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Ca 2+ is inhibitory in the standard assay in the 10-100/zM range, and the inhibition is competitive with Mg 2÷ or Mn2÷. 2° Topography on Microsomal Membrane. The resistance to extraction from membranes with high salt, EDTA, or low detergent concentrations and the inactivation that coincides with detergent-mediated solubilization of membrane bilayers suggest that CPT is an integral membrane protein. 17 Since CDPcholine is impermeable to intact microsomal membranes, one would anticipate that the active site of CPT faces the cytosol. Evidence supporting this notion includes inactivation by membrane-impermeant proteases or mercury-dextran. 21-23 Reversibility. Incubation of microsomes with CMP inhibits the incorporation of [14C]CDPcholine into PC by stimulation of the back-reaction of CPT. 24 CMP at 0.5 mM inhibits rat liver microsomal CPT 50% when assayed by the method described above. The back-reaction can be used to generate endogenous diacylglycerol. The Km for CMP in the backreaction ranges from 0.18 to 0.35 mM depending on the source. 11,25Pitfalls associated with this procedure have been described in an earlier volume of this series./4 21 R. Coleman and R. M. Bell, J. Cell Biol. 76, 245 (1978). 22 D. E. Vance, P. C. Choy, S. B. Farren, P. Lira, and W. J. Schneider, Nature (London) 270, 268 (1977). 23 L. M. Ballas and R. M. Bell, Biochim. Biophys. Acta 602, 578 (1980). 24 G. Goracci, P. Gresele, G. Arienti, P. Porrovecchio, G. Nenci, and G. Porcellati, Lipids 18, 179 (1983). 2~ G. Goracci, E. Francescangeli, L. Horrocks, and G. Porcellati, Biochim. Biophys. Acta 664, 373 (1981).
[32] C h o l i n e - a n d E t h a n o l a m i n e p h o s p h o t r a n s f e r a s e s
from
Saccharomyces cerevisiae By RUSSELL H. HJELMSTAD and ROBERT M. BELL Introduction Phosphatidylcholine (PC) and phosphatidylethanolamine (PE), the principal phospholipids of eukaryotic membranes, are synthesized from the common precursor sn-1,2-diacylglycerol and CDPcholine or CDPethanolamine, respectively, in reactions catalyzed by membrane-bound amino alcohol phosphotransferases. 1-3 Comparative enzymological studies in l E. P. Kennedy and S. B. Weiss, J. Biol. Chem. 222, 193 (1956).
METHODSIN ENZYMOLOGY,VOL. 209
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Saccharomyces cerevisiae 4 and higher eukaryotic cells 3 have suggested the presence of distinct microsomal cholinephosphotransferase (EC 2.7.8.2) and ethanolaminephosphotransferase (EC 2.7.8.1) enzymes. In addition, a distinct cholinephosphotransferase may function in 1-alkyl-2acetyloglycerophosphocholine(platelet-activating factor, PAF) 5synthesis. The yeast S. cerevisiae provides an excellent genetic system in which to pursue in-depth studies on the structure, function, and regulation of choline- and ethanolaminephosphotransferases. Mutants defective in cholinephosphotransferase (cpt mutants) and ethanolaminephosphotransferase (ept mutants) activities were isolated, 6'7 and the corresponding structural genes for two amino alcohol phosphotransferases have been cloned, sequenced, and used to generate chromosomal null mutations. 6-9 The CPT1 gene product is primarily a cholinephosphotransferase, whereas the EPT1 gene product catalyzes both choline- and ethanolaminephosphotransferase reactions. The inferred gene products (CPT1: 407 amino acids, 46,000 Da; EPTI: 391 amino acids, 44,525 Da) bear 54% amino acid identity, are highly hydrophobic, and have similar predicted secondary structures including seven putative membrane-spanning segments. Together, the CPT1 and EPT1 gene products make up the complete set of amino alcohol phosphotransferases active in yeast, 1°permitting the study of the individual gene products in membranes prepared from strains bearing a null mutation in the cognate gene. The mixed micellar assays described here were developed to perform detailed enzymological studies of the individual CPT1 and EPTI gene products.
Assay Method Principle. Yeast choline- and ethanolaminephosphotransferase activities arc determined as the amount of 32p incorporatcd from [32p]CDPcholine or [32p]CDPethanolamine into PC or PE, respectively. The activities are measured in Triton X-100 mixed micelles containing sn-I,2-dioleoyl2 C. R. H. Raetz and J. D. Esko, in "The Enzymes" (P. D. Boyer, ed.), 3rd Ed., vol. 16, p. 207. Academic Press, New York, 1983. 3 R. M. Bell, Annu. Rev. Biochem. 49, 459 (1980). 4 A. K. Percy, M. A. Carson, J. F. Moore, and C. J. Waechter, Arch. Biochem. Biophys. 230, 69 (1984). 5 D. S. Woodard, T. Lee, and F. Snyder, J. Biol. Chem. 262, 2520 (1987). 6 R. H. Hjelmstad and R. M. Bell, J. Biol. Chem. 262, 3909 (1987). 7 R. H. Hjelmstad and R. M. Bell, J. Biol. Chem. 263, 19748 (1988). s R. H. Hjelmstad and R. M. Bell, J. Biol. Chem. 265, 1755 (1990). 9 R. H. Hjelmstad and R. M. Bell, J. Biol. Chem. 266, 5094 (1991). ~0R. H. Hjeimstad and R. M. Bell, J. Biol. Chem. 266, 4357 (1991).
