[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
42
De N o v o PYRIMIDINE BIOSYNTHESIS
[6]
Reagents. The standard assay mixture contained 0.1 M Tris.HC1 (pH 8.5), 10 mM carbamyl phosphate, 50 mM L-aspartate (pH 8.5), and enzyme to yield a final volume of 1 ml.
Procedure. The reaction was started by adding either carbamyl phosphate or enzyme after preincubation of the reaction mixture for 1 min at 25 °. The incubation time for the assay was usually 10 min. The reaction was terminated by the addition of 1 ml of 1 M perchloric acid, and part of the total reaction mixture was taken for the colorimetric carbamyl aspartate assay. A blank containing the same components but omitting enzyme source was used as the control. Definition o f Unit and Specific Activity. A unit of enzyme activity is defined as that amount of enzyme which catalyzes the formation of 1 /zmole of carbamyl aspartate or phosphate per minute under standard assay conditions. The protein concentration was determined using the method of Oyama and Eagle. 2 Method 2. The [14C]carbamyl phosphate assay of Bethell et al. a was used when more rigorous kinetic analysis was performed.
Assay Reagents and Procedure. The 0.5- or 1-ml reaction mixture contained 5 mM Tris.HCl with varying levels of substrates, inhibitors, and aspartate transcarbamylase. The final pH of the reaction mixture was 8.5. Reaction was performed at 25 °. Incubation time ranged from 10 to 20 min. The reaction was stopped by addition of 0.5 or 1 ml of 1 M perchloric acid. After acidification the test tubes were processed according to the procedure of Bethell et al. 3 Controls without enzyme were routinely run and gave reproducible background values. The specific activity of [!~C]carbamyl phosphate used was selected so that the counts per minute in each experimental sample were greater than 500. The initial velocity was linear up to about 40% substrate consumption when carbamyl phosphate was limiting, or to about 20% substrate consumption when aspartate was the limiting substrate. All experiments were performed within these ranges. 2 V. I. Oyama and H. Eagle, Proc. Soc. Exp. Biol. Med. 91, 305 (1956). a M. R. Bethell, K. E. Smith, J. S. White, and M. E. Jones, Proc. Natl. Acad. Sci. U.S.A. 60, 1442 (1968).
[6]
ASPARTATECARBAMYLTRANSFERASE
43
Growth of Cells for E n z y m e Source Cells of Streptococcus faecalis R (ATCC #8043) were grown as arginine-adapted culture and harvested as described by Jones. 4 Purification Procedure
Step 1. Disruption of Cells and Extraction o f Enzyme. Cells were disrupted with a Braun Model MSK Mechanical Cell homogenizer (Brownwill Scientific) using 0.17-0.18 mm glass beads, according to the method of Bleiweis et al. 5 Frozen S. faecalis cells (0.52 kg) were thawed, suspended in 1.56 liter of ice-cold, 20 mM potassium phosphate buffer at pH 6.6, and held at 4 °. Tri-n-butyl phosphate (14 ml) was added as antifoaming agent. For each single operation, 30 ml of diluted cell paste and 30 g of glass beads were mixed in a 75-ml glass flask. The stoppered flask was shaken for 3 min at 4000 oscillations/rain in a stream of liquid CO2 delivered at a rate sufficient to prevent heating of the chamber. After the disruption step was complete, the entire homogenate was centrifuged with a Sorvall GSA rotor at 10,000 rpm at 4 ° for 30 min; the supernatant was collected and dialyzed against three changes of 16 liters of 20 mM potassium phosphate buffer (pH 6.6) at 4 °. The dialyzed homogenate had a total volume of 1950 ml. Step 2. Streptomycin Sulfate Precipitation. Streptomycin sulfate solution (324 ml of a 5% solution in H~0) was added drop by drop to 1950 ml of homogenate which had a protein concentration of 25.8 mg/ml. After the addition of streptomycin, the entire solution was kept overnight at 4 ° without disturbance to allow for the complete precipitation of nucleic acids. The precipitated protein was collected as in step 1 and discarded. Step 3. pH 4.8 Precipitation. Approximately 30 ml of 0.5 M acetic acid were required to bring the pH of 1 liter of the streptomycin sulfate supernatant to 4.8. The acetic acid must be added as quickly as thorough mixing permits. The time usually required was 10-20 min. As soon as the acid addition was complete, the precipitate was centrifuged down as in step I. The precipitate was then resuspended in 0.1 M potassium phosphate buffer (pH 6.6) using a tissue homogenizer with a Teflon pestle. Normally a volume of buffer about 5% the volume of streptomycin sulfate supernatant was used. The resuspended pH 4.8 precipitate 4 M. E. Jones, in "Methods in Enzymology"(S. P. Colowickand N. O. Kaplan, eds.), Vol. 5, p. 903. AcademicPress, New York. 5 A. S. Bleiweis,W. W. Karakawa,and R. M. Krause,J. Bacteriol. 88, 1198(1964).
