PROTEIN
EXPRESSION
AND
PURIFICATION
1,
45-48
(1990)
Purification of Hamster Dihydroorotate Synthetase Using Procion Blue-Sepharose’ Linda
Crofts,
Yin Peide, Amanda
Woodhouse,
Elizabeth
M. Algar,’
and Richard
I. Christopherson
Department of Biochemistry, University of Sydney, Sydney, New South Wales 2006, Australia
Received
February
23, 1990,
and in revised
form
May
4, 1990
Dihydroorotate (DHO) synthetase is a trifunctional protein that catalyzes the first three reactions of de nova pyrimidine biosynthesis. A single-step procedure for purification of DHO synthetase from mutant hamster cells that overproduce this protein has been developed. The synthetase is adsorbed from a postmitochondrial supernatant to a column of Procion blue-sepharose 4B and, after the column is washed, the synthetase is eluted as a single peak with 0.4 M KCl. Pooled fractions from the trailing side of this peak yield DHO synthetase with a specific activity for aspartate transcarbamylase of 14 ~mol/min/mgprotein, representing a purification factor of 8.5-fold and a recovery of 28% from the postmitochondrial supernatant. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the DHO synthetase was of high purity. A further 34% of the DHO synthetase from the leading side of the eluted peak contained a minor proportion of a proteolytic fragment. Similar results were obtained with an established four-step purification procedure. o 1990 Academic
Press, Inc.
Dihydroorotate (DH0)4 synthetase5 is part of a trifunctional protein that catalyzes the first three reac-
i This work was supported by National Health and Medical Research Council Project Grant 860690. * School of Science, Griffith University, Nathan, Queensland 4111, Australia. 3 To whom requests for reprints should be addressed. 4 Abbreviations used: CAP, carbamyl phosphate; CA-Asp, N-carbamyl-r,-aspartate; DHO, L-dihydroorotate; GDH, a stabilization buffer containing 30% (v/v) glycerol, 2 mM dithiothreitol, and 10 mM K * Hepes, pH 7.2. ’ DHO synthetase, also known as CAD, is the trifunctional protein containing the first three enzymatic activities of the pyrimidine pathway: carbamyl phosphate synthetase (EC 6.3.5.5), aspartate transcarbamylase (EC 2.1.3.2), and dihydroorotase (EC 3.5.2.3). 1046-5928/90 $3.00 Copyright 0 1990 by Academic Press, All rights of reproduction in any form
tions of de nouo pyrimidine karyotes (1). HCO;
+ 2ATP + L-Gln
%
biosynthesis
carbamyl
in higher eu-
phosphate
+ 2ADP + L-Glu +L-Asp_ N-carbamylL-aspartate
-
+H+
L-dihydroorotate
+ H,O
[l]
DHO synthetase has been partially purified in low yield from Drosophila melanogaster (2), bullfrog eggs (3), mouse Ehrlich ascites carcinoma (l), and rat liver (4). Mori and Tatibana (5) subsequently purified DHO synthetase from rat liver in six steps. This trifunctional protein has been purified in large amounts from an SV40-transformed hamster cell line (165-23) which overproduces the protein by loo-fold (6). The procedure of Coleman et al. involves four steps from the postmitochondrial supernatant: streptomycin sulfate precipitation, ribonuclease A digestion, ammonium sulfate precipitation, and fractionation on a Bio-Gel A-5m column, resulting in a purification factor of 5.2-fold. We have developed a single-step purification procedure using dye-ligand chromatography which yields DHO synthetase with a purity comparable to that obtained using the procedure of Coleman et al. (6). EXPERIMENTAL
PROCEDURES
Soybean trypsin inhibitor, benzamidine, and highmolecular-weight protein markers were purchased from
Sigma Chemical
Co. (St. Louis, MO). Dilithium
[l”C]-
CAP (7.94 Ci/mol) was purchased from New England Nuclear (Boston, MA). Sodium [14C]bicarbonate (57.0 Ci/mol) was from Amersham, UK. [14CJCA-Asp and [14C]DH0 (57.0 Ci/mol) were synthesized from [14C]bicarbonate as previously described (7). All other chemicals were of analytical reagent grade. 45
Inc. reserved.
46
CROFTS
Mr
205,000 116,000
97,400 66,000 45,000 29,000
-> u 0 ;;:,“p”:
_= z
= 5
s
* 2
P Gi
FIG. 1.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of pooled and concentrated fractions from Procion blue-Sepharose 4B. PMS, postmitochondrial supernatant (110 pg of protein); Std, molecular weight standards (myosin was run separately); Pools I-IV (7 pg of protein each). Proteins were stained with Coomassie blue R-250. Further details appear under Experimental Procedures.
