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Fatty acid synthesis in aorta Isolation of fatty acid synthetase from chicken aorta
103
Afherosclerosis, 26 (1977) 103-115 @ Elsevier/North-Holland Biomedical Press
FATTY
ACID SYNTHESIS
ISOLATION
OF FATTY
IN AORTA
ACID SYNTHETASE
ALAN C. WILSON, MAHDI MURTADHA Marrs McLean Department 77030 (U.S.A.)
FROM CHICKEN AORTA
and SALIH J. WAKIL
of Biochemistry,
Baylor
College
of Medicine,
Houston,
Texas
(Received 14 May, 1976) (Accepted 4 August, 1976)
Summary
Fatty acid synthesis by subcellular fractions of human aorta was studied by measuring the incorporation of either radioactive acetyl-CoA or malonyl-CoA into long chain fatty acids. The high speed supematant fraction contained fatty acid synthetase and was capable of de nom fatty acid synthesis. The fatty acid synthetase from chicken aorta was purified 800-fold from the high speed supematant and was judged to be 10% pure at this level. Its molecular weight was estimated to be 450,000 on the basis of agarose gel filtration chromatography, while under dissociating conditions a molecular weight of 220,000 was obtained on sodium dodecyl sulphate disc gel electrophoresis. Fatty acid synthesis was dependent on acetyl-CoA, malonyl-CoA and NADPH. The major product was free palmitic acid. In enzymatic and physical characteristics the chicken aorta fatty acid synthetase strongly resembles the synthetase isolated from chicken liver. The two enzymes cross-react immunochemically and this homology provides the possibility of studying the synthesis and degradation of the aorta synthetase during the development of atherosclerosis. ~~ _____ Key words:
Aorta Human
___~~__
Chicken
-Fatty
acid
chain
elongation
-Fatty
acid
synthetase
-
_.___
Introduction
The development of atherosclerosis is characterized by the focal accumulation of lipids in the vessel walls. The origin of this lipid may be either from the This
work
was
supported
by
United
States
Public
Health
Service
grant
HL-17269-01
(1)
P-3
l-A-3.
104
plasma lipoproteins or from endogenous synthesis in the arterial wall. Increased fatty acid synthesis has been reported to occur in atherosclerotic aortas from rabbit, pigeon, monkey, and humans [l-6]. Whereat [7] isolated subcellular fractions from rabbit aorta and, measuring [l-14C]acetate incorporation, identified mitochondria as the major site of fatty acid synthesis. Filipovic and his co-workers [8] using calf arterial segments, found increased incorporation of [ l-‘4C]acetate into triglyceride fatty acids in hypoxia and proposed that mitochondrial fatty acid chain elongation was the predominant process. Howard [9] investigated fatty acid synthesis by subcellular fractions of monkey aorta and concluded that aorta is able to carry out both de nouo synthesis and elongation of fatty acids. Aortic high-speed supernatant extracts synthesized palmitic and stearic acids completely from malonyl-CoA. Both mitochondria and microsomes elongated fatty acids with acetyl-CoA, microsomes also elongated fatty acids with malonyl-CoA. In order to understand the reasons for the apparent increased synthesis of fatty acids, the mechanisms regulating fatty acid synthesis in the arