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[18] Cyclopropane fatty acid synthase from Escherichia coli
[18]
CYCLOPROPANE FATTY ACID SYNTHASE FROM E. coli
133
19% random coil. The removal of lipids results in the disorganization of the complex as judged from both the total enzyme inactivation and the changes in the helical conformation. Urea induces a conformational transition at the 3 M concentration; cholate and sodium dodecyl sulfate have little effect on the a-helical structure although both agents induce a loss of enzyme activity in a similar manner to that induced by urea. All data 8,1a'14 support the interpretation that phospholipids play a fundamental role in the conformation of the active site and the secondary structure, whereas triacylglycerols contribute to the general support of the oligomeric enzyme.
[18] C y c l o p r o p a n e F a t t y A c i d S y n t h a s e f r o m Escherichia coli B y F R E D E R I C K R . T A Y L O R , D E N N I S W . GROGAN, and J O H N E . C R O N A N , JR.
Cyclopropane fatty acids (CFA) 1 are found in the phospholipids of many eubacteria 2 and have also been reported in a few eukaryotic organisms.2 In bacteria, Law a and his co-workers showed that CFAs are formed by methylenation of the double bond of unsaturated fatty acids. These workers also demonstrated an enzyme, CFA synthase, in Clostridium butyricum that catalyzed methylenation of the unsaturated fatty acid moieties of phospholipids using the methyl carbon ofS-adenosyl-L-methionine (SAM) as the methylene donor. This synthase is one of the few enzymes known to act on the nonpolar portion of phospholipids dispersed in a vesicle. The substrate of the enzyme is the double bond of a phospholipid unsaturated fatty acid residue) This double bond must be 9-11 carbon atoms removed from the glycerol backbone of the phospholipid molecule; 4,5 therefore, the site of action is well within the hydrophobic region of the lipid bilayer. For these reasons, CFA synthase is an unusually interesting system for the study of proteinlipid interactions. It binds to substrate phospholipid vesicles. This binding greatly stabilizes the enzyme and is exploited in its purification. 6 H. Goldfine, Adv. Microbiol. Physiol. 8, 1 (1972). 2 W. W. Christie, Top. Lipid Chem. 1, 1 (1970). a j. H. Law, Acc. Chem. Res. 4, 199 (1971). 4 L. A. Marinari, H. Goldfine, and C. Panos, Biochemistry 13, 1978 (1974). 5 j. B. Ohlrogge, F. D. Gunstone, I. A. Ismail, and W. E. M. Lands, Biochim. Biophys. Acta 431, 257 (1976). F. R. Taylor, and J. E. Cronan, Biochemistry 15, 3292 (1979).
METHODS IN ENZYMOLOGY,VOL. 71
Copyright © 1981by AcademicPress, Inc. All rights of reproductionin any form reserved. ISBH 0-12-181971-X
134
FATTY ACID SYNTHESIS
[18]
Assay Principle. The assay is based on the incorporation label from [methyl-aH]S-adenosyl-L-methionine (SAM) into phospholipid. 6,7 After incubation the reaction mixture is pipetted onto a filter paper disk. The disks are washed in trichloroacetic acid (which precipitates phospholipids, but not SAM). CFA synthase activity is then quantitated by scintillation counting of the disk. Reagents [Methyl-3H]S-adenosylmethionine (commercially available) 25 Ci/ftmol; final concentration 0.5 mm Phospholipid dispersion, 0.1 mg, final concentration 1 mg/ml Buffer, potassium phosphate (pH 7.5), final concentration 20 mM S-adenosyl-L-homocysteine hydrolase (SAHase), 1 unit/ml final concentration Partial Purification of SAH Nucleotidase (SAHase). This enzyme was purified from E. coil B by essentially the method of Duerre. s The SAHase preparations purified by ammonium sulfate fractionation and DEAEcellulose chromatography were free of CFA synthase activity. It was found that less purified preparations could be freed of CFA synthase with full retention of SAHase activity by heating to 45° for 15 rain. The preparations were stored at - 2 0 ° in 50 mM Tris-HCl (pH 7.5) buffer and were stable for at least 1 year. SAHase activity is assayed as described by Duerre, 8 except that Nelson's test 9 is used to measure the production of reducing sugar. One unit of SAHase activity is defined as 1 ~tmol of reducing sugar formed from SAH per minute at 37° and pH 7.5. SAHase activity can also be assayed indirectly by relief of the inhibition of CFA synthase by added SAH. Preparation of Phospholipid Dispersions. Phospholipids deficient in CFA (hence rich in unsaturates) are extracted either from E. coil wild-type cells grown to early log phase or from stationary phase cells of a mutant strain deficient in CFA synthase. ~° The cells are grown in a broth medium, harvested, and the phospholipids extracted as described by Ames. H The phospholipids are purified free of neutral lipids by the solvent precipitation method of Law and Essen ~2 or by chromatography on a silicic acid column. ~aThe resulting phospholipids are dried under a stream of nitrogen 7 H. Goldflne, J. LipidRes. 7, 146 (1966). s j. A. Duerre, J. Biol. Chem. 237, 3737 (1962). N. Nelson, J. Biol. Chem. 153, 375 (1944). 10 F. R. Taylor, and J. E. Cronan, Jr. J. Bacteriol. 125, 518 (1976). 11 G. F. Ames, J. Bacteriol. 95, 833 (1968). 12 j. H. Law, and B. Essen, this series, Vol. 14, p. 665. la j. C. Rittmer, and M. A. Wells, this series, Vol. 14, p. 483.
