. The homogenate was acidified to 0.5 N with ice-cold 2.0 N HCIO4, the mixture was rehomogenized, the precipitate was sedimented by centrifugation at 15,000g for 10min and the supernatant fluid removed. The acid-insoluble precipitate was washed twice with ice-cold 0.3 N HC104 and the perchloric acid extracts were pooled. In those experiments in which acid-soluble nucleotides were isolated, the nucleotides were removed from solution by adsorption to charcoal (Kusama & Roberts, 1963) and the charcoal was sedimented by centrifugation. The charcoal was washed with water and extracted 3 times with 10 ml of ethanolH20-concentrated NH4OH (2:2:1) at 37°C to elute the adsorbed nucleotides; the eluates were pooled and dried under a stream of warm air. The residue, containing the acid-soluble nucleotides, was hydrolyzed in 1 N HCIO4 for 1 hr in a boiling water bath. The hydrolysate, containing the pyrimidine nucleoside monophosphates, was neutralized with KOH; chilled in ice, and centrifuged to remove KCIO4; the supernatant solutions were concentrated by freeze-drying. In those experiments in which (14C) orotic acid was isolated, the acid-soluble fraction was neutralized with KOH, the precipitate of KC104 was removed by centrifugation, and the neutralized acid-soluble fraction was diluted to 25 ml with water. The diluted acid-soluble fraction was then saturated with carrier monosodium orotate at 95-100°C, and the (1"C) orotate was isolated by cocrystallization with carrier orotate as described previously (Tremblay et al., 1976). Routinely, embryos were harvested from the seawater incubation medium by centrifugation before they were homogenized. To test the possibility that some (14C) orotic acid was released into the seawater during the course of the incubation, (14C) orotic acid was isolated from seawater after the embryos had been removed by centrifugation, and the amount of radioactivity recovered from seawater was compared with that recovered from the embryos. The results of such an experiment showed that the (14C) orotic acid recovered from seawater was 8.2 + 2.2~o (hl = 15) of the total (14C) orotic acid synthesized by the embryos. Treatment of acid-soluble fraction with orotate phosphoribosyltransferase and orotidine-5'-phosphate decarboxylase To determine whether the 1*C-labeled metabolite of (~4C) NaHCO3 cocrystallizing with monosodium orotate was (~'*C)orotate, the acid-soluble fraction of embryos was subjected to an enzyme treatment which converts orotic acid to UMP. The acid-soluble fraction prepared from sea urchin embryos which had been cultured in the presence of 15 m M ( C1,~)-J NaHCO3 (366 dis/min/nmole) and 10raM 6-azauridine, was adjusted to pH 8.0 and incubated with MgC12 (5raM), phosphoribosyl pyrophosphate (5mM), and orotate phosphoribosyltransferase and orotidine-5'phosphate decarboxylase (3 units*, mixed enzymes prepared from yeast) in a final vol of 2.8 ml. Following incubation for 4 hr at 25°C, the reaction was stopped by placing the test tubes in a boiling water bath for 5 min. The precipitated protein was centrifuged and the supematant decanted. The reaction mixture was diluted to 25 ml with water and the t4C-labeled metabolite of (14C) NaHCO3 cocrystallizing with carrier monosodium orotate was isolated as described above.

Pyrimidine biosynthesis in embryos of the sea urchin

Extraction and hydrolysis of RNA The acid-insoluble fraction of the incubation mixture was washed twice with ice-cold 0.3N HCIO4, and the lipids were removed by extractions, in succession, with 10 ml of ethanol buffered with 1% potassium acetate, 10 ml of ethanol-chloroform (3:1), 10 ml of ethanol-ether (3:1), and finally 10ml of ether. The acid-insoluble, lipid-free residue so obtained was suspended in 2 ml of 0.3 N KOH and incubated at 37°C for 1 hr to hydrolyze the RNA (Schneider, 1957). After hydrolysis, the mixture was cooled in tap water and adjusted to pH 2 by the addition of 1 N HC104; the mixture was chilled in ice and centrifuged to remove the precipitate of DNA and protein. The supernatant fluid was neutralized with KOH, kept overnight in the refrigerator, and centrifuged to remove the precipitate of KC104. The supernatant fluid, containing ribonucleotides, was freeze-dried and the residue dissolved in 1 ml of 0.05 N HCI for chromatographic fractionation. The nudeotides of RNA were separated by ion-exchange chromatography according to the procedure of Katz & Comb (1963); ascending thin layer chromatography on cellulose, in a solvent system of 95% ethanol: 1 M ammonium acetate, 75:30 (Ciardi & Anderson, 1968), was used to corroborate the identity of the UMP fractions from ion-exchange chromatography.

Stepwise decomposition of uracil Sea urchin embryos were incubated for 2 hr with (t4C) NaHCO3 (0.9 mM, 15795 dis/min/nmole); at the end of the incubation period, UMP was isolated from the acid-soluble fraction of the incubation mixture by the procedures described above. The chromatographic fractions containing UMP were combined and dried, and the residue was dissolved in I ml of 12N HC104 in a stoppered glass test tube. The solution was heated at 75°C for 1 hr to hydrolyze U M P to uracil. The hydrolysate was diluted with 10 ml of water and uracil was isolated by adsorption to charcoal and subsequent thin layer chromatography on cellulose in the solvent system 95~o ethanol: 1 M ammonium acetate, 75:30. Following development, the band containing uracil was detected under ultraviolet light, the cellulose was scraped off, and the uracil was eluted from the cellulose with water. The method employed for the stepwise degradation of uracil was a modification of the methods of Kusama & Roberts (1963) and Pillarisetty & Karasek (1970); the procedure, with its modifications, is described in detail by Tremblay et al. (1976). Briefly, the sample of uracil was sequentially oxidized in 3 stages: the first oxidation liberated the C-4 carbon as CO2, the second liberated the C-2 carbon as CO2 in a urease-dependent reaction sequence, and in the last stage the C-5 and C-6 carbons were oxidized to CO2. At the end of each stage of oxidation, the CO2 was collected in KOH and the amount of radioisotope contained in the CO2 indicated the distribution of ~4C from bicarbonate among the carbon atoms of the pyrimidine ring.

