[311
DESATURATION OF LONG-CHAIN FATTY ACIDS
253
inhibitory. The curves which describe the rate as a function of substrate concentration (V/S) are hyperbolic at a fixed concentration of the amine and varying concentrations of acetyl-CoA. They are also hyperbolic when acetyl-CoA is the fixed substrate and the varying substrate is a water-soluble amine or a normal, primary amine of 6-10 carbon atoms. With amines of 12-16 carbon atoms, substrate inhibition ensues at concentrations greater than the critical micellar concentration of these substrates (above about 0.5 mM with dodecylamine, above 0.3 mM with tetradecylamine, and 0.2 with hexadecylamine). Values of Km and V.... are calculated from kinetic curves of the type employed for a bisubstrate reaction.17 Substrate Specificity. The enzyme transfers the aeetyl group of acetylCoA only. Butyryl-CoA and palmitoyl-CoA are not utilized; neither is acetyl-AMP or acetyl pantetheine. The following amines are acceptors of the acetyl group of acetyl-CoA: normal amines of 6-16 carbon atoms; glucosamine, histamine, tryptamine, hydroxytyramine, serotonin, norepinephrine, and p-nitro aniline. The following amines are not substrates: normal amines of 1-4 carbon atoms, polyamines of the spermidine group, amino acids, secondary or tertiary amines, or sphingosine bases. This contrasts with the microsomal transferase which utilizes sphingosine bases and long-chain amines, but not water-soluble amines. 13,18 Inhibitors. The reaction is inhibited by coenzyme A, a product of the reaction. Other inhibitors are butyryl- or pahnitoyl-CoA, and SH inhibitors. 1, W. W. Cleland, Biochim. Biophys. Acta 67, 104, 173, and 188 (1963). 18y. Barenholz and S. Gatt, this volume [29].
[31] D e s a t u r a t i o n o f L o n g - C h a i n F a t t y A c i d s b y A n i m a l L i v e r
By P. W. HOLLOWAY F a t t y acyl-CoA -4- NADH -t- 02 ~ unsaturated fatty acyl-CoA + NAD + + H:O Olefinic bonds may be introduced into saturated or unsaturated fatty acid chains by a process of desaturation. The enzyme system catalyzing this reaction, the fatty acyl coenzyme A desaturase, is found associated with the microsomal fraction of homogenates of liver, adipose tissue, adrenals, and testicles. 1 This report will be concerned only with the liver ' R. R. Brenner, Lipids 6, 567 (1971).
254
MISCELLANEOUS ENZYMES
[31]
microsomal desaturase and, primarily, with the desaturation of stearylCoA by hen liver microsomes.
Assay Method Principle. Stearyl-CoA desaturase activity is estimated from the conversion of [1-14C]stcaryl-CoA into [1-14C]oleate. '-' The incubation mixture is saponified and the liberated [1-1~C]stearate and [1-i~C]oleate are separated by argentation thin-layer chromatography. An alternative assay based upon formation of ~H20 from [9,103H2]stearyl-CoA has been described2, 4 Reagents
Potassium phosphate buffer, 1 M, pH 7.2 at 37 ° N A D H , 5 m M in 10 m M potassium phosphate buffer, pH 7.2 at 0% The N A D H solution is prepared fresh daily. [1-~C]Stearyl-CoA, 0.5 m M in 10 m M potassium phosphate buffer pH 6.0 at 0% The [1-14C]stearyl-CoA of high specific activity is prepared by the method of Kornberg and Pricer ~ or may be purchased from New England Nuclear, Boston, Mass. The radioactive material is diluted to approximately 3 ~Ci/~mole with nonlabeled stearyl-CoA obtained from P L Biochemicals, Inc., Milwaukee, Wis. Ethanolic KOH, 10% Aqueous KOH, saturated H2SO~, 2 M Pentane, redistilled or pesticide grade Chloroform, reagent grade Hexane, reagent grade Diethyl ether, reagent grade Methanol, reagent grade Etherial diazomethane prepared fresh daily. A diazomethane generator is easily made from a 50-ml round bottom flask. The neck of the flask is extended to 15 cm by a piece of 18 mm glass tubing 2 When microsomal preparations are used the radioactivity is found distributed among many lipid products. The amount of oleyl-CoA formed is determined after conversion of all fatty acyl residues to their corresponding methyl esters. Hence, it is convenient to refer to the product as [124C]oleate although at the end of the incubation period, this radioactivity is distributed among oleyl-CoA, oleic acid, and other lipids. B. R. Talamo and K. Bloch, Anal. Biochem. 29, 300 (1969). 4A. R. Johnson and M. I. Gurr, Lipids 6, 78 (1971). A. Kornberg and W. E. Pricer, Jr., J. Biol. Chem. 204, 329 (1953).