[18]
CYCLOPROPANE FATTY ACID SYNTHASE FROM
E. coli
135
and then dispersed into 1 mM EDTA in glass-distilled water (1 ml/10 mg of lipid) by sonication for 1 min resulting in monolamellar vesicles or by vigorous agitation (vortex mixer) and homogenization resulting in multilamellar liposomes. After dispersion the phospholipid concentration is determined by either the hydroxamate test for lipid esters t4 or by phosphate analysis. 15 Hydrogenation of unsaturated phospholipids (50-100 mg of phospholipid) as performed with 5 mg of Adams catalyst (PtO2) in 20 ml of tetrahydrofuran-methanol (1 : 1) or chloroform-methanol (1 : 1) in an apparatus made from two 125 ml sidearm Erlenmeyer filter flasks connected via the sidearm with thick-wall rubber tubing. One flask contained the phospholipid solution and the catalyst, and the second flask contained 50 ml of 5 N HC1. The first flask was stoppered with a silicone stopper pierced by a glass tube, to the external end of which was wired a rubber policeman. The second flask was capped with a rubber septum. A stabilized NaBH4 solution TM was injected into the acid of the second flask; this resulted in the immediate evolution of Hz (monitored by expansion of the rubber policeman). The apparatus was shaken at room temperature until no further uptake of H2 occurred. Hydrogenation under these conditions has no effect on cyclopropane rings. 2 Liposomes of these phospholipids are made as described above except the solution is heated to 60-70 ° periodically during homogenization. Assay Procedure. To the buffer is added the [all]SAM and then the phospholipid dispersion and enzyme fraction. The buffer is added first to neutralize the dilute sulfuric acid in which the SAM is stored and thus prevent acid precipitation of the phospholipids. It is important that the [all]SAM be mixed with carrier SAM (if needed) just before use. Storage of concentrated [all]SAM solutions results in formation of a trichloroacetic acid (TCA) insoluble product, which leads to high background values. After incubation at 37° for 30 min, the entire reaction mixture is pipetted onto a 2.4 cm disk of Whatman No. 3 MM filter paper mounted on a pin. The filter disks are dried in a stream of hot air for 20 sec and immersed in TCA (10% w/v) for 5 min at room temperature. The disks are then placed in a boiling solution of 5% (w/v) TCA solution, washed in two changes of distilled water for 15 min each, then dried, and assayed for radioactivity after addition of scintillation fluid. The assay is linear with protein from 0.02 to 5 mg of crude supernatant protein and is linear with time for at least 1 hr. A unit of CFA synthase activity is defined as 1 pmol of CFA formed per minute at 37°. 14 B. Shapiro, Biochem. J. 53, 663 (1953). ~5 B. N. Ames, this series, Vol. 8, p. 115. ~e L. F. Fieser, and M. Fieser, "Reagents for Organic Synthesis," Vol. I, pp. 1045-1055. Wiley, New York.
