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[16]Carbamyl-phosphate synthetase (glutamine): Aspartate carbamyltransferase of Neurospora
[16]
CPSAsE (GLUTAMINE): ATCAsE oF Neurospora
105
[16]Carbamyl-phosphate Synthetase (Glutamine): Aspartate Carbamyltransferase o f Neurospora B y LARRY G . W I L L I A M S a n d R O W L A N D H . DAVIS
Mg2 ÷
2 ATP + L-glutamine + COs
, carbamyl phosphate + L-glutamate + 2 ADP + P~
Carbamyl phosphate + L-aspartate ~ L-ureidosuccinate + Pi
In Neurospora the initial enzymic activities of pyrimidine synthesis, carbamyl-phosphate synthetase (CPSase) and aspartate carbamyltransferase (ATCase), are determined by a single genetic locus and copurify as a single enzyme complex. 1 Neurospora also possesses a second CPSase which synthesizes carbamyl phosphate (carbamyl-P) specifically for use in arginine metabolism. 2 Although the arginine- and pyrimidinespecific CPSase have the same substrate requirements, they are under control of separate genetic loci and can clearly be distinguished on the basis of feedback inhibitors, derepression conditions, stabilizing factors, and molecular weight. The CPSase referred to in this article is the pyrimdine-specific CPSase (that is, its product is normally not available to the arginine pathway), which copurifies with ATCase. A peculiarity of the CPSase activity is its cold lability in the absence of its feedback effector, UTP. Assay Method for Carbamyl-Phosphate Synthetase
Principle. CPSase activity is measured by the conversion of the labile product, carbamyl-P, to a stable compound which can be determined colorimetrically. The method currently used is to add NH4CI to the reaction mixture when it is completed, and to boil it. This converts carbamyl-P almost quantitatively to urea. 3'4 Alternatively, stable derivatives can be formed in the course of the reaction by trapping carbamyl-P enzymically. This requires addition of aspartate and ATCase or of ornithine and ornithine carbamoyltransferase to the CPSase reaction mixture to form ureidosuccinate or citrulline, respectively. 2'3 Urea, ureidosuccinate, and citrulline can all be measured by the same colorimetric procedure for determination of carbamyl groups. 1 L. G. Williams, S. Bernhardt, and R. H. Davis, Biochemistry 9, 4329 (1970). 2 L. G. Williams and R. H. Davis, J. Bacteriol. 103, 335 (1970). 3 R. H. Davis, Biochim. Biophys. Acta 107, 44 (1965). 4 j. Yashphe and L. Gorini, J. Biol. Chem. 240, 1681 (1965). METHODS IN ENZYMOLOGY, VOL. LI
Copyright© 1978by AcademicPress,Inc. All rightsof reproductionin any formreserved. ISBN 0-12-181951-5
106
De Novo PYRIMIDINEBIOSYNTHESIS
[16]
Reagents Tris-acetate buffer, 1 M, pH 7.5 L-glutamine, 0.06 M MgC12, 0.12 M NH4C1, 1 M (freshly prepared) KHCO3 Disodium ATP Procedure. The reaction mixture (0.50 ml) contains 0.05 ml Lglutamine, 0.05 ml MgCl2, 0.25 ml H~O, 15/~moles KHCOa, 6 ~moles ATP, and 0.10 ml enzyme preparation. Control mixtures lacking either ATP or KHCO3 are also prepared. Where crude extracts with low activity are assayed, it may be desirable to increase the volume of extract and decrease the volume of H20 added to the reaction mixture. Crude extracts must also be desalted by gel filtration. Following addition of enzyme, the reaction is incubated at 25 ° for 30 min. The reaction is terminated by addition of 0.30 ml of NH4C1, followed by 10 min in a boiling water bath. Precipitate, if any, is removed by low-speed centrifugation, and a portion of the supernatant is tested for the presence of urea by a modified Gerhart-Pardee colorimetric assay. 5 Modifications were a final 20-min incubation for color development at 25 ° and then placement of the reaction tubes in ice water to prevent loss of developed color and formation of spurious color. Color is read in a Klett-Summerson colorimeter using a No. 54 filter. This procedure will detect as little as 5 nmoles urea. An enzyme unit is the quantity of enzyme that forms 1 /.tmole of carbamyl-P per minute under the conditions above.