[31]
DESATURATION OF LONG-CHAIN FATTY ACIDS
255
and a 25-cm length of 8 mm diameter glass tubing is attached to the neck at a 45 ° angle (pointing up) 5 cm above the flask. This side arm is now bent down through a 45 ° angle 5 cm from the flask in order to be parallel to the neck of the flask, but pointing down. This generator is free from ground glass joints which can explosively decompose diazomethane. Into the flask is placed 2 g of N,N'-dimethyl-N,N'-dinitrosoterephthalamide, 20 ml of diethyl ether, and 5 ml of methanol. The flask is cooled in ice and when cold 2 ml of saturated aqueous KOH is added. After 10 minutes (or longer, the mixture may be kept in ice for several hours) the side arm of the flask is placed in an 18-ram test tube, cooled in ice, and the flask is stoppered with a rubber stopper. Upon warming the flask with water at 50 ° the diazomethane distills over and collects in the 18-mm test tube. It is used within 30 minutes or is returned to the generator and redistilled. AgNO3 impregnated silica gel H thin-layer plates 0.5 mm thick. Four 20 X 20 cm plates may be prepared from 30 g of silica gel H and 75 ml 4% aqueous AgN03. The slurry is spread with a plastic spreader (metal corrodes). The plates are dried at 110° for 2 hours and are stored, if necessary, in a desiccator in the dark. If stored, they are reactivated at 110 ° for 1 hour. Standard thin-layer chromatography (TLC) mixture containing 25 mg of methyl oleate and 60 mg of methyl stearate per milliliter of chloroform 2,7-Dichlorofluorcsceine, 0.2% in 95% ethanol
Procedure. Incubations are performed in open 16 X 150 mm test tubes, and the following reagents are added to the test tubes in ice: water to 0.5 ml, 30 ~l phosphate buffer, 20 ~l [1-14C]stearyl-CoA, 10 ~l NADH, and enzyme protein 100--500 ~g. The reaction is started by transferring the test tubes to a water bath at 37 ° and gently shaking them for 15 seconds. After 15 minutes at 37 ° the test tubes are placed in ice and to each tube is rapidly added 0.2 ml of ethanolic KOH. The tubes are transferred to a boiling water bath and are capped with glass marbles. After 20 minutes the tubes are removed and cooled to room temperature. If necessary the incubation tubes may be stored at --20 ° until next day. The reaction mixture is acidified with 0.2 ml 2 M H2SO,, 0.1 ml of methanol is added, and the fatty acids are extracted with three 3-ml portions of pentane. It may be necessary to centrifuge the tubes to separate the pentane and aqueous layers. The combined pentane extracts, in a 15-ml glass conical centrifuge tube, are evaporated to dryness in a stream of nitrogen at room temperature. The residue is methylated with 2-3 drops
256
MISCELLANEOUS ENZYMES
[31]
of etherial diazomethane (an excess, yellow color). After 5 minutes at room temperature the excess diazomethane is removed in a stream of nitrogen. The fatty acid methyl esters are dissolved in 50 ~l of chloroform and 5 ~l are removed for radioactive assay to check on the efficiency of extraction (usually 95%). A 20-~1 portion is applied as a single spot to a AgNO.~ impregnated silica gel H plate. Either 4 or 5 samples may be run on a single 20 X 20 cm plate together with two 5-~1 aliquots of the standard TLC mixture applied at both sides of the plate. The plate is developed with hexane-diethyl ether (9:1 v/v). After development the plate is airdried and the edges of the plate, where the standards are located, are sprayed lightly with dichlorofluorescein. The positions of the standard methyl oleate and stearate (Ri 0.35 and 0.65, respectively) are located under ultraviolet light, and the silica gel from the areas corresponding