Pyrimidine reducing enzymes of rat liver' RONALD0.HAL LOCK^ A N D ESTHER W. YAMADA
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Department ofBiochemistw, University ofManitoba, Win~lr~ipeg, Manitoba R3E OW3 Received July 28, 1975 Hallock, R. 0. & Yamada, E. W. (1976) Pyrimidine reducing enzymes of rat liver. Can. 9. Biochsm. 54. 178-184 Dihydrouracil dehydrogenase (NABPf) (EC 1.3.1.2) was partially purified from the cytosol fraction of rat liver and fractionated by disc gel electrophoresis. A major and minor band were visualized by staining for enzyme activity. The substrate specificity of these bands was investigated. It was found that both bands were two to three times more active with dihydrsthymine as substrate than with dihydrouracil in the presence of N A B P and the optimum pH of 7.4. Mitochondria1ti-actions containing most of the NADPI-dependent uracil reductase of rat liver cells were fractionated by centrifugation in sucrose density gradients. Two procedures involving linear or discontinuous gradients were used. By both, good separation of NADH- and NADPHdependent reductases was achieved. Marker enzyme studies supported the view that the NADH-dependent enzyme is located principally in mitochondria whereas the NADPHdependent enzyme is mainly in plasma and endoplasmic reticulum membranes. For the WABHdependent reductase the apparent Km for thymine at pH 7.4 was 1.39 times that found for uracil whereas for the NADPH-dependent enzyme the apparent K , values were similar for the two substrates at this pH. Dihydrouracil was the principal product isolated by paper chromatography from the reaction mixture containinga partially purified fraction of mitochondria, uracil and NABH at pH 7.4. This fraction also catalyzed the formation of radioactive carbon dioxide from [Z14C]uracil. The propc~rtia~n of C 0 2 formed by the mitochondria was about of that formed by the original homogenate. HaIlock, W. 0 . & Yarnada, E. W. (1976) Pyrimidine reducing enzymes of rat liver. Can. 9. Bioc-hem.54, 178-1 8.4 La dihydrouracile deshydrsgenase (NADP+}(EC 1.3.1.2) est partiellement purifiee dans la fraction cytosolique du foie de rat et fractionnee par electrsphorese sur disque de gel. Une bande majeure et une bande mineure soar visualisCes par coloration pour deceler I'activite enzymatique. Nous recherchons la specificite de ces bandes a i'egard du substrat. kes deux bandes sont deux a trois fois plus actives avec Iadihydrothymine comme substrat qu'avec %edihydrouracile en prtsence de NADP+ et au pH optimum de 7.4. Les fractions rnitochondriales contenant la plus grande partie de Ba NADW-uracile reductase des cellules hepatiques du rat sont fractionnees par centrifugation dans des gradients de densite de saccharose. Nous utilisons deux techniques impliquant des gradients lineaires ou discontinus. Les deux techniques wsus donnent une bonne separation des NADH- et des NADPH-reductases. kes etudes avec rnarqueur enzymatique confirme I'hypothese que la NADH-reductase est BscalisCe surtout dans les mitochondries tandis que la NADPW-reductase se retrouve surtout dans Bes membranes plasmiques et celles du reticulum endoplasmique. ke Km apparent de la NADM-reductase pour la thymine a pH 7.4 kgale 1.39 fois celui pour I'uracile tandis que pour la NADPH-reductase et ies vaIeurs du K , sont semblables pour les deux substrats a c e pH. Le dihydrouracile est le principal produit isole par chromatographie skar papier du milieu de reaction contenant une fraction partiellement purifiee de mitochondries, de I'uracile et du NADH h pH 7.4. Cette fraction catalyse aussi la formation de CO, radisactif a partir de [2-"C]uracile. La proportion de CO, formee par les mitochondries &ale environ 10% celle forrnee par I'homogknat original. [Traduit par le journal]
Intrsduetisn Soluble dihydrouracil dehydrogenase (NADP9) (EC 1.3. 1.2),3 located in the cytssol fraction of mammafian liver, has been the subject of a number lSupported by the Medical Research Council of Canada. 'Submitted in partial fulfillment of the requirements for the degree of Master of Science. 35 ,$-Dihydrouracil: NABP+ oxidoreductase (EC 1.3.1.2).
of investigations (1-5) but, a s yet, little information is available regarding the particulate form of this enzyme (6) which is designated uracil reductase4 herein. These enzymes differ in a number of ways other than in subcellular distribution. Dihydrouracil dehydrogenase catalyzes a reversible reaction and is specific f a r NABP+ (1, 3, 4) although NAD9 is a cofactor a t high, nonpkysioIogical pH values ( 5 ) . Ht $Also referred to as uracil hydrogenase (6).
I79
HALLOCK A N D YAMADA
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is activated by ATP (2) or ATP plus Mga+ (6); both uracil and thymine are substrates (1, 3, 6-$). It was concluded by Barrett et a/. (8)that the same enzyme acts on both substrates. Subsequently, two enzyme bands with dihydrouracil dehydrogenase activity were separated by disc gel electrophoresis of cytosol fractions (5). The substrate and coenzyme specificities of these bands comprise the first part of present studies. In contrast to dihydrouracil dehydrogenase, NABH as well as NADPH are cofactors at physiological pH for uracil reductase of particulate fractions (6). ATP plus MgB inhibit enzyme activity and the reaction catalyzed is essentially nonreversible. In the second part of the present work, mitochondria1 fractions were purified further; uracil reductase of these fractions was then characterized as to the products of the reaction. activity towards thymine and coenzyme specificity.
Pahydrouracil Dehydrogenase Enzyme Assays Activity was assayed as described previously (6) with dihydrothymine replacing dihydrouracil where specified in the text. The final pH of the reaction mixture was adjusted to 8.8 or 7.4, the pH optima of the NAD+- and N ADP+-dependentactivities, respectively (5). The extinction coefficients per micromole of dihydrothymine oxidized were found to be 13.18 in the presence of NADP+ and 16.26 in the presence of NAD+, both at 295 nm. Disc Gel Electrophoresis This procedure was as outlined before (5). Partially purified enzyme fractions were prepared from rat liver cytosol and fractionated by electrophoresis. The gels were stained for enzyme activity and densitometer tracings of the stained gels were obtained as outlined previously (5). Umcil Reductase Plar$ca tion oj' Mr'tcpehondrial Fractions Male Holtzman rats weighing between 300 and 350g were decapitated, their livers perfused, weighed, and homogenized in eight volumes of sucrose-Mg2+ (0.24 M sucrose - 3 mM hlgCl, - 5 d l 4 /3-mercaptoethanol) as described previously (homogenate A) and subcellular fractions were prepared (6). The heavy mitochondria1 (HM)S fraction was wash& once with sucrose-Mga+, suspended in sucrose-EDTA (0.25 M sucrose - 1 rmW EDTA (pH 7.0) - 5 mM 0-mercaptcdethanol) and purified further by either procedure 1 or 2 below. Procedure I-In early experiments, 3 ml HM fraction were layered on 25.5 ml of a sucrose density gradient, linear from 1.91 to 0.5 M sucrose, and centrifuged for 1 h at 70800 x g in a Spinco SW 25.1 swing-out bucket rotor. Fractions of 1.5 mI were then collected. Procedure 2-To achieve larger yields of comparable purity in a shorter time, the discontinuous sucrose density gradient described by Sarzala et al. 49) was modified. An aliquot of HM fraction was layered on top of a gradient consisting of 20-6596 (w/v) sucrose, neutralized with 1M Tris-HC1 bufTer (pH 7.0), in the volumes shown in Fig. 1.