Assay Method for Aspartate Carbamyltransferase Principle. ATCase activity is measured by colorimetric determination of the product, L-ureidosuccinate. Reagents Glycine.NaOH buffer, 1.0 M, pH 9.0 L-aspartic acid, 0.10 M, pH 7.0 Dilithium carbamyl-P, 0.02 M (dissolved just before use) HCIO4, 2.0 M
The reaction mixture (1.0 ml) contains 0.15 ml glycine buffer, 0.10 ml aspartate, 0.25 ml carbamyl-P, 0.30 ml H20, and 0.20 ml enzyme preparation. Control incubation mixtures lack aspartate. The reaction is 5j. C. Gerhart and A. B. Pardee, J. Biol. Chem. 237, 891 (1962).
[16]
CPSASE (GLUTAMINE): ATCASE OF Neurospora
107
initiated by addition of enzyme. Following 15-min incubation at 25 ° the reaction is stopped by addition of 0.20 ml of HCIO4. The protein precipitate is removed by centrifugation, and portions of the supernatant are assayed for ureidosuccinate. The colorimetric assay employed is the same as that previously described for urea except that the incubation time for color development is 30 min. The assay can detect as little as l0 nmoles ureidosuccinate. An enzyme unit is the quantity of enzyme that forms 1 /xmole of ureidosuccinate per minute under the conditions stated above. Purification Procedure 1 Mycelium from an arg-3, pyr-1 double-mutant strain is the source of the enzyme complex. The arg-3 mutation eliminates arginine-specific CPSase activity, while the pyr-1 mutation permits an approximately 5fold derepression of synthesis of the CPSase-ATCase complex under conditions of pyrimidine starvation. 2 Mycelia of arg-3, pyr-1 are grown from conidia (10 e per milliliter) in standard Neurospora medium containing Vogel's salts, 15 g sucrose, 100 mg uridine, and 200 mg arginine per liter. Growth takes place on a reciprocating shaker at 25 ° in 700 ml medium in 2500-ml, low-form culture flasks. After 18-24 hr growth, mycelia are harvested without packing, rinsed in distilled water, and resuspended in the same volume of fresh medium lacking uridine. After 4 to 5 hr, derepression of pyrimidine enzymes has taken place. The standard buffer system used in enzyme extraction and purification is 0.05 M potassium phosphate, pH 7.3, containing l × 10-3 M L-glutamine and 2 × 10-4M Cleland's reagent. Where noted, UTP, an inhibitor of cold inactivation of CPSase activity, is added to the standard buffer. All purification steps are conducted at room temperature, 22-25 °, unless otherwise noted. Each purification step is devised to give maximum yield of the more labile CPSase activity while maintaining its sensitivity to feedback inhibition. See the accompanying purification table.
Step I. Extraction. Mycelia are harvested on cheesecloth and pressed between paper towels to form a damp pad. Approximately 80 g of damp mycelial pad, an equal volume of standard buffer containing 7.5 × l0 -4 M UTP, and 16 g of fine sand are ground to a slurry with mortar and pestle at 0 °. Sand and insoluble material are removed by 15-rain centrifugation at 12,000 g and 5 °. The crude supernatant (110 ml) is then centrifuged 30 rain at 50,000 g at 5 °. After removal of the heavy lipid pellicle (by pipet), the high-speed supernatant, containing all the recoverable CPSase and ATCase activity, is decanted from the residue and maintained at 0 °.
108
De Novo PYRIMIDINE BIOSYNTHESIS
[16]
r~
Y
L)
r~
z O < r~
(.)
