[30] Ethanolamine-phosphate cytidylyltransferase

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CYTIDYLYLTRANSFERASES

[30]

even in the presence of detergents, has a high tendency to bind to glass and plastic surfaces. This can result in large losses when the enzyme is diluted and transferred to other containers. The use of siliconized glass tubes significantly reduces the surface binding. We use Surfasil (Pierce), diluted 1 : 10 in hexane, to coat the inside of glass tubes.

[30] E t h a n o l a m i n e - P h o s p h a t e

Cytidylyltransferase

B y LILIAN B. M. TIJBURG, PIETER S. VERMEULEN,

and LAMBERT M. G. VAN GOLDE Introduction CTP + phosphoethanolaminc~ CDPcthanolamine + PPi CTP:phosphoethanolamine cytidylyltransfcrase~ (EC 2.7.7.14, ethanolamine-phosphate cytidylyltransfcrase) is generally considered the key regulatory enzyme of the biosynthesis of phosphatidylethanolamine via the CDPethanolamine pathway.2,3 The assay and partial purification of this cytosolic enzyme have previously becn describcd by Sundlcr. 4 Recently, we have modified the original purification procedure to achieve a 1200fold purification of the enzyme.

Assay

Principle.The assay isbased on thc convcrsion of phospho[ 1,2-~4C]ethanolamine to CDP[l,2-14C]ethanolamine. Radioactivc substrate and reaction product are separated by thin-layer chromatography, which is followed by determination of the radioactivity in CDPethanolamine. Reagents

Tris buffer, 40 mM, adjusted to pH 7.8 with 1 M HCI, containing 20 mM MgC12 Dithiothreitol, 100 mM, freshly prepared l E. P. Kennedy and S. B. Weiss, J. Biol. Chem. 222, 193 (1956). 2 R. Sundler and B./~kesson, J. Biol. Chem. 250, 3359 (1975). 3 L. B. M. Tijburg, M. J. H. Geelen, and L. M. G. van Golde, Biochim. Biophys. Acta 1004, 1 (1989). 4 R. Sundler, J. Biol. Chem. 250, 8585 (1975). METHODS IN ENZYMOLOGY, VOL. 209

Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

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CTP, 40 raM, adjusted to pH 7 with 1 M NaOH Phospho[1,2:4C]ethanolamine, 10 mM (0.5 mCi/mmol) Because labeled phosphoethanolamine is not commercially available, we prepare the 14C derivative from [1,2-14C]ethanolamine (4 mCi/mmol). Ethanolamine kinase that is required for this procedure is partially purified from rat liver, as described by Porter and K e n t ) Rat livers (30 g) are homogenized in 4 volumes of 0.15 M KC1, 20 mM Tris-HCl (pH 7.5), 2 mM mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride, and 1 mM EDTA. Following acidification of the cytosol to pH 5.2 and subsequent centrifugation, the enzyme is isolated by (NH4)2SO4 precipitation) The (NH4)zSO4-precipitable fraction (30-45%) is redissolved in 20 ml of 10 mM Tris-HCl (pH 7.5), 2 mM mercaptoethanol, and 1 mM EDTA at a protein concentration of approximately 25 mg/ml and, subsequently, dialyzed against the same buffer. Partially purified ethanolamine kinase from 30 g rat liver is sufficient for the preparation of 100/zCi phosphoethanolamine. Five microcuries of [1,2-14C]ethanolamine is added to a 40-ml tube, and the solvent is evaporated under a stream of nitrogen. The reaction mixture contains the following in a total volume of 5 ml: 10 mM TrisHCI (pH 8.5), I0 mM MgCI2, 4 mM ATP, 0.25 mM (4 mCi/mmol) [1,214C]ethanolamine, and 1 ml partially purified ethanolamine kinase. The reaction is carried out for 2 hr at 37° and terminated by the addition of 18.75 ml methanol-chloroform (2 : 1, v/v). 6 After 60 min, 6.25 ml chloroform and 6.25 ml water are added, and the mixture is stirred for 5 min. After the phases have been separated by centrifugation, the upper phase, containing labeled phosphoethanolamine and unreacted ethanolamine, is collected. The lower chloroform phase is washed twice with 10 ml methanol-water (5:4, v/v). The aqueous extracts of 20 incubations are combined and evaporated to dryness under reduced pressure using a rotary evaporator. The residue is dissolved in 25 ml distilled water, and the pH of the solution is adjusted to pH 8 with 1 M NaOH. The mixture is applied to a Dowex l-X4 (200-400 mesh, formate form) column (1.5 × 25 cm). The column is flushed with 100 ml water to remove labeled ethanolamine; subsequently, phosphoethanolamine is eluted with 200 ml of 20 mM formic acid. The fractions containing labeled phosphoethanolamine are combined and evaporated to dryness under a stream of nitrogen at 40°. The residue, containing pure phosphoethanolamine, is dissolved in water at a concentration of 5/xCi/ml. Unlabeled phosphoethanolamine is added to give the desired specific activity. The yield is 70-80%. The purity of [~4C]phospho5 T. J. Porter and C. Kent, this volume [15]. 6 L. B. M. Tijburg, M. Houweling, M. J. H. Geelen, and L. M. G. van Golde, Biochim. Biophys. Acta 959, 1 (1988).