5Abbreviation: HM, heavy mitochondrial.
130000 g 30 rnin
FIG. 1. Purification of crude mitochondrial fractions (HM) by discontinuous sucrose density gradient centrifugation (procedure 2). Layering was done in a cold room in the volumes specified. Centrifugation was at 4 "g: in an International model B-60 ultracentrifuge. The tubes were centrifuged at 130080 x g for 30 rnin in rotor 5B-283 in an International model B-60 preparative ultracentrifuge. Three layers (Mi, M,, and M,) were separated; they were removed by means of a U-shaped syringe needle with a blunt end. All fractions were dialyzed routinely for 16 h at 2 4 @ in either buffer A (0.05 M potassium phosphate buffer (pH 7.0) - 5 rnhl P-mercaptoethanol - 1 nalM EDTA (pH 7.0)) or, when 5 '-nucleotidase was being assayed, in buffer B (buffer A with 0.05 M Tris-HC1 buffer (pH 7,O) instead of the Pi buffer). Marker Enzymes Glutamate dehydrogenase and 5 '-nucleotidase were assayed as described before (6). A unit of 5 '-nuclestidase activity is defined as that quantity that catalyzes the release of 1 pmol of Pi per hour. For glutamate dehydrogenase, one enzyme unit catalyzes the formation of 1 pmol of NADH per hour. Specific activity is given in enzyme units per milligram protein. Uracil Reductase Activities were assayed as described before (6) at pH 7.4 with either uracil or thymine as substrate and with NADH or NADPH present. Enzyme fractions were stored frozen at-78 @ in small aliquots and thawed only once before use. Unless specified otherwise, enzyme fractions were suspended in 5.4 M glycerol in ice just before use. One unit of enzyme activity is defined as the quantity that catalyzes the formation of 1 pmol of dihydrouracil or dihydrothymine per hour, The extinction coefficients per micromole of substrate reduced were the same as those for the assay of dihydrouracil dehydrogenase. Protein was determined by the method of Lowry et al. (10). Enzyme activity was a linear function of time up to 14 min and of protein concentration up to 1.8 mg of protein. The spectrophotometric assay method dQueener et ab. (1 1) was not used because of the protein density of the subcellular fractions as well as the fact that saturating levels of uracil (or thymine) interfered with the measurements. Paper Chromatography of Reaction Products To the standard assay mixture for NADH-dependent uracil reductase4 was added 0.6 pmol of [2-84C]uracil (0.0035 pCi). In these experiments, enzyme fractions were not suspended in glycerol because it interfered with the separation of reaction products. Duplicate tubes were incubated at 37 "C for 30 min. The reaction was stopped by boiling for 3 min. Control tubes contained no enzyme
TABLE 1. Summary of purification of dihydrouracil dehydrogenasea
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Specific activity (units/mg protein)
Stepandtreatment
Volume (ml)
Homogemate Supernatant Ammonium sulfate
146 91 63
Bihydrouracil NAB+ NADP+ 0.061 0.123 8.267
0.079 0.134 0.283
Dihydrothymine NAB
+
0.070 48.134 0.291
NADP
+
0.082 8.844 8.342
"Livers from two rats weighing 200 and 201 g were pooled.
which was added just before boiling. The tubes were centrifuged at 1 6 W x g for 20 min in a Sowall RC-2B refrigerated centrifuge. Fifty-microliter aliquots of the supernatant were then applied to Whatman No. I filter paper strips (1.8 x 38 em) which were developed by descending chromatcdgraphy in eollidine saturated with water as described by Fritzson ( 12). Chromatograms were air-dried and then viewed under U V light to detect uracil (I?,= 0.72). They were sprayed with p-dimethylaminobenzaldehyde and HCI (13) to detect dihydrouracil (I?,= 0.53) and carbamyl@-alanine (R,= 0.02). Other strips were dipped in winhydrin and heated to $8 'T for 10 min (13) to detect p-alanine (Rf=0.02). To detect radioactivity, still other chromatograms were cut into I-cm segments. Each segment was eluted with 500 pl of water and then 15 ml of toluene-based scintillation fluid containing 8.4% (w/v) omnifluor and 30% (v/v) ethanol were added. Radioactivity was counted in a Beckman LS-250 scintillation spectrometer at an efficiency of 80%. P)etermbnation ofLc~beNedCarbon Dioxide A modification of the method of Canellakis (2) was used. Mitochondria fraction M, was incubated in a final volume of 3 ml in the following (in micromoles): potassium phosphate buffer (pH 7.4t, 75; uracil, 0.7143 40.1 pCi); NAD+, 8.4: 108 p1 of absolute ethanol; 30 pg of alcohol dehydrogenase (Nutritional Biochem.); 10 mg Pa, protein. Duplicate samples were incubated at 37 "@ in a shaker bath in 10-rnl Erlenmeyer flasks equipped with rubber wells (Kontes Co.) to which 0.3 ml hyamine hydroxide was added to absorb radioactive CO,. Flasks were preincubated for 15 min before the addition of radioactive uracil. After inckebation for 30 min. 0.3 ml of 30% (wlv) trichloroacetic acid was added to stop the reaction. Flasks were incubated for an additional hour to ensure the complete absorption of C0,. To scintillation vials containing toluene-based scintillation fluid (4g omnifluor per liter toluene), 0.2 mB hyamine hydroxide from the wells of the reaction flasks was added. Radioactivity was determined as before. Appropriate controls lacking M, were included.