The dye-matrix screening kit was from Amicon, Australia. Procion blue MX-4GD (Imperial Chemical Industries, Australia) was a kind gift from Dr. R. K. Scopes, La Trobe University. Sepharose 2B, 4B, and 6B were obtained from Pharmacia Fine Chemicals, Sweden. GelBond PAG film was from LKB, Sweden. BioGel A-5m (200-400 mesh) was from Bio-Rad Laboratories (Richmond, CA). Polyethyleneimine-cellulose thinlayer chromatograms (20 X 20 cm) were from Machery-Nagel and Co., Germany. Dulbecco’s modification of Eagle’s medium and sterile penicillin-streptomycin (5000 IU/ml and 5000 pg/ml) were obtained from Flow Laboratories, UK. Fetal calf serum (batch 971 4001) was supplied by the Commonwealth Serum Laboratories, Australia. Cell culture. A mutant hamster cell line (165-23) producing 100 times the normal level of DHO synthetase and able to grow in the presence of high concentrations of N-phosphonacetyl-L-aspartate was obtained from Dr. George Stark (Imperial Cancer Research Fund, UK). Cells were grown in Dulbecco’s modified Eagle’s medium in loo-mm plastic culture dishes as described by Coleman et al. (6). Confluent cells adhering to each dish were washed with 1.0 ml of TD buffer (0.14 M NaCl, 5 mM KCl, 1 mM Na,HPO,) and 50 mM TrisHCl, pH 7.4). Cells to be propagated were removed by digestion for 1 min with 1.0 ml of 0.5% (w/v) trypsin and resuspended in TD buffer (0.14 M NaCl, 5 mM KCl, 1 mM Na,HPO, and 50 mM Tris-HCl, pH 7.4) prior to inoculation. For purification of DHO synthetase, cells were washed with 1.0 ml of cold PBS (137 mM NaCl, 9.7 mM NazHPO,, pH 7.4) and scraped from dishes with PBS containing 0.1 mM EDTA, 2.5 mM benzamidine, and soybean trypsin inhibitor (0.25 mg/ml). Cells were resuspended and washed in the same medium prior to freezing at -80°C or extraction.
ET
AL.
Enzyme assays. Carbamyl phosphate synthetase was assayed by measuring the incorporation of [14C]bicarbonate into acid-stable i4C-labeled CA-Asp and DHO by coupling the synthesis of [14C]CAP to endogenous aspartate transcarbamylase and dihydroorotase of DHO synthetase; pyruvate kinase was used to regenerate ATP. Aspartate transcarbamylase was measured by the conversion of [14C]CAP to acid-stable products, dihydroorotase activity was determined in the reverse direction using [14C]DH0 as substrate, and [14C]CA-Asp was isolated by chromatography on polyethyleneiminecellulose (8,9). Acid-stable radioactivity in aqueous samples from assays of carbamyl phosphate synthetase and aspartate transcarbamylase was counted at an efficiency of 91.0% with a Beckman LS 3800 scintillation counter after mixing with 9 vol of scintillation cocktail (5 g PPO, 0.1 g POPOP/liter toluene:Teric X-10; 2:1, v/v). [14C]CA-Asp from dihydroorotase assays isolated on polyethyleneimine-cellulose was counted at an efficiency of 81.2% in cocktail containing 3.0 g PPO and 0.1 g POPOP/liter toluene. Dye-ligand chromatography. To develop a simpler procedure for purification of DHO synthetase from mutant hamster cells, 40 triazine dye-Sepharose adsorbents were screened for their ability to bind this trifunctional protein. Using a screening strategy described by Scopes (10, 11) and a rapid batch-wise centrifugation procedure, navy B- (Procion blue), brown B-, and violet B-Sepharose 4B were found to bind most of the trifunctional protein; orange D-Sepharose 4B interacted weakly with the synthetase while retaining other proteins. Of the “positive” adsorbents, DHO synthetase could be eluted with 1 M KC1 only from Procion blueSepharose and this adsorbent was chosen for further development. The triazine dye Procion blue (MX-4GD) was coupled to Sepharose 2B, 4B, or 6B by the method of Atkinson et al. (12). To 30 g of Sepharose suspended in water (105 ml) a solution of Procion blue (30 ml, 1.5%, w/v) was added, followed by 4 M NaCl (15 ml) and 10 M NaOH (1.98 ml). The slurry was stirred for 2 days at room temperature, treated with 2-mercaptoethanol in alkali to react with the second chlorine group, and then washed extensively. For enzyme inhibition studies, the reactive chlorine groups of free Procion blue were replaced with amino groups by treating the dye with 2 M ammonium chloride (pH 8.5) for 4 h at room temperature (12). Analysis of free and blocked Procion blue by reverse-phase high-pressure liquid chromatography showed the presence of at least six different chemical species. Purification of DHO synthetase on Procion blueSepharose 4B. Hamster cells (8.4 g) were swollen for 15 min in 2 vol of hypotonic buffer (5 mM MgCl,, 2 mM 15 IIIM KCl, 6 mM L-Gln, 6 CaCl,, 1 mM dithiothreitol, mM ~-Asp,
