- Email: [email protected]
[24] Phosphatidate phosphatase from yeast mitochondria
[24]
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[24] P h o s p h a t i d a t e P h o s p h a t a s e f r o m Y e a s t M i t o c h o n d r i a By GEORGE M. CARMAN and JENNIFER J. QUINLAN
Introduction Phosphatidate phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) catalyzes the conversion of phosphatidate to diacylglycerol) Phosphatidate ~ diacylglycerol + Pi
In the yeast Saccharomyces cerevisiae phosphatidate phosphatase is associated with the membrane and cytosolic fractions of the ceU. 2'3 The enzyme plays an important role in the biosynthesis of phospholipids and triacylglycerols in S. cerevisiae. 4 A 91-kDa form of phosphatidate phosphatase has been purified from the total membrane fraction5 and is described elsewhere in this series.6 Immunoblot analysis of cell extracts using antibodies specific for the 91-kDa form ofphosphatidate phosphatase revealed the existence of a 45-kDa form of the enzyme. 7 This immunoblot analysis also revealed that the 91-kDa enzyme is a proteolysis product of a 104kDa enzyme. 7 The mitochondrial fraction contains the 45-kDa enzyme, whereas the microsomal fraction contains the 45- and 104-kDa enzymes. 7 The 45-kDa phosphatidate phosphatase is induced in yeast cells by inositol supplementation, whereas the 104-kDa enzyme is not affected by inositol. 7 Both forms of the enzyme are induced when cells enter the stationary phase of growth. 7 The phosphatidate phosphatase 45-kDa enzyme has been purified from yeast mitochondria by a procedure similar to that used to purify the phosphatidate phosphatase 91-kDa enzyme from total membranes. 5,6 We describe here the purification and properties of the 45-kDa form of the enzyme.
i M. Kates, Can. J. Biochem. 35, 575 (1955). 2 K. Hosaka and S. Yamashita, Biochim. Biophys. Acta 796, 102 (1984). 3 K. R. Morlock, Y.-P. Lin, and G. M. Carman, J. Bacteriol. 170, 3561 (1988). 4 G. M. Carman and S. A. Henry, Annu. Rev. Biochem. 58, 635 (1989). 5 y . . p . Lin and G. M. Carman, J. Biol. Chem. 264, 864l (1989). 6 G. M. Carman and Y.-P. Lin, this series, Vol. 197, p. 548. 7 K. R. Morlock, J. J. McLaughlin, Y.-P. Lin, and G. M. Carman, J. Biol. Chem. 266, 3586 (1991).
METHODS IN ENZYMOLOGY, VOL. 209
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Preparation of Substrates [y-32p]Phosphatidate is synthesized enzymatically f r o m [y-32P]ATP and diacylglycerol using Escherichia coli diacylglycerol kinase (Lipidex, Inc., Westfield, NJ) under the assay conditions described by Walsh and Bell. 8 The labeled substrate is purified by thin-layer c h r o m a t o g r a p h y . 3
Assay Method Phosphatidate p h o s p h a t a s e activity is routinely m e a s u r e d by following the release of water-soluble [32p]pi f r o m the chloroform-soluble 0.5 m M [3,-32p]phosphatidate [1000-2000 counts/rain (cpm)/nmol] in 50 m M T r i s - m a l e a t e buffer (pH 7.0) containing 5 m M Triton X-100, l0 m M 2-mercaptoethanol, 2 m M MgCl2, and e n z y m e protein in a total volume of 0.1 ml at 300. 3'6 One unit of enzymatic activity is defined as the amount of e n z y m e that catalyzes the formation of 1 nmol of product per minute. Protein is determined by the method of Bradford. 9 Growth of Yeast S a c c h a r o m y c e s cerevisiae strain ade5 M A T a l° is used as a representative wild-type strain Ira2 for e n z y m e purification. Cells are grown in I % yeast extract, 2% peptone, and 2% glucose (w/v) at 28 ° to late exponential phase, h a r v e s t e d b y centrifugation, and stored at - 8 0 ° as d e s c r i b e d J TM We have not been able to purify phosphatidate p h o s p h a t a s e f r o m the wildtype strain $288C, which has b e e n used to purify other yeast phospholipid biosynthetic e n z y m e s J 3-18