to methyl stearate, methyl oleate, and origin from each assay spot is scraped from the thin-layer chromatogram into scintillation vials containing a toluene solution of scintillator. The radioactivity in the three areas derived from each assay spot is determined. The percentage desaturation in an incubation is calculated from the ratio of radioactivity in the oleate region compared to the total radioactivity recovered from the thin-layer chromatogram of that assay spot. With conversions of 20% or less the reaction is usually linear with time; however, for precise determination of desaturase activity several protein concentrations and incubation times should be used. From the percentage desaturation the amount of oleate formed may be calculated. A tube lacking protein is used as a control. It should be noted that there is seldom any radioactive material remaining at the origin of the thin-layer chromatogram, any radioactivity in this position could be attributed to hydroxy esters but in this laboratory has always indicated faulty saponification or methylation. Units. One unit of desaturase is defined as the amount of protein catalyzing the formation of 1 ~mole of oleate per minute under the conditions of the assay. Specific activity is expressed in milliunits per milligram of protein. Resolution of Desaturase
Isolation o/ Hen Liver Microsomes. Livers from old laying hens are collected in ice at the slaughterhouse. The livers may be kept on ice overnight or processed the same day. Three-hundred grams of liver are blended for 30 seconds with 450 ml of 0.1 M potassium phosphate buffer (pH 7.2 at 0 °) in a Waring blender at 5 ° . The products of two such
[311
D E S A T U R A T I O N OF LONG-CHAIN FATTY ACIDS
257
blendings are combined and centrifuged in a Sorvall GSA rotor at 9,000 rpm (13,200 g max) for 20 minutes. The supernatant is poured off through four layers of cheesecloth and centrifuged in two Beckman No. 30 rotors at 30,000 rpm (78,000 g average) for 120 minutes. The supernatant is poured off and the microsomal pellets are removed (avoiding the central dark area), and homogenized in a Potter-EIvehjem Teflon homogenizer in 0.25 M sucrose. Yield from 300 g of hen liver 3.5 g of crude microsomal protein. Specific activity 0.15-0.50 milliunits per milligram of protein. The microsomes may be kept several weeks at --20 ° with no loss of desaturase activity. Preparation o] Particulate Fraction (P'0- A "solubilizing solution" is prepared from 240 ml of glycerol, 80 ml 1 M KC1, 80 ml 1 M potassium citrate (pH 7.7 at 0°), and 40 ml 10% sodium deoxycholate (pH 8.5 at 20°). To 42 ml of solubilizing solution at 0 ° is added 0.3 ml of 0.2 M dithiothreitol and 30 ml of microsomal suspension (60 mg of protein/ml). The mixture is stirred gently for 20 minutes at 5 ° and centrifuged at 105,000 g (average) for 60 minutes. The supernatant fluid is filtered through glass wool and diluted, with stirring, with 0.75 volume of 0.1 mM dithiothreitol at 0 °. The mixture is stirred for 20 minutes at 5 ° and centrifuged at 105,000 g (average) for 60 minutes. The supernatant fluid is isolated and diluted with 1.25 volumes of 0.1 mM dithiothreitol at 0 °. The mixture, after stirring for 20 minutes at 5 °, is centrifuged at 105,000 g (average) for 60 minutes and the resulting pellets are suspended in 10 mM potassium phosphate (pH 7.2 at 0 °) containing 0.1 mM dithiothreitol. This particulate fraction is designated "P~ fraction" and may be stored at --20 ° for several weeks with no loss of desaturase activity. Sephadex G-200 Chromatography. The P o fraction is diluted to 20 ml with 0.25 M sucrose at 0% The suspension is homogenized