10 mM benzamidine,
1 mg/ml
soybean
tryp-
PURIFICATION
OF
DIHYDROOROTATE
TABLE
Comparison of Purification
1
of DHO Synthetase Using Either Procion Blue-Sepharose 4B or Bio-Gel A-5m Aspartate
Volume (ml) Postmitochondrial I
Pool
II
Pool Pool
III IV
Protein concentration bdd
supernatant
Total protein (md 100
& Pool
47
SYNTHETASE
(2) 1.2
(4.2) 0.95 1.2
(2993
(f-50)
(E7) 1.9 (0.82) 0.80 -=
3.3 (0.97) 2.4 (3.4) 0.76 -
Total activity (gmol/min) 160 (97) (2) (YZ, 11 6.9
transcarbamylase Specific activity (amol/min/mg)
(::W) (::I 14 (5.4) 14 -
Purification factor
t:, 10.6 (8.5) (E) 8.8 -
Note. A postmitochondrial supernatant from mutant hamster cells was loaded directly onto a column of Procion blue-Sepharose 4B (1 X 41 cm) and eluted as described under Experimental Procedures or subjected to the procedure of Coleman et al. (6) completed by elution from a column of Bio-Gel A-5m (1 X 20 cm). Values pertaining to the latter established procedure obtained in our laboratory appear in parentheses. Fractions pooled from the Procion blue-Sepharose column were: Pool I, 350-360 ml; Pool II, 360-375 ml; Pool III, 375-390 ml; Pool IV, 390-410 ml. The protein constituents of these pools are shown in Fig. 1. Pools for the Bio-Gel A-5m column were made according to Coleman et al. (6). The purification of trifunctional DHO synthetase was measured by aspartate transcarbamylase activity. a The protein concentration was too low to be accurately measured.
sin inhibitor, 10 mM triethanolamine, 1 mM EDTA, and 0.3 InM UTP, pH 7.0). The swollen cells were homogenized in hypotonic buffer for 5 strokes, glycerol was added to a final concentration of 30% (v/v), and the cells were homogenized for a further 15 strokes. The homogenate was centrifuged at 20,OOOg for 20 min in a Sorvall SS-34 rotor at 4°C. The postmitochondrial supernatant (26 ml) was removed and immediately applied to a column of Procion blue-Sepharose 4B (1 X 41 cm) equilibrated with 5 mM MgCl, in GDH buffer (30% (v/v) glycerol, 2 InM dithiothreitol, 10 InM KS Hepes, pH 7.2). Unbound protein was removed with this solvent (122 ml) at a flow rate of 12 ml/h; further protein was removed with GDH lacking MgCl, (118 ml). The column was then washed with GDH containing 0.2 M KC1 (80 ml), which eluted a minor proportion of DHO synthetase. A major proportion of the trifunctional synthetase was then eluted with GDH containing 0.4 M KC1 (80 ml); increasing KC1 to 0.5 M (80 ml) resulted in a small peak of DHO synthetase. A further increase to 1 M KC1 (183 ml) eluted only minor peaks for DHO synthetase and protein. Appropriate fractions were pooled and concentrated and KC1 was removed under nitrogen gas using an Amicon Model 8050 ultrafiltration cell fitted with an XM 300 membrane and an auxiliary reservoir (800 ml). Protein concentrations were measured using the BioRad microassay (13); standards contained O-20 pg/ml of bovine albumin. Electrophoresis. Protein in samples was precipitated with 5% (w/v) trichloroacetic acid and redissolved in sample buffer (1% (w/v) sodium dodecyl sulfate, 1% (v/ v) mercaptoethanol, 0.15 M Tris-HCl, pH 8.8). Acrylam-
ide slabs consisted of a 3% (w/v) stacking gel and a 5% (w/v) resolving gel containing gel buffer (0.4% (w/v) sodium dodecyl sulfate, 1.5 M Tris-HCl, pH 8.8); electrode buffer contained 1% (w/v) sodium dodecyl sulfate and 0.375 M Tris-glycine, pH 8.3. The gel was poured to a thickness of 0.5 mm between GelBond PAG film attached to one glass plate and slot molds attached to the other plate. Samples (6 ~1) were pipetted into the slots; standards contained a mixture of carbonic anhydrase (M, 29,000), ovalbumin (iW, 45,000), bovine albumin (M, 66,000), phosphorylase b (M, 97,400), P-galactosidase (M, 116,000), myosin (M, 205,000), and 0.01% (w/v) bromphenol blue. Electrophoresis was performed with an LKB Multiphor II electrophoresis unit at 40 mA for 6 h. The gel was then fixed in 20% (w/v) trichloroacetic acid, stained in 0.1% (w/v) Coomassie blue R-250 in methanol:acetic acidwater (45:10:45), and destained in methanol:acetic acidwater (35:10:55). Stained gels were scanned using an RFT Transidyne scanning densitometer at 590 nm. RESULTS
