Purification and Characterization of Diacylglycerol Pyrophosphate Phosphatase from Saccharomyces cerevisiae
1996; Elsevier BV; Volume: 271; Issue: 4 Linguagem: Inglês
10.1074/jbc.271.4.1868
ISSN1083-351X
AutoresWen‐I Wu, Yongsheng Liu, Bettina Riedel, Josef Wissing, Anthony S. Fischl, George Carman,
Tópico(s)Alkaline Phosphatase Research Studies
ResumoDiacylglycerol pyrophosphate (DGPP) phosphatase is a novel membrane-associated enzyme that catalyzes the dephosphorylation of the β phosphate of DGPP to yield phosphatidate and Pi. DGPP phosphatase was purified 33,333-fold from Saccharomyces cerevisiae by a procedure that included Triton X-100 solubilization of microsomal membranes followed by chromatography with DE53, Affi-Gel Blue, hydroxylapatite, and Mono Q. The procedure resulted in the isolation of an apparent homogeneous protein with a subunit molecular mass of 34 kDa. DGPP phosphatase activity was associated with the 34-kDa protein. DGPP phosphatase had a broad pH optimum between 6.0 and 8.5 and was dependent on Triton X-100 for maximum activity. The enzyme was inhibited by divalent cations, NaF, and pyrophosphate and was relatively insensitive to thioreactive agents. The turnover number (molecular activity) for the enzyme was 5.8 × 103 min-1 at pH 6.5 and 30°C. DGPP phosphatase exhibited typical saturation kinetics with respect to DGPP (Km = 0.55 mol %). The Kmvalue for DGPP was 3-fold greater than its cellular concentration (0.18 mol %). DGPP phosphatase also catalyzed the dephosphorylation of phosphatidate, but this dephosphorylation was subsequent to the dephosphorylation of the β phosphate of DGPP. The dependence of activity on phosphatidate (Km = 2.2 mol %) was cooperative (Hill number = 2.0). DGPP was the preferred substrate for the enzyme with a specificity constant (VMAX /Km) 10-fold greater than that for phosphatidate. In addition, DGPP potently inhibited (Ki = 0.35 mol %) the dephosphorylation of phosphatidate by a competitive mechanism whereas phosphatidate did not inhibit the dephosphorylation of DGPP. DGPP was neither a substrate nor an inhibitor of pure phosphatidate phosphatase from S. cerevisiae. DGPP was synthesized from phosphatidate via the phosphatidate kinase reaction. Diacylglycerol pyrophosphate (DGPP) phosphatase is a novel membrane-associated enzyme that catalyzes the dephosphorylation of the β phosphate of DGPP to yield phosphatidate and Pi. DGPP phosphatase was purified 33,333-fold from Saccharomyces cerevisiae by a procedure that included Triton X-100 solubilization of microsomal membranes followed by chromatography with DE53, Affi-Gel Blue, hydroxylapatite, and Mono Q. The procedure resulted in the isolation of an apparent homogeneous protein with a subunit molecular mass of 34 kDa. DGPP phosphatase activity was associated with the 34-kDa protein. DGPP phosphatase had a broad pH optimum between 6.0 and 8.5 and was dependent on Triton X-100 for maximum activity. The enzyme was inhibited by divalent cations, NaF, and pyrophosphate and was relatively insensitive to thioreactive agents. The turnover number (molecular activity) for the enzyme was 5.8 × 103 min-1 at pH 6.5 and 30°C. DGPP phosphatase exhibited typical saturation kinetics with respect to DGPP (Km = 0.55 mol %). The Kmvalue for DGPP was 3-fold greater than its cellular concentration (0.18 mol %). DGPP phosphatase also catalyzed the dephosphorylation of phosphatidate, but this dephosphorylation was subsequent to the dephosphorylation of the β phosphate of DGPP. The dependence of activity on phosphatidate (Km = 2.2 mol %) was cooperative (Hill number = 2.0). DGPP was the preferred substrate for the enzyme with a specificity constant (VMAX /Km) 10-fold greater than that for phosphatidate. In addition, DGPP potently inhibited (Ki = 0.35 mol %) the dephosphorylation of phosphatidate by a competitive mechanism whereas phosphatidate did not inhibit the dephosphorylation of DGPP. DGPP was neither a substrate nor an inhibitor of pure phosphatidate phosphatase from S. cerevisiae. DGPP was synthesized from phosphatidate via the phosphatidate kinase reaction. INTRODUCTIONDiacylglycerol pyrophosphate (DGPP) 1The abbreviations used are: DGPPdiacylglycerol pyrophosphatePAphosphatidate. is a novel phospholipid metabolite recently identified from the plant Catharanthus roseus by Wissing and Behrbohm(1.Wissing J.B. Behrbohm H. FEBS Lett. 1993; 315: 95-99Google Scholar). DGPP contains a pyrophosphate group attached to diacylglycerol (Fig. 1). This compound was previously observed by several workers (2.Heim S. Bauleke A. Wylegalla C. Wagner K.G. Plant Sci. 1987; 49: 159-165Google Scholar, 3.Sommarin M. Sandelius A.S. Biochim. Biophys. Acta. 1988; 958: 268-278Google Scholar, 4.Memon A.R. Boss W.F. J. Biol. Chem. 1990; 265: 14817-14821Google Scholar) as the phospholipid product of a lipid kinase reaction in plants that was not identified correctly(1.Wissing J.B. Behrbohm H. FEBS Lett. 1993; 315: 95-99Google Scholar). It is now known that DGPP is synthesized from PA and ATP through the reaction catalyzed by the novel enzyme PA kinase(1.Wissing J.B. Behrbohm H. FEBS Lett. 1993; 315: 95-99Google Scholar). PA kinase is a ubiquitous membrane-associated enzyme found in the plant kingdom(5.Wissing J.B. Behrbohm H. Plant Physiol. 