[CANCER RESEARCH 32, 1837—1841,September 1972J

SUMMARY

Particles of certain rat tissue preparations contain aphosphate ester hydrolase that destroys carbamyl phosphate.This activity is similar to that of the alkaline phosphatases.Both have the same molar activities, pH curves, and particulatelocalization. Both are activated when solubilized with 1-butanol and are inhibited by adenine nucleotides and inorganicphosphate. Their reactions are not additive in the same assay.They have the same distributions in rat tissues and tumors,with highest activities in adult kidney and in fetal and adultsmall intestine. The two activities also increase in parallelduring development from fetal to adult kidney.

Aspartate transcarbamylase activity in homogenatescontaining the hydrolase can be measured qualitatively byinhibition of the hydrolase or by raising the carbamylphosphate concentration to allow for its hydrolysis during theassay.

INTRODUCFION

Aspartate transcarbamylase (EC 2.1 .3 .2 , carbamoylphosphate :L-aspartate carbamoyltransferase) has usually beenreported only in soluble fractions of rat tissues (8, 11, 18), butit has been found in a particulate fraction of humanleukocytes (14). A survey was made of aspartate transcarbamylase activities in both homogenates and soluble fractionsof rat tissues to find other active particulate fractions thatmight contribute in a special way to the pyrimidine synthesiscontrolled by this enzyme (5). This revealed that little or noactivity could be obtained under the usual conditions of assayin homogenates of certain tissues, among them a mammarytumor, although the soluble fractions of these same tissueswere highly active. Hydrolysis of carbamyl phosphate by analkaline phosphatase was responsible.

MATERIALS AND METHODSInbred rats of the NEDH strain were used routinely in these

experiments. Fetal animals were used between the 19th and22nd days of gestation. Estimation of their precise age wasbased on the correlation between body weight and ageaccording to the method of Gonzalez (2). The transplanted

â€T̃his investigation was supported by USPHS Grants AM00567 and

AM-K6-2018 from the National Institute of Arthritis and MetabolicDiseases, by Grant HD-04532 from the National Institute of ChildHealth and Human Development, and by United States Atomic EnergyCommission Contract AT(30-1)-3779 with the New England DeaconessHospital.

Received August 18, 1971 ; accepted May 19, 1972.

mammary tumors that were grown in male Fischer rats (SA,1C, 3230AC, and 8B) or in NEDH rats (205, 21 1, and Walker)all have been described elsewhere (10).

Fresh tissues were prepared as 10% homogenates in 0.15 MKC1 at 5°with a glass Teflon homogenizer and were diluted asindicated. Supernatant and particulate preparations wereobtained from the 10% homogenates after SO mm ofcentrifugation at 100,000 X g in a Spinco Model L preparativecentrifuge. The particulate fraction was rinsed 2 or 3 timeswith 0.15 M KC1 and then was resuspended (with gentlehomogenization) in the original volume of 0.1 5 M KC1.Carbamyl phosphate-'@ C was either prepared from14C-labeled potassium cyanate (New England Nuclear, Boston,

Mass.) and potassium phosphate by the procedure of Spectoret al. (15) or was purchased as the dilithium salt from NewEngland Nuclear. The ‘@ C-labeled carbamyl phosphate wasdiluted with commercial dffithium carbamyl phosphate(Sigma Chemical Co., St. Louis, Mo.) to give a specific activitybetween 4,000 and 10,000 cpm/pmole. Radioactivity wasdetermined in a Packard Tri-Carb scintillation counter in 10 mlof Aquafluor (New England Nuclear), with 69% countingefficiency for ‘@ C. Carbamyl phosphate solutions of givenmolarity were prepared fresh daily in ice-chilled water, and theconcentrations and specific activities were checked bydetermination of alkali-labile phosphate (6).

The procedure described by Morton (1 3) was followed forthe solubilization of phosphomonoesterase with 1-butanol.Twenty % homogenates were shaken for 15 mm at 37°with1-butanol equal (v/w) to the tissue weight. The preparationswere centrifuged at room temperature for I S mm. The yellowwater-soluble fraction was separated, and the residue wasresuspended in 0.15 M KC1 to the original volume. Bothfractions were then diluted for assay.

Enzyme Assays. Aspartate transcarbamylase determinationswere based on the method described by Lowenstein andCohen (1 1). From the counts fixed in carbamyl aspartate andfrom the specific activity of the carbamyl phosphate used, theassay results were calculated as units (pmoles carbamylaspartate formed per mm at 37°)per g, wet tissue weight.