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[30]

ethanolamine is more than 99%, as assessed by thin-layer chromatography on silica gel G plates (Merck, Darmstadt, Germany) eluted with methanol-0.5% NaCl-ammonium hydroxide (50 : 50 : 5, v/v). Procedure. The assay mixture contains the following in a final volume of 100/zl: Tris-HC1 (2/zmol, 50/zl), MgCI2 (1 /zmol), dithiothreitol (0.5 /zmol, 5 tzl), CTP (0.2 /zmol, 5 ~1), phospho[1,2J4C]ethanolamine (0.I tzmol, 10 txl), bovine serum albumin (25 tzg), and distilled water (0-30/.d). The mixture is prewarmed at 37°, and the assay is started by the addition of enzyme (0-30/zl). The reaction is carried out for 15 min and stopped by immersing the tube for 2 min into a boiling water bath. The tubes are centrifuged for 5 min at 5000 g. Fifty microliters of the supernatant is spotted on a silica gel G thin-layer chromatography plate, and 0.3 tzmol CDPethanolamine and 1 tzmol phosphoethanolamine are applied as carrier. The plates are eluted with methanol-0.5% NaCl-ammonium hydroxide (50 : 50 : 5, v/v). After the solvent has evaporated from the plate, ethanolamine-containing compounds are visualized by spraying with a solution of 0.2% (w/v) ninhydrin in 96% ethanol, followed by heating for 5 rain at 100°. CDPethanolamine is scraped from the plate into a scintillation vial, and the radioactivity is determined by liquid scintillation counting. The reaction is linear with protein concentration between 0 and 150 txg cytosolic protein and linear with time for at least 30 min. One unit of enzyme activity is defined as 1 ~mol of CDPethanolamine formed per minute. Purification Procedure

Ammonium Sulfate Precipitation. Livers from 6 rats (body weight 200-250 g), previously perfused free of blood with saline, are homogenized with 5 strokes of a motor-driven Potter-Elvehjem homogenizer in 4 volumes of 150 mM NaC1, 20 mM Tris-HC1 (pH 7.8), 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mg/liter benzamidine, 1 mg/liter leupeptin, and 0.7 mg/liter pepstatin A. All procedures are performed at 4°. The homogenate is centrifuged at 10,000 g for 20 min and the pellet discarded. After centrifugation at 105,000 g for 70 min, the resulting supernatant is collected and the pH readjusted to pH 7.2 with 0.1 M NaOH. The solution is brought to 25% of saturation with solid (NH4)2SO 4 o v e r a period of 10 min with rapid stirring. After 15 min, the precipitate is removed by centrifugation at 20,000 g for 20 rain, and further (NH4)2SO4 is added to bring the supernatant to 40% saturation. After stirring for I0 min, the mixture is subjected to centrifugation as described above. The pellet is dissolved in approximately 80 ml of 20 mM Tris-HC1 (pH 7.6), 1 mM EDTA, and 1 mM dithiothreitol (buffer A) and desalted by dialyzing against the same buffer.

[30]

261

ETHANOLAMINE-PHOSPHATE CYTIDYLYLTRANSFERASE

DE-52 Chromatography. After the solution has been clarified by centrifugation at 20,000 g for 5 min, the sample is loaded onto a DE-52 column (2.65 x 50 cm), equilibrated with buffer A, at a flow rate of 55 ml/hr. The column is flushed with 400 ml buffer A. The enzyme is eluted from the column with a 500-ml linear salt gradient of 0 to 0.3 M NaCl in buffer A, followed by 100 ml of 0.3 M NaCl in the same buffer. Phosphoethanolamine cytidylyltransferase activity elutes at a salt concentration of around 0.15 M NaCl. The activity peak is pooled and dialyzed overnight against 20 mM Tris-HC1 (pH 7.6), 1 mM EDTA, 1 mM dithiothreitol, and 10% (v/v) ethylene glycol (buffer B). Matrex Gel Red A Chromatography. The pooled enzyme is applied to a column of Matrex Gel Red A (I .3 × 39 cm), equilibrated with buffer B. After a 150-ml wash with buffer B, the enzyme is eluted with 450 ml of a linear gradient of 0 to 1.0 M KC1 in the same buffer, at a flow rate of 20 ml/hr. This procedure yields an activity peak eluting between 0.3 and 0.5 M KCI (Fig. 1). Octyl-Sepharose CL-4B Chromatography. Fractions from the Matrex Gel Red A column containing cytidylyltransferase activity are pooled and directly pumped onto an Octyl-Sepharose CL-4B column (2.6 × 20 cm). The column is flushed with 75 ml 0.5 M NaCl, 25 mM Tris-HCl (pH 7.5),

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FIO. 1. Elution of CTP : phosphoethanolamine cytidylyltransferase from a Matrex Gel Red A column. Fractions o f 6 ml were collected, and the enzyme activity was determined. For experimental details, see text.

