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Home , Dihydroorotase, Pyrimidine, Uridine, Uridine monophosphate

J. Nutr. Sci. Vitaminol., 37, 517-528, 1991

Effect of Dietary Protein on -Metabolizing in Rats

Masae KANEKO, Shigeko FUJIMOTO, Mariko KIKUGAWA, Yasuhide KONTANI, and Nanaya TAMAKI* Laboratory of Nutritional Chemistry, Faculty of Nutrition, Kobe-Gakuin University, Nishi-ku, Kobe 651-21, Japan (Received February 25, 1991)

Summary The effect of dietary protein on pyrimidine-metabolizing enzymes was studied in the rat. The activities of dihydropyrimidine

and ƒÀ-ureidopropionase in the livers of rats fed a protein free diet were significantly decreased, while the activity of dihydropyrim

idinase was unaffected. Protein deficiency (5%) also decreased the activity of ƒÀ-ureidopropionase. On the other hand, a high-protein diet

(60%) increased the level of ƒÀ-ureidopropionase. The activities of ƒÀ- alanine-oxoglutarate aminotransferase (aminobutyrate aminotransferase) and D-3-aminoisobutyrate-pyruvate aminotransferase ((R)-3-amino-2-

methylpropionate-pyruvate aminotransferase), which are present in mi tochondria, depended on the amount of protein in the diet.

ions supplemented in the diet and given by injection did not affect the activities of rat liver pyrimidine-metabolizing enzymes (dihydropyrimi

dine dehydrogenase, , ƒÀ-ureidopropionase, ƒÀ-alanine - oxoglutarate aminotransferase and D-3-aminoisobutyrate-pyruvate ami

notransferase). Dietary resulted in the accumulation of in the liver, but did not affect the activities of pyrimidine-metabolizing enzymes.

Key Words dihydropyrimidine dehydrogenase, dihydropyrimidinase, ƒÀ- ureidopropionase, ƒÀ-alanine-oxoglutarate aminotransferase, D-3-amino

isobutyrate-pyruvate aminotransferase, pyrimidine

The de novo of (UMP) involves six enzymatic reactions and appears to be encoded by only three structural genes in animals. The active sites of the first three enzymes, carbamoyl- synthase, aspartate transcarbamylase, and , are on a single large polypeptide

* To whom correspondence should be addressed .

Abbreviations: ƒÀ-AlaAT I, ƒÀ-alanine-oxoglutarate aminotransferase; ƒÀ-AlaAT II, D-3

- aminoisobutyrate-pyruvate aminotransferase.

517 518 M. KANEKO et al.

that aggregates to form the native multienzymatic protein (1-3) . The active sites of the fifth and sixth enzymes of the pathway , orotate phosphoribosyl- and

orotidylate decarboxylase, are also composed of a single polypeptide chain (4) . Carbamoyl-phosphate synthetase is the rate-limiting in the de novo UMP

- biosynthetic pathway and is the site of feedback inhibition by

(UTP) and activation by 5-phosphoribose 1-bisphosphate (5, 6). Weber et al. (7) found that the incorporation of into DNA inceased after partial hep atectomy, and with hepatoma growth, but decreased in rat livers over time after

birth. On the other hand, the catabolism of thymidine to CO2 increased with

post-natal time and was inactivated after partial hepatectomy and in rapidly

growing neoplasms (7). Uracil and are metabolized to ƒÀ-alanine and ƒÀ- aminoisobutyrate, respectively, by dihydropyrimidine dehydrogenase , dihydropy rimidinase and ƒÀ-ureidopropionase in cytosol , as ilustrated in Fig. 1. ƒÀ-Alanine and ƒÀ-aminoisobutyrate are transported into mitochondria (8 , 9), where they are further metabolized to acetyl-CoA and propionyl-CoA , respectively, by ƒÀ-alanine - oxoglutarate aminotransferase (ƒÀ-AlaAT I), D-3-aminoisobutyrate-pyruvate ami notransferse (ƒÀ-AlaAT II) and methylmalonate semialdehyde dehydrogenase in