Preliminary experiments showed that of the 40 dyeligand adsorbents tested, DHO synthetase bound with high affinity, but reversibly, only to Procion blueSepharose 4B. Inclusion of 5 InM MgCI, in the GDH buffer resulted in almost quantitative retention of DHO synthetase by Procion blue-Sepharose 4B. Divalent metal ions form chelate bridges between triazine dyes and adsorbed proteins (10,ll). Specific elution of the trifunctional protein from the adsorbent was tried with
48
CROFTS
all substrates of the three enzymatic activities without success. From experiments where bound DHO synthetase was eluted with a KC1 gradient in GDH and by step-wise increases in KCl, the column washing and elution procedure described under Experimental Procedures was optimized. Procion blue-Sepharose 2B, 4B, and 6B were synthesized, but the separation obtained with Procion blue-Sepharose 4B was optimal. DHO synthetase does not elute in the void volume after addition of 0.4 M KC1 but it is sieved by Sepharose 4B. Thus the purification obtained can be attributed to a combination of adsorption and molecular sieve chromatography. Appropriate fractions from the elution profile were subjected to slab gel electrophoresis (Fig. 1). Pool II (360-375 ml) and Pool III (375-390 ml) were of high purity. Pool I contained a contaminant of slightly lower molecular weight which may be formed by proteolysis of DHO synthetase (6). DHO synthetase could be maintained as a single polypeptide (M, 210,000, Fig. 1) during the purification procedure when all manipulations were performed rapidly and at 0-4’C. For comparitive purposes, DHO synthetase was also purified by the procedure of Coleman et al. (6) from the same cells; results from both procedures are presented in Table 1. The specific activity of aspartate transcarbamylase purified by passage through Procion blue-Sepharose 4B of 14 ~mol/min/mg protein for Pools II and III is similar to the activity obtained for Pool I by the established procedure. The ratios of aspartate transcarbamylase and dihydroorotase activities relative to carbamyl phosphate synthetase activity are increased over those for the postmitochondrial supernatant ((carbamyl phosphate synthetase):(aspartate transcarbamylase):(dihydroorotase), 1:70:4.6), indicating that there is some loss of carbamyl phosphate synthetase activity in either procedure. The ratio obtained for Pool II from the Procion blue-Sepharose column was 1:105:6.0 and that for Pool I from the Bio-Gel A-5m column was 1:104:9.0. Densitometric scans of slab gels stained with Coomassie blue (Fig. 1) confirmed that the two procedures yielded DHO synthetase of comparable purity.
DISCUSSION DHO synthetase can be rapidly purified in a single step by passage through a column of Procion blueSepharose 4B; 67% of the aspartate transcarbamylase activity applied to the column eluted with 0.4 M KCl. The chemical structure of Procion blue does not resemble that of any of the substrates of DHO synthetase and
ET
AL.
it was therefore not surprising that the synthetase could not be*specifically eluted with a substrate. Free Procion blue with the reactive chloro groups displaced by amino groups inhibits carbamyl phosphate synthetase but not aspartate transcarbamylase or dihydroorotase activities.‘j The purification factor of 8.5-8.8 obtained with Procion blue-Sepharose 4B (Pools II and III, Table 1) is comparable to the factor of 8.5 (Table 1) obtained by the established procedure (6), but silver staining of the purest fractions revealed the presence of a number of minor contaminating proteins6 (cf. Fig. 1). These minor contaminants would persist in a significant relative abundance if DHO synthetase were purified from “wildtype” cells. An additional purification step would be required to purify DHO synthetase from normal cells. ACKNOWLEDGMENTS We thank the Procion preliminary
Dr. Robert blue used experiments.
Scopes in this
for helpful study and
advice Elizabeth
and
for providing Isaac for some
REFERENCES 1. Shoaf,
W. T.,
and
Jones,
M. E. (1973)
Biochemistry
12,
4039-
P. P. (1975)
Proc.
4051. 2. Jarry, 3. Kent, Natl.
B. (1976)
FEBS
I&t.
70,
71-75.
R. J., Lin, R., Sallach, H. J., and Cohen, Acad. Sci. USA 72, 1712-1716.
4. Mori, 5.
M., and Tatibana, M. (1973) Biochem. Biophys. Res. Commun. 54,1525-1531. Mori, M., and Tatibana, M. (1978) in “Methods in Enzymology” (Hoffee, P. A., and Jones, M. E., Eds.), Vol. 51, pp. 111-121, Academic Press, New York.
6. Coleman, P. F., Suttle, D. P., and Stark, G. R. (1977) J. Biol. Chem. 252,6379-6385. 7. Christopherson, R. I., Schmalzl, K. J., Szabados, E., Goodridge, 8.
