- Email: [email protected]
[5] Aspartate transcarbamoylase (Escherichia coli): Preparation of subunits
[5]
S U B U N I T S IN A T C A S E
35
and 0.2 mM UMP stabilized the large subunit for 4 weeks at 4 °. Combination of dialyzed subunits results in their immediate reassociation into holoenzyme. E n z y m i c Properties o f Subunits. The ot subunit catalyzes the synthesis of carbamoyl phosphate from ammonia but not glutamine. The rate of carbamoyl-phosphate synthesis by the separated a subunit is sigmoidal, and the Hill plot yielded an interaction coefficient of 1.5. The s0,~ value for ATP was 0.3 mM which is close to that obtained for the ammoniadependent activity of the native enzyme. The/3 subunit exhibits glutaminase activity. Ornithine, IMP, and phosphoribosyl-pyrophosphate clearly stimulate the ammonia activity of the large subunit. This activity, however, was only slightly inhibited by high concentrations of UMP. The addition of the small subunit restores the ability to utilize glutamine as well as normal sensitivity to UMP.
[5] A s p a r t a t e T r a n s c a r b a m o y l a s e (Escherichia Preparation of Subunits
co/i):
By YING R. YANG, MARC W, KIRSCHNER,and H. K. SCHACHMAN
Carbamoylphosphate + L-aspartate~ carbamoylaspartate Aspartate transcarbamoylase (ATCase, EC 2.1.3.2: carbamoyl phosphate: L-aspartate carbamoyltransferase) catalyzes the first reaction unique to pyrimidine biosynthesis; in E. coli regulation is achieved in part by feedback inhibition of the enzyme by CTP, an end-product of the pathway. 1"~ The purified enzyme when treated with the mercurial, pmercuribenzoate, is known to dissociate into two types of subunits, one responsible for catalysis, termed the catalytic subunit, and the other which binds both the inhibitor, CTP, and the activator, ATP. 3 After separation of the two types of subunits and removal of the mercurial, the enzyme can be reconstituted by mixing the catalytic and regulatory subunits. The procedure for preparation of the discrete subunits after dissociation of the enzyme with p-mercuribenzoate was described in detail by Gerhart and Holoubek. 4 A modified procedure devised by Kirschner 5 1R. A. Yates and A. B. Pardee,J. Biol. Chem. 221,757 (1956). 2j. C. Gerhart and A. B. Pardee,J. Biol. Chem, 237, 891 (1962). a j. C. Gerhart and H. K. Schachman,Biochemistry 4, 1054(1965). 4j. C. Gerhart and H. Holoubek,J. Biol. Chem. 242, 2886 (1967). 5 M. W. Kirschner, Ph.D. Thesis, Universityof California, Berkeley(1971). METHODS
IN
ENZYMOLOGY, VOL. LI
Copyright ~) 1978 by Academic Press, lnc, All rights of reproduction in any form reserved, ISBN 0-12-181951-5
36
De Novo PYRIMIDINE BIOSYNTHESIS
[5]
was found to give higher yields of regulatory subunit and to reduce the time required for the separation. An alternative procedure involving heat treatment of the enzyme has also been used for the preparation of catalytic subunit, e The method described here is slightly modified from Kirschner's method and provides high yields of purified catalytic and regulatory subunits. It has been used routinely in our laboratory with ATCase from both E. coli and Salmonella typhimurium. Reagents
100 mg purified ATCase 6.3 mg 1-(3-chloromercuri-2-methoxypropyl)urea, known as neohydrin or chloromecodrin (ICN, K & K Laboratories, Inc.) 20 g DEAE-cellulose (Schleicher & Schuell Co.) 0.01 M Tris-Cl, 0.1 M KCI, pH 8.7 0.01 M Tris-Cl, 0.5 M KCI, pH 8.7 20 mM K2HPO4, pH 8.7 3.6 M ammonium sulfate containing 5 mM 2-mercaptoethanol (the pH of the solution is adjusted with KOH to a value of 7 as measured with indicator paper) 25 mM Tris-C1 containing 2 m M 2-mercaptoethanol and 0.2 mM zinc acetate, pH 8.0 Dissociation of E n z y m e into Subunits A sample of I00 m g of ATCase at 15-20 mg/ml is dialyzed overnight at 4 ° against two changes of 500 ml of 0.01 M Tris-Cl containing 0. I M KCI at p H 8.7 in order to reduce the concentration of 2-mercaptoethanol and E D T A in the stock enzyme solution. One milliliterof neohydrin solution, containing 6.3 rng of neohydrin in 0.01 M Tris-Cl at p H 8.7, is added to the enzyme solution which had been equilibrated at room temperature. The addition is performed rapidly, and the solution is mixed by gently inverting the tube several times. After 15 min of incubation at room temperature the reaction mixture is examined by zone electrophoresis on cellulose polyacetate strips in order to test whether the enzyme is completely dissociated. Electrophoresis Zone electrophorcsis is used routinely before the mercurial-treated enzyme is loaded on the DEAE-cellulose column. Samples are applied in 20 m M potassium phosphate buffer at p H 8.7 onto a cellulose polyace-