the mitochondrial matrix (9-11). Dihydropyrimidine dehydrogenase has been

Fig. 1. Degradation pathway of pyrimidine. 1, dihydropyrimidine dehydrogenase

[EC 1.3.1.1]; 2, dihydropyrimidinase [EC 3.5.2.2]; 3, ƒÀ-ureidopropionase [EC

3.5.1.6]; 4, aminobutyrate aminotransferase [EC 2.6 .1.19]; 5, (R)-3-amino-2

- methylpropionate-pyruvate aminotransferase [EC 2 .6.1.40]; 6, methylmalonate semialdehyde dehydrogenase [EC 1.2.1.27]. Abbreviations: ƒÀ-Ala , ƒÀ-alanine; ƒÀ- AIB, ƒÀ-aminoisobutyrate.

J Nutr. Sci. Vitaminol. PROTEIN LEVEL ON PYRIMIDINE 519

identified as the rate-limiting enzyme of pyrimidine catabolism (12, 13). In the spectrum of hepatomas, dihydropyrimidine dehydrogenase activity decreased in parallel with increased growth rate (7, 14). In the livers of rats fed a protein-deficient diet, DNA synthesis and activity of thymidine kinase decreased to less than half of control values (15). In contrast, incorporation of [6-14C]-orotate into RNA and activity significantly increased (15). Ammonium ions have been shown to stimulate pyrimidine bio synthesis as a result of synthesis by mitochondria carbamoyl phosphate synthetase (16-18). In order to define the relevance of normal and abnormally increased uracil for hepatic pyrimidine catabolism, we investigated the effect of dietary protein, ammonium ions and uridine on pyrimidine-catabolizing enzymes in the rat liver.

MATERIALS AND METHODS

Chemicals. All chemicals used were of analytical grade and were purchased

from Nacalai Tesque (Kyoto) unless otherwise stated. Uridine was a product of Sigma Chemicals. 5-Bromo-5,6- was synthesized from 5,6-dihydro

uracil by direct bromination (19). ƒÀ-[2-14C]alanine was purchased from New England Nuclear. Materials for the animals' diets were obtained from Oriental Ltd., Tokyo.

Animals. Male albino rats (Sprague-Dawley, 130-150g) were housed in individual screen-bottom cages in a room maintained at 23•}1•Ž with 50% hu

midity under controlled lighting conditions (12h light-dark cycle). The animals were fed a commercial stock diet and ad libitum for 1 week before the experiment to acclimatize them to the new environment. Acclimatized rats

showing progressive weight gain were selected and separated into groups. All

animals were sacrificed between 10:00a.m. and noon except those used in the ammonium acetate injection experiments. Low and high protein diets. The compositions of the low and high-protein

diets are shown in Table 1. The animals received 20g of feed per day. Ammonium chloride and uridine diets, and ammonium acetate injection. The

composition of ammonium chloride and uridine diets is shown in Table 2. The 20% casein diet was used as the control.

Ammonium acetate was dissolved in physiological saline to be prepared to 3.7 M and 3.7mol/kg of body weight was intraperitoneally injected 4 or 8 times at 1-h intervals. One hour after the last injection, the animals were sacrificed.

Enzyme assays: dihydropyrimidine dehydrogenase. The livers were homog enized in 10vol. buffer A (10mM potassium phosphate, pH 7.4, containing 5mM 2

- mercaptoethanol and 2.5mM MgCl2). After centrifugation, the supernatant was heated to 50•Ž for 1min, and then cooled to 4•Ž. The precipitate was discarded

after centrifugation and the supernatant was adjusted to pH 4.85 with 5% acetate. After centrifugation, the supernatant was neutralized with 0.5M KOH and treated

Vol. 37, No. 5, 1991 520 M. KANEKO et al.

