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[20] Phosphatidylinositol 4-Kinase from Yeast B y GEORGE M. CARMAN, CHARLES J. BELUNIS,
and JOSEPH T. NICKELS, JR. Introduction Phosphatidylinositol 4-kinase ( 1-phosphatidylinositol kinase) catalyzes the reaction of phosphatidylinositol with ATP to form phosphatidylinositol Phosphatidylinositol + ATP--~ phosphatidylinositol 4-phosphate + ADP 4-phosphate.~ Phosphatidylinositol 4-kinase (ATP : 1-phosphatidyl-lDm y o - i n o s i t o l 4-phosphotransferase, EC 2.7.1.67) is the first e n z y m e in the phosphorylation sequence of phosphatidylinositol leading to the formation of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in S a c c h a r o m y c e s cerevisiae, z The synthesis and turnover of the polyphosphoinositides in S. cerevisiae as well as in higher cukaryotes play an important role in cell g r o w t h J -7 Phosphatidylinositol 4-kinase is associated with the microsomal, 8 plasma membrane, 9 and cytosolic 1° fractions of S. cerevisiae. Phosphatidylinositol 4-kinase has been purified to near homogeneity from the microsomal fraction of S. cerevisiae by standard protein purification procedures. 8 We describe here the purification and properties of the enzyme. Assay Method Phosphatidylinositol 4-kinase activity is measured for 10 min by following the phosphorylation of 0.4 m M phosphatidylinositol with 5 m M [7-32p]ATP [3000-5000 counts/min (cpm)/nmol] in the presence 50 m M I M. Colodzin and E. P. Kennedy, J. Biol. Chem. 240, 3771 0965). 2 S. Stciner and R. L. Lester, Biochim. Biophy#. Acta 2,60, 82 0972). 3 R. T. Talwalkar and R. L. Lester, Biochim. Biophys. Acta 306, 412 0973).
4 K. Kaibuchi, A. Miyajima, K.-I. Arai, and K. Matsumoto, Proc. Natl. Acad. Sci. U.S.A. 83, 8172 0986). 5 C. Dahl, H.-P. Biemann, and J. Dahl, Proc. Natl. Acad. Sci. U.S.A. 84, 4012 (1987). 6 I. Uno, K. Fukami, H. Kato, T. Takenawa, and T. Ishikawa, Nature (London) 333, 188 (1988). 7 M. J. Berridge, Biochim. Biophys. Acta 907, 33 (1987).
s C. J. Belunis, M. Bae-Lee, M. J. Kelley, and G. M. Carman, J. Biol. Chem. 263, 18897 (1988). 9 A. J. Kinney and G. M. Carman, J. Bacteriol. 172, 4115 (1990). J0R. T. Talwalker and R. L. Lester, Biochim. Biophys. Acta 360, 306 (1974). METHODS IN ENZYMOLOGY,VOL. 209
Copyright © 1992by AcademicPress, Inc. All rights of reproductionin any formreserved.
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Tris-maleate buffer (pH 8.0) containing 25 mM Triton X-100, 30 mM MgCI2, and enzyme protein in a total volume of 0.1 ml at 30°) t The reaction is terminated by the addition of 0.5 ml of 0.1 N HC1 in methanol. Chloroform (1 ml) and 1 M MgCIz (1.5 ml) are added, the system is mixed, and the phases are separated by a 2-min centrifugation at 100 g at room temperature. A 0.5-ml sample of the chloroform phase is removed and taken to dryness on an 80° water bath. Betafluor (4 ml, National Diagnostics, Somerville, NJ) is added to the sample, and radioactivity is determined by scintillation counting. The chloroform-soluble phospholipid product of the reaction, phosphatidylinositol 4-phosphate, is analyzed by thin-layer chromatography with standard phosphatidylinositol 4-phosphate using the solvent system chloroform-methanol-2.5 M ammonium hydroxide ( 9 : 7 : 2 , v-v).11 Alternatively, activity is measured using 0.4 mM phosphatidyl[2-3H]inositol (2000 cpm/nmol) and 5 mM ATP as substrates. A unit of enzymatic activity is defined as the amount of enzyme that catalyzes the formation of 1 nmol of product per minute. Protein is determined by the method of Bradford) 2 Buffers which are identical to those containing the protein samples are used as blanks. The presence of Triton X-100 does not interfere with the protein determination, provided the blank contains a final concentration of detergent identical to that of the sample.