R. J., Harsanyi, M. C., Sant, M. E., Algar, A., Sharma, S. C., Bubb, W. A., and Lyons, S. D. (1989) Biochemistry 28,463-470. Christopherson, R. I., Yu, M.-L., and Jones, M. E. (1981) Anal. Biochem. 111,240-249.
9. Christopherson,
R. I., Matsuura, T., and Jones, M. E. (1978) Anal. Biochem. 89, 225-234. R. K. (1984) “Protein Purification: Principles and Prac10. Scopes, tice,” pp. 125-131, Springer-Verlag, New York. 11. Scopes, R. K. (1984) “Amicon 40 Column Screening Kit Instructions,” Amicon Corp., Danvers, MA. T., Hammond, P.M., Hartwell, R. D., Hughes, P., Sca12. Atkinson, wen, M. D., Sherwood, R. F., Small, D. A. P., B&on, C. J., Harvey, M. J., and Lowe, C. R. (1981) Biochem. Sot. Trans. 9, 290293.
13. Bradford, ‘A. ments.
M. M. (1976)
Woodhouse
and
Anal.
Biochem.
R. I. Christopherson,
72,
248-254.
unpublished
experi-
EXPRESSION
AND
PURIFICATION
1,
45-48
(1990)
Purification of Hamster Dihydroorotate Synthetase Using Procion Blue-Sepharose’ Linda
Crofts,
Yin Peide, Amanda
Woodhouse,
Elizabeth
M. Algar,’
and Richard
I. Christopherson
Department of Biochemistry, University of Sydney, Sydney, New South Wales 2006, Australia
Received
February
23, 1990,
and in revised
form
May
4, 1990
Dihydroorotate (DHO) synthetase is a trifunctional protein that catalyzes the first three reactions of de nova pyrimidine biosynthesis. A single-step procedure for purification of DHO synthetase from mutant hamster cells that overproduce this protein has been developed. The synthetase is adsorbed from a postmitochondrial supernatant to a column of Procion blue-sepharose 4B and, after the column is washed, the synthetase is eluted as a single peak with 0.4 M KCl. Pooled fractions from the trailing side of this peak yield DHO synthetase with a specific activity for aspartate transcarbamylase of 14 ~mol/min/mgprotein, representing a purification factor of 8.5-fold and a recovery of 28% from the postmitochondrial supernatant. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the DHO synthetase was of high purity. A further 34% of the DHO synthetase from the leading side of the eluted peak contained a minor proportion of a proteolytic fragment. Similar results were obtained with an established four-step purification procedure. o 1990 Academic
Press, Inc.
Dihydroorotate (DH0)4 synthetase5 is part of a trifunctional protein that catalyzes the first three reac-
i This work was supported by National Health and Medical Research Council Project Grant 860690. * School of Science, Griffith University, Nathan, Queensland 4111, Australia. 3 To whom requests for reprints should be addressed. 4 Abbreviations used: CAP, carbamyl phosphate; CA-Asp, N-carbamyl-r,-aspartate; DHO, L-dihydroorotate; GDH, a stabilization buffer containing 30% (v/v) glycerol, 2 mM dithiothreitol, and 10 mM K * Hepes, pH 7.2. ’ DHO synthetase, also known as CAD, is the trifunctional protein containing the first three enzymatic activities of the pyrimidine pathway: carbamyl phosphate synthetase (EC 6.3.5.5), aspartate transcarbamylase (EC 2.1.3.2), and dihydroorotase (EC 3.5.2.3). 1046-5928/90 $3.00 Copyright 0 1990 by Academic Press, All rights of reproduction in any form
tions of de nouo pyrimidine karyotes (1). HCO;
+ 2ATP + L-Gln
%
biosynthesis
carbamyl
in higher eu-
phosphate
+ 2ADP + L-Glu +L-Asp_ N-carbamylL-aspartate
-
+H+
L-dihydroorotate
+ H,O
[l]
DHO synthetase has been partially purified in low yield from Drosophila melanogaster (2), bullfrog eggs (3), mouse Ehrlich ascites carcinoma (l), and rat liver (4). Mori and Tatibana (5) subsequently purified DHO synthetase from rat liver in six steps. This trifunctional protein has been purified in large amounts from an SV40-transformed hamster cell line (165-23) which overproduces the protein by loo-fold (6). The procedure of Coleman et al. involves four steps from the postmitochondrial supernatant: streptomycin sulfate precipitation, ribonuclease A digestion, ammonium sulfate precipitation, and fractionation on a Bio-Gel A-5m column, resulting in a purification factor of 5.2-fold. We have developed a single-step purification procedure using dye-ligand chromatography which yields DHO synthetase with a purity comparable to that obtained using the procedure of Coleman et al. (6). EXPERIMENTAL
PROCEDURES
Soybean trypsin inhibitor, benzamidine, and highmolecular-weight protein markers were purchased from
Sigma Chemical
Co. (St. Louis, MO). Dilithium
[l”C]-
CAP (7.94 Ci/mol) was purchased from New England Nuclear (Boston, MA). Sodium [14C]bicarbonate (57.0 Ci/mol) was from Amersham, UK. [14CJCA-Asp and [14C]DH0 (57.0 Ci/mol) were synthesized from [14C]bicarbonate as previously described (7). All other chemicals were of analytical reagent grade. 45
Inc. reserved.