J. P. Rosenbuschand K. Weber,J. Biol. Chem. 246, 1644(1971).
[5]
SUBUNITS IN ATCASE
37
tate strip (Gelman Sepraphore III), and electrophoresis is performed for 10 rain at 250 V in a Microzone Electrophoresis Cell (Model R101, Beckman Instruments, Inc., Spinco Division). The protein is fixed and stained by immersion of the membrane in a solution of Ponceau-S (Beckman Spinco) for 5 min, and then the membrane is rinsed well in 5% acetic acid and dried at room temperature for storage. Typical patterns for the native enzyme and the mixture of separated catalytic and regulatory subunits are shown in the top two strips in Fig. 1. The catalytic subunit yields a narrow band which migrates slightly faster than that corresponding to ATCase. In contrast, the regulatory subunit in the unfractionated mixture exhibits a very broad zone and migrates much more slowly than ATCase. At this stage the regulatory subunit has probably lost some of its zinc ions which may have been replaced in part by mercury. 7 As a consequence, there is a greater tendency for the regulatory dimers to dissociate into monomers, a and this reversible equilibrium leads to a broader zone than that observed when zinc ions are present in the preparation of the regulatory subunit (see bottom pattern in Fig. 1). Separation of Catalytic and Regulatory Subunits b y DEAE-Cellulose Chromatography A column (0.9 cm x 25 cm) is packed with DEAE-cellulose which had been pre-equilibrated with 0.01 M Tris-C1 containing 0.1 M KCI at pH 8.7. The packed column is washed with 100 ml of the same buffer. Fibrous-form resin gives a flow rate between 20-30 ml/hr whereas preswollen microgranular form (Whatman DE 52) usually yields a much slower flow rate. Both produce satisfactory separations of the subunits. The sample of mercurial-treated ATCase is applied to the column and then is washed with 45 ml of the same buffer (0.01 M Tris-C1 containing 0.1 M KC1 at pH 8.7). This procedure elutes the regulatory subunit. The column is then washed with 100 ml of 0.01 M Tris-C1 containing 0.5 M KC1 at pH 8.7 in order to elute the catalytic subunit. Fractions of 3 ml volume are collected and examined spectrophotometrically at 280 nm in order to locate the two subunits. A typical elution pattern is shown in Fig. 2. The yield of regulatory subunit is greater than 90% of the theoretical value and that for the catalytic subunit is generally 85-90% of the theoretical yield. Concentrations of the catalytic and regulatory subunits r M. E. Nelbach, V. P. Piglet, Jr., J. C. Gerhart, and H. K. Schachman, BiochemistD, 11, 315 (1972). s j. A. Cohlberg, V. P. Pigiet, Jr., and H. K. Schachman, Biochernistry, 11, 3396(1972).
38
De Novo PYRIMIDINE BIOSYNTHESIS
[5]
Origin
¢
FIG. 1. Electrophoresis of ATCase dissociation products and purified subunits on cellulose polyacetate strip. Patterns from top to bottom are: (1) native ATCase; (2) ATCase treated with neohydrin to cause complete dissociation into catalytic and regulatory subunits; (3) partially dissociated ATCase with the broad band at the right corresponding to undissociated enzyme, ATCase-like molecules lacking one regulatory subunit, and free catalytic subunit; (4) purified catalytic subunit; and (5) zinc acetate treated regulatory subunit.