44
De Novo PYRIMIDINE BIOSYNTHESIS
[6]
was then dialyzed overnight at 4 ° against two 3-liter changes of 0.05 M phosphate buffer (pH 6.6). The pH 4.8 precipitate did not immediately dissolve completely when suspended in the 0.1 M buffer, but it completely dissolved during the dialysis. The dialyzed pH 4.8 precipitate solution was usually stored frozen at - 2 0 ° until it could be further purified. The enzyme is very stable in this condition.
Step 4. Hydroxylapatite Chromatography. A column, 16 cm in diameter, made in a Biichner funnel with fritted disc (porosity C, 3000ml capacity), was packed with hydroxylapatite gel and equilibrated with 1 mM potassium phosphate buffer (pH 6.6) at 4 °. The column height can v~ry from 5 to 9.5 cm without affecting the resolution of the column. Flow rate of the column was kept at about 200 ml/hr. The protein sample obtained from step 3, dialyzed against 1 mM potassium phosphate buffer (pH 6.6) at 4 °, was applied to the column. The amount of protein applied was 4 - 5 nag of protein/ml of bed volume. The column was eluted with about two column volumes of 40 mM potassium phosphate buffer (pH 6.6), which removed large amounts of proteins without ATCase activity. The column was then eluted with 50 mM potassium phosphate buffer (pH 6.6) to elute ATCase activity. Usually 3-4 column volumes of 50 mM potassium .phosphate buffer were sufficient to elute the enzyme, but sometimes a severe "tailing effect" was seen and as many as 6 column volumes of 50 mM phosphate buffer were required in order to elute all of the ATCase activity from the column. The fractions with ATCase activity were pooled and concentrated using an Amicon ultrafiltration cell fitted with a PM-10 membrane to yield a solution containing 6-8 mg of protein/ml. The concentrated protein solution had an appreciable amount of insoluble material which was centrifuged down and discarded since it contained no ATCase activity. The clear supernatant solution was dialyzed against 3 changes of 50 mM potassium phosphate buffer (pH 6.6) at 4 °, and was stored at 4 °. The hydroxylapatite column could be regenerated by washing with 4-5 column volumes of 0.4 M potassium phosphate buffer (pH 6.6) and used again several times. Step 5. Ammonium Sulfate Fractionation. The 43 ml of enzyme solution (6 mg of protein/ml) from step 4 were brought to 43% saturation with saturated ammonium sulfate solution. This was done by very slow addition of the saturated solution at a rate of 2 ml/5 rain with thorough mixing. The suspension was then kept at 4 ° for 1 hr undisturbed, and after it was centrifuged at 1700 rpm with a Sorvall SS-34 rotor for 10 min, the precipitate was discarded. The supernatant was then kept at 4 ° undisturbed for 1 day, after which it was centrifuged again; the precipitate was collected and dissolved in 4 ml of cold 50 mM potassium phosphate buffer (pH 6.6). This solution, which contained most of the
[6]
ASPARTATE CARBAMYLTRANSFERASE
45
ATCase activity, was dialyzed against two changes of 1 liter of l0 mM potassium phosphate buffer (pH 6.6) containing 0.28 mM KCl and 2 mM mercaptoethanol.
Step 6. DEAE-Sephadex Column Chromatography. A 1.5 x 15 cm DEAE-Sephadex A-50 column (Pharmacia) was packed at 4 ° and was equilibrated with 10 mM potassium phosphate buffer (pH 6.6) containing 0.28 M KC1 and 2 mM mercaptoethanol; the pressure drop over the bed was kept near 17-18 cm throughout the eluting process, and the flow rate was about 26 ml/hr. A 5-ml sample containing 13 mg of protein/ml (from step 5) was carefully layered on top of the column, after which a 300-ml linear KC1 elution gradient, from 0.28 to 0.47 M, was used. Under the conditions described above, two peaks with ATCase activity were invariably obtained regardless of the amount of protein sample applied on the column (10-67 rag). This phenomenon could also be demonstrated with a DEAE-cellulose column (Bio-Rad Laboratories). Rechromatography of peak I (the peak that is eluted earlier) and peak II (the second peak) established that peak I was an "artificial" peak. 6 Therefore, only fractions from peak II were collected for further purification. Later it was found that the appearance of peak I could be totally prevented by running the DEAE-Sephadex or DEAE-ceUulose column at a much faster flow rate, so the enzyme did not remain in the column too long (-< 2 hr for the DEAE-cellulose column, or -
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
42
De N o v o PYRIMIDINE BIOSYNTHESIS
[6]
Reagents. The standard assay mixture contained 0.1 M Tris.HC1 (pH 8.5), 10 mM carbamyl phosphate, 50 mM L-aspartate (pH 8.5), and enzyme to yield a final volume of 1 ml.
