[27]
CDPDIACYLGLYCEROL SYNTHASE FROM E. coli
237
[27] Purification of CDPdiacylglycerol Synthase from E s c h e r i c h i a coli B y CARL P . SPARROW
Introduction CDPdiacylglycerol synthase (CTP:phosphatidate cytidylyltransferase; EC 2.7.7.41, phosphatidate cytidylyltransferase) catalyzes the activation of phosphatidic acid by CTP to form CDPdiacylglycerol and inorganic pyrophosphate. This activity is essential for all phospholipid biosynthesis in Escherichia coli 1 and for the biosynthesis of acidic phospholipids in mammalian cells. 2 The enzymes of phospholipid synthesis are usually membrane bound and present in relatively low levels, making their purification difficult. The purification of E. coli CDPdiacylglycerol synthase is greatly aided by enzyme overproduction driven by an expression plasmid) Starting with cells that overproduce the enzyme 50-fold, essentially homogeneous enzyme can be obtained after chromatographic procedures that yield 160fold purification. 4 The enzyme can be partially purified from wild-type cells using the same procedures. Assay of CDPdiacylglycerol Synthase Activity The assay follows the conversion of [a-3Ep]dCTP to chloroform-soluble material, dependent on phosphatidic acid. The standard assay mixture contains 100 mM potassium phosphate, pH 7.4, 0.2% (w/v) Triton X-100, 1 mM phosphatidic acid, 1 mg/ml of bovine serum albumin, 10 mM MgC12, 5 mM [a-32p]dCTP ( - 2 Ci/mol) and 0.25 mM dithiothreitol. Stock solutions of each component are stored separately and then mixed together on the day of assay. To prevent precipitation of magnesium phosphatidate it is essential to mix the Triton X-100 and phosphatidic acid prior to addition of the magnesium chloride. The reaction is performed in 13 × 100 mm Pyrex tubes with Teflon-faced screw caps (Coming, Coming, NY). The reaction is initiated by adding l0/.d of enzyme source to 40 ~l of a mixture of the other assay components. t C. R. H. Raetz, Annu. Rev. Genet. 20, 253 (1986). 2 j. D. Esko and C. R. H. Raetz, in "The Enzymes" (P. D. Boyer, ed.), 3rd Ed., Vol., 16, p. 208. Academic Press, New York, 1983. 3 T. Icho, C. P. Sparrow, and C. R. H. Raetz, J. Biol. Chem. 260, 12078 0985). 4 C. P. Sparrow and C. R. H. Raetz, J. Biol. Chem. 260, 12084 (1985).
METHODSIN ENZYMOLOGY,VOL. 209
Copyright~ 1992by AcademicPress,Inc. All fightsof reproductionin any formreserved.
238
CYTIDYLYLTRANSFERASES
[2 7]
After 10 min at 30°, the reaction is stopped by adding 2 ml of chloroform-methanol (1 : 1, v/v). The tubes can be stored in this state indefinitely if they are tightly capped. The extraction is finished by adding 0.85 ml of 1 M NaCI, pH 2. This creates a two-phase system. 5 The samples are mixed well and spun briefly to separate the phases. The upper phase, the interface, and any interfacial precipitate is aspirated and discarded. One milliliter of the lower phase (which is 50% of the total lower phase) is transferred to a glass vial, the solvent is evaporated, and radioactivity is quantitated by liquid scintillation spectrometry. One unit of activity is defined as the amount of enzyme needed to convert 1 nmol of dCTP to chloroform-soluble material per minute. Under these conditions, the reaction is linear with time and enzyme up to about 5-10 nmol of product per 10 min. The reaction is linear to larger conversions with partially purified enzyme, presumably because there are fewer activities competing for the dCTP substrate. The most appropriate blank for the assay is a tube containing enzyme but no phosphatidic acid. Similar or identical blanks are obtained from tubes containing phosphatidic acid but no enzyme. The standard assay is sensitive to the molecular species of phosphatidic acid used. Dioleoylphosphatidic acid, palmitoyloleoylphosphatidic acid, or phosphatidic a c i d made from egg phosphatidylcholine give optimal results, whereas dipalmitoylphosphatidic acid gives rates less than 10% of optimum. 