ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

178,

475-485

(1977)

Pig Liver Fatty Acid Synthetase: Purification Properties1 IN-CHEOL Department

KIM,

CLIFFORD

of Biochemistry,

J. UNKEFER,*

Michigan

AND

State Uniuersity,

Received

and Physicochemical

WILLIAM

East Lansing,

C. DEAL, Michigan

JR.

48824

May 17, 1976

This paper reports the first detailed study of the physicochemical properties of a fatty acid synthetase multienzyme complex from a mammalian liver. Fatty acid synthetase from pig liver was purified by a procedure including the following main steps: (i) preparation of a clarified supernatant solution (50,000 g), (ii) ammonium sulfate fractionation, (iii) DEAE-cellulose chromatography to separate 11 S catalase from the 13 S fatty acid synthetase, (iv) a preparative sucrose density gradient step to remove a 7 S impurity, and (v) a calcium phosphate gel step to remove an unusual yellow 16 S heme protein to yield a colorless preparation. The purified fatty acid synthetase was colorless and showed a single symmetrical peak in sucrose density gradient and conventional sedimentation velocity experiments. Fatty acid synthetase was very stable at 4°C in the presence of 1 mM dithiothreitol and 25% sucrose. Extrapolation to zero protein concentration yielded values of s&= 13.3 S and D&,,w = 2.60 k 10 -’ cm*/s for the sedimentation and diffusion coefficients of the enzyme. Frictional coefficient values of 1.55 and 1.56 x 10m7 cm, respectively, were calculated from the values for the sedimentation and diffusion coefficients. Based on these frictional coefficient values, the Stokes radius of the enzyme was calculated to be 82.4 A. Sedimentation and diffusion coefficient data yielded a molecular weight value of M, (s/D) = 478,000 and sedimentation equilibrium data yielded a value of M, = 476,000. Preliminary intrinsic viscosity measurements at 20°C gave a value of 7.3 ml/g, indicating that the enzyme is somewhat asymmetric. This is supported by the value of 1.58 calculated for the frictional ratio and by the fact that the values for the sedimentation and diffusion coefficients are both slightly lower than expected for a globular protein of molecular weight 478,000. The enzyme possesses about 90 SH groups per molecule, assuming a molecular weight of 478,000. The ultraviolet absorption spectrum of the enzyme shows a maximum at 280 nm and an unusual shoulder at 290 nm. The fluorescence spectrum of the enzyme is dominated by tryptophan fluorescence and, over the excitation range of 260-300 nm, there is a single emission maximum at 344 nm.

The fatty acid synthetase multienzyme complex has been purified from livers of several avian and mammalian sources, including rat (2), pigeon (3), chicken (4), and human (51, but thus far, no mammalian liver fatty acid synthetase has been exten-

sively characterized physically. It is important to characterize the mammalian liver fatty acid synthetase for two reasons. First, the liver is the main organ responsible for fatty acid synthesis for @iglyceride formation and for phospholipid formation for use in membranes. Second, the structural and regulatory properties of enzymes from livers of sources lower on the evolutionary scale are frequently different from those of higher-order mammals, like man, in which we are greatly interested. Recently, Dutler et al. (1) reported the purification of fatty acid synthetase from

’ Supported in part by grants from the National Institute of Arthritis, Metabolism and Digestive Diseases (Grant No. AM-15345) and the Michigan State University Agricultural Experiment Station (Hatch 932; Publication No. 7426). 2 Participant in the Undergraduate Research Program of the Department of Biochemistry, Michigan State University. 475 Copyright All rights

0 1977 by Academic

of reproduction

Press,

Inc.

in any form reserved.

476

KIM,

UNKEFER

pig liver. They carried out an analysis of the stereospecifkity of the catalytic reaction and also estimated the molecular weight of the enzyme to be about 500,000 from gel filtration experiments; no further structural studies were carried out on the enzyme. In order to investigate the physical properties of fatty acid synthetase of pig liver, we first attempted the purification according to Dutler et al. (1); however, we were not able to obtain a pure, colorless fatty acid synthetase preparation in good yield using these procedures. This prompted us to develop a new purification procedure retaining some of the steps from their procedures. In this communication, we report our purification procedure and physicochemical properties of the enzyme. MATERIALS

AND

METHODS

Chemicals. NADPH, acetyl-CoA, and malonylCoA were products of P-L Biochemicals, Inc. DEAEcellulose3 was obtained from Schleicher and Schuell, Inc. All other chemicals were reagent grade or the highest purity obtainable. Liver. Pig livers were obtained from a local slaughterhouse within 1 h after slaughter and were frozen in dry ice. The livers were stored frozen at -40°C. Protein determination. Protein concentration was determined according to Massey and Deal (6) with bovine serum albumin as the standard. Enzyme assays. Enzyme assays were carried out at 23°C. The decrease in absorbance at 340 nm was monitored with a Cilford 240 spectrophotometer. Pig liver fatty acid synthetase was analyzed routinely using the partial reaction assay of Kim and Deal (7) in which a mixture of enantiomers of truns-l-decalone, a substrate analog model compound (11, serves as the substrate. The assay solution (8) for analysis of the overall fatty acid synthetase reaction contained 0.1 M potassium phosphate buffer, pH 6.9, 0.05 mM acetyl CoA, 0.05 mM malonyl CoA, 0.1% bovine serum albumin, 0.2 mM NADPH, and an appropriate amount of en-

