110
[14]
FATTY ACID SYNTHESIS
[14] Acyl Carrier Protein from Euglena g r a c i l i s By R. K. DINELLO and M. L. ERNST-FONBERG
Assay Method Principle. Acyl carrier protein (ACP) which carries the carboxylic acid substrates of fatty acid synthesis as thiolesters during the sequence of fatty acid biosynthesis was first described by Vagelos and co-workers. 1 ACP is measured by the malonyl-pantetheine-14CO._, exchange reaction ~,3 which uses the first three enzymes in fatty acid biosynthesis to fix free 14C0~ into an acid-stable radioactive product, malonyl-pantetheine. acyl transacylase
Acyl-pantotheine -t- A C P - S H (
~ acyl-ACP + pantotheine-SH malonyl transacylase
Malonyl-pantotheine + A C P - S H (
~ malonyl-ACP -t- pantotheine-SH
condensing enzyme
Acyl-ACP + malonyl-ACP (
' /~-ketoaeyl-ACP Jr 14C02 -t- A C P - S H
Reagents Imidazole-HC1, 1.0 M, pH 6.2, 0.2 M in 2-mercaptoethanol Euglena fatty acid synthase, type II ACP solution (1-4 ~M) NaH14CQ, 250 m M (0.2-0.6 mCi/mmole) Perchloric acid, 10% Malonyl pantetheine, 2 9.0 m M Caproyl pantetheine, 2 2.0 m M
Procedure. To test tubes secured in a 30 ° circulating water bath add 25 ~l each of imidazole buffer, caproyl-pantetheine, and malonyl-pantetheine; Euglena fatty acid synthase, type II, 25 or 50 ~l (an amount which fixes 500-1000 cpm without added A C P ) ; ACP, 25-50 ~l; water sufficient for a final volume of 250 ~1; and NaHI~CO~, 25 ~1, to initiate reaction. The reaction is done in a fume hood, and the tubes are stoppered during the assay with serum caps containing two 18 gauge steel needles which are used at the termination of reaction to purge the test tubes with 1p. W. Majerus, A. W. Alberts, and P. R. Vagelos, Proc. Nat. Acad. Sci. U.S. 51, 1231 (1964). 2p. W. Majerus, A. W. Alberts, and P. R. Vagelos, Vol. XIV [6]. R. K. DiNello and M. L. Ernst-Fonberg, J. Biol. Chem. 248, 1707 (1973).
[14]
CARRIER PROTEIN FROM Euglena gracilis
111
air for 15 minutes to remove the ~CO~ which is trapped in 40% KOH. Reaction is halted after 15 minutes by the injection of 25 gl of 10% perchlorate through the serum caps. The tubes are centrifuged in a clinical centrifuge for 10 minutes, and 25 ~l aliquots of the supernatants are spotted on filter paper strips (Whatman No. 1, 1 X 2 cm). Following drying, the papers are placed in counting vials with 15 ml of toluene containing 15.1 g of 2,5-diphenyloxazole/gallon and measured in a liquid scintillation counter. Euglena Fatty Acid Synthase, Type II. All procedures are carried out at 4 °. Euglena gracilis strain Z3, 50 g, are suspended in 50 ml of 0.05 M imidazole-HCl, pH 7.0, 0.01 M in 2-mercaptoethanol in a 100-ml beaker. Cells are disrupted by ultrasound in 30 second bursts while the temperature is maintained at 2-5 ° with a methanol-ice bath. Cell breakage is monitored microscopically. The mixture is centrifuged in a Sorvall RC-2B centrifuge, 45,000 g for 70 minutes, and the supernatant is collected and adjusted to 35% (NH4)._,S04 saturation by the addition of 20.84 g/100 ml over a 15-minute period with stirring. After stirring for an additional 30 minutes, the precipitate is removed by eentrifugation for 20 minutes at 20,000 g. The supernatant is adjusted to 70% (NH4) 2S04 saturation by the addition of 23.61 g/100 ml over 15 minutes. After 30 minutes of stirring, the precipitate containing the desired enzymes is collected as before while the supernatant is discarded. The precipitate is dissolved in 8 ml of the imidazole buffer described above and stored in 0.6 ml aliquots at --50 ° until use. At the time of use, the preparation is desalted on a small Sephadex G-25 column equilibrated in and eluted with 0.05 M imidazole-HC1, pH 7.0, 0.01 M