The incubation mixture for the measurement of carbamylphosphate hydrolysis contained (per ml) 0.1 M diethanolaminebuffer, pH 9.3, 5 mM carbamyl phosphate, and 0.2 ml of a 1%tissue homogenate. One pmole of Mg@ per ml was added toassays of kidney where indicated. Aliquots (1 .0 ml) werepipetted into 1.0 ml of 10% cold trichloroacetic acid (at zerotime and after 7.5 and 1S mm at 37°)and then were placed inan ice bath and were flushed with a steady stream of CO2 for

SEPTEMBER 1972 I 837

Carbamyl Phosphate Hydrolysis in Rat Tissues and Tumorsby an Alkaline'

AnnemarieHerzfeldandW. EugeneKnox

Department of Biological Chemistry, Harvard Medical School, and the Cancer ResearchInstitute, New England DeaconessHospital, Boston,Massachusetts 02215

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TissueHomogenatesSupernatantsMaximal

velocityobserved

(@moles/min/g)Carbamyl

phosphateconcentration

(mM)Maximal

velocityobserved

(pmoles/min/g)Carbamyl

phosphateconcentration

(mM)Walker

tumorLiver (NEDH)RNC 205 tumorKidneyIntestine2.91

±0.59 (9)0.69 ±0.12 (5)1.40 ±0.34 (4)00.62 (1)a0.96 (1)―2.5

5.010.012.530.01.95

±0.48 (7)0.70 ±0.16 (16)1.21 ±0.35 (4)0.76 ±0.13 (4)1.03 (1)2.5

2.55.010.010.0

Annemarie Herzfeld and W. Eugene Knox

10 mm. Aliquots of 0.5 ml were then counted in 10 ml ofAqua.fluor. In every experiment, the spontaneous hydrolysis ofcarbamyl phosphate under identical incubation conditions andin the concentrations used was measured at each time point.These blank values (0.02 to 0.05 jimole carbamyl phosphatehydrolyzed per mm per ml) were subtracted from theexperimental values. Results are expressed as imoles carbamylphosphate hydrolyzed per mm at 37°per g, wet weight, oftissue.

Alkaline phosphatase was determined by the colorimetricprocedure described by Lowry et al. (12). p-Nitrophenylphosphate (1 .6 X i0@ M) (disodium trihydrate, Calbiochem,Los Angeles, Calif.); 0.1 M diethanolamine buffer, pH 9.3; and0.01 to 0.05 ml of 0.5 to 2% tissue homogenate in a totalvolume of 1.5 ml were incubated at 37°. After 15 mm, 1.5 mlof cold 0.25 N NaOH were added. The mixture wascentrifuged if it was not clear, and the color was readimmediately at 410 nm in a Zeiss PMQ II spectrophotometer.The molar extinction coefficient for standard p-nitrophenolunder those conditions was 1.5 X i04 . Results are expressed as@imolesof p-nitrophenol formed per mm at 37°per g, wetweight, of tissue.

For pH profiles of alkaline phosphatase and carbamylphosphate hydrolysis, a series of 0.1 M buffers were used asfollows: pH 4.5 to 5.4, sodium acetate; pH 6.0 to 6.9,2-(N-morpholino)ethanesulfomc acid; pH 7 to 8,N-2-hydroxyethylpiperazine-JV'-2-ethanesulfonic acid; pH 8 to8.5 , N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid(all from Calbiochem); and pH 8.7 to 11, diethanolamine-HC1.

RESULTS

With the usual concentration of S.0 mM carbamylphosphate as substrate, activity was proportional to theamounts of tissue supernatant fractions used. However,homogenates of several rat tissues did not have as muchaspartate transcarbamylase activity as the supematantfractions prepared from them. The activity in kidneyhomogenate was one-sixth of that in its supernatant (Chart1A). There was a similar discrepancy between the low activityin the homogenate of mammary tumor 205 and the high

Table 1

activity in its supernatant, although there was no suchdiscrepancy between the activities of homogenate andsupernatant fractions of the Walker tumor (Chart lB). Withthe addition of several times as much carbamyl phosphate tothe assay, the activity in the homogenate approached that ofthe already saturated reaction in the supernatant fractions.Homogenates of tissues showing this lessened reaction hadeven lower activity on a gram basis when more homogenatewas present in the reaction (Chart IA). Kidney homogenate orits particulate fraction, when added to other assays, alsoinactivated the aspartate transcarbamylase reaction in a solubleliver fraction and inactivated the ornithine transcarbamylasereaction of Streptococcus faecalis extracts (1 7). Both of thesereactions require carbamyl phosphate as substrate.