4 This fact is the cause of the disagreement in the literature concerning the level of CDPdiacylglycerol synthase activity in wild-type crude extracts. 4'6 Purification of CDPdiacylglycerol Synthase A summary of the purification of CDPdiacylglycerol synthase is given in Table I. The behavior of CDPdiacylglycerol synthase during column chromatography is influenced greatly by detergents and by the presence of EDTA. Although the physical basis of these effects is unknown, the detergents are useful tools for purifying the enzyme. Furthermore, CDPdiacylglycerol synthase is fairly stable in the presence of dithiothreitol, even at room temperature, and therefore the columns are run on the laboratory bench (20°-24°). After elution, column fractions are stored on ice. Tris buffers are prepared with chloride as the counterion. Concentrations in units of percent are all (w/v). Protein is assayed after precipitation as described by Peterson, 7 except that samples containing high concentra5 E. G. Bligh and J. J. Dyer, Can. J. Biochem. Physiol. 37, 911 (1959). 6 K. E. Langley and E. P. Kennedy, J. Bacteriol. 136, 85 (1978). 7 G. L. Peterson, Anal. Biochem. 83, 346 (1977).
CDPDIACYLGLYCEROL SYNTHASEFROME. coil
[27]
239
TABLE I PURIFICATION OF CDPDIACYLGLYCEROLSYNTHASEFROM Escherichia coil a
Step
Total protein (mg)
Specific activity (units/mg)
Yield (%)
Requirements for step b
Crude extract Membranes Solubilized membranes First DEAE column Second DEAE column Hydroxylapatite column
1656 676 589 288 10.3 2.2
192 466 346 607 9,102 30,786
(100) 99 64 55 30 21
(Plasmid) Mg2÷ EDTA, OG c EDTA Zwittergent 3-12 Pi gradient, OG
Adapted from C. P. Sparrow and C. R. H. Raetz, J. Biol. Chem. 260, 12084 (1985). b See text for detailed discussion of requirements. c OG, n-Octyl-/3-o-glucopyranoside. a
tions of Zwittergent cannot be precipitated. For these samples appropriate blanks must be assayed to subtract the signal generated by dithiothreitol. Growth of Cells and Preparation of Crude Extract. The strain DH1/ pCD100 overproduces CDPdiacylglycerol synthase about 50-fold. 3 This strain is grown in LB mediums and harvested in log phase. The cell paste (22 g) is resuspended in 9 volumes of 0.1 M potassium phosphate, pH 7.4, containing 10 mM magnesium sulfate, and the cells are broken by passage through a Mantlin-Gaulin press (9000 psi). The material is centrifuged at 5000 g for 15 min at 4° to remove unbroken cells. The supernatant is the crude extract. Membrane Preparation and Solubilization of Enzyme. The crude extract is ultracentrifuged at 180,000 g for 90 min at 4°, and the membrane pellet is resuspended in 30-50 ml of 10 mM Tris, pH 8.0, containing 0.25 mM dithiothreitol. Resuspension can be accomplished using either a Teflon-glass homogenizer or a needle and syringe. CDPdiacylglycerol synthase is solubilized by adjusting the membrane fraction to 6-7 membrane protein/ml in I0 mM Tris, pH 8.5, 10 mM EDTA, 0.25 mM dithiothreitol, 1.5% Triton X-100, 1.5% n-octyl-/3-D-glucopyranoside (OG), and 10% glycerol. This mixture is stirred for 1 hr on ice and then spun at 180,000 g for 60 min. The supernatant is the solubilized membrane fraction. DEAE-cellulose Column Chromatography. CDPdiacylglycerol synthase binds to DEAE-cellulose (DE-52, Whatman, Clifton, N J) in the absence of EDTA but not in the presence of EDTA. This property is exploited in the following purification. The solubilized membrane fraction 8 j. H. Miller, "Experiments in Molecular Genetics." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1972.