3 Abbreviations used: DR, trans-1-decalone redup tase; DTT, dithiothreitol; FAS, fatty acid synthetase; PED, 0.2 M potassium phosphate buffer, pH 7.4, containing 1 mM dithiothreitol and 1 mM EDTA; TED, 0.05 M Tris-HCl buffer, pH 7.4, containing 1 mM EDTA and 1 mM dithiothreitol; DEAE, diethylaminoethyl; uv, ultraviolet; CoA, coenzyme A, SDS, sodium dodecyl sulfate; DTNB, 5,5’-dithiobis(2-nitrobenzoic acid).

AND

DEAL

zyme and deionized water to make the assay volume up to 0.4 ml. The reaction was initiated by addition of malonyl CoA and the activity was measured from the initial slope. A unit of activity is the amount of enzyme that catalyzes the oxidation of 1 pmol of NADPH per minute. Specific activity is expressed as units of enzymic activity per milligram of protein. Absorption and emission spectra. Absolute and difference spectra of fatty acid synthetase preparations were obtained with a Cary 15 spectrophotometer, using as a blank the buffer in which the enzyme was dialyzed. The extinction coefficient of fatty acid synthetase at 280 nm was also determined with a Cary 15 spectrophotometer; the protein concentration was determined using the tannic acid precipitation method (6). Difference spectra1 analysis was used to detect and monitor a specific yellow-colored contaminant; we have found this colored contaminant to be a heme protein (14). Emission spectra of fatty acid synthetase were obtained at 23°C with an Aminco-Bowman spectrofluorophotometer with a xenon light source and recorded on a strip-chart recorder. The excitation wavelengths ranged from 260 to 300 nm. Sucrose density gradient centrifugation. Sucrose density gradient centrifugation was performed according to the method of Martin and Ames (9) with either a Beckman SW27 or SW50.1 rotor in a Beckman Model L3-50 preparative ultracentrifuge at 20°C. The 5 and 20% sucrose solutions were prepared in 0.2 M potassium phosphate buffer, pH 7.4, containing 1 mM EDTA and 1 mM dithiothreitol (PED buffer). Linear gradients of 5 to 20% sucrose solution were prepared in cellulose nitrate tubes using an Isco gradient former (Model 570). The volume per tube was 37 ml for the SW27 rotor and 5 ml for the SW50.1 rotor. Volumes of 1.5 or 0.1 ml of enzyme in PED buffer were layered on the top of each tube (1 x 3.5 in.) for the SW27 or SW50.1 rotor (0.5 x 2 in. tube), respectively. For the SW27 rotor, centrifugation was performed at 27,000 rpm for 18 h and for the SW50.1 rotor for 4 h at 47,000 rpm. The gradients were fractionated and read at 280 nm using an Isco automatic density gradient fractionator (Model 183) with an Isco Model UA-5 absorbance monitor. Fractionation of SW27 tubes was carried out with a flow rate of 3 ml/min and fractions of 1.5 ml were collected. For the SW50.1 rotor, the flow rate was 0.37 ml/min and 0.185-ml fractions were collected. Ultracentrifugal analysis. All experiments were performed in a Beckman Model E analytical ultracentrifuge equipped with a phase plate as a schlieren diaphragm. Photographs were measured with a Bausch and Lomb microcomparator. Sedimentation velocity experiments were run with 12-mm double-sector cells at 48,000 rpm in an

LIVER

FATTY

ACID

SYNTHETASE

AN-D rotor. A sample volume of 0.28 ml was added to the right sector and 0.30 ml of buffer was added to the left sector. Diffusion experiments were carried out with a standard 12-mm double-sector synthetic boundary cell at 4300 rpm using an An-D rotor. A solvent volume of 0.41 ml was used in the left sector and a sample volume of 0.18 ml was used in the right sector. The diffusion coefficient was calculated by the height-area method (10). For all ofthese studies, purified fatty acid synthetase was dialyzed against PED buffer overnight at 4°C. The density of the solvent was measured with a hydrometer at the same temperature as that used in the centrifuge studies. Both sedimentation and diffusion coefficients were corrected to 20°C. The sedimentation and diffusion coefficient data were analyzed by the method of least squares using a computer program developed in this laboratory. The deviations presented with the data are standard deviations. The partial specific volume of the enzyme was assumed to be 0.74 ml/g, based on amino acid analysis of a 24-h hydrolysate. The molecular weight was calculated from the values of sedimentation and diffusion coefficients according to the Svedberg equation (11). The frictional coefficient and Stokes radius were calculated (11) from the values of diffusion coefficients and sedimentation coefficients. The frictional ratio was calculated (11) from these values. A meniscus depletion sedimentation equilibrium experiment (12) was used to obtain the weight-average molecular weight. A 12-mm double-sector cell with sapphire windows and interference window holders was used. Fatty acid synthetase was dialyzed overnight at 4°C against TED buffer containing 0.1 M KCl. A 0.12-ml volume of sample solution was added to the right sector of the cell with 0.01 ml of fluorocarbon (FC-43) and 0.13 ml of solvent was added to the left sector of the cell. The experiment was carried out in an An-G rotor at 10,600 rpm at 21.5”C for 24 h. The Rayleigh interference pictures were taken after 23 h, with spectroscopic II-G plates. The initial protein concentration was 1.2 mgiml. The log of the fringe displacement, log (Y,-Y,,) was plotted against the square of the distance (r*) and the weight-average molecular weight was calculated from the slope of the line. Viscosity determination. The relative viscosities of solutions were measured using a Cannon-Ubbelohde semimicro dilution capillary viscometer with a flow time of approximately 275 s for water at 20°C. The temperature was maintained at 20°C. Before doing viscosity experiments, the fatty acid synthetase was dialyzed overnight at 4°C against 0.2 M potassium phosphate buffer, pH 7.4, containing 1 mM EDTA and 20 mM DTT. The dialyzed buffer was filtered through a VM6 Millipore filter (pore size of 0.45 pm) under air pressure. The dialyzed enzyme