in 2-mercapt0ethanol. Purification of Acyl Carries Protein All procedures are carried out at 4 ° unless noted otherwise. A total of 2045 g of Euglena gracilis strain Z are disrupted in eight portions. About 250 g of frozen cells are thawed in 250 ml of 0.05 M imidazoleHC1, pH 7.1, 0.07 M in 2-mercaptoethanol. The suspension is saturated with N~ and kept at 2-5 ° in a methanol-ice bath. The cells are broken by ultrasound in about 14 bursts of 1 minute. Disruption is monitored microscopically. The mixture is centrifuged in a Sorvall RC-2 centrifuge at 15,000 g for 80 minutes. Tile combined supernatants are adjusted to 70% (NH~)..SO~ saturation by the addition of 47.23 g/100 ml. Following 1 hour of stirring, the precipitate is removed by centrifugation at 22,000 g for 20 minutes. The ACP containing supernatant is brought to 95% (NH~)2S04 saturation by
112
FATTY ACID SYNTHESIS
[14]
the slow addition of 17.9 g/100 ml over a period of 2 hours. After stirring the covered container for 12 hours, the precipitate is collected as described above and dissolved in 48 ml of 0.01 M potassium phosphate buffer, pH 6.2, containing 0.01 M 2-mercaptoethanol (buffer A). The solution is dialyzed against 1200 ml of the same buffer through three changes of 2 hours each. The protein solution is centrifuged to remove any denatured protein and allowed to come to room temperature. It is then applied to a DEAEcellulose column (2.5 X 29 cm) equilibrated with buffer A at room temperature (10 mg of protein/ml of column bed). After the protein solution has soaked in, the column is washed with 4 column volumes of buffer A, and is then eluted with 9 column volumes of a linear gradient from 0.1 to 0.4 M LiC1 in the same buffer. Starting with the application of the gradient, 10 ml fractions are collected and assayed for ACP (fractions 73 through 83 under these conditions) and for absorbance at 280 and 4164 nm. The appropriate fractions are pooled and assayed for protein. The solution is divided into 2 ml aliquots in test tubes which are thrust into a 90 ° water bath for 4 minutes and are then cooled immediately in an ice bath. The resulting suspension is centrifuged to remove any precipitate and is then assayed for protein. The solution (104 ml) is dialyzed against three changes of 1200 ml each of 0.01 M potassium phosphate buffer, pH 7.5, 0.01 M in 2-mercaptoethanol (buffer B). A DEAE-cellulose column, 1.2 X 5.0 cm (1 ml column bed/rag protein), is equilibrated in the same buffer. Immediately prior to application the protein solution is brought to room temperature, and the column is run at room temperature. After the sample has soaked in, the column is washed with 50 ml of buffer B. It is then eluted with 50 ml of buffer B adjusted to contain 0.1 M LiC1. Finally, a 250-ml linear gradient from 0.1 to 0.15 M LiC1 in buffer B is applied and 4 ml fractions are collected. Fractions are assayed as before, and the broad peak of ACP activity (fractions 28 through 51 under these conditions) is pooled. The pooled fractions are adjusted to pH 6.2 with 1 N HC1 and dialyzed against three changes of buffer A, 1 liter each. The solution is concentrated on a DEAE-cellulose column (0.8 X 1.2 cm) equilibrated with buffer A, and eluted with the same buffer adjusted to 0.35 M LiCl. The protein containing fractions are pooled (4.5 ml under these conditions). The final step in the purification is acrylamide gel electrophoresis. Gels of 17.5% are run using the pH 9.4 system described by Davis. 5 This 4Euglena ACP tends to copurify with cytochrome 552, a heine protein with a
molecular weight of 13,500 and an isoelectric point near 5.5. 5B. J. Davis, Ann. N.Y. Acad. Sci. 121,404 (1964).