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PHOSPHOTRANSFERASES
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glycerol and dioleoyl-PC. The product [32p]PC o r [32p]PE is extracted into acidified organic solvent and quantitated by scintillation counting. Delivery of the water-insoluble diacylglycerol substrate in the homogeneous micellar phase overcomes problems encountered in previous assays 4'6 arising from the physical properties of this substrate and effectively dilutes endogenous diacylglycerols present in yeast membrane preparations. Endogenous phospholipids are similarly subject to surface dilution, and complete dependence on a phospholipid activator is observed. The concentration dependencies of lipid substrates and cofactors as well as water-soluble components can thus be systematically studied using the mixed micellar assays. Reagents. 4-Morpholinepropanesulfonic acid (MOPS), CDPcholine, CDPethanolamine, and MgCI2 are from Sigma Chemical Company (St. Louis, MO). Triton X-100 (10% aqueous detergent in sealed ampules) is purchased from Pierce Chemical Company (Rockford, IL). L-a-Dioleoylphosphatidylcholine is obtained from Avanti Polar Lipids (Birmingham, AL). sn-l,2-Dioleoylglycerol is prepared from L-a-dioleoyl-PC by phospholipase C digestion1~ and quantitated as detailed elsewhere in this volume. 12 Other reagents are obtained or prepared as previously des c r i b e d . 6'7'1° Both dioleoylglycerol (-25-30 mM) and dioleoyl-PC (20 mg/ ml, 25.4 mM) are stored as chloroform solutions at - 2 0 ° under argon. Radiolabeled Substrates. [methyl-14C]CDPcholine and [ethanolamine1,2-14C]CDPethanolamine are commercially available but are expensive, and the available specific activity of the ~4Ccompounds is limiting for some experiments. We have developed inexpensive and reliable procedures for the synthesis of [32p]CDPcholine and [32p]CDPethanolamine from [32p]p i . Complete experimental protocols for these radiochemical syntheses have been published. 6,1° Briefly, choline [32p]phosphate or ethanolamine [32P]phosphate is prepared from [32p]p i by including choline or ethanolamine and yeast choline kinase in a standard enzymatic [y-3ZP]ATP-generating system. The product amino alcohol [32p]phosphates are purified by anion-exchange chromatography. The ethanolamine [32P]phosphate is converted to the benzyl carbamate derivative, and the protected amine is purified by cation-exchange chromatography. The choline [32p]phosphate and ethanolamine [32p]phosphate benzyl carbamates are then used in nucleophilic displacement reactions with CMPmorpholidate to give [32p]CDPcholine and [32p]CDPethanolamine benzyl carbamates. The latter product is deprotected, and both [32p]CDPamino alcohols are purified by anion-exchange chromatography. 6'~° li R. D. Mavis, R. M. Bell, and P. R. Vagelos, J. Biol. Chem. 247, 2835 (1972). 12 j. p. Walsh and R. M. Bell, this volume [17].
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The concentrations of CDPcholine and CDPethanolamine are determined by the phosphate assay of Ames and Dubin 13 and ultraviolet absorbance measurements at 280 nm using an extinction coefficient of 12.8 mM-1 cm-1 for the CMP moieties. Both substrates may be prepared at initial radiochemical specific activities such that they remain useful for periods of 3 months. The more stable CDP[methyl-3H]choline can also be prepared using a modification of this method. 6 [methyl-3H]Choline may be purchased from New England Nuclear (Boston, MA). The enzymatic and chemical synthesis of radiolabeled substrates may be generalized to prepare [3zp]CDPdimethylethanolamine and [32p]CDPmonomethylethanolamine. 10The yeast choline kinase utilizes the dimethylethanolamine and monomethylethanolamine substrates poorly but will efficiently convert them to their corresponding [32p]phosphates in the presence of regenerating ATP. Enzyme Preparations. Membranes are prepared from yeast cultures and the protein concentration determined as previously described. 7 Aliquots stored at - 7 0 ° (1-I0 mg/ml protein) are thawed immediately prior to use and diluted using isolation buffer (20%, w/v, glycerol, 50 mM MOPS, pH 7.5, and 1 mM EDTA) to the appropriate concentration such that the desired amount may be delivered in a volume of 10/xl. Membranes containing solely the CPT1 gene product activity are prepared from strain HJ051 which bears a chromosomal insertional/deletional mutation in the EPT1 locus generated by gene disruption. 7 Similarly, membranes exclusively containing the EPT1 gene product activity are derived from HJ001 which contains an insertional mutation in the CPT1 locus. 8 The wild-type parental strain DBY7466 contains a mixture of both enzymes. Both of these strains have been shown to exhibit true null phenotypes with respect to the disrupted genes. 8'9 Assay Procedure. The cholinephosphotransferase assay contains the following in a total volume of 200/zl: 50 mM M O P S - N a O H (pH 7.5), 20 mM MgC12, 0.45% (w/v, 6.5 mM) Triton X-100, 10 mol % sn-l,2dioleoylglycerol, 10 mol % dioleoyl-PC, and 0.5 mM [32p]CDPcholine. The ethanolaminephosphotransferase contains precisely the same constituents except that 0.1 mM [32p]CDPethanolamine is used rather than the [32p]CDPcholine. The mole fractions of lipid components in the micellar phase are estimated as the mole lipid/total mole detergent ratio, neglecting the monomer concentration of Triton X-100 (0.3 mM), which is less than 5% of the total Triton X-100 concentration.14 A 4 × assay mix is prepared which contains 1.8% (w/v) Triton X-100 13 B. N. Ames and D. T. Dubin, J. Biol. Chem. 235, 769 (1960). 14 A. Helenius, D. R. McCaslin, E. Fries, and C. Tanford, this series, Vol. 56, p. 734.