PROPERTIES

477

was centrifuged in a Sorvall SS-34 rotor at 16,000 rpm for 20 min. Flow times were determined for a given sample until an average deviation of less than 0.4 s was obtained. The intrinsic viscosity value was obtained by extrapolation of the reduced viscosity, (n/n,, - 1)/c to zero protein concentration. Bubble problems were kept to a minimum in the viscosity studies through use of a special apparatus developed in this laboratory.4 This apparatus, which contains a special valve and tubing system, attaches to the viscometer and facilitates pumping of the solutions up into the viscometer under a gentle air pressure, thereby minimizing the formation of bubbles. SH analysis. Sulfhydryl group content was determined according to the procedure of Habeeb (13), except that fatty acid synthetase was dialyzed at room temperature for 3 h against a 2% SDS solution in 0.08 M sodium phosphate buffer, pH 8.0, containing 0.05% EDTA. pH and conductivity. The pH values of buffers were measured at 23°C with a Radiometer PM-4 pH meter equipped with a GK 2321C electrode. Conductivities of solutions were measured with a Radiometer CDM 2e conductivity meter and Type 114 cell (cell constant, 0.56). RESULTS .

Purlficatton

.

Procedure

Unless indicated otherwise, all purification steps were carried out at 4°C and centrifugation was performed in the Sorvall GSA rotor at 11,000 rpm for 25 min or in the Sorvall SS-34rotor at 16,000 rpm for 15 min. Step 1. Clarified crude extract. Frozen pig liver (800 g) was cut into small pieces with a stainless steel knife and homogenized for 1 min at maximum speed in a Waring Blendor in 2 vol (1600 ml) of cold 0.05 M Tris-HCl buffer, pH 7.4, containing 1 mM EDTA. The homogenized solution was centrifuged in a GSA rotor. The supernatant solution was further centrifuged in polycarbonate tubes in a Beckman Model L3-50 preparative ultracentrifuge at 21,000 rpm for 2 h using a Beckman Type 21 rotor. A clear, dark-red supernatant was obtained (1200 ml); this is designated the “clarified crude extract.” It was always prepared fresh. Step 2. Ammonium sulfate precipitation. Saturated ammonium sulfate (25 ml/ * Blatti, S. P., and Deal, W. C., Jr. (19671, unpublished results.

478

KIM,

UNKEFER

100 ml of extract), pH 7.4, was added to the clarified crude extract to 0.2 saturation, gently stirred for 15 min, and centrifuged in a GSA rotor. The small amount of precipitated protein was discarded. The supernatant solutions were pooled. Additional saturated ammonium sulfate solution was added (20 ml/100 ml of supernatant) to bring the supernatant solution to 0.33 saturation, gently stirred for 15 min, and centrifuged in a GSA rotor. The pellet was collected and suspended in the same volume of homogenization buffer as the original clarified crude extract (about 1200 ml). The recovery of decalone reductase activity averaged about 65 to 75% up to this point. Saturated ammonium sulfate solution (50 ml/100 ml of solution) was added to 0.33 saturation, and the solution was stirred gently for 15 min and then centrifuged in the GSA rotor. The precipitate was resuspended in 200 ml of the homogenization buffer and dialyzed at 4°C overnight against 4 liters of TED buffer with one buffer change. The decalone reductase activity was stable during overnight dialysis at 4°C under these conditions; also, the enzyme activity was quite stable even at room temperature if the buffer contained at least 1 mM dithiothreitol. Very little decalone reductase activity is lost in these repeated ammonium sulfate precipitations, and these steps remove a considerable amount of foreign protein and reduce the amount of DEAE required in later steps. The dialyzed enzyme was centrifuged in a SS-34 rotor to remove denatured protein, diluted to 1000ml with TED buffer at room temperature, and applied to DEAE-cellulose columns. Step 3. DEAE-cellulose column chromatography. This step was carried out at

