[61
RAT LIVER FATTY ACID SYNTHASE
37
Sephadex G-200 is 145,000. SDS-gel electrophoresis gives rise to two stained protein bands of 30,000 and 35,000 daltons which have approximately equal staining intensities. Thus, the carboxyltransferase appears to be composed of two polypeptide chains of differing size and to have an A2B2 subunit structure. Maximal activity in the pH range 4.5-8.0 is achieved with D-biotin methyl ester and D-biotinol acetate; D-biotinol and biocytin are approximately 50% as active. D-Biotin, D-homobiotin, and D-norbiotin are less active exhibiting greatest activity at pH 4.5 and being far less active at pH 8.0. 1-Biotin and D-2'-thiobiotin are inactive. Acknowledgments
This work was supported by research grants from The National Institutes of Health, USPHS (AM-14574 and AM-14575), and The American Heart Association, Inc. The authors are indebted to Mr. Eberhard Zwergel for his superb technical assistance.
[5] F a t t y A c i d S y n t h a s e f r o m R a t L i v e r B y CARL M.
NEPOKROEFF,M. R. LAKSHMANAN, and JOHN W. POaTER
The fatty acid synthase complex exists in mammalian as well as avian liver in the soluble portion of the cell. 1 Negligible activity for the de novo synthesis of fatty acids is associated with the microsomal and mitochondrial fractions. The purification of the soluble fatty acid synthase complex of rat liver to homogeneity has been achieved, ~ and this complex has been found to have a molecular weight of approximately 540,000. Assay Method
The fatty acid synthase complex may be assayed by either radiochemical or spectrophotometric methods. In the r~dioisotopic method, incorporation of 1-14C-labeled acetyl-CoA into fatty acids is measured in the presence of malonyl-CoA and NADPH. This method is reliable for either crude or purified enzyme preparations. (For the methods of prepa1j. W. Porter, S. Kumar, and R. E. Dugan, Progr. Biochem. Pharmacol. 6, 1 (1971). D. N, Burton, A. G. Haavik, and J. W. Porter, Arch. Biochem. Biophys. 126, 141 (1968).
38
FATTY ACID SYNTHESIS
[5]
ration of 1-14C-labeled acetyl-CoA, unlabeled acetyl-CoA, malonyl-CoA, and details of the radioassay, see Hsu et al. 2a) The spectrophotometric method measures the malonyl-CoA- and acetyl-CoA-dependent rate of oxidation of N A D P H at 340 nm and is best suited for purified enzyme preparations. However, it can be used with crude preparations providing appropriate corrections are made. Spectrophotometric Assay. The reaction mixture (total volume 1.0 ml; final pH 7.0) contains 500 t~moles potassium phosphate buffer, pH 7.0; 33 nmoles of acetyl-CoA; 100 nmoles of malonyl-CoA; 100 nmoles of N A D P H ; 1 ~mole of E D T A ; 1 ~mole of fl-mercaptoethanol; and enzyme protein (5-10 ~g of DEAE-cellulose purified fatty acid synthase or 50-100 ~g of the 105,000 g supernatant protein fraction). The reaction is initiated by the addition of enzyme to the mixture of substrates previously equilibrated at 30 ° for 5 minutes. It is important to preincubate the enzyme in order to obtain maximum enzymatic activity2 The oxidation of N A D P H is followed at 340 nm, while the cuvette chamber is maintained at 30 °. Full-scale deflection of the recorder tracing is set at 0.2 absorbance unit. The initial slope of the recorder tracing is used to calculate the rate of f a t t y acid synthesis. A correction is made for the rate of N A D P H oxidation in the absence of malonyl-CoA. It should be'noted that butyryl-CoA has been reported to be a better primer than acetyl-CoA in the mammalian fatty acid synthase system. 4 The fatty acid synthase assay system has also been modified and successfully employed for the determination of malonyl-CoA in tissue extracts2 Units. A unit of enzymatic activity is defined as the amount of enzyme protein required to synthesize 1 nmole of palmitic acid (equivalent to the oxidation of 14 nmoles of N A D P H ) per minute under the conditions of the assay. The specific activity is defined as the number of activity units per milligram of protein. Protein is determined by the biuret method of Gornall et al2
Purification of the Enzyme System All solutions used in the preparation of the enzyme system are prepared with deionized water and all buffers contain potassium phosphate, 2, R. Y. Hsu, P. H. W. Butterworth, and J. W. Porter, Vol. XIV [4], 33. 3Preincubation of either purified enzyme or crude liver homogenate fractions in 500 mM potassium phosphate buffer, pH 7.0, and 5 mM dithiothreitol at 37o for at least 15 minutes is essential for maximum enzymatic activity. 4C. H. Lin and S. Kumar, J. Biol. Chem. 247, 604 (1972). 5R. W. Guynn, D. Veloso, and R. L. Veech, J. Biol. Chem. 247, 7325 (1972). ~A. G. Gornall, C. J. Bardawill, and M. M. David, d. Biol. Chem. 117, 751 (1949).