240
CYTIDYLYLTRANSFERASES
[2 7]
(-600 mg protein) is loaded onto a DE-52 column, 4 cm wide and 7 cm high, which is preequilibrated with 10 mM Tris, pH 8.0, 10 mM EDTA, 2.0% Triton X-100, 10% glycerol, and 0.25 mM dithiothreitol. The column is washed with the same buffer, at a flow rate of 8 ml/min. Because only the unretained material is desired, a few fractions of large volume are collected: typically, the first 50 ml can be discarded, and the next 120-150 ml contains the CDPdiacylglycerol synthase activity. This is the DEAE run-through fraction. This material can be stored frozen with no loss of activity. The run-through fraction is dialyzed at 4° against at least 20 volumes of buffer T, which is 10 mM Tris, pH 8.0, 1% Triton X-100, and 0.25 mM dithiothreitol. This material is loaded onto a second DE-52 column, 2.6 cm wide and 4 cm high, preequilibrated in buffer T. This column is run at 5 ml/min. After loading, the column is washed sequentially with I0 ml of buffer T, then 10 ml of buffer LT, which is 10 mM Tris, pH 8.0, 0.2% Triton X-100, and 0.25 mM dithiothreitol. Significant amounts of protein, but little CDPdiacylglycerol synthase, is then removed from the column by washing with 60 ml of buffer LT containing 70 mM KCI followed by 20 ml of buffer LT. The CDPdiacylglycerol synthase is then step-eluted with 63 ml of 10 mM Tris, pH 8.0, 4% Zwittergent 3-12 (Calbiochem, San Diego, CA), 25 mM KC1, and 0.25 mM dithiothreitol. The first 10-15 ml of eluant contains no activity and is discarded; the next 50 ml is collected in one fraction, called the Zwittergent enzyme preparation. Hydroxylapatite Column Chromatography. The Zwittergent enzyme preparation is diluted 1 : 1 with buffer T and loaded onto a hydroxylapatite column (Bio-Rad, Richmond, CA, BioSil A, 200-400 mesh). The column, 1.4 cm wide and 3.4 cm high, is preequilibrated with buffer T and run at 4 ml/min. After loading, the column is washed with 10 ml of buffer T, and then the detergent is changed by washing with 5 ml of 10 mM Tris, pH 8.0, 10 mM EDTA, 1.2% OG, and 0.25 mM dithiothreitol. The load and wash are collected as one fraction. A phosphate elution is begun with 38 ml of 0.2 M phosphate, pH 8.0, 10 mM EDTA, 1.2% OG, and 0.25 mM dithiothreitol, and collected as one fraction. (The stock phosphate buffer contains sodium and potassium cations in a ratio of 1 : 2.) Then a linear phosphate gradient of 110 ml, 0.2-0.9 M, elutes the CDPdiacylglycerol synthase. The gradient solutions also contain 10 mM EDTA, 1.2% OG, and 0.25 mM dithiothreitol. Fractions are collected and analyzed for CDPdiacylglycerol synthase and total protein content. A typical run of this column is shown in Fig. 1. The latter half of the peak of CDPdiacylglycerol synthase activity is usually pure. In some preparations the early fractions are contaminated with a protein that migrates slightly slower than CDPdi-
[27]
60
CDPDIACYLGLYCEROL SYNTHASEFROM E. coil
.................... Column Load and Run-Through
- ......
241
1200
200raM =Phosphate Wash
'.2.. 40
800
'-
,
.~
~
e.