23°C. Two columns (4.1 x 33 cm) of DEAEcellulose were preequilibrated with TED buffer and their outlet tubes were connected to two separate flow cells of an Isco Model UA-5 absorbance monitor; then 500 ml of diluted dialyzed enzyme was applied to each of the columns. About 300 ml of TED buffer was then applied to the columns. A linear KC1 gradient ranging from

AND

DEAL

0 to 0.23 M, in TED buffer, was used to elute the enzyme. The total volume of the gradient (3.1 liters) was approximately seven times the bed volume (440 ml) of the DEAE-cellulose. The eluted fractions were collected using an Isco fraction collector (Model 328). The elution of protein was followed by analysis of absorbance at 280 nm using an Isco UA-5 absorbance monitor and recorder. The flow rate was controlled at about 7 ml/min by keeping the bed head between 55 and 70 cm. A typical elution profile is presented in Fig. 1. Decalone reductase starts to appear at about 0.09 M KCl, and the peak of enzyme elution is about at 0.13 M KCl. This DEAE-cellulose step is the most important step for the removal of catalase. This step yields about 50% pure enzyme. Fresh, unused, acid-base-washed DEAE-cellulose was found to give better capacity, resolution, and reproducibility. The capacity of DEAE-cellulose is at least twofold lower at 4°C than at room temperature. At least 1 mM dithiothreitol should be present in the gradient to maintain enzyme activity. With all other conditions the same, replac-

FIG. 1. Profile of pig liver fatty acid synthetase on DEAE-cellulose. Five hundred milliliters (about 300 mg) of dialyzed enzyme preparation in TED buffer was adsorbed onto a DEAE-cellulose (4.1 x 33-cm) column. Then 300 ml of TED buffer was added. Next, a linear KC1 gradient of 0 to 0.23 M in TED buffer was used as eluant. The gradient was formed using 3.1 liters of TED buffer and 3.1 liters of TED buffer containing, in addition, 0.23 M KCl. The flow rate was 8 ml/min, and 20-ml fractions were collected. The elution of the protein was monitored by absorption at 280 nm using an Isco Model UA-5 absorbance monitor, and the concentration of the KC1 was measured with a conductivity meter. Decalone reductase activity (DR ACT.) was assayed using 50-~1 aliquots of eluant. DR activity is expressed as the change of absorbance at 340 nm per 12 s per 50 ~1 of eluant.

LIVER

FATTY

ACID

SYNTHETASE

PROPER’I’IES

479

0.1 -

FIG. 2. Sucrose density gradient centrifugation of pig liver fatty acid synthetase after the DEAE step. A 1.5ml (about IO-mg) sample of an enzyme preparation taken through the DEAE-cellulose step was applied to each tube, which contained a linear 5 to 20% sucrose solution in PED buffer. Centrifugation was at 27,000 ‘pm for 18 h at 20°C. The gradients were fractionated and read at 280 nm at room temperature. Fractions of 1 ml were collected. The enzyme activity is expressed as the absorbance change at 340 nm per 10 s per 1 ~1 of sample for decalone reductase (DR ACT.) or per 10 ~1 of sample for the overall fatty acid synthetase activity (FAS ACT.).

ing 1 mM dithiothreitol by 10 mM mercaptoethanol markedly decreases (as much as twofold) the capacity of the DEAE-cellulose column. The fractions which had decalone reductase activity were pooled, cooled to 4”C, and kept there until the next step. Saturated ammonium sulfate solution was added to 0.33 saturation, and the solution was stirred gently for 15 min, and then centrifuged in a GSA rotor. The resulting yellow pellet was suspended in 5 mM potassium phosphate buffer, pH 7.2, and desalted, in two separate batches, on a Sephadex G-25 column (2.4 x 35 cm) at room temperature. This preparation is bright yellow.

out as soon as possible in order to avoid inactivation of the enzyme. The pellet was suspended in a minimal volume of PED buffer and dialyzed against 500 ml of PED buffer for at least 2 h at 4°C. The calcium phosphate gel step is used solely to remove a yellow substance, a hemoprotein (14). This step is critical for getting a colorless preparation of fatty acid synthetase. Lower ratios of calcium phosphate gel to protein are not effective in removing the yellow substance and higher ratios decrease the yield. Step 5. Sucrose density gradient centrifugation. This step was done at 23°C. The

enzyme solution from the previous step was diluted to 9 ml. Then 1.5 ml (concenStep 4. Calcium phosphate gel treattration ~10 mg/ml) was applied to each of ment. This step was performed at 23°C. A six sucrose density gradient tubes (3 x 1.5 suspension of calcium phosphate gel at a in.>. The enzyme was then centrifuged at concentration of 28 mg/ml was added (2 mg 20°C for 18 h at 27,000 r-pm in a Beckman of gel/mg of protein) to the above desalted Model L3-50 as described in Materials and preparation. After stirring gently for Methods. A typical profile of the sucrose about 3 min, th.is suspension was centri- density gradient fractionation is shown in fuged for about 7 min at 16,000 rpm at 4°C Fig. 2. in a SS-34 rotor. The supernatant solution As indicated in Fig. 2, there were two was brought quickly to 0.5 saturation by protein peaks: a faster moving, 13 S fatty adding saturated ammonium sulfate solu- acid synthetase peak, and a slower movtion, stirred for 15 min, and centrifuged in ing, 7 S peak. The resolution and relative a SS-34 rotor. This step should be carried areas under the curves of these two peaks