Table 1. Compositions of the experimental diets .

a Sucrose e corn starch=2 :1. b Minerals were (in g/kg of diet): CaHPO4•E2H2O 8 .736; KH2PO4 15.432; NaH2PO4 5,610; NaCl 2 .796; Ca-lactate 21,054; Fe-citrate 1908;

MgSO4 4.302; ZnCO3 0.066; MnSO4•E4-6H2O 0 .072; CuSO4•E5H2O 0.018; KI 0.006. c Vitamins were (in IU/kg of diet); retinyl acetate 10 ,000; cholecalciferol 2,000, and (in mg/kg diet): ƒ¿-tocopheryl acetate 100; menadione 104; thiamin hydrochloride 24; riboflavin 80; pyridoxine hydrochloride 16; cyanocobalamin 0 .01; ascorbic acid 600; D-biotin 0.4; folic acid 4; pantothenate 100; p-aminobenzoic acid 100; niacin

120; chloride 4,000.

Table 2. Compositions of the diets supplemented with ammonium chloride and uridine.

1 Diets used for the experiments on the effect of ammonium ions . 2 Diets used for the experiments on the effect of uridine . a Sucrose: corn starch=2:1. b Salt mixture and vitamin mixture are shown in Table 1.

with ammonium sulfate. The precipitate obtained at 30-50% saturation was

dissolved in a minimum volume of buffer A and used for enzyme analysis . Dihydropyrimidine dehydrogenase activity was followed by measuring the rate

of the disappearance of NADPH at 37•Ž (20) . The standard assay mixture contained 50mM potassium phosphate, pH 7 .4, including 0.15mM uracil and 0.15 mM NADPH in a total volume of 3 .0ml.

Dihydropyrimidinase. The livers were homogenized in 10vol . 10mM potas sium phosphate, pH 7.0, containing 10mM 2-mercaptoethanol . After centrifu gation, the supernatant was used for analyses of dihydropyrimidinase and ,ƒÀ-

J. Nutr. Sci. Vitaminol. PROTEIN LEVEL ON PYRIMIDINE METABOLISM 521

ureidopropionase activities. Dihydropyrimidinase was measured by the method of Brooks et al. (21). The enzyme activity was assessed by measuring the rate of disappearance of 5-bromo

- 5,6-dihydrouracil at 225nm in a cuvette with a 1.0cm light path at 37•Ž. The extinction coefficient of 5-bromo-5,6-dihydrouracil at 225nm was 3,24•~103M-1•E

cm-1, at pH 8.2, in 50mM Tris-HCl buffer pH 8.2 and 0.17mM 5-bromo-5,6 - dihydrouracil in a total volume of 3.0ml. ƒÀ-Ureidopropionase . ƒÀ-Ureidopropionase activity was measured with respect

to the rate of formation of (22). The standard reaction mixture con tained 0.1M sodium phosphate, pH 7.0, including bovine serum albumin (0.1%),

10mM MgCl2, 1mM EDTA, 5mM 2-mercaptoethanol, and 2mM N-carbamoyl-ƒÀ- alanine. Incubation was carried out in a shaking water bath for 30min at 37•Ž.ƒÀ

-AlaAT I . The livers were homogenized in 10vol. 10mM potassium phos

phate, pH 7.5, containing 1mM EDTA, 2mM 2-mercaptoethanol and 40ƒÊM pyridoxal 5•L-phosphate. The homogenate was briefly centrifuged. The supernatant was used to analyze ƒÀ-AlaAT I and ƒÀ-AlaAT II activities. The activity of ƒÀ-AlaAT I was determined by the amount of malonate

semialdehyde produced from ƒÀ-alanine with 2-oxoglutarate according to methods

previously described (23). The reaction mixture contained 50mM sodium borate

(pH 8.8), 5mM 2-mercaptoethanol, 0.5mM pyridoxal 5•L-phosphate, 1mM ƒÀ- [2-14C]alanine (specific activity 37 GBq/mol) and l0mM 2-oxoglutarate in a final volume of 1.0ml. The incubation was carried out in a shaking water bath for 30 min at 37•Ž. The reaction was terminated by adding 0.5ml 2M HCl and the tube