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(27.7 mM), 200 mM M O P S - N a O H (pH 7.5), 80 mM MgCI2, and 10 mol % each of dioleoylglycerol and dioleoyl-PC; 50/~l is prepared for each assay to be performed. Based on the amount of Triton X-100 to be used in the 4 x solution [/~mol Triton X-100 = 27.7 mM x volume 4 x solution (ml)], volumes of dioleoylglycerol and dioleoyl-PC stock solutions in chloroform are pipetted into a glass screw-capped test tube using a Hamilton syringe such that 10 mol % of each are present (/~mol lipid = 0.1 x /xmol Triton X-100). The chloroform is dried under a stream of dry nitrogen. The remaining constituents of the 4 × stock solution are then added to the dried lipids using stock solutions (10%, w/v, Triton X-100, I M MOPS-NaOH, pH 7.5, and 1 M MgCl2). Solubilization of the lipids into detergent is achieved by alternating vigorous vortexing, bath sonication, and shaking in a 37° water bath until the solution is clear and no visible lipid film remains on the bottom of the tube. [32p]CDPcholine and [32p]CDPethanolamine are prepared as 4 x substrate solutions of 2.0 and 0.4 raM, respectively. The radiolabeled compounds are stored as aqueous solutions at - 2 0 ° at concentrations of approximately 5 mM. Their radioactive specific activity is initially 500-1000 mCi/mmol. The 4 x stock solutions are prepared using the 32p. labeled compounds and unlabeled CDPamino alcohols (also stored as ~5 mM solutions) such that the final specific activity is 40 mCi/mmol. For some experiments, higher specific activities are required and may be easily prepared by varying the relative amounts of labeled and unlabeled compounds used to prepare the 4 x substrate solution. To perform the assay, 50 ~l of the 4 x assay mix is dispensed into each individual assay tube. Water is then added to bring the volume of each tube to 140/.d. Membranes (10/~l, 5-100/~g protein) are then added to each tube followed by brief vortexing. The mixture is allowed to stand at room temperature for 5 min, after which the reaction is initiated by the addition of 50/xl of 4 x [32p]CDPcholine or [32p]CDPethanolamine. After 10 min at 25°, the reaction is terminated by the addition of 0.6 ml of 1% perchloric acid and 3 ml of methanol-chloroform (2 : l, v/v). Phases are broken by addition of 1 ml each of chloroform and 1% perchloric acid followed by brief centrifugation at high speed in a clinical centrifuge. The upper phase is removed, and the lower phase is washed with two successive 2-ml portions of 1% perchloric acid, each wash being followed by repeated centrifugation and removal of the upper phase. One milliliter of the final lower chloroform phase is pipetted into a scintillation vial, the chloroform is removed by heating in a heating block, and the radioactivity is determined by scintillation counting in 4 ml of Aquasol-2 (New England Nuclear). Greater than 99% of the chloroform-soluble radioactivity produced in the choline- and ethanolaminephosphotransferase assays comi-
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grates with authentic PC and PE, respectively, on analysis by thin-layer chromatography. ~0Both assays are linear with respect to time and protein in the ranges of 0-30 min and 0-I00/zg of membrane protein. Variation of water-soluble components can be accommodated in the assay by utilizing the volume ordinarily added as water to the assay. When variation of lipid components is desired, the lipid to be varied is omitted from the 4 × assay mix and is instead delivered to each assay tube in chloroform. After removal of chloroform by drying, the variable lipid is solubilized by the 4 x assay mix containing the nonvariable lipid, detergent, and water-soluble components by vortexing and shaking as described above. We have observed that solubilization of dioleoylglycerol into Triton X-100 mixed micelles is more easily achieved in the presence of phospholipid and that when this is not possible, greater solubilization efforts are required.
Properties of the Yeast CPT1 and EPTI Gene Products DiacylglycerolDependencies. Both the CPTI and EPTI gene products exhibit Michaelis-Menten kinetics with respect to the mole fraction of dioleoylglycerol present in the micellar phase. It is essential to express the concentration of surface active components in this way since mixed micellar assays exhibit "surface dilution kinetics.''~5 Because purified preparations of these enzymes are not yet available, the crude membrane preparations used to measure their activitiescontain appreciable endogenous diacylglycerol which is also subject to surface dilution.As the amount of membrane protein added to the assay is decreased, the mole fraction of endogenous diacylglycerol in the assay becomes negligible, and complete dependence on exogenous diacylglycerol is achieved. Less than 20/~g of membrane protein should be used in experiments in which dilution of endogenous diacylglycerol is important. Phospholipid Activator Requirement. Nonlinearity with protein concentration in both assays suggested the presence of a dissociable enzyme activator substance present in the membranes; this requirement could be satisfied by exogenous PC. l0 Like endogenous diacylglycerol, endogenous phospholipid is effectively diluted into the micellar phase, and its mole fraction becomes negligible at low membrane protein concentrations; absolute requirements for a phospholipid activator can be demonstrated for the activities of both the CPT1 and EPT1 gene products. Less than 10/zg of membrane protein must be used to achieve complete dependence on 15 E. A. Dennis, in "The Enzymes" (P. D. Boyer, ed.), 3rd Ed., Vol. 16, p. 307. Academic Press, New York, 1983.