For several different tissues (Table 1), the aspartatetranscarbamylase activity obtained in homogenates was at leastas great as that in the supernatant, provided sufficientcarbamyl phosphate was added to the reaction. In these cases,activity was proportional to the amount of enzyme added. The

Chart 1. Aspartate transcarbamylase activities in soluble fractionsand homogenates of rat kidney (A) and tumors (B) with increasingcarbamyl phosphate concentration. A, assays with 0.25 ml of solublefraction (a) and homogenate (.) and with 0.5 ml of homogenate (X) ofkidney; B, assays with 0.25 ml of mammary tumor 205 (a and .) andWalker tumor (1'. and 0); (a and tx), soluble fractions; (. and a),homogenates.

CARBAMYLPHOSPHATE(mM)

Carbamyl phosphate requirements for aspartate transcarbamylase activities in some tissues ofadult ratsActivities (mean ±S.D.) for the number of determinations in a tissue (in parentheses) are given, with

the carbamyl phosphate concentrations necessary to produce these maximal activities in the usual assay(see “Materialsand Methods―).

a For homogenates of these tissues, even the concentrations of carbamyl phosphate that were usedwere not saturating (see also Chart 1).

1838 CANCER RESEARCH VOL. 32

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Hydrolytic activity(pmoles/min/g)withCarbamyl

p-NitrophenylphosphatephosphateHomogenate

ParticlesSoluble fraction71

8080 92

691-Butanol-treated

homogenateSoluble extract156

104125 85

CarbamylPhosphate Hydrolysis in Tissues

concentration of carbamyl phosphate required for theoptimum reaction rate in homogenates of kidney and intestinewas particularly high (above 30 mM) and, even for thesupematant fractions of these tissues, more than the usualcarbamyl phosphate concentration was necessary in order toobtain maximal activity that was proportional to the amountof enzyme used. It was notable that Walker tumor hadsignificantly more activity in the homogenate than in thesupernatant when measured under conditions for optimalactivity. However, in the other tissues, the activity in thehomogenates was substantially accounted for by that found inthe supernatant fractions.

The stability of carbamyl phosphate under the conditions ofthe aspartate transcarbamylase reaction was measured by itsdisappearance as described in “Materialsand Methods.―Therewas a small spontaneous hydrolysis of carbamyl phosphateamounting to 0.04 j.zmole/mmn/ml reaction mixture; this wasindependent of the presence of aspartate in the mixture.Disappearance of carbamyl phosphate was greatly increased bythe addition of kidney or intestine homogenates. Withsufficiently diluted homogenate preparations, thedisappearance of carbamyl phosphate was linear with time(Chart 2) and proportional to the amount of kidney added, upto at least 0.6 j.tmole of carbamyl phosphate removed per mmat 37°with 10 mg of homogenized kidney tissue. In moreconcentrated kidney homogenates, the release of inorganicphosphate from carbamyl phosphate was measured (6), andthis established a stoichiometric relationship between thehydrolytic formation of ‘@ CO2 and the concomitant inorganicphosphate release from carbamyl phosphate in the reaction.The hydrolase activity in the kidney homogenate persistedaf@terdialysis but was lost after the homogenate was boiled forS mm.

Because of the chemical nature of the reaction, itsoccurrence at alkaline pH, and its high activity in those tissuesknown to contain much alkaline phosphatase, the propertiesof the carbamyl phosphate hydrolase and the alkalinephosphatase were compared in the same tissue preparations.Half-maximal velocities occurred with substrate concentrations

Ui

>--J00>-1:

0.I-)

E

Chart 2. Spontaneous and enzymatic hydrolysis of carbamylphosphate (CP) by rat kidney homogenate. Reaction mixtures of 1 mlcontained 0.2 ml of 1% kidney homogenate (@) or no enzyme (X ); thedifference is shown by (.).

of S X l0@ M carbamyl phosphate (one-tenth that in theaspartate transcarbamylase reaction mixtures) and 1 X 10@ Mp-nitrophenyl phosphate. Both reactions were maximal nearthe alkaline pH optimum of the aspartate transcarbamylasereaction, and their pH curves were generally similar (Chart 3).Under the conditions used (pH 9.3), carbamyl phosphatehydrolysis was about two-thirds of the molar rate obtainedwith p-nitrophenyl phosphate for a given preparation. Bothactivities centrifuged almost wholly with the particulatefraction at 100,000 X g, and both were solubiized by butanoltreatment, with some increase in total activity (Table 2).