1993; 102: 1243-1249Google Scholar). The enzyme has been purified from suspension-cultured C. roseus cells (5.Wissing J.B. Behrbohm H. Plant Physiol. 1993; 102: 1243-1249Google Scholar) and characterized with respect to its enzymological and kinetic properties(6.Wissing J.B. Kornak B. Funke A. Riedel B. Plant Physiol. 1994; 105: 903-909Google Scholar).Metabolic labeling studies using C. roseus have shown that DGPP is rapidly metabolized by a membrane-associated phosphatase, which has been named DGPP phosphatase. 2B. Riedel, W.-I. Wu, G. M. Carman, and J. B. Wissing, manuscript in preparation. DGPP phosphatase catalyzes the dephosphorylation of DGPP to form PA and Pi.2 In addition to being present in plant cells, DGPP phosphatase activity is also present in membrane fractions of Escherichia coli, Saccharomyces cerevisiae, and rat liver.2 Whereas it is unclear what role DGPP plays in phospholipid metabolism and cell growth, PA, the product of the DGPP phosphatase reaction, plays a major role in lipid metabolism. PA is the precursor of all phospholipids and triacylglycerol(7.Kennedy E.P. Op den Kamp J.A.F. Roelofsen B. Wirtz K.W.A. Lipids and Membranes: Past, Present and Future. Elsevier Science Publishers B. V., Amsterdam1986: 171-206Google Scholar, 8.Carman G.M. Henry S.A. Annu. Rev. Biochem. 1989; 58: 635-669Google Scholar). In addition, PA regulates the activity of several lipid-dependent enzymes (9.Bae-Lee M. Carman G.M. J. Biol. Chem. 1990; 265: 7221-7226Google Scholar, 10.Moritz A. DeGraan P.N.E. Gispen W.H. Wirtz K.W.A. J. Biol. Chem. 1992; 267: 7207-7210Google Scholar, 11.Jones G.A. Carpenter G. J. Biol. Chem. 1993; 268: 20845-20850Google Scholar, 12.Bhat B.G. Wang P. Coleman R.A. J. Biol. Chem. 1994; 269: 13172-13178Google Scholar) and has mitogenic effects in animal cells (13.Moolenaar W.H. Kruijer W. Tilly B.C. Verlaan I. Bierman A.J. de Laat S.W. Nature. 1986; 323: 171-173Google Scholar, 14.Yu C.-L. Tsai M.-H. Stacey D.W. Cell. 1988; 52: 63-71Google Scholar, 15.Gomez-Munoz A. Martin A. O'Brien L. Brindley D.N. J. Biol. Chem. 1994; 269: 8937-8943Google Scholar). Thus, the discovery of DGPP phosphatase activity in a wide range of organisms suggests that this enzyme may play an important role in phospholipid metabolism and cell growth.A purified preparation of DGPP phosphatase is required for defined studies on the mechanism and regulation of this novel enzyme of phospholipid metabolism. Owing to its amenable molecular genetic system, we are using the yeast S. cerevisiae as a model eucaryote to study DGPP phosphatase. We report in this paper the purification of DGPP phosphatase to apparent homogeneity. The purified enzyme was characterized with respect to its enzymological and kinetic properties. Moreover, we demonstrated that S. cerevisiae synthesized DGPP via the PA kinase reaction.EXPERIMENTAL PROCEDURESMaterialsAll chemicals were reagent grade. Growth medium supplies were purchased from Difco. Radiochemicals were from DuPont NEN. Scintillation counting supplies and acrylamide for electrophoresis were from National Diagnostics. Nucleotides, pyrophosphate, glycerol 3-phosphate, inositol 1-phosphate, N-ethylmaleimide, p-chloromercuriphenylsulfonic acid, phenylmethanesulfonyl fluoride, Triton X-100, and bovine serum albumin were purchased from Sigma. Phospholipids were purchased from Avanti Polar Lipids and Sigma. DE53 (DEAE-cellulose) was purchased from Whatman. Affi-Gel Blue, hydroxylapatite (Bio-Gel HT), molecular mass standards for SDS-polyacrylamide gel electrophoresis, electrophoresis reagents, and protein assay reagent were purchased from Bio-Rad. Mono Q was purchased from Pharmacia Biotech Inc. Silica gel 60 thin-layer chromatography plates were from EM Science. E. coli diacylglycerol kinase was obtained from Lipidex Inc.MethodsStrain and Growth ConditionsStrain MATaade5(16.Culbertson M.R. Henry S.A. Genetics. 1975; 80: 23-40Google Scholar), which shows normal regulation of phospholipid metabolism(17.Greenberg M. Goldwasser P. Henry S.A. Mol. & Gen. Genet. 