Measurement of 14CO2 eenerated from (7-1"C) orotic acid The decarboxylation of (7-t4C) orotic acid was monitored by measuring the liberation of 14CO2. Embryos were allowed to develop in a 600ml beaker for 3hr; from the magnetically-stirred beaker 5 ml aliquots of embryos were transferred to centrifuge tubes. The embryos were sedimented by gentle centrifugation in a clinical centrifuge and the supernatant seawater was removed by aspiration. Some of the aliquots of embryos were used for the determination of protein. The embryos were suspended in seawater supplemented with varying concentrations of 6-azauridine, * One unit catalyzes the formation of 1/~mole of citrulline per min at 37°C.

573

and the suspension was transferred to a 25 ml Erlenmeyer flask with a Pasteur pipet. The flask was sealed with a rubber cap fitted with a plastic center-well containing 0.3 ml of 20°/O KOH and a filter paper wick. A solution containing 1 #mole of (7-1"C) orotic acid (1980dis/min/ m o l e ) was then injected through the rubber cap to bring the vol to 5 ml and start the reaction. Following a 2 hr incubation at room temperature, the reaction was terminated by the injection of 1.7 ml of 2 N HC10,, and 14CO2 was distilled into the KOH during an additional 30 min incubation at 37°C. The center-well and its contents were transferred to a scintillation vial and diluted with 1.7 ml of water followed by 6.5 ml of Aquasol, and the quantity of radioactivity was measured in a liquid scintillation spectrometer.

Assay of carbamoylphosphate synthetase Sea urchin eggs were suspended in 4 vol of homogenizing medium containing the following components at the indicated concentrations: glycerol, 20°,/0; dimethyl sulfoxide, 20%; Tris-HC1, pH 7.9, 40 mM; dithiothreitol, 1 mM; mercaptoethanol, 5mM; EDTA, l mM; and glutamine, 5 mM. The eggs, kept at 0°C in an ice-water bath, were disrupted by sonication during two 10 sec pulses at ½ power in a Biosonik sonicator. The unfractionated sonieate was used as the enzyme source. The activity of carbamoylphosphate synthetase was measured by converting the (14C) carbamoylphosphate synthesized from (t4C) NaHCO3 to (14C) citrulline in a system containing excess ornithine and commercial ornithine carbamoyltransferase. The reaction mixture contained, in a vol of 1 ml, the following components at the indicated final concentrations: Tris-HCl, pHT.9, 75mM; MgC12, 15mM; L-glutamine, 10mM; N-acetyl-L-glutamic acid, 5mM; ATP, 10mM; L-ornithine, 10mM; ornithine carbamoyltransferase (Sigma), 3 units*; (t4C) NaHCO 3 (15,795 dis/min/nmole), 10 mM; and 0.4 ml of enzyme preparation. Reagents were adjusted to pH 7.9 with KOH or HC1. The reaction was started by addition of the enzyme source, allowed to proceed for 30 min at 30°C, and terminated by the addition of 0.25 ml of 30% trichloroacetic acid. The precipitated protein was sedimented by centrifugation and the unreacted t'CO2 was removed by gassing with CO2. An aliquot of 10 or 25/~1 of the reaction mixture was spotted on Whatman No. 1 paper and the Q4C) citruiline was isolated by descending chromatography with 955/o ethanol: 1 M ammonium acetate, 75:30. Citrulline was visualized by spraying with Ehrlich's reagent and identified by reference to a citrulline standard. The area of the chromatogram containing Q'C) citrulline was cut out and transferred to a scintillation vial, eluted with 2 ml of water, mixed with 6.5 ml of Aquasol, and the content of radioactivity was measured in a liquid scintillation spectrometer. The radioactive compound cochromatographing with citrulline on a companion chromatogram was subjected to arsenolysis, an ornithine carbamoyltransferase-dependent reaction which releases t'CO2 from the ureido group of (1"C) citrulline. The incubation mixture contained, in 2ml, 150~mole of sodium arsenate, pH 5.8, 30 units of Streptococcus ornithine carbamoyltransferase, and the eluant from the chromatogram. The reaction was carried out in a closed vessel, and after an incubation of 60 min at 37°C, 0.5 ml of 12 N H2SO, was injected to drive off the 1"CO2 released by arsenolysis. The 1*CO2 was trapped in a plastic center-well containing 0.3 ml of 20~o KOH during an additional 30 min incubation, following which the center-well and its contents were transferred to a scintillation vial for counting.

Assay of aspartate carbamoyltransferase The activity of aspartate carbamoyltransferase was measured in a reaction mixture of 0.5 ml containing the following components at the indicated final concert-

574

DAWD E. CRANDALLAND GEORGEC. TREMBLAY

trations: glycine-potassium hydroxide buffer, pH9.5, 0.1 M; potassium aspartate, pH 9.5, 20mM; (~4C) carbamoyl-phosphate (8.8 dis/min/nmole), 16 mM; and 0.2 ml of the enzyme preparation. The enzyme source was an unfractionated 20% homogenate of sea urchin eggs. The reaction was started by the addition of (14C) carbamoylphosphate, allowed to proceed for 30min at 37°C, and terminated by the addition of 0.25 ml of 1 N HC1. An aliquot of 0.5 ml of the acidified reaction mixture was transferred to a scintillation vial, and the contents were baked to dryness over a boiling water bath; this operation hydrolyzes the unreacted (~4C) carbamoylphosphate, releasing 14CO2which is driven off. The residue, containing the (i4C) carbamoylaspartate, was dissolved in 2 ml of water and mixed with 6.5ml of Aquasol; the amount of radioisotope was measured in a liquid scintillation spectrometer.