[51
RAT LIVER FATTY ACID SYNTHASE
39
pH 7.0, 1 mM EDTA and either 1 mM dithiothreitol or 1 mM fl-mercaptoethanol, unless otherwise stated. Preparation o] Liver Supernatant Solution. Since the concentration of fatty acid synthase in liver is related to the nutritional state of the rat 7 it is important to begin the preparation with rats which have been fasted and refed. Hence, male rats weighing about 150 g each are starved for 48 hours, then fed a fat-free diet s for 48 hours. The rats are killed by decapitation and the livers are quickly removed and placed on ice. The chilled livers are homogenized in 1.5 volumes of a phosphate-bicarbonate buffer (70 mM K H C Q , 85 mM K2HPO4, 9 mM KH2PO4, and 1 mM dithiothreitol), pH 8.0, in a Potter-Elvehjem type of homogenizer with a motor-driven Teflon pestle. Large batches of liver are more conveniently homogenized in a Waring blender for 15 seconds at full speed. The homogenate is centrifuged at 20,000 g for 10 minutes; the supernatant solution (postmitochondrial fraction) is retained, and then it is recentrifuged at 105,000 g for 60 minutes. The supernatant solution (105,000 g fraction) contains the fatty acid synthase. This solution can be stored at --15 ° for at least 2 months without appreciable loss of enzymatic activity. The purification procedure for the fatty acid synthase that follows is a modification of the method originally described by Burton et al. 2 This procedure is carried out at room temperature unless otherwise specified. First Ammonium Sul]ate Fractionation. The frozen rat liver 105,000 g supernatant solution (40 ml) is thawed at room temperature. Saturated ammonium sulfate solution, pH 7.0, containing 3 mM EDTA and 1 mM fl-mercaptoethanol is added to a saturation of 20%. After stirring for 15 minutes the mixture is centrifuged and the precipitate discarded. The supernatant solution is brought to 35% saturation with ammonium sulfate. The precipitated protein is collected by centrifugation and retained for further fractionation. Calcium Phosphate Gel Adsorption. The protein fraction from the previous step (approximately 500 mg protein) is dissolved and then diluted to approximately 150 ml with 5 mM potassium phosphate buffer. The protein fraction is solubilized by gentle stirring with a glass rod in a small volume of buffer. Foaming is avoided insofar as possible since the enzyme is susceptible to surface denaturation. 2 Calcium phosphate gel equal to half the weight of the protein is added with stirring. The 7D. N. Burton, J. M. Collins, A. L. Kennan, and J. W. Porter, J. Biol. Chem. 244, 4510 (1969). s Fat-free diet is obtained from Nutritional Biochemical Corporation, Cleveland, Ohio.
40
FATTY ACID SYNTHESIS
[6]
suspension is centrifuged immediately for 2-3 minutes at 5000 g and the supernatant solution is retained. Since the fatty acid synthase is not stable in a low ionic strength buffer,~ it is imperative that this procedure be performed rapidly and that the next purification step, DEAE-cellulose chromatography, follow immediately. DEAE-Cellulose Chromatography. The supernatant solution from the previous step is adsorbed to a column of DEAE-cellulose (10.3 X 3.5 cm) which has previously been washed with 50 mM potassium phosphate buffer. Most of the protein is removed from the column by washing with the above buffer. Washing with the buffer is continued until the light absorption at 280 nm decreases to approximately 0.05 unit. The enzyme is then eluted from the column with 160 mM potassium phosphate buffer. This chromatographic procedure is performed at room temperature with a flow rate of approximately 5-10 ml/minute. All steps of the chromatographic procedure are performed rapidly since the enzyme is not stable in low ionic strength buffer. Elution of the protein is monitored spectrophotometrically at 280 nm and 5 ml eluate fractions are collected. Those fractions exhibiting absorbancies greater than 0.50 unit are combined and retained. Second Ammonium Sul]ate Fractionation. The material eluted from DEAE-cellulose is brought to 33% saturation with ammonium sulfate and the precipitated protein is collected by centrifugation. This protein fraction is dissolved in a minimum volume (1-2 ml) of cold 0.5 M potassium phosphate buffer, pH 7.0, and dialyzed against the same buffer overnight at 4 °. The enzyme at this stage is referred to as DEAE-cellulose purified fatty acid synthase. A yield of 30-40 mg of the fatty acid synthase can be expected from 40 ml (approximately 2000 mg of total soluble rat liver protein) of the crude 105,000 g supernatant fraction. Since the rat liver enzyme is sensitive to surface denaturation," as are the rat mammary gland and pigeon liver enzymes, this preparation should be handled with care. The results of a typical purification of this enzyme are shown in Table I. The purification of DEAE-cellulose purified fatty acid synthase from the 105,000 g supernatant fraction requires approximately 5 hours. Sucrose Density Gradient CentriJugation. The results of ultracentrifugation studies by Collins et al2 on DEAE-cellulose purified fatty acid synthase isolated from livers of fasted, diabetic, or normal rats showed the presence of a contaminating protein with a sedimentation coefficient of 7 S. The relative concentration of this component is negligible, however, in enzyme isolated from animals in the refed state and is therefore deJ. M. Collins, M. C. Craig, C. M. Nepokroeff, A. L. Kennan, and J. W. Porter,
Arch, Biochem. Biophys. 143, 343 (1971).