and to it is added 1 ml of sodium Tricine buffer (pH 8.0 at 0°), 0.1 ml of 0.2 M dithiothreitol, and 1 ml of 10% sodium deoxycholate. The mixture is rehomogenized, sonicated for 2 seconds at 0 ° with a Biosonik II sonicator (Bronwill Scientific, Rochester, N.Y.) using the 4-ram tip at 20% output and applied to a column (40 X 430 ram) of Sephadex G-200 stabilized by a 5-mm layer of Sephadex G-75 on the top. The column is equilibrated at 5 ° with 20 mM sodium bicarbonate (pH 7.7 at 0 °) containing 0.1 mM dithiothreitol and 0.2% sodium deoxycholate. The column is eluted overnight at 5 ° with the same buffer and 16 ml (approximately 20 minutes) fractions are collected. This procedure completely resolves cytochrome b~ from the majority of the protein applied to the column. The fractions which contain cytochrome b~ are located by measurement of reduced minus oxidized difference spectra. A 1-ml aliquot is removed
258
MISCELLANEOUS ENZYMES
[31]
from each fraction, 7.4 ~g of purified rat liver microsomal NADH-cytochrome bs reductase 6,~ are added, and the mixture is diluted to 2.5 ml with 10 mM potassium phosphate buffer (pH 7.2 at 37°). The lnixture is divided equally between two euvettes and a base line of equal light absorbanee is established at room temperature in a Cary 14 recording spectrophotometer with 0 . 1 0 D full-scale sensitivity. To the sample cuvette is added 0.1 ~mole NADH and the NADH-redueed minus oxidized speetruln is recorded. The difference in absorbanee between 424 and 410 nm is a measure of eytoehrome b~. Cytoehrome bs is usually eluted in tubes 16-23. The material eluted from the Sephadex G-200 column immediately after the void volume (tubes 10-15) is applied to a column (40 X 180 ram) of Sephadex G-25 equilibrated at 5 ° with 10 mM potassium phosphate buffer (pH 7.2 at 0 °) containing 0.1 mM dithiothreitol. This proeedure removes the deoxyehol~te. The cloudy eluate is poured into an equal volume of stirred saturated ammonium sulfate solution at 5 ° and the precipitate is collected by centrifugation. The pellets are suspended in a small volume of 10 mM potassium phosphate buffer (pH 7.2 at 0 °) containing 0.1 mM dithiothreitol. This fraction is designated the "P3 fraction" and may be kept for up to 2 weeks at --20 ° with no loss of activity. Before it is assayed for desaturase activity the thawed P:~ fraction is clarified by a 2-second sonieation with a Biosonik II using the 4-mm tip at 20% output. The P:~ fraction when assayed for desaturase activity is inactive; desaturase activity can however be restored to a high level by addition of NADH-cytochrome b~ reductase, eytochrome b~ isolated by a detergent procedure, ~,9 lipid dispersion, and sodium deoxycholate (Table I). The recovery of desaturase activity is shown in Table II. The Sephadex G-200 column retains some protein and lipid material with the result that the top of the column gradually becomes impermeable to the solubilized P~ fraction. It is advisable to remove the Sephadex G-75 and repack the column, using the same Sephadex G-200, after four separations have been performed. After eight separations have been performed on one batch of Sephadex G-200 the column should be repacked with fresh Sephadex G-200. Preparation of Lipid Dispersion. In 3 ml of benzene are dissolved 17.5 mg of purified egg yolk phosphatidyleholineTM and 4.7 mg of oleie S. Takesue and T. Omura, J. Biochem. (Tokyo) 67, 267 (1970). Tp. W. Holloway, Biochemistry 10, 1556 (1971). SL. Spatz and P. Strittmatter, Proc. Nat. Acad. Sci. U.S. 68, 1042 (1971). ~P. W. Holloway and J. T. Katz, Biochemistry 11, 3689 (19711). ~*B. J. Litman, Biochemistry 12, 2545 (1973).