was immediately transferred to an ice bath. After adding 0.02ml 1M ƒÀ-alanine and 2ml 0.2% 2,4-dinitrophenylhydrazine (in 2M HCl) the mixture was allowed to stand at 37•Ž for 15min and the dinitrophenylhydrazone formed was extracted by

shaking with 5.0ml toluene. After brief centrifugation, the radioactivity of a 2-ml aliquot of the extract was measured with a Packard Tri-Garb liquid scintillation

spectrometer (460 CD type). ƒÀ-AlaAT II. ƒÀ-AlaAT II activity was assayed according to the above method

for ƒÀ-AlaAT I activity except that 10mM pyruvate was used as the amino acceptor.

Protein measurement. Protein concentration was determined by the method

of Lowry et al. (24), using bovine serum albumin as a standard. Analytical assays. The livers were quickly removed after the rats were sac rificed; they were then sliced manually using a stainless-steel razor. The sliced

tissues were immediately freeze-clamped, weighed, and homogenized in 5vol. of 0.6 M perchloric acid. After centrifugation, the supernatant was neutralized with 4M

KOH. Uracil and uridine were separated by HPLC using a linear

gradient from 50mM potassium phosphate buffer, pH 5.6, to 20% methanol for 40 min, with a reverse-phase C 18 analytical column (, 5ƒÊm, 0.39•~15cm). The wavelength was set at 254nm.

Statistical analysis. One-way analysis of variance was used to compare the

groups. When a significant difference (p<0.05) was found among groups, the

Vol. 37, No, 5, 1991 522 M. KANEKO et al. statistical significance of difference between values of the data was assessed by Student's t-test.

RESULTS

Effect of amount of dietary protein on pyrimidine-catabolizing enzymes Figure 2 and Table 3 show the activities of dihydropyrimidine dehydrogenase, dihydropyrimidinase, ƒÀ-ureidopropionase, ƒÀ-AlaAT I and ƒÀ-AlaAT II, and the effect of dietary protein. In Experiment I (Fig. 2 and Table 3), weight gains

(mean•}SE) of rats fed protein-free, 5% casein and 20% casein diets for 1 week were 24.7•}1.3g, 10.0•}1.2g and 40•}4.6g, respectively, and liver wet weights were 4.57•}0.19g, 5.80•}0,06g and 9.27•}0.71g, respectively. In Experiment II

(Fig. 2 and Table 3), weight gains of rats fed 5%, 20%, 40% and 60% casein diets for 4 weeks were 12.7•}4.4g, 113.3•}11.1g, 110.3•}3.8g and 112.7•}5.5g, re spectively, and liver wet weights were 5.63•}0.15g, 10.90•}0.58g, 10.67•}0.77g and 9.23•}0.67g, respectively. When enzyme activity was expressed as nmol/min per g of tissue, dihydropy rimidine dehydrogenase, dihydropyrimidinase and ƒÀ-ureidopropionase activities in

Table 3. Effect of dietary protein on rat liver ƒÀ-AlaAT I and ƒÀ-AlaAT II.

The activities of ƒÀ-AlaAT I and ƒÀ-AlaAT II were determined as described in

MATERIALS AND METHODS. Each value is the mean±SE of three or four separate

experiments. * p<0.05, ** p<0.01 compared to 20% casein.

Fig. 2. Effect of dietary protein on rat liver pyrimidine-metabolizing enzymes. (A)

Dihydropyrimidine dehydrogenase, (B) dihydropyrimidinase and (C) ƒÀ-

ureidopropionase. Protein concentrations in the precipitate obtained at 30-50%

ammonium sulfate saturation (A) and in the supernatant after centrifugation of

the homogenate (B and C) were estimated as described in MATERIALS AND

METHODS. Each value is the mean•}SE. * p<0.05, ** p<0.01 compared to

control (20% casein diet).