Growth of Yeast Saccharomyces cerevisiae strain ade5 MATa 13is used as a representative wild-type strain for enzyme purification. Cells are grown in 1% yeast extract, 2% peptone, and 2% glucose (w/v) at 28 ° to late exponential phase, harvested by centrifugation, and stored at -80°. 14'15 We use wild-type strain $288C for the purification of phosphatidylinositol synthase, 14a5 phosphatidylserine synthase,~6'17 and CDPdiacylglycerol synthase. 18,19We are unable to purify phosphatidylinositol 4-kinase activity from strain $288C. The reason for this is unclear. 11 M. A. McKenzie and G. M. Carman, J. Food Biochem. 6, 77 (1982). 12 M. M. Bradford, Anal. Biochem. 72, 248 (1976). 13 M. R. Culbertson and S. A. Henry, Genetics 80, 23 (1975). 14 A. S. Fischl and G. M. Carman, J. Bacteriol. 154, 304 (1983). 15 G. M. Carman and A. S. Fischl, this volume [36]. 16 M. Bae-Lee and G. M. Carman, J. Biol. Chem. 259, 10857 (1984). 17 G. M. Carman and M. Bae-Lee, this volume [35]. 18 M. J. Kelley and G. M. Carman, J. Biol. Chem. 262, 14563 (1987). 19 G. M. Carman and M. J. Kelley, this volume [28].
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Purification Procedure All steps are performed at 5°.
Step 1: Preparation of Cell Extract. Cells (200 g) are disrupted with glass beads (diameter, 0.5 mm) with a BioSpec Products (Bartlesville, OK) Bead-Beater in 50 mM Tris-HCl buffer (pH 7.5) containing 1 mM Na2EDTA, 0.3 M sucrose, and 10 mM 2-mercaptoethanol as described previously.14.15 Glass beads and unbroken cells are removed by centrifugation at 1500 g for 15 min to obtain the cell extract. Step 2: Preparation of Microsomes. Microsomes are collected from the cell extract by differential centrifugation) 4'15 Microsomal pellets are washed with 50 mM Tris-HCl buffer (pH 8.0) containing 10 mM MgC12, 10 mM 2-mercaptoethanol, and 10% glycerol. Microsomes can be frozen at - 8 0 ° until the enzyme is purified. Step 3: Preparation of Triton X-IO0 Extract. Microsomes are suspended in 50 mM Tris-HCl buffer (pH 8.0) containing 10 mM MgCI2, 10 mM 2-mercaptoethanol, 10% glycerol, and 1% Triton X-100 (v/v) 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 1.5 hr to obtain the Triton X-100 extract (supernatant). The Triton X-100 extract is immediately used for the next purification step since, at this stage, the enzyme is labile. The addition of protease inhibitors does not improve enzyme stability. Step 4:DE-52 Chromatography. A DE-52 (Whatman, Clifton, NJ) column (2.5 x 5 cm) is equilibrated with 50 mM Tris-HC1 buffer (pH 8.0) containing 10 mM MgCI2, 10 mM 2-mercaptoethanol, 10% glycerol, and 0.5% Triton X-100. The Triton X-100 extract is applied to the column at a flow rate 20 ml/hr. The column is washed with 6 column volumes of equilibration buffer followed by elution of the enzyme with equilibration buffer containing 0.1 M NaCI. Fractions containing phosphatidylinositol 4-kinase activity are pooled and immediately used for the next step in the purification scheme. The enzyme is still labile at this stage. Step 5: Hydroxylapatite Chromatography. A hydroxylapatite (BioGel HT, Bio-Rad, Richmond, CA) column (2.5 × 3 cm) is equilibrated with 10 mM potassium phosphate buffer (pH 8.0) containing 10 mM MgCI2, 10 mM 2-mercaptoethanol, 10% glycerol, and 0.2% Triton X-100. The DE52 purified enzyme is diluted 2 : 1 with equilibration buffer and applied to the column at a flow rate of 10 ml/hr. The column is washed with 6 column volumes of equilibration buffer followed by elution of the enzyme with 8 column volumes of a linear potassium phosphate gradient (0.01-0.3 M) in equilibration buffer at a flow rate of 10 ml/hr. The peak of phosphatidylinositol 4-kinase activity elutes from the column at a phosphate concentration
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of about 0.15 M. The most active fractions are pooled and used for the next step in the purification. At this stage in the purification, phosphatidylinositol 4-kinase activity is completely stable. Step 6: Octyl-Sepharose Chromatography. An octyl-Sepharose (Pharmacia LKB Biotechnology, Piscataway, NJ) column (1.5 × 16 cm) is equilibrated with 50 mM Tris-HCl buffer (pH 8.0) containing 10 mM MgCI2, 10 mM 2-mercaptoethanol, 10% glycerol, and 3 M NaCI. Solid NaCI is added to the hydroxylapatite-purified enzyme to a final concentration of 3 M, and the enzyme is applied to the octyl-Sepharose column at a flow rate of 20 ml/hr. It is necessary to add 3 M NaCI and to omit Triton