46
CROFTS
Mr
205,000 116,000
97,400 66,000 45,000 29,000
-> u 0 ;;:,“p”:
_= z
= 5
s
* 2
P Gi
FIG. 1.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of pooled and concentrated fractions from Procion blue-Sepharose 4B. PMS, postmitochondrial supernatant (110 pg of protein); Std, molecular weight standards (myosin was run separately); Pools I-IV (7 pg of protein each). Proteins were stained with Coomassie blue R-250. Further details appear under Experimental Procedures.
The dye-matrix screening kit was from Amicon, Australia. Procion blue MX-4GD (Imperial Chemical Industries, Australia) was a kind gift from Dr. R. K. Scopes, La Trobe University. Sepharose 2B, 4B, and 6B were obtained from Pharmacia Fine Chemicals, Sweden. GelBond PAG film was from LKB, Sweden. BioGel A-5m (200-400 mesh) was from Bio-Rad Laboratories (Richmond, CA). Polyethyleneimine-cellulose thinlayer chromatograms (20 X 20 cm) were from Machery-Nagel and Co., Germany. Dulbecco’s modification of Eagle’s medium and sterile penicillin-streptomycin (5000 IU/ml and 5000 pg/ml) were obtained from Flow Laboratories, UK. Fetal calf serum (batch 971 4001) was supplied by the Commonwealth Serum Laboratories, Australia. Cell culture. A mutant hamster cell line (165-23) producing 100 times the normal level of DHO synthetase and able to grow in the presence of high concentrations of N-phosphonacetyl-L-aspartate was obtained from Dr. George Stark (Imperial Cancer Research Fund, UK). Cells were grown in Dulbecco’s modified Eagle’s medium in loo-mm plastic culture dishes as described by Coleman et al. (6). Confluent cells adhering to each dish were washed with 1.0 ml of TD buffer (0.14 M NaCl, 5 mM KCl, 1 mM Na,HPO,) and 50 mM TrisHCl, pH 7.4). Cells to be propagated were removed by digestion for 1 min with 1.0 ml of 0.5% (w/v) trypsin and resuspended in TD buffer (0.14 M NaCl, 5 mM KCl, 1 mM Na,HPO, and 50 mM Tris-HCl, pH 7.4) prior to inoculation. For purification of DHO synthetase, cells were washed with 1.0 ml of cold PBS (137 mM NaCl, 9.7 mM NazHPO,, pH 7.4) and scraped from dishes with PBS containing 0.1 mM EDTA, 2.5 mM benzamidine, and soybean trypsin inhibitor (0.25 mg/ml). Cells were resuspended and washed in the same medium prior to freezing at -80°C or extraction.
ET
AL.
Enzyme assays. Carbamyl phosphate synthetase was assayed by measuring the incorporation of [14C]bicarbonate into acid-stable i4C-labeled CA-Asp and DHO by coupling the synthesis of [14C]CAP to endogenous aspartate transcarbamylase and dihydroorotase of DHO synthetase; pyruvate kinase was used to regenerate ATP. Aspartate transcarbamylase was measured by the conversion of [14C]CAP to acid-stable products, dihydroorotase activity was determined in the reverse direction using [14C]DH0 as substrate, and [14C]CA-Asp was isolated by chromatography on polyethyleneiminecellulose (8,9). Acid-stable radioactivity in aqueous samples from assays of carbamyl phosphate synthetase and aspartate transcarbamylase was counted at an efficiency of 91.0% with a Beckman LS 3800 scintillation counter after mixing with 9 vol of scintillation cocktail (5 g PPO, 0.1 g POPOP/liter toluene:Teric X-10; 2:1, v/v). [14C]CA-Asp from dihydroorotase assays isolated on polyethyleneimine-cellulose was counted at an efficiency of 81.2% in cocktail containing 3.0 g PPO and 0.1 g POPOP/liter toluene. Dye-ligand chromatography. To develop a simpler procedure for purification of DHO synthetase from mutant hamster cells, 40 triazine dye-Sepharose adsorbents were screened for their ability to bind this trifunctional protein. Using a screening strategy described by Scopes (10, 11) and a rapid batch-wise centrifugation procedure, navy B- (Procion blue), brown B-, and violet B-Sepharose 4B were found to bind most of the trifunctional protein; orange D-Sepharose 4B interacted weakly with the synthetase while retaining other proteins. Of the “positive” adsorbents, DHO synthetase could be eluted with 1 M KC1 only from Procion blueSepharose and this adsorbent was chosen for further development. The triazine dye Procion blue (MX-4GD) was coupled to Sepharose 2B, 4B, or 6B by the method of Atkinson et al. (12). To 30 g of Sepharose suspended in water (105 ml) a solution of Procion blue (30 ml, 1.5%, w/v) was added, followed by 4 M NaCl (15 ml) and 10 M NaOH (1.98 ml). The slurry was stirred for 2 days at room temperature, treated with 2-mercaptoethanol in alkali to react with the second chlorine group, and then washed extensively. For enzyme inhibition studies, the reactive chlorine groups of free Procion blue were replaced with amino groups by treating the dye with 2 M ammonium chloride (pH 8.5) for 4 h at room temperature (12). Analysis of free and blocked Procion blue by reverse-phase high-pressure liquid chromatography showed the presence of at least six different chemical species. Purification of DHO synthetase on Procion blueSepharose 4B. Hamster cells (8.4 g) were swollen for 15 min in 2 vol of hypotonic buffer (5 mM MgCl,, 2 mM 15 IIIM KCl, 6 mM L-Gln, 6 CaCl,, 1 mM dithiothreitol, mM ~-Asp,