480

KIM,

UNKEFER

varied from experiment to experiment. We cannot exclude the possibility that some or all of the 7 S peak material may be related to fatty acid synthetase in some way, such as being a precursor or degradation or dissociation product. Further evaluation of this possibility is needed. The separation between the two peaks depended on the amount of enzyme applied to each tube. When the resolution between the two peaks was poor, the sucrose density gradient step was repeated a second time to completely remove the 7 S impurity. The 13 S fractions which had activity were pooled and precipitated at 4°C by addition of saturated ammonium sulfate solution to 0.5 saturation. After centrifugation in a SS34rotor, the white pellet was suspended in a small volume (about 4 ml) of PED buffer and dialyzed at 4°C overnight against 500 ml of PED buffer, with one change of buffer. After centrifugation in a SS-34 rotor to remove a slight amount of precipitated protein, the homogeneous colorless, fatty acid synthetase solution was stored at 4°C. The purification steps are summarized in Table I. Stability

and Storage

Stability tests of pig liver fatty acid synthetase (3 mg/ml) were run at 4°C over a period of 15 days by assaying the activity of various fatty acid synthetase solutions containing or lacking 0.2 mM NADPH or 25% sucrose, or both. All solutions also

AND

DEAL

contained D’M’, EDTA, and 0.1 M potassium phosphate at pH 7.4. The decalone reductase (DR) activity was completely stable over this period in all solutions tested (Table II). Over the first 7 days, the overall fatty acid synthetase activity was completely stable in either 0.2 mM NADPH or 25% sucrose; those lacking either NADPH or sucrose dropped about 13-16%. After 11 days, full activity was retained only in the solution containing 25% sucrose; the activity dropped 20% in solutions containing 0.2 mM NADPH and about 33% in the solutions containing no NADPH and no sucrose (Table II). Other experiments showed that 0.2 mM NADH had no effect on the stability. Homogeneity

of Fatty Acid &vzthetase

Schlieren patterns from sedimentation velocity experiments in the analytical Model E ultracentrifuge showed only a single Gaussian peak. This is consistent with, but not proof of, homogeneity. Absorption Spectra

Spectra

and

Fluorescence

For these studies, the stabilizing reducing agent, dithiothreitol, was removed from the fatty acid synthetase solutions by chromatography on Sephadex G-25 (2.4 x 35 cm) in order to obtain the absorption spectra and fluorescence spectra of the enzyme without interference. As indicated in Fig. 3, fatty acid synthetase does not absorb in the visible range from 475 nm down to about 370 nm, but in the ultraviolet

TABLE I PURIFICATION OF FATTY ACID SYNTHETASE FROM PIG LIVER (800 g)”

Clarified crude extract AmSO,-dialyzed enzyme DEAE-cellulose-G25 step (before Ca-phosphate step) Enzyme after Ca-phosphate treatment FAS (after sucrose density gradienV

Total volume (ml)

Total protein h-rid

Total units (units)

1190 1000 100

70091 6300 470

1850 1290 710

130 9

152.5 89.1

692 622.7

Specific activitp (units/mg)

Yield (%o)

0.026 0.205 1.51

100 69.7 38.4

4.54 6.99

37.4 33.7

a See text for details. b Baaed on the partial reaction assay of decalone reductase activity, not overall fatty acid synthetase activity (see Materials and Methods). c The specific activity for the overall fatty acid synthetase reaction was 0.28 unitslmg of protein for this pure enzyme.

LIVER

FATTY

ACID

SYNTHETASE TABLE

STABILITY

II ACID SYNTHETASE”

OF FATTY

Activity

Addition

No addition No addition 0.2 mM NADPH 0.2 mM NADPH 25% sucrose 25% sucrose

481

PROPERTIES

Days

FAS DR FAS DR FAS DR

0

3

5

7

11

15

0.31 4.80 0.35 4.04 0.33 4.97

0.32 4.80 0.36 4.13 0.32 4.18

0.32 4.70 0.36 4.94 0.35 4.3

0.27 4.80 0.36 4.63 0.38 4.36

0.21 4.30 0.29 4.32 0.34 4.67

0.24 4.89 0.30 4.4 0.32 4.73

(1Fatty acid synthetase solutions (3.3 mg; 3 mg/ml) were kept at 4°C in 0.1 M potassium phosphate, 7.4, containing 1 mM EDTA and 1 mM dithiothreitol and assayed at the indicated times. All values specific activities. See text for further details. Decalone reductase activity was measured at pH 6.9.

range, it has a major absorption peak at 280 nm and a shoulder at 290 nm. This shoulder was also noted by Dutler et al. (1). The nature of this chromophore component is not known, although it may be due to tryptophan. The fluorescence emission spectra of fatty acid synthetase were obtained at various excitation wavelengths ranging from 260 to 300 nm. As shown in Fig. 4, fatty acid synthetase showed a typical tryptophan fluorescence peak at 344 nm under the above conditions. The fluorescence of tyrosine was apparently too weak to show. Maximal fluorescence occurred with an excitation wavelength of 285 nm. Sulfbydryl