400
..:, ~_,_. 0 0
=1 60
', i --..----~ 120 180 Column Effluent, ml
800
--
E
400
.. ,,5
o n
t
~ 240
0
Jo
FIG. 1. Hydroxylapatite chromatography of CDPdiacylglycerolsynthase. The Zwittergent-eluted enzyme from the second DEAE column is chromatographed on hydroxylapatite as described in the text. Fractions are assayed for protein and CDPdiacylglycerolsynthase activity. The width of the plateaus for protein concentration indicate the volume of the fraction. The bar at the lower right indicates the fractions pooled. [Adapted from C. P. Sparrow and C. R. H. Raetz, J. Biol. Chem. 260, 12084 (1985).]
acylglycerol synthase on sodium dodecyl sulfate (SDS)-polyacrylamide gels.
Handling and Storage of Purified CDPdiacylglycerol Synthase The pooled peak fractions from the hydroxylapatite column can be concentrated by ultrafiltration on Amicon (Danvers, MA) PM10 membranes. This concentrates the OG as well as the enzyme. The pure e n z y m e can be dialyzed, but to maintain e n z y m e activity the dialyzing buffer must contain 1% OG, 0.25 m M dithiothreitol, and at least 200 m M salt (e.g., NaC1, potassium phosphate). Purified e n z y m e can be stored frozen for months if quick-frozen in liquid nitrogen. Activity will be lost if the e n z y m e is diluted into assay cocktail (phosphate buffer containing albumin) prior to freezing. The Zwittergent-eluted e n z y m e fraction from the second D E A E column loses all activity on freezing.
242
CYTIDYLYLTRANSFERASES
[28]
Characteristics of Purified CDPdiacylglycerol Synthase Pure CDPdiacylglycerol synthase has an apparent minimum subunit molecular mass of about 27,000 daltons as judged by SDS-polyacrylamide gel electrophoresis. 4 This is close to the mass of 27,570 daltons predicted from the gene sequence) The reaction mechanism of CDPdiacylglycerol synthase is sequential. 4 The enzyme does not hydrolyze CDPdiacylglycerol.4 The apparent kinetic constants are as follows: Km for phosphatidic acid, 0.28 mM; Km for dCTP, 0.58 mM; Vmax, 55 /xmol/min/mg.4 These constants may be somewhat dependent on assay conditions, because CDPdiacylglycerol synthase displays surface dilution kinetics. 4 CDPdiacylglycerol synthase will utilize either CTP or dCTP but no other nucleotide triphosphates.4 The enzyme is also specific for long-chain phosphatidic acid; 1-acyl-sn-glycero-3-phosphate is not a substrate, and phosphatidic acids with acyl chains shorter than 16 carbons are poor substrates. 4 The enzyme has a broad pH optimum between pH 7 and 8. CDPdiacylglycerol synthase can be reconstituted into phospholipid vesicles that retain activity. 4 The forward reaction of CDPdiacylglycerol synthase does not go to completion; under standard assay conditions, the equilibrium constant is about 0.2. 4 The reaction can be driven closer to completion in vitro by the addition of inorganic pyrophosphatase. For example, optimal conditions for synthesizing radioactive dCDPdiacylglycerol are 100 mM Tris, pH 7.5, 200 mM KC1, I mg/ml bovine serum albumin, 1 mM phosphatidic acid, 0.3% Triton X-100, l0 mM MgC12, 10/~M [ot-32p]dCTP, 150 units/ml pure CDPdiacylglycerol synthase, and 30 units/ml of inorganic pyrophosphatase. After 3 hr at 30°, up to 80% of the [a-aEp]dCTP will be converted to [a-32p]dCDPdiacylglycerol. The radioactive lipid product can be purified as previously described for unlabeled CDPdiacylglycerol.4
[28] C D P d i a c y l g l y c e r o l S y n t h a s e f r o m Y e a s t
By
GEORGE M. CARMAN and MICHAE L J. KE L L E Y
Introduction CDPdiacylglycerol synthase (CTP:phosphatidate cytidylyltransferase; EC 2.7.7.41) catalyzes the conversion of phosphatidate to CDPdiacylMETHODS IN ENZYMOLOGY,VOL. 209
Copyright © 1992by Academic Press, Inc. All rightsof reproductionin any form reserved.