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TABLE I KINETIC PARAMETERSOF AMINO ALCOHOL PHOSPHOTRANSFERASEACTIVITIES OF CPT1 AND EPT1 GENE PRODUCTSa Vmax (nmol/min/mg protein)
Km (/xM) Gene product(s) (strain)
ChoPT
DMEPT
MMEPT
EthPT
109
27
26
20
0.78
120 100
29 137
29 b
22 __
0.62 0.20
Wild-type mixture (DBY746) EPT1 (HJ001) CPT1 (HJ051)
ChoPT DMEPT
MMEPT
EthPT
0.44
0.30
1.26
0.42 0.07
0,27 --
1.35 --
Kinetic parameters were obtained from double-reciprocal plots. ChoPT, Cholinephosphotransferase; DMEPT, dimethylethanolaminephosphotransferase; MMEPT, monomethylethanolaminephosphotransferase; EthPT, ethanolaminephosphotransferase. From Hjelmstad and Bell. I° b Could not be evaluated.
exogenous phospholipid. Several naturally occurring phospholipids satisfy the phospholipid activator requirement of these enzymes to varying extents. 10 CDPamino Alcohol Specificities. The activities of the CPT1 and EPT1 gene products have been evaluated using a series of CDPamino alcohol substrates including CDPcholine, CDPdimethylethanolamine, CDPmonomethylethanolamine, and CDPethanolamine. ~° The CPT1 gene product TABLE II DIFFERENTIAL PROPERTIES OF CPT1 AND E P T I GENE PRODUCTS EPT1 Gene product
Property (units) K m, dioleoylglycerol (mol %) Kin, CDPcholine (p,M) K m, CDPethanolamine (/zM) Ka, Mg2÷ (mM) Phospholipid activation by PE (%)~ Inhibition by CMP (% remaining activity) b
Ethanolaminephosphotransferase activity
Cholinephosphotransferase activity
CPT1 Gene product,
3.3 -22 2.6 13
2.9 120 -2.6 65
8.0 110 -0.7 65
16
71
18
cholinephosphotransferase activity
a Activity obtained using 10 mol % PE is expressed as a percentage of that measured when 10 mol % PC is used. b Value reported represents the percent remaining activity in the presence of 1 mM CMP relative to no addition.
[33]
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279
primarily utilizes CDPcholine and CDPethanolamine, whereas the EPT1 gene product utilizes all four derivatives to appreciable extents (Table I). The CPT1 gene product does, however, have measurable activity when CDPethanolamine or CDPmonomethylethanolamine are employed. Monomethylethanolamine- and dimethylethanolaminephosphotransferase activities solely represent alternative substrate specificities of the CPT1 and EPT1 gene products since cptl eptl double-mutant strains completely lack these activities.~° Mg 2+ Dependencies. The activities of both the CPT1 and EPTI gene products exhibit absolute requirements for a divalent metal ion. Differential Properties o f the CPT1 and EPT1 Gene Products. The properties of the CPT1 and EPT1 gene product-dependent cholinephosphotransferase activities and the EPT1 gene product-dependent ethanolaminephosphotransferase activity are summarized in Table II. Intrinsic properties of the two enzymes which are independent of the CDPamino alcohol employed include the apparent K mfor dioleoylglycerol, the apparent K a for M g 2+ , and inhibition by CMP. The latter property, which occurs by an undetermined mechanism, is useful in assessing the relative contributions of the CPTI and EPT1 gene products to total cholinephosphotransferase activity in membranes containing a mixture of the two enzymes, l° The CPT1 and EPT1 gene products also exhibit differing properties with respect to phospholipid activation; however, this difference is dependent on the CDPamino alcohol employed, and the cholinephosphotransferase activities of both enzymes are indistinguishable with respect to this property.
[33] 1 - A l k y l - 2 - a c e t y l - s n - g l y c e r o l C h o l i n e p h o s p h o t r a n s f e r a s e By TEN-CHING LEE and FRED SNYDER
Introduction A dithiothreitol (DTT)-insensitive cholinephosphotransferase (EC 2.7.8.16, 1-alkyl-2-acetyl-sn-glycerol cholinephosphotransferase) catalyzes the transfer of phosphocholine from CDPcholine to l-alkyl-2-acetylsn-glycerols (Fig. 1) in the final step of the biosynthesis of platelet-activating factor (PAF) in the de novo pathway ~'2;in fact the term DTT-insensitive I W. Renooij and F. Snyder, Biochim. Biophys. Acta 663, 545 (1981). 2 D. S. Wo odard , T.-C. Lee, and F. Snyder, J. Biol. Chem. 262, 2520 (1987).
METHODS IN ENZYMOLOGY, VOL. 209
Copyright© 1992by AcademicPress, Inc. All rights of reproduction in any form reserved.