[5]
SUBUNITS IN ATCASE
39
°7 I 1 1.5
-
1,0
-
0.5
-
0
P
o~ 0
6
U::::_':a 10
- - -.I.d. 20
30
4O
Fraction number FIG. 2. Chromatographic separation of subunits on DEAE-cellulose. A sample of 100 mg of neohydrin-dissociated ATCase was loaded on a 0.9 x 20 cm column containing DEAE-cellulose equilibrated with 0.01 M Tris-Cl containing 0.1 M KCI at pH 8.7. The elution of the regulatory subunit with this buffer is seen by the peak in optical density at 280 nm in the fractions 4 to 7. After fraction 15 was collected the KCI concentration was increased to 0.5 M which led to the elution of the more highly charged catalytic subunit in fractions 19 to 25. The flow rate was 20-30 ml/hr, and each fraction was 3 ml.
are determined spectrophotometrically based on extinction coefficients (cm 2 mg -1) of 0.72 and 0.30, respectively, at 280 nm. Characterization of the Purified Subunits The fractions (4 to 7) containing regulatory subunit are pooled and 2mercaptoethanol and zinc acetate are added to give a final concentration of 10 mM and 2 mM, respectively. Addition of zinc acetate prior to the thiol causes the formation of a white precipitate which disappears when the reducing agent is added; only a slight loss of regulatory subunit occurs in this process. If the 2-mercaptoethanol is added before the zinc acetate there is no precipitation. The protein solution is then dialyzed against 500 ml of 25 mM Tris-Cl containing 2 mM 2-mercaptoethanol and 0.2 mM zinc acetate at pH 8.0. For storage of the regulatory subunit the protein is precipitated by dialysis against 3.6 M ammonium sulfate containing 5 mM 2-mercaptoethanol at pH 7.0. The fractions (19 to 25) containing catalytic subunit are pooled and 2mercaptoethanol is added to give a concentration of 10 mM. Precipitation of the protein is performed by dialyzing it overnight at 4 ° against 3.6 M ammonium sulfate containing 5 mM 2-mercaptoethanol at pH 7.0.
40
De Novo PYRIMIDINEBIOSYNTHESIS
[5]
The precipitated protein is collected by centrifugation and resuspended in any desired buffer. Both the catalytic and regulatory subunits are characterized in terms of purity and extent of aggregation by sedimentation velocity experiments and by electrophoresis in polyacrylamide gels. The catalytic subunit gives a single symmetrical boundary in the ultracentrifuge with a sedimentation coefficient about 5.8 S. The boundary for the regulatory subunit in the sedimentation velocity experiment migrates slowly (sedimentation coefficient is 2.8 S), and it is significantly broader than that for the catalytic subunit. This broadening is to be expected since the regulatory subunit has a much lower molecular weight than the catalytic subunit, s The catalytic subunit exhibits a single band in polyacrylamide gel electrophoresis which migrates with a much larger mobility than the intact enzyme. Similarly a single band is observed for the purified regulatory subunit which has a slightly greater mobility than the intact enzyme. The band for the regulatory subunit is usually much broader than that observed for the freshly prepared sample. On prolonged storage the regulatory subunit exhibits a broad smear on electrophoresis but a much sharper band can be obtained by increasing the N , N methylene-bis-acrylamide in the gel or by adding a small amount (0.6 mM) of zinc acetate in the gel and the lower buffer (L. Davis, unpublished). The broadening is probably attributable to the associationdissociation equilibrium exhibited by the regulatory subunit which is observed in the absence of zinc ions. s