[~]
41
RAT LIVER FATTY ACID S Y N T H A S E
TABLE I PURIFICATION OF RAT LIVER FATTY ACID SYNTHASE
Fraction
Total protein (mg)
Enzymatic activity (units a)
Specific activity (unitsa/mg protein)
105,000 g supernatant (NH4) ~SO4, 20-33 % DEAE-cellulose Sucrose density gradient
2000 500 40 10b
10,000 6,000 2,640 750
5 12 66 75
a Units are reported in nanomoles of palmitate formed per minute at 30°. b Recovery of protein after gradient centrifugation is actually not as low as indicated. In order to insure that the enzyme fraction was completely devoid of minor components, only the leading half of the protein profile was recovered after each centrifugation. Total recovery of enzyme from the gradients is usually 90-95 %. tected only by immunologic methods. This 7 S component can be removed by two successive sucrose density gradient centrifugations in a Spinco SW 25.1 rotor at 4 ° and 58,000 g for a period of 46 hours. The linear gradients contain 5-20% (w/v) of sucrose and 500 m M potassium phosphate buffer. After centrifugation the bottom of the tube is punctured and the solution is collected dropwise in 1 ml fractions. Protein in the gradient is monitored spectrophotometrically at 280 nm. The fatty acid synthase sediments as a single peak of protein. The faster sedimenting half of the protein profile from the gradient is concentrated by ammonium sulfate precipitation and the protein fraction is dialyzed against 500 m M potassium phosphate buffer. This fraction is again subjected to a second identical gradient centrifugation. The protein profile from the second centrifugation is fractionated as before and the faster sedimenting half of the profile is retained. This fraction is dialyzed overnight against 500 m M potassium phosphate buffer and then stored in the same buffer. This concentration of buffer is necessary to preserve the structural integrity of the rat liver multienzyme complex. The puri'fied enzyme fraction is referred to as the sucrose density gradient (SDG) purified fatty acid synthase. This enzyme has been shown to be homogeneous by immunologic criteria. TM Storage o] Enzyme. The purified rat liver fatty acid synthase can be stored in the 500 m M potassium phosphate buffer containing 1 m M dithiothreitol at 0-4 ° for 4-5 days with negligible loss of activity. The 1, M. C. Craig, C. M. Nepokroeff, M. R. Lakshmanan, and J. W. Porter, Arch. Biochem. Biophys. 152, 619 (1972).
42
FATTY ACID SYNTHESIS
[5]
TABLE II PARTIAL AMINO ACID COMPOSITIONS OF RAT AND PIGEON LIVER FATTY ACID SYNTHASES a
Amino acid
Rat (moles/mole of enzyme)
Pigeon (moles/mole of enzyme)
Aspartic acid Threonine Serine Proline Glutamic acid Glycine Alanine Valine Isoleucim Leucine Tyrosine Phenylalanine
280 189 248 194 355 274 306 256 141 456 76 118
254 137 193 137 344 240 250 240 165 340 76 99
Reproduced from Burton et al. 2 synthase can also be stored frozen (--20 °) in the same buffer. Under this condition the enzyme complex is stable for at least 1 month. Whether the synthase is stored at 4 ° or at --20 °, it is imperative that the preparation be preincubated prior to assay2 Characterization of the Fatty Acid Synthase P h y s i c o c h e m i c a l C h a r a c t e r i z a t i o n ? The DEAE-cellulose purified rat liver fatty acid synthase sediments essentially as a single component in the Spinco model E analytical ultracentrifuge when dissolved in 0.5 M potassium phosphate buffer. The s2o,~ value is 12.3 S. A trace of a slower sedimenting component represents either half-molecular weight subunits of the fatty acid synthase or the 7 S minor component. The DEAE-cellulose purified enzyme complex has also been shown to be homogeneous by SDG centrifugation. The molecular weight of the rat liver fatty acid synthase has been determined to be 5.4 X l0 ~ g/mole by sedimentationdiffusion analysis in 0.5 M potassium phosphate buffer. 2 The amino acid composition of rat and pigeon liver fatty acid synthases are very similar (Table II), but these enzymes are immunologically dissimilar since they do not cross-react. 11 The rat liver enzyme has a high sulfhydryl content (90-92 groups/mole) and a 4'-phosphopante-
11M. C. Craig, C. M. Nepokroeff, and J. W. Porter, unpublished results.