136
FATTY ACID SYNTHESIS
[18]
Purification of CFA Synthase Cell Disruption and Ammonium Sulfate Fractionation. Cyclopropane fatty acid synthase has been purified from either freshly grown (then frozen) E. coli K 12 or commercially grown E. coli B stationary phase cells. The freshly grown cells have higher activities and give more consistent results. The cell paste (20 g) is thawed and homogenized in 20 ml of 50 mM potassium phosphate buffer, pH 7.6, containing MgC12 (5 mM) and about 1 mg ofdeoxyribonuclease I. This and all subsequent steps are done at 0-4 °. The cells were disrupted by two passages through a French pressure cell at 11,000 psi. The resulting lysate is cleared of large particulate material by centrifugation at 10,000g for 10 min, and the supernatant is retained. The centrifugation supernatant was diluted to a protein concentration of 10 mg/ml, and ammonium sulfate is slowly added to 40% of saturation. After equilibration the precipitate is collected by centrifugation at 10,000g for 15 min and dissolved in the phosphate buffer. Residual ammonium sulfate is removed either by dialysis or by gel filtration. Flotation. The CFA synthase binds to and is stabilized by vesicles of phospholipids containing unsaturated fatty acids, n To purify CFA synthase the ammonium sulfate-fractionated enzyme is exposed first to vesicles made ofphospholipids containing only saturated fatty acids (hydrogenated phospholipids). The synthase does not bind to such vesicles, but, since other proteins do, this provides a negative purification step; CFA synthase is then bound to vesicles made of phospholipids that contain unsaturated fatty acids and separated from the bulk of the other proteins by equilibrium sucrose gradient centrifugation. 6 Ammonium sulfate-purified enzyme, 60% sucrose, and a suspension of multilamellar liposomes (made of hydrogenated phospholipids) are mixed to give a solution containing final concentrations of sucrose, protein, and liposomes of 30% (w/v), 10 mg/ml, and 4 mg/ml, respectively, in 50 mM potassium phosphate (pH 7.5). After incubation at 37° for 15 min, 4 ml of this mixture are placed in a centrifuge tube and sequentially overlaid with 0.5 ml of phosphate buffer containing 25% (w/v) sucrose (d = 1.09 g/ml), 0.5 ml of phosphate buffer containing 20% (w/v) sucrose (d = 1.08 g/ml), and 0.1 ml of buffer. The tube is then centrifuged at 80,000 g for I-2 hr. After centrifugation, the lipid is visible as an opalescent band in or on the surface of the 20% (and sometimes in the 25%) sucrose layer (Fig. 1). This layer was removed by puncturing the side of the tube and removing the band with a syringe. The protein remaining in the 30% sucrose layer is removed by puncturing the bottom of the tube. This fraction is mixed with liposomes made of unsaturated phospholipids, overlaid with 25% and 20% sucrose layer, and recentrifuged. The lipid band containing CFA synthase is removed as before.
[18]
CYCLOPROPANEFATTY ACID SYNTHASE FROM E. coil no phospholipid
l mcj/ml phospholipid
I Protein (%)
Activity (%) I
20%
I.I
7.t I
2.8
25%
2.9
~.2 I
5.3
~%
96.0
91.7 ]
91.9
Sucrose
137
I Protein (%)
Activity {%) I
[26.3 I
I 31.5
FIG. 1. Distribution of cyclopropane fatty acid synthase activity and protein in sucrose step gradients in the absence (left) or presence (right) of liposomes (1 mg/ml) formed from phospholipids containing unsaturated fatty acids. The enzyme preparation (10 mg/ml ammonium sulfate purified enzyme) was mixed with liposomes in 30% sucrose overlaid first with 25% sucrose and then with 20% sucrose as described. After centrifugation, the 20 and 25% layers were collected through the side of the tube whereas the 30% layer was collected through the bottom of the tube. The rationale o f the double flotation step procedure is that CFA synthase does not bind to liposomes made from saturated phospholipid whereas other proteins do. This step is therefore a negative purification step and leaves CFA synthase in the 30% sucrose layer. The synthase is then bound to liposomes o f substrate (unsaturated) phospholipids and resolved from the bulk o f the protein by the second centrifugation. To remove the lipids from the CFA synthase, the vesicle-bound synthase is mixed with sufficient sucrose and KCl to give concentrations 0f40% (w/v) and 1 M, respectively. This mixture is centrifuged at 50,000 g for 1 hr. Centrifugation results in the lipid being layered in the surface of the solution, and lipid-free CFA synthase is obtained by puncturing the bottom o f the tube. The e n z y m e is very unstable in the absence of phospholipid and is best stored in the presence o f 1-2 mg o f phospholipid per milliliter at - 7 0 ° . Purity of CFA Synthase Preparations. Using the double flotation technique, 500- to 600-fold purification CFA synthase have been obtained. 6 If the negative purification step (the first flotation) is omitted, then the resulting e n z y m e is purified only 20 to 50-fold. Preparations obtained by double flotation vesicles are not homogeneous, they contain at least 15 proteins of differing molecular weight as estimated by gel electrophoresis in the presence o f sodium dodecyl sulfate. 6 The molecular weight o f the active CFA synthase is estimated to be 80,000 to 100,000. ~ The E. coli gene coding for CFA synthase has been cloned using in vivo techniques. 17 Such strains 17D. W. Grogan and J. E. Cronan, Jr., unpublished observations. Purified preparations with specific activities of 5 × 105 U/mg protein have been obtained from these strains using the procedure given in the table [modified by omission of the (NH4)~SO4step].