PHOSPHOTRANSFERASES
[32]
Ca 2+ is inhibitory in the standard assay in the 10-100/zM range, and the inhibition is competitive with Mg 2÷ or Mn2÷. 2° Topography on Microsomal Membrane. The resistance to extraction from membranes with high salt, EDTA, or low detergent concentrations and the inactivation that coincides with detergent-mediated solubilization of membrane bilayers suggest that CPT is an integral membrane protein. 17 Since CDPcholine is impermeable to intact microsomal membranes, one would anticipate that the active site of CPT faces the cytosol. Evidence supporting this notion includes inactivation by membrane-impermeant proteases or mercury-dextran. 21-23 Reversibility. Incubation of microsomes with CMP inhibits the incorporation of [14C]CDPcholine into PC by stimulation of the back-reaction of CPT. 24 CMP at 0.5 mM inhibits rat liver microsomal CPT 50% when assayed by the method described above. The back-reaction can be used to generate endogenous diacylglycerol. The Km for CMP in the backreaction ranges from 0.18 to 0.35 mM depending on the source. 11,25Pitfalls associated with this procedure have been described in an earlier volume of this series./4 21 R. Coleman and R. M. Bell, J. Cell Biol. 76, 245 (1978). 22 D. E. Vance, P. C. Choy, S. B. Farren, P. Lira, and W. J. Schneider, Nature (London) 270, 268 (1977). 23 L. M. Ballas and R. M. Bell, Biochim. Biophys. Acta 602, 578 (1980). 24 G. Goracci, P. Gresele, G. Arienti, P. Porrovecchio, G. Nenci, and G. Porcellati, Lipids 18, 179 (1983). 2~ G. Goracci, E. Francescangeli, L. Horrocks, and G. Porcellati, Biochim. Biophys. Acta 664, 373 (1981).
[32] C h o l i n e - a n d E t h a n o l a m i n e p h o s p h o t r a n s f e r a s e s
from
Saccharomyces cerevisiae By RUSSELL H. HJELMSTAD and ROBERT M. BELL Introduction Phosphatidylcholine (PC) and phosphatidylethanolamine (PE), the principal phospholipids of eukaryotic membranes, are synthesized from the common precursor sn-1,2-diacylglycerol and CDPcholine or CDPethanolamine, respectively, in reactions catalyzed by membrane-bound amino alcohol phosphotransferases. 1-3 Comparative enzymological studies in l E. P. Kennedy and S. B. Weiss, J. Biol. Chem. 222, 193 (1956).
METHODSIN ENZYMOLOGY,VOL. 209
Copyright© 1992by AcademicPress, Inc. All fightsof reproductionin any formreserved.
[32]
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273
Saccharomyces cerevisiae 4 and higher eukaryotic cells 3 have suggested the presence of distinct microsomal cholinephosphotransferase (EC 2.7.8.2) and ethanolaminephosphotransferase (EC 2.7.8.1) enzymes. In addition, a distinct cholinephosphotransferase may function in 1-alkyl-2acetyloglycerophosphocholine(platelet-activating factor, PAF) 5synthesis. The yeast S. cerevisiae provides an excellent genetic system in which to pursue in-depth studies on the structure, function, and regulation of choline- and ethanolaminephosphotransferases. Mutants defective in cholinephosphotransferase (cpt mutants) and ethanolaminephosphotransferase (ept mutants) activities were isolated, 6'7 and the corresponding structural genes for two amino alcohol phosphotransferases have been cloned, sequenced, and used to generate chromosomal null mutations. 6-9 The CPT1 gene product is primarily a cholinephosphotransferase, whereas the EPT1 gene product catalyzes both choline- and ethanolaminephosphotransferase reactions. The inferred gene products (CPT1: 407 amino acids, 46,000 Da; EPTI: 391 amino acids, 44,525 Da) bear 54% amino acid identity, are highly hydrophobic, and have similar predicted secondary structures including seven putative membrane-spanning segments. Together, the CPT1 and EPT1 gene products make up the complete set of amino alcohol phosphotransferases active in yeast, 1°permitting the study of the individual gene products in membranes prepared from strains bearing a null mutation in the cognate gene. The mixed micellar assays described here were developed to perform detailed enzymological studies of the individual CPT1 and EPTI gene products.
Assay Method Principle. Yeast choline- and ethanolaminephosphotransferase activities arc determined as the amount of 32p incorporatcd from [32p]CDPcholine or [32p]CDPethanolamine into PC or PE, respectively. The activities are measured in Triton X-100 mixed micelles containing sn-I,2-dioleoyl2 C. R. H. Raetz and J. D. Esko, in "The Enzymes" (P. D. Boyer, ed.), 3rd Ed., vol. 16, p. 207. Academic Press, New York, 1983. 3 R. M. Bell, Annu. Rev. Biochem. 49, 459 (1980). 4 A. K. Percy, M. A. Carson, J. F. Moore, and C. J. Waechter, Arch. Biochem. Biophys. 230, 69 (1984). 5 D. S. Woodard, T. Lee, and F. Snyder, J. Biol. Chem. 262, 2520 (1987). 6 R. H. Hjelmstad and R. M. Bell, J. Biol. Chem. 262, 3909 (1987). 7 R. H. Hjelmstad and R. M. Bell, J. Biol. Chem. 263, 19748 (1988). s R. H. Hjelmstad and R. M. Bell, J. Biol. Chem. 265, 1755 (1990). 9 R. H. Hjelmstad and R. M. Bell, J. Biol. Chem. 266, 5094 (1991). ~0R. H. Hjeimstad and R. M. Bell, J. Biol. Chem. 266, 4357 (1991).