262

CYTIDYLYLTRANSFERASES

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TABLE I PURIFICATION OF ETHANOLAMINE-PHOSPHATE CYTIDYLYLTRANSFERASE FROM RAT LIVER

Fraction

Protein (mg)

Total activity (units)

Specific activity (units/mg × 103)

Purification (-fold)

Recovery (%)

Cytosol 25-40% (NH4)2SO4 DE-52 Matrex Gel Red A Octyi-Sepharose Matrex Gel Blue A

4880 1190 209 58 3.04 0.37

18.4 16.4 14.4 13.1 4.73 1.62

3.77 13.8 70.1 228 1560 4380

1 3.7 19 60 414 1162

100 89 78 71 25 8.8

0.5 mM EDTA, 1 mM dithiothreitol and 10% (v/v) ethyleneglycol at a flow rate of 40 ml/hr. Subsequently, the column is batchwise eluted with 75 ml of 15 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, and 1 mM dithiothreitol (buffer C) containing 10% ethyleneglycol, followed by 75 ml buffer C with 45% and 75 ml of buffer C with 70% ethyleneglycol at flow rates of 40, 30, and 20 ml/hr, respectively. Matrex Gel Blue A Pseudoaffinity Chromatography. Enzyme activity recovered from the Octyl-Sepharose column is pooled and dialyzed against 25 mM Tris-HC1 (pH 8.2), 0.5 mM EDTA, 1 mM dithiothreitol and 5% (v/v) ethyleneglycol (buffer D). The enzyme is applied to a Matrex Gel Blue A column (2.6 × 20 cm) at a flow rate of 35 ml/hr. After loading the sample the pump is stopped for 15 min to allow proteins to bind to the column. The column is then flushed with 100 ml of buffer D. Since cytidylyltransferase does not bind to the column, the enzyme activity is recovered in the flushing step. This procedure results in one activity peak of pure phosphoethanolamine cytidylyltransferase. Fractions containing cytidylyltransferase activity are collected and concentrated approximately 10 times by dialysis against 20 volumes of 20 mM Tris-HCl (pH 7.8), 0.5 mM EDTA, 2 mM dithioerythritol, 5% (v/v) ethyleneglycol, and 30% (g/v) polyethyleneglycol-6000. Concentrated enzyme is dialyzed against the same buffer omitting polyethyleneglycol-6000 and supplemented with 10% (v/v) glycerol. The enzyme preparation is stored at - 70°. The results of a typical purification procedure of CTP : phosphoethanolamine cytidylyltransferase are summarized in Table I. Polyacrylamide gel electrophoresis (PAGE) under nondenaturing conditions, followed by sodium dodecyl sulfate-PAGE, reveals one single band at 49 kDa con-

[30]

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263

taining the cytidylyltransferase activity. The specific activity of the 1160fold purified fraction is a factor of 3.8 higher than that reported previously.4 Properties

pH Optimum. Phosphoethanolamine cytidylyltransferase has a sharp optimum at pH 7.8 and a broader one, with lower maximal activity, around pH 6. 4 Stability. The enzyme has a limited stability below pH 7. The activity of purified enzyme is dependent on the presence of dithiothreitol in the assay mixture. 4 In the 105,000 g supernatant the enzyme is quite stable toward freezing and thawing. Purified enzyme can be stabilized by the addition of 10% (v/v) glycerol or 2% bovine serum albumin and stored at 20° for at least 4 weeks without significant loss of activity. Molecular Weight. The enzyme has a molecular weight of 100,000 to 120,000 as estimated by Superose 12 gel filtration. This value is in line with that reported by Sundler using Sephadex G-200 chromatography. 4 Kinetic Properties. In contrast to CTP : phosphocholine cytidylyltransferase, which is an ambiquitous enzyme, 3 phosphoethanolamine cytidylyltransferase is localized predominantly in the cytosol. Although a striking feature of phosphocholine cytidylyltransferase is its requirement of lipid for significant activity, 7 addition of lipids does not stimulate the activity of cytosolic phosphoethanolamine cytidylyltransferase. The Michaelis constants for CTP and phosphoethanolamine are 53 and 65 /zM, respectively. An ordered sequential reaction mechanism has been proposed, with CTP being the first substrate to bind to the enzyme and CDPethanolamine being the last product to be released. 4 In addition to CTP, phosphoethanolamine cytidylyltransferase can also use deoxyCTP as a substrate and form deoxyCDPethanolamine. 8 -

7 p. A. Weinhold and D. A. Feldman, this volume [29]. 8 E. P. Kennedy, L. F. Borkenhagen, and S. W. Smith, J. Biol. Chem. 234, 1998 (1959).