1982; 186: 157-163Google Scholar, 18.Poole M.A. Homann M.J. Bae-Lee M. Carman G.M. J. Bacteriol. 1986; 168: 668-672Google Scholar, 19.Klig L.S. Homann M.J. Carman G.M. Henry S.A. J. Bacteriol. 1985; 162: 1135-1141Google Scholar, 20.Klig L.S. Homann M.J. Kohlwein S. Kelley M.J. Henry S.A. Carman G.M. J. Bacteriol. 1988; 170: 1878-1886Google Scholar), was used for the purification of DGPP phosphatase. Cultures were maintained on YEPD medium (1% yeast extract, 2% peptone, 2% glucose) plates containing 2% Bacto-agar. For enzyme purification, cells were grown in YEPD medium at 30°C to late exponential phase, harvested by centrifugation, and stored at −80°C as described previously (21.Fischl A.S. Carman G.M. J. Bacteriol. 1983; 154: 304-311Google Scholar).Preparation of EnzymesPA kinase was purified from suspension-cultured C. roseus cells as described by Wissing and Behrbohm(5.Wissing J.B. Behrbohm H. Plant Physiol. 1993; 102: 1243-1249Google Scholar). PA phosphatase was purified from S. cerevisiae as described by Lin and Carman(22.Lin Y.-P. Carman G.M. J. Biol. Chem. 1989; 264: 8641-8645Google Scholar). The total membrane fraction (22.Lin Y.-P. Carman G.M. J. Biol. Chem. 1989; 264: 8641-8645Google Scholar) of S. cerevisiae was used for the assay of PA kinase activity.Preparation of DGPP Standard and Labeled SubstratesDGPP standard was synthesized enzymatically from PA and ATP using purified C roseus PA kinase as described by Wissing and Behrbohm(1.Wissing J.B. Behrbohm H. FEBS Lett. 1993; 315: 95-99Google Scholar). Purified PA kinase was also used to prepare 32P-labeled DGPP. [α-32P]DGPP was synthesized from [32P]PA and ATP. [β-32P]DGPP was prepared from PA and [γ-32P]ATP. 32P-Labeled DGPP was purified by thin-layer chromatography on potassium oxalate-treated plates using the solvent system chloroform/acetone/methanol/glacial acetic acid/water (50:15:13:12:4). [32P]PA was synthesized from diacylglycerol and [γ-32P]ATP using E. coli diacylglycerol kinase(23.Walsh J.P. Bell R.M. J. Biol. Chem. 1986; 261: 6239-6247Google Scholar). Labeled PA was purified by thin-layer chromatography using the solvent system chloroform/methanol/water (65:25:4).ElectrophoresisPolyacrylamide gel electrophoresis under nondenaturing conditions (24.Davis B. Ann. N. Y. Acad. Sci. 1964; 121: 404-427Google Scholar) was performed at 5°C in 6% slab gels containing 0.5% Triton X-100. SDS-polyacrylamide gel electrophoresis (25.Laemmli U.K. Nature. 1970; 227: 680-685Google Scholar) was performed with 9% slab gels. Molecular mass standards were phosphorylase b (92.5 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), and soybean trypsin inhibitor (21.5 kDa). Proteins on SDS-polyacrylamide gels were stained with silver(26.Merril C.R. Dunau M.L. Goldman D. Anal. Biochem. 1981; 110: 201-207Google Scholar).Enzyme Assays and Product IdentificationThe optimal conditions for assay of DGPP phosphatase activity were determined in detail with purified enzyme. DGPP phosphatase activity was measured by monitoring the release of water-soluble 32Pi from 0.1 mM [β-32P]DGPP (10,000-20,000 cpm/nmol) in 50 mM Tris-maleate buffer (pH 6.5) containing 2 mM Triton X-100, 10 mM 2-mercaptoethanol, and enzyme protein in a total volume of 0.1 ml. Purified DGPP phosphatase was routinely diluted 2,000-fold for enzyme assays. The reaction was terminated by addition of 0.5 ml of 0.1 N HCl in methanol. Chloroform (1 ml) and 1 M MgCl2 (1 ml) were added, the system was mixed, and the phases were separated by 2 min of centrifugation at 100 × g. Ecoscint H (4 ml) was added to a 0.5-ml sample of the aqueous phase, and radioactivity was determined by scintillation counting. Alternatively, DGPP phosphatase activity was measured by monitoring the formation of [32P]PA from [α-32P]DGPP (10,000-20,000 cpm/nmol) under the assay conditions described above. The chloroform-soluble phospholipid product of the reaction, PA, was analyzed with standard PA and DGPP by thin-layer chromatography on potassium oxalate-treated plates using the solvent system chloroform/acetone/methanol/glacial acetic acid/water (50:15:13:12:4). The positions of the labeled phospholipids on the chromatograms were determined by autoradiography. The amount of labeled phospholipids was determined by scintillation counting.PA kinase activity was measured with 40 mM imidazole-HCl buffer (pH 6.1), 10 mM MgCl2, 100 mM NaCl, 0.5 mM dithiothreitol, 10 mM NaF, 5 mM β-glycerol phosphate, 0.1 mM MnCl2, 6.9 mM Triton X-100, 0.6 mM PA, 1 mM [γ-32P]ATP (100,000 cpm/nmol), and 0.3 mg/ml membrane protein(1.Wissing J.B. Behrbohm H. FEBS Lett. 1993; 315: 95-99Google Scholar, 5.Wissing J.B. Behrbohm H. Plant Physiol. 