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PHOSPHOTRANSFERASES
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glycerol and dioleoyl-PC. The product [32p]PC o r [32p]PE is extracted into acidified organic solvent and quantitated by scintillation counting. Delivery of the water-insoluble diacylglycerol substrate in the homogeneous micellar phase overcomes problems encountered in previous assays 4'6 arising from the physical properties of this substrate and effectively dilutes endogenous diacylglycerols present in yeast membrane preparations. Endogenous phospholipids are similarly subject to surface dilution, and complete dependence on a phospholipid activator is observed. The concentration dependencies of lipid substrates and cofactors as well as water-soluble components can thus be systematically studied using the mixed micellar assays. Reagents. 4-Morpholinepropanesulfonic acid (MOPS), CDPcholine, CDPethanolamine, and MgCI2 are from Sigma Chemical Company (St. Louis, MO). Triton X-100 (10% aqueous detergent in sealed ampules) is purchased from Pierce Chemical Company (Rockford, IL). L-a-Dioleoylphosphatidylcholine is obtained from Avanti Polar Lipids (Birmingham, AL). sn-l,2-Dioleoylglycerol is prepared from L-a-dioleoyl-PC by phospholipase C digestion1~ and quantitated as detailed elsewhere in this volume. 12 Other reagents are obtained or prepared as previously des c r i b e d . 6'7'1° Both dioleoylglycerol (-25-30 mM) and dioleoyl-PC (20 mg/ ml, 25.4 mM) are stored as chloroform solutions at - 2 0 ° under argon. Radiolabeled Substrates. [methyl-14C]CDPcholine and [ethanolamine1,2-14C]CDPethanolamine are commercially available but are expensive, and the available specific activity of the ~4Ccompounds is limiting for some experiments. We have developed inexpensive and reliable procedures for the synthesis of [32p]CDPcholine and [32p]CDPethanolamine from [32p]p i . Complete experimental protocols for these radiochemical syntheses have been published. 6,1° Briefly, choline [32p]phosphate or ethanolamine [32P]phosphate is prepared from [32p]p i by including choline or ethanolamine and yeast choline kinase in a standard enzymatic [y-3ZP]ATP-generating system. The product amino alcohol [32p]phosphates are purified by anion-exchange chromatography. The ethanolamine [32P]phosphate is converted to the benzyl carbamate derivative, and the protected amine is purified by cation-exchange chromatography. The choline [32p]phosphate and ethanolamine [32p]phosphate benzyl carbamates are then used in nucleophilic displacement reactions with CMPmorpholidate to give [32p]CDPcholine and [32p]CDPethanolamine benzyl carbamates. The latter product is deprotected, and both [32p]CDPamino alcohols are purified by anion-exchange chromatography. 6'~° li R. D. Mavis, R. M. Bell, and P. R. Vagelos, J. Biol. Chem. 247, 2835 (1972). 12 j. p. Walsh and R. M. Bell, this volume [17].
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The concentrations of CDPcholine and CDPethanolamine are determined by the phosphate assay of Ames and Dubin 13 and ultraviolet absorbance measurements at 280 nm using an extinction coefficient of 12.8 mM-1 cm-1 for the CMP moieties. Both substrates may be prepared at initial radiochemical specific activities such that they remain useful for periods of 3 months. The more stable CDP[methyl-3H]choline can also be prepared using a modification of this method. 6 [methyl-3H]Choline may be purchased from New England Nuclear (Boston, MA). The enzymatic and chemical synthesis of radiolabeled substrates may be generalized to prepare [3zp]CDPdimethylethanolamine and [32p]CDPmonomethylethanolamine. 10The yeast choline kinase utilizes the dimethylethanolamine and monomethylethanolamine substrates poorly but will efficiently convert them to their corresponding [32p]phosphates in the presence of regenerating ATP. Enzyme Preparations. Membranes are prepared from yeast cultures and the protein concentration determined as previously described. 7 Aliquots stored at - 7 0 ° (1-I0 mg/ml protein) are thawed immediately prior to use and diluted using isolation buffer (20%, w/v, glycerol, 50 mM MOPS, pH 7.5, and 1 mM EDTA) to the appropriate concentration such that the desired amount may be delivered in a volume of 10/xl. Membranes containing solely the CPT1 gene product activity are prepared from strain HJ051 which bears a chromosomal insertional/deletional mutation in the EPT1 locus generated by gene disruption. 7 Similarly, membranes exclusively containing the EPT1 gene product activity are derived from HJ001 which contains an insertional mutation in the CPT1 locus. 8 The wild-type parental strain DBY7466 contains a mixture of both enzymes. Both of these strains have been shown to exhibit true null phenotypes with respect to the disrupted genes. 8'9 Assay Procedure. The cholinephosphotransferase assay contains the following in a total volume of 200/zl: 50 mM M O P S - N a O H (pH 7.5), 20 mM MgC12, 0.45% (w/v, 6.5 mM) Triton X-100, 10 mol % sn-l,2dioleoylglycerol, 10 mol % dioleoyl-PC, and 0.5 mM [32p]CDPcholine. The ethanolaminephosphotransferase contains precisely the same constituents except that 0.1 mM [32p]CDPethanolamine is used rather than the [32p]CDPcholine. The mole fractions of lipid components in the micellar phase are estimated as the mole lipid/total mole detergent ratio, neglecting the monomer concentration of Triton X-100 (0.3 mM), which is less than 5% of the total Triton X-100 concentration.14 A 4 × assay mix is prepared which contains 1.8% (w/v) Triton X-100 13 B. N. Ames and D. T. Dubin, J. Biol. Chem. 235, 769 (1960). 14 A. Helenius, D. R. McCaslin, E. Fries, and C. Tanford, this series, Vol. 56, p. 734.