J. Nutr. Sci. Vitaminol. PROTEIN LEVEL ON PYRIMIDINE METABOLISM 523

(A) Dihydropyrimidine dehydrogenase

(B) Dihydropyrirnidinase

(C) ƒÀ-Ureidopropionase

Vol. 37, No. 5, 1991 524 M. KANEKO et al.

the livers of rats fed protein-free and low-protein diets were significantly lower than control (Fig. 2). Specific activities of ƒÀ-ureidopropionase in the livers of rats fed

protein-free and 5% casein diets were 38% and 66%, respectively, of the control

(20% casein) value. The specific activity of dihydropyrimidine dehydrogenase, a key enzyme of pyrimidine catabolism, was less in the protein-free group than in the

20% casein group, but was not significantly different from the 5% casein group . The specific activity of dihydropyrimidinase was unaffected by the protein-free and 5% casein diets.

High-protein diets (40% and 60% casein) did not significantly alter the activity of dihydropyrimidine dehydrogenase or dihydropyrimidinase. The activity

of ƒÀ-ureidopropionase increased in the livers of rats fed the 60% casein diet. ƒÀ-Alanine and ƒÀ-aminoisohutyrate form in cytosol from uracil and thymine , respectively, transfer into mitochondria, and are further metabolized to malonate semialdehyde and methylmalonate semialdehyde, respectively (9, 10). ƒÀ-Alanine is a substrate for both ƒÀ-AlaAT I and ƒÀ-AlaAT II (22, 23), and ƒÀ-aminoisobutyrate metabolized from thymine is a substrate for ƒÀ-AlaAT II (22). The effect of dietary protein on rat liver ƒÀ-AlaAT I and ƒÀ-AIlAT II is shown in Table 3. Both ƒÀ-AlaAT I and ƒÀ-AlaAT II activities were increased by high-protein diets and decreased by protein-deficient diets.

Effect of ammonium ions on pyrimidine-catabolizing enzymes in vivo Weight gains of rats fed control and 2% ammonium chloride diets for 1 week were 31.6•}3.5g and 20.6•}2.0g, respectively, and liver wet weights were 9.82•}

0.12g and 8.34•}0.20g, respectively. The stimulation of pyrimidine biosynthesis by ammonium ions (16-18) activates pyrimidine catabolism. However, the activities of pyrimidine-catabolizing enzymes were not influenced by ammonium chloride diets (Table 4). When ammonium acetate was injected intraperitoneally 4 or 8

Table 4. Effect of ammonium chloride diet on rat liver pyrimidine-degrading enzymes.

Five rats were used in each experimental subgroup. Dihydropyrimidine

dehydrogenase, dihydropyrimidinase, ƒÀ-ureidopropionase, ƒÀ-AlaAT I and ƒÀ-AlaAT

II activities were determined as described in MATERIALS AND METHODS. Each

value is the mean•}SE.

J. Nutr. Sci. Vitaminol. PROTEIN LEVEL ON PYRIMIDINE METABOLISM 525

Table 5. Effect of dietary uridine on uridine and uracil contents, and on pyrimidine-catabolizing rat liver enzymes.

Four rats were used in each experimental subgroup. 1The contents of uridine and

uracil were estimated as described in MATERIALS AND METHODS and are expressed

as nmol/g of tissue. 2 The activities of dihydropyrimidine dehydrogenase , dihydropyrimidinase, ƒÀ-ureidopropionase, ƒÀ-AlaAT I and ƒÀ-AlaAT II were expressed

as ƒÊmol/min per g of tissue. Each value is a mean•}SE. ** p<001 compared to

control.

times as described in MATERIALS AND METHODS, the ammonium ions did not alter

these five pyrimidine catabolizing enzymes (data not shown).

Effect of uridine diet on the content of uracil, uridine, and pyrimidine-metabolizing enzymes in the rat liver

Weight gains of rats fed control and 5% uridine diets for 1 week were 30 .3•} 3.2g and 15.7•}6.6g, respectively, and liver wet weight were 8.56•}0.39g and 8.65•}0.49g, respectively.