1993; 102: 1243-1249Google Scholar). NaF, β-glycerol phosphate, and MnCl2 were included in the reaction mixture to inhibit phosphatase reactions. The 32P-labeled chloroform-soluble phospholipid product of the reaction, DGPP, was analyzed with standard DGPP by thin-layer chromatography as described above for the DGPP phosphatase assay.PA phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) was measured with 50 mM Tris maleate buffer (pH 7.0), 10 mM 2-mercaptoethanol, 2 mM MgCl2, 1 mM Triton X-100, 0.1 mM [32P]PA, and enzyme protein(27.Carman G.M. Lin Y.-P. Methods Enzymol. 1991; 197: 548-553Google Scholar). A unit of enzymatic activity was defined as the amount of enzyme that catalyzed the formation of 1 μmol of product/min unless otherwise indicated. Specific activity was defined as units/mg of protein.Protein DeterminationProtein concentration was determined by the method of Bradford (28.Bradford M.M. Anal. Biochem. 1976; 72: 248-254Google Scholar) using bovine serum albumin as the standard. Buffers that were identical to those containing protein samples were used as blanks. Protein concentration of samples after Mono Q chromatography was determined by scanning densitometry of silver-stained SDS-polyacrylamide gels using bovine serum albumin as the standard. Protein was monitored during purification on columns by measuring the absorbance at 405 nm.Preparation of Triton X-100/Phospholipid-mixed MicellesPhospholipids in chloroform were transferred to a test tube, and solvent was removed in vacuo for 40 min. Triton X-100/phospholipid-mixed micelles were prepared by adding Triton X-100 to dried lipids. The total phospholipid concentration in Triton X-100/phospholipid-mixed micelles did not exceed 15 mol % to ensure that the structure of the mixed micelles were similar to the structure of pure Triton X-100(29.Lichtenberg D. Robson R.J. Dennis E.A. Biochim. Biophys. Acta. 1983; 737: 285-304Google Scholar, 30.Robson R.J. Dennis E.A. Acct. Chem. Res. 1983; 16: 251-258Google Scholar).Analysis of Kinetic DataKinetic data were analyzed according to the Michaelis-Menten and Hill equations using the EZ-FIT enzyme kinetic model fitting program (31.Perrella F. Anal. Biochem. 1988; 174: 437-447Google Scholar). EZ-FIT uses the Nelder-Mead Simplex and Marquardt/Nash nonlinear regression algorithms sequentially and tests for the best fit of the data among different kinetic models.Mass Analyses of DGPP and PAPhospholipids were extracted from cells using method IIIB described by Hanson and Lester (32.Hanson B.A. Lester R.L. J. Lipid Res. 1980; 21: 309-315Google Scholar) and dried in vacuo. The solvent mixture used in method IIIB is 95% ethanol/water/diethylether/pyridine/ammonium hydroxide (15:15:5:1:0.018). The sample was dissolved in chloroform/methanol/water (15:15:5). Samples were subjected to analytical normal phase high performance liquid chromatography using a 5-μm Kromasil silica column (150 × 4.6 mm, inner diameter) equilibrated with chloroform/methanol/30% ammonium hydroxide (80:19.5:0.5). Elution was carried out using a 14-min linear gradient (0-30%) of methanol/water/30% ammonium hydroxide (80:19.5:0.5) followed by holding the same solvent at 30% for an additional 14 min. The identity of DGPP was determined by comparing its elution profile with that of authentic DGPP (1.Wissing J.B. Behrbohm H. FEBS Lett. 1993; 315: 95-99Google Scholar) using an evaporative light scattering detector. A flow rate of 1 ml/min was used throughout. The cellular concentration of DGPP was calculated relative to the concentration of the major phospholipids in the extract. The cellular concentration of PA was determined as described previously(33.Wu W. Lin Y.-P. Wang E. Merrill Jr., A.H. Carman G.M. J. Biol. Chem. 1993; 268: 13830-13837Google Scholar).Purification of DGPP PhosphataseAll steps were performed at 5°C.Step 1: Preparation of Cell ExtractThe cell extract was prepared from 200 g (wet weight) of cells by disruption with glass beads with a Bead-Beater (Biospec Products) in buffer A (50 mM Tris-maleate (pH 7.0), 1 mM Na2EDTA, 0.3 M sucrose, 10 mM 2-mercaptoethanol, and 0.5 mM phenylmethanesulfonyl fluoride) as described previously(21.Fischl A.S. Carman G.M. J. Bacteriol. 1983; 154: 304-311Google Scholar). Unbroken cells and glass beads were removed by centrifugation at 1,500 × g for 5 min.Step 2: Preparation of MicrosomesMicrosomes were isolated from the cell extract by differential centrifugation(21.Fischl A.S. Carman G.M. J. Bacteriol. 