Purification Procedure All steps are p e r f o r m e d at 5 ° . Step I: Preparation o f Cell Extract. Cells (180 g) are disrupted with glass beads with a Bead-Beater (BioSpec Products, Bartlesville, O K ) in
8 j. p. Walsh and R. M. Bell, J. Biol. Chem. 2,61, 6239 (1986). 9 M. M. Bradford, Anal. Biochem. 72, 248 (1976). l0 M. R. Culbertson and S. A. Henry, Genetics 80, 23 (1975). II L. S. Klig, M. J. Homann, G. M. Carman, and S. A. Henry, J. Bacteriol. 162, 1135 (1985). 12M. L. Greenberg, L. S. Klig, V. A. Letts, B. S. Loewy, and S. A. Henry, J. Bacteriol. 153, 791 (1983). 13A. S. Fischl and G. M. Carman, J. Bacteriol. 154, 304 (1983). 14G. M. Carman and A. S. Fisehl, this volume [36]. 15M. Bae-Lee and G. M. Carman, J. Biol. Chem. 259, 10857 (1984). 16G. M. Carman and M. Bae-Lee, this volume [35]. 17M. J. Kelley and G. M. Carman, J. Biol. Chem. 262, 14563 (1987). ,s G. M. Carman and M. J. Kelley, this volume [28].
[24]
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50 mM Tris-maleate buffer (pH 7.0) containing 1 mM Na2EDTA, 0.3 M sucrose, and l0 mM 2-mercaptoethanol as described previously. 13.14Glass beads and unbroken cells are removed by centrifugation at 1500 g for 15 min to obtain the cell extract. Step 2: Preparation of Mitochondrial Fraction. Crude mitochondria are collected from the cell extract by centrifugation at 32,000 g for l0 min. Mitochondrial pellets are washed with 50 mM Tris-maleate buffer (pH 7.0) containing l0 mM MgCI2, l0 mM 2-mercaptoethanol, and 20% glycerol. Mitochondria are routinely frozen at - 8 0 ° until the enzyme is purified. Step 3: Preparation of Sodium Cholate Extract. Mitochondria are suspended in 50 mM Tris-maleate buffer (pH 7.0) containing 10 mM MgC12, l0 mM 2-mercaptoethanol, 20% glycerol, and 1% sodium cholate at a final protein concentration of 10 mg/ml. The suspension is incubated for 1 hr on a rotary shaker at 150 rpm. After the incubation, the suspension is centrifuged at 100,000 g for 90 min to obtain the sodium cholate extract. Step 4:DE-53 Chromatography. A DE-53 (Whatman, Clifton, N J) column (1.5 × 5.6 cm) is equilibrated with 5 column volumes of 50 mM Tris-maleate buffer (pH 7.0) containing 10 mM MgC12, l0 mM 2-mercaptoethanol, and 20% glycerol followed by 1 column volume of the same buffer containing 1% sodium cholate (v/v). The enzyme is applied to the column followed by washing of the column with 4 column volumes of equilibration buffer containing 1% sodium cholate. The enzyme is eluted from the column with 10 column volumes of a linear NaCl gradient (0-0.3 M) in the same buffer. The peak of phosphatidate phosphatase activity elutes from the column at a NaC1 concentration of about 0.1 M. Step 5: Affi-Gel Blue Chromatography. An Affi-Gel Blue (Bio-Rad, Richmond, CA) column (1.0 × 6 cm) is equilibrated with 5 column volumes of 50 mM Tris-maleate buffer (pH 7.0) containing l0 mM MgCl2, 10 mM 2-mercaptoethanol, 20% glycerol, and 0.1 M NaCl followed by equilibration with 1 column volume of the same buffer containing 1% sodium cholate. The enzyme preparation from the previous step is applied to the column, which is then washed with 3.5 column volumes of equilibration buffer containing 0.3 M NaC1 and 1% sodium cholate. Phosphatidate phosphatase is then eluted from the column with 9 column volumes of a linear NaCl gradient (0.3-1.0 M) in the same buffer at a flow rate of 30 ml/hr. The peak of phosphatidate phosphatase activity elutes from the column at a NaC1 concentration of about 0.55 M. The enzyme preparation is desalted by dialysis against equilibration buffer. Step 6: Hydroxylapatite Chromatography. A hydroxylapatite (BioGel HT, Bio-Rad) column (1.5 × 2.3 cm) is equilibrated with 10 mM potassium phosphate buffer (pH 7.0) containing 5 mM MgCI2, 10 mM 2-mercaptoethanol, 20% glycerol, and I% sodium cholate. Dialyzed enzyme from the