Content and Optimum

1,O111

pH

Spectrophotometric titration of the sulfhydryl groups with DTNB indicated the presence of about 90 SH groups per molecule of enzyme, assuming a molecular weight of 478,000. The pH profile of the overall pig liver fatty acid synthetase reaction was measured in 0.1 M potassium phosphate buffer over the range 6.0 to 7.3. As shown in Fig. 5, a pH optimum ranging from 6.5 to 6.8 was observed. Viscosity, Sedimentation, Studies

I

pH are

and Diffusion

It was rather difficult to obtain good viscosity data for pig liver fatty acid synthetase. The enzyme seems to denature fairly easily, particularly at concentrations above about 10 mg/ml or below about 5 mg/ml. Even in the most reproducible experiments, the apparent intrinsic viscosity, which was about 7 to 7.6 ml/g, in-

FIG. 3. Absolute absorption spectrum of pig liver fatty acid synthetase in the uv and visible regions. Just prior to use, the stock enzyme solution (19 mg/ ml) was run through a Sephadex G-25 column (2.4 x 35 cm) to remove dithiothreitol. Then the enzyme was diluted with elution buffer to a concentration of 0.70 mg/ml. Fatty acid synthetase (0.7 mg/ml) in 0.2 M potassium phosphate buffer, pH 7.4, containing 1 mM EDTA was added to the sample cuvette. The reference cuvette contained elution buffer solution. The spectrum was obtained with a Cary 15 spectrophotometer at 23°C using quartz microcuvettes. No absorption was observed at wavelengths greater than 350 nm.

creased with decreasing protein concentration, contrary to expectations, and contrary to the behavior seen with most other proteins. At the lower protein concentra-

482

KIM, UNKEFER

AND DEAL

2 -

300nm

1

260nm

-

FIG. 4. Ultraviolet fluorescence emission spectra for pig liver fatty acid synthetase. Emission spectra at different excitation wavelengths were obtained at 23°C using an AmincoBowman spectrofluorometer equipped with a grating-type excitation monochrometer. The apparent emission between 600 and 800 nm is an artifact resulting from the second-order reflection from the emission in the 345-nm region. For this experiment, the fatty acid synthetase was dialyzed against PED buffer overnight at 4°C and then diluted with dialysate buffer to a concentration of 0.11 mg/ml. The various wavelengths used for excitation are indicated on the graph.

zo.ooL&-J 6.0

7.0

65

Z4

PH

FIG. 5. Effect of pH on the overall fatty acid synthetase activity. Each assay mixture contained 0.1 M potassium phosphate buffer at the specified pH. Assays were done at 25°C as described under Materials and Methods.

tions (below 4 mg/ml), the apparent intrinsic viscosity increased drastically. Surprisingly, the flow times at a given concentration in this range were quite reproducible and did not increase with time. This argues against, but does not exclude, the possibility of this being an artifact resulting from precipitation or adsorption of the enzyme to the walls of the viscometer, such as was observed with acetyl-CoA carboxylase (15). Other possibilities are that the enzyme swells or dissociates into asymmetric subunits at these lower concentrations. Our tentative best estimate of

FIG. 6. Concentration dependence of the sedimentation coeffkient of pig liver fatty acid synthetase. Fatty acid synthetase was dialyzed against PED buffer at 4°C overnight. Sedimentation experiments were performed at 23°C at 48,000 rpm using double-sector cells.

the intrinsic viscosity of this enzyme is about 7.3 ml/g. As shown in Fig. 6, the sedimentation coefficient of pig liver fatty acid synthetase has a typical slight dependence on protein concentration. The extrapolation to zero protein concentration gives a value of s&,~ = 13.30 -c 0.07 S. The data in Fig. 6 fit a regression curve of the form se = so + kc, where so = 13.30 & 0.07 S, and k = -0.08 * 0.01 S/mg/ml. The plot of diffusion coefficient values as a function of protein concentration is given in Fig. 7. This curve has a slight positive slope; normally, a negative slope would be expected. The extraphlation to

LIVER

FATTY

ACID

SYNTHETASE

483

PROPERTIES

TABLE III zero protein concentration yields a value = 2.60 + 0.04 x lop7 cm* s-l. PHYSICOCHEMICAL PROPERTIES OF PIG LIVER FATTY of- @o,w ACID SYNTHETASE~ These data fit a regression curve of the Parameters Values formD = Do + Izc, whereDO = 2.60 & 0.04 F and K = 0.022 * 0.006 F/mg/ml. From Molecular weight (from s$,,,,, and 478,00@ these values for sedimentation and diffuQ&v) sion coefficients, respectively, values of Molecular weight (from sedimen476,OOW tation equilibrium) 1.55 and 1.56 x lo-’ cm were calculated 13.30 s for the frictional coefficient. The value of s&m 2.60 Fd the Stokes radius calculated from both %3.W 1.55 x 10-7 the sedimentation and the diffusion coef- f,, frictional coefficient (from s) 1.56 x lo-’ fD, frictional coefficient (from D) ficient was found to be 82.4 A; similarly, 82.4 A r, Stokes radius the frictional ratio was calculated to be f/h,, frictional ratio 1.58 [nl, intrinsic viscosity SH groups per molecule (assumingM = 478,000) E!&$~&, extinction coefficient p, Scheraga-Mandelkern shape factor