[31]
DESATURATION OF LONG-CHAIN FATTY ACIDS
259
TABLE I REGENERATION Of DESATURASE ACTIVITY Components a
Stearyl-CoA desaturase (nmoles oleate formed)
P3 P3 + Fp Ps + Fp + deoxycholate P3 + F p + c y t b ~ P3 + Fp + cyt b5 + deoxycholate Pa -t- Fp -t- cyt b5 A- lipid Pa -{- Fp -4- cyt b5 A- deoxycholate + lipid Fp -4- cyt b5 "4- deoxycholate A- lipid
DESATURATION OF LONG-CHAIN FATTY ACIDS
253
inhibitory. The curves which describe the rate as a function of substrate concentration (V/S) are hyperbolic at a fixed concentration of the amine and varying concentrations of acetyl-CoA. They are also hyperbolic when acetyl-CoA is the fixed substrate and the varying substrate is a water-soluble amine or a normal, primary amine of 6-10 carbon atoms. With amines of 12-16 carbon atoms, substrate inhibition ensues at concentrations greater than the critical micellar concentration of these substrates (above about 0.5 mM with dodecylamine, above 0.3 mM with tetradecylamine, and 0.2 with hexadecylamine). Values of Km and V.... are calculated from kinetic curves of the type employed for a bisubstrate reaction.17 Substrate Specificity. The enzyme transfers the aeetyl group of acetylCoA only. Butyryl-CoA and palmitoyl-CoA are not utilized; neither is acetyl-AMP or acetyl pantetheine. The following amines are acceptors of the acetyl group of acetyl-CoA: normal amines of 6-16 carbon atoms; glucosamine, histamine, tryptamine, hydroxytyramine, serotonin, norepinephrine, and p-nitro aniline. The following amines are not substrates: normal amines of 1-4 carbon atoms, polyamines of the spermidine group, amino acids, secondary or tertiary amines, or sphingosine bases. This contrasts with the microsomal transferase which utilizes sphingosine bases and long-chain amines, but not water-soluble amines. 13,18 Inhibitors. The reaction is inhibited by coenzyme A, a product of the reaction. Other inhibitors are butyryl- or pahnitoyl-CoA, and SH inhibitors. 1, W. W. Cleland, Biochim. Biophys. Acta 67, 104, 173, and 188 (1963). 18y. Barenholz and S. Gatt, this volume [29].
[31] D e s a t u r a t i o n o f L o n g - C h a i n F a t t y A c i d s b y A n i m a l L i v e r
By P. W. HOLLOWAY F a t t y acyl-CoA -4- NADH -t- 02 ~ unsaturated fatty acyl-CoA + NAD + + H:O Olefinic bonds may be introduced into saturated or unsaturated fatty acid chains by a process of desaturation. The enzyme system catalyzing this reaction, the fatty acyl coenzyme A desaturase, is found associated with the microsomal fraction of homogenates of liver, adipose tissue, adrenals, and testicles. 1 This report will be concerned only with the liver ' R. R. Brenner, Lipids 6, 567 (1971).
254
MISCELLANEOUS ENZYMES
[31]
microsomal desaturase and, primarily, with the desaturation of stearylCoA by hen liver microsomes.
Assay Method Principle. Stearyl-CoA desaturase activity is estimated from the conversion of [1-14C]stcaryl-CoA into [1-14C]oleate. '-' The incubation mixture is saponified and the liberated [1-1~C]stearate and [1-i~C]oleate are separated by argentation thin-layer chromatography. An alternative assay based upon formation of ~H20 from [9,103H2]stearyl-CoA has been described2, 4 Reagents
Potassium phosphate buffer, 1 M, pH 7.2 at 37 ° N A D H , 5 m M in 10 m M potassium phosphate buffer, pH 7.2 at 0% The N A D H solution is prepared fresh daily. [1-~C]Stearyl-CoA, 0.5 m M in 10 m M potassium phosphate buffer pH 6.0 at 0% The [1-14C]stearyl-CoA of high specific activity is prepared by the method of Kornberg and Pricer ~ or may be purchased from New England Nuclear, Boston, Mass. The radioactive material is diluted to approximately 3 ~Ci/~mole with nonlabeled stearyl-CoA obtained from P L Biochemicals, Inc., Milwaukee, Wis. Ethanolic KOH, 10% Aqueous KOH, saturated H2SO~, 2 M Pentane, redistilled or pesticide grade Chloroform, reagent grade Hexane, reagent grade Diethyl ether, reagent grade Methanol, reagent grade Etherial diazomethane prepared fresh daily. A diazomethane generator is easily made from a 50-ml round bottom flask. The neck of the flask is extended to 15 cm by a piece of 18 mm glass tubing 2 When microsomal preparations are used the radioactivity is found distributed among many lipid products. The amount of oleyl-CoA formed is determined after conversion of all fatty acyl residues to their corresponding methyl esters. Hence, it is convenient to refer to the product as [124C]oleate although at the end of the incubation period, this radioactivity is distributed among oleyl-CoA, oleic acid, and other lipids. B. R. Talamo and K. Bloch, Anal. Biochem. 29, 300 (1969). 4A. R. Johnson and M. I. Gurr, Lipids 6, 78 (1971). A. Kornberg and W. E. Pricer, Jr., J. Biol. Chem. 204, 329 (1953).