Discussion The procedure presented here is analogous to that of Gerhart and Holoubek 4 except that neohydrin is used in place ofp-mercuribenzoate for the dissociation of ATCase and DEAE-cellulose is used for the chromatographic separation of the subunits instead of DEAE-Sephadex. With these changes the required column size and the time of separation are reduced, Within a few hours the purified subunits may be separated in high yield from 100 nag of ATCase. Moreover, both subunits are obtained in relatively concentrated solutions directly from the column. The only difficulty experienced with this procedure results from storage of the neohydrin for long periods at room temperature. With such preparations the dissociation of ATCase proceeds slowly, and the prolonged reaction leads to the precipitation of the catalytic subunit. Incomplete dissociation of the enzyme leads to the formation of an
[6]
ASPARTATECARBAMYLTRANSFERASE
41
ATCase-like species lacking one regulatory subunit 9- n in addition to the two types of subunits and some undissociated ATCase. Such a mixture is shown in the third pattern in Fig. 1. This difficulty can be avoided by purifying the neohydrin according to the following procedure. One gram of neohydrin is dissolved in 100 ml of 0.05 M K O H (pH about 11-12) and filtered through Whatman No. 50 filter paper. The filtrate is titrated drop by drop with concentrated HC1 to pH 2.0 at 4 °. A precipitate forms in several minutes and is collected on Whatman No. 50 filter paper. The extraction is then repeated, and the white precipitate is recovered and dried in a desiccator under vacuum. The dried purified neohydrin is stored in a freezer and used when needed for the dissociation of the enzyme. Acknowledgments This work was supportedby NIH researchgrantG M 12159from the NationalInstitute of General Medical Sciences, and by grant PCM-76-23308 from the National Science Foundation. 9 y . R. Yang, J. M, Syvanen, G. M. Nagel, and H. K. Schachman, Proc. Natl. Acad. Sci. U.S.A. 71,918 (1974). 10 M. Bothwell and H. K. Schachman, Proc. Natl. Acad. Sci. U.S.A. 71, 3221 (1974). n D. R. Evans, S. C. Pastra-Landis, and W. N, Lipscomb, Proc. Natl. Acad. Sci. U.S.A. 71, 1351 (1974).
[6] A s p a r t a t e
Carbamyltransferase
(Streptococcus
faecalis) B y T A - Y U A N C H A N G , LANSING M . PRESCOTT a n d MARY E L L E N JONES
Aspartate + carbamyl phosphate x-xcarbamyl aspartate + Pl + H+
Assay M e t h o d s
Method I. A colorimetric measurement of carbamyl aspartate production developed by Prescott and Jones I was used in e n z y m e purification analysis and specific activity determination. This assay in our hands was reproducible and linear from 0.01 to 0.2 /~molc of carbamyl aspartate with optical density values ranging from 0.04 to 0.65 at 466 nm. 1 L. M. Prescott and M. E. Jones, Anal. Biochem. 32, 408 (1969). METHODS IN ENZYMOLOGY, VOL. LI
Copyright© 1978by AcademicPress,Inc. All rightsof reproductionin any formreserved. ISBN 0-12-181951-5
S U B U N I T S IN A T C A S E
35
and 0.2 mM UMP stabilized the large subunit for 4 weeks at 4 °. Combination of dialyzed subunits results in their immediate reassociation into holoenzyme. E n z y m i c Properties o f Subunits. The ot subunit catalyzes the synthesis of carbamoyl phosphate from ammonia but not glutamine. The rate of carbamoyl-phosphate synthesis by the separated a subunit is sigmoidal, and the Hill plot yielded an interaction coefficient of 1.5. The s0,~ value for ATP was 0.3 mM which is close to that obtained for the ammoniadependent activity of the native enzyme. The/3 subunit exhibits glutaminase activity. Ornithine, IMP, and phosphoribosyl-pyrophosphate clearly stimulate the ammonia activity of the large subunit. This activity, however, was only slightly inhibited by high concentrations of UMP. The addition of the small subunit restores the ability to utilize glutamine as well as normal sensitivity to UMP.