X-100 from the equilibration buffer for the enzyme to bind to the octylSepharose column. The column is washed with 2 column volumes of equilibration buffer followed by 6 column volumes of equilibration buffer without NaCI. Phosphatidylinositol 4-kinase activity is then eluted from the column with 8 column volumes of a linear Triton X- 100 gradient (0-1%) in 50 mM Tris-HCl buffer (pH 8.0) containing 10 mM MgCI2, 10 mM 2-mercaptoethanol, and 10% glycerol. The peak of activity elutes from the column at a Triton X-100 concentration of about 0.5%. The most active fractions are pooled and used for the next step in the purification. Step 7: Mono Q Chromatography I. A anion-exchange Mono Q (Pharmacia LKB Biotechnology) column (0.5 x 5 cm) is equilibrated with 50 mM Tris-HC1 buffer (pH 8.0) containing 10 mM MgC12, 10 mM 2-mercaptoethanol, 10% glycerol, and 0.5% Triton X-100. Enzyme from the previous step is applied to the column at a flow rate of 60 ml/hr. The column is washed with 4 column volumes of equilibration buffer followed by 2 column volumes of equilibration buffer containing 0.2 M NaC1. The enzyme is then eluted from the column with l0 column volumes of a linear NaCl gradient (0.2-0.5 M) in equilibration buffer. The peak of enzyme activity elutes at a NaC1 concentration of about 0.22 M. Fractions containing activity are pooled and used for the next step. Step 8: Mono Q Chromatography //. A second Mono Q column (0.5 x 5 cm) is equilibrated with 50 mM Tris-HC1 buffer (pH 8.0) containing l0 mM MgCl2, l0 mM 2-mercaptoethanol, 10% glycerol, 0.5% Triton X-100, and 60 mM NaC1. The enzyme from the previous Mono Q column is diluted 4: I with equilibration buffer and applied to the column at a flow rate of 45 ml/hr. The column is washed with 7 column volumes of equilibration buffer followed by enzyme elution with 20 column volumes of a linear NaC1 gradient (0.06-0.3 M) in equilibration buffer. Phosphatidylinositol 4-kinase activity elutes at a NaC1 concentration of about 0.2 M. Fractions containing activity are pooled and stored at - 8 0 °. The purified enzyme is completely stable for at least 3 months of storage at - 80 °.
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TABLE I PURIFICATION OF PHOSPHATIDYLINOSlTOL 4-KINASE FROM S a c c h a r o m y c e s cerevisiae a
Purification step
Total units (nmol-min)
1. Cell extract 2. Microsomes 3. Triton X-100 extract 4. DE-52 5. Hydroxylapatite 6. Octyl-Sepharose 7. Mono Q I 8. Mono Q II
5970 4418 3104 1086 1032 743 558 380
Protein (mg) 10,119 2,559 794 175 57 5.64 0.24 0.08
Specific activity (units/mg) 0.59 1.72 3.9 6.2 18.1 131.7 2325 4750
Purification (-fold) 1 2.9 6.6 10.5 30.6 223.2 3940.6 8050.8
Yield (%) 100 74 52 18 17.2 12.4 9.3 6.3
a Data from Belunis et al. 8
A summary of the purification of phosphatidylinositol 4-kinase is presented in Table I. The overall purification of phosphatidylinositol 4-kinase over the cell extract is 8051-fold, with an activity yield of 6.3%. Improved Purification Procedure. The purification scheme described above includes octyl-Sepharose chromatography. This step takes approximately 12 hr to perform and results in proteolytic degradation of the enzyme. 2° We have modified the purification scheme by omitting octylSepharose chromatography and Mono Q I chromatography (Steps 6 and 7 above, respectively). 2°The elution conditions for Mono Q II chromatography (Step 8) have been changed. 2° Following hydroxylapatite chromatography (Step 5), the enzyme preparation is desalted by dialysis against 50 mM Tris-HCl buffer (pH 8.0) containing 10 mM MgC12, 10 mM 2-mercaptoethanol, 10% glycerol, and 0.5% Triton X-100 (equilibration buffer). The dialyzed enzyme is applied to a Mono Q column (0.5 × 5 cm) that is equilibrated with equilibration buffer containing 60 mM NaC1. The column is washed with 10 column volumes of equilibration buffer containing 60 mM NaC1 followed by enzyme elution with 35 column volumes of a linear NaCI gradient (0.06-0.3 M) in equilibration buffer. Fractions containing phosphatidylinositol 4-kinase activity are pooled and stored at - 8 0 °. The Mono Q chromatography step results in a 370-fold increase in specific activity over the hydroxylapatite chromatography step with an activity yield of 75%. Overall the enzyme is purified 9000-fold relative to the cell extract with a final specific activity of 4400 nmol/ min/mg. 2o R. J. Buxeda, J. T. Nickels, Jr., C. J. Belunis, and G. M. Carman, J. Biol. Chem. 266, 13859 (1991).