1983; 154: 304-311Google Scholar). Microsomes were washed and resuspended in buffer B (50 mM Tris-maleate (pH 7.0), 10 mM MgCl2, 10 mM 2-mercaptoethanol, 20% glycerol, and 0.5 mM phenylmethanesulfonyl fluoride). These membranes were routinely frozen at −80°C until used for purification.Step 3: Preparation of Triton X-100 ExtractMicrosomes were suspended in buffer B containing 1% Triton X-100 at a final protein concentration of 10 mg/ml. The suspension was incubated for 1 h on a rotary shaker at 150 rpm. After the incubation, the suspension was centrifuged at 100,000 × g for 1.5 h to obtain the Triton X-100 extract (supernatant).Step 4: DE53 ChromatographyA DE53 column (2.5 × 20.5 cm) was equilibrated with buffer C (50 mM Tris-maleate (pH 7.0), 10 mM MgCl2, 10 mM 2-mercaptoethanol, 20% glycerol, and 1% Triton X-100). The Triton X-100 extract was applied to the column at a flow rate of 60 ml/h. The column was washed with one column volume of buffer C followed by elution of DGPP phosphatase activity in 6-ml fractions with nine column volumes of a linear NaCl gradient (0-0.25 M) in buffer C. The peak of DGPP phosphatase activity eluted from the column at the beginning of the NaCl gradient. The most active fractions containing activity were pooled and used for the next step in the purification scheme.Step 5: Affi-Gel Blue ChromatographyAn Affi-Gel Blue column (2.0 × 16 cm) was equilibrated with buffer C. DE53-purified enzyme was applied to the column at a flow rate of 30 ml/h. The column was washed with one column volume of buffer C followed by two column volumes of buffer C containing 0.3 M NaCl. DGPP phosphatase activity was eluted from the column in 3.5-ml fractions with 10 column volumes of a linear NaCl gradient (0.3-0.9 M) in buffer C. The peak of DGPP phosphatase activity eluted from the column at a NaCl concentration between 0.3 and 0.4 M. The most active fractions were pooled and the enzyme preparation was desalted by dialysis against buffer D (10 mM potassium phosphate (pH 7.0), 10 mM MgCl2, 10 mM 2-mercaptoethanol, 20% glycerol, and 1% Triton X-100).Step 6: Hydroxylapatite ChromatographyA hydroxylapatite column (1.5 × 8.5 cm) was equilibrated with buffer D. The desalted Affi-Gel Blue-purified enzyme was applied to the column at a flow rate of 20 ml/h. The column was washed with one column volume of buffer D, and DGPP phosphatase activity was eluted from the column in 2-ml fractions with 20 column volumes of a linear potassium phosphate gradient (10-150 mM) in buffer D. Two peaks of DGPP phosphatase activity eluted from the column. The first peak (peak I) of activity eluted from the column at the beginning of the gradient. The second peak (peak II) of activity eluted at a phosphate concentration of about 24 mM. The most active fractions from each peak were pooled and dialyzed against buffer C.Step 7: Mono Q I ChromatographyA Mono Q column (0.5 × 5 cm) was equilibrated with buffer C. Hydroxylapatite-purified enzyme from peak I was applied to the column at a flow rate of 24 ml/h. The column was washed with two column volumes of buffer C. DGPP phosphatase activity was eluted from the column in 1-ml fractions with 40 column volumes of a linear NaCl gradient (0-0.3 M) in buffer C. The peak of DGPP phosphatase activity eluted from the column at a NaCl concentration of about 0.12 M. Fractions containing activity were pooled and stored at −80°C. The purified enzyme was completely stable for at least 2 months.Step 8: Mono Q II ChromatographyA second Mono Q column (0.5 × 5 cm) was equilibrated with buffer C. Hydroxylapatite-purified enzyme from peak II was applied to the column at a flow rate of 24 ml/h. The column was washed with two column volumes of buffer C. DGPP phosphatase activity was eluted from the column in 1-ml fractions with 40 column volumes of a linear NaCl gradient (0-0.3 M) in buffer C. The peak of DGPP phosphatase activity eluted from the column at the beginning of the NaCl gradient. Fractions containing activity were pooled and stored at −80°C. The purified enzyme was completely stable for at least 2 months.DISCUSSIONWe undertook the purification of DGPP phosphatase from S. cerevisiae to facilitate well defined studies on the biochemical regulation of this novel enzyme of phospholipid metabolism. Purification of DGPP phosphatase required the solubilization of the enzyme from microsomal membranes with Triton X-100 followed by conventional chromatography steps performed in the presence of Triton X-100. The presence of Triton X-100 in the buffers used for enzyme purification was required to prevent aggregation of this integral membrane protein. The hydroxylapatite chromatography step