[5] A s p a r t a t e T r a n s c a r b a m o y l a s e (Escherichia Preparation of Subunits
co/i):
By YING R. YANG, MARC W, KIRSCHNER,and H. K. SCHACHMAN
Carbamoylphosphate + L-aspartate~ carbamoylaspartate Aspartate transcarbamoylase (ATCase, EC 2.1.3.2: carbamoyl phosphate: L-aspartate carbamoyltransferase) catalyzes the first reaction unique to pyrimidine biosynthesis; in E. coli regulation is achieved in part by feedback inhibition of the enzyme by CTP, an end-product of the pathway. 1"~ The purified enzyme when treated with the mercurial, pmercuribenzoate, is known to dissociate into two types of subunits, one responsible for catalysis, termed the catalytic subunit, and the other which binds both the inhibitor, CTP, and the activator, ATP. 3 After separation of the two types of subunits and removal of the mercurial, the enzyme can be reconstituted by mixing the catalytic and regulatory subunits. The procedure for preparation of the discrete subunits after dissociation of the enzyme with p-mercuribenzoate was described in detail by Gerhart and Holoubek. 4 A modified procedure devised by Kirschner 5 1R. A. Yates and A. B. Pardee,J. Biol. Chem. 221,757 (1956). 2j. C. Gerhart and A. B. Pardee,J. Biol. Chem, 237, 891 (1962). a j. C. Gerhart and H. K. Schachman,Biochemistry 4, 1054(1965). 4j. C. Gerhart and H. Holoubek,J. Biol. Chem. 242, 2886 (1967). 5 M. W. Kirschner, Ph.D. Thesis, Universityof California, Berkeley(1971). METHODS
IN
ENZYMOLOGY, VOL. LI
Copyright ~) 1978 by Academic Press, lnc, All rights of reproduction in any form reserved, ISBN 0-12-181951-5
36
De Novo PYRIMIDINE BIOSYNTHESIS
[5]
was found to give higher yields of regulatory subunit and to reduce the time required for the separation. An alternative procedure involving heat treatment of the enzyme has also been used for the preparation of catalytic subunit, e The method described here is slightly modified from Kirschner's method and provides high yields of purified catalytic and regulatory subunits. It has been used routinely in our laboratory with ATCase from both E. coli and Salmonella typhimurium. Reagents
100 mg purified ATCase 6.3 mg 1-(3-chloromercuri-2-methoxypropyl)urea, known as neohydrin or chloromecodrin (ICN, K & K Laboratories, Inc.) 20 g DEAE-cellulose (Schleicher & Schuell Co.) 0.01 M Tris-Cl, 0.1 M KCI, pH 8.7 0.01 M Tris-Cl, 0.5 M KCI, pH 8.7 20 mM K2HPO4, pH 8.7 3.6 M ammonium sulfate containing 5 mM 2-mercaptoethanol (the pH of the solution is adjusted with KOH to a value of 7 as measured with indicator paper) 25 mM Tris-C1 containing 2 m M 2-mercaptoethanol and 0.2 mM zinc acetate, pH 8.0 Dissociation of E n z y m e into Subunits A sample of I00 m g of ATCase at 15-20 mg/ml is dialyzed overnight at 4 ° against two changes of 500 ml of 0.01 M Tris-Cl containing 0. I M KCI at p H 8.7 in order to reduce the concentration of 2-mercaptoethanol and E D T A in the stock enzyme solution. One milliliterof neohydrin solution, containing 6.3 rng of neohydrin in 0.01 M Tris-Cl at p H 8.7, is added to the enzyme solution which had been equilibrated at room temperature. The addition is performed rapidly, and the solution is mixed by gently inverting the tube several times. After 15 min of incubation at room temperature the reaction mixture is examined by zone electrophoresis on cellulose polyacetate strips in order to test whether the enzyme is completely dissociated. Electrophoresis Zone electrophorcsis is used routinely before the mercurial-treated enzyme is loaded on the DEAE-cellulose column. Samples are applied in 20 m M potassium phosphate buffer at p H 8.7 onto a cellulose polyace-