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previous step is applied to the column. The column is washed with 2 column volumes of equilibration buffer followed by elution of phosphatidate phosphatase with 8 column volumes of a linear potassium phosphate gradient (10-150 mM) in equilibration buffer. The concentration of MgC12 is increased to 10 mM in the elution buffer. The peak of activity elutes from the column at a potassium phosphate concentration of about 95 mM. The most active fractions are pooled and used for the next step in the purification. Step 7: Mono Q Chromatography. An anion-exchange Mono Q (Pharmacia LKB Biotechnology, Piscataway, NJ) column (0.5 × 5 crn) is equilibrated with 6 column volumes of 50 mM Tris-maleate buffer (pH 7.0) containing 10 mM MgCI2, 10 mM 2-mercaptoethanol, 20% glycerol, 1% sodium cholate, and 0.1 M NaC1. The hydroxylapatite-purified enzyme is applied to the column. The column is washed with 8 column volumes of equilibration buffer followed by 2 column volumes of a linear NaC1 gradient (0.1-0.17 M) in equilibration buffer. The enzyme is then eluted from the column with 10 column volumes of a linear NaCI gradient (0.17-0.4 M) in equilibration buffer. The peak of enzyme activity elutes at a NaCI concentration of about 0.2 M. Phosphatidate phosphatase activity is completely stable for at least 3 months of storage at - 8 0 °. Enzyme Purity. A summary of the purification of the phosphatidate phosphatase 45-kDa enzyme is presented in Table I. The enzyme is purified 800-fold to a final specific activity of 2400 nmol/min/mg with an activity yield of 0.2%. The -fold purification and final specific activity of the 45kDa form of phosphatidate phosphatase are considerably lower when
TABLE I PURIFICATION OF PHOSPHATIDATE PHOSPHATASEFROM MITOCHONDRIAL FRACTION OF S a c c h a r o m y c e s cereoisiae a
Purification step
Total units (nmol/min)
1. Cell extract 2. Mitochondria 3. Sodium cholate extract 4. DE-53 5. Affi-Gel Blue 6. Hydroxylapatite 7. Mono Q
30,000 6,000 4,200 2,610 1,200 480 60
Data from Morlock et al. 7
Protein (mg) 10,000 909 350 52 4 1 0.025
Specific activity (units/rag) 3 6.6 12 50 300 480 2400
Purification (-fold) 1 2.2 4 16.6 100 160 800
Yield (%) 100 20 14 8.7 4 1.6 0.2
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compared to the 91-kDa form of the e n z y m e : '6 This is a reflection of the low activity yield of the 45-kDa enzyme during each step of the purification. It is not necessary to use the final Superose 12 chromatography step 5,6to obtain a nearly homogeneous protein preparation. Phosphatidate phosphatase activity is associated with the 45-kDa protein on sodium dodecyl sulfate-polyacrylamide gels. 7 Phosphatidate phosphatase 45-kDa enzyme is present during the purification of the phosphatidate phosphatase 91-kDa enzyme purified from total membranes. The 45-kDa enzyme is removed from the 91-kDa enzyme preparation during the final Superose 12 chromatography step. 5