Assay ofdihydro-orotase and dihydro-orotate dehydrogenase The coupled activity of dihydro-orotase and dihydroorotate dehydrogenase was measured by the conversion of (~4C) carbamoylaspartate to (~4C) orotic acid. The reaction mixture contained, in a vol of 1 ml, the following components at the indicated final concentrations: Tris-HC1, pH 7.5, 50 raM; (14C) carbamoylaspartate (8389dis/min/ nmole), 0.12mM; and 0.5ml of enzyme preparation, an unfractionated 20% homogenate of sea urchin eggs. Following incubation at 30°C for 1 hr, the reaction was stopped by the addition of 0.33 ml of 2 N HC104, the precipitated protein was sedimented by centrifugation, and the supernatant fluid was decanted, Following neutralization of the acid-soluble fraction with KOH and removal of the KC104 by centrifugation, the solution was diluted to 25 ml with water and (14C) orotic acid was isolated by cocrystallization with carrier sodium orotate. The (~4C) orotate was recrystallized from water to constant specific activity.

urchin embryo was obtained by the demonstration of the incorporation of (1*C) NaHCO3, (ureido-14C) carbamoylaspartate, and (6-14C) orotic acid into the pyrimidine nucleotides of RNA. Chromatographic resolution of the nucleotides released by mild alkaline hydrolysis of RNA revealed that both pyrimidine and purine nucleotides were labeled by (t4C) NaHCO3, while (ureido-~4C) carbamoylaspartate and (6-~4C) orotic acid served as precursors only to the pyrimidine nucleotides (Fig. 2). Further evidence that the incorporation of (14C) N a H C O 3 into pyrimidines occurs via the orotate pathway was obtained upon localization of the radioisotope within the pyrimidine ring. Consistent with incorporation of (14C) NaHCO3 through the orotate pathway, stepwise oxidation of the pyrimidine ring revealed that 90% of the radioisotope was localized in the C-2 position of uracil (Fable 1). The dependence on urease of the release of the C-2 carbon as

21

Determination of protein Protein was measured by the biuret method (Layne, 1957), using a Klett-Summerson Photoelectric Colorimeter, with bovine serum albumin as the standard. REsuLTs

Incorporation of 14C-labeled precursors into pyrimidines Evidence for the occurrence of the orotate pathway for the de novo biosynthesis of pyrimidines in the sea

z0

L5

AMP A O

~U~P (14C1 CARBAMOYLASPARTATE

Assay of orotate phosphoribosyltransferase and orotidine-5'phosphate decarboxylase The combined activity of orotate phosphoribosyltransferase and orotidine-5'-phosphate decarboxylase was assayed by trapping the 14CO2 liberated by the decarboxylation of (7-~*C) orotic acid. The reaction was carried out in a closed vessel sealed with a rubber cap holding a plastic center-well containing 0.3 ml of 20% KOH and a filter paper wick. The complete reaction mixture, in a vol of 1 ml, contained the following components at the indicated final concentrations: MgC12, 5raM; 5'-phosphoribosyl-l'pyrophosphate, 5 mM; (7-~4C) orotic acid (21326 dis/min/ nmole), 0.06 mM; Tris-HC1, pH 7.5, 50mM; and 0.5 ml of enzyme preparation. The enzyme source was prepared by homogenizing sea urchin eggs in 4 vols of water. All reagents were adjusted to pH 7.5 with KOH or HCI before addition. Following incubation at 30°C for 30min, the reaction was stopped by an injection of 0.33 ml of 2 N HC104 through the rubber cap, and 14CO2 was distilled for 30 min at 37°C. The plastic center-well and its contents were transferred to a scintillation vial, diluted with 1.7 ml of water followed by 6.5 ml of Aquasol and the amount of radioactivity was measured in a liquid scintillation spectrometer.

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Fig. 2. Incorporation of (14C) NaHCOa, (14C) carbamoylaspartate, and (6-14C) orotic acid into RNA. Fertilized eggs were allowed to develop for 3 hr in millipore-filtered seawater and were then cultured for an additional 2 hr in seawater containing the following 14C-labeled precursors: (a) (14C) NaHCOa, 0.4 mM (11000 dis/min/nmole); (b) (t4C)carbamoylaspartate, 0.44mM (8389dis/min/ nmole); (c) (6-14C) orotic acid, 0.04raM (85855dis/rain/ nmole). Following the incubation, the acid-insoluble fraction and RNA hydrolysate were prepared as described under Materials and Methods, and an aliquot of the RNA hydrolysate containing approximately 600 #g of RNA was fractionated by ion-exchange chromatography.

Pyrimidine biosynthesis in embryos of the sea urchin

575

Table 1. Incorporation of (~4C) NaHCO3 into the C-2 position of uracil. Sea urchin embryos were incubated with 0.9 mM (14C) NaHCO3 (15795 dis/min/umole); (14C) uracil was isolated from the acid-soluble nucleotides and subjected to stepwise oxidation as described under Materials and Methods. Commercial (2-~4C) uracil was used as a standard to test the efficacy of the method and the results are included for comparison

Description (2-14C) Uracil standard Assay 1 Assay 2 Assay 3 (no urease) (14C) Uracil sample Assay 1 Assay 2 Assay 3 (no urease)

Radioactivity added

Location of radioisotope C-4 C-2 (C-5 + C - 6 ) (~o of recovered 14C)

Recovery of radioactivity

(~o)

67606 67606 67606

0.2 0.2 0.2

98.2 98.5 5.2

1.6 1.3 --

88 88 --

5312 5312 5312

1.0 1.2 1.3

87.3 94.2 22.0

11.7 4.6 --

116 107 --

14C02 is indicative of the specificity of this oxidative procedure.