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Enzyme Purity. Electrophoretic analysis using native polyacrylamide21 and sodium dodecyl sulfate-polyacrylamide22 gels indicates that the purified phosphatidylinositol 4-kinase preparation is nearly homogeneous. Native gels (6% polyacrylamide) contain 0.5% Triton X-100. Phosphatidylinositol 4-kinase activity is associated with the protein bands found in native polyacrylamide8 and sodium dodecyl sulfate-polyacrylamide gels. 8'2° The subunit molecular weight of the enzyme is 45,000. z° On storage, the enzyme is degraded to molecular weights of 35,000 and 30,000 but retains full activity. 8,z° Product Identification A standard phosphatidylinositol 4-kinase reaction is carded out with either [y-32p]ATP or phosphatidyl[2-3H]inositol as the labeled substrate. The chloroform-soluble product of the reaction is analyzed by one-dimensional paper chromatography on EDTA-treated SG81 paper z3 with chloroform-acetone-methanol-glacial acetic acid-water (40 : 15 : 13 : 12 : 8, v/v) and chloroform-methanol-2.5 M ammonium hydroxide (9 : 7 : 2, v/v) as the solvent systems. The only phospholipid product of the reaction using either labeled substrate comigrates precisely with authentic phosphatidylinositol 4-phosphate. 8'2° The 3H-labeled product of the reaction is isolated and used as the substrate for phosphoinositide-specific phospholipase C, and the inositol phosphate product is identified by high-performance liquid chromatography. 8,24 The inositol phosphate hydrolysis product of the phospholipase C reaction is identified as inositol 1,4-bisphosphate. 8 In addition, the 32p-labeled product of the reaction is isolated, then deacylated with methylamine reagent, and the water-soluble product is analyzed by high-performance liquid chromatography. 25 The product is identified as glycerophosphoinositol 4-phosphate, confirming that the product of the enzyme reaction is phosphatidylinositol 4-phosphate. 2° Properties of Phosphatidylinositol 4-Kinase Maximum phosphatidylinositol 4-kinase activity is dependent on magnesium ions (27 mM) and Triton X-100 (25 mM) at the pH optimum of 8.0: The activation energy for the reaction is 31.5 kcal/mol, and the 21 B. Davis, Ann. N.Y. Acad. Sci. 121, 404 (1964). 22 U. K. Laemmli, Nature (London) 227, 680 (1970). 23 S. Steiner and R. L. Lester, J. Bacteriol. 109, 81 (1972). 24 C. A. Hansen, S. Mah, and J. R. Williamson, J. Biol. Chem. 261, 8100 (1986). 25 D. L. Lips, P. W. Majerus, F. R. Gorga, A. T. Young, and T. L. Benjamin, J. Biol. Chem. 264, 8759 (1989).
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enzyme is thermally labile above 30°. 8 Phosphatidylinositol 4-kinase activity is inhibited by calcium ions and thioreactive agents but is not affected by various nucleotides including adenosine. 8 Kinetic experiments are conducted with a mixed micelle substrate of Triton X-100 and phosphatidylinositol. 8,2° The enzyme shows saturation kinetics with respect to the bulk (Km of 70 ~M) 8 and surface (Km of 0.4 mol%) 2° concentrations of phosphatidylinositol. The K m value for MgATP is 0.3-0.5 mM, 8,2° and the Vmaxis 4750 nmol/min/mg. 8 The turnover number for the enzyme is 166 min -~. Results of kinetic and isotopic exchange reactions indicate that phosphatidylinositol 4-kinase catalyzes a sequential Bi-Bi reaction mechanism. 8,2° The enzyme binds to phosphatidylinositol prior to ATP, and phosphatidylinositol 4-phosphate is the first product released in the reaction. 8 Synthetic Uses Pure phosphatidylinositol 4-kinase can be used to synthesize radiolabeled phosphatidylinositol 4-phosphate. 832p-Labeled phosphatidylinositol 4-phosphate is prepared from phosphatidylinositol and [y-32p]ATP, and 3H-labeled phosphatidylinositol 4-phosphate is prepared from phosphatidyl[2-3H]inositol and ATP. Acknowledgments This work was supported by U.S. Public Health Service Grant GM-35655 from the National Institutes of Health, New Jersey State funds, and the Charles and Johanna Busch Memorial Fund.