FIG. 7. Effect of-concentration on the diffusion coefficient of pig liver fatty acid synthetase. The stock solution of fatty acid synthetase (28.6 mg/ml) used for the sedimentation velocity experiments was also used for these experiments. The experiments were performed with a double-sector synthetic boundary cell at 4300 rpm at 23°C. The point at the lowest concentration (open circle) was not included in the least-squares analysis because the precision of the data was significantly lower than that of the remaining points. 30 l-

z I

2c l-

::

1.3 ml/g About 90 1.23 2.14 x 10"

a See text for details. * Calculated from the Svedberg equation using the s and D values listed. c Calculated from meniscus depletion sedimentation equilibrium experiments. d The units of F (Fick) are 10m7cm2 s’.

1.58 using both sedimentation and diffusion coefficient data. The values of weight-average molecular weight calculated from the Svedberg equation and from the meniscus-depletion sedimentation equilibrium experiment (Fig. 8) were 478,000 and 476,000, respectively. Using the Scheraga-Mandelkern equation (16), the shape factor, p, was calculated from the intrinsic viscosity and sedimentation coefficient data to be 2.14 x lo-“. Table III summarizes the physicochemical properties of pig liver fatty acid synthetase.

+ 0

DISCUSSION

Physical

1.0

It’ (CW FIG. 8. Meniscus depletion sedimentation equilibrium analysis of fatty acid synthetase. The initial protein concentration was 1.2 mg/ml. The experiment was carried out for 24 h at 10,600 rpm at 21.5”C.

Properties

These studies provide the first detailed physical characerization of a mammalian liver fatty acid synthetase. The data show that pig liver fatty acid synthetase has a molecular weight of 478,000, based on sedimentation equilibrium studies and on sedimentation and diffusion coefficient determinations. The enzyme appears to be asymmetric judging from its value of intrinsic viscosity. To the best of our

484

KIM, UNKEFER

knowledge, this intrinsic viscosity study is the first reported for fatty acid synthetase from any source. The exact quantitative value is still somewhat uncertain due to difficulties resulting from slight instability of the enzyme in the viscosity experiments. However, it seems safe to draw the qualitative conclusion that the intrinsic viscosity (7.1-7.6 ml/g) is significantly and sufficiently higher than the usual value of 3 to 4 ml/g for typical globular proteins to ‘suggest significant asymmetry. Furthermore, strong support for asymmetry of the enzyme comes from the frictional ratio of 1.58 found from sedimentation coefficient data and from diffusion coefficient data; most typical globular proteins have a value of about 1.25. Also, the values for sedimentation coefficient (13.3 S) and for the diffusion coefficient (2.6 F) of pig liver fatty acid synthetase are considerably smaller than would be expected for a globular protein of molecular weight of 478,000 (about 15.5 S and 2.9 F, respectively). The physical parameters of pigeon liver fatty acid synthetase were originally reported (17) to be s$,,~ = 14.7 S, D$&, = 2.5. x lop7 cm %, and M,(S/D) = 5.33 x 105. The most recent report (19) lists the same values except for a lower molecular weight (4.5 x 109) based on three values (3.92 to 5.22 x lo”) from sedimentation equilibrium experiments (20). A molecular weight value (4) of 5.01 x lo5 and an s&, value (18) of 13.02 S have been reported for the chicken liver enzyme.

AND DEAL

vins and are yellow, so the existence of a yellow color in the initial preparations did not cause much concern. However, attempts to demonstrate spectral properties compatible with flavins were unsuccessful. The yellow substance could be separated from pig liver fatty acid synthetase by calcium phosphate gel treatment. Neither activated charcoal nor bentonite were suitable for this purpose since they adsorbed fatty acid synthetase as well as the yellow substance. From further extensive studies of the yellow substance in our pig liver fatty acid synthetase preparations, we found (14) it to be a new, highly unusual hemoprotein. Its most unusual feature is that the dithionite-reduced protein exhibits a strong Soret band at 450 nm, which is higher than that of other known hemoproteins except P-450. However, it is not P-450. Further studies are in progress on this hemoprotein. Removal