CDPDIACYLGLYCEROL SYNTHASE FROM E. coli
237
[27] Purification of CDPdiacylglycerol Synthase from E s c h e r i c h i a coli B y CARL P . SPARROW
Introduction CDPdiacylglycerol synthase (CTP:phosphatidate cytidylyltransferase; EC 2.7.7.41, phosphatidate cytidylyltransferase) catalyzes the activation of phosphatidic acid by CTP to form CDPdiacylglycerol and inorganic pyrophosphate. This activity is essential for all phospholipid biosynthesis in Escherichia coli 1 and for the biosynthesis of acidic phospholipids in mammalian cells. 2 The enzymes of phospholipid synthesis are usually membrane bound and present in relatively low levels, making their purification difficult. The purification of E. coli CDPdiacylglycerol synthase is greatly aided by enzyme overproduction driven by an expression plasmid) Starting with cells that overproduce the enzyme 50-fold, essentially homogeneous enzyme can be obtained after chromatographic procedures that yield 160fold purification. 4 The enzyme can be partially purified from wild-type cells using the same procedures. Assay of CDPdiacylglycerol Synthase Activity The assay follows the conversion of [a-3Ep]dCTP to chloroform-soluble material, dependent on phosphatidic acid. The standard assay mixture contains 100 mM potassium phosphate, pH 7.4, 0.2% (w/v) Triton X-100, 1 mM phosphatidic acid, 1 mg/ml of bovine serum albumin, 10 mM MgC12, 5 mM [a-32p]dCTP ( - 2 Ci/mol) and 0.25 mM dithiothreitol. Stock solutions of each component are stored separately and then mixed together on the day of assay. To prevent precipitation of magnesium phosphatidate it is essential to mix the Triton X-100 and phosphatidic acid prior to addition of the magnesium chloride. The reaction is performed in 13 × 100 mm Pyrex tubes with Teflon-faced screw caps (Coming, Coming, NY). The reaction is initiated by adding l0/.d of enzyme source to 40 ~l of a mixture of the other assay components. t C. R. H. Raetz, Annu. Rev. Genet. 20, 253 (1986). 2 j. D. Esko and C. R. H. Raetz, in "The Enzymes" (P. D. Boyer, ed.), 3rd Ed., Vol., 16, p. 208. Academic Press, New York, 1983. 3 T. Icho, C. P. Sparrow, and C. R. H. Raetz, J. Biol. Chem. 260, 12078 0985). 4 C. P. Sparrow and C. R. H. Raetz, J. Biol. Chem. 260, 12084 (1985).
METHODSIN ENZYMOLOGY,VOL. 209
Copyright~ 1992by AcademicPress,Inc. All fightsof reproductionin any formreserved.
238
CYTIDYLYLTRANSFERASES
[2 7]
After 10 min at 30°, the reaction is stopped by adding 2 ml of chloroform-methanol (1 : 1, v/v). The tubes can be stored in this state indefinitely if they are tightly capped. The extraction is finished by adding 0.85 ml of 1 M NaCI, pH 2. This creates a two-phase system. 5 The samples are mixed well and spun briefly to separate the phases. The upper phase, the interface, and any interfacial precipitate is aspirated and discarded. One milliliter of the lower phase (which is 50% of the total lower phase) is transferred to a glass vial, the solvent is evaporated, and radioactivity is quantitated by liquid scintillation spectrometry. One unit of activity is defined as the amount of enzyme needed to convert 1 nmol of dCTP to chloroform-soluble material per minute. Under these conditions, the reaction is linear with time and enzyme up to about 5-10 nmol of product per 10 min. The reaction is linear to larger conversions with partially purified enzyme, presumably because there are fewer activities competing for the dCTP substrate. The most appropriate blank for the assay is a tube containing enzyme but no phosphatidic acid. Similar or identical blanks are obtained from tubes containing phosphatidic acid but no enzyme. The standard assay is sensitive to the molecular species of phosphatidic acid used. Dioleoylphosphatidic acid, palmitoyloleoylphosphatidic acid, or phosphatidic a c i d made from egg phosphatidylcholine give optimal results, whereas dipalmitoylphosphatidic acid gives rates less than 10% of optimum. 