138
FATTY ACID SYNTHESIS
[18]
PURIFICATION OF CYCLOPROPANE FATTY ACID SYNTHASE a
Step
Protein (mg)
Specific activity (unit/rag)
Cell extract Centrifugation (NH4)2SO4 Double flotation
74,100 27,500 9,400 39. l
24.3 49. l 95.3 14,094
a
The synthase was purified from 500 Escherichia coli B.
g
Yield (%) (100) 76 50 6.5
Purification (fold) (l) 2 4 580
of frozen cell paste of commercially grown
show elevated CFA synthase levels and should greatly facilitate purification of this enzyme. 17 A summary of a sample purification is given in the table. Properties of Purified CFA Synthase Purified preparations of CFA synthase are very labile. In the absence of lipid, all activity is lost in <30 min at 37°. In the presence of phospholipid vesicles (2 mg/ml) the enzyme is greatly stabilized (no loss in activity in 30 min at 37°) whereas S-adenosylmethionine (1 raM) preserves about 50% of the activity under similar conditions. The purified enzyme has a Michaelis constant for SAM of 90/~M. S-Adenosylhomocysteine is a competitive inhibitor with a Ki of 220/~M. 6 CFA synthase is saturated with phospholipid vesicles at a liposome concentration of about 0.67 mM (0.5 mg/ml) of phospholipid, e Phospholipids dispersed by sonication are somewhat more effective on a weight basis. This is probably due to the greater external surface area per weight of lipid of single bilayer vesicles. However, the heterogeneous site of sonicated vesicles made from E. coli lipids and the unusual mechanism of action of CFA synthase (see below) precludes calculation of a Michaelis "constant. Liposomes made of hydrogenated lipids are inactive as substrates and fail to inhibit the reaction of the enzyme with liposomes made of unsaturated phospholipids. The CFA synthase is sensitive to sulfhydryl reagents. Dithiobis (nitrobenzoic acid) (DTNB), N-ethylmaleimide, and p-hydroxymercuribenzoate all inhibit the enzyme; however, iodoacetic acid does not. The most specific of these reagents, DTNB, inhibits the enzyme by over 90% at a concentration of 0.5 raM. Inhibition by DTNB is completely reversed by addition of a reducing reagent such as dithiothreitol at 2 raM. The CFA synthase is destabilized and inhibited by high salt concentrations. This is probably due to dissociation of the enzyme from stabilizing lipids. The finding that CFA synthase requires the presence of lipid for maintenance
[19]
UROPYGIAL GLAND FATTY ACID SYNTHASE m R N A
139
of activity explains the activity losses encountered during standard protein chromatographic fractionation procedures since such methods tend to resolve protein and lipid. Owing to these considerations we developed methods for the purification of CFA synthase that avoided conventional chromatographic steps. The commonly used detergents (Tritons, Tweens, Brijs, etc.) destroy enzyme activity. Two detergents, the monooleate and the monolaurate esters of sorbitol, stabilize the enzyme. Unfortunately, these detergents inhibit the enzyme assay and disperse poorly, thus limiting their usefulness. In vesicles composed of a mixture of these lipids, 6 CFA synthase reacts equally well with each of the phospholipid species present in E. coli (phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin). The enzyme is equally active on vesicles of phospholipid in either the ordered or disordered states of the lipid phase transition6 and appears able to react with phosphatidylethanolamine molecules of both the outer and inner leaflets of single bilayer phospholipid vesicles. 6
[19] Isolation, T r a n s l a t i o n in Vitro, a n d P a r t i a l Purification of M e s s e n g e r R N A for F a t t y Acid S y n t h a s e f r o m Uropygial Gland 1
By ALAN G. GOODRIDGE, SIDNEY M. MORRIS, JR., and TAMAR GOLDFLAM Fatty acid synthase is one of the set of lipogenic enzymes whose activities are inhibited by starvation and stimulated by refeeding starved animals.~ In both avian and mammalian liver, changes in lipogenic enzyme activities are a function of altered enzyme protein concentration, which, in turn, are primarily functions of selective alterations in the rates of synthesis of these enzymes.S-6 In maintenance cultures of chick embryo i This work was supported in part by Grant AM 21594 from the National Institute of Arthritis, Metabolism, and Digestive Diseases. We thank Ms. Sally Mansbacher for excellent technical assistance. 2 j. j. Volpe and P. R. Vagelos, Physiol. Rev. 56, 339 (1976). a j. j. Volpe and J. C. Marasa, Biochim. Biophys. Acta 380, 454 (1975). 4 M. C. Craig, C. M. Nepokroeff, M. R. Lakshmanan, and J. W. Porter, Arch. Biochem. Biophys. 152, 619 (1972). 5 Z. E. Zehner, V. C. Joshi, and S. J. Wakil, J. Biol. Chem. 252, 7015 (1977). 6 p. W. F. Fischer and A. G. Goodridge, Arch. Biochem. Biophys. 190, 332 (1978).