of Catalase and the 7 S Peak

It is crucial that catalase, a green-colored protein, be separated in the DEAEcellulose step since it is about the same size (11.3 S) as fatty acid synthetase (13.3 S). With a linear 0 to 0.23 M KC1 gradient, catalase elutes from the DEAE column just ahead of fatty acid synthetase, and complete separation is obtained only if unusually large volumes (more than five to six times the DEAE bed volume) of the eluting gradient are used. The properties of the two enzymes are so similar that a discontinuous salt gradient does not give separation on DEAE-cellulose. The Yellow Substance Fatty acid synthetase begins to elute In our hands, an earlier method (1) for with 0.09 M salt at either 4 or 23°C and purification of fatty acid synthetase from with either KC1 or NaCl as the salt spepig liver yielded slightly yellow enzyme cies. We chose to run the DEAE-cellulose preparations, and the final two steps (gel column at 23°C because the apparent capacity was much filtration on Sepharose 4B and a second DEAE-cellulose chromatography on DEAE) lowered the greater at room temperature than at 4°C yield without significantly enhancing the (0.6 g of DEAE-cellulose required per purification. Also, as might be expected, gram of liver at 23°C versus 2.0 g/g of the method (2) used for rat liver did not liver at 4°C). The enzyme is quite stable at 23°C in the presence of 1 mM DTT. The yield pure enzyme when used for isolation repeated ammonium sulfate fractionation of the enzyme from pig liver. Some fatty acid synthetases [e.g., the prior to the DEAE-cellulose column reyeast enzyme (21, 22)l contain bound fla- moved much unwanted protein and de-

LIVER

FATTY

ACID

SYNTHETASE

creased the amount of DEAE-cellulose required. After the DEAE-cellulose column, the major heterogeneity in the preparation was due to a 7 S substance or group of substances and to the yellow 16 S substance described previously. The colorless 7 S material was removed by preparative sucrose density gradient sedimentation experiments. The relative velocity amount of 7 S material varied from liver to liver. Purification

Procedure:

Other Aspects

The major changes in this purification procedure from the original methods of Dutler et al. (1) are homogenization with a Waring Blendor, a change in the salt gradient elution in the DEAE-cellulose chromatography step, introduction of a density gradient sedimentation velocity step, and introduction of a calcium phosphate gel step. Our procedure gives about twice the yield of the method of Dutler et al. (l), and the enzyme is completely colorless. Since we use a different substrate for the partial reaction assay, our results with partial reaction assays cannot be compared directly with theirs (1). However, pure enzyme preparations from the two precedures appear to have about the same specific activity in the overall fatty acid synthetase reaction. REFERENCES 1. DUTLER, H., COON, M. J., KULL, A., VOGEL, H., WALDVOGE, G., AND PRELOG, V. (1971) Eur. J. Biochem. 22, 203-212. 2. BURTON, D. N., HAAVIK, A. G., AND PORTER, J. W. (1968) Arch. Biochem. Biophys. 126, 141154. 3. Hsu, R. Y., WASSON, G., AND PORTER, J. W. (1965) J. Biol. Chem. 240, 3736-3746.

PROPERTIES

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4. Hsu, R. Y., AND YUN, S. (1970) Biochemistry 9, 239-244. 5. RONCARI, D. A. K. (1974)Canad. J. Biochem. 52, 221-230. 6. MASSEY, T. H., AND DEAL, W. C., JR. (1973) J. Biol. Chem. 248, 56-62. 7. KIM, I. C., AND DEAL, W. C., JR. (1976) Submitted for publication. 8. LYNEN, F. (1969) in Methods in Enzymology, Vol. 14, pp. 17-33, Academic Press, New York. 9. MARTIN, R. G., AND AMES, B. N. (1961) J. Biol. Chem. 236, 1372-1379. 10. CHERVENKA, C. H. (1969) A Manual of Methods for the Analytical Ultracentrifuge, p. 83, Spinco Division of Beckman Instruments, Inc., Palo Alto, Calif. 11. SVEDBERG, T., AND PEDERSEN, K. 0. (1940) The Ultracentrifuge, pp. 22, 36, 39, Oxford University Press, Oxford, England. 12. CHERVENKA, C. H. (1969) A Manual of Methods for the Analytical Ultracentrifuge, p. 56, Spinco Division of Beckman Instruments, Inc., Palo Alto, Calif. 13. HABEEB, A. F. A. S. (1972) In Methods in Enzymology, Vol. 25, p. 457, Academic Press, New York. 14. KIM, I. C., AND DEAL, W. C., JR. (1976) Biochemistry, in press. 15. Moss, J., AND LANE, M. D. (1972) J. Biol. Chem. 247, 4944-4951. 16. SCHERAGA, H. A., AND MANDELKERN, L.(1953) J. Amer. Chem. Sot. 75, 179-184. 17. YANG, P. C., BOCK, R. M., Hsu, R. Y., AND PORTER, J. W. (1965) Biochim. Biophys. Acta 110, 608-615. 18. YUN, S. L., AND Hsu, R. Y. (1972) J. Biol. Chem. 247, 2689-2698. 19. Hsu, R. Y., BUTTERWORTH, P. H. W., AND PORTER, J. W. (1969) in Methods in Enzymology, Vol. 14, pp. 33-39, Academic Press, New York. 20. YANG, P. C., BUTTERWORTH, P. H. W., BOCK, R. M., AND PORTER, J. W. (1967) J. Biol. Chem. 242, 3501-3507. 21. LYNEN, F. (1961) Fed. Proc. 20, 941-951. 22. LYNEN, F. (1967) Biochem. J. 102, 381-400.