Bihydrouvaci! BeBaydrsgenwss3 Disc GPIElec~rophoresis Table 1 shows a typical experiment in which dihydroumci%dehydrogenase was partially purified from the cytosol fraction of rat liver. Routinely, a purification of about fourfold was obtained. The activity of the ammonium sulfate fraction with dihydrothymine as substrate was comparable to that
FIG.2. Relationship between staining intensity and protein concentration of enzyme bands separated by disc gel electrophoresis and stained B'or enzyme activity. The areas under the peaks for the major component (band 1) and the minor component (band 2) were determined from densitometer tracings of the stained gels by disc integrator attachment to give the intensity of staining in arbitrary units. Enzyme fractions purified fivefold from rat liver cytosoi were incubated in media containing NADP+ at pH 7.4 with dihydrothymine band 1 (@) and band 2 (0t; with dihydrouracil band 1 (V)and band 2 4 BB.
found with dihydrouracil, both at pH 8.8 with NAD' as coenzyme, but was 28% higher at pH 7.4 and N ADP' as coenzyme. Figure 2 gives a plot of activity Lverslas concentration of the major (band I ) and minor (band 2) enzyme components after disc gel electrophoresis and staining for activity by our nitroblue tetrazolium method ( 5 ) . By this procedure, only two active enzyme bands appear although several nonactive protein bands are known t o be present ( 5 ) . With NAK)Pf as coenzyme, b ~ t bands h showed two to three times more activity with dihydrothymine as substrate compared to dihydrouracil. Band 1 was always considerably more active with either substrate than was band 2. As before ( 5 ) . the relative mobilities (It%,)of the two bands were the same with either NAD' or NADP'; it was now found that the W,, values were also the same with dihydrothymine replacing dihy-
HALLOCK A N D Y.$MADA
TABLE2. Distribution of thymine reductase activity in subcellular fractionsQ Glutamate dehydrogenase
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Thymine f NADH Fraction Homogenate A Crude nuclei HM
Lb
Thymine + NADPH
5'-Nuclestidase
Specific activity
Total
(%)
Specific activity
Total (%)
Specific activity
Total (%)
Specific activity
Total (%)
0.088 0.123 0.264 0.107
100 24.1 51.0 6.0
1.12 1.93 2.86 B .55
168 38.3 63.0 7.0
8.045 0.889 0.064 0.140
100 29.9 33.2 23.2
1.45 2.80 2.08 3.21
1Ml 32.7 31.7 10.8
0
0.020
17.9
0.96
16rnx.g
supernatant Recovery (%)
0.020
-
12.4 93.5
108.3
104.2
33.4 108.6
T h e averages from two to four experimends are given. bLight mitochondria and lysasornes.
TABLE3. Enzyme distribution in mitochondria1 fractionsQ HM
Enzyme
Thymine-NADH reductase Uracil-NADH reductase Glutamate dehydrogenase Thymine-NADPH reductase Uracil-NABPM reductase 5'-Nucleotidase
Mr
-
M2
--
-
M3 -
Specific activity
Total (%)b
Specific activity
Total
(x)
Specific activity
Total (%)
Specific Total activity
0.214 0.156 2.860
51 .0 47.7 63.0
0.064 0.052 0
3.1 3.8 0
0.482 0.535 6.078
37.0 41.0 55.8
8.144 0.047 0.980
4.$ 2.4 4.4
0.064 0.055 2.080
33.2 35.8 31.7
0.330 0.324 2.350
21.4 28.4 9.2
0.078 0.013 1.480
9.9 7.0 16.9
0.033 0.031 0.930
3.3 2.4 2.9
(z)
QHMfraction was centrifuged in sucrose density gradients (procedure 2). Averages from two to four experiments are given. bCornpared to the total units in homogenate A taken as IOOY,.
drouracil with either coenzyme present. However, contrary to what was found when NADP+ was the coenzyme, both bands were as active with dihydrothymine a s with dihydrourcacil when NAD' was present. The physiological significance of the NAD'-linked activity cannot be assessed at present mainly because of the high pH optimum of this reaction. Urcacij Rcdrrcttrse Table 2 shows that the subcellular distribution of NADH-dependent reductase activity with thymine as substrate followed the distribution of the mitochondria%marker enzyme glutamate dehydrogenase. It is similar to that found earlier for the WADH-dependent uracil reductase activity (6). It is also apparent from Table 2 that the subcellular distribution of the WADPH-dependent reductase activity with thymine as substrate, as was found previously with uracil (6), corresponded closely with that of the marker enzyme 5 '-nucleotidase, which is associated with plasma and endsplasmic reticulum membranes (15-18). The distribution of enzyme activities after cen-
trifugation of the HMS fraction in discontinuous sucrose density gradients (procedure 2) is shown in Table 3. Fraction M, contained the major portion of the NADH-dependent reductase activity, with either thymine o r uracil a s substrate, as well as most of the glutamate dehydrogenase activity. The specific activity of M, with uracil a s substrate was usually higher than that found with thymine. The NADH-dependent enzyme was solubilized in 8.1 M P i buffer (pH 7.4) (14) rather than in glycerol, and the apparent K , values at pH 7.4 were estimated to be 0.085 mM for uracil, 0.118 mM for thymine, and 0.103 mM for NADH, with either substrate present. The K , value for uracil is Bower than that reported earlier for the less pure HM fraction (B), Centrifugation of the HM fraction by procedure 2 also I-esulted in enrichment of the NADPHdependent reductase a s well a s 5 '-nucleotidase, but in fraction Fa, rather than M, (Table 3). Fraction M, was almost as active with uracil as with thymine. The reductase was solubilized a s above and its afinity for the two substrates determined. The apparent K,, values (at pH 7.4) were estimated to be 0.118 mikf for uracil, 0.113 mM for thymine, and
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CAN. I. BIOCHEM. VOE. 54, 1976
FKG.3. Enzyme distribution in HM fractions (heavy mitochondria) centrifuged in linear sucrose density gradients (procedure 1). Activity is expressed as micromoles of dihydrouracil (DHU) or dihydrothymine (DHT) formed per hour per tube with uracil (U) or thymine (T) as substrate; 1.5 ml fractions were collected. A unit of glutamate dehydrogenase activity is expressed as the micromoles of NADH formed per hour per tube; a 3-ml aliquot cc~ntaining75.6 mg protein s f the HM fraction was used. The arrow indicates the direction of sedimentation. 0.065 m M for NABPH, with either substrate present. The Km value for NADPH is lower than that found earlier for less pure fractions (6). Figure 3 shows that fractionation of the HM fraction by centrifugation in a linear sucrose density gradient (procedure 1) gave less well defined separation of the NABM- and NADPH-dependent reductases. Notable, however, was the finding that activity with uracil or thymine as substrate peaked in the same fraction. Again the NADH-dependent activity coincided with that of glutamate dehydrogenase whereas the NADPH-dependent activity was in the position routinely marked by 5 '-nucleotidase (6).
Weaction Products Mitochondria1 fraction M2 was incubated in the standard assay mixture containing [2-14C]uraciland NABH. Incubation was for 30 min at 37 "C. The reaction products were separated by paper chromatography as described in Experimental. Fifty percent of the radioactivity was recovered as uracil, 30% as dihydrouracil and 7.5% as a ninhydrinpositive spot close to the origin (Fig. 4B). Whether the last was P-ahnine or residual enzyme protein was uncertain. Recovery of total radioactivity initially added to the reaction mixture was 85-98% in two separate experiments.