J. P. Rosenbuschand K. Weber,J. Biol. Chem. 246, 1644(1971).
[5]
SUBUNITS IN ATCASE
37
tate strip (Gelman Sepraphore III), and electrophoresis is performed for 10 rain at 250 V in a Microzone Electrophoresis Cell (Model R101, Beckman Instruments, Inc., Spinco Division). The protein is fixed and stained by immersion of the membrane in a solution of Ponceau-S (Beckman Spinco) for 5 min, and then the membrane is rinsed well in 5% acetic acid and dried at room temperature for storage. Typical patterns for the native enzyme and the mixture of separated catalytic and regulatory subunits are shown in the top two strips in Fig. 1. The catalytic subunit yields a narrow band which migrates slightly faster than that corresponding to ATCase. In contrast, the regulatory subunit in the unfractionated mixture exhibits a very broad zone and migrates much more slowly than ATCase. At this stage the regulatory subunit has probably lost some of its zinc ions which may have been replaced in part by mercury. 7 As a consequence, there is a greater tendency for the regulatory dimers to dissociate into monomers, a and this reversible equilibrium leads to a broader zone than that observed when zinc ions are present in the preparation of the regulatory subunit (see bottom pattern in Fig. 1). Separation of Catalytic and Regulatory Subunits b y DEAE-Cellulose Chromatography A column (0.9 cm x 25 cm) is packed with DEAE-cellulose which had been pre-equilibrated with 0.01 M Tris-C1 containing 0.1 M KCI at pH 8.7. The packed column is washed with 100 ml of the same buffer. Fibrous-form resin gives a flow rate between 20-30 ml/hr whereas preswollen microgranular form (Whatman DE 52) usually yields a much slower flow rate. Both produce satisfactory separations of the subunits. The sample of mercurial-treated ATCase is applied to the column and then is washed with 45 ml of the same buffer (0.01 M Tris-C1 containing 0.1 M KC1 at pH 8.7). This procedure elutes the regulatory subunit. The column is then washed with 100 ml of 0.01 M Tris-C1 containing 0.5 M KC1 at pH 8.7 in order to elute the catalytic subunit. Fractions of 3 ml volume are collected and examined spectrophotometrically at 280 nm in order to locate the two subunits. A typical elution pattern is shown in Fig. 2. The yield of regulatory subunit is greater than 90% of the theoretical value and that for the catalytic subunit is generally 85-90% of the theoretical yield. Concentrations of the catalytic and regulatory subunits r M. E. Nelbach, V. P. Piglet, Jr., J. C. Gerhart, and H. K. Schachman, BiochemistD, 11, 315 (1972). s j. A. Cohlberg, V. P. Pigiet, Jr., and H. K. Schachman, Biochernistry, 11, 3396(1972).
38
De Novo PYRIMIDINE BIOSYNTHESIS
[5]
Origin
¢
FIG. 1. Electrophoresis of ATCase dissociation products and purified subunits on cellulose polyacetate strip. Patterns from top to bottom are: (1) native ATCase; (2) ATCase treated with neohydrin to cause complete dissociation into catalytic and regulatory subunits; (3) partially dissociated ATCase with the broad band at the right corresponding to undissociated enzyme, ATCase-like molecules lacking one regulatory subunit, and free catalytic subunit; (4) purified catalytic subunit; and (5) zinc acetate treated regulatory subunit.
[5]
SUBUNITS IN ATCASE
39
°7 I 1 1.5
-
1,0
-
0.5
-
0
P
o~ 0
6
U::::_':a 10
- - -.I.d. 20
30
4O
Fraction number FIG. 2. Chromatographic separation of subunits on DEAE-cellulose. A sample of 100 mg of neohydrin-dissociated ATCase was loaded on a 0.9 x 20 cm column containing DEAE-cellulose equilibrated with 0.01 M Tris-Cl containing 0.1 M KCI at pH 8.7. The elution of the regulatory subunit with this buffer is seen by the peak in optical density at 280 nm in the fractions 4 to 7. After fraction 15 was collected the KCI concentration was increased to 0.5 M which led to the elution of the more highly charged catalytic subunit in fractions 19 to 25. The flow rate was 20-30 ml/hr, and each fraction was 3 ml.
are determined spectrophotometrically based on extinction coefficients (cm 2 mg -1) of 0.72 and 0.30, respectively, at 280 nm. Characterization of the Purified Subunits The fractions (4 to 7) containing regulatory subunit are pooled and 2mercaptoethanol and zinc acetate are added to give a final concentration of 10 mM and 2 mM, respectively. Addition of zinc acetate prior to the thiol causes the formation of a white precipitate which disappears when the reducing agent is added; only a slight loss of regulatory subunit occurs in this process. If the 2-mercaptoethanol is added before the zinc acetate there is no precipitation. The protein solution is then dialyzed against 500 ml of 25 mM Tris-Cl containing 2 mM 2-mercaptoethanol and 0.2 mM zinc acetate at pH 8.0. For storage of the regulatory subunit the protein is precipitated by dialysis against 3.6 M ammonium sulfate containing 5 mM 2-mercaptoethanol at pH 7.0. The fractions (19 to 25) containing catalytic subunit are pooled and 2mercaptoethanol is added to give a concentration of 10 mM. Precipitation of the protein is performed by dialyzing it overnight at 4 ° against 3.6 M ammonium sulfate containing 5 mM 2-mercaptoethanol at pH 7.0.