resulted in two distinct peaks of DGPP phosphatase activity. Each peak of activity was purified separately by Mono Q chromatography. The seven-step purification procedure reported for the enzyme from hydroxylapatite peak I resulted in a DGPP phosphatase preparation that was essentially homogeneous as evidenced by native and SDS-polyacrylamide gel electrophoresis. DGPP phosphatase was identified in this preparation as a 34-kDa protein. Overall, DGPP phosphatase was purified 33,333-fold relative to the activity in the cell extract to a final specific activity of 150 μmol/min/mg. The enzyme from hydroxylapatite peak II was purified 24,666-fold over the activity in the cell extract to a final specific activity of 111 μmol/min/mg. This near homogeneous enzyme preparation also contained the 34-kDa protein, which was associated with DGPP phosphatase activity. The reason for the different chromatographic properties of the two DGPP phosphatase preparations is unclear. Additional studies are required to address these differences. The molecular mass of native DGPP phosphatase in Triton X-100 micelles cannot be determined until the interaction of the enzyme with detergent has been quantitated(40.Tanford C. Nozaki Y. Reynolds J.A. Makino S. Biochemistry. 1974; 13: 2369-2376Google Scholar).The fold purifications and final specific activities of the DGPP phosphatase preparations described here were higher than any other phospholipid-dependent enzyme purified from S. cerevisiae (reviewed in (8.Carman G.M. Henry S.A. Annu. Rev. Biochem. 1989; 58: 635-669Google Scholar) and (39.Paltauf F. Kohlwein S.D. Henry S.A. Jones E.W. Pringle J.R. Broach J.R. The Molecular and Cellular Biology of the Yeast Saccharomyces: Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992: 415-500Google Scholar)). That such a high degree of purification was required to obtain purified DGPP phosphatase indicated that the abundance of this enzyme in S. cerevisiae was relatively low when compared with other phospholipid-dependent enzymes.Pure DGPP phosphatase derived from hydroxylapatite peak I was used for the biochemical characterization of the enzyme. The enzyme had a broad pH optimum and required Triton X-100 for maximum activity. The enzyme did not have a divalent cation requirement, and activity was insensitive to Na2EDTA. Divalent cations, especially Mn2+ ions, potently inhibited DGPP phosphatase activity. DGPP phosphatase was also inhibited by NaF but was relatively insensitive to thioreactive compounds. Pyrophosphate and, to a lesser extent, ADP (which contains a pyrophosphate group) inhibited DGPP phosphatase activity. We did not test whether these compounds were substrates for DGPP phosphatase. Pyrophosphate is the specific substrate for inorganic pyrophosphatase in S. cerevisiae(41.Bailey K. Webb E.C. Biochem. J. 1944; 38: 394-398Google Scholar, 42.Heppel L.A. Hilmoe R.J. J. Biol. Chem. 1951; 192: 87-94Google Scholar). DGPP phosphatase differed from inorganic pyrophosphatase in that inorganic pyrophosphatase is a cytosolic enzyme, which is dependent on Mg2+ ions for activity(41.Bailey K. Webb E.C. Biochem. J. 1944; 38: 394-398Google Scholar, 42.Heppel L.A. Hilmoe R.J. J. Biol. Chem. 1951; 192: 87-94Google Scholar).DGPP phosphatase catalyzed the dephosphorylation of the β phosphate of DGPP yielding PA and Pi. DGPP phosphatase activity was not inhibited by its product PA. This lack of product inhibition was consistent with the length of time in which the reaction was linear and the enzyme catalyzing the near quantitative conversion of DGPP to PA. Whereas the enzyme was not inhibited by PA, DGPP phosphatase catalyzed the dephosphorylation of PA. The dephosphorylation of PA was subsequent to the dephosphorylation of the β phosphate of DGPP. Moreover, the dephosphorylation of PA by DGPP phosphatase was potently inhibited by DGPP. DGPP was a competitive inhibitor with respect to PA and the Ki value for DGPP was similar to the Km value for DGPP (Table 3). Taken together, these results suggested that the DGPP and PA binding sites on the enzyme were the same.Tabled 1DGPP phosphatase activity followed surface dilution kinetics (35.Carman G.M. Deems R.A. Dennis E.A. J. Biol. Chem. 1995; 270: 18711-18714Google Scholar) using Triton X-100/phospholipid-mixed micelles. The role of Triton X-100 in the assay of phospholipid-dependent enzymes is to form a mixed micelle with the phospholipid substrate and, therefore, provide a surface for catalysis(35.Carman G.M. Deems R.A. Dennis E.A. J. Biol. Chem. 1995; 270: 18711-18714Google Scholar). Indeed, DGPP phosphatase activity was dependent on the surface concentration