[61
RAT LIVER FATTY ACID SYNTHASE
43
theine content of 1.05 moles/mole of enzyme. Negligible amounts of flavin (0.006-0.024 mole of flavin per mole of enzyme) are found. Kinetics. The pH optimum for the purified rat liver fatty acid synthase complex is 7.0. The Km value for acetyl-CoA is 4.4 fdl//.2 The apparent K,, value for malonyl-CoA is 10 ~.M.11a Immunochemical Characterization TM (a) Preparation of antiserum. The SDG-purified fatty acid synthase is used as the antigen in order to obtain antiserum that is specific for only the fatty acid synthase. The enzyme purified by two successive SDG centrifugations is mixed with an equal volume of incomplete Freund's adjuwmt and iniected into the foot pads of rabbits. Each rabbit receives 2 mg of fatty acid synthase protein and a second injection is given 10 days later. The rabbits are bled at 2-week intervals after the second injection, sera are pooled, and the -/-globulin fraction is isolated as described by Levy and Sober. 12 The serum from rabbits which are injected only with incomplete Freund's adjuvant is prepared in the same way and used as control serum. All sera are stored at --15 °. (b) Immunodiffusion and precipitation reactions. Ouchterlony microdouble diffusion is carried out on microscope slides in 0.5% agarose containing 1.0% (w/v) sodium chloride. The agarose gels are developed for 24 hours at 24 °. Immunoprecipitation reactions are performed for 1 hour at 37 ° in 10 mM potassium phosphate buffer, pH 7.0, containing 1 mM dithiothreitol. The samples are then incubated at 4 ° overnight. Precipitates are collected by centrifugation and then washed twice with cold 0.9% (w/v) sodium chloride containing 10 mM potassium phosphate buffer, pH 7.0. Quantitative precipitin analyses and equivalence point determinations are performed as outlined by Kabat and Mayer. 13 Our recent studies have shown that rat liver fatty acid synthase is immunologically homogeneous only after the DEAE-cellulose purified enzyme is subiected to two successive SDG centrifugations. TM Ouchterlony double diffusion patterns show that only one precipitin band is obtained with antiserum prepared against fatty acid synthase purified by SDG centrifugation. However, antiserum prepared against DEAE-cellulose purified enzyme gives a second minor band. The minor precipitin band 1,~ S. S. Katiyar, Dept. of Physiological Chemistry, University of Wisconsin, Madison (unpublished). 1, H. B. Levy and H. A. Sober, Proc. Soc. Exp. Biol. Med. 103, 250 (1960). The ammonium sulfate method of preparation of the -~-globulin fraction was also successfully employed. See M. Goldman, "Fluorescent Antibody Methods." Academic Press, New York, 1968. 1, E. A. Kabat and M. M. Mayer, in "Experimental Immunochemistry," 2nd ed., p. 22. Thomas, Springfield, Illinois, 1971.
44
FATTY ACID SYNTHESIS
[6]
is the result of the reaction of antibody with the 7 S component present in the DEAE-cellulose purified fatty acid synthase2 The immunodiffusion studies also show that the same species of fatty acid synthase is present in the livers of rats fed a normal diet, fat-free diet, or fasted. The rat liver fatty acid synthase reacts with its antiserum to produce a typical quantitative precipitin reaction. At the equivalence point, 33.5 mg of antiserum protein were required to precipitate 1 mg of purified fatty acid synthase. The equivalence point is essentially the same for enzyme obtained from rats fed a normal diet or refed a fat-free diet. Antiserum prepared against fatty acid synthase has been successfully employed in imlnunochemical studies to evaluate the relative importance of synthetic and degradative rates on changes in content of rat liver fatty acid synthase under different nutritional conditions. 1° The results of these immunochemical studies are discussed in the next section. Control of the Rate of Synthesis of the Fatty Acid Synthase Rat liver fatty acid synthase is markedly affected by the nutritional and hormonal states of the animal. Thus, the level of the enzyme is greatly reduced on fasting and increased to supranormal levels on rcfeeding a fat-free diet to previously fasted animals. This increase has been shown to result from an adaptive increase in the rate of synthesis of the enzyme.7 The rates of synthesis and degradation of rat liver fatty acid synthase have been determined as a function of nutritional state in order to determine the relative importance of each in controlling the quantity of this enzyme in the liver. 1° The amount of fatty acid synthase has been determined in these studies by enzyme isolation and by the immunochemical technique. The results of these studies have established the major factor controlling the level of this enzyme to be the rate of its synthesis. However, changes in the rate of degradation may be important in the early stages of fasting. Recently, it has been shown that insulin is essential for the dietary induction of rat liver fatty acid synthase to supranormal levels) 4 Diabetic rats synthesize negligible amounts of this enzyme whereas insulin administration brings about a dramatic increase in its synthesis. Glucago~ and cyclic AMP block the dietary induction of the fatty acid synthase. Hence, it is important to consider both the hormonal and dietary status of animals in studies which concern the isolation and the properties of rat liver fatty acid synthase. 14M. R. Lakshmanan, C. M. Nepokroeff, and J. W. Porter, Proc. Nat. Acad. Sci. U.S. 69, 3516 (1972).