[31]
DESATURATION OF LONG-CHAIN FATTY ACIDS
255
and a 25-cm length of 8 mm diameter glass tubing is attached to the neck at a 45 ° angle (pointing up) 5 cm above the flask. This side arm is now bent down through a 45 ° angle 5 cm from the flask in order to be parallel to the neck of the flask, but pointing down. This generator is free from ground glass joints which can explosively decompose diazomethane. Into the flask is placed 2 g of N,N'-dimethyl-N,N'-dinitrosoterephthalamide, 20 ml of diethyl ether, and 5 ml of methanol. The flask is cooled in ice and when cold 2 ml of saturated aqueous KOH is added. After 10 minutes (or longer, the mixture may be kept in ice for several hours) the side arm of the flask is placed in an 18-ram test tube, cooled in ice, and the flask is stoppered with a rubber stopper. Upon warming the flask with water at 50 ° the diazomethane distills over and collects in the 18-mm test tube. It is used within 30 minutes or is returned to the generator and redistilled. AgNO3 impregnated silica gel H thin-layer plates 0.5 mm thick. Four 20 X 20 cm plates may be prepared from 30 g of silica gel H and 75 ml 4% aqueous AgN03. The slurry is spread with a plastic spreader (metal corrodes). The plates are dried at 110° for 2 hours and are stored, if necessary, in a desiccator in the dark. If stored, they are reactivated at 110 ° for 1 hour. Standard thin-layer chromatography (TLC) mixture containing 25 mg of methyl oleate and 60 mg of methyl stearate per milliliter of chloroform 2,7-Dichlorofluorcsceine, 0.2% in 95% ethanol
Procedure. Incubations are performed in open 16 X 150 mm test tubes, and the following reagents are added to the test tubes in ice: water to 0.5 ml, 30 ~l phosphate buffer, 20 ~l [1-14C]stearyl-CoA, 10 ~l NADH, and enzyme protein 100--500 ~g. The reaction is started by transferring the test tubes to a water bath at 37 ° and gently shaking them for 15 seconds. After 15 minutes at 37 ° the test tubes are placed in ice and to each tube is rapidly added 0.2 ml of ethanolic KOH. The tubes are transferred to a boiling water bath and are capped with glass marbles. After 20 minutes the tubes are removed and cooled to room temperature. If necessary the incubation tubes may be stored at --20 ° until next day. The reaction mixture is acidified with 0.2 ml 2 M H2SO,, 0.1 ml of methanol is added, and the fatty acids are extracted with three 3-ml portions of pentane. It may be necessary to centrifuge the tubes to separate the pentane and aqueous layers. The combined pentane extracts, in a 15-ml glass conical centrifuge tube, are evaporated to dryness in a stream of nitrogen at room temperature. The residue is methylated with 2-3 drops
256
MISCELLANEOUS ENZYMES
[31]
of etherial diazomethane (an excess, yellow color). After 5 minutes at room temperature the excess diazomethane is removed in a stream of nitrogen. The fatty acid methyl esters are dissolved in 50 ~l of chloroform and 5 ~l are removed for radioactive assay to check on the efficiency of extraction (usually 95%). A 20-~1 portion is applied as a single spot to a AgNO.~ impregnated silica gel H plate. Either 4 or 5 samples may be run on a single 20 X 20 cm plate together with two 5-~1 aliquots of the standard TLC mixture applied at both sides of the plate. The plate is developed with hexane-diethyl ether (9:1 v/v). After development the plate is airdried and the edges of the plate, where the standards are located, are sprayed lightly with dichlorofluorescein. The positions of the standard methyl oleate and stearate (Ri 0.35 and 0.65, respectively) are located under ultraviolet light, and the silica gel from the areas corresponding to methyl stearate, methyl oleate, and origin from each assay spot is scraped from the thin-layer chromatogram into scintillation