CARRI]~R PROTEIN FROM Euglena gracilis
[14]
113
TABLE I ACP PURIFICATION
Step
Total protein (mg)
Total activity (unit) a
Specific activity
Purification (-fold)
Crude extract 70-95 % (NH4) 2SO.2fraction DEAE-cellulose, pH 6.2 90 ° for 4 minutes DEAE-cellulose, pH 7.5 Acrylamide gel electrophoresis
74,000 1,440 59 48.5 6.3 2.8
5,904 b 5,904 6,962 4,800 3,070 2,340
0.08 4.1 118.00 99.00 487.00 835.00
0 51 1,475 1,238 6,088 10,438
A unit of activity is defined as the incorporation of 1 nmole of 14CO~in 30 minutes. b The activity in the crude extract was too dilute to measure. For the sake of estimating the extent of purification of the protein, the total activity found in the (NH4)~SO4 precipitate was taken as representing that present in the crude extract. procedure m a y be conducted in a preparative or analytical apparatus. When analytical gels in glass tubes are run, the rigid gel is extracted most easily from the tube by wrapping the tube in cloth and gently breaking it with a hammer. In all instances, it is important to remove the A C P from the p H 9.4 environment as soon as possible either by altering the p H of the eluted protein in a preparative gel or by immediately extracting the protein from an analytical gel. In the latter case, the appropriate gel bands are excised and homogenized in buffer A. The homogenate is centrifuged and washed several times with buffer A. All supernatants are combined and dialyzed against 10 volumes of buffer A for three changes. The protein solution is concentrated on a DEAE-cellulose column as described earlier and stored at - - 2 0 ° or below in buffer A (see Table I ) .
Properties of Acyl Carrier Protein
Purity. Aerylamide (17.5%) gel electrophoresis of the final material shows a single band of protein in a p H 9.4 system 5 and in a p H 6.5 system2 The preparation shows three bands after the second D E A E - c e l lulose column. The purified A C P must be stored in the presence of 0.01 M 2-mercaptoethanol, otherwise disulfide bond formation occurs with the formation of A C P dimer. 6 D. Rodbard and A. Chrambach, Anal. Biochem. 40, 95 (1971).
114
FATTY ACID SYNTHESIS
[14]
Properties. The molecular weight is estimated to be 10,400 from amino acid composition and from gel filtration. There is one mole of 4'-phosphopantetheine per mole of ACP. It contains 92 amino acids (trytophan was not measured) among which there are no histidine, cystine, or tyrosine residues. Euglena ACP is not stable to acid precipitation nor can it tolerate long periods at pH values above 8.
[14]
FATTY ACID SYNTHESIS
[14] Acyl Carrier Protein from Euglena g r a c i l i s By R. K. DINELLO and M. L. ERNST-FONBERG
Assay Method Principle. Acyl carrier protein (ACP) which carries the carboxylic acid substrates of fatty acid synthesis as thiolesters during the sequence of fatty acid biosynthesis was first described by Vagelos and co-workers. 1 ACP is measured by the malonyl-pantetheine-14CO._, exchange reaction ~,3 which uses the first three enzymes in fatty acid biosynthesis to fix free 14C0~ into an acid-stable radioactive product, malonyl-pantetheine. acyl transacylase
Acyl-pantotheine -t- A C P - S H (
~ acyl-ACP + pantotheine-SH malonyl transacylase
Malonyl-pantotheine + A C P - S H (
~ malonyl-ACP -t- pantotheine-SH
condensing enzyme
Acyl-ACP + malonyl-ACP (
' /~-ketoaeyl-ACP Jr 14C02 -t- A C P - S H
Reagents Imidazole-HC1, 1.0 M, pH 6.2, 0.2 M in 2-mercaptoethanol Euglena fatty acid synthase, type II ACP solution (1-4 ~M) NaH14CQ, 250 m M (0.2-0.6 mCi/mmole) Perchloric acid, 10% Malonyl pantetheine, 2 9.0 m M Caproyl pantetheine, 2 2.0 m M
Procedure. To test tubes secured in a 30 ° circulating water bath add 25 ~l each of imidazole buffer, caproyl-pantetheine, and malonyl-pantetheine; Euglena fatty acid synthase, type II, 25 or 50 ~l (an amount which fixes 500-1000 cpm without added A C P ) ; ACP, 25-50 ~l; water sufficient for a final volume of 250 ~1; and NaHI~CO~, 25 ~1, to initiate reaction. The reaction is done in a fume hood, and the tubes are stoppered during the assay with serum caps containing two 18 gauge steel needles which are used at the termination of reaction to purge the test tubes with 1p. W. Majerus, A. W. Alberts, and P. R. Vagelos, Proc. Nat. Acad. Sci. U.S. 51, 1231 (1964). 2p. W. Majerus, A. W. Alberts, and P. R. Vagelos, Vol. XIV [6]. R. K. DiNello and M. L. Ernst-Fonberg, J. Biol. Chem. 248, 1707 (1973).