10 mM benzamidine,
1 mg/ml
soybean
tryp-
PURIFICATION
OF
DIHYDROOROTATE
TABLE
Comparison of Purification
1
of DHO Synthetase Using Either Procion Blue-Sepharose 4B or Bio-Gel A-5m Aspartate
Volume (ml) Postmitochondrial I
Pool
II
Pool Pool
III IV
Protein concentration bdd
supernatant
Total protein (md 100
& Pool
47
SYNTHETASE
(2) 1.2
(4.2) 0.95 1.2
(2993
(f-50)
(E7) 1.9 (0.82) 0.80 -=
3.3 (0.97) 2.4 (3.4) 0.76 -
Total activity (gmol/min) 160 (97) (2) (YZ, 11 6.9
transcarbamylase Specific activity (amol/min/mg)
(::W) (::I 14 (5.4) 14 -
Purification factor
t:, 10.6 (8.5) (E) 8.8 -
Note. A postmitochondrial supernatant from mutant hamster cells was loaded directly onto a column of Procion blue-Sepharose 4B (1 X 41 cm) and eluted as described under Experimental Procedures or subjected to the procedure of Coleman et al. (6) completed by elution from a column of Bio-Gel A-5m (1 X 20 cm). Values pertaining to the latter established procedure obtained in our laboratory appear in parentheses. Fractions pooled from the Procion blue-Sepharose column were: Pool I, 350-360 ml; Pool II, 360-375 ml; Pool III, 375-390 ml; Pool IV, 390-410 ml. The protein constituents of these pools are shown in Fig. 1. Pools for the Bio-Gel A-5m column were made according to Coleman et al. (6). The purification of trifunctional DHO synthetase was measured by aspartate transcarbamylase activity. a The protein concentration was too low to be accurately measured.
sin inhibitor, 10 mM triethanolamine, 1 mM EDTA, and 0.3 InM UTP, pH 7.0). The swollen cells were homogenized in hypotonic buffer for 5 strokes, glycerol was added to a final concentration of 30% (v/v), and the cells were homogenized for a further 15 strokes. The homogenate was centrifuged at 20,OOOg for 20 min in a Sorvall SS-34 rotor at 4°C. The postmitochondrial supernatant (26 ml) was removed and immediately applied to a column of Procion blue-Sepharose 4B (1 X 41 cm) equilibrated with 5 mM MgCl, in GDH buffer (30% (v/v) glycerol, 2 InM dithiothreitol, 10 InM KS Hepes, pH 7.2). Unbound protein was removed with this solvent (122 ml) at a flow rate of 12 ml/h; further protein was removed with GDH lacking MgCl, (118 ml). The column was then washed with GDH containing 0.2 M KC1 (80 ml), which eluted a minor proportion of DHO synthetase. A major proportion of the trifunctional synthetase was then eluted with GDH containing 0.4 M KC1 (80 ml); increasing KC1 to 0.5 M (80 ml) resulted in a small peak of DHO synthetase. A further increase to 1 M KC1 (183 ml) eluted only minor peaks for DHO synthetase and protein. Appropriate fractions were pooled and concentrated and KC1 was removed under nitrogen gas using an Amicon Model 8050 ultrafiltration cell fitted with an XM 300 membrane and an auxiliary reservoir (800 ml). Protein concentrations were measured using the BioRad microassay (13); standards contained O-20 pg/ml of bovine albumin. Electrophoresis. Protein in samples was precipitated with 5% (w/v) trichloroacetic acid and redissolved in sample buffer (1% (w/v) sodium dodecyl sulfate, 1% (v/ v) mercaptoethanol, 0.15 M Tris-HCl, pH 8.8). Acrylam-
ide slabs consisted of a 3% (w/v) stacking gel and a 5% (w/v) resolving gel containing gel buffer (0.4% (w/v) sodium dodecyl sulfate, 1.5 M Tris-HCl, pH 8.8); electrode buffer contained 1% (w/v) sodium dodecyl sulfate and 0.375 M Tris-glycine, pH 8.3. The gel was poured to a thickness of 0.5 mm between GelBond PAG film attached to one glass plate and slot molds attached to the other plate. Samples (6 ~1) were pipetted into the slots; standards contained a mixture of carbonic anhydrase (M, 29,000), ovalbumin (iW, 45,000), bovine albumin (M, 66,000), phosphorylase b (M, 97,400), P-galactosidase (M, 116,000), myosin (M, 205,000), and 0.01% (w/v) bromphenol blue. Electrophoresis was performed with an LKB Multiphor II electrophoresis unit at 40 mA for 6 h. The gel was then fixed in 20% (w/v) trichloroacetic acid, stained in 0.1% (w/v) Coomassie blue R-250 in methanol:acetic acidwater (45:10:45), and destained in methanol:acetic acidwater (35:10:55). Stained gels were scanned using an RFT Transidyne scanning densitometer at 590 nm. RESULTS