=o
0
7
g,
¢¢ O
;> e~
0
~ M M 4 ~
[16]
CPSASE (GLUTAM1NE):ATCASE OF Neurospora
109
Step 2. First Ammonium Sulfate Fractionation. Solid (NH4)2SO4 is immediately dissolved in the high-speed supernatant (2.5 g/10 ml). Following 5 min of stirring to dissolve the (NH4)~SO, and standing an additional 5 min at 0 °, the protein precipitate is collected by 25-min centrifugation at 12,000 g at 5 °. The supernatant is discarded. The precipitate is dissolved in 8 ml standard buffer (to about 15 mg of protein per milliliter) and desalted by passage of 4-ml portions through Sephadex G-25 gel filtration columns (3.3 cm diam. x 4.5 cm) equilibrated with the same buffer, at room temperature. Step 3. Calcium Phosphate Gel Fractionation. Calcium phosphate gel (21 mg dry weight/ml) is prepared by a standard procedure. 6 The desalted protein solution from step 2 (about 20 ml) is stirred for 3-5 min with 4.0-4.5 ml of calcium phosphate gel at room temperature. The gel is sedimented by a brief centrifugation and discarded. An additional 13 to 14 ml of calcium phosphate gel are added to the supernatant. The second gel addition absorbs most of the CPSase and ATCase activity. The gel is collected by a brief low-speed centrifugation. The supernatant is discarded. Protein is eluted from the gel by successive washes (suspension of the gel in buffer with 5-rain stirring, followed by centrifugation) with 10-ml portions of standard buffer (containing 1 × 10-4 M UTP) at pH 7.3, 7.8, and 8.1. The amount of calcium phosphate gel required to give optimum purification varies slightly from preparation to preparation. Therefore, a trial experiment must be carried out on a small portion of each preparation to determine the volumes of gel needed. Step 4. Second (NH4)2S04 Fractionation. The three washes of the calcium phosphate gel are combined and cooled to 0 °, and protein is precipitated by the addition of solid (NH4)2SO4 (3 g/10 ml). Following 5min standing at 0 ° the precipitate is recovered by 15-min centrifugation at 50,000 g at 5 °. Step 5. Agarose Gel Filtration. A Biogel agarose 1.5 m column (1.2 x 37.5 cm) is equilibrated at room temperature by the flow of 200 ml of standard buffer through the column. The precipitate of the second (NH4)2SO4 fractionation is immediately redissolved in 1.25 ml of standard buffer and applied to the top of the gel column. A constant flow rate of buffer through the column is maintained by use of a Mariotte flask. The void volume of the column is 20 ml. About 85% of the recoverable enzyme activities are recovered in fractions eluting between 21 and 29 ml, which are then pooled. 6 D. Keilin and E. F. Hartree, Proc. R. Soc. London, Ser. B 124, 397 (1938).
110
De Novo PYRIMIDINEBIOSYNTHESIS
[16]
Step 6. DEAE-Ceilulose Fractionation. DEAE-cellulose (Biorad), washed with 0.5 N NaOH, 0.5 N HCI, and H~O, is used to prepare a small column (1.1 x 4.0 cm). Following equilibration of the column at room temperature with 30 ml of standard buffer containing 1 x 10-4, UTP, the pooled fractions from step 5 are immediately applied to the column. After the protein solution has entered the column bed the column is washed with 15 ml of standard buffer (1 x 10-4M in UTP) and then eluted with a 100-ml exponential gradient of 0-0.5 M NaCI prepared in standard buffer (1 x 10-4 M in UTP). Fractions of 3.0 ml are collected, CPSase assays are run on a small portion of each fraction, and those fractions having appreciable activity are pooled. The pooled fractions are concentrated by (NH4)2SO4 precipitation as described in step 4. The precipitate is redissolved in 1-2 ml of standard buffer containing 5 × 10-4M UTP and stored at - 8 0 °. Optimum recovery of CPSase activity is obtained if the purification is conducted over a 2-day period. The first day the preparation is taken through step 2 of the procedure. It is then frozen overnight at - 8 0 ° in standard buffer 5 × 10-4 M in UTP, and the purification is completed the next day. Properties