Carbamoylphosphate synthetase Carbamoylphosphate synthetase activity was demonstrated in cell-free extracts of Arbacia eggs. Maximal activity required the addition of ATP; the addition of ornithine and ornithine carbamoyltransferase was necessary to convert (14C) carbamoylphosphate to (14C) citrulline (Table 2). The pH optimum for this system was found to be 7.9. Under the reaction conditions employed, 40-45~o of the acid-stable radioactivity was found to be in (~4C) citrulline. A series of paper strips, each spotted with a 25 #1 aliquot of the acidified reaction mixture, was developed in 95~o ethanol: 1 M ammonium acetate, 75:30. One of the strips was spotted with 20 nmoles each of carrier citrulline and carbamoylaspartate; after development, the location of the compounds was visualized by spraying with Ehrlich's reagent. A companion strip was analyzed for localization of radioactivity on a Nuclear Chicago Actigraph III strip counter; these two records are shown together in Fig. 3. Three radioactive peaks can be seen, one each corresponding to carbamoylaspartate and citrulline, and one peak between these two. A third companion strip was cut into 1 cm sections which were analyzed for their content of radioactivity in a liquid scintillation spectrometer; 100~o of the acid-stable radioactivity was recovered from this strip. To verify further the identity of the radioactive compound migrating with carrier citrulline, the (t4C) citrulline was eluted from the chromatogram with water and subjected to arsenolysis (Reichard, 1957), a reaction which releases ~CO2 from the ureido group of (14C) citrulline. Of the radioactivity which co-chromatographed with citrulline, 78~o was recovered as 14CO2; when ornithine carbamoyltransferase was left out of the reaction mixture, only 13~ of the radioactivity was released as t4CO2. These data confirm the identity of the radioactive compound migrating with carrier citruUine as (~4C) citrulline and establish the presence of carbamoylphosphate synthetase activity in sea urchin eggs. N o attempt was made to identify the radioactive compound of peak "A" (Fig. 3).

Aspartate carbamoyltransferase Aspartate carbamoyltransferase activity was demonstrated in sonicates of sea urchin eggs incubated with (14C) carbamoylphosphate and aspartate. The pH optimum of the reaction was 9.5. The level of activity was found to be 14.3 nmoles of carbamoylaspartate synthesized per min/mg protein at 37°C, and the production of carbamoylaspartate was linear with enzyme concentration at least up to 8 mg of protein per ml of reaction mixture. Table 2. Carbamoylphosphate synthetase in sonicates of unfertilized sea urchin eggs

Conditions Complete Minus sonicate of eggs Minus ATP Minus ornithine and ornithine carbamoyltransferase

Enzyme activity nmoles of (14C) NaHCO3 incorporated into Citrulline per 30 min-10 mg Protein 30.9 0.01 4.1 7.2

The complete incubation mixture contained, in a vol of I ml, the following components at the indicated final concentrations: Tris-HC1, pH7.9, 75mM; MgC12, 15mM; L-glutamine, 10mM; N-acetyl-L-glutamate, 5 mM; ATP, 10 raM; phosphoenol pyruvate, 2.5 mM; pyruvate kinase, 10 units; L-ornithine, 10 mM; ornithine carbamoyltransferase (Sigma), 3 units; (14C) NaHCOa (15795 dis/min/nmole), 10mM; and 0.4ml of enzyme preparation. The enzyme source was prepared by sonicating degelled sea urchin eggs in 4 vols of medium containing the following components at the indicated concentration: glycerol, 20%; dimethyl sulfoxide, 20%; Tris-HCl, pH 7.9, 40mM; dithiothreitoi, 1 mM; mercaptoethanol, 5 mM; EDTA, 1 raM; and glutamine, 5 raM. The reaction proceeded for 30 min at 30°C and was terminated by the addition of 0.25 ml of 30°/0 trichloroacetic acid. Following removal of precipitated protein by centrifugation, the acidified reaction mixture was gassed with C02 to remove unreacted 14C02. (14C) CitruUine was isolated from the reaction mixture by descending paper chromatography in 95~o ethanol-lM ammonium acetate, 75:30.

576

D A V I D E. CRANDALL AND GEORGE C . TREMBLAY

(each incubation vessel contained 60 m o l e s of (7-14C) orotic acid, 21620dis/min/nmole) was recovered as t4CO2; generation of 1"CO2 in the absence of enzyme source was only 0.08% that observed with the enzyme. Omitting phosphoribosyl pyrophosphate from the reaction mixture reduced the recovery of radioactivity as ~4CO2 by 89?/0, consistent with the conversion of orotic acid to U M P via orotate phosphoribosyltransferase and orotidine-5'-phosphate decarboxylase.

ORIGIN

Incorporation of (14C) NaHCO 3 into orotic acid Corixlmoyl .

~lx=rfote

Citrulli~

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Q

Fig. 3. Isolation of (~4C) citrulline by paper chromatography. An aliquot of 25 #1 of acidified reaction mixture (components of the reaction mixture are listed in the legend to Table 2) was applied to Whatman No. 1 paper and developed in the solvent system 95% ethanol: 1 M ammonium acetate, 75:30; on a companion strip, 20 nmole each of carrier carbamoylaspartate and citrulline were spotted. Following development, the ureido compounds were visualized by spraying with Ehrlich's reagent, and the strip containing radioactivity was analyzed on a Nuclear Chicago Actigraph III strip counter.

A series of experiments was performed to determine the optimal conditions for measuring the incorporation of (1*C) NaHCO 3 into orotic acid. Sea urchin eggs were fertilized and allowed to develop on a rotating table shaker for 3 hr, at which time they were concentrated by allowing the embryos to settle and decanting the supernatant seawater; 5 ml aliquots of embryos were taken from the concentrated embryo culture, which was kept in even suspension by a magnetic stirrer, and transferred to beakers with seawater containing (1"C) NaHCO3 and the chemicals specific to the individual experiments. An additional 2 hr incubation followed; this time interval in early development was chosen because the rate of uptake of (14C) NaHCO3 by sea urchin embryos, having increased during the first stages of cleavage, reached a plateau at about 3 hr after fertilization and did not change through the blastula stage (Crandall, 1975). r~

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Dihydro-orotase and dihydro-orotate dehydrogenase The presence of the enzymes dihydro-orotase and dihydro-orotate dehydrogenase was detected in homogenates of sea urchin eggs by measuring the conversion of (14C) carbamoylaspartate to (14C) orotic acid. In two experiments, an average of 13.1% of the added (14C) carbamoylaspartate (each incubation vessel contained 0.12/maole of (14C) carbamoylaspartate, 8250 dis/min/nmole) was incorporated into orotic acid, and when the enzyme source was omitted f r o m the incubation mixture, this incorporation dropped to 0.2% of the added isotope. No attempt was made to assay the activities of these two enzymes at optimal conditions, the object of these experiments being merely to demonstrate the presence of the enzymes in sea urchin eggs.