Formation s f C 0 , Mitochondria1fraction M2 was incubated in reaction media at pH 7.4 containing [2-14C]uracil.Figure 4A shows that the formation of radioactive GO, increased with increasing uracil concentrations (at constant specific radioactivity) and began to level off at 10 mM. Linearity between CO, produced and time was observed for 38 min with 1.6 - 10 mg of mitochondria1 protein per flask. The amount of uracil degraded by mitochondria was estimated to be about 10% of that formed by the original hornogenate, both with NAD+ as coenzyme.
Discussion With regard to the substrate specificity of dihydrouracil dehydrogenase3 of cytosol fractions, in two separate reports, thymine was found to be degraded at */, and */, the rate of uracil (3, 19) with NABPH as coenzyme. Thymine inhibited the catabolism of uracil much more than vice versa (8)For the reverse reaction (3), dihydrothymine appeared to be oxidized at a much more rapid rate than dihydrouracil although no mention of this was made in the text. In the present work, the major and minor
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HALLOCK AND YAMADA
D I S T A N C E (ern x lo-')
FIG.4. (A) Relationship between nadioactive C 0 , formed (cpm) and [2-'4C]uracil concentration. Fraction M, (10 rng protein) was incubated for 30 rnin at 37 "C at pH 7.4. (B) Radioactivity (cprn) of reaction products separated by paper chromatography. Fraction M, (10 mg protein) was incubated for 30 min at 37 "62 in the standard reaction mixture for uracil reductase in the presence of [2-14C]uraciIand NABH.
bands of dih ydrouraciI deh ydrogenase, separated from rat liver cytosol fractions by disc gel electrophoresis, did not differ in substrate specificity; both bands were t w o t o three times more active towards dihydrsthymine a s towards dihydrouracil with NADP+ a s cofactor. It is possible that the earlier preparations of dihydrouracli dehydrogenase contained the NADPW-dependent reductase of membranes; this would explain, in part at least, the anomalous results a s t o the substrates preferred (3, 8, 19). Tentatively then, our resuits are not in conflict with the conclusions of Barrett et cal. (8) that the same dihydrouracil dehydrogenase of cytosol fractions acts o n both uracil and thymine but it is apparent that more extensive enzyme purification and characterization are needed. That there are at lease two separate uracil reductases4 in particulate fractions of rat liver is apparent. Besides coenzyme specificity, intracellular location and apparent Km values for thymine and uracil, the reductases have recently been found to differ most notably in sensitivity t o activation by P,, E D T A o r EGTA, and inhibition by calcium ions (14).
That mitochondria1 fractions catalyze the degradation of uracil t o form CO, to only a limited extent is shown in the present work. The results suggest that the degradative enzymes beyond uracil reductase are limiting in amount in mitochondria s o that the dihydroumcil formed must be extruded mainly into the cytsso! for further degradation. Such an
efflux of dihydrouracil may perhaps be the reason for the virtual nonreversibility of the reaction catalyzed by uracil reductase of these organelles with dihydrouracil a s substrate; significant activity in the reverse direction was found only after solubilization of mitochondria by glycerol 46). The high concentration of uracil ( 10 rnM)required fipr maximal formation of C 0 2 , in contrast to the low K , for uracil of uracil reductase of mitochondria, indicates that the reduction of uracil is not the ratelimiting step of uracil degradation in mitochondria and perhaps not even in the cytosol(20,2 19. A factor to be considered is the high K , value of 11.75 x M reported for dihydrouracil hydrase with dihydrouracil a s substrate; this enzyme was purified 200-fold from calf liver (229. This Km value is much - 2.0 x M found higher than those of 1.5 x for di hydrouracil dehydrogenase of rat liver cytosol with dihydrouracil a s substrate (2,6); an even lower K , for uracil, estimated t o be less than 4 x 101'WM, was reported for the cytosol enzyme (2). However, n o conclusions can be drawn a s yet from these data since the enzymes were from different sources and only partially purified. 1. Canellakis, E. S. (1456)J. Biol. Cl~etn. 221,3 15-322 2. Fritzson, P. (1960)J. Biol. Chern. 235,719-725 3. Grisolia, S. &Cardoso, S. S. (1957)Bioclzim. Biophys. Acfn 25,430-43 1 4. Goedde, H. W., Agarwal, B. P. & Eickhoff, K. (1978) Z . Physiol. Chem. 451 ,945-95 1 5, Hallock, R. 8 . & Yamada, E. W. (1973) Anal. Biochem. 56,84-90
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CAN. J . BIOCHEM. VOL. 54, 1976
IS. Bosman, H. B., Hagopian, A. & Eylar, E. H. (1948) 6. Smith, A. E. & Yarnada, E. W. (1971) J . Bicd. Chem. Arch. Biochern. Biophys. %28,51-69 246,3610-3617 16. Song, C. S., Nisselbaum, J . S., Landler, B. & 7. Bresnick, E. (1964) Tes. Rep. Biol. Aged. 22,431-439 Bodansky, 0. (1968) Biorhim. Biophys. Acso 150, 8. Barrett, H. W., Munavalli, S. N. & Newrnark, P. ( 1964) Bioclaim. Bioplays. Acra 91 , 199-204 380-303 17. Song, C. S. & Bodansky, 0 .(1967)J. Biol. Chem. 242, 9. Sarzaia, M. G.. Van Gslde, L. M. C.. De Kruyff, B. & 694-699 Van Deenen, L. L. M. ( 1970) Biochirn. Biophys. A C S ~ 18. Evans, W. H . B 1869) FEBS &eft.3,237-2463 202,106-3 19 18. Lowry, 0. H.,Rosebrough, N. J.. Farr, A. L. & 19. Newmark, P., Stephens,.!. D. & Barnett, H. W. (1962) Randall, W. J . (1951) 5. Bicd. Chem. 193,265-275 Bischina. Biophys. Actu 62,414-416 1 8 . Qiaeener, S. F., Morris, H . P. & Weber, G . (1971) 20. Sanno, Y., Hslzer. M. & Schimke, R. T. (1978) J. Ctasace~aR e s . 36, 1004-1009 Biol. Chern. 245,5668-5474 82. Fritzson, P. & Pihl, A. (1957) J. Biol. Chprn. 226, 2 1. Fritzson. P. ( 1957)5. B i d . C'hem. 226,223-228 22. Wallach, D. P. $L Grisslia, S. (1957) J. Biol. Clzem. 229-235 226.277-288 93. Fink, R. M . , Cline, W. E., McGaughey, C. & Fink, K. (1956) A n d . Chem. 28,4-6 14. Yarnada, E. W. (1975) Can. J. Biochem. submitted for publication