Stability and Purity. The CPSase activity of the enzyme complex is quite labile in the absence of stabilizers. In a buffered solution over 90% of this activity is irreversibly lost after only 45 min at 0 °, while 50% of the activity is lost after 3 hr at 25 °. Addition of glutamine to the buffer greatly slows loss of activity at room temperature (5% loss after 3 hr at 25%), while addition of UTP retards cold inactivation (25% loss after 3 hr at 00). 2 The enzyme can be stored for several months if frozen at - 8 0 ° in the presence of 5 × 10-4 M UTP and at a protein concentration about 0.5 mg/ml. The final enzyme preparation appears to be about 50% pure based on sucrose gradient data where concomitant shifts in the major protein peak and the peak of enzyme activities occur in response to the presence or absence of UTP. 1 CPSase-ATCase Enzyme Complex. The purification procedure is designed for maximum yield of CPSase; however, ATCase activity always copurifies with CPSase. The two activities cannot be separated by (NH4)2SO4 fractionation or calcium phosphate gel adsorption. The distribution of the two activities from gel filtration columns, from ionexchange columns, and from sucrose density gradients is identical. In no step of the procedure was it possible to isolate a second fraction which
[17]
CPSASE U: ATCASE: DIHYROOROTASECOMPLEX
11 1
had only one of the two activities. Difference in yield between the two activities with some purification steps apparently is due to differential inactivation of the two active sites.
Molecular Weight. 1 On the basis of elution patterns from agarose gel filtration columns and sedimentation patterns in sucrose density gradients the enzyme complex is estimated to have a molecular weight of 650,000 __ 50,000. On sucrose density gradients the complex sediments at a value of 21 S in the absence of UTP and 15 S in the presence of UTP, suggesting a dissociation of enzyme subunits in the presence of the feedback inhibitor. Other Properties. UTP, an end-product of the pyrimidine synthetic pathway, gave 50% inhibition of CPSase activity at a concentration of 1 × 10-4 M and complete inhibition at 1 × 10-a M. UMP was about twothirds as effective, but the cytidine nucleotides had no inhibiting effect. 2 None of the pyrimidine nucleotides had any effect on the Vmax of ATCase activity. At pH 7.5 ATCase exhibits about 5% of the activity present at its pH optimum of 9.1. 7 The Km of ATCase for carbamyl-P decreases from 83 p314 at pH 9.1 to 32 p2k/at pH 7.5. The Km of ATCase for carbamyl phosphate is doubled in the presence of 5 × 10-4 M UTP. ~ 7 R. H. Davis, in "Organizational Biosynthesis" (H. J. Vogel, J. O. Lampen, and V. Bryson, eds.), p, 302. Academic Press, New York, 1%7. L. G. Williams, Genetics 77, 270 (1974).
[17] A M u l t i e n z y m e C o m p l e x o f C a r b a m o y l - p h o s p h a t e Synthase (Glutamine):Aspartate Carbamoyltransferase:Dihydoorotase (Rat Ascites Hepatoma Cells and Rat Liver) B y MASATAKA MORI a n d MASAMITI TATIBANA L-Glutamine + 2 ATP + HCO-a + H20 --~ carbamoyl phosphate + 2 ADP + P~ + L-glutamate Carbamoyl phosphate + L-aspartate ~ carbamoyl-L-aspartate + P~ Carbamoyl-L-aspartate ~-- L-dihydroorotate + H20
Carbamoyl-P ~ synthase (glutamine) (EC 2.7.2.9) of higher animals, which was first discovered by Tatibana and Ito z'a in hematopoietic 1 Abbreviations used are: Carbamoyl-P, carbamoyl phosphate; Me2SO, dimethyl sulfoxide; Hepes, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; PP-ribose-P, 5-phosphoribosyl 1-pyrophosphate, z M. Tatibana and K. Ito, Bioehem. Biophys. Res. Commun. 26, 221 (1%7). a M. Tatibana and K. Ito, J. Biol. Chem, 244, 5403 (1%9). METHODS IN ENZYMOLOGY, VOL. LI
Copyright © 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181951-5