vials containing a toluene solution of scintillator. The radioactivity in the three areas derived from each assay spot is determined. The percentage desaturation in an incubation is calculated from the ratio of radioactivity in the oleate region compared to the total radioactivity recovered from the thin-layer chromatogram of that assay spot. With conversions of 20% or less the reaction is usually linear with time; however, for precise determination of desaturase activity several protein concentrations and incubation times should be used. From the percentage desaturation the amount of oleate formed may be calculated. A tube lacking protein is used as a control. It should be noted that there is seldom any radioactive material remaining at the origin of the thin-layer chromatogram, any radioactivity in this position could be attributed to hydroxy esters but in this laboratory has always indicated faulty saponification or methylation. Units. One unit of desaturase is defined as the amount of protein catalyzing the formation of 1 ~mole of oleate per minute under the conditions of the assay. Specific activity is expressed in milliunits per milligram of protein. Resolution of Desaturase
Isolation o/ Hen Liver Microsomes. Livers from old laying hens are collected in ice at the slaughterhouse. The livers may be kept on ice overnight or processed the same day. Three-hundred grams of liver are blended for 30 seconds with 450 ml of 0.1 M potassium phosphate buffer (pH 7.2 at 0 °) in a Waring blender at 5 ° . The products of two such
[311
D E S A T U R A T I O N OF LONG-CHAIN FATTY ACIDS
257
blendings are combined and centrifuged in a Sorvall GSA rotor at 9,000 rpm (13,200 g max) for 20 minutes. The supernatant is poured off through four layers of cheesecloth and centrifuged in two Beckman No. 30 rotors at 30,000 rpm (78,000 g average) for 120 minutes. The supernatant is poured off and the microsomal pellets are removed (avoiding the central dark area), and homogenized in a Potter-EIvehjem Teflon homogenizer in 0.25 M sucrose. Yield from 300 g of hen liver 3.5 g of crude microsomal protein. Specific activity 0.15-0.50 milliunits per milligram of protein. The microsomes may be kept several weeks at --20 ° with no loss of desaturase activity. Preparation o] Particulate Fraction (P'0- A "solubilizing solution" is prepared from 240 ml of glycerol, 80 ml 1 M KC1, 80 ml 1 M potassium citrate (pH 7.7 at 0°), and 40 ml 10% sodium deoxycholate (pH 8.5 at 20°). To 42 ml of solubilizing solution at 0 ° is added 0.3 ml of 0.2 M dithiothreitol and 30 ml of microsomal suspension (60 mg of protein/ml). The mixture is stirred gently for 20 minutes at 5 ° and centrifuged at 105,000 g (average) for 60 minutes. The supernatant fluid is filtered through glass wool and diluted, with stirring, with 0.75 volume of 0.1 mM dithiothreitol at 0 °. The mixture is stirred for 20 minutes at 5 ° and centrifuged at 105,000 g (average) for 60 minutes. The supernatant fluid is isolated and diluted with 1.25 volumes of 0.1 mM dithiothreitol at 0 °. The mixture, after stirring for 20 minutes at 5 °, is centrifuged at 105,000 g (average) for 60 minutes and the resulting pellets are suspended in 10 mM potassium phosphate (pH 7.2 at 0 °) containing 0.1 mM dithiothreitol. This particulate fraction is designated "P~ fraction" and may be stored at --20 ° for several weeks with no loss of desaturase activity. Sephadex G-200 Chromatography. The P o fraction is diluted to 20 ml with 0.25 M sucrose at 0% The suspension is homogenized and to it is added 1 ml of sodium Tricine buffer (pH 8.0 at 0°), 0.1 ml of 0.2 M dithiothreitol, and 1 ml of 10% sodium deoxycholate. The mixture is rehomogenized, sonicated for 2 seconds at 0 ° with a Biosonik II sonicator (Bronwill Scientific, Rochester, N.Y.) using