4 This fact is the cause of the disagreement in the literature concerning the level of CDPdiacylglycerol synthase activity in wild-type crude extracts. 4'6 Purification of CDPdiacylglycerol Synthase A summary of the purification of CDPdiacylglycerol synthase is given in Table I. The behavior of CDPdiacylglycerol synthase during column chromatography is influenced greatly by detergents and by the presence of EDTA. Although the physical basis of these effects is unknown, the detergents are useful tools for purifying the enzyme. Furthermore, CDPdiacylglycerol synthase is fairly stable in the presence of dithiothreitol, even at room temperature, and therefore the columns are run on the laboratory bench (20°-24°). After elution, column fractions are stored on ice. Tris buffers are prepared with chloride as the counterion. Concentrations in units of percent are all (w/v). Protein is assayed after precipitation as described by Peterson, 7 except that samples containing high concentra5 E. G. Bligh and J. J. Dyer, Can. J. Biochem. Physiol. 37, 911 (1959). 6 K. E. Langley and E. P. Kennedy, J. Bacteriol. 136, 85 (1978). 7 G. L. Peterson, Anal. Biochem. 83, 346 (1977).
CDPDIACYLGLYCEROL SYNTHASEFROME. coil
[27]
239
TABLE I PURIFICATION OF CDPDIACYLGLYCEROLSYNTHASEFROM Escherichia coil a
Step
Total protein (mg)
Specific activity (units/mg)
Yield (%)
Requirements for step b
Crude extract Membranes Solubilized membranes First DEAE column Second DEAE column Hydroxylapatite column
1656 676 589 288 10.3 2.2
192 466 346 607 9,102 30,786
(100) 99 64 55 30 21
(Plasmid) Mg2÷ EDTA, OG c EDTA Zwittergent 3-12 Pi gradient, OG
Adapted from C. P. Sparrow and C. R. H. Raetz, J. Biol. Chem. 260, 12084 (1985). b See text for detailed discussion of requirements. c OG, n-Octyl-/3-o-glucopyranoside. a
tions of Zwittergent cannot be precipitated. For these samples appropriate blanks must be assayed to subtract the signal generated by dithiothreitol. Growth of Cells and Preparation of Crude Extract. The strain DH1/ pCD100 overproduces CDPdiacylglycerol synthase about 50-fold. 3 This strain is grown in LB mediums and harvested in log phase. The cell paste (22 g) is resuspended in 9 volumes of 0.1 M potassium phosphate, pH 7.4, containing 10 mM magnesium sulfate, and the cells are broken by passage through a Mantlin-Gaulin press (9000 psi). The material is centrifuged at 5000 g for 15 min at 4° to remove unbroken cells. The supernatant is the crude extract. Membrane Preparation and Solubilization of Enzyme. The crude extract is ultracentrifuged at 180,000 g for 90 min at 4°, and the membrane pellet is resuspended in 30-50 ml of 10 mM Tris, pH 8.0, containing 0.25 mM dithiothreitol. Resuspension can be accomplished using either a Teflon-glass homogenizer or a needle and syringe. CDPdiacylglycerol synthase is solubilized by adjusting the membrane fraction to 6-7 membrane protein/ml in I0 mM Tris, pH 8.5, 10 mM EDTA, 0.25 mM dithiothreitol, 1.5% Triton X-100, 1.5% n-octyl-/3-D-glucopyranoside (OG), and 10% glycerol. This mixture is stirred for 1 hr on ice and then spun at 180,000 g for 60 min. The supernatant is the solubilized membrane fraction. DEAE-cellulose Column Chromatography. CDPdiacylglycerol synthase binds to DEAE-cellulose (DE-52, Whatman, Clifton, N J) in the absence of EDTA but not in the presence of EDTA. This property is exploited in the following purification. The solubilized membrane fraction 8 j. H. Miller, "Experiments in Molecular Genetics." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1972.