276
PHOSPHOTRANSFERASES
[32]
(27.7 mM), 200 mM M O P S - N a O H (pH 7.5), 80 mM MgCI2, and 10 mol % each of dioleoylglycerol and dioleoyl-PC; 50/~l is prepared for each assay to be performed. Based on the amount of Triton X-100 to be used in the 4 x solution [/~mol Triton X-100 = 27.7 mM x volume 4 x solution (ml)], volumes of dioleoylglycerol and dioleoyl-PC stock solutions in chloroform are pipetted into a glass screw-capped test tube using a Hamilton syringe such that 10 mol % of each are present (/~mol lipid = 0.1 x /xmol Triton X-100). The chloroform is dried under a stream of dry nitrogen. The remaining constituents of the 4 × stock solution are then added to the dried lipids using stock solutions (10%, w/v, Triton X-100, I M MOPS-NaOH, pH 7.5, and 1 M MgCl2). Solubilization of the lipids into detergent is achieved by alternating vigorous vortexing, bath sonication, and shaking in a 37° water bath until the solution is clear and no visible lipid film remains on the bottom of the tube. [32p]CDPcholine and [32p]CDPethanolamine are prepared as 4 x substrate solutions of 2.0 and 0.4 raM, respectively. The radiolabeled compounds are stored as aqueous solutions at - 2 0 ° at concentrations of approximately 5 mM. Their radioactive specific activity is initially 500-1000 mCi/mmol. The 4 x stock solutions are prepared using the 32p. labeled compounds and unlabeled CDPamino alcohols (also stored as ~5 mM solutions) such that the final specific activity is 40 mCi/mmol. For some experiments, higher specific activities are required and may be easily prepared by varying the relative amounts of labeled and unlabeled compounds used to prepare the 4 x substrate solution. To perform the assay, 50 ~l of the 4 x assay mix is dispensed into each individual assay tube. Water is then added to bring the volume of each tube to 140/.d. Membranes (10/~l, 5-100/~g protein) are then added to each tube followed by brief vortexing. The mixture is allowed to stand at room temperature for 5 min, after which the reaction is initiated by the addition of 50/xl of 4 x [32p]CDPcholine or [32p]CDPethanolamine. After 10 min at 25°, the reaction is terminated by the addition of 0.6 ml of 1% perchloric acid and 3 ml of methanol-chloroform (2 : l, v/v). Phases are broken by addition of 1 ml each of chloroform and 1% perchloric acid followed by brief centrifugation at high speed in a clinical centrifuge. The upper phase is removed, and the lower phase is washed with two successive 2-ml portions of 1% perchloric acid, each wash being followed by repeated centrifugation and removal of the upper phase. One milliliter of the final lower chloroform phase is pipetted into a scintillation vial, the chloroform is removed by heating in a heating block, and the radioactivity is determined by scintillation counting in 4 ml of Aquasol-2 (New England Nuclear). Greater than 99% of the chloroform-soluble radioactivity produced in the choline- and ethanolaminephosphotransferase assays comi-
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277
grates with authentic PC and PE, respectively, on analysis by thin-layer chromatography. ~0Both assays are linear with respect to time and protein in the ranges of 0-30 min and 0-I00/zg of membrane protein. Variation of water-soluble components can be accommodated in the assay by utilizing the volume ordinarily added as water to the assay. When variation of lipid components is desired, the lipid to be varied is omitted from the 4 × assay mix and is instead delivered to each assay tube in chloroform. After removal of chloroform by drying, the variable lipid is solubilized by the 4 x assay mix containing the nonvariable lipid, detergent, and water-soluble components by vortexing and shaking as described above. We have observed that solubilization of dioleoylglycerol into Triton X-100 mixed micelles is more easily achieved in the presence of phospholipid and that when this is not possible, greater solubilization efforts are required.
Properties of the Yeast CPT1 and EPTI Gene Products DiacylglycerolDependencies. Both the CPTI and EPTI gene products exhibit Michaelis-Menten kinetics with respect to the mole fraction of dioleoylglycerol present in the micellar phase. It is essential to express the concentration of surface active components in this way since mixed micellar assays exhibit "surface dilution kinetics.''~5 Because purified preparations of these enzymes are not yet available, the crude membrane preparations used to measure their activitiescontain appreciable endogenous diacylglycerol which is also subject to surface dilution.As the amount of membrane protein added to the assay is decreased, the mole fraction of endogenous diacylglycerol in the assay becomes negligible, and complete dependence on exogenous diacylglycerol is achieved. Less than 20/~g of membrane protein should be used in experiments in which dilution of endogenous diacylglycerol is important. Phospholipid Activator Requirement. Nonlinearity with protein concentration in both assays suggested the presence of a dissociable enzyme activator substance present in the membranes; this requirement could be satisfied by exogenous PC. l0 Like endogenous diacylglycerol, endogenous phospholipid is effectively diluted into the micellar phase, and its mole fraction becomes negligible at low membrane protein concentrations; absolute requirements for a phospholipid activator can be demonstrated for the activities of both the CPT1 and EPT1 gene products. Less than 10/zg of membrane protein must be used to achieve complete dependence on 15 E. A. Dennis, in "The Enzymes" (P. D. Boyer, ed.), 3rd Ed., Vol. 16, p. 307. Academic Press, New York, 1983.