Table 5 shows the liver uridine and uracil contents , and the effects of the uridine diet. When the animals were fed the 5% uridine diet for 1 week, the level of uracil increased 68-fold, but the content of uridine was unaffected. Uridine is metabolized to uracil in the serosal secretions after uridine crosses the mucosal epithelial layer of the small intestine (25). Pyrimidine-metabolizing enzymes such as dihydropyrimidine dehydrogenase, dihydropyrimidinase and ƒÀ-ureidopropion ase, were also unaffected by the uridine diet as was the case of ammonium acetate injection.

DISCUSSION

The incorporation of [3H]-orotate into RNA increased with activation of uridine kinase on a protein-deficient regimen (15). On the other hand, the activity of dihydropyrimidine dehydrogenase, the rate-limiting enzyme in the degradation of uridine, decreased in animals on the protein-free diet (Fig. 2). These adaptations would favor the preservation of function in liver cells during protein malnutrition . Protein-deficient and protein-free diets inactivated the ƒÀ-ureidopropionase of py rimidine-metabolizing enzymes (Fig. 2). As ƒÀ-ureidopropionase can also be rate

Vol. 37, No. 5, 1991 526 M. KANEKO et al.

limiting (26, 27), it may have an important role in pyrimidine metabolism . Earlier reports by Dagg et al. (28) and Barrett et al. (29) suggest that dihydropyrimidine dehydrogenase, dihydropyrimidinase and ƒÀ-ureidopropionase might normally

function in a complex. However, we recently purified rat liver ƒÀ-ureidopropionase

which lacked dihydropyrimidine dehydrogenase and dihydropyrimidinase (22) . Moreover, judging from the effect of dietary protein on enzyme activities (Fig . 2), ƒÀ-ureidopropionase might be controlled by regulatory mechanisms . Detailed studies are necessary to clarify the regulatory effect of ƒÀ-ureidopropionase in

pyrimidine metabolism under limited conditions. It is well known that L-amino acid aminotransferase activity is affected by

dietary protein: it is low in the livers of animals fed a protein-free diet and high in the livers of animals fed a high-protein diet (30-32). ƒÀ-Alanine and ƒÀ-aminoiso

butyrate formed from uracil and thymine, respectively, do not form proteins. ƒÀ- AlaAT I activity in the rat liver was significantly increased by prednisolone injection, while ƒÀ-AlaAT II activity was not (10, 23). Though ƒÀ-alanine and ƒÀ-

aminoisobutyrate do not form proteins, both ƒÀ-AlaAT I and ƒÀ-AlaAT II enzyme activities might be regulated by dietary protein.

Excess ammonium ions administered by injection or diet did not affect the activities of pyrimidine-metabolizing enzymes under our experimental conditions . This suggests that activation of ƒÀ-AlaAT I and ƒÀ-AlaAT II in the livers of rats fed a high-protein diet does not depend on the accumulation of . This follows from the data shown in Table 5, i.e. that dietary uridine does not activate either aminotransferase.

Intracellular pyrimidines are regulated by , salvage synthesis and catabolism. The concentrations of UTP, 5-phosphoribose 1-bisphosphate and aspartate regulate de novo pyrimidine synthesis (5, 6). The stimulation of pyrimi dine synthesis by ammonium ions (16-18) did not activate pyrimidine degradation

(Table 4). Moreover, as shown in Table 5, dietary uridine accumulated as uracil in cells but did not influence the pyrimidine-metabolic enzymes. These data suggest that these catabolizing enzymes could not adapt to eliminate the excess uracil . Moreover, recently we found that the decreased dihydropyrimidine dehydrogenase activity in the livers of rats fed a vitamin B2-deficient diet did not affect the uridine, uracil and ƒ°UMP (the sum of acid-soluble uracil 5•L-) pool in the liver

(33). Therefore, pyrimidine de novo and salvage synthetic enzymes are more effectively regulated by metabolites and nutrients than are the catabolic enzymes .