RAT LIVER FATTY ACID SYNTHASE
37
Sephadex G-200 is 145,000. SDS-gel electrophoresis gives rise to two stained protein bands of 30,000 and 35,000 daltons which have approximately equal staining intensities. Thus, the carboxyltransferase appears to be composed of two polypeptide chains of differing size and to have an A2B2 subunit structure. Maximal activity in the pH range 4.5-8.0 is achieved with D-biotin methyl ester and D-biotinol acetate; D-biotinol and biocytin are approximately 50% as active. D-Biotin, D-homobiotin, and D-norbiotin are less active exhibiting greatest activity at pH 4.5 and being far less active at pH 8.0. 1-Biotin and D-2'-thiobiotin are inactive. Acknowledgments
This work was supported by research grants from The National Institutes of Health, USPHS (AM-14574 and AM-14575), and The American Heart Association, Inc. The authors are indebted to Mr. Eberhard Zwergel for his superb technical assistance.
[5] F a t t y A c i d S y n t h a s e f r o m R a t L i v e r B y CARL M.
NEPOKROEFF,M. R. LAKSHMANAN, and JOHN W. POaTER
The fatty acid synthase complex exists in mammalian as well as avian liver in the soluble portion of the cell. 1 Negligible activity for the de novo synthesis of fatty acids is associated with the microsomal and mitochondrial fractions. The purification of the soluble fatty acid synthase complex of rat liver to homogeneity has been achieved, ~ and this complex has been found to have a molecular weight of approximately 540,000. Assay Method
The fatty acid synthase complex may be assayed by either radiochemical or spectrophotometric methods. In the r~dioisotopic method, incorporation of 1-14C-labeled acetyl-CoA into fatty acids is measured in the presence of malonyl-CoA and NADPH. This method is reliable for either crude or purified enzyme preparations. (For the methods of prepa1j. W. Porter, S. Kumar, and R. E. Dugan, Progr. Biochem. Pharmacol. 6, 1 (1971). D. N, Burton, A. G. Haavik, and J. W. Porter, Arch. Biochem. Biophys. 126, 141 (1968).
38
FATTY ACID SYNTHESIS
[5]
ration of 1-14C-labeled acetyl-CoA, unlabeled acetyl-CoA, malonyl-CoA, and details of the radioassay, see Hsu et al. 2a) The spectrophotometric method measures the malonyl-CoA- and acetyl-CoA-dependent rate of oxidation of N A D P H at 340 nm and is best suited for purified enzyme preparations. However, it can be used with crude preparations providing appropriate corrections are made. Spectrophotometric Assay. The reaction mixture (total volume 1.0 ml; final pH 7.0) contains 500 t~moles potassium phosphate buffer, pH 7.0; 33 nmoles of acetyl-CoA; 100 nmoles of malonyl-CoA; 100 nmoles of N A D P H ; 1 ~mole of E D T A ; 1 ~mole of fl-mercaptoethanol; and enzyme protein (5-10 ~g of DEAE-cellulose purified fatty acid synthase or 50-100 ~g of the 105,000 g supernatant protein fraction). The reaction is initiated by the addition of enzyme to the mixture of substrates previously equilibrated at 30 ° for 5 minutes. It is important to preincubate the enzyme in order to obtain maximum enzymatic activity2 The oxidation of N A D P H is followed at 340 nm, while the cuvette chamber is maintained at 30 °. Full-scale deflection of the recorder tracing is set at 0.2 absorbance unit. The initial slope of the recorder tracing is used to calculate the rate of f a t t y acid synthesis. A correction is made for the rate of N A D P H oxidation in the absence of malonyl-CoA. It should be'noted that butyryl-CoA has been reported to be a better primer than acetyl-CoA in the mammalian fatty acid synthase system. 4 The fatty acid synthase assay system has also been modified and successfully employed for the determination of malonyl-CoA in tissue extracts2 Units. A unit of enzymatic activity is defined as the amount of enzyme protein required to synthesize 1 nmole of palmitic acid (equivalent to the oxidation of 14 nmoles of N A D P H ) per minute under the conditions of the assay. The specific activity is defined as the number of activity units per milligram of protein. Protein is determined by the biuret method of Gornall et al2
Purification of the Enzyme System All solutions used in the preparation of the enzyme system are prepared with deionized water and all buffers contain potassium phosphate, 2, R. Y. Hsu, P. H. W. Butterworth, and J. W. Porter, Vol. XIV [4], 33. 3Preincubation of either purified enzyme or crude liver homogenate fractions in 500 mM potassium phosphate buffer, pH 7.0, and 5 mM dithiothreitol at 37o for at least 15 minutes is essential for maximum enzymatic activity. 4C. H. Lin and S. Kumar, J. Biol. Chem. 247, 604 (1972). 5R. W. Guynn, D. Veloso, and R. L. Veech, J. Biol. Chem. 247, 7325 (1972). ~A. G. Gornall, C. J. Bardawill, and M. M. David, d. Biol. Chem. 117, 751 (1949).