Properties of Phosphatidate Phosphatase Maximum activity is observed between pH 6 and 7. The enzyme is dependent on magnesium ions, with maximum activity at 1 mM. The requirement for magnesium ions cannot be substituted by manganese, cobalt, or calcium ions. Maximal phosphatidate phosphatase activity is also dependent on the addition of 5 mM Triton X- 100 (molar ratio of Triton X-100 to phosphatidate of 10: 1). Phosphatidate phosphatase is unstable at temperatures above 30°, with total inactivation occurring after heating for 20 min at 50 °. The enzyme is inhibited by p-chloromercuriphenylsulfonic acid (1 mM), N-ethylmaleimide (1 mM), phenylglyoxal (IC50 4.3 mM), and propranolol (IC50 0.95 mM). The kinetic properties of the phosphatidate phosphatase 45-kDa enzyme have been examined with uniform mixed micelles containing Triton X-100 and phosphatidate. 7 Phosphatidate phosphatase displays saturation kinetics with respect to the bulk and surface concentrations of phosphatid a t e . 7 At a surface concentration of phosphatidate of 9 mol %, the K m value for the bulk concentration of phosphatidate is 94/zM. At a bulk concentration of phosphatidate of 0.5 mM, the Km value for the surface concentration of phosphatidate is 2.9 mol %. These results are consistent with the phosphatidate phosphatase 45-kDa enzyme following "surface dilution" kinetics) 9 The properties of the purified phosphatidate phosphatase 45-kDa enzyme are similar to those of the 91-kDa enzyme5'7'2° with respect to pH optimum, cofactor requirement, kinetic properties using Triton X-100phosphatidate mixed micelles, temperature stability, and inhibition to various compounds. However, the 45- and 91-kDa forms of phosphatidate 19 R. A. Deems, B. R. Eaton, and E. A. Dennis, J. Biol. Chem. 250, 9013 (1975). 2o y._p. Lin and G. M. Carman, J. Biol. Chem. 265, 166 (1990).
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phosphatase differ with respect to their isoelectric points and peptide fragments resulting from V8 proteolysis and cyanogen bromide cleavage. 7
Acknowledgments This work was supported by U.S. Public Health Service Grant GM-28140 from the National Institutesof Health, N e w Jersey State funds, and the Charles and Johanna Busch Memorial Fund.
[25] P h o s p h a t i d y l g l y c e r o p h o s p h a t e P h o s p h a t a s e f r o m Escherichia coli By WILLIAM DOWHAN and CINDEE R. FUNK
Introduction Phosphatidylglycerophosphate --->phosphatidylglycerol + Pi Phosphatidic acid ~ diacylglycerol + Pi Lysophosphatidic acid---> 1-acyl-sn-glycerol + Pi
(1) (2) (3)
Chang and Kennedy I reported a membrane-associated phosphatidylglycerophosphate phosphatase activity [reaction (1)] in Escherichia coli that was distinct from other phosphatases with specificities limited to water-soluble substrates. Icho and Raetz 2 determined that reaction (1) in crude extracts of E. coli was dependent on at least two gene products. The pgpA gene (mapping at 10 min) product (PGP-A) only catalyzes reaction (1), whereas the pgpB gene (mapping at 28 min) product (PGP-B) catalyzes all three reactions. Neither of these genes appears to encode the biosynthetic phosphatidylglycerophosphate phosphatase activity since strains with both genes inactivated by gene interruption 3 still synthesize near-normal in vivo levels of phosphatidylglycerol. Cell lysates made from double mutants still have about 50% of the normal levels of activity catalyzing reaction (1) when assayed at 30°; this residual activity (PGP-C) went unrecognized because, unlike the activity contributed by PGP-A and PGPB, it is both temperature- and detergent-sensitive) These extracts also contain significant levels of activity [reaction (3)] which is independent of the PGP-C activity, suggesting that a second lysophosphatidic acid I y._y. Chang and E. P. Kennedy, J. Lipid Res. 8, 456 (1967). 2 T. Icho and C. R. H. Raetz, J. Bacteriol. 153, 722 (1983). a C. R. Funk and W. Dowhan, J. Bacteriol. 174, in press (1992).