40
De Novo PYRIMIDINEBIOSYNTHESIS
[5]
The precipitated protein is collected by centrifugation and resuspended in any desired buffer. Both the catalytic and regulatory subunits are characterized in terms of purity and extent of aggregation by sedimentation velocity experiments and by electrophoresis in polyacrylamide gels. The catalytic subunit gives a single symmetrical boundary in the ultracentrifuge with a sedimentation coefficient about 5.8 S. The boundary for the regulatory subunit in the sedimentation velocity experiment migrates slowly (sedimentation coefficient is 2.8 S), and it is significantly broader than that for the catalytic subunit. This broadening is to be expected since the regulatory subunit has a much lower molecular weight than the catalytic subunit, s The catalytic subunit exhibits a single band in polyacrylamide gel electrophoresis which migrates with a much larger mobility than the intact enzyme. Similarly a single band is observed for the purified regulatory subunit which has a slightly greater mobility than the intact enzyme. The band for the regulatory subunit is usually much broader than that observed for the freshly prepared sample. On prolonged storage the regulatory subunit exhibits a broad smear on electrophoresis but a much sharper band can be obtained by increasing the N , N methylene-bis-acrylamide in the gel or by adding a small amount (0.6 mM) of zinc acetate in the gel and the lower buffer (L. Davis, unpublished). The broadening is probably attributable to the associationdissociation equilibrium exhibited by the regulatory subunit which is observed in the absence of zinc ions. s
Discussion The procedure presented here is analogous to that of Gerhart and Holoubek 4 except that neohydrin is used in place ofp-mercuribenzoate for the dissociation of ATCase and DEAE-cellulose is used for the chromatographic separation of the subunits instead of DEAE-Sephadex. With these changes the required column size and the time of separation are reduced, Within a few hours the purified subunits may be separated in high yield from 100 nag of ATCase. Moreover, both subunits are obtained in relatively concentrated solutions directly from the column. The only difficulty experienced with this procedure results from storage of the neohydrin for long periods at room temperature. With such preparations the dissociation of ATCase proceeds slowly, and the prolonged reaction leads to the precipitation of the catalytic subunit. Incomplete dissociation of the enzyme leads to the formation of an
[6]
ASPARTATECARBAMYLTRANSFERASE
41
ATCase-like species lacking one regulatory subunit 9- n in addition to the two types of subunits and some undissociated ATCase. Such a mixture is shown in the third pattern in Fig. 1. This difficulty can be avoided by purifying the neohydrin according to the following procedure. One gram of neohydrin is dissolved in 100 ml of 0.05 M K O H (pH about 11-12) and filtered through Whatman No. 50 filter paper. The filtrate is titrated drop by drop with concentrated HC1 to pH 2.0 at 4 °. A precipitate forms in several minutes and is collected on Whatman No. 50 filter paper. The extraction is then repeated, and the white precipitate is recovered and dried in a desiccator under vacuum. The dried purified neohydrin is stored in a freezer and used when needed for the dissociation of the enzyme. Acknowledgments This work was supportedby NIH researchgrantG M 12159from the NationalInstitute of General Medical Sciences, and by grant PCM-76-23308 from the National Science Foundation. 9 y . R. Yang, J. M, Syvanen, G. M. Nagel, and H. K. Schachman, Proc. Natl. Acad. Sci. U.S.A. 71,918 (1974). 10 M. Bothwell and H. K. Schachman, Proc. Natl. Acad. Sci. U.S.A. 71, 3221 (1974). n D. R. Evans, S. C. Pastra-Landis, and W. N, Lipscomb, Proc. Natl. Acad. Sci. U.S.A. 71, 1351 (1974).
[6] A s p a r t a t e
Carbamyltransferase
(Streptococcus
faecalis) B y T A - Y U A N C H A N G , LANSING M . PRESCOTT a n d MARY E L L E N JONES
Aspartate + carbamyl phosphate x-xcarbamyl aspartate + Pl + H+
Assay M e t h o d s
Method I. A colorimetric measurement of carbamyl aspartate production developed by Prescott and Jones I was used in e n z y m e purification analysis and specific activity determination. This assay in our hands was reproducible and linear from 0.01 to 0.2 /~molc of carbamyl aspartate with optical density values ranging from 0.04 to 0.65 at 466 nm. 1 L. M. Prescott and M. E. Jones, Anal. Biochem. 32, 408 (1969). METHODS IN ENZYMOLOGY, VOL. LI
Copyright© 1978by AcademicPress,Inc. All rightsof reproductionin any formreserved. ISBN 0-12-181951-5