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Department ofBiochemistw, University ofManitoba, Win~lr~ipeg, Manitoba R3E OW3 Received July 28, 1975 Hallock, R. 0. & Yamada, E. W. (1976) Pyrimidine reducing enzymes of rat liver. Can. 9. Biochsm. 54. 178-184 Dihydrouracil dehydrogenase (NABPf) (EC 1.3.1.2) was partially purified from the cytosol fraction of rat liver and fractionated by disc gel electrophoresis. A major and minor band were visualized by staining for enzyme activity. The substrate specificity of these bands was investigated. It was found that both bands were two to three times more active with dihydrsthymine as substrate than with dihydrouracil in the presence of N A B P and the optimum pH of 7.4. Mitochondria1ti-actions containing most of the NADPI-dependent uracil reductase of rat liver cells were fractionated by centrifugation in sucrose density gradients. Two procedures involving linear or discontinuous gradients were used. By both, good separation of NADH- and NADPHdependent reductases was achieved. Marker enzyme studies supported the view that the NADH-dependent enzyme is located principally in mitochondria whereas the NADPHdependent enzyme is mainly in plasma and endoplasmic reticulum membranes. For the WABHdependent reductase the apparent Km for thymine at pH 7.4 was 1.39 times that found for uracil whereas for the NADPH-dependent enzyme the apparent K , values were similar for the two substrates at this pH. Dihydrouracil was the principal product isolated by paper chromatography from the reaction mixture containinga partially purified fraction of mitochondria, uracil and NABH at pH 7.4. This fraction also catalyzed the formation of radioactive carbon dioxide from [Z14C]uracil. The propc~rtia~n of C 0 2 formed by the mitochondria was about of that formed by the original homogenate. HaIlock, W. 0 . & Yarnada, E. W. (1976) Pyrimidine reducing enzymes of rat liver. Can. 9. Bioc-hem.54, 178-1 8.4 La dihydrouracile deshydrsgenase (NADP+}(EC 1.3.1.2) est partiellement purifiee dans la fraction cytosolique du foie de rat et fractionnee par electrsphorese sur disque de gel. Une bande majeure et une bande mineure soar visualisCes par coloration pour deceler I'activite enzymatique. Nous recherchons la specificite de ces bandes a i'egard du substrat. kes deux bandes sont deux a trois fois plus actives avec Iadihydrothymine comme substrat qu'avec %edihydrouracile en prtsence de NADP+ et au pH optimum de 7.4. Les fractions rnitochondriales contenant la plus grande partie de Ba NADW-uracile reductase des cellules hepatiques du rat sont fractionnees par centrifugation dans des gradients de densite de saccharose. Nous utilisons deux techniques impliquant des gradients lineaires ou discontinus. Les deux techniques wsus donnent une bonne separation des NADH- et des NADPH-reductases. kes etudes avec rnarqueur enzymatique confirme I'hypothese que la NADH-reductase est BscalisCe surtout dans les mitochondries tandis que la NADPW-reductase se retrouve surtout dans Bes membranes plasmiques et celles du reticulum endoplasmique. ke Km apparent de la NADM-reductase pour la thymine a pH 7.4 kgale 1.39 fois celui pour I'uracile tandis que pour la NADPH-reductase et ies vaIeurs du K , sont semblables pour les deux substrats a c e pH. Le dihydrouracile est le principal produit isole par chromatographie skar papier du milieu de reaction contenant une fraction partiellement purifiee de mitochondries, de I'uracile et du NADH h pH 7.4. Cette fraction catalyse aussi la formation de CO, radisactif a partir de [2-"C]uracile. La proportion de CO, formee par les mitochondries &ale environ 10% celle forrnee par I'homogknat original. [Traduit par le journal]
Intrsduetisn Soluble dihydrouracil dehydrogenase (NADP9) (EC 1.3. 1.2),3 located in the cytssol fraction of mammafian liver, has been the subject of a number lSupported by the Medical Research Council of Canada. 'Submitted in partial fulfillment of the requirements for the degree of Master of Science. 35 ,$-Dihydrouracil: NABP+ oxidoreductase (EC 1.3.1.2).
of investigations (1-5) but, a s yet, little information is available regarding the particulate form of this enzyme (6) which is designated uracil reductase4 herein. These enzymes differ in a number of ways other than in subcellular distribution. Dihydrouracil dehydrogenase catalyzes a reversible reaction and is specific f a r NABP+ (1, 3, 4) although NAD9 is a cofactor a t high, nonpkysioIogical pH values ( 5 ) . Ht $Also referred to as uracil hydrogenase (6).
I79
HALLOCK A N D YAMADA
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is activated by ATP (2) or ATP plus Mga+ (6); both uracil and thymine are substrates (1, 3, 6-$). It was concluded by Barrett et a/. (8)that the same enzyme acts on both substrates. Subsequently, two enzyme bands with dihydrouracil dehydrogenase activity were separated by disc gel electrophoresis of cytosol fractions (5). The substrate and coenzyme specificities of these bands comprise the first part of present studies. In contrast to dihydrouracil dehydrogenase, NABH as well as NADPH are cofactors at physiological pH for uracil reductase of particulate fractions (6). ATP plus MgB inhibit enzyme activity and the reaction catalyzed is essentially nonreversible. In the second part of the present work, mitochondria1 fractions were purified further; uracil reductase of these fractions was then characterized as to the products of the reaction. activity towards thymine and coenzyme specificity.
Pahydrouracil Dehydrogenase Enzyme Assays Activity was assayed as described previously (6) with dihydrothymine replacing dihydrouracil where specified in the text. The final pH of the reaction mixture was adjusted to 8.8 or 7.4, the pH optima of the NAD+- and N ADP+-dependentactivities, respectively (5). The extinction coefficients per micromole of dihydrothymine oxidized were found to be 13.18 in the presence of NADP+ and 16.26 in the presence of NAD+, both at 295 nm. Disc Gel Electrophoresis This procedure was as outlined before (5). Partially purified enzyme fractions were prepared from rat liver cytosol and fractionated by electrophoresis. The gels were stained for enzyme activity and densitometer tracings of the stained gels were obtained as outlined previously (5). Umcil Reductase Plar$ca tion oj' Mr'tcpehondrial Fractions Male Holtzman rats weighing between 300 and 350g were decapitated, their livers perfused, weighed, and homogenized in eight volumes of sucrose-Mg2+ (0.24 M sucrose - 3 mM hlgCl, - 5 d l 4 /3-mercaptoethanol) as described previously (homogenate A) and subcellular fractions were prepared (6). The heavy mitochondria1 (HM)S fraction was wash& once with sucrose-Mga+, suspended in sucrose-EDTA (0.25 M sucrose - 1 rmW EDTA (pH 7.0) - 5 mM 0-mercaptcdethanol) and purified further by either procedure 1 or 2 below. Procedure I-In early experiments, 3 ml HM fraction were layered on 25.5 ml of a sucrose density gradient, linear from 1.91 to 0.5 M sucrose, and centrifuged for 1 h at 70800 x g in a Spinco SW 25.1 swing-out bucket rotor. Fractions of 1.5 mI were then collected. Procedure 2-To achieve larger yields of comparable purity in a shorter time, the discontinuous sucrose density gradient described by Sarzala et al. 49) was modified. An aliquot of HM fraction was layered on top of a gradient consisting of 20-6596 (w/v) sucrose, neutralized with 1M Tris-HC1 bufTer (pH 7.0), in the volumes shown in Fig. 1.