[21] P h o s p h a t i d y l i n o s i t o l - 4 - P h o s p h a t e 5 - K i n a s e s f r o m Human Erythrocytes
By CHANTAL E. BAZENET and RICHARD A. ANDERSON Introduction Polyphosphoinositides play a crucial role in cellular signal transduction. Phosphatidylinositol (PI) undergoes a series of phosphorylations to give phosphatidylinositol phosphate (PIP) and further phosphatidylinositol bisphosphate (PIP2), which produces two second messengers, 1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) , mediating Ca 2÷ mobilization in response to many hormones, neurotransmitters, and growth METHODS IN ENZYMOLOGY, VOL. 209
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
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[20] Phosphatidylinositol 4-Kinase from Yeast B y GEORGE M. CARMAN, CHARLES J. BELUNIS,
and JOSEPH T. NICKELS, JR. Introduction Phosphatidylinositol 4-kinase ( 1-phosphatidylinositol kinase) catalyzes the reaction of phosphatidylinositol with ATP to form phosphatidylinositol Phosphatidylinositol + ATP--~ phosphatidylinositol 4-phosphate + ADP 4-phosphate.~ Phosphatidylinositol 4-kinase (ATP : 1-phosphatidyl-lDm y o - i n o s i t o l 4-phosphotransferase, EC 2.7.1.67) is the first e n z y m e in the phosphorylation sequence of phosphatidylinositol leading to the formation of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in S a c c h a r o m y c e s cerevisiae, z The synthesis and turnover of the polyphosphoinositides in S. cerevisiae as well as in higher cukaryotes play an important role in cell g r o w t h J -7 Phosphatidylinositol 4-kinase is associated with the microsomal, 8 plasma membrane, 9 and cytosolic 1° fractions of S. cerevisiae. Phosphatidylinositol 4-kinase has been purified to near homogeneity from the microsomal fraction of S. cerevisiae by standard protein purification procedures. 8 We describe here the purification and properties of the enzyme. Assay Method Phosphatidylinositol 4-kinase activity is measured for 10 min by following the phosphorylation of 0.4 m M phosphatidylinositol with 5 m M [7-32p]ATP [3000-5000 counts/min (cpm)/nmol] in the presence 50 m M I M. Colodzin and E. P. Kennedy, J. Biol. Chem. 240, 3771 0965). 2 S. Stciner and R. L. Lester, Biochim. Biophy#. Acta 2,60, 82 0972). 3 R. T. Talwalkar and R. L. Lester, Biochim. Biophys. Acta 306, 412 0973).
4 K. Kaibuchi, A. Miyajima, K.-I. Arai, and K. Matsumoto, Proc. Natl. Acad. Sci. U.S.A. 83, 8172 0986). 5 C. Dahl, H.-P. Biemann, and J. Dahl, Proc. Natl. Acad. Sci. U.S.A. 84, 4012 (1987). 6 I. Uno, K. Fukami, H. Kato, T. Takenawa, and T. Ishikawa, Nature (London) 333, 188 (1988). 7 M. J. Berridge, Biochim. Biophys. Acta 907, 33 (1987).
s C. J. Belunis, M. Bae-Lee, M. J. Kelley, and G. M. Carman, J. Biol. Chem. 263, 18897 (1988). 9 A. J. Kinney and G. M. Carman, J. Bacteriol. 172, 4115 (1990). J0R. T. Talwalker and R. L. Lester, Biochim. Biophys. Acta 360, 306 (1974). METHODS IN ENZYMOLOGY,VOL. 209
Copyright © 1992by AcademicPress, Inc. All rights of reproductionin any formreserved.
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Tris-maleate buffer (pH 8.0) containing 25 mM Triton X-100, 30 mM MgCI2, and enzyme protein in a total volume of 0.1 ml at 30°) t The reaction is terminated by the addition of 0.5 ml of 0.1 N HC1 in methanol. Chloroform (1 ml) and 1 M MgCIz (1.5 ml) are added, the system is mixed, and the phases are separated by a 2-min centrifugation at 100 g at room temperature. A 0.5-ml sample of the chloroform phase is removed and taken to dryness on an 80° water bath. Betafluor (4 ml, National Diagnostics, Somerville, NJ) is added to the sample, and radioactivity is determined by scintillation counting. The chloroform-soluble phospholipid product of the reaction, phosphatidylinositol 4-phosphate, is analyzed by thin-layer chromatography with standard phosphatidylinositol 4-phosphate using the solvent system chloroform-methanol-2.5 M ammonium hydroxide ( 9 : 7 : 2 , v-v).11 Alternatively, activity is measured using 0.4 mM phosphatidyl[2-3H]inositol (2000 cpm/nmol) and 5 mM ATP as substrates. A unit of enzymatic activity is defined as the amount of enzyme that catalyzes the formation of 1 nmol of product per minute. Protein is determined by the method of Bradford) 2 Buffers which are identical to those containing the protein samples are used as blanks. The presence of Triton X-100 does not interfere with the protein determination, provided the blank contains a final concentration of detergent identical to that of the sample.