of DGPP. The reaction of the dephosphorylation of PA also followed surface dilution kinetics. However, in contrast to the typical kinetic behavior (saturation kinetics) the enzyme exhibited toward DGPP, the dependence of activity on the surface concentration of PA was cooperative.DGPP was clearly the preferred substrate for pure DGPP phosphatase based on the relative values for VMAX and Km (Table 3). Moreover, the specificity constant (VMAX /Km) for DGPP was 10-fold higher than that for PA (Table 3). Although the cellular concentration of DGPP was 16-fold lower than that of PA (Table 3), an argument can be made for DGPP being the the preferred substrate for the enzyme in vivo. PA, at a concentration 20-fold higher than that of DGPP, did not inhibit DGPP phosphatase activity, and DGPP potently inhibited the enzyme's ability to catalyze the dephosphorylation of PA. Moreover, the Ki value for DGPP was very close to its cellular concentration (Table 3). Our studies, however, do not rule out the possibility that the enzyme could use PA as a substrate in vivo.The enzyme in S. cerevisiae which is responsible for the dephosphorylation of PA is PA phosphatase(22.Lin Y.-P. Carman G.M. J. Biol. Chem. 1989; 264: 8641-8645Google Scholar). PA phosphatase has been purified and extensively characterized from S. cerevisiae(22.Lin Y.-P. Carman G.M. J. Biol. Chem. 1989; 264: 8641-8645Google Scholar, 33.Wu W. Lin Y.-P. Wang E. Merrill Jr., A.H. Carman G.M. J. Biol. Chem. 1993; 268: 13830-13837Google Scholar, 38.Lin Y.-P. Carman G.M. J. Biol. Chem. 1990; 265: 166-170Google Scholar, 43.Morlock K.R. McLaughlin J.J. Lin Y.-P. Carman G.M. J. Biol. Chem. 1991; 266: 3586-3593Google Scholar, 44.Quinlan J.J. Nickels Jr., J.T. Wu W. Lin Y.-P. Broach J.R. Carman G.M. J. Biol. Chem. 1992; 267: 18013-18020Google Scholar, 45.Wu W.-I. Carman G.M. J. Biol. Chem. 1994; 269: 29495-29501Google Scholar). DGPP phosphatase differed from PA phosphatase with respect to molecular mass, substrate specificity, cofactor requirement, and sensitivity to thioreactive agents. For example, PA phosphatase requires Mg2+ ions for activity and is inhibited by N-ethylmaleimide(22.Lin Y.-P. Carman G.M. J. Biol. Chem. 1989; 264: 8641-8645Google Scholar, 43.Morlock K.R. McLaughlin J.J. Lin Y.-P. Carman G.M. J. Biol. Chem. 1991; 266: 3586-3593Google Scholar).If DGPP phosphatase was to play an important role in phospholipid metabolism, it was important for us to demonstrate that DGPP was synthesized in S. cerevisiae. Indeed, we demonstrated that S. cerevisiae possessed PA kinase activity and DGPP was identified in growing cells. PA kinase activity (7 pmol/min/mg) in S. cerevisiae was very low when compared with the activity (3.3 nmol/min/mg) in membranes from C. roseus(5.Wissing J.B. Behrbohm H. Plant Physiol. 1993; 102: 1243-1249Google Scholar). This low level of activity may be a reflection of a low level of PA kinase expression, the product's rapidly hydrolyzation by DGPP phosphatase, and/or not knowing the correct assay conditions for the enzyme. Additional studies are needed to characterize PA kinase activity in S. cerevisiae. DGPP was previously not identified in S. cerevisiae. In previous studies (reviewed in (39.Paltauf F. Kohlwein S.D. Henry S.A. Jones E.W. Pringle J.R. Broach J.R. The Molecular and Cellular Biology of the Yeast Saccharomyces: Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992: 415-500Google Scholar)), a relatively large percentage of unidentified phospholipids have been labeled as “others.” Perhaps DGPP was a minor phospholipid among the unidentified phospholipids described in previous studies. DGPP accounted for only 0.18 mol % of the major phospholipids in S. cerevisiae. The identification of DGPP was dependent on the procedure used to extract phospholipids. Method IIIB of Hanson and Lester (32.Hanson B.A. Lester R.L. J. Lipid Res. 1980; 21: 309-315Google Scholar) was the best method to extract DGPP. This method is commonly used for the extraction of polar lipids such as polyphosphoinositides and sphingolipids(32.Hanson B.A. Lester R.L. J. Lipid Res. 1980; 21: 309-315Google Scholar). This method also minimizes creation of artifacts such as lysolipids(32.Hanson B.A. Lester R.L. J. Lipid Res. 1980; 21: 309-315Google Scholar). The cellular concentration of DGPP was 3-fold lower than the Km value (0.55 mol %) for DGPP. Thus, DGPP phosphatase activity would be expected to be very sensitive to changes in the cellular concentration of DGPP.It is unclear what roles DGPP and DGPP phosphatase play in phospholipid metabolism. One could speculate that DGPP is the precursor of the PA used