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Fig. 4. The effect of the concentration of bicarbonate on

Orotate phosphoribosyltransferase and orotidine-5'- the incorporation of (14C) NaHCO3 into orotic acid. phosphate decarboxylase Embryos were allowed to develop for 3 hr after fertilization In order to demonstrate the activities of orotate phosphoribosyltransferase and orotidine-5'-phosphate decarboxylase in sea urchin eggs, the decarboxylation of orotic acid was measured by trapping the ~4CO2 evolved from this reaction in a system containing (7-14C) orotic acid, phosphoribosyl pyrophosphate, and an homogenate of sea urchin eggs. In two experiments, an average of 98% of the added radioactivity

and were then incubated for an additional 2 hr with 10 mM 6-azauridine and varying concentrations of (14C) NaHCO 3 (366 dis/min/nmole). The embryos were harvested by centrifugation, suspended in 2 ml of water and homogenized, and an aliquot was taken for the determination of protein content. The homogenate was acidified, and (14C) orotic acid was isolated from the acid-soluble fraction by cocrystallization with carrier orotate. The figure combines data from 2 experiments.

Pyrimidine biosynthesis in embryos of the sea urchin 0 o

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Fig. 5. The effect of 6-azauridine on the incorporation of (14C) NaHCO3 into orotic acid and RNA. The experimental conditions were the same as those described in the legend to Fig. 4 except that the concentration of (14C) NaHCO3 was 15 mM for those incubations from which Q'C) orotic acid was isolated and 0.4 mM for those incubations from which the RNA hydrolysate was prepared; the concentration of 6-azauridine was varied as indicated. To prepare the RNA hydrolysate, the acid-insoluble fraction was washed with organic solvents to remove lipid and hydrolyzed in 0.3 N KOH. Following acidification of the RNA hydrolysate, the precipitated macromolecules were sedimented by centrifugation and the supernatant fraction was neutralized with KOH. Aliquots of the neutralized supernatant fraction, containing the RNA hydrolysate, were measured for absorbancy at 260 nm, and the content of radioisotope was determined in a liquid scintillation spectrometer.

In order to maximize the recovery of t*C-labeled orotic acid synthesized from (t4C) NaHCO3, the incubations were performed at saturating concentrations of (taC) NaHCO3, and in the presence of 6-azauridine, an inhibitor of the conversion of orotic acid to U M P (Handschumacher & Pasternak, 1958). A study of the effect of increasing concentrations of (14C) NaHCO3 on the amount of (14C) orotate isolated from sea urchin embryos demonstrated that bicarbonate was saturating at a concentration of 15 mM (Fig. 4), and this concentration was employed routinely in further experiments. Measurements of the effect of 6-azauridine on the incorporation of (14C) NaHCO3 into orotic acid and RNA demonstrated that 6-azauridine at a concentration of 10raM was sufficient to promote maximum accumulation of Q*C) orotate and minimum incorporation of isotope into RNA (Fig. 5); 10 mM 6-azauridine was used routinely in further experiments. Chromatographic resolution of the nucleotides of RNA, performed as described above, revealed that 6-azauridine specifically inhibited incorporation of (t*C) NaHCO3 into the pyrimidine components of RNA; residual t*C-labelling of RNA from (14C) NaHCO3 in the presence of 6-azauridine was largely due to labelling of purines (CrandaU, 1975).

577

Proof that the 14C-labeled metabolite of (1"C) NaHCO3 cocrystallizing with carrier mono-sodium orotate was (14C) orotic acid was obtained by subjecting the acid-soluble fraction of sea urchin embryos to an enzyme treatment that converted orotic acid to UMP, as described under Materials and Methods. When the acid-soluble fraction was incubated with the yeast enzymes orotate phosphoribosyltransferase and orotidine-5'-phosphate decarboxylase, and phosphoribosyl pyrophosphate, the amount of radioisotope isolated with carrier orotate was reduced by 789/0; when phosphoribosyl pyrophosphate, a substrate necessary for the conversion of orotic acid to orotidine monophosphate, was omitted from the reaction mixture, the reduction in isolated radioisotope did not occur (Table 3). These data confirm the identity of the ~4C-labeled metabolite cocrystallizing with carrier orotate as Q*C) orotic acid. A study of the effect of pyrimidine and purine nucleosides on the incorporation of (:*C) NaHCO3 into orotic acid demonstrated that uridine, or a metabolite of uridine, inhibited the incorporation up to 55%, while guanosine and adenosine had only a slight effect, if any (Fig. 6). We explored the possibility that the decrease in the incorporation of (t*C) NaHCO3 into orotic acid in the presence of uridine was caused by an antagonism by uridine of the metabolic block imposed by 6-azauridine. This possibility was eliminated by examining the ability of uridine to prevent the inhibitory action of 6-azauridine on the conversion of orotic acid to UMP. Using the generation of 14CO2 from (carboxyl-~*C) orotic acid as a measure of the conversion of orotic acid to U M P by the embryos, the concentration of 6-azauridine routinely employed (10mM)was found to inhibit the conversion of orotic acid to U M P by 97~, and the Table 3. Identification of the metabolite of (t4C) NaHCO 3 coerystallizing with carrier orotate Conditions of incubation Untreated control Complete Complete minus phosphoribosyl pyrophosphate

Specific activity of isolated orotate ~o of untreated (counts/min/50 rag) control 772 172

100 22

791

102

The acid-soluble fraction prepared from sea urchin embryos which had been incubated with (t'C) NaHCO3, 15 mM (366 dis/min/nmole) and 6-azauridine, 10 mM, was adjusted to pH 8.0 and divided into 3 aliquots for incubation with the following components: the complete reaction mixture contained MgCl2, 5mM; phosphoribosyl pyrophosphate, 5 mM; orotate phosphoribosyltransferase and orotidine-5'-phosphate decarboxylase, 3 units (from yeast, 1 unit catalyzes the conversion of I Fmole orotic acid to UMP/hr at 25°C); final vol was 2.8 ml. One aliquot, the untreated control, was incubated only with Water, a second with the complete system, and a third with the complete system minus phospboribosyl pyrophosphate. Following incubation at 25°C for 4 hr, the reaction was stopped by placing the test tubes in a boiling water bath for 5 min. The precipitated protein was centrifuged and the supernatant decanted; the reaction mixture was diluted to 25 ml with water and (t'C) orotic acid was isolated by cocrystallization with 200 mg of monosodium orotate.