METHODS IN ENZYMOLOGY, VOL. 209
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
PHOSPHATIDATE
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[24] P h o s p h a t i d a t e P h o s p h a t a s e f r o m Y e a s t M i t o c h o n d r i a By GEORGE M. CARMAN and JENNIFER J. QUINLAN
Introduction Phosphatidate phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) catalyzes the conversion of phosphatidate to diacylglycerol) Phosphatidate ~ diacylglycerol + Pi
In the yeast Saccharomyces cerevisiae phosphatidate phosphatase is associated with the membrane and cytosolic fractions of the ceU. 2'3 The enzyme plays an important role in the biosynthesis of phospholipids and triacylglycerols in S. cerevisiae. 4 A 91-kDa form of phosphatidate phosphatase has been purified from the total membrane fraction5 and is described elsewhere in this series.6 Immunoblot analysis of cell extracts using antibodies specific for the 91-kDa form ofphosphatidate phosphatase revealed the existence of a 45-kDa form of the enzyme. 7 This immunoblot analysis also revealed that the 91-kDa enzyme is a proteolysis product of a 104kDa enzyme. 7 The mitochondrial fraction contains the 45-kDa enzyme, whereas the microsomal fraction contains the 45- and 104-kDa enzymes. 7 The 45-kDa phosphatidate phosphatase is induced in yeast cells by inositol supplementation, whereas the 104-kDa enzyme is not affected by inositol. 7 Both forms of the enzyme are induced when cells enter the stationary phase of growth. 7 The phosphatidate phosphatase 45-kDa enzyme has been purified from yeast mitochondria by a procedure similar to that used to purify the phosphatidate phosphatase 91-kDa enzyme from total membranes. 5,6 We describe here the purification and properties of the 45-kDa form of the enzyme.
i M. Kates, Can. J. Biochem. 35, 575 (1955). 2 K. Hosaka and S. Yamashita, Biochim. Biophys. Acta 796, 102 (1984). 3 K. R. Morlock, Y.-P. Lin, and G. M. Carman, J. Bacteriol. 170, 3561 (1988). 4 G. M. Carman and S. A. Henry, Annu. Rev. Biochem. 58, 635 (1989). 5 y . . p . Lin and G. M. Carman, J. Biol. Chem. 264, 864l (1989). 6 G. M. Carman and Y.-P. Lin, this series, Vol. 197, p. 548. 7 K. R. Morlock, J. J. McLaughlin, Y.-P. Lin, and G. M. Carman, J. Biol. Chem. 266, 3586 (1991).
METHODS IN ENZYMOLOGY, VOL. 209
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Preparation of Substrates [y-32p]Phosphatidate is synthesized enzymatically f r o m [y-32P]ATP and diacylglycerol using Escherichia coli diacylglycerol kinase (Lipidex, Inc., Westfield, NJ) under the assay conditions described by Walsh and Bell. 8 The labeled substrate is purified by thin-layer c h r o m a t o g r a p h y . 3
Assay Method Phosphatidate p h o s p h a t a s e activity is routinely m e a s u r e d by following the release of water-soluble [32p]pi f r o m the chloroform-soluble 0.5 m M [3,-32p]phosphatidate [1000-2000 counts/rain (cpm)/nmol] in 50 m M T r i s - m a l e a t e buffer (pH 7.0) containing 5 m M Triton X-100, l0 m M 2-mercaptoethanol, 2 m M MgCl2, and e n z y m e protein in a total volume of 0.1 ml at 300. 3'6 One unit of enzymatic activity is defined as the amount of e n z y m e that catalyzes the formation of 1 nmol of product per minute. Protein is determined by the method of Bradford. 9 Growth of Yeast S a c c h a r o m y c e s cerevisiae strain ade5 M A T a l° is used as a representative wild-type strain Ira2 for e n z y m e purification. Cells are grown in I % yeast extract, 2% peptone, and 2% glucose (w/v) at 28 ° to late exponential phase, h a r v e s t e d b y centrifugation, and stored at - 8 0 ° as d e s c r i b e d J TM We have not been able to purify phosphatidate p h o s p h a t a s e f r o m the wildtype strain $288C, which has b e e n used to purify other yeast phospholipid biosynthetic e n z y m e s J 3-18