Carbamyl phosphate hydrolase and alkaline phosphataseactivities were not additive and were in fact decreased whenassayed with both substrates present in the same reactionmixture (Table 3). There were parallel inhibitions of bothactivities by adenine nucleotides and by inorganic phosphate.One difference between the 2 reactions was limited to kidneyhomogenates; 1 mM MgCl2 increased the rate of carbamylphosphate hydrolysis but decreased the hydrolysis ofp-nitrophenyl phosphate (Table 3). There was no significanteffect of MgCl2 on the reactions in the other tissues examined.

E

0

0

a>.I

I-

I')CO

U)

E

Chart 3. The pH curves showing the hydrolysis of carbamylphosphate (a) and p-nitrophenyl phosphate (.) by female rat kidneyhomogenates. Each point is the mean of 2 assays, each with 2 enzymeconcentrations. Buffers used at the various pH's are givenin “Materialsand Methods.―

Table2Distribution of carbamyl phosphate and p-nitrophenyl phosphate

hydrolytic activities in rat kidneyHomogenates were separated at 100,000 x g. Treatment with

1-butanol is described in “Materialsand Methods.―

pH

TIME (mm)

SEPTEMBER 1972 I839

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Hydrolytic activity(j@moles/min/g)withCarbamyl

p-NitrophenylphosphatephosphateAdditions(5

X 10@ M) (1.6 X 10@M)None42±

13@@(12)b 70±24(16)Carbamylphosphate,C155

X iO@Mp-Nitrophenylphosphate,8C5

x 10-sMATP,5X103M193ADP,

5 x 10@ M2016AMP,5X103M54Inorganic

phosphate, 5 X l0@ M 1712MgC12,l03M58±17(11) 44± 10(5)

Hydrolytic activity(@moles/min/g)withCarbamyl

p-NitrophenylTissuephosphatephosphateKidney,―

fetal0 6.7 ± 1 6b(4)CKidney,'@adultFemale55

±19 (20) 70 ±24(16)Male40±15 (12) 46 ±14(4)Intestine,

fetal41 ±9 (3)31Intestine,adult43 ±6 (4) 62 ± 9(3)Liver,

fetal0 2.3 ±0.5(4)Liver,adult2±1(4) 1.0±0.4(10)Spleen2

1.4 ±0.5(4)Brain0,0 2.7 ± 0.9(4)Lung0

4.9± 1.4(4)Pancreas0,0 2.0 ±0.4(3)Adrenal0Neoplasms

RNC2OS30±9(4)5to30@(4)7A(generation 11)1 1 ±6 (4) 2 ±0.5(4)RNC211 (generation13A)3105A

(generation 37)0, 2 3 ±1(3)Walker(generation1A)13230

AC (generation 21)2, 1.4 6.5 (2)

Annemarie Herzfeld and W. Eugene Knox

Table 3Inhibition of carbamyl phosphate hydrolase and alkaline phosphatase

in kidney homogenatesin female rats E

a

0

a

I

U)CO

U,

E

Birth6 (4 22 30 38AGE(days)

Chart 4. Developmental formation of carbamyl phosphate hydrolase(•)and alkaline phosphatase (a) in rat kidney. The same animals wereassayed for both activities; 1 mM Mg@ was added to the carbamylphosphate assays. Vertical bars (representing 1 S.D.) distinguish pointsthat are the means of 3 or more animals. Adult values are from females.There was no sex difference at 38 days of age.

The distribution of the 2 hydrolyzing activities inhomogenates of different normal (adult and fetal) tissues andin 6 neoplastic tissues is shown in Table 4. The 2 activitieswere approximately equal and parallel in all the tissues tested.Both activities were fractionally higher in the kidneys offemale rats than in males, but sex differences were not notedin other tissues. Highest activities were found in small intestineand adult kidney, tissues that were already known (9) to berich in alkaline phosphatase. Both activities were also presentin fetal intestine at levels almost as high as in adults. Fetalkidney showed essentially no hydrolyzing activity of eitherkind. Relatively little of either activity was found in themammary tumors examined, except in Tumor 205, as alreadydescribed.