578

DAVID E. CRANDALL AND GEORGE C . TREMBLAY

J 0 I,-

into orotic acid in the presence of uridine represents end-product inhibition of the de novo biosynthesis of pyrimidines and not an antagonism by uridine of the action of 6-azauridine on the accumulation of orotic acid. This interpretation is supported by the observation that uridine is effective in reducing the incorporation of (14C) NaHCO3 into orotic acid in the absence of 6-azauridine; although the quantity of (~4C) orotate isolated was greatly reduced in the absence of 6-azauridine, uridine at a concentration of 10mM inhibited the incorporation of (14C) NaHCO3 into orotic acid by 45%. These observations are consistent with the interpretation that uridine or its metabolites regulate the de novo biosynthesis of orotic acid through end-product inhibition in the sea urchin embryo.

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Fig. 6. The effect of pyrimidine and purine nucleosides on the incorporation of (14C) NaHCO3 into orotic acid. The experimental conditions were the same as those described in the legend to Fig. 4 except that the concentration of (14C) NaHCO3 (366dis/min/nmole) was 15 mM, the concentration of 6-azauridine was 10 mM, and the concentrations of the purine or pyrimidine nucleosides were varied as indicated. inhibitory action of 6-azauridine was only slightly reduced by concentrations of uridine as high as 20 mM (Table 4). Thus, it would appear that reduction in the rate of incorporation of (~4C) NaHCO3

Sea urchin embryos possess the full complement of enzymes of the orotate pathway for the de novo biosynthesis of pyrimidines, and this pathway is active during early embryonic development, when heavy demands for pyrimidine nucleotides are imposed by the rapid rate of DNA synthesis. The occurrence of the orotate pathway is demonstrated by the incorporation of (14C) NaHCO3, (14C) carbamoylaspartate, and (6-14C) orotic acid into U M P and CMP of RNA in the developing embryo, and also by demonstration of the catalytic activity of the component enzymes of the pathway in cell-free extracts of sea urchin eggs. In addition, 90% of the radioactivity incorporated into uracil when sea urchin embryos were incubated with (1~C) bicarbonate was recovered in the C-2 position, consistent with a reaction sequence beginning with the synthesis of carbamoylphosphate. The activity of carbamoylphosphate synthetase observed in cell-free extracts of unfertilized eggs was 30.9 nmoles carbamoylphosphate synthesized during

Table 4. The effect of uridine and 6-azauridine on the decarboxylation of (7-14C) orotic acid 6-Azauridine (mM)

Uridine (mM)

(7-14C) Orotic acid decarboxylated (nmoles)

% Inhibition

0 10 10 10 10 10 10 10

0 0 O.5 1 5 10 15 20

0.78 0.02 0.09 0.09 0.15 0.23 0.19 0.19

0 97 88 88 81 71 76 76

Embryos were allowed to develop for 3 hr after fertilization, when they were harvested by gentle centrifugation, resuspended in seawater containing uridine and 6-azauridine at the indicated concentrations, and transferred to a 25 ml flask. The flask was sealed with a rubber cap fitted with a plastic center-well containing 0.3 ml of 20% KOH and a filter paper wick, and a solution containing 1/~mole of (7-14C) orotic acid (1980dis/min/nmole) was injected through the rubber cap. After an additional 2 hr incubation, the reaction was terminated by the injection of 1.7 ml of 2 N HCIO4, and t4COz was distilled into the KOH during an additional 30min incubation at 37°C. The center-well was transferred to a scintillation vial and its contents were diluted with 1.7 ml of water followed by 6.5 ml of Aquasol, and radioactivity was measured in a liquid scintillation spectrometer. Each flask contained embryos equivalent to 10.5 mg of embryo protein.

Pyrimidine biosynthesis in embryos of the sea urchin

579

Table 5. Activity of carbamoylphosphate synthetase in invertebrate tissues, and in normal and neoplastic mammalian tissues. The activities of carbamoylphosphate synthetase tabulated here were, in some cases, recalculated to permit presentation in this form. If assays employing different conditions yielded varying results, the highest value was chosen for inclusion here. The enzymic activity of invertebrate tissues was assayed at 30°C except as noted, and that of mammalian tissues at 37°C

Organism Strophocheilus oblongus (land snail; Tramell & Campbell, 1970) Otala lactea (land snail; Tramell & Campbell, 1971) Helix aspersa (land snail; Tramell & Campbell, 1971) Lumbricus terrestris (earthworm; Tramell & Campbell, 1971) Bipalium kewense (land planarian; Tramell & Campbell, 1971) Aldrichina grahami (blowfly; Abe & Miura, 1972) Nezara viridula (green vegetable bug; Powles et al., 1972) Bilimulus dealbatus (land snail; Horne, 1973) Arbacia punctulata (sea urchin; this study) Mouse (Tatibana & Ito, 1969) Rat (Yip & Knox, 1970) Rat (Yip & Knox, 1970) Rat (Yip & Knox, 1970; Hager & Jones, 1967b) Rat (Yip & Knox, 1970) Mouse (Hager & Jones, 1967a)