Growth of Yeast Saccharomyces cerevisiae strain ade5 MATa 13is used as a representative wild-type strain for enzyme purification. Cells are grown in 1% yeast extract, 2% peptone, and 2% glucose (w/v) at 28 ° to late exponential phase, harvested by centrifugation, and stored at -80°. 14'15 We use wild-type strain $288C for the purification of phosphatidylinositol synthase, 14a5 phosphatidylserine synthase,~6'17 and CDPdiacylglycerol synthase. 18,19We are unable to purify phosphatidylinositol 4-kinase activity from strain $288C. The reason for this is unclear. 11 M. A. McKenzie and G. M. Carman, J. Food Biochem. 6, 77 (1982). 12 M. M. Bradford, Anal. Biochem. 72, 248 (1976). 13 M. R. Culbertson and S. A. Henry, Genetics 80, 23 (1975). 14 A. S. Fischl and G. M. Carman, J. Bacteriol. 154, 304 (1983). 15 G. M. Carman and A. S. Fischl, this volume [36]. 16 M. Bae-Lee and G. M. Carman, J. Biol. Chem. 259, 10857 (1984). 17 G. M. Carman and M. Bae-Lee, this volume [35]. 18 M. J. Kelley and G. M. Carman, J. Biol. Chem. 262, 14563 (1987). 19 G. M. Carman and M. J. Kelley, this volume [28].
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Purification Procedure All steps are performed at 5°.
Step 1: Preparation of Cell Extract. Cells (200 g) are disrupted with glass beads (diameter, 0.5 mm) with a BioSpec Products (Bartlesville, OK) Bead-Beater in 50 mM Tris-HCl buffer (pH 7.5) containing 1 mM Na2EDTA, 0.3 M sucrose, and 10 mM 2-mercaptoethanol as described previously.14.15 Glass beads and unbroken cells are removed by centrifugation at 1500 g for 15 min to obtain the cell extract. Step 2: Preparation of Microsomes. Microsomes are collected from the cell extract by differential centrifugation) 4'15 Microsomal pellets are washed with 50 mM Tris-HCl buffer (pH 8.0) containing 10 mM MgC12, 10 mM 2-mercaptoethanol, and 10% glycerol. Microsomes can be frozen at - 8 0 ° until the enzyme is purified. Step 3: Preparation of Triton X-IO0 Extract. Microsomes are suspended in 50 mM Tris-HCl buffer (pH 8.0) containing 10 mM MgCI2, 10 mM 2-mercaptoethanol, 10% glycerol, and 1% Triton X-100 (v/v) 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 1.5 hr to obtain the Triton X-100 extract (supernatant). The Triton X-100 extract is immediately used for the next purification step since, at this stage, the enzyme is labile. The addition of protease inhibitors does not improve enzyme stability. Step 4:DE-52 Chromatography. A DE-52 (Whatman, Clifton, NJ) column (2.5 x 5 cm) is equilibrated with 50 mM Tris-HC1 buffer (pH 8.0) containing 10 mM MgCI2, 10 mM 2-mercaptoethanol, 10% glycerol, and 0.5% Triton X-100. The Triton X-100 extract is applied to the column at a flow rate 20 ml/hr. The column is washed with 6 column volumes of equilibration buffer followed by elution of the enzyme with equilibration buffer containing 0.1 M NaCI. Fractions containing phosphatidylinositol 4-kinase activity are pooled and immediately used for the next step in the purification scheme. The enzyme is still labile at this stage. Step 5: Hydroxylapatite Chromatography. A hydroxylapatite (BioGel HT, Bio-Rad, Richmond, CA) column (2.5 × 3 cm) is equilibrated with 10 mM potassium phosphate buffer (pH 8.0) containing 10 mM MgCI2, 10 mM 2-mercaptoethanol, 10% glycerol, and 0.2% Triton X-100. The DE52 purified enzyme is diluted 2 : 1 with equilibration buffer and applied to the column at a flow rate of 10 ml/hr. The column is washed with 6 column volumes of equilibration buffer followed by elution of the enzyme with 8 column volumes of a linear potassium phosphate gradient (0.01-0.3 M) in equilibration buffer at a flow rate of 10 ml/hr. The peak of phosphatidylinositol 4-kinase activity elutes from the column at a phosphate concentration
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of about 0.15 M. The most active fractions are pooled and used for the next step in the purification. At this stage in the purification, phosphatidylinositol 4-kinase activity is completely stable. Step 6: Octyl-Sepharose Chromatography. An octyl-Sepharose (Pharmacia LKB Biotechnology, Piscataway, NJ) column (1.5 × 16 cm) is equilibrated with 50 mM Tris-HCl buffer (pH 8.0) containing 10 mM MgCI2, 10 mM 2-mercaptoethanol, 10% glycerol, and 3 M NaCI. Solid NaCI is added to the hydroxylapatite-purified enzyme to a final concentration of 3 M, and the enzyme is applied to the octyl-Sepharose column at a flow rate of 20 ml/hr. It is necessary to add 3 M NaCI and to omit Triton