METHODS IN ENZYMOLOGY, VOL, 71
Copyright © 1981 by Academic Press, Inc. All fights of reproduction in any form reserved. ISBN 0-12-181971-X
CYCLOPROPANE FATTY ACID SYNTHASE FROM E. coli
133
19% random coil. The removal of lipids results in the disorganization of the complex as judged from both the total enzyme inactivation and the changes in the helical conformation. Urea induces a conformational transition at the 3 M concentration; cholate and sodium dodecyl sulfate have little effect on the a-helical structure although both agents induce a loss of enzyme activity in a similar manner to that induced by urea. All data 8,1a'14 support the interpretation that phospholipids play a fundamental role in the conformation of the active site and the secondary structure, whereas triacylglycerols contribute to the general support of the oligomeric enzyme.
[18] C y c l o p r o p a n e F a t t y A c i d S y n t h a s e f r o m Escherichia coli B y F R E D E R I C K R . T A Y L O R , D E N N I S W . GROGAN, and J O H N E . C R O N A N , JR.
Cyclopropane fatty acids (CFA) 1 are found in the phospholipids of many eubacteria 2 and have also been reported in a few eukaryotic organisms.2 In bacteria, Law a and his co-workers showed that CFAs are formed by methylenation of the double bond of unsaturated fatty acids. These workers also demonstrated an enzyme, CFA synthase, in Clostridium butyricum that catalyzed methylenation of the unsaturated fatty acid moieties of phospholipids using the methyl carbon ofS-adenosyl-L-methionine (SAM) as the methylene donor. This synthase is one of the few enzymes known to act on the nonpolar portion of phospholipids dispersed in a vesicle. The substrate of the enzyme is the double bond of a phospholipid unsaturated fatty acid residue) This double bond must be 9-11 carbon atoms removed from the glycerol backbone of the phospholipid molecule; 4,5 therefore, the site of action is well within the hydrophobic region of the lipid bilayer. For these reasons, CFA synthase is an unusually interesting system for the study of proteinlipid interactions. It binds to substrate phospholipid vesicles. This binding greatly stabilizes the enzyme and is exploited in its purification. 6 H. Goldfine, Adv. Microbiol. Physiol. 8, 1 (1972). 2 W. W. Christie, Top. Lipid Chem. 1, 1 (1970). a j. H. Law, Acc. Chem. Res. 4, 199 (1971). 4 L. A. Marinari, H. Goldfine, and C. Panos, Biochemistry 13, 1978 (1974). 5 j. B. Ohlrogge, F. D. Gunstone, I. A. Ismail, and W. E. M. Lands, Biochim. Biophys. Acta 431, 257 (1976). F. R. Taylor, and J. E. Cronan, Biochemistry 15, 3292 (1979).
METHODS IN ENZYMOLOGY,VOL. 71
Copyright © 1981by AcademicPress, Inc. All rights of reproductionin any form reserved. ISBH 0-12-181971-X
134
FATTY ACID SYNTHESIS
[18]
Assay Principle. The assay is based on the incorporation label from [methyl-aH]S-adenosyl-L-methionine (SAM) into phospholipid. 6,7 After incubation the reaction mixture is pipetted onto a filter paper disk. The disks are washed in trichloroacetic acid (which precipitates phospholipids, but not SAM). CFA synthase activity is then quantitated by scintillation counting of the disk. Reagents [Methyl-3H]S-adenosylmethionine (commercially available) 25 Ci/ftmol; final concentration 0.5 mm Phospholipid dispersion, 0.1 mg, final concentration 1 mg/ml Buffer, potassium phosphate (pH 7.5), final concentration 20 mM S-adenosyl-L-homocysteine hydrolase (SAHase), 1 unit/ml final concentration Partial Purification of SAH Nucleotidase (SAHase). This enzyme was purified from E. coil B by essentially the method of Duerre. s The SAHase preparations purified by ammonium sulfate fractionation and DEAEcellulose chromatography were free of CFA synthase activity. It was found that less purified preparations could be freed of CFA synthase with full retention of SAHase activity by heating to 45° for 15 rain. The preparations were stored at - 2 0 ° in 50 mM Tris-HCl (pH 7.5) buffer and were stable for at least 1 year. SAHase activity is assayed as described by Duerre, 8 except that Nelson's test 9 is used to measure the production of reducing sugar. One unit of SAHase activity is defined as 1 ~tmol of reducing sugar formed from SAH per minute at 37° and pH 7.5. SAHase activity can also be assayed indirectly by relief of the inhibition of CFA synthase by added SAH. Preparation of Phospholipid Dispersions. Phospholipids deficient in CFA (hence rich in unsaturates) are extracted either from E. coil wild-type cells grown to early log phase or from stationary phase cells of a mutant strain deficient in CFA synthase. ~° The cells are grown in a broth medium, harvested, and the phospholipids extracted as described by Ames. H The phospholipids are purified free of neutral lipids by the solvent precipitation method of Law and Essen ~2 or by chromatography on a silicic acid column. ~aThe resulting phospholipids are dried under a stream of nitrogen 7 H. Goldflne, J. LipidRes. 7, 146 (1966). s j. A. Duerre, J. Biol. Chem. 237, 3737 (1962). N. Nelson, J. Biol. Chem. 153, 375 (1944). 10 F. R. Taylor, and J. E. Cronan, Jr. J. Bacteriol. 125, 518 (1976). 11 G. F. Ames, J. Bacteriol. 95, 833 (1968). 12 j. H. Law, and B. Essen, this series, Vol. 14, p. 665. la j. C. Rittmer, and M. A. Wells, this series, Vol. 14, p. 483.