CPSAsE (GLUTAMINE): ATCAsE oF Neurospora
105
[16]Carbamyl-phosphate Synthetase (Glutamine): Aspartate Carbamyltransferase o f Neurospora B y LARRY G . W I L L I A M S a n d R O W L A N D H . DAVIS
Mg2 ÷
2 ATP + L-glutamine + COs
, carbamyl phosphate + L-glutamate + 2 ADP + P~
Carbamyl phosphate + L-aspartate ~ L-ureidosuccinate + Pi
In Neurospora the initial enzymic activities of pyrimidine synthesis, carbamyl-phosphate synthetase (CPSase) and aspartate carbamyltransferase (ATCase), are determined by a single genetic locus and copurify as a single enzyme complex. 1 Neurospora also possesses a second CPSase which synthesizes carbamyl phosphate (carbamyl-P) specifically for use in arginine metabolism. 2 Although the arginine- and pyrimidinespecific CPSase have the same substrate requirements, they are under control of separate genetic loci and can clearly be distinguished on the basis of feedback inhibitors, derepression conditions, stabilizing factors, and molecular weight. The CPSase referred to in this article is the pyrimdine-specific CPSase (that is, its product is normally not available to the arginine pathway), which copurifies with ATCase. A peculiarity of the CPSase activity is its cold lability in the absence of its feedback effector, UTP. Assay Method for Carbamyl-Phosphate Synthetase
Principle. CPSase activity is measured by the conversion of the labile product, carbamyl-P, to a stable compound which can be determined colorimetrically. The method currently used is to add NH4CI to the reaction mixture when it is completed, and to boil it. This converts carbamyl-P almost quantitatively to urea. 3'4 Alternatively, stable derivatives can be formed in the course of the reaction by trapping carbamyl-P enzymically. This requires addition of aspartate and ATCase or of ornithine and ornithine carbamoyltransferase to the CPSase reaction mixture to form ureidosuccinate or citrulline, respectively. 2'3 Urea, ureidosuccinate, and citrulline can all be measured by the same colorimetric procedure for determination of carbamyl groups. 1 L. G. Williams, S. Bernhardt, and R. H. Davis, Biochemistry 9, 4329 (1970). 2 L. G. Williams and R. H. Davis, J. Bacteriol. 103, 335 (1970). 3 R. H. Davis, Biochim. Biophys. Acta 107, 44 (1965). 4 j. Yashphe and L. Gorini, J. Biol. Chem. 240, 1681 (1965). METHODS IN ENZYMOLOGY, VOL. LI
Copyright© 1978by AcademicPress,Inc. All rightsof reproductionin any formreserved. ISBN 0-12-181951-5
106
De Novo PYRIMIDINEBIOSYNTHESIS
[16]
Reagents Tris-acetate buffer, 1 M, pH 7.5 L-glutamine, 0.06 M MgC12, 0.12 M NH4C1, 1 M (freshly prepared) KHCO3 Disodium ATP Procedure. The reaction mixture (0.50 ml) contains 0.05 ml Lglutamine, 0.05 ml MgCl2, 0.25 ml H~O, 15/~moles KHCOa, 6 ~moles ATP, and 0.10 ml enzyme preparation. Control mixtures lacking either ATP or KHCO3 are also prepared. Where crude extracts with low activity are assayed, it may be desirable to increase the volume of extract and decrease the volume of H20 added to the reaction mixture. Crude extracts must also be desalted by gel filtration. Following addition of enzyme, the reaction is incubated at 25 ° for 30 min. The reaction is terminated by addition of 0.30 ml of NH4C1, followed by 10 min in a boiling water bath. Precipitate, if any, is removed by low-speed centrifugation, and a portion of the supernatant is tested for the presence of urea by a modified Gerhart-Pardee colorimetric assay. 5 Modifications were a final 20-min incubation for color development at 25 ° and then placement of the reaction tubes in ice water to prevent loss of developed color and formation of spurious color. Color is read in a Klett-Summerson colorimeter using a No. 54 filter. This procedure will detect as little as 5 nmoles urea. An enzyme unit is the quantity of enzyme that forms 1 /.tmole of carbamyl-P per minute under the conditions above.