X-100 from the equilibration buffer for the enzyme to bind to the octylSepharose column. The column is washed with 2 column volumes of equilibration buffer followed by 6 column volumes of equilibration buffer without NaCI. Phosphatidylinositol 4-kinase activity is then eluted from the column with 8 column volumes of a linear Triton X- 100 gradient (0-1%) in 50 mM Tris-HCl buffer (pH 8.0) containing 10 mM MgCI2, 10 mM 2-mercaptoethanol, and 10% glycerol. The peak of activity elutes from the column at a Triton X-100 concentration of about 0.5%. The most active fractions are pooled and used for the next step in the purification. Step 7: Mono Q Chromatography I. A anion-exchange Mono Q (Pharmacia LKB Biotechnology) column (0.5 x 5 cm) is equilibrated with 50 mM Tris-HC1 buffer (pH 8.0) containing 10 mM MgC12, 10 mM 2-mercaptoethanol, 10% glycerol, and 0.5% Triton X-100. Enzyme from the previous step is applied to the column at a flow rate of 60 ml/hr. The column is washed with 4 column volumes of equilibration buffer followed by 2 column volumes of equilibration buffer containing 0.2 M NaC1. The enzyme is then eluted from the column with l0 column volumes of a linear NaCl gradient (0.2-0.5 M) in equilibration buffer. The peak of enzyme activity elutes at a NaC1 concentration of about 0.22 M. Fractions containing activity are pooled and used for the next step. Step 8: Mono Q Chromatography //. A second Mono Q column (0.5 x 5 cm) is equilibrated with 50 mM Tris-HC1 buffer (pH 8.0) containing l0 mM MgCl2, l0 mM 2-mercaptoethanol, 10% glycerol, 0.5% Triton X-100, and 60 mM NaC1. The enzyme from the previous Mono Q column is diluted 4: I with equilibration buffer and applied to the column at a flow rate of 45 ml/hr. The column is washed with 7 column volumes of equilibration buffer followed by enzyme elution with 20 column volumes of a linear NaC1 gradient (0.06-0.3 M) in equilibration buffer. Phosphatidylinositol 4-kinase activity elutes at a NaC1 concentration of about 0.2 M. Fractions containing activity are pooled and stored at - 8 0 °. The purified enzyme is completely stable for at least 3 months of storage at - 80 °.
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TABLE I PURIFICATION OF PHOSPHATIDYLINOSlTOL 4-KINASE FROM S a c c h a r o m y c e s cerevisiae a
Purification step
Total units (nmol-min)
1. Cell extract 2. Microsomes 3. Triton X-100 extract 4. DE-52 5. Hydroxylapatite 6. Octyl-Sepharose 7. Mono Q I 8. Mono Q II
5970 4418 3104 1086 1032 743 558 380
Protein (mg) 10,119 2,559 794 175 57 5.64 0.24 0.08
Specific activity (units/mg) 0.59 1.72 3.9 6.2 18.1 131.7 2325 4750
Purification (-fold) 1 2.9 6.6 10.5 30.6 223.2 3940.6 8050.8
Yield (%) 100 74 52 18 17.2 12.4 9.3 6.3
a Data from Belunis et al. 8
A summary of the purification of phosphatidylinositol 4-kinase is presented in Table I. The overall purification of phosphatidylinositol 4-kinase over the cell extract is 8051-fold, with an activity yield of 6.3%. Improved Purification Procedure. The purification scheme described above includes octyl-Sepharose chromatography. This step takes approximately 12 hr to perform and results in proteolytic degradation of the enzyme. 2° We have modified the purification scheme by omitting octylSepharose chromatography and Mono Q I chromatography (Steps 6 and 7 above, respectively). 2°The elution conditions for Mono Q II chromatography (Step 8) have been changed. 2° Following hydroxylapatite chromatography (Step 5), the enzyme preparation is desalted by dialysis against 50 mM Tris-HCl buffer (pH 8.0) containing 10 mM MgC12, 10 mM 2-mercaptoethanol, 10% glycerol, and 0.5% Triton X-100 (equilibration buffer). The dialyzed enzyme is applied to a Mono Q column (0.5 × 5 cm) that is equilibrated with equilibration buffer containing 60 mM NaC1. The column is washed with 10 column volumes of equilibration buffer containing 60 mM NaC1 followed by enzyme elution with 35 column volumes of a linear NaCI gradient (0.06-0.3 M) in equilibration buffer. Fractions containing phosphatidylinositol 4-kinase activity are pooled and stored at - 8 0 °. The Mono Q chromatography step results in a 370-fold increase in specific activity over the hydroxylapatite chromatography step with an activity yield of 75%. Overall the enzyme is purified 9000-fold relative to the cell extract with a final specific activity of 4400 nmol/ min/mg. 2o R. J. Buxeda, J. T. Nickels, Jr., C. J. Belunis, and G. M. Carman, J. Biol. Chem. 266, 13859 (1991).