[18]
CYCLOPROPANE FATTY ACID SYNTHASE FROM
E. coli
135
and then dispersed into 1 mM EDTA in glass-distilled water (1 ml/10 mg of lipid) by sonication for 1 min resulting in monolamellar vesicles or by vigorous agitation (vortex mixer) and homogenization resulting in multilamellar liposomes. After dispersion the phospholipid concentration is determined by either the hydroxamate test for lipid esters t4 or by phosphate analysis. 15 Hydrogenation of unsaturated phospholipids (50-100 mg of phospholipid) as performed with 5 mg of Adams catalyst (PtO2) in 20 ml of tetrahydrofuran-methanol (1 : 1) or chloroform-methanol (1 : 1) in an apparatus made from two 125 ml sidearm Erlenmeyer filter flasks connected via the sidearm with thick-wall rubber tubing. One flask contained the phospholipid solution and the catalyst, and the second flask contained 50 ml of 5 N HC1. The first flask was stoppered with a silicone stopper pierced by a glass tube, to the external end of which was wired a rubber policeman. The second flask was capped with a rubber septum. A stabilized NaBH4 solution TM was injected into the acid of the second flask; this resulted in the immediate evolution of Hz (monitored by expansion of the rubber policeman). The apparatus was shaken at room temperature until no further uptake of H2 occurred. Hydrogenation under these conditions has no effect on cyclopropane rings. 2 Liposomes of these phospholipids are made as described above except the solution is heated to 60-70 ° periodically during homogenization. Assay Procedure. To the buffer is added the [all]SAM and then the phospholipid dispersion and enzyme fraction. The buffer is added first to neutralize the dilute sulfuric acid in which the SAM is stored and thus prevent acid precipitation of the phospholipids. It is important that the [all]SAM be mixed with carrier SAM (if needed) just before use. Storage of concentrated [all]SAM solutions results in formation of a trichloroacetic acid (TCA) insoluble product, which leads to high background values. After incubation at 37° for 30 min, the entire reaction mixture is pipetted onto a 2.4 cm disk of Whatman No. 3 MM filter paper mounted on a pin. The filter disks are dried in a stream of hot air for 20 sec and immersed in TCA (10% w/v) for 5 min at room temperature. The disks are then placed in a boiling solution of 5% (w/v) TCA solution, washed in two changes of distilled water for 15 min each, then dried, and assayed for radioactivity after addition of scintillation fluid. The assay is linear with protein from 0.02 to 5 mg of crude supernatant protein and is linear with time for at least 1 hr. A unit of CFA synthase activity is defined as 1 pmol of CFA formed per minute at 37°. 14 B. Shapiro, Biochem. J. 53, 663 (1953). ~5 B. N. Ames, this series, Vol. 8, p. 115. ~e L. F. Fieser, and M. Fieser, "Reagents for Organic Synthesis," Vol. I, pp. 1045-1055. Wiley, New York.