REFERENCES

1) Hoogenraad, N. J., Levine, R. L., and Kretchmer, N. (1971): Copurification of carbamyol phosphate synthetase and aspartate transcarbamoylase from mouse spleen. Biochem. Biophys. Res. Commun., 44, 981-988. 2) Shoaf, W. T., and Jones, M. E. (1971): Initial steps in pyrimidine synthesis in Ehrlich ascites carcinoma. Biochem. Biophys. Res. Commun., 45, 796-802.

J. Nutr. Sci. Vituminol. PROTEIN LEVEL ON PYRIMIDINE METABOLISM 527

3) Shoaf, W. T., and Jones, M. K (1973): Uridylic acid synthesis in Ehrlich ascites

carcinoma: Properties, subcellular distribution, and nature of enzyme complexes of the

six biosynthetic enzymes. , 12, 4039-4051.

4) Grobner, W., and Kelley, W. N. (1975): Effect of and its metabolic

derivatives on the configuration of human orotate phosphoribosyl transferase and

5•L-phosphate decarboxylase. Biochem. Pharmacol., 24, 379-384.

5) Tatibana, M., and Sigesada, K. (1972): Two carbamyl phosphate synthetases of

mammals: Specific roles in control of pyrimidine and biosynthesis. Adv. Enzyme

Regul., 10, 249-271.

6) Tatibana, M., and Shigesada, K. (1972): Control of pyrimidine biosynthesis in

mammalian tissues. V. Regulation of glutamine-dependent carbamylphosphate syn

thetase: Activation by 5-phosphoribosyl 1-pyrophosphate and inhibition by uridine

triphosphate. J. Biochem., 72, 549-560.

7) Weber, G., Queener, S. F., and Ferdinandus, J. A. (1971). Control of gene expression

in carbohydrate, pyrimidine and DNA metabolism. Adv. Enzyme Regul., 9, 63-95.

8) Griffith, O. W. (1986): ƒÀ-Amino acids: Mammalian metabolism and utility as ƒ¿-

amino acid analogues. Annu. Rev. Biochem., 55, 855-878.

9) Tamaki, N., Fujimoto, S., Mizota, C., and Kikugawa, M. (1987): Submitochondrial

localization of rat liver ƒÀ-alanine-oxoglutarate aminotransferase. J Nutr. Sci. Vit

aminol., 33, 439-449.

10) Mizota, C., Fujimoto, S., Kikugawa, M., Kimura, Y., and Tamaki, N. (1988): Effect

of pyridoxine deficiency and prednisolone on ƒÀ-alanine-oxoglutarate aminotransferase

and D-3-aminoisobutyrate aminotransferase in rat liver and kidney. J Nutr. Sci.

Vitaminol., 34, 223-236.

11) Goodwin, G. W., Rougraff, P. M., Davis, E. J., and Harris, R. A. (1989): Purification

and characterization of methylmalonate-semialdehyde dehydrogenase from rat liver.

J. Biol. Chem., 264, 14965-14971.

12) Canellakis, E. S. (1956): Pyrimidine metabolism: I. Enzymatic pathways of uracil and

thymine degradation. J. Biol. Chem., 221, 315-322.

13) Fritzson, P. (1957): The catabolism of 14C-labeled uracil, dihydrouracil, and ƒÀ-

ureidopropionic acid in rat liver slices. J. Biol. Chem., 226, 223-228.

14) Queener, S. F., Morris, H. P., and Weber, G. (1971): Dihydrouracil dehydrogenase

activity in normal, differentiating, and regenerating liver and in hepatomas. Cancer

Res., 31, 1004-1009.

15) Dallman, P. R., and Manies, E. C. (1973): Protein deficiency: Contrasting effects on

DNA and RNA metabolism in rat liver. J. Nutr., 103, 1311-1318.