Assay Method for Aspartate Carbamyltransferase Principle. ATCase activity is measured by colorimetric determination of the product, L-ureidosuccinate. Reagents Glycine.NaOH buffer, 1.0 M, pH 9.0 L-aspartic acid, 0.10 M, pH 7.0 Dilithium carbamyl-P, 0.02 M (dissolved just before use) HCIO4, 2.0 M
The reaction mixture (1.0 ml) contains 0.15 ml glycine buffer, 0.10 ml aspartate, 0.25 ml carbamyl-P, 0.30 ml H20, and 0.20 ml enzyme preparation. Control incubation mixtures lack aspartate. The reaction is 5j. C. Gerhart and A. B. Pardee, J. Biol. Chem. 237, 891 (1962).
[16]
CPSASE (GLUTAMINE): ATCASE OF Neurospora
107
initiated by addition of enzyme. Following 15-min incubation at 25 ° the reaction is stopped by addition of 0.20 ml of HCIO4. The protein precipitate is removed by centrifugation, and portions of the supernatant are assayed for ureidosuccinate. The colorimetric assay employed is the same as that previously described for urea except that the incubation time for color development is 30 min. The assay can detect as little as l0 nmoles ureidosuccinate. An enzyme unit is the quantity of enzyme that forms 1 /xmole of ureidosuccinate per minute under the conditions stated above. Purification Procedure 1 Mycelium from an arg-3, pyr-1 double-mutant strain is the source of the enzyme complex. The arg-3 mutation eliminates arginine-specific CPSase activity, while the pyr-1 mutation permits an approximately 5fold derepression of synthesis of the CPSase-ATCase complex under conditions of pyrimidine starvation. 2 Mycelia of arg-3, pyr-1 are grown from conidia (10 e per milliliter) in standard Neurospora medium containing Vogel's salts, 15 g sucrose, 100 mg uridine, and 200 mg arginine per liter. Growth takes place on a reciprocating shaker at 25 ° in 700 ml medium in 2500-ml, low-form culture flasks. After 18-24 hr growth, mycelia are harvested without packing, rinsed in distilled water, and resuspended in the same volume of fresh medium lacking uridine. After 4 to 5 hr, derepression of pyrimidine enzymes has taken place. The standard buffer system used in enzyme extraction and purification is 0.05 M potassium phosphate, pH 7.3, containing l × 10-3 M L-glutamine and 2 × 10-4M Cleland's reagent. Where noted, UTP, an inhibitor of cold inactivation of CPSase activity, is added to the standard buffer. All purification steps are conducted at room temperature, 22-25 °, unless otherwise noted. Each purification step is devised to give maximum yield of the more labile CPSase activity while maintaining its sensitivity to feedback inhibition. See the accompanying purification table.
Step I. Extraction. Mycelia are harvested on cheesecloth and pressed between paper towels to form a damp pad. Approximately 80 g of damp mycelial pad, an equal volume of standard buffer containing 7.5 × l0 -4 M UTP, and 16 g of fine sand are ground to a slurry with mortar and pestle at 0 °. Sand and insoluble material are removed by 15-rain centrifugation at 12,000 g and 5 °. The crude supernatant (110 ml) is then centrifuged 30 rain at 50,000 g at 5 °. After removal of the heavy lipid pellicle (by pipet), the high-speed supernatant, containing all the recoverable CPSase and ATCase activity, is decanted from the residue and maintained at 0 °.
108
De Novo PYRIMIDINE BIOSYNTHESIS
[16]
r~
Y
L)
r~
z O < r~
(.)