136
FATTY ACID SYNTHESIS
[18]
Purification of CFA Synthase Cell Disruption and Ammonium Sulfate Fractionation. Cyclopropane fatty acid synthase has been purified from either freshly grown (then frozen) E. coli K 12 or commercially grown E. coli B stationary phase cells. The freshly grown cells have higher activities and give more consistent results. The cell paste (20 g) is thawed and homogenized in 20 ml of 50 mM potassium phosphate buffer, pH 7.6, containing MgC12 (5 mM) and about 1 mg ofdeoxyribonuclease I. This and all subsequent steps are done at 0-4 °. The cells were disrupted by two passages through a French pressure cell at 11,000 psi. The resulting lysate is cleared of large particulate material by centrifugation at 10,000g for 10 min, and the supernatant is retained. The centrifugation supernatant was diluted to a protein concentration of 10 mg/ml, and ammonium sulfate is slowly added to 40% of saturation. After equilibration the precipitate is collected by centrifugation at 10,000g for 15 min and dissolved in the phosphate buffer. Residual ammonium sulfate is removed either by dialysis or by gel filtration. Flotation. The CFA synthase binds to and is stabilized by vesicles of phospholipids containing unsaturated fatty acids, n To purify CFA synthase the ammonium sulfate-fractionated enzyme is exposed first to vesicles made ofphospholipids containing only saturated fatty acids (hydrogenated phospholipids). The synthase does not bind to such vesicles, but, since other proteins do, this provides a negative purification step; CFA synthase is then bound to vesicles made of phospholipids that contain unsaturated fatty acids and separated from the bulk of the other proteins by equilibrium sucrose gradient centrifugation. 6 Ammonium sulfate-purified enzyme, 60% sucrose, and a suspension of multilamellar liposomes (made of hydrogenated phospholipids) are mixed to give a solution containing final concentrations of sucrose, protein, and liposomes of 30% (w/v), 10 mg/ml, and 4 mg/ml, respectively, in 50 mM potassium phosphate (pH 7.5). After incubation at 37° for 15 min, 4 ml of this mixture are placed in a centrifuge tube and sequentially overlaid with 0.5 ml of phosphate buffer containing 25% (w/v) sucrose (d = 1.09 g/ml), 0.5 ml of phosphate buffer containing 20% (w/v) sucrose (d = 1.08 g/ml), and 0.1 ml of buffer. The tube is then centrifuged at 80,000 g for I-2 hr. After centrifugation, the lipid is visible as an opalescent band in or on the surface of the 20% (and sometimes in the 25%) sucrose layer (Fig. 1). This layer was removed by puncturing the side of the tube and removing the band with a syringe. The protein remaining in the 30% sucrose layer is removed by puncturing the bottom of the tube. This fraction is mixed with liposomes made of unsaturated phospholipids, overlaid with 25% and 20% sucrose layer, and recentrifuged. The lipid band containing CFA synthase is removed as before.
[18]
CYCLOPROPANEFATTY ACID SYNTHASE FROM E. coil no phospholipid
l mcj/ml phospholipid
I Protein (%)
Activity (%) I
20%
I.I
7.t I
2.8
25%
2.9
~.2 I
5.3
~%
96.0
91.7 ]
91.9
Sucrose
137
I Protein (%)
Activity {%) I
[26.3 I
I 31.5
FIG. 1. Distribution of cyclopropane fatty acid synthase activity and protein in sucrose step gradients in the absence (left) or presence (right) of liposomes (1 mg/ml) formed from phospholipids containing unsaturated fatty acids. The enzyme preparation (10 mg/ml ammonium sulfate purified enzyme) was mixed with liposomes in 30% sucrose overlaid first with 25% sucrose and then with 20% sucrose as described. After centrifugation, the 20 and 25% layers were collected through the side of the tube whereas the 30% layer was collected through the bottom of the tube. The rationale o f the double flotation step procedure is that CFA synthase does not bind to liposomes made from saturated phospholipid whereas other proteins do. This step is therefore a negative purification step and leaves CFA synthase in the 30% sucrose layer. The synthase is then bound to liposomes o f substrate (unsaturated) phospholipids and resolved from the bulk o f the protein by the second centrifugation. To remove the lipids from the CFA synthase, the vesicle-bound synthase is mixed with sufficient sucrose and KCl to give concentrations 0f40% (w/v) and 1 M, respectively. This mixture is centrifuged at 50,000 g for 1 hr. Centrifugation results in the lipid being layered in the surface of the solution, and lipid-free CFA synthase is obtained by puncturing the bottom o f the tube. The e n z y m e is very unstable in the absence of phospholipid and is best stored in the presence o f 1-2 mg o f phospholipid per milliliter at - 7 0 ° . Purity of CFA Synthase Preparations. Using the double flotation technique, 500- to 600-fold purification CFA synthase have been obtained. 6 If the negative purification step (the first flotation) is omitted, then the resulting e n z y m e is purified only 20 to 50-fold. Preparations obtained by double flotation vesicles are not homogeneous, they contain at least 15 proteins of differing molecular weight as estimated by gel electrophoresis in the presence o f sodium dodecyl sulfate. 6 The molecular weight o f the active CFA synthase is estimated to be 80,000 to 100,000. ~ The E. coli gene coding for CFA synthase has been cloned using in vivo techniques. 17 Such strains 17D. W. Grogan and J. E. Cronan, Jr., unpublished observations. Purified preparations with specific activities of 5 × 105 U/mg protein have been obtained from these strains using the procedure given in the table [modified by omission of the (NH4)~SO4step].