The 2 hydrolyzing activities developed with age in kidneyfrom the very low fetal levels to high adult levels according tothe same time pattern (Chart 4). Both were virtually absentuntil the late suckling stage (Day 16) and rose rapidly duringthe next week, then increased more gradually toward adultlevels. The presence of optimal Mg@, which in this tissueincreased the activity of carbamyl phosphate hydrolase relativeto that of alkaline phosphatase, did not otherwise alter therelationship between the 2 activities.

DISCUSSION

The potent hydrolysis of carbamyl phosphate by certaintissues at alkaline pH has not been previously described.Grisolia et aL (3, 4) described hydrolysis of carbamylphosphate by an enzyme extracted from brain thatpreferentially cleaved acetyl phosphate. The pH optimum waspH 4, and the reaction apparently did not occur at alkalinepH's. The carbamyl phosphate hydrolysis that we found alsocannot be identified with the reverse reaction of a carbamyl

a Mean ±S.D.b Numbers in parentheses, number of determinations.C Same as control.

Table 4Hydrolytic activities in homogenates ofnormal and

neoplastic rat tissuesAlkaline phosphatase assays of kidney, intestine, and neoplasm RNC

205 used 0.5% homogenates in 1.5-ml reaction mixtures. Up to 20times more concentrated homogenates were tested for all other tissues.Assays for carbamyl phosphate hydrolysis contained 0.2 ml of 1%homogenates/ml incubation mixture of kidney, intestine, and RNC205, and up to 20-fold more of other, less active tissues. Adult tissueswere used except as indicated.

a Measured without the addition of MgCl2 , which increasedcarbamyl phosphate hydrolysis and decreased p-nitrophenyl phosphatehydrolysis only in kidney (Table 3).

b Mean ±S.D.C Numbers in parentheses, number of determinations.

d Alkaline phosphatase increased, but carbamyl phosphate hydrolasedid not increase, with tumor size.

1840 CANCER RESEARCH VOL. 32

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Carbamyl Phosphate Hydrolysis in Tissues

phosphate kinase, like that found in bacterial systems (7),since the present activity is not accelerated by ADP but isinhibited by both ADP and ATP.

The activity we found closely resembles that of the alkalinephosphatases. Its activity is low at pH 4 and maximal at pH9.2 to 9.5, in parallel with the hydrolysis of p-nitrophenylphosphate. The molar activities of both reactions are of thesame order of magnitude under optimal conditions. Both arepresent almost wholly in the particulate fraction of kidney,and both are solubilized (with increased yield) by 1-butanoltreatment. The 2 reactions do not occur additively in the sameassay but are mutually inhibitory. Carbamyl phosphatehydrolysis is inhibited by inorganic phosphate and adeninenucleotides to an extent very similar to the inhibitions ofalkaline phosphatase. The activity found by us is notprominent in brain, as is the enzyme found by Grisolia, but isprominent in kidney and in fetal and adult intestine, which arealso rich in alkaline phosphatase. Other tissues that aredeficient in one of these activities also lack the other. Bothactivities are higher in female than in male rat kidneys, asalready described for alkaline phosphatase ( 16). The 2 enzymeactivities in kidney develop in parallel with age. Extracts ofboth kidney and intestine hydrolyze carbamyl phosphate inproportion to the total alkaline phosphatase present, althoughthese 2 alkaline phosphatases can be differentially inhibited byphenylalanine (1). In all of these comparisons, there is onlythe minor discrepancy that, in kidney, Mg'@slightly stimulatescarbamyl phosphate hydrolysis and inhibits alkalinephosphatase activity. Carbamyl phosphate appears to be asubstrate for the alkaline phosphatases in these rat tissues.

The significance of the carbamyl phosphate hydrolyzingactivity for the moment lies only in its interference with theactivity of aspartate transcarbamylase or other enzymesforming or using carbamyl phosphate. The physiological roleof the carbamyl phosphate hydrolyzing activity is unknown, asis, of course, the physiological role of alkaline phosphatase.The list of tissues in Table 4 extends the relativeconcentrations of alkaline phosphatase to more than the 9 rattissues that are included in the other most complete listing ofthis frequently studied enzyme (9). The tissue comparisonsindicate that alkaline phosphatase (and carbamyl phosphatehydrolysis) is relatively low in most tumors.