Tissue

Carbamoylphosphate synthetase activity nmole/g tissue/hr

hepatopancreas

150-500

hepatopancreas

132

hepatopancreas

236

gut

652

whole body

70

larvae

64*

gut

500

hepatopancreas eggs

72 (feeding) 236 (estivating) 960

spleen brain heart fetal liver Walker carcinosarcoma 256 Ehrlich ascites cells

744 7.2 13.8 351:128 630 99

* Assayed at 28°C.

a 30 min incubation per 10 mg of egg protein. This rate translates into 0.22 pmole/hr/egg, or 0.96 pmole/ hr/g degelled eggs (the protein content of the Arbacia punctulata egg is 36 ng (Fry & Gross, 1970), and there are 4.3 x 10° degelled eggs/g (Harvey, 1956). Thus, the level of carbamoylphosphate synthetase activity in sea urchin eggs is the highest reported for an invertebrate tissue, as can be seen in Table 5. It is considerably higher than the activity of invertebrate tissues which do not produce urea and is in the range of activities reported for urea-producing invertebrate tissues. Some values for carbamoylphosphate synthetase activities in normal and neoplastic mammalian tissues are also included in Table 5, for comparison. It should be noted that the activities of the invertebrate tissues were assayed at 30°C, a temperature rarely, if ever, encountered in the environment; therefore, a direct estimate of the rate of biosynthesis of carbamoylphosphate in vivo is not possible from these data. In the assays of carbamoylphosphate synthetase from sea urchin eggs, maximum levels of (14C) citrulline production required the addition of ATP, ornithine, and ornithine carbamoyltransferase. Omission of these components from the reaction mixture decreased, but did not abolish, the synthesis of (14C) citrulline. It remains to be determined whether the carbamoylphosphate synthetase (CPSase) of the sea urchin has properties similar to those of CPSase-I (NH 3 as exclusive nitrogen source, activated by N-acetylglutamate, localized in the mitochondrion), CPSase-II (glutamine as nitrogen source, no require-

ment for N-acetylglutamate, localized in the cytosol), or CPSase-III (glutamine as nitrogen source, requirement for N-acetyl-L-glutamate as cofactor, localized in the mitochondrion). Preliminary studies indicate that extensive purification of the enzyme will be necessary before the characteristics of the enzyme can be determined with confidence (Crandall, 1975). The effect of pyrimidine nucleotides on carbamoylphosphate synthetase has been studied in only two invertebrates, with opposite results: in the blowfly, Aldrichina grahami, several pyrimidine nucleotides, at concentrations of 2 mM, were found to inhibit carbamoylphosphate synthetase (Abe & Miura, 1972), but the enzyme from the snail, Strophocheilus, probably the best characterized carbamoylphosphate synthetase from an invertebrate, was not affected by UTP (Tramell & Campbell, 1970). In the present study, the orotate pathway for the de novo biosynthesis of pyrimidines in sea urchin embryos has been found to be subject to end-product regulation by uridine. The incorporation of (14C) NaHCO3 into orotic acid was inhibited by 55% by uridine at a concentration of 20 mM. It is likely that the activity of the orotate pathway observed in the absence of exogenous uridine is already depressed by the action of endogenous pyrimidine nucleotides, and that the total range of possible activity, from uninhibited to maximally inhibited, is broader than the values reported here. Although inhibition by pyrimidine nucleotides of enzymes of the orotate pathway has been demonstrated in eell-free preparations, only a few other

580

DAVID E. CRANDALLAND GEORGEC. TREMBLAY

reports have shown that this mechanism operates in intact cells. Uridine was found to inhibit the incorporation of (1"C) NaI-ICO3 into orotic acid in slices of several mammalian tissues (Smith et al., 1973), and in minces of chick oviduct (Gulen et al., 1974), and the results indicate the site of inhibition to be carbamoylphosphate synthetase. The effect of purine nucleosides on the incorporation of (1"C) NaHCO3 was investigated because of recent reports that purines inhibit the biosynthesis of pyrimidines; adenosine and guanosine were more effective than uridine in decreasing the incorporation of ('4C) NaHCO3 into orotic acid in minces of rat mammary gland and in minces of chick oviduct (Gulen et al., 1974), and adenosine caused pyrimidine starvation in cultured fibroblasts and lymphoid cells (Ishii & Green, 1973; Green & Chan, 1973). In the sea urchin embryo, however, purine nucleosides appear to have no effect on the activity of the orotate pathway.

REFERENCES

ABE A., & MIURAK. (1972) Some properties of carbamylphosphate synthase partially purified from the larvae of blowfly, Aldrichina grahami. Agric. Biol. Chem. 36, 446-450. ANDERSONW. A. (1969) Nuclear and cytoplasmic DNA synthesis during early emhryogenesis of Paracentrotus lividus. J. Ultrastruct. Res. 26, 95-110. BlmOI, S. H. & CAMPBELLJ. W. (1965) Arginine and urea biosynthesis in the earthworm, Lumbricus terrestris. Comp. Biochem. Physiol. 15, 51-71. CmRDIJ. E. & ANDERSONE. P. (1968) Separation of purine and pyrimidine derivatives by thin-layer chromatography. Anal. Biochera. 22, 398--408. C-MANDALLD. E. (1975) Pyrimidine metabolism and its regulation in the sea urchin (Arbacia punctulata). Ph.D. thesis, University of Rhode Island, Kingston, RI. FRY B. J. & GROSSP. R. (1970) Patterns and rates of protein synthesis in sea urchin embryos---II. The calculation of absolute rates. Dev. Biol. 21, 125-146. GnJDICEG. (1973) Developmental Biology of the Sea Urchin Embryo. Academic Press, New York. GREEN H., & CHAN T. (1973) Pyrimidine starvation induced by adenosine in fibroblasts and lymphoid cells: role of adenosine deaminase. Science, N.Y. 182, 836-837. GULENS., SMrm P. C. & ~ L A Y G. C. (1974) Evidence for the control of pyrimidine biosynthesis in tissue minces by purines. Biochera. biophys. Res. Commun. 56, 934--939. HAGERS. E. & JONESM. E. (1967a) Initial steps in pyrimidine synthesis in Ehrlich ascites carcinoma in vitro--II. The glutamine-dependent carbamylphosphate synthetase. J. biol. Chem. 242, 5667-5673. HAcrERS. E. & JONESM. E. (1967b) A glutamlne-dependent enzyme for the synthesis of carbamylphosphate for pyrimidine biosynthesis in fetal rat liver. J. biol. Chem. 242, 5674-5680. H~~crn~ R. E. & PASTERNAKC. A. (1958) Inhibition of orotidylic acid decarboxylase, a primary site of carcinostasis by 6-azauracil. Biochim. biophys. Acta 30, 451-452. HARvEv E. B. (1956) The American Arbacia and Other Sea Urchins. Princeton University Press, Princeton, New Jersey. HEATH R. L. & HART J. L. (1970) Biosynthesis de novo