[14]
CARRIER PROTEIN FROM Euglena gracilis
111
air for 15 minutes to remove the ~CO~ which is trapped in 40% KOH. Reaction is halted after 15 minutes by the injection of 25 gl of 10% perchlorate through the serum caps. The tubes are centrifuged in a clinical centrifuge for 10 minutes, and 25 ~l aliquots of the supernatants are spotted on filter paper strips (Whatman No. 1, 1 X 2 cm). Following drying, the papers are placed in counting vials with 15 ml of toluene containing 15.1 g of 2,5-diphenyloxazole/gallon and measured in a liquid scintillation counter. Euglena Fatty Acid Synthase, Type II. All procedures are carried out at 4 °. Euglena gracilis strain Z3, 50 g, are suspended in 50 ml of 0.05 M imidazole-HCl, pH 7.0, 0.01 M in 2-mercaptoethanol in a 100-ml beaker. Cells are disrupted by ultrasound in 30 second bursts while the temperature is maintained at 2-5 ° with a methanol-ice bath. Cell breakage is monitored microscopically. The mixture is centrifuged in a Sorvall RC-2B centrifuge, 45,000 g for 70 minutes, and the supernatant is collected and adjusted to 35% (NH4)._,S04 saturation by the addition of 20.84 g/100 ml over a 15-minute period with stirring. After stirring for an additional 30 minutes, the precipitate is removed by eentrifugation for 20 minutes at 20,000 g. The supernatant is adjusted to 70% (NH4) 2S04 saturation by the addition of 23.61 g/100 ml over 15 minutes. After 30 minutes of stirring, the precipitate containing the desired enzymes is collected as before while the supernatant is discarded. The precipitate is dissolved in 8 ml of the imidazole buffer described above and stored in 0.6 ml aliquots at --50 ° until use. At the time of use, the preparation is desalted on a small Sephadex G-25 column equilibrated in and eluted with 0.05 M imidazole-HC1, pH 7.0, 0.01 M in 2-mercapt0ethanol. Purification of Acyl Carries Protein All procedures are carried out at 4 ° unless noted otherwise. A total of 2045 g of Euglena gracilis strain Z are disrupted in eight portions. About 250 g of frozen cells are thawed in 250 ml of 0.05 M imidazoleHC1, pH 7.1, 0.07 M in 2-mercaptoethanol. The suspension is saturated with N~ and kept at 2-5 ° in a methanol-ice bath. The cells are broken by ultrasound in about 14 bursts of 1 minute. Disruption is monitored microscopically. The mixture is centrifuged in a Sorvall RC-2 centrifuge at 15,000 g for 80 minutes. Tile combined supernatants are adjusted to 70% (NH~)..SO~ saturation by the addition of 47.23 g/100 ml. Following 1 hour of stirring, the precipitate is removed by centrifugation at 22,000 g for 20 minutes. The ACP containing supernatant is brought to 95% (NH~)2S04 saturation by
112
FATTY ACID SYNTHESIS
[14]
the slow addition of 17.9 g/100 ml over a period of 2 hours. After stirring the covered container for 12 hours, the precipitate is collected as described above and dissolved in 48 ml of 0.01 M potassium phosphate buffer, pH 6.2, containing 0.01 M 2-mercaptoethanol (buffer A). The solution is dialyzed against 1200 ml of the same buffer through three changes of 2 hours each. The protein solution is centrifuged to remove any denatured protein and allowed to come to room temperature. It is then applied to a DEAEcellulose column (2.5 X 29 cm) equilibrated with buffer A at room temperature (10 mg of protein/ml of column bed). After the protein solution has soaked in, the column is washed with 4 column volumes of buffer A, and is then eluted with 9 column volumes of a linear gradient from 0.1 to 0.4 M LiC1 in the same buffer. Starting with the application of the gradient, 10 ml fractions are collected and assayed for ACP (fractions 73 through 83 under these conditions) and for absorbance at 280 and 4164 nm. The appropriate fractions are pooled and assayed for protein. The solution is divided into 2 ml aliquots in test tubes which are thrust into a 90 ° water bath for 4 minutes and are then cooled immediately in an ice bath. The resulting suspension is centrifuged to remove any precipitate and is then assayed