Procedure. The reaction was started by adding either carbamyl phosphate or enzyme after preincubation of the reaction mixture for 1 min at 25 °. The incubation time for the assay was usually 10 min. The reaction was terminated by the addition of 1 ml of 1 M perchloric acid, and part of the total reaction mixture was taken for the colorimetric carbamyl aspartate assay. A blank containing the same components but omitting enzyme source was used as the control. Definition o f Unit and Specific Activity. A unit of enzyme activity is defined as that amount of enzyme which catalyzes the formation of 1 /zmole of carbamyl aspartate or phosphate per minute under standard assay conditions. The protein concentration was determined using the method of Oyama and Eagle. 2 Method 2. The [14C]carbamyl phosphate assay of Bethell et al. a was used when more rigorous kinetic analysis was performed.
Assay Reagents and Procedure. The 0.5- or 1-ml reaction mixture contained 5 mM Tris.HCl with varying levels of substrates, inhibitors, and aspartate transcarbamylase. The final pH of the reaction mixture was 8.5. Reaction was performed at 25 °. Incubation time ranged from 10 to 20 min. The reaction was stopped by addition of 0.5 or 1 ml of 1 M perchloric acid. After acidification the test tubes were processed according to the procedure of Bethell et al. 3 Controls without enzyme were routinely run and gave reproducible background values. The specific activity of [!~C]carbamyl phosphate used was selected so that the counts per minute in each experimental sample were greater than 500. The initial velocity was linear up to about 40% substrate consumption when carbamyl phosphate was limiting, or to about 20% substrate consumption when aspartate was the limiting substrate. All experiments were performed within these ranges. 2 V. I. Oyama and H. Eagle, Proc. Soc. Exp. Biol. Med. 91, 305 (1956). a M. R. Bethell, K. E. Smith, J. S. White, and M. E. Jones, Proc. Natl. Acad. Sci. U.S.A. 60, 1442 (1968).
[6]
ASPARTATECARBAMYLTRANSFERASE
43
Growth of Cells for E n z y m e Source Cells of Streptococcus faecalis R (ATCC #8043) were grown as arginine-adapted culture and harvested as described by Jones. 4 Purification Procedure
Step 1. Disruption of Cells and Extraction o f Enzyme. Cells were disrupted with a Braun Model MSK Mechanical Cell homogenizer (Brownwill Scientific) using 0.17-0.18 mm glass beads, according to the method of Bleiweis et al. 5 Frozen S. faecalis cells (0.52 kg) were thawed, suspended in 1.56 liter of ice-cold, 20 mM potassium phosphate buffer at pH 6.6, and held at 4 °. Tri-n-butyl phosphate (14 ml) was added as antifoaming agent. For each single operation, 30 ml of diluted cell paste and 30 g of glass beads were mixed in a 75-ml glass flask. The stoppered flask was shaken for 3 min at 4000 oscillations/rain in a stream of liquid CO2 delivered at a rate sufficient to prevent heating of the chamber. After the disruption step was complete, the entire homogenate was centrifuged with a Sorvall GSA rotor at 10,000 rpm at 4 ° for 30 min; the supernatant was collected and dialyzed against three changes of 16 liters of 20 mM potassium phosphate buffer (pH 6.6) at 4 °. The dialyzed homogenate had a total volume of 1950 ml. Step 2. Streptomycin Sulfate Precipitation. Streptomycin sulfate solution (324 ml of a 5% solution in H~0) was added drop by drop to 1950 ml of homogenate which had a protein concentration of 25.8 mg/ml. After the addition of streptomycin, the entire solution was kept overnight at 4 ° without disturbance to allow for the complete precipitation of nucleic acids. The precipitated protein was collected as in step 1 and discarded. Step 3. pH 4.8 Precipitation. Approximately 30 ml of 0.5 M acetic acid were required to bring the pH of 1 liter of the streptomycin sulfate supernatant to 4.8. The acetic acid must be added as quickly as thorough mixing permits. The time usually required was 10-20 min. As soon as the acid addition was complete, the precipitate was centrifuged down as in step I. The precipitate was then resuspended in 0.1 M potassium phosphate buffer (pH 6.6) using a tissue homogenizer with a Teflon pestle. Normally a volume of buffer about 5% the volume of streptomycin sulfate supernatant was used. The resuspended pH 4.8 precipitate 4 M. E. Jones, in "Methods in Enzymology"(S. P. Colowickand N. O. Kaplan, eds.), Vol. 5, p. 903. AcademicPress, New York. 5 A. S. Bleiweis,W. W. Karakawa,and R. M. Krause,J. Bacteriol. 88, 1198(1964).