=o
0
7
g,
¢¢ O
;> e~
0
~ M M 4 ~
[16]
CPSASE (GLUTAM1NE):ATCASE OF Neurospora
109
Step 2. First Ammonium Sulfate Fractionation. Solid (NH4)2SO4 is immediately dissolved in the high-speed supernatant (2.5 g/10 ml). Following 5 min of stirring to dissolve the (NH4)~SO, and standing an additional 5 min at 0 °, the protein precipitate is collected by 25-min centrifugation at 12,000 g at 5 °. The supernatant is discarded. The precipitate is dissolved in 8 ml standard buffer (to about 15 mg of protein per milliliter) and desalted by passage of 4-ml portions through Sephadex G-25 gel filtration columns (3.3 cm diam. x 4.5 cm) equilibrated with the same buffer, at room temperature. Step 3. Calcium Phosphate Gel Fractionation. Calcium phosphate gel (21 mg dry weight/ml) is prepared by a standard procedure. 6 The desalted protein solution from step 2 (about 20 ml) is stirred for 3-5 min with 4.0-4.5 ml of calcium phosphate gel at room temperature. The gel is sedimented by a brief centrifugation and discarded. An additional 13 to 14 ml of calcium phosphate gel are added to the supernatant. The second gel addition absorbs most of the CPSase and ATCase activity. The gel is collected by a brief low-speed centrifugation. The supernatant is discarded. Protein is eluted from the gel by successive washes (suspension of the gel in buffer with 5-rain stirring, followed by centrifugation) with 10-ml portions of standard buffer (containing 1 × 10-4 M UTP) at pH 7.3, 7.8, and 8.1. The amount of calcium phosphate gel required to give optimum purification varies slightly from preparation to preparation. Therefore, a trial experiment must be carried out on a small portion of each preparation to determine the volumes of gel needed. Step 4. Second (NH4)2S04 Fractionation. The three washes of the calcium phosphate gel are combined and cooled to 0 °, and protein is precipitated by the addition of solid (NH4)2SO4 (3 g/10 ml). Following 5min standing at 0 ° the precipitate is recovered by 15-min centrifugation at 50,000 g at 5 °. Step 5. Agarose Gel Filtration. A Biogel agarose 1.5 m column (1.2 x 37.5 cm) is equilibrated at room temperature by the flow of 200 ml of standard buffer through the column. The precipitate of the second (NH4)2SO4 fractionation is immediately redissolved in 1.25 ml of standard buffer and applied to the top of the gel column. A constant flow rate of buffer through the column is maintained by use of a Mariotte flask. The void volume of the column is 20 ml. About 85% of the recoverable enzyme activities are recovered in fractions eluting between 21 and 29 ml, which are then pooled. 6 D. Keilin and E. F. Hartree, Proc. R. Soc. London, Ser. B 124, 397 (1938).
110
De Novo PYRIMIDINEBIOSYNTHESIS
[16]
Step 6. DEAE-Ceilulose Fractionation. DEAE-cellulose (Biorad), washed with 0.5 N NaOH, 0.5 N HCI, and H~O, is used to prepare a small column (1.1 x 4.0 cm). Following equilibration of the column at room temperature with 30 ml of standard buffer containing 1 x 10-4, UTP, the pooled fractions from step 5 are immediately applied to the column. After the protein solution has entered the column bed the column is washed with 15 ml of standard buffer (1 x 10-4M in UTP) and then eluted with a 100-ml exponential gradient of 0-0.5 M NaCI prepared in standard buffer (1 x 10-4 M in UTP). Fractions of 3.0 ml are collected, CPSase assays are run on a small portion of each fraction, and those fractions having appreciable activity are pooled. The pooled fractions are concentrated by (NH4)2SO4 precipitation as described in step 4. The precipitate is redissolved in 1-2 ml of standard buffer containing 5 × 10-4M UTP and stored at - 8 0 °. Optimum recovery of CPSase activity is obtained if the purification is conducted over a 2-day period. The first day the preparation is taken through step 2 of the procedure. It is then frozen overnight at - 8 0 ° in standard buffer 5 × 10-4 M in UTP, and the purification is completed the next day. Properties