for protein. The solution (104 ml) is dialyzed against three changes of 1200 ml each of 0.01 M potassium phosphate buffer, pH 7.5, 0.01 M in 2-mercaptoethanol (buffer B). A DEAE-cellulose column, 1.2 X 5.0 cm (1 ml column bed/rag protein), is equilibrated in the same buffer. Immediately prior to application the protein solution is brought to room temperature, and the column is run at room temperature. After the sample has soaked in, the column is washed with 50 ml of buffer B. It is then eluted with 50 ml of buffer B adjusted to contain 0.1 M LiC1. Finally, a 250-ml linear gradient from 0.1 to 0.15 M LiC1 in buffer B is applied and 4 ml fractions are collected. Fractions are assayed as before, and the broad peak of ACP activity (fractions 28 through 51 under these conditions) is pooled. The pooled fractions are adjusted to pH 6.2 with 1 N HC1 and dialyzed against three changes of buffer A, 1 liter each. The solution is concentrated on a DEAE-cellulose column (0.8 X 1.2 cm) equilibrated with buffer A, and eluted with the same buffer adjusted to 0.35 M LiCl. The protein containing fractions are pooled (4.5 ml under these conditions). The final step in the purification is acrylamide gel electrophoresis. Gels of 17.5% are run using the pH 9.4 system described by Davis. 5 This 4Euglena ACP tends to copurify with cytochrome 552, a heine protein with a
molecular weight of 13,500 and an isoelectric point near 5.5. 5B. J. Davis, Ann. N.Y. Acad. Sci. 121,404 (1964).
CARRI]~R PROTEIN FROM Euglena gracilis
[14]
113
TABLE I ACP PURIFICATION
Step
Total protein (mg)
Total activity (unit) a
Specific activity
Purification (-fold)
Crude extract 70-95 % (NH4) 2SO.2fraction DEAE-cellulose, pH 6.2 90 ° for 4 minutes DEAE-cellulose, pH 7.5 Acrylamide gel electrophoresis
74,000 1,440 59 48.5 6.3 2.8
5,904 b 5,904 6,962 4,800 3,070 2,340
0.08 4.1 118.00 99.00 487.00 835.00
0 51 1,475 1,238 6,088 10,438
A unit of activity is defined as the incorporation of 1 nmole of 14CO~in 30 minutes. b The activity in the crude extract was too dilute to measure. For the sake of estimating the extent of purification of the protein, the total activity found in the (NH4)~SO4 precipitate was taken as representing that present in the crude extract. procedure m a y be conducted in a preparative or analytical apparatus. When analytical gels in glass tubes are run, the rigid gel is extracted most easily from the tube by wrapping the tube in cloth and gently breaking it with a hammer. In all instances, it is important to remove the A C P from the p H 9.4 environment as soon as possible either by altering the p H of the eluted protein in a preparative gel or by immediately extracting the protein from an analytical gel. In the latter case, the appropriate gel bands are excised and homogenized in buffer A. The homogenate is centrifuged and washed several times with buffer A. All supernatants are combined and dialyzed against 10 volumes of buffer A for three changes. The protein solution is concentrated on a DEAE-cellulose column as described earlier and stored at - - 2 0 ° or below in buffer A (see Table I ) .
Properties of Acyl Carrier Protein
Purity. Aerylamide (17.5%) gel electrophoresis of the final material shows a single band of protein in a p H 9.4 system 5 and in a p H 6.5 system2 The preparation shows three bands after the second D E A E - c e l lulose column. The purified A C P must be stored in the presence of 0.01 M 2-mercaptoethanol, otherwise disulfide bond formation occurs with the formation of A C P dimer. 6 D. Rodbard and A. Chrambach, Anal. Biochem. 40, 95 (1971).
114
FATTY ACID SYNTHESIS
[14]
Properties. The molecular weight is estimated to be 10,400 from amino acid composition and from gel filtration. There is one mole of 4'-phosphopantetheine per mole of ACP. It contains 92 amino acids (trytophan was not measured) among which there are no histidine, cystine, or tyrosine residues. Euglena ACP is not stable to acid precipitation nor can it tolerate long periods at pH values above 8.