the 4-ram tip at 20% output and applied to a column (40 X 430 ram) of Sephadex G-200 stabilized by a 5-mm layer of Sephadex G-75 on the top. The column is equilibrated at 5 ° with 20 mM sodium bicarbonate (pH 7.7 at 0 °) containing 0.1 mM dithiothreitol and 0.2% sodium deoxycholate. The column is eluted overnight at 5 ° with the same buffer and 16 ml (approximately 20 minutes) fractions are collected. This procedure completely resolves cytochrome b~ from the majority of the protein applied to the column. The fractions which contain cytochrome b~ are located by measurement of reduced minus oxidized difference spectra. A 1-ml aliquot is removed
258
MISCELLANEOUS ENZYMES
[31]
from each fraction, 7.4 ~g of purified rat liver microsomal NADH-cytochrome bs reductase 6,~ are added, and the mixture is diluted to 2.5 ml with 10 mM potassium phosphate buffer (pH 7.2 at 37°). The lnixture is divided equally between two euvettes and a base line of equal light absorbanee is established at room temperature in a Cary 14 recording spectrophotometer with 0 . 1 0 D full-scale sensitivity. To the sample cuvette is added 0.1 ~mole NADH and the NADH-redueed minus oxidized speetruln is recorded. The difference in absorbanee between 424 and 410 nm is a measure of eytoehrome b~. Cytoehrome bs is usually eluted in tubes 16-23. The material eluted from the Sephadex G-200 column immediately after the void volume (tubes 10-15) is applied to a column (40 X 180 ram) of Sephadex G-25 equilibrated at 5 ° with 10 mM potassium phosphate buffer (pH 7.2 at 0 °) containing 0.1 mM dithiothreitol. This proeedure removes the deoxyehol~te. The cloudy eluate is poured into an equal volume of stirred saturated ammonium sulfate solution at 5 ° and the precipitate is collected by centrifugation. The pellets are suspended in a small volume of 10 mM potassium phosphate buffer (pH 7.2 at 0 °) containing 0.1 mM dithiothreitol. This fraction is designated the "P3 fraction" and may be kept for up to 2 weeks at --20 ° with no loss of activity. Before it is assayed for desaturase activity the thawed P:~ fraction is clarified by a 2-second sonieation with a Biosonik II using the 4-mm tip at 20% output. The P:~ fraction when assayed for desaturase activity is inactive; desaturase activity can however be restored to a high level by addition of NADH-cytochrome b~ reductase, eytochrome b~ isolated by a detergent procedure, ~,9 lipid dispersion, and sodium deoxycholate (Table I). The recovery of desaturase activity is shown in Table II. The Sephadex G-200 column retains some protein and lipid material with the result that the top of the column gradually becomes impermeable to the solubilized P~ fraction. It is advisable to remove the Sephadex G-75 and repack the column, using the same Sephadex G-200, after four separations have been performed. After eight separations have been performed on one batch of Sephadex G-200 the column should be repacked with fresh Sephadex G-200. Preparation of Lipid Dispersion. In 3 ml of benzene are dissolved 17.5 mg of purified egg yolk phosphatidyleholineTM and 4.7 mg of oleie S. Takesue and T. Omura, J. Biochem. (Tokyo) 67, 267 (1970). Tp. W. Holloway, Biochemistry 10, 1556 (1971). SL. Spatz and P. Strittmatter, Proc. Nat. Acad. Sci. U.S. 68, 1042 (1971). ~P. W. Holloway and J. T. Katz, Biochemistry 11, 3689 (19711). ~*B. J. Litman, Biochemistry 12, 2545 (1973).
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DESATURATION OF LONG-CHAIN FATTY ACIDS
259
TABLE I REGENERATION Of DESATURASE ACTIVITY Components a
Stearyl-CoA desaturase (nmoles oleate formed)
P3 P3 + Fp Ps + Fp + deoxycholate P3 + F p + c y t b ~ P3 + Fp + cyt b5 + deoxycholate Pa -t- Fp -t- cyt b5 A- lipid Pa -{- Fp -4- cyt b5 A- deoxycholate + lipid Fp -4- cyt b5 "4- deoxycholate A- lipid