[51
RAT LIVER FATTY ACID SYNTHASE
39
pH 7.0, 1 mM EDTA and either 1 mM dithiothreitol or 1 mM fl-mercaptoethanol, unless otherwise stated. Preparation o] Liver Supernatant Solution. Since the concentration of fatty acid synthase in liver is related to the nutritional state of the rat 7 it is important to begin the preparation with rats which have been fasted and refed. Hence, male rats weighing about 150 g each are starved for 48 hours, then fed a fat-free diet s for 48 hours. The rats are killed by decapitation and the livers are quickly removed and placed on ice. The chilled livers are homogenized in 1.5 volumes of a phosphate-bicarbonate buffer (70 mM K H C Q , 85 mM K2HPO4, 9 mM KH2PO4, and 1 mM dithiothreitol), pH 8.0, in a Potter-Elvehjem type of homogenizer with a motor-driven Teflon pestle. Large batches of liver are more conveniently homogenized in a Waring blender for 15 seconds at full speed. The homogenate is centrifuged at 20,000 g for 10 minutes; the supernatant solution (postmitochondrial fraction) is retained, and then it is recentrifuged at 105,000 g for 60 minutes. The supernatant solution (105,000 g fraction) contains the fatty acid synthase. This solution can be stored at --15 ° for at least 2 months without appreciable loss of enzymatic activity. The purification procedure for the fatty acid synthase that follows is a modification of the method originally described by Burton et al. 2 This procedure is carried out at room temperature unless otherwise specified. First Ammonium Sul]ate Fractionation. The frozen rat liver 105,000 g supernatant solution (40 ml) is thawed at room temperature. Saturated ammonium sulfate solution, pH 7.0, containing 3 mM EDTA and 1 mM fl-mercaptoethanol is added to a saturation of 20%. After stirring for 15 minutes the mixture is centrifuged and the precipitate discarded. The supernatant solution is brought to 35% saturation with ammonium sulfate. The precipitated protein is collected by centrifugation and retained for further fractionation. Calcium Phosphate Gel Adsorption. The protein fraction from the previous step (approximately 500 mg protein) is dissolved and then diluted to approximately 150 ml with 5 mM potassium phosphate buffer. The protein fraction is solubilized by gentle stirring with a glass rod in a small volume of buffer. Foaming is avoided insofar as possible since the enzyme is susceptible to surface denaturation. 2 Calcium phosphate gel equal to half the weight of the protein is added with stirring. The 7D. N. Burton, J. M. Collins, A. L. Kennan, and J. W. Porter, J. Biol. Chem. 244, 4510 (1969). s Fat-free diet is obtained from Nutritional Biochemical Corporation, Cleveland, Ohio.
40
FATTY ACID SYNTHESIS
[6]
suspension is centrifuged immediately for 2-3 minutes at 5000 g and the supernatant solution is retained. Since the fatty acid synthase is not stable in a low ionic strength buffer,~ it is imperative that this procedure be performed rapidly and that the next purification step, DEAE-cellulose chromatography, follow immediately. DEAE-Cellulose Chromatography. The supernatant solution from the previous step is adsorbed to a column of DEAE-cellulose (10.3 X 3.5 cm) which has previously been washed with 50 mM potassium phosphate buffer. Most of the protein is removed from the column by washing with the above buffer. Washing with the buffer is continued until the light absorption at 280 nm decreases to approximately 0.05 unit. The enzyme is then eluted from the column with 160 mM potassium phosphate buffer. This chromatographic procedure is performed at room temperature with a flow rate of approximately 5-10 ml/minute. All steps of the chromatographic procedure are performed rapidly since the enzyme is not stable in low ionic strength buffer. Elution of the protein is monitored spectrophotometrically at 280 nm and 5 ml eluate fractions are collected. Those fractions exhibiting absorbancies greater than 0.50 unit are combined and retained. Second Ammonium Sul]ate Fractionation. The material eluted from DEAE-cellulose is brought to 33% saturation with ammonium sulfate and the precipitated protein is collected by centrifugation. This protein fraction is dissolved in a minimum volume (1-2 ml) of cold 0.5 M potassium phosphate buffer, pH 7.0, and dialyzed against the same buffer overnight at 4 °. The enzyme at this stage is referred to as DEAE-cellulose purified fatty acid synthase. A yield of 30-40 mg of the fatty acid synthase can be expected from 40 ml (approximately 2000 mg of total soluble rat liver protein) of the crude 105,000 g supernatant fraction. Since the rat liver enzyme is sensitive to surface denaturation," as are the rat mammary gland and pigeon liver enzymes, this preparation should be handled with care. The results of a typical purification of this enzyme are shown in Table I. The purification of DEAE-cellulose purified fatty acid synthase from the 105,000 g supernatant fraction requires approximately 5 hours. Sucrose Density Gradient CentriJugation. The results of ultracentrifugation studies by Collins et al2 on DEAE-cellulose purified fatty acid synthase isolated from livers of fasted, diabetic, or normal rats showed the presence of a contaminating protein with a sedimentation coefficient of 7 S. The relative concentration of this component is negligible, however, in enzyme isolated from animals in the refed state and is therefore deJ. M. Collins, M. C. Craig, C. M. Nepokroeff, A. L. Kennan, and J. W. Porter,
Arch, Biochem. Biophys. 143, 343 (1971).