5Abbreviation: HM, heavy mitochondrial.
130000 g 30 rnin
FIG. 1. Purification of crude mitochondrial fractions (HM) by discontinuous sucrose density gradient centrifugation (procedure 2). Layering was done in a cold room in the volumes specified. Centrifugation was at 4 "g: in an International model B-60 ultracentrifuge. The tubes were centrifuged at 130080 x g for 30 rnin in rotor 5B-283 in an International model B-60 preparative ultracentrifuge. Three layers (Mi, M,, and M,) were separated; they were removed by means of a U-shaped syringe needle with a blunt end. All fractions were dialyzed routinely for 16 h at 2 4 @ in either buffer A (0.05 M potassium phosphate buffer (pH 7.0) - 5 rnhl P-mercaptoethanol - 1 nalM EDTA (pH 7.0)) or, when 5 '-nucleotidase was being assayed, in buffer B (buffer A with 0.05 M Tris-HC1 buffer (pH 7,O) instead of the Pi buffer). Marker Enzymes Glutamate dehydrogenase and 5 '-nucleotidase were assayed as described before (6). A unit of 5 '-nuclestidase activity is defined as that quantity that catalyzes the release of 1 pmol of Pi per hour. For glutamate dehydrogenase, one enzyme unit catalyzes the formation of 1 pmol of NADH per hour. Specific activity is given in enzyme units per milligram protein. Uracil Reductase Activities were assayed as described before (6) at pH 7.4 with either uracil or thymine as substrate and with NADH or NADPH present. Enzyme fractions were stored frozen at-78 @ in small aliquots and thawed only once before use. Unless specified otherwise, enzyme fractions were suspended in 5.4 M glycerol in ice just before use. One unit of enzyme activity is defined as the quantity that catalyzes the formation of 1 pmol of dihydrouracil or dihydrothymine per hour, The extinction coefficients per micromole of substrate reduced were the same as those for the assay of dihydrouracil dehydrogenase. Protein was determined by the method of Lowry et al. (10). Enzyme activity was a linear function of time up to 14 min and of protein concentration up to 1.8 mg of protein. The spectrophotometric assay method dQueener et ab. (1 1) was not used because of the protein density of the subcellular fractions as well as the fact that saturating levels of uracil (or thymine) interfered with the measurements. Paper Chromatography of Reaction Products To the standard assay mixture for NADH-dependent uracil reductase4 was added 0.6 pmol of [2-84C]uracil (0.0035 pCi). In these experiments, enzyme fractions were not suspended in glycerol because it interfered with the separation of reaction products. Duplicate tubes were incubated at 37 "C for 30 min. The reaction was stopped by boiling for 3 min. Control tubes contained no enzyme
TABLE 1. Summary of purification of dihydrouracil dehydrogenasea
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Specific activity (units/mg protein)
Stepandtreatment
Volume (ml)
Homogemate Supernatant Ammonium sulfate
146 91 63
Bihydrouracil NAB+ NADP+ 0.061 0.123 8.267
0.079 0.134 0.283
Dihydrothymine NAB
+
0.070 48.134 0.291
NADP
+
0.082 8.844 8.342
"Livers from two rats weighing 200 and 201 g were pooled.
which was added just before boiling. The tubes were centrifuged at 1 6 W x g for 20 min in a Sowall RC-2B refrigerated centrifuge. Fifty-microliter aliquots of the supernatant were then applied to Whatman No. I filter paper strips (1.8 x 38 em) which were developed by descending chromatcdgraphy in eollidine saturated with water as described by Fritzson ( 12). Chromatograms were air-dried and then viewed under U V light to detect uracil (I?,= 0.72). They were sprayed with p-dimethylaminobenzaldehyde and HCI (13) to detect dihydrouracil (I?,= 0.53) and carbamyl@-alanine (R,= 0.02). Other strips were dipped in winhydrin and heated to $8 'T for 10 min (13) to detect p-alanine (Rf=0.02). To detect radioactivity, still other chromatograms were cut into I-cm segments. Each segment was eluted with 500 pl of water and then 15 ml of toluene-based scintillation fluid containing 8.4% (w/v) omnifluor and 30% (v/v) ethanol were added. Radioactivity was counted in a Beckman LS-250 scintillation spectrometer at an efficiency of 80%. P)etermbnation ofLc~beNedCarbon Dioxide A modification of the method of Canellakis (2) was used. Mitochondria fraction M, was incubated in a final volume of 3 ml in the following (in micromoles): potassium phosphate buffer (pH 7.4t, 75; uracil, 0.7143 40.1 pCi); NAD+, 8.4: 108 p1 of absolute ethanol; 30 pg of alcohol dehydrogenase (Nutritional Biochem.); 10 mg Pa, protein. Duplicate samples were incubated at 37 "@ in a shaker bath in 10-rnl Erlenmeyer flasks equipped with rubber wells (Kontes Co.) to which 0.3 ml hyamine hydroxide was added to absorb radioactive CO,. Flasks were preincubated for 15 min before the addition of radioactive uracil. After inckebation for 30 min. 0.3 ml of 30% (wlv) trichloroacetic acid was added to stop the reaction. Flasks were incubated for an additional hour to ensure the complete absorption of C0,. To scintillation vials containing toluene-based scintillation fluid (4g omnifluor per liter toluene), 0.2 mB hyamine hydroxide from the wells of the reaction flasks was added. Radioactivity was determined as before. Appropriate controls lacking M, were included.