Carbamyl phosphate hydrolysis is so rapid in homogenatesof kidney and intestine that even very large additions ofcarbamyl phosphate may not satisfy conditions for optimalaspartate transcarbamylase assays. Aspartate transcarbamylaseactivity is more readily observable in such tissues in the solublefractions that lack most of the hydrolase, and aspartatetranscarbamylase has, in fact, usually been measured only inthe soluble fractions of tissues. Without special precautions tocontrol the hydrolase, it is not possible to determine whethersignificant amounts of aspartate transcarbamylase activity alsoreside in the particles of some of these tissues, as appears to bethe case in the Walker tumor (Table 1).

ACKNOWLEDGMENTS

The authors wish to thank Dr. Morris Yip, who kindly synthesizedcarbamyl phosphate-' 4C and provided purified ornithinetranscarbamylase, and we also thank Mr. Bruce B. Duncan for histechnical assistance.

REFERENCES

1. Fishmann, W. H., Green, S., and Inglis, N. I. Organ SpecificBehavior Exhibited by Rat Intestine and Liver AlkalinePhosphatase. Biochim. Biophys. Acta, 62: 363—375, 1962.

2. Gonzalez, A. W. A. The Prenatal Growth of the Albino Rat. Anat.Record,52: 117—138,1932.

3. Grisolia, S., Caravaca, J., and Joyce, B. K. Purification andProperties of Brain Carbamyl and Acyl Phosphatase. Biochim.Biophys. Acta, 29: 432—433, 1958.

4. Grisolia, S., and Marshall, R. 0. Enzymic Decomposition of theActive Intermediate in Citrulline Synthesis. Biochim. Biophys.Acta, 14: 446—447, 1954.

5. Herzfeld, A., and Knox, W. E. Aspartate TranscarbamylaseConcentrations in Relation to Growth Rates of Fetal, Adult, andNeoplastic Rat Tissues. Cancer Res., 32: 1842—1847, 1972.

6. Jones, M. E. Carbamyl Phosphate Synthesis and Utilization.Methods Enzymol., 5: 903—925, 1962.

7. Jones, M. E., and Lipmann, F. Chemical and Enzymatic Synthesisof Carbamyl Phosphate. Proc. NaIl. Acad. Sci. U. S., 46:1194—1205,1960.

8. Kim, S., and Cohen, P. P. Transcarbamylase Activity in Fetal Liverand in Liver of Partially Hepatectomized Parabiotic Rats. Arch.Biochem. Biophys., 109: 421—428, 1965.

9. Knox, W. E. Enzyme Patterns in Fetal, Adult and Neoplastic RatTissues. Basel, Switzerland: S. Karger, 1971.

10. Knox, W. E., Linder, M., and Friedell, G. H. A Series ofTransplantable Rat Mammary Tumors with Graded Differentiation,Growth Rate, and Glutaminase Content. Cancer Res., 30:283—287, 1970.

11. Lowenstein, J. M., and Cohen, P. P. Studies on the Biosynthesis ofCarbamylaspartic Acid. J. Biol. Chem., 220: 57—70,1956.

12. Lowry, 0. H., Roberts, N. R., Wu, M. L., Hixon, W. S., andCrawford, E. J. The Quantitative Histochemistry of Brain. II.Enzyme Measurements. J. Biol. Chem., 207: 19—37,1954.

13. Morton, R. K. The Purification of Alkaline Phosphatases of AnimalTissues. Biochem. J., 57: 595—603,1954.

14. Smith, L. H., Jr., and Baker, F. A. Pyrimidine Metabolism in Man.I. The Biosynthesis of Orotic Acid. J. Clin. Invest., 38: 798—809,1959.

15. Spector, L., Jones, M. E., and Lipmann, F. Carbamyl Phosphate.Methods Enzymol., 3: 653—655,1957.

16. Wacker, G. R., Zarkowsky, H. S., and Burch, H. S. Changes inKidney Enzymes of Rats after Birth. Am. J. Physiol., 200:367—369,1961.

17. Yip, M. C. M., and Knox, W. E. Glutamine-dependentCarbamoylphosphate Synthetase: Properties and Distribution inNormal and Neoplastic Rat Tissues. J. Biol. Chem., 245:2199—2204,1970.

18. Young, J. E., Prager, M. D., and Atkins, I. C. ComparativeActivities of Aspartate Transcarbamylase in Various Tissues of theRat. Proc. Soc. Exptl. Biol. Med., 125: 860—862,1969.

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