Purification Procedure All steps are p e r f o r m e d at 5 ° . Step I: Preparation o f Cell Extract. Cells (180 g) are disrupted with glass beads with a Bead-Beater (BioSpec Products, Bartlesville, O K ) in
8 j. p. Walsh and R. M. Bell, J. Biol. Chem. 2,61, 6239 (1986). 9 M. M. Bradford, Anal. Biochem. 72, 248 (1976). l0 M. R. Culbertson and S. A. Henry, Genetics 80, 23 (1975). II L. S. Klig, M. J. Homann, G. M. Carman, and S. A. Henry, J. Bacteriol. 162, 1135 (1985). 12M. L. Greenberg, L. S. Klig, V. A. Letts, B. S. Loewy, and S. A. Henry, J. Bacteriol. 153, 791 (1983). 13A. S. Fischl and G. M. Carman, J. Bacteriol. 154, 304 (1983). 14G. M. Carman and A. S. Fisehl, this volume [36]. 15M. Bae-Lee and G. M. Carman, J. Biol. Chem. 259, 10857 (1984). 16G. M. Carman and M. Bae-Lee, this volume [35]. 17M. J. Kelley and G. M. Carman, J. Biol. Chem. 262, 14563 (1987). ,s G. M. Carman and M. J. Kelley, this volume [28].
[24]
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50 mM Tris-maleate buffer (pH 7.0) containing 1 mM Na2EDTA, 0.3 M sucrose, and l0 mM 2-mercaptoethanol as described previously. 13.14Glass beads and unbroken cells are removed by centrifugation at 1500 g for 15 min to obtain the cell extract. Step 2: Preparation of Mitochondrial Fraction. Crude mitochondria are collected from the cell extract by centrifugation at 32,000 g for l0 min. Mitochondrial pellets are washed with 50 mM Tris-maleate buffer (pH 7.0) containing l0 mM MgCI2, l0 mM 2-mercaptoethanol, and 20% glycerol. Mitochondria are routinely frozen at - 8 0 ° until the enzyme is purified. Step 3: Preparation of Sodium Cholate Extract. Mitochondria are suspended in 50 mM Tris-maleate buffer (pH 7.0) containing 10 mM MgC12, l0 mM 2-mercaptoethanol, 20% glycerol, and 1% sodium cholate at a final protein concentration of 10 mg/ml. The suspension is incubated for 1 hr on a rotary shaker at 150 rpm. After the incubation, the suspension is centrifuged at 100,000 g for 90 min to obtain the sodium cholate extract. Step 4:DE-53 Chromatography. A DE-53 (Whatman, Clifton, N J) column (1.5 × 5.6 cm) is equilibrated with 5 column volumes of 50 mM Tris-maleate buffer (pH 7.0) containing 10 mM MgC12, l0 mM 2-mercaptoethanol, and 20% glycerol followed by 1 column volume of the same buffer containing 1% sodium cholate (v/v). The enzyme is applied to the column followed by washing of the column with 4 column volumes of equilibration buffer containing 1% sodium cholate. The enzyme is eluted from the column with 10 column volumes of a linear NaCl gradient (0-0.3 M) in the same buffer. The peak of phosphatidate phosphatase activity elutes from the column at a NaC1 concentration of about 0.1 M. Step 5: Affi-Gel Blue Chromatography. An Affi-Gel Blue (Bio-Rad, Richmond, CA) column (1.0 × 6 cm) is equilibrated with 5 column volumes of 50 mM Tris-maleate buffer (pH 7.0) containing l0 mM MgCl2, 10 mM 2-mercaptoethanol, 20% glycerol, and 0.1 M NaCl followed by equilibration with 1 column volume of the same buffer containing 1% sodium cholate. The enzyme preparation from the previous step is applied to the column, which is then washed with 3.5 column volumes of equilibration buffer containing 0.3 M NaC1 and 1% sodium cholate. Phosphatidate phosphatase is then eluted from the column with 9 column volumes of a linear NaCl gradient (0.3-1.0 M) in the same buffer at a flow rate of 30 ml/hr. The peak of phosphatidate phosphatase activity elutes from the column at a NaC1 concentration of about 0.55 M. The enzyme preparation is desalted by dialysis against equilibration buffer. Step 6: Hydroxylapatite Chromatography. A hydroxylapatite (BioGel HT, Bio-Rad) column (1.5 × 2.3 cm) is equilibrated with 10 mM potassium phosphate buffer (pH 7.0) containing 5 mM MgCI2, 10 mM 2-mercaptoethanol, 20% glycerol, and I% sodium cholate. Dialyzed enzyme from the