Bihydrouvaci! BeBaydrsgenwss3 Disc GPIElec~rophoresis Table 1 shows a typical experiment in which dihydroumci%dehydrogenase was partially purified from the cytosol fraction of rat liver. Routinely, a purification of about fourfold was obtained. The activity of the ammonium sulfate fraction with dihydrothymine as substrate was comparable to that
FIG.2. Relationship between staining intensity and protein concentration of enzyme bands separated by disc gel electrophoresis and stained B'or enzyme activity. The areas under the peaks for the major component (band 1) and the minor component (band 2) were determined from densitometer tracings of the stained gels by disc integrator attachment to give the intensity of staining in arbitrary units. Enzyme fractions purified fivefold from rat liver cytosoi were incubated in media containing NADP+ at pH 7.4 with dihydrothymine band 1 (@) and band 2 (0t; with dihydrouracil band 1 (V)and band 2 4 BB.
found with dihydrouracil, both at pH 8.8 with NAD' as coenzyme, but was 28% higher at pH 7.4 and N ADP' as coenzyme. Figure 2 gives a plot of activity Lverslas concentration of the major (band I ) and minor (band 2) enzyme components after disc gel electrophoresis and staining for activity by our nitroblue tetrazolium method ( 5 ) . By this procedure, only two active enzyme bands appear although several nonactive protein bands are known t o be present ( 5 ) . With NAK)Pf as coenzyme, b ~ t bands h showed two to three times more activity with dihydrothymine as substrate compared to dihydrouracil. Band 1 was always considerably more active with either substrate than was band 2. As before ( 5 ) . the relative mobilities (It%,)of the two bands were the same with either NAD' or NADP'; it was now found that the W,, values were also the same with dihydrothymine replacing dihy-
HALLOCK A N D Y.$MADA
TABLE2. Distribution of thymine reductase activity in subcellular fractionsQ Glutamate dehydrogenase
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Thymine f NADH Fraction Homogenate A Crude nuclei HM
Lb
Thymine + NADPH
5'-Nuclestidase
Specific activity
Total
(%)
Specific activity
Total (%)
Specific activity
Total (%)
Specific activity
Total (%)
0.088 0.123 0.264 0.107
100 24.1 51.0 6.0
1.12 1.93 2.86 B .55
168 38.3 63.0 7.0
8.045 0.889 0.064 0.140
100 29.9 33.2 23.2
1.45 2.80 2.08 3.21
1Ml 32.7 31.7 10.8
0
0.020
17.9
0.96
16rnx.g
supernatant Recovery (%)
0.020
-
12.4 93.5
108.3
104.2
33.4 108.6
T h e averages from two to four experimends are given. bLight mitochondria and lysasornes.
TABLE3. Enzyme distribution in mitochondria1 fractionsQ HM
Enzyme
Thymine-NADH reductase Uracil-NADH reductase Glutamate dehydrogenase Thymine-NADPH reductase Uracil-NABPM reductase 5'-Nucleotidase
Mr
-
M2
--
-
M3 -
Specific activity
Total (%)b
Specific activity
Total
(x)
Specific activity
Total (%)
Specific Total activity
0.214 0.156 2.860
51 .0 47.7 63.0
0.064 0.052 0
3.1 3.8 0
0.482 0.535 6.078
37.0 41.0 55.8
8.144 0.047 0.980
4.$ 2.4 4.4
0.064 0.055 2.080
33.2 35.8 31.7
0.330 0.324 2.350
21.4 28.4 9.2
0.078 0.013 1.480
9.9 7.0 16.9
0.033 0.031 0.930
3.3 2.4 2.9
(z)
QHMfraction was centrifuged in sucrose density gradients (procedure 2). Averages from two to four experiments are given. bCornpared to the total units in homogenate A taken as IOOY,.
drouracil with either coenzyme present. However, contrary to what was found when NADP+ was the coenzyme, both bands were as active with dihydrothymine a s with dihydrourcacil when NAD' was present. The physiological significance of the NAD'-linked activity cannot be assessed at present mainly because of the high pH optimum of this reaction. Urcacij Rcdrrcttrse Table 2 shows that the subcellular distribution of NADH-dependent reductase activity with thymine as substrate followed the distribution of the mitochondria%marker enzyme glutamate dehydrogenase. It is similar to that found earlier for the WADH-dependent uracil reductase activity (6). It is also apparent from Table 2 that the subcellular distribution of the WADPH-dependent reductase activity with thymine as substrate, as was found previously with uracil (6), corresponded closely with that of the marker enzyme 5 '-nucleotidase, which is associated with plasma and endsplasmic reticulum membranes (15-18). The distribution of enzyme activities after cen-
trifugation of the HMS fraction in discontinuous sucrose density gradients (procedure 2) is shown in Table 3. Fraction M, contained the major portion of the NADH-dependent reductase activity, with either thymine o r uracil a s substrate, as well as most of the glutamate dehydrogenase activity. The specific activity of M, with uracil a s substrate was usually higher than that found with thymine. The NADH-dependent enzyme was solubilized in 8.1 M P i buffer (pH 7.4) (14) rather than in glycerol, and the apparent K , values at pH 7.4 were estimated to be 0.085 mM for uracil, 0.118 mM for thymine, and 0.103 mM for NADH, with either substrate present. The K , value for uracil is Bower than that reported earlier for the less pure HM fraction (B), Centrifugation of the HM fraction by procedure 2 also I-esulted in enrichment of the NADPHdependent reductase a s well a s 5 '-nucleotidase, but in fraction Fa, rather than M, (Table 3). Fraction M, was almost as active with uracil as with thymine. The reductase was solubilized a s above and its afinity for the two substrates determined. The apparent K,, values (at pH 7.4) were estimated to be 0.118 mikf for uracil, 0.113 mM for thymine, and
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CAN. I. BIOCHEM. VOE. 54, 1976
FKG.3. Enzyme distribution in HM fractions (heavy mitochondria) centrifuged in linear sucrose density gradients (procedure 1). Activity is expressed as micromoles of dihydrouracil (DHU) or dihydrothymine (DHT) formed per hour per tube with uracil (U) or thymine (T) as substrate; 1.5 ml fractions were collected. A unit of glutamate dehydrogenase activity is expressed as the micromoles of NADH formed per hour per tube; a 3-ml aliquot cc~ntaining75.6 mg protein s f the HM fraction was used. The arrow indicates the direction of sedimentation. 0.065 m M for NABPH, with either substrate present. The Km value for NADPH is lower than that found earlier for less pure fractions (6). Figure 3 shows that fractionation of the HM fraction by centrifugation in a linear sucrose density gradient (procedure 1) gave less well defined separation of the NABM- and NADPH-dependent reductases. Notable, however, was the finding that activity with uracil or thymine as substrate peaked in the same fraction. Again the NADH-dependent activity coincided with that of glutamate dehydrogenase whereas the NADPH-dependent activity was in the position routinely marked by 5 '-nucleotidase (6).
Weaction Products Mitochondria1 fraction M2 was incubated in the standard assay mixture containing [2-14C]uraciland NABH. Incubation was for 30 min at 37 "C. The reaction products were separated by paper chromatography as described in Experimental. Fifty percent of the radioactivity was recovered as uracil, 30% as dihydrouracil and 7.5% as a ninhydrinpositive spot close to the origin (Fig. 4B). Whether the last was P-ahnine or residual enzyme protein was uncertain. Recovery of total radioactivity initially added to the reaction mixture was 85-98% in two separate experiments.