240
CYTIDYLYLTRANSFERASES
[2 7]
(-600 mg protein) is loaded onto a DE-52 column, 4 cm wide and 7 cm high, which is preequilibrated with 10 mM Tris, pH 8.0, 10 mM EDTA, 2.0% Triton X-100, 10% glycerol, and 0.25 mM dithiothreitol. The column is washed with the same buffer, at a flow rate of 8 ml/min. Because only the unretained material is desired, a few fractions of large volume are collected: typically, the first 50 ml can be discarded, and the next 120-150 ml contains the CDPdiacylglycerol synthase activity. This is the DEAE run-through fraction. This material can be stored frozen with no loss of activity. The run-through fraction is dialyzed at 4° against at least 20 volumes of buffer T, which is 10 mM Tris, pH 8.0, 1% Triton X-100, and 0.25 mM dithiothreitol. This material is loaded onto a second DE-52 column, 2.6 cm wide and 4 cm high, preequilibrated in buffer T. This column is run at 5 ml/min. After loading, the column is washed sequentially with I0 ml of buffer T, then 10 ml of buffer LT, which is 10 mM Tris, pH 8.0, 0.2% Triton X-100, and 0.25 mM dithiothreitol. Significant amounts of protein, but little CDPdiacylglycerol synthase, is then removed from the column by washing with 60 ml of buffer LT containing 70 mM KCI followed by 20 ml of buffer LT. The CDPdiacylglycerol synthase is then step-eluted with 63 ml of 10 mM Tris, pH 8.0, 4% Zwittergent 3-12 (Calbiochem, San Diego, CA), 25 mM KC1, and 0.25 mM dithiothreitol. The first 10-15 ml of eluant contains no activity and is discarded; the next 50 ml is collected in one fraction, called the Zwittergent enzyme preparation. Hydroxylapatite Column Chromatography. The Zwittergent enzyme preparation is diluted 1 : 1 with buffer T and loaded onto a hydroxylapatite column (Bio-Rad, Richmond, CA, BioSil A, 200-400 mesh). The column, 1.4 cm wide and 3.4 cm high, is preequilibrated with buffer T and run at 4 ml/min. After loading, the column is washed with 10 ml of buffer T, and then the detergent is changed by washing with 5 ml of 10 mM Tris, pH 8.0, 10 mM EDTA, 1.2% OG, and 0.25 mM dithiothreitol. The load and wash are collected as one fraction. A phosphate elution is begun with 38 ml of 0.2 M phosphate, pH 8.0, 10 mM EDTA, 1.2% OG, and 0.25 mM dithiothreitol, and collected as one fraction. (The stock phosphate buffer contains sodium and potassium cations in a ratio of 1 : 2.) Then a linear phosphate gradient of 110 ml, 0.2-0.9 M, elutes the CDPdiacylglycerol synthase. The gradient solutions also contain 10 mM EDTA, 1.2% OG, and 0.25 mM dithiothreitol. Fractions are collected and analyzed for CDPdiacylglycerol synthase and total protein content. A typical run of this column is shown in Fig. 1. The latter half of the peak of CDPdiacylglycerol synthase activity is usually pure. In some preparations the early fractions are contaminated with a protein that migrates slightly slower than CDPdi-
[27]
60
CDPDIACYLGLYCEROL SYNTHASEFROM E. coil
.................... Column Load and Run-Through
- ......
241
1200
200raM =Phosphate Wash
'.2.. 40
800
'-
,
.~
~
e.