222
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previous step is applied to the column. The column is washed with 2 column volumes of equilibration buffer followed by elution of phosphatidate phosphatase with 8 column volumes of a linear potassium phosphate gradient (10-150 mM) in equilibration buffer. The concentration of MgC12 is increased to 10 mM in the elution buffer. The peak of activity elutes from the column at a potassium phosphate concentration of about 95 mM. The most active fractions are pooled and used for the next step in the purification. Step 7: Mono Q Chromatography. An anion-exchange Mono Q (Pharmacia LKB Biotechnology, Piscataway, NJ) column (0.5 × 5 crn) is equilibrated with 6 column volumes of 50 mM Tris-maleate buffer (pH 7.0) containing 10 mM MgCI2, 10 mM 2-mercaptoethanol, 20% glycerol, 1% sodium cholate, and 0.1 M NaC1. The hydroxylapatite-purified enzyme is applied to the column. The column is washed with 8 column volumes of equilibration buffer followed by 2 column volumes of a linear NaC1 gradient (0.1-0.17 M) in equilibration buffer. The enzyme is then eluted from the column with 10 column volumes of a linear NaCI gradient (0.17-0.4 M) in equilibration buffer. The peak of enzyme activity elutes at a NaCI concentration of about 0.2 M. Phosphatidate phosphatase activity is completely stable for at least 3 months of storage at - 8 0 °. Enzyme Purity. A summary of the purification of the phosphatidate phosphatase 45-kDa enzyme is presented in Table I. The enzyme is purified 800-fold to a final specific activity of 2400 nmol/min/mg with an activity yield of 0.2%. The -fold purification and final specific activity of the 45kDa form of phosphatidate phosphatase are considerably lower when
TABLE I PURIFICATION OF PHOSPHATIDATE PHOSPHATASEFROM MITOCHONDRIAL FRACTION OF S a c c h a r o m y c e s cereoisiae a
Purification step
Total units (nmol/min)
1. Cell extract 2. Mitochondria 3. Sodium cholate extract 4. DE-53 5. Affi-Gel Blue 6. Hydroxylapatite 7. Mono Q
30,000 6,000 4,200 2,610 1,200 480 60
Data from Morlock et al. 7
Protein (mg) 10,000 909 350 52 4 1 0.025
Specific activity (units/rag) 3 6.6 12 50 300 480 2400
Purification (-fold) 1 2.2 4 16.6 100 160 800
Yield (%) 100 20 14 8.7 4 1.6 0.2
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compared to the 91-kDa form of the e n z y m e : '6 This is a reflection of the low activity yield of the 45-kDa enzyme during each step of the purification. It is not necessary to use the final Superose 12 chromatography step 5,6to obtain a nearly homogeneous protein preparation. Phosphatidate phosphatase activity is associated with the 45-kDa protein on sodium dodecyl sulfate-polyacrylamide gels. 7 Phosphatidate phosphatase 45-kDa enzyme is present during the purification of the phosphatidate phosphatase 91-kDa enzyme purified from total membranes. The 45-kDa enzyme is removed from the 91-kDa enzyme preparation during the final Superose 12 chromatography step. 5