[~]
41
RAT LIVER FATTY ACID S Y N T H A S E
TABLE I PURIFICATION OF RAT LIVER FATTY ACID SYNTHASE
Fraction
Total protein (mg)
Enzymatic activity (units a)
Specific activity (unitsa/mg protein)
105,000 g supernatant (NH4) ~SO4, 20-33 % DEAE-cellulose Sucrose density gradient
2000 500 40 10b
10,000 6,000 2,640 750
5 12 66 75
a Units are reported in nanomoles of palmitate formed per minute at 30°. b Recovery of protein after gradient centrifugation is actually not as low as indicated. In order to insure that the enzyme fraction was completely devoid of minor components, only the leading half of the protein profile was recovered after each centrifugation. Total recovery of enzyme from the gradients is usually 90-95 %. tected only by immunologic methods. This 7 S component can be removed by two successive sucrose density gradient centrifugations in a Spinco SW 25.1 rotor at 4 ° and 58,000 g for a period of 46 hours. The linear gradients contain 5-20% (w/v) of sucrose and 500 m M potassium phosphate buffer. After centrifugation the bottom of the tube is punctured and the solution is collected dropwise in 1 ml fractions. Protein in the gradient is monitored spectrophotometrically at 280 nm. The fatty acid synthase sediments as a single peak of protein. The faster sedimenting half of the protein profile from the gradient is concentrated by ammonium sulfate precipitation and the protein fraction is dialyzed against 500 m M potassium phosphate buffer. This fraction is again subjected to a second identical gradient centrifugation. The protein profile from the second centrifugation is fractionated as before and the faster sedimenting half of the profile is retained. This fraction is dialyzed overnight against 500 m M potassium phosphate buffer and then stored in the same buffer. This concentration of buffer is necessary to preserve the structural integrity of the rat liver multienzyme complex. The puri'fied enzyme fraction is referred to as the sucrose density gradient (SDG) purified fatty acid synthase. This enzyme has been shown to be homogeneous by immunologic criteria. TM Storage o] Enzyme. The purified rat liver fatty acid synthase can be stored in the 500 m M potassium phosphate buffer containing 1 m M dithiothreitol at 0-4 ° for 4-5 days with negligible loss of activity. The 1, M. C. Craig, C. M. Nepokroeff, M. R. Lakshmanan, and J. W. Porter, Arch. Biochem. Biophys. 152, 619 (1972).
42
FATTY ACID SYNTHESIS
[5]
TABLE II PARTIAL AMINO ACID COMPOSITIONS OF RAT AND PIGEON LIVER FATTY ACID SYNTHASES a
Amino acid
Rat (moles/mole of enzyme)
Pigeon (moles/mole of enzyme)
Aspartic acid Threonine Serine Proline Glutamic acid Glycine Alanine Valine Isoleucim Leucine Tyrosine Phenylalanine
280 189 248 194 355 274 306 256 141 456 76 118
254 137 193 137 344 240 250 240 165 340 76 99
Reproduced from Burton et al. 2 synthase can also be stored frozen (--20 °) in the same buffer. Under this condition the enzyme complex is stable for at least 1 month. Whether the synthase is stored at 4 ° or at --20 °, it is imperative that the preparation be preincubated prior to assay2 Characterization of the Fatty Acid Synthase P h y s i c o c h e m i c a l C h a r a c t e r i z a t i o n ? The DEAE-cellulose purified rat liver fatty acid synthase sediments essentially as a single component in the Spinco model E analytical ultracentrifuge when dissolved in 0.5 M potassium phosphate buffer. The s2o,~ value is 12.3 S. A trace of a slower sedimenting component represents either half-molecular weight subunits of the fatty acid synthase or the 7 S minor component. The DEAE-cellulose purified enzyme complex has also been shown to be homogeneous by SDG centrifugation. The molecular weight of the rat liver fatty acid synthase has been determined to be 5.4 X l0 ~ g/mole by sedimentationdiffusion analysis in 0.5 M potassium phosphate buffer. 2 The amino acid composition of rat and pigeon liver fatty acid synthases are very similar (Table II), but these enzymes are immunologically dissimilar since they do not cross-react. 11 The rat liver enzyme has a high sulfhydryl content (90-92 groups/mole) and a 4'-phosphopante-
11M. C. Craig, C. M. Nepokroeff, and J. W. Porter, unpublished results.