44
De Novo PYRIMIDINE BIOSYNTHESIS
[6]
was then dialyzed overnight at 4 ° against two 3-liter changes of 0.05 M phosphate buffer (pH 6.6). The pH 4.8 precipitate did not immediately dissolve completely when suspended in the 0.1 M buffer, but it completely dissolved during the dialysis. The dialyzed pH 4.8 precipitate solution was usually stored frozen at - 2 0 ° until it could be further purified. The enzyme is very stable in this condition.
Step 4. Hydroxylapatite Chromatography. A column, 16 cm in diameter, made in a Biichner funnel with fritted disc (porosity C, 3000ml capacity), was packed with hydroxylapatite gel and equilibrated with 1 mM potassium phosphate buffer (pH 6.6) at 4 °. The column height can v~ry from 5 to 9.5 cm without affecting the resolution of the column. Flow rate of the column was kept at about 200 ml/hr. The protein sample obtained from step 3, dialyzed against 1 mM potassium phosphate buffer (pH 6.6) at 4 °, was applied to the column. The amount of protein applied was 4 - 5 nag of protein/ml of bed volume. The column was eluted with about two column volumes of 40 mM potassium phosphate buffer (pH 6.6), which removed large amounts of proteins without ATCase activity. The column was then eluted with 50 mM potassium phosphate buffer (pH 6.6) to elute ATCase activity. Usually 3-4 column volumes of 50 mM potassium .phosphate buffer were sufficient to elute the enzyme, but sometimes a severe "tailing effect" was seen and as many as 6 column volumes of 50 mM phosphate buffer were required in order to elute all of the ATCase activity from the column. The fractions with ATCase activity were pooled and concentrated using an Amicon ultrafiltration cell fitted with a PM-10 membrane to yield a solution containing 6-8 mg of protein/ml. The concentrated protein solution had an appreciable amount of insoluble material which was centrifuged down and discarded since it contained no ATCase activity. The clear supernatant solution was dialyzed against 3 changes of 50 mM potassium phosphate buffer (pH 6.6) at 4 °, and was stored at 4 °. The hydroxylapatite column could be regenerated by washing with 4-5 column volumes of 0.4 M potassium phosphate buffer (pH 6.6) and used again several times. Step 5. Ammonium Sulfate Fractionation. The 43 ml of enzyme solution (6 mg of protein/ml) from step 4 were brought to 43% saturation with saturated ammonium sulfate solution. This was done by very slow addition of the saturated solution at a rate of 2 ml/5 rain with thorough mixing. The suspension was then kept at 4 ° for 1 hr undisturbed, and after it was centrifuged at 1700 rpm with a Sorvall SS-34 rotor for 10 min, the precipitate was discarded. The supernatant was then kept at 4 ° undisturbed for 1 day, after which it was centrifuged again; the precipitate was collected and dissolved in 4 ml of cold 50 mM potassium phosphate buffer (pH 6.6). This solution, which contained most of the
[6]
ASPARTATE CARBAMYLTRANSFERASE
45
ATCase activity, was dialyzed against two changes of 1 liter of l0 mM potassium phosphate buffer (pH 6.6) containing 0.28 mM KCl and 2 mM mercaptoethanol.
Step 6. DEAE-Sephadex Column Chromatography. A 1.5 x 15 cm DEAE-Sephadex A-50 column (Pharmacia) was packed at 4 ° and was equilibrated with 10 mM potassium phosphate buffer (pH 6.6) containing 0.28 M KC1 and 2 mM mercaptoethanol; the pressure drop over the bed was kept near 17-18 cm throughout the eluting process, and the flow rate was about 26 ml/hr. A 5-ml sample containing 13 mg of protein/ml (from step 5) was carefully layered on top of the column, after which a 300-ml linear KC1 elution gradient, from 0.28 to 0.47 M, was used. Under the conditions described above, two peaks with ATCase activity were invariably obtained regardless of the amount of protein sample applied on the column (10-67 rag). This phenomenon could also be demonstrated with a DEAE-cellulose column (Bio-Rad Laboratories). Rechromatography of peak I (the peak that is eluted earlier) and peak II (the second peak) established that peak I was an "artificial" peak. 6 Therefore, only fractions from peak II were collected for further purification. Later it was found that the appearance of peak I could be totally prevented by running the DEAE-Sephadex or DEAE-ceUulose column at a much faster flow rate, so the enzyme did not remain in the column too long (-< 2 hr for the DEAE-cellulose column, or -