138
FATTY ACID SYNTHESIS
[18]
PURIFICATION OF CYCLOPROPANE FATTY ACID SYNTHASE a
Step
Protein (mg)
Specific activity (unit/rag)
Cell extract Centrifugation (NH4)2SO4 Double flotation
74,100 27,500 9,400 39. l
24.3 49. l 95.3 14,094
a
The synthase was purified from 500 Escherichia coli B.
g
Yield (%) (100) 76 50 6.5
Purification (fold) (l) 2 4 580
of frozen cell paste of commercially grown
show elevated CFA synthase levels and should greatly facilitate purification of this enzyme. 17 A summary of a sample purification is given in the table. Properties of Purified CFA Synthase Purified preparations of CFA synthase are very labile. In the absence of lipid, all activity is lost in <30 min at 37°. In the presence of phospholipid vesicles (2 mg/ml) the enzyme is greatly stabilized (no loss in activity in 30 min at 37°) whereas S-adenosylmethionine (1 raM) preserves about 50% of the activity under similar conditions. The purified enzyme has a Michaelis constant for SAM of 90/~M. S-Adenosylhomocysteine is a competitive inhibitor with a Ki of 220/~M. 6 CFA synthase is saturated with phospholipid vesicles at a liposome concentration of about 0.67 mM (0.5 mg/ml) of phospholipid, e Phospholipids dispersed by sonication are somewhat more effective on a weight basis. This is probably due to the greater external surface area per weight of lipid of single bilayer vesicles. However, the heterogeneous site of sonicated vesicles made from E. coli lipids and the unusual mechanism of action of CFA synthase (see below) precludes calculation of a Michaelis "constant. Liposomes made of hydrogenated lipids are inactive as substrates and fail to inhibit the reaction of the enzyme with liposomes made of unsaturated phospholipids. The CFA synthase is sensitive to sulfhydryl reagents. Dithiobis (nitrobenzoic acid) (DTNB), N-ethylmaleimide, and p-hydroxymercuribenzoate all inhibit the enzyme; however, iodoacetic acid does not. The most specific of these reagents, DTNB, inhibits the enzyme by over 90% at a concentration of 0.5 raM. Inhibition by DTNB is completely reversed by addition of a reducing reagent such as dithiothreitol at 2 raM. The CFA synthase is destabilized and inhibited by high salt concentrations. This is probably due to dissociation of the enzyme from stabilizing lipids. The finding that CFA synthase requires the presence of lipid for maintenance
[19]
UROPYGIAL GLAND FATTY ACID SYNTHASE m R N A
139
of activity explains the activity losses encountered during standard protein chromatographic fractionation procedures since such methods tend to resolve protein and lipid. Owing to these considerations we developed methods for the purification of CFA synthase that avoided conventional chromatographic steps. The commonly used detergents (Tritons, Tweens, Brijs, etc.) destroy enzyme activity. Two detergents, the monooleate and the monolaurate esters of sorbitol, stabilize the enzyme. Unfortunately, these detergents inhibit the enzyme assay and disperse poorly, thus limiting their usefulness. In vesicles composed of a mixture of these lipids, 6 CFA synthase reacts equally well with each of the phospholipid species present in E. coli (phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin). The enzyme is equally active on vesicles of phospholipid in either the ordered or disordered states of the lipid phase transition6 and appears able to react with phosphatidylethanolamine molecules of both the outer and inner leaflets of single bilayer phospholipid vesicles. 6
[19] Isolation, T r a n s l a t i o n in Vitro, a n d P a r t i a l Purification of M e s s e n g e r R N A for F a t t y Acid S y n t h a s e f r o m Uropygial Gland 1
By ALAN G. GOODRIDGE, SIDNEY M. MORRIS, JR., and TAMAR GOLDFLAM Fatty acid synthase is one of the set of lipogenic enzymes whose activities are inhibited by starvation and stimulated by refeeding starved animals.~ In both avian and mammalian liver, changes in lipogenic enzyme activities are a function of altered enzyme protein concentration, which, in turn, are primarily functions of selective alterations in the rates of synthesis of these enzymes.S-6 In maintenance cultures of chick embryo i This work was supported in part by Grant AM 21594 from the National Institute of Arthritis, Metabolism, and Digestive Diseases. We thank Ms. Sally Mansbacher for excellent technical assistance. 2 j. j. Volpe and P. R. Vagelos, Physiol. Rev. 56, 339 (1976). a j. j. Volpe and J. C. Marasa, Biochim. Biophys. Acta 380, 454 (1975). 4 M. C. Craig, C. M. Nepokroeff, M. R. Lakshmanan, and J. W. Porter, Arch. Biochem. Biophys. 152, 619 (1972). 5 Z. E. Zehner, V. C. Joshi, and S. J. Wakil, J. Biol. Chem. 252, 7015 (1977). 6 p. W. F. Fischer and A. G. Goodridge, Arch. Biochem. Biophys. 190, 332 (1978).
METHODS IN ENZYMOLOGY, VOL, 71
Copyright © 1981 by Academic Press, Inc. All fights of reproduction in any form reserved. ISBN 0-12-181971-X