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TABLE I KINETIC PARAMETERSOF AMINO ALCOHOL PHOSPHOTRANSFERASEACTIVITIES OF CPT1 AND EPT1 GENE PRODUCTSa Vmax (nmol/min/mg protein)
Km (/xM) Gene product(s) (strain)
ChoPT
DMEPT
MMEPT
EthPT
109
27
26
20
0.78
120 100
29 137
29 b
22 __
0.62 0.20
Wild-type mixture (DBY746) EPT1 (HJ001) CPT1 (HJ051)
ChoPT DMEPT
MMEPT
EthPT
0.44
0.30
1.26
0.42 0.07
0,27 --
1.35 --
Kinetic parameters were obtained from double-reciprocal plots. ChoPT, Cholinephosphotransferase; DMEPT, dimethylethanolaminephosphotransferase; MMEPT, monomethylethanolaminephosphotransferase; EthPT, ethanolaminephosphotransferase. From Hjelmstad and Bell. I° b Could not be evaluated.
exogenous phospholipid. Several naturally occurring phospholipids satisfy the phospholipid activator requirement of these enzymes to varying extents. 10 CDPamino Alcohol Specificities. The activities of the CPT1 and EPT1 gene products have been evaluated using a series of CDPamino alcohol substrates including CDPcholine, CDPdimethylethanolamine, CDPmonomethylethanolamine, and CDPethanolamine. ~° The CPT1 gene product TABLE II DIFFERENTIAL PROPERTIES OF CPT1 AND E P T I GENE PRODUCTS EPT1 Gene product
Property (units) K m, dioleoylglycerol (mol %) Kin, CDPcholine (p,M) K m, CDPethanolamine (/zM) Ka, Mg2÷ (mM) Phospholipid activation by PE (%)~ Inhibition by CMP (% remaining activity) b
Ethanolaminephosphotransferase activity
Cholinephosphotransferase activity
CPT1 Gene product,
3.3 -22 2.6 13
2.9 120 -2.6 65
8.0 110 -0.7 65
16
71
18
cholinephosphotransferase activity
a Activity obtained using 10 mol % PE is expressed as a percentage of that measured when 10 mol % PC is used. b Value reported represents the percent remaining activity in the presence of 1 mM CMP relative to no addition.
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279
primarily utilizes CDPcholine and CDPethanolamine, whereas the EPT1 gene product utilizes all four derivatives to appreciable extents (Table I). The CPT1 gene product does, however, have measurable activity when CDPethanolamine or CDPmonomethylethanolamine are employed. Monomethylethanolamine- and dimethylethanolaminephosphotransferase activities solely represent alternative substrate specificities of the CPT1 and EPT1 gene products since cptl eptl double-mutant strains completely lack these activities.~° Mg 2+ Dependencies. The activities of both the CPT1 and EPTI gene products exhibit absolute requirements for a divalent metal ion. Differential Properties o f the CPT1 and EPT1 Gene Products. The properties of the CPT1 and EPT1 gene product-dependent cholinephosphotransferase activities and the EPT1 gene product-dependent ethanolaminephosphotransferase activity are summarized in Table II. Intrinsic properties of the two enzymes which are independent of the CDPamino alcohol employed include the apparent K mfor dioleoylglycerol, the apparent K a for M g 2+ , and inhibition by CMP. The latter property, which occurs by an undetermined mechanism, is useful in assessing the relative contributions of the CPTI and EPT1 gene products to total cholinephosphotransferase activity in membranes containing a mixture of the two enzymes, l° The CPT1 and EPT1 gene products also exhibit differing properties with respect to phospholipid activation; however, this difference is dependent on the CDPamino alcohol employed, and the cholinephosphotransferase activities of both enzymes are indistinguishable with respect to this property.
[33] 1 - A l k y l - 2 - a c e t y l - s n - g l y c e r o l C h o l i n e p h o s p h o t r a n s f e r a s e By TEN-CHING LEE and FRED SNYDER
Introduction A dithiothreitol (DTT)-insensitive cholinephosphotransferase (EC 2.7.8.16, 1-alkyl-2-acetyl-sn-glycerol cholinephosphotransferase) catalyzes the transfer of phosphocholine from CDPcholine to l-alkyl-2-acetylsn-glycerols (Fig. 1) in the final step of the biosynthesis of platelet-activating factor (PAF) in the de novo pathway ~'2;in fact the term DTT-insensitive I W. Renooij and F. Snyder, Biochim. Biophys. Acta 663, 545 (1981). 2 D. S. Wo odard , T.-C. Lee, and F. Snyder, J. Biol. Chem. 262, 2520 (1987).
METHODS IN ENZYMOLOGY, VOL. 209
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