Preliminary experiments showed that of the 40 dyeligand adsorbents tested, DHO synthetase bound with high affinity, but reversibly, only to Procion blueSepharose 4B. Inclusion of 5 InM MgCI, in the GDH buffer resulted in almost quantitative retention of DHO synthetase by Procion blue-Sepharose 4B. Divalent metal ions form chelate bridges between triazine dyes and adsorbed proteins (10,ll). Specific elution of the trifunctional protein from the adsorbent was tried with
48
CROFTS
all substrates of the three enzymatic activities without success. From experiments where bound DHO synthetase was eluted with a KC1 gradient in GDH and by step-wise increases in KCl, the column washing and elution procedure described under Experimental Procedures was optimized. Procion blue-Sepharose 2B, 4B, and 6B were synthesized, but the separation obtained with Procion blue-Sepharose 4B was optimal. DHO synthetase does not elute in the void volume after addition of 0.4 M KC1 but it is sieved by Sepharose 4B. Thus the purification obtained can be attributed to a combination of adsorption and molecular sieve chromatography. Appropriate fractions from the elution profile were subjected to slab gel electrophoresis (Fig. 1). Pool II (360-375 ml) and Pool III (375-390 ml) were of high purity. Pool I contained a contaminant of slightly lower molecular weight which may be formed by proteolysis of DHO synthetase (6). DHO synthetase could be maintained as a single polypeptide (M, 210,000, Fig. 1) during the purification procedure when all manipulations were performed rapidly and at 0-4’C. For comparitive purposes, DHO synthetase was also purified by the procedure of Coleman et al. (6) from the same cells; results from both procedures are presented in Table 1. The specific activity of aspartate transcarbamylase purified by passage through Procion blue-Sepharose 4B of 14 ~mol/min/mg protein for Pools II and III is similar to the activity obtained for Pool I by the established procedure. The ratios of aspartate transcarbamylase and dihydroorotase activities relative to carbamyl phosphate synthetase activity are increased over those for the postmitochondrial supernatant ((carbamyl phosphate synthetase):(aspartate transcarbamylase):(dihydroorotase), 1:70:4.6), indicating that there is some loss of carbamyl phosphate synthetase activity in either procedure. The ratio obtained for Pool II from the Procion blue-Sepharose column was 1:105:6.0 and that for Pool I from the Bio-Gel A-5m column was 1:104:9.0. Densitometric scans of slab gels stained with Coomassie blue (Fig. 1) confirmed that the two procedures yielded DHO synthetase of comparable purity.
DISCUSSION DHO synthetase can be rapidly purified in a single step by passage through a column of Procion blueSepharose 4B; 67% of the aspartate transcarbamylase activity applied to the column eluted with 0.4 M KCl. The chemical structure of Procion blue does not resemble that of any of the substrates of DHO synthetase and
ET
AL.
it was therefore not surprising that the synthetase could not be*specifically eluted with a substrate. Free Procion blue with the reactive chloro groups displaced by amino groups inhibits carbamyl phosphate synthetase but not aspartate transcarbamylase or dihydroorotase activities.‘j The purification factor of 8.5-8.8 obtained with Procion blue-Sepharose 4B (Pools II and III, Table 1) is comparable to the factor of 8.5 (Table 1) obtained by the established procedure (6), but silver staining of the purest fractions revealed the presence of a number of minor contaminating proteins6 (cf. Fig. 1). These minor contaminants would persist in a significant relative abundance if DHO synthetase were purified from “wildtype” cells. An additional purification step would be required to purify DHO synthetase from normal cells. ACKNOWLEDGMENTS We thank the Procion preliminary
Dr. Robert blue used experiments.
Scopes in this
for helpful study and
advice Elizabeth
and
for providing Isaac for some
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