[61
RAT LIVER FATTY ACID SYNTHASE
43
theine content of 1.05 moles/mole of enzyme. Negligible amounts of flavin (0.006-0.024 mole of flavin per mole of enzyme) are found. Kinetics. The pH optimum for the purified rat liver fatty acid synthase complex is 7.0. The Km value for acetyl-CoA is 4.4 fdl//.2 The apparent K,, value for malonyl-CoA is 10 ~.M.11a Immunochemical Characterization TM (a) Preparation of antiserum. The SDG-purified fatty acid synthase is used as the antigen in order to obtain antiserum that is specific for only the fatty acid synthase. The enzyme purified by two successive SDG centrifugations is mixed with an equal volume of incomplete Freund's adjuwmt and iniected into the foot pads of rabbits. Each rabbit receives 2 mg of fatty acid synthase protein and a second injection is given 10 days later. The rabbits are bled at 2-week intervals after the second injection, sera are pooled, and the -/-globulin fraction is isolated as described by Levy and Sober. 12 The serum from rabbits which are injected only with incomplete Freund's adjuvant is prepared in the same way and used as control serum. All sera are stored at --15 °. (b) Immunodiffusion and precipitation reactions. Ouchterlony microdouble diffusion is carried out on microscope slides in 0.5% agarose containing 1.0% (w/v) sodium chloride. The agarose gels are developed for 24 hours at 24 °. Immunoprecipitation reactions are performed for 1 hour at 37 ° in 10 mM potassium phosphate buffer, pH 7.0, containing 1 mM dithiothreitol. The samples are then incubated at 4 ° overnight. Precipitates are collected by centrifugation and then washed twice with cold 0.9% (w/v) sodium chloride containing 10 mM potassium phosphate buffer, pH 7.0. Quantitative precipitin analyses and equivalence point determinations are performed as outlined by Kabat and Mayer. 13 Our recent studies have shown that rat liver fatty acid synthase is immunologically homogeneous only after the DEAE-cellulose purified enzyme is subiected to two successive SDG centrifugations. TM Ouchterlony double diffusion patterns show that only one precipitin band is obtained with antiserum prepared against fatty acid synthase purified by SDG centrifugation. However, antiserum prepared against DEAE-cellulose purified enzyme gives a second minor band. The minor precipitin band 1,~ S. S. Katiyar, Dept. of Physiological Chemistry, University of Wisconsin, Madison (unpublished). 1, H. B. Levy and H. A. Sober, Proc. Soc. Exp. Biol. Med. 103, 250 (1960). The ammonium sulfate method of preparation of the -~-globulin fraction was also successfully employed. See M. Goldman, "Fluorescent Antibody Methods." Academic Press, New York, 1968. 1, E. A. Kabat and M. M. Mayer, in "Experimental Immunochemistry," 2nd ed., p. 22. Thomas, Springfield, Illinois, 1971.
44
FATTY ACID SYNTHESIS
[6]
is the result of the reaction of antibody with the 7 S component present in the DEAE-cellulose purified fatty acid synthase2 The immunodiffusion studies also show that the same species of fatty acid synthase is present in the livers of rats fed a normal diet, fat-free diet, or fasted. The rat liver fatty acid synthase reacts with its antiserum to produce a typical quantitative precipitin reaction. At the equivalence point, 33.5 mg of antiserum protein were required to precipitate 1 mg of purified fatty acid synthase. The equivalence point is essentially the same for enzyme obtained from rats fed a normal diet or refed a fat-free diet. Antiserum prepared against fatty acid synthase has been successfully employed in imlnunochemical studies to evaluate the relative importance of synthetic and degradative rates on changes in content of rat liver fatty acid synthase under different nutritional conditions. 1° The results of these immunochemical studies are discussed in the next section. Control of the Rate of Synthesis of the Fatty Acid Synthase Rat liver fatty acid synthase is markedly affected by the nutritional and hormonal states of the animal. Thus, the level of the enzyme is greatly reduced on fasting and increased to supranormal levels on rcfeeding a fat-free diet to previously fasted animals. This increase has been shown to result from an adaptive increase in the rate of synthesis of the enzyme.7 The rates of synthesis and degradation of rat liver fatty acid synthase have been determined as a function of nutritional state in order to determine the relative importance of each in controlling the quantity of this enzyme in the liver. 1° The amount of fatty acid synthase has been determined in these studies by enzyme isolation and by the immunochemical technique. The results of these studies have established the major factor controlling the level of this enzyme to be the rate of its synthesis. However, changes in the rate of degradation may be important in the early stages of fasting. Recently, it has been shown that insulin is essential for the dietary induction of rat liver fatty acid synthase to supranormal levels) 4 Diabetic rats synthesize negligible amounts of this enzyme whereas insulin administration brings about a dramatic increase in its synthesis. Glucago~ and cyclic AMP block the dietary induction of the fatty acid synthase. Hence, it is important to consider both the hormonal and dietary status of animals in studies which concern the isolation and the properties of rat liver fatty acid synthase. 14M. R. Lakshmanan, C. M. Nepokroeff, and J. W. Porter, Proc. Nat. Acad. Sci. U.S. 69, 3516 (1972).