Stability and Purity. The CPSase activity of the enzyme complex is quite labile in the absence of stabilizers. In a buffered solution over 90% of this activity is irreversibly lost after only 45 min at 0 °, while 50% of the activity is lost after 3 hr at 25 °. Addition of glutamine to the buffer greatly slows loss of activity at room temperature (5% loss after 3 hr at 25%), while addition of UTP retards cold inactivation (25% loss after 3 hr at 00). 2 The enzyme can be stored for several months if frozen at - 8 0 ° in the presence of 5 × 10-4 M UTP and at a protein concentration about 0.5 mg/ml. The final enzyme preparation appears to be about 50% pure based on sucrose gradient data where concomitant shifts in the major protein peak and the peak of enzyme activities occur in response to the presence or absence of UTP. 1 CPSase-ATCase Enzyme Complex. The purification procedure is designed for maximum yield of CPSase; however, ATCase activity always copurifies with CPSase. The two activities cannot be separated by (NH4)2SO4 fractionation or calcium phosphate gel adsorption. The distribution of the two activities from gel filtration columns, from ionexchange columns, and from sucrose density gradients is identical. In no step of the procedure was it possible to isolate a second fraction which
[17]
CPSASE U: ATCASE: DIHYROOROTASECOMPLEX
11 1
had only one of the two activities. Difference in yield between the two activities with some purification steps apparently is due to differential inactivation of the two active sites.
Molecular Weight. 1 On the basis of elution patterns from agarose gel filtration columns and sedimentation patterns in sucrose density gradients the enzyme complex is estimated to have a molecular weight of 650,000 __ 50,000. On sucrose density gradients the complex sediments at a value of 21 S in the absence of UTP and 15 S in the presence of UTP, suggesting a dissociation of enzyme subunits in the presence of the feedback inhibitor. Other Properties. UTP, an end-product of the pyrimidine synthetic pathway, gave 50% inhibition of CPSase activity at a concentration of 1 × 10-4 M and complete inhibition at 1 × 10-a M. UMP was about twothirds as effective, but the cytidine nucleotides had no inhibiting effect. 2 None of the pyrimidine nucleotides had any effect on the Vmax of ATCase activity. At pH 7.5 ATCase exhibits about 5% of the activity present at its pH optimum of 9.1. 7 The Km of ATCase for carbamyl-P decreases from 83 p314 at pH 9.1 to 32 p2k/at pH 7.5. The Km of ATCase for carbamyl phosphate is doubled in the presence of 5 × 10-4 M UTP. ~ 7 R. H. Davis, in "Organizational Biosynthesis" (H. J. Vogel, J. O. Lampen, and V. Bryson, eds.), p, 302. Academic Press, New York, 1%7. L. G. Williams, Genetics 77, 270 (1974).
[17] A M u l t i e n z y m e C o m p l e x o f C a r b a m o y l - p h o s p h a t e Synthase (Glutamine):Aspartate Carbamoyltransferase:Dihydoorotase (Rat Ascites Hepatoma Cells and Rat Liver) B y MASATAKA MORI a n d MASAMITI TATIBANA L-Glutamine + 2 ATP + HCO-a + H20 --~ carbamoyl phosphate + 2 ADP + P~ + L-glutamate Carbamoyl phosphate + L-aspartate ~ carbamoyl-L-aspartate + P~ Carbamoyl-L-aspartate ~-- L-dihydroorotate + H20
Carbamoyl-P ~ synthase (glutamine) (EC 2.7.2.9) of higher animals, which was first discovered by Tatibana and Ito z'a in hematopoietic 1 Abbreviations used are: Carbamoyl-P, carbamoyl phosphate; Me2SO, dimethyl sulfoxide; Hepes, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; PP-ribose-P, 5-phosphoribosyl 1-pyrophosphate, z M. Tatibana and K. Ito, Bioehem. Biophys. Res. Commun. 26, 221 (1%7). a M. Tatibana and K. Ito, J. Biol. Chem, 244, 5403 (1%9). METHODS IN ENZYMOLOGY, VOL. LI
Copyright © 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181951-5