Properties of Phosphatidate Phosphatase Maximum activity is observed between pH 6 and 7. The enzyme is dependent on magnesium ions, with maximum activity at 1 mM. The requirement for magnesium ions cannot be substituted by manganese, cobalt, or calcium ions. Maximal phosphatidate phosphatase activity is also dependent on the addition of 5 mM Triton X- 100 (molar ratio of Triton X-100 to phosphatidate of 10: 1). Phosphatidate phosphatase is unstable at temperatures above 30°, with total inactivation occurring after heating for 20 min at 50 °. The enzyme is inhibited by p-chloromercuriphenylsulfonic acid (1 mM), N-ethylmaleimide (1 mM), phenylglyoxal (IC50 4.3 mM), and propranolol (IC50 0.95 mM). The kinetic properties of the phosphatidate phosphatase 45-kDa enzyme have been examined with uniform mixed micelles containing Triton X-100 and phosphatidate. 7 Phosphatidate phosphatase displays saturation kinetics with respect to the bulk and surface concentrations of phosphatid a t e . 7 At a surface concentration of phosphatidate of 9 mol %, the K m value for the bulk concentration of phosphatidate is 94/zM. At a bulk concentration of phosphatidate of 0.5 mM, the Km value for the surface concentration of phosphatidate is 2.9 mol %. These results are consistent with the phosphatidate phosphatase 45-kDa enzyme following "surface dilution" kinetics) 9 The properties of the purified phosphatidate phosphatase 45-kDa enzyme are similar to those of the 91-kDa enzyme5'7'2° with respect to pH optimum, cofactor requirement, kinetic properties using Triton X-100phosphatidate mixed micelles, temperature stability, and inhibition to various compounds. However, the 45- and 91-kDa forms of phosphatidate 19 R. A. Deems, B. R. Eaton, and E. A. Dennis, J. Biol. Chem. 250, 9013 (1975). 2o y._p. Lin and G. M. Carman, J. Biol. Chem. 265, 166 (1990).
224
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[25]
phosphatase differ with respect to their isoelectric points and peptide fragments resulting from V8 proteolysis and cyanogen bromide cleavage. 7
Acknowledgments This work was supported by U.S. Public Health Service Grant GM-28140 from the National Institutesof Health, N e w Jersey State funds, and the Charles and Johanna Busch Memorial Fund.
[25] P h o s p h a t i d y l g l y c e r o p h o s p h a t e P h o s p h a t a s e f r o m Escherichia coli By WILLIAM DOWHAN and CINDEE R. FUNK
Introduction Phosphatidylglycerophosphate --->phosphatidylglycerol + Pi Phosphatidic acid ~ diacylglycerol + Pi Lysophosphatidic acid---> 1-acyl-sn-glycerol + Pi
(1) (2) (3)
Chang and Kennedy I reported a membrane-associated phosphatidylglycerophosphate phosphatase activity [reaction (1)] in Escherichia coli that was distinct from other phosphatases with specificities limited to water-soluble substrates. Icho and Raetz 2 determined that reaction (1) in crude extracts of E. coli was dependent on at least two gene products. The pgpA gene (mapping at 10 min) product (PGP-A) only catalyzes reaction (1), whereas the pgpB gene (mapping at 28 min) product (PGP-B) catalyzes all three reactions. Neither of these genes appears to encode the biosynthetic phosphatidylglycerophosphate phosphatase activity since strains with both genes inactivated by gene interruption 3 still synthesize near-normal in vivo levels of phosphatidylglycerol. Cell lysates made from double mutants still have about 50% of the normal levels of activity catalyzing reaction (1) when assayed at 30°; this residual activity (PGP-C) went unrecognized because, unlike the activity contributed by PGP-A and PGPB, it is both temperature- and detergent-sensitive) These extracts also contain significant levels of activity [reaction (3)] which is independent of the PGP-C activity, suggesting that a second lysophosphatidic acid I y._y. Chang and E. P. Kennedy, J. Lipid Res. 8, 456 (1967). 2 T. Icho and C. R. H. Raetz, J. Bacteriol. 153, 722 (1983). a C. R. Funk and W. Dowhan, J. Bacteriol. 174, in press (1992).
METHODS IN ENZYMOLOGY, VOL. 209
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