of purines and pyrimidines in Mesoceswides (Cestoda) 11. J. Parasitol. 56, 340-345. HORNEF. R. (1973) Urea metabolism in an estivating terrestrial snail Bulimulus dealbatus. Am. J. Physiol. 224, 781-787. ISHn K. & GREEN H. (1973) Lethality of adenosine for cultured mammalian cells by interference with pyrimidine biosynthesis. J. cell Sci. 13, 429-439. JONESM. E. (1972) Regulation of uridylic acid biosynthesis in eukaryotic cells. In Current Topics in Cellular Regulation (Edited by HORECKERB. L. & STADTMANE. R.) Vol. 6, pp. 227-265. Jo~_ks M. E., SPECrORL. & LIPMArCNF. (1955) Carbamylphosphate, the carbamyl donor in enzymatic citrulline synthesis. J. Am. Chem. Soc. 77, 819-820. Kmz S. & Com~ D. G. (1963) A new method for the determination of the base composition of ribonucleic acid. J. biol. Chem. 238, 3065-3067. KLrRELECB. (1972) Lack of carbamylphosphate synthesis in some parasitic platyhelminths. Comp. Biochem. Physiol. 4313, 769-780. KUSAMAK. & ROBERTSE. (1963) Carbon dioxide incorporation into the uracil of mouse liver and Ehrlich ascites tumor cells. Biochemistry 2, 573-576. LAYSE E. (1957) Spectrophotometric and turbidometric methods for measuring protein. III. Biuret method. In Methods in Enzymolooy (Edited by COLOWICKS. P. & KAPLANN. O.) Vol. III, pp. 450-451, Academic Press, New York. LONGO F. T. & PLUNKETTW. (1973) The onset of DNA synthesis and its relation to morphogenetic events of the pronuclei in activated eggs of the sea urchin Arbacia punctulata. Dev. Biol. 30, 56457. MORIUCHI A., KOGA K., YAMADAJ. & AKUMES. (1972) DNA synthesis and activities of pyrimidine-synthesizing enzymes in the silk gland of Bombyx mori. J. Insect Physiol. 18, 1463-1476. NORBY S. (1973) The biochemical genetics of rudimentary mutants of Drosophila melanoaaster. I. Aspartate carbamyltransferase levels in complementing strains. Hereditas 73, 11-16. O'DoNoVAN G. A. & NEUHARDJ. (1970) Pyrimidine metabolism in microorganisms. Bacteriol. Rev. 34, 278-343. PmLAmSET~ RAO J. & KARASEKM. A. (1970) Pyrimidine biosynthesis de novo in skin. J. biol. Chem. 245, 358-362. POREMBSKAZ., GORZHOWSKIB. & JEZEWSKAM. M. (1966) Utilization of t4C-orotate in the biosynthesis of pyrimidines in Helix pomatia and Celerio euphorbiae. Acta BIOchim. Pol. 13, 107-111. POWLESM. A., JANSSENSP. A. & GILMOURD. (1972) Urea formation in the green vegetable bug Nezara viridula. J. Insect Physiol. 18, 2343-2358. REICHARD P. (1957) Ornithine carbamyltransferase from rat liver. Acta Chem. Scand. 11, 523-536. RINALDIA. M. & MONROYA. (1969) Polyribosome formation and RNA synthesis in the early post-fertilization stages of the sea urchin egg. Dev. Biol. 19, 73-86. SCHNEIDERW. C. (1957) Determination of nucleic acids in tissues by pentose analysis, In Methods in Enzymology (Edited by COLOWlCKS. P. & KAPLANN. O.) Vol. III, pp. 680--684, Academic Press, New York. SMITHP. C., KNott C. E. & TREMBLAYG. C. (1973) Detection of the feedback control of pyrimidine biosynthesis in slices of several rat tissues. Biochem. biophys. Res. Commun. 55, 1141-1146. STEARNSL. W. (1974) Sea Urchin Development: Cellular and Molecular Aspects. Dowden, Hutchinson & Ross, Stroudsburg, Pennsylvania. TATIBANAi . & ITO K. (1969) Control of pyrimidine biosynthesis in mammalian tissues. I. Partial purification and characterization of glutamine--utilizing carbamylphosphate synthetase of mouse spleen and its tissue distribution. J. biol. Chem. 244, 5403-5413.

Pyrimidine biosynthesis in embryos of the sea urchin TRAMELLP. & CAMPBELLJ. W. (1970) Carbamylphosphate synthesis in a land snail, Strophocheilus oblonous. J. biol. Chem. 245, 663445641. TRAMELL P. R. & CAMPBELLJ. W. (1971) Carbamylphosphate synthesis in invertebrates. Comp. Biochem. Physiol. 40B, 395-406.

581

TI~r,mLAV G. C., Jmt~,mz U. & CRANDALLD. E. (1976) Pyrimidine biosynthesis and its regulation in the developing rat brain. J. Neurochem. 26, 57-64. YIP M. C. & KNOX W. E. (1970) Glutamine-dependent carbamylphosphate synthetase: Properties and distribution in normal and neoplastic rat tissues. J. biol. Chem. 245, 2199-2204.