16) Pauch, J. G., Keppler, D. O. R., and Gerok, W. (1977): Increased do novo pyrimidine

synthesis in liver induced by ammonium ions in amounts surpassing the urea

cycle capacity. Eur. J Biochem., 76, 157-163.

17) Tremblay, G. C., Crandall, D. E., Knott, C. E., and Alfant, M. (1977):

biosynthesis in rat liver: Studies on the source of carbamoylphosphate. Arch. Biochem.

Biophys., 178, 264-277.

18) Monks, A., Chisena, C. A., and Cysyk, R. L. (1985): Influence of ammonium ions on

hepatic de novo pyrimidine biosynthesis. Arch. Biochem. Biophys., 236, 1-10.

19) Zee-Cheng, K.-Y., Robins, R. K., and Cheng, C. C. (1961): Pyrimidines: III. 5,6

- Dihydropyrimidines. J Org. Chem., 26, 1877-1884.

Vol. 37, No. 5, 1991 528 M. KANEKO et al.

20) Fritzson, P. (1960): Properties and assay of dihydrouracil dehydrogenase of rat liver.

J Biol. Chem., 235, 719-725.

21) Brooks, K. P., Jones, E. A., Kim, B. D., and Sander, E. G. (1983): Bovine liver

dihydropyrimidine : Purification, properties, and characterization as a

metaloenzyme. Arch. Biochem. Biophys., 226, 469-483.

22) Tamaki, N., Mizutani, N., Kikugawa, M., Fujimoto, S., and Mizota, C. (1987):

Purification and properties of ƒÀ-ureidopropionase from the rat liver. Eur. J Biochem.,

169, 21-26.

23) Fujimoto, S., Mizutani, N., Mizota, C., and Tamaki, N. (1986): The level of ƒÀ-alanine

aminotransfease activity in regenerating and differentiating rat liver. Biochim. Biophys.

Acta, 882, 106-112.

24) Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951): Protein

measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265-275.

25) Brook, J. B., and Hastewell, J. G. (1988): The transport and metabolism of naturally

occurring pyrimidine by isolated rat jejunum. J. Physiol., 395, 349-361.

26) Sanno, Y., Holzer, M., and Schimke, R. T. (1970): Studies of a affecting

pyrimidine degradation in inbred mice. J. Biol. Chem., 245, 5668-5676. 27) Traut, T. W., and Loechel, S. (1984): Pyrimidine catabolism: Individual characteri

zation of the three sequential enzymes with a new assay. Biochemistry, 23, 2533-2539.

28) Dagg, C. P., Coleman, D. L., and Fraser, G. M. (1964): A gene affecting the rate of

pyrimidine degradation in mice. Genetics, 49, 979-989. 29) Barrett, H. W., Munavalli, S. N., and Newmark, P. (1964): Synthetic pyrimidine as

inhibitors of uracil and thymine degradation by rat-liver supernatant. Biochim.

Biophys. Acta, 91, 199-204.

30) Krebs, H. A. (1971): Some aspects of the regulation of fuel supply in omnivorous

animals. Adv. Enzyme Regul., 10, 379-420.

31) Pitot, H. C., and Peraino, C. (1964): Studies on the induction and repression of

enzymes in rat liver: I. Induction of threonine dehydrase and ornithine-ƒÂ-transaminase

by oral intubation of casein hydrolysate. J. Biol. Chem., 239, 1783-1788.

32) Cheung, G. P., Cotropia, J. P., and Sallech, H. J. (1969): The effects of dietary protein

on the hepatic enzymes of metabolism in the rabbit. Arch. Biochem. Biophys.,

129, 672-683.

33) Fujimoto, S., Matsuda, K., Kikugawa, M., Kaneko, M., and Tamaki, N. (1991): Role

of flavin in rat liver dihydropyrimidine dehydrogenase activity. J. Nutr. Sci. Vita

minol., 37, 89-98.

J. Nutr. Set. Vitaminol.