Formation s f C 0 , Mitochondria1fraction M2 was incubated in reaction media at pH 7.4 containing [2-14C]uracil.Figure 4A shows that the formation of radioactive GO, increased with increasing uracil concentrations (at constant specific radioactivity) and began to level off at 10 mM. Linearity between CO, produced and time was observed for 38 min with 1.6 - 10 mg of mitochondria1 protein per flask. The amount of uracil degraded by mitochondria was estimated to be about 10% of that formed by the original hornogenate, both with NAD+ as coenzyme.
Discussion With regard to the substrate specificity of dihydrouracil dehydrogenase3 of cytosol fractions, in two separate reports, thymine was found to be degraded at */, and */, the rate of uracil (3, 19) with NABPH as coenzyme. Thymine inhibited the catabolism of uracil much more than vice versa (8)For the reverse reaction (3), dihydrothymine appeared to be oxidized at a much more rapid rate than dihydrouracil although no mention of this was made in the text. In the present work, the major and minor
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HALLOCK AND YAMADA
D I S T A N C E (ern x lo-')
FIG.4. (A) Relationship between nadioactive C 0 , formed (cpm) and [2-'4C]uracil concentration. Fraction M, (10 rng protein) was incubated for 30 rnin at 37 "C at pH 7.4. (B) Radioactivity (cprn) of reaction products separated by paper chromatography. Fraction M, (10 mg protein) was incubated for 30 min at 37 "62 in the standard reaction mixture for uracil reductase in the presence of [2-14C]uraciIand NABH.
bands of dih ydrouraciI deh ydrogenase, separated from rat liver cytosol fractions by disc gel electrophoresis, did not differ in substrate specificity; both bands were t w o t o three times more active towards dihydrsthymine a s towards dihydrouracil with NADP+ a s cofactor. It is possible that the earlier preparations of dihydrouracli dehydrogenase contained the NADPW-dependent reductase of membranes; this would explain, in part at least, the anomalous results a s t o the substrates preferred (3, 8, 19). Tentatively then, our resuits are not in conflict with the conclusions of Barrett et cal. (8) that the same dihydrouracil dehydrogenase of cytosol fractions acts o n both uracil and thymine but it is apparent that more extensive enzyme purification and characterization are needed. That there are at lease two separate uracil reductases4 in particulate fractions of rat liver is apparent. Besides coenzyme specificity, intracellular location and apparent Km values for thymine and uracil, the reductases have recently been found to differ most notably in sensitivity t o activation by P,, E D T A o r EGTA, and inhibition by calcium ions (14).
That mitochondria1 fractions catalyze the degradation of uracil t o form CO, to only a limited extent is shown in the present work. The results suggest that the degradative enzymes beyond uracil reductase are limiting in amount in mitochondria s o that the dihydroumcil formed must be extruded mainly into the cytsso! for further degradation. Such an
efflux of dihydrouracil may perhaps be the reason for the virtual nonreversibility of the reaction catalyzed by uracil reductase of these organelles with dihydrouracil a s substrate; significant activity in the reverse direction was found only after solubilization of mitochondria by glycerol 46). The high concentration of uracil ( 10 rnM)required fipr maximal formation of C 0 2 , in contrast to the low K , for uracil of uracil reductase of mitochondria, indicates that the reduction of uracil is not the ratelimiting step of uracil degradation in mitochondria and perhaps not even in the cytosol(20,2 19. A factor to be considered is the high K , value of 11.75 x M reported for dihydrouracil hydrase with dihydrouracil a s substrate; this enzyme was purified 200-fold from calf liver (229. This Km value is much - 2.0 x M found higher than those of 1.5 x for di hydrouracil dehydrogenase of rat liver cytosol with dihydrouracil a s substrate (2,6); an even lower K , for uracil, estimated t o be less than 4 x 101'WM, was reported for the cytosol enzyme (2). However, n o conclusions can be drawn a s yet from these data since the enzymes were from different sources and only partially purified. 1. Canellakis, E. S. (1456)J. Biol. Cl~etn. 221,3 15-322 2. Fritzson, P. (1960)J. Biol. Chern. 235,719-725 3. Grisolia, S. &Cardoso, S. S. (1957)Bioclzim. Biophys. Acfn 25,430-43 1 4. Goedde, H. W., Agarwal, B. P. & Eickhoff, K. (1978) Z . Physiol. Chem. 451 ,945-95 1 5, Hallock, R. 8 . & Yamada, E. W. (1973) Anal. Biochem. 56,84-90
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184
CAN. J . BIOCHEM. VOL. 54, 1976
IS. Bosman, H. B., Hagopian, A. & Eylar, E. H. (1948) 6. Smith, A. E. & Yarnada, E. W. (1971) J . Bicd. Chem. Arch. Biochern. Biophys. %28,51-69 246,3610-3617 16. Song, C. S., Nisselbaum, J . S., Landler, B. & 7. Bresnick, E. (1964) Tes. Rep. Biol. Aged. 22,431-439 Bodansky, 0. (1968) Biorhim. Biophys. Acso 150, 8. Barrett, H. W., Munavalli, S. N. & Newrnark, P. ( 1964) Bioclaim. Bioplays. Acra 91 , 199-204 380-303 17. Song, C. S. & Bodansky, 0 .(1967)J. Biol. Chem. 242, 9. Sarzaia, M. G.. Van Gslde, L. M. C.. De Kruyff, B. & 694-699 Van Deenen, L. L. M. ( 1970) Biochirn. Biophys. A C S ~ 18. Evans, W. H . B 1869) FEBS &eft.3,237-2463 202,106-3 19 18. Lowry, 0. H.,Rosebrough, N. J.. Farr, A. L. & 19. Newmark, P., Stephens,.!. D. & Barnett, H. W. (1962) Randall, W. J . (1951) 5. Bicd. Chem. 193,265-275 Bischina. Biophys. Actu 62,414-416 1 8 . Qiaeener, S. F., Morris, H . P. & Weber, G . (1971) 20. Sanno, Y., Hslzer. M. & Schimke, R. T. (1978) J. Ctasace~aR e s . 36, 1004-1009 Biol. Chern. 245,5668-5474 82. Fritzson, P. & Pihl, A. (1957) J. Biol. Chprn. 226, 2 1. Fritzson. P. ( 1957)5. B i d . C'hem. 226,223-228 22. Wallach, D. P. $L Grisslia, S. (1957) J. Biol. Clzem. 229-235 226.277-288 93. Fink, R. M . , Cline, W. E., McGaughey, C. & Fink, K. (1956) A n d . Chem. 28,4-6 14. Yarnada, E. W. (1975) Can. J. Biochem. submitted for publication