400
..:, ~_,_. 0 0
=1 60
', i --..----~ 120 180 Column Effluent, ml
800
--
E
400
.. ,,5
o n
t
~ 240
0
Jo
FIG. 1. Hydroxylapatite chromatography of CDPdiacylglycerolsynthase. The Zwittergent-eluted enzyme from the second DEAE column is chromatographed on hydroxylapatite as described in the text. Fractions are assayed for protein and CDPdiacylglycerolsynthase activity. The width of the plateaus for protein concentration indicate the volume of the fraction. The bar at the lower right indicates the fractions pooled. [Adapted from C. P. Sparrow and C. R. H. Raetz, J. Biol. Chem. 260, 12084 (1985).]
acylglycerol synthase on sodium dodecyl sulfate (SDS)-polyacrylamide gels.
Handling and Storage of Purified CDPdiacylglycerol Synthase The pooled peak fractions from the hydroxylapatite column can be concentrated by ultrafiltration on Amicon (Danvers, MA) PM10 membranes. This concentrates the OG as well as the enzyme. The pure e n z y m e can be dialyzed, but to maintain e n z y m e activity the dialyzing buffer must contain 1% OG, 0.25 m M dithiothreitol, and at least 200 m M salt (e.g., NaC1, potassium phosphate). Purified e n z y m e can be stored frozen for months if quick-frozen in liquid nitrogen. Activity will be lost if the e n z y m e is diluted into assay cocktail (phosphate buffer containing albumin) prior to freezing. The Zwittergent-eluted e n z y m e fraction from the second D E A E column loses all activity on freezing.
242
CYTIDYLYLTRANSFERASES
[28]
Characteristics of Purified CDPdiacylglycerol Synthase Pure CDPdiacylglycerol synthase has an apparent minimum subunit molecular mass of about 27,000 daltons as judged by SDS-polyacrylamide gel electrophoresis. 4 This is close to the mass of 27,570 daltons predicted from the gene sequence) The reaction mechanism of CDPdiacylglycerol synthase is sequential. 4 The enzyme does not hydrolyze CDPdiacylglycerol.4 The apparent kinetic constants are as follows: Km for phosphatidic acid, 0.28 mM; Km for dCTP, 0.58 mM; Vmax, 55 /xmol/min/mg.4 These constants may be somewhat dependent on assay conditions, because CDPdiacylglycerol synthase displays surface dilution kinetics. 4 CDPdiacylglycerol synthase will utilize either CTP or dCTP but no other nucleotide triphosphates.4 The enzyme is also specific for long-chain phosphatidic acid; 1-acyl-sn-glycero-3-phosphate is not a substrate, and phosphatidic acids with acyl chains shorter than 16 carbons are poor substrates. 4 The enzyme has a broad pH optimum between pH 7 and 8. CDPdiacylglycerol synthase can be reconstituted into phospholipid vesicles that retain activity. 4 The forward reaction of CDPdiacylglycerol synthase does not go to completion; under standard assay conditions, the equilibrium constant is about 0.2. 4 The reaction can be driven closer to completion in vitro by the addition of inorganic pyrophosphatase. For example, optimal conditions for synthesizing radioactive dCDPdiacylglycerol are 100 mM Tris, pH 7.5, 200 mM KC1, I mg/ml bovine serum albumin, 1 mM phosphatidic acid, 0.3% Triton X-100, l0 mM MgC12, 10/~M [ot-32p]dCTP, 150 units/ml pure CDPdiacylglycerol synthase, and 30 units/ml of inorganic pyrophosphatase. After 3 hr at 30°, up to 80% of the [a-aEp]dCTP will be converted to [a-32p]dCDPdiacylglycerol. The radioactive lipid product can be purified as previously described for unlabeled CDPdiacylglycerol.4
[28] C D P d i a c y l g l y c e r o l S y n t h a s e f r o m Y e a s t
By
GEORGE M. CARMAN and MICHAE L J. KE L L E Y
Introduction CDPdiacylglycerol synthase (CTP:phosphatidate cytidylyltransferase; EC 2.7.7.41) catalyzes the conversion of phosphatidate to CDPdiacylMETHODS IN ENZYMOLOGY,VOL. 209
Copyright © 1992by Academic Press, Inc. All rightsof reproductionin any form reserved.