for phospholipid synthesis or neutral lipid synthesis, DGPP is the precursor of the PA which acts as a signaling molecule, and/or DGPP itself is a signaling molecule. The activity of DGPP phosphatase could regulate the levels of DGPP and PA in the cell. Since DGPP phosphatase also dephosphorylated PA, the enzyme may play a role in regulating diacylglycerol levels. The studies reported here provide the foundation for future molecular genetic studies directed toward understanding the roles DGPP and DGPP phosphatase play in phospholipid metabolism and cell growth. INTRODUCTIONDiacylglycerol pyrophosphate (DGPP) 1The abbreviations used are: DGPPdiacylglycerol pyrophosphatePAphosphatidate. is a novel phospholipid metabolite recently identified from the plant Catharanthus roseus by Wissing and Behrbohm(1.Wissing J.B. Behrbohm H. FEBS Lett. 1993; 315: 95-99Google Scholar). DGPP contains a pyrophosphate group attached to diacylglycerol (Fig. 1). This compound was previously observed by several workers (2.Heim S. Bauleke A. Wylegalla C. Wagner K.G. Plant Sci. 1987; 49: 159-165Google Scholar, 3.Sommarin M. Sandelius A.S. Biochim. Biophys. Acta. 1988; 958: 268-278Google Scholar, 4.Memon A.R. Boss W.F. J. Biol. Chem. 1990; 265: 14817-14821Google Scholar) as the phospholipid product of a lipid kinase reaction in plants that was not identified correctly(1.Wissing J.B. Behrbohm H. FEBS Lett. 1993; 315: 95-99Google Scholar). It is now known that DGPP is synthesized from PA and ATP through the reaction catalyzed by the novel enzyme PA kinase(1.Wissing J.B. Behrbohm H. FEBS Lett. 1993; 315: 95-99Google Scholar). PA kinase is a ubiquitous membrane-associated enzyme found in the plant kingdom(5.Wissing J.B. Behrbohm H. Plant Physiol. 1993; 102: 1243-1249Google Scholar). The enzyme has been purified from suspension-cultured C. roseus cells (5.Wissing J.B. Behrbohm H. Plant Physiol. 1993; 102: 1243-1249Google Scholar) and characterized with respect to its enzymological and kinetic properties(6.Wissing J.B. Kornak B. Funke A. Riedel B. Plant Physiol. 1994; 105: 903-909Google Scholar).Metabolic labeling studies using C. roseus have shown that DGPP is rapidly metabolized by a membrane-associated phosphatase, which has been named DGPP phosphatase. 2B. Riedel, W.-I. Wu, G. M. Carman, and J. B. Wissing, manuscript in preparation. DGPP phosphatase catalyzes the dephosphorylation of DGPP to form PA and Pi.2 In addition to being present in plant cells, DGPP phosphatase activity is also present in membrane fractions of Escherichia coli, Saccharomyces cerevisiae, and rat liver.2 Whereas it is unclear what role DGPP plays in phospholipid metabolism and cell growth, PA, the product of the DGPP phosphatase reaction, plays a major role in lipid metabolism. PA is the precursor of all phospholipids and triacylglycerol(7.Kennedy E.P. Op den Kamp J.A.F. Roelofsen B. Wirtz K.W.A. Lipids and Membranes: Past, Present and Future. Elsevier Science Publishers B. V., Amsterdam1986: 171-206Google Scholar, 8.Carman G.M. Henry S.A. Annu. Rev. Biochem. 1989; 58: 635-669Google Scholar). In addition, PA regulates the activity of several lipid-dependent enzymes (9.Bae-Lee M. Carman G.M. J. Biol. Chem. 1990; 265: 7221-7226Google Scholar, 10.Moritz A. DeGraan P.N.E. Gispen W.H. Wirtz K.W.A. J. Biol. Chem. 1992; 267: 7207-7210Google Scholar, 11.Jones G.A. Carpenter G. J. Biol. Chem. 1993; 268: 20845-20850Google Scholar, 12.Bhat B.G. Wang P. Coleman R.A. J. Biol. Chem. 1994; 269: 13172-13178Google Scholar) and has mitogenic effects in animal cells (13.Moolenaar W.H. Kruijer W. Tilly B.C. Verlaan I. Bierman A.J. de Laat S.W. Nature. 1986; 323: 171-173Google Scholar, 14.Yu C.-L. Tsai M.-H. Stacey D.W. Cell. 1988; 52: 63-71Google Scholar, 15.Gomez-Munoz A. Martin A. O'Brien L. Brindley D.N. J. Biol. Chem. 1994; 269: 8937-8943Google Scholar). Thus, the discovery of DGPP phosphatase activity in a wide range of organisms suggests that this enzyme may play an important role in phospholipid metabolism and cell growth.A purified preparation of DGPP phosphatase is required for defined studies on the mechanism and regulation of this novel enzyme of phospholipid metabolism. Owing to its amenable molecular genetic system, we are using the yeast S. cerevisiae as a model eucaryote to study DGPP phosphatase. We report in this paper the purification of DGPP phosphatase to apparent homogeneity. The purified enzyme was characterized with respect to its enzymological and kinetic properties. Moreover, we demonstrated that S. cerevisiae synthesized DGPP via the PA kinase reaction.
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