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Enzyme Purity. Electrophoretic analysis using native polyacrylamide21 and sodium dodecyl sulfate-polyacrylamide22 gels indicates that the purified phosphatidylinositol 4-kinase preparation is nearly homogeneous. Native gels (6% polyacrylamide) contain 0.5% Triton X-100. Phosphatidylinositol 4-kinase activity is associated with the protein bands found in native polyacrylamide8 and sodium dodecyl sulfate-polyacrylamide gels. 8'2° The subunit molecular weight of the enzyme is 45,000. z° On storage, the enzyme is degraded to molecular weights of 35,000 and 30,000 but retains full activity. 8,z° Product Identification A standard phosphatidylinositol 4-kinase reaction is carded out with either [y-32p]ATP or phosphatidyl[2-3H]inositol as the labeled substrate. The chloroform-soluble product of the reaction is analyzed by one-dimensional paper chromatography on EDTA-treated SG81 paper z3 with chloroform-acetone-methanol-glacial acetic acid-water (40 : 15 : 13 : 12 : 8, v/v) and chloroform-methanol-2.5 M ammonium hydroxide (9 : 7 : 2, v/v) as the solvent systems. The only phospholipid product of the reaction using either labeled substrate comigrates precisely with authentic phosphatidylinositol 4-phosphate. 8'2° The 3H-labeled product of the reaction is isolated and used as the substrate for phosphoinositide-specific phospholipase C, and the inositol phosphate product is identified by high-performance liquid chromatography. 8,24 The inositol phosphate hydrolysis product of the phospholipase C reaction is identified as inositol 1,4-bisphosphate. 8 In addition, the 32p-labeled product of the reaction is isolated, then deacylated with methylamine reagent, and the water-soluble product is analyzed by high-performance liquid chromatography. 25 The product is identified as glycerophosphoinositol 4-phosphate, confirming that the product of the enzyme reaction is phosphatidylinositol 4-phosphate. 2° Properties of Phosphatidylinositol 4-Kinase Maximum phosphatidylinositol 4-kinase activity is dependent on magnesium ions (27 mM) and Triton X-100 (25 mM) at the pH optimum of 8.0: The activation energy for the reaction is 31.5 kcal/mol, and the 21 B. Davis, Ann. N.Y. Acad. Sci. 121, 404 (1964). 22 U. K. Laemmli, Nature (London) 227, 680 (1970). 23 S. Steiner and R. L. Lester, J. Bacteriol. 109, 81 (1972). 24 C. A. Hansen, S. Mah, and J. R. Williamson, J. Biol. Chem. 261, 8100 (1986). 25 D. L. Lips, P. W. Majerus, F. R. Gorga, A. T. Young, and T. L. Benjamin, J. Biol. Chem. 264, 8759 (1989).
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enzyme is thermally labile above 30°. 8 Phosphatidylinositol 4-kinase activity is inhibited by calcium ions and thioreactive agents but is not affected by various nucleotides including adenosine. 8 Kinetic experiments are conducted with a mixed micelle substrate of Triton X-100 and phosphatidylinositol. 8,2° The enzyme shows saturation kinetics with respect to the bulk (Km of 70 ~M) 8 and surface (Km of 0.4 mol%) 2° concentrations of phosphatidylinositol. The K m value for MgATP is 0.3-0.5 mM, 8,2° and the Vmaxis 4750 nmol/min/mg. 8 The turnover number for the enzyme is 166 min -~. Results of kinetic and isotopic exchange reactions indicate that phosphatidylinositol 4-kinase catalyzes a sequential Bi-Bi reaction mechanism. 8,2° The enzyme binds to phosphatidylinositol prior to ATP, and phosphatidylinositol 4-phosphate is the first product released in the reaction. 8 Synthetic Uses Pure phosphatidylinositol 4-kinase can be used to synthesize radiolabeled phosphatidylinositol 4-phosphate. 832p-Labeled phosphatidylinositol 4-phosphate is prepared from phosphatidylinositol and [y-32p]ATP, and 3H-labeled phosphatidylinositol 4-phosphate is prepared from phosphatidyl[2-3H]inositol and ATP. Acknowledgments This work was supported by U.S. Public Health Service Grant GM-35655 from the National Institutes of Health, New Jersey State funds, and the Charles and Johanna Busch Memorial Fund.
[21] P h o s p h a t i d y l i n o s i t o l - 4 - P h o s p h a t e 5 - K i n a s e s f r o m Human Erythrocytes
By CHANTAL E. BAZENET and RICHARD A. ANDERSON Introduction Polyphosphoinositides play a crucial role in cellular signal transduction. Phosphatidylinositol (PI) undergoes a series of phosphorylations to give phosphatidylinositol phosphate (PIP) and further phosphatidylinositol bisphosphate (PIP2), which produces two second messengers, 1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) , mediating Ca 2÷ mobilization in response to many hormones, neurotransmitters, and growth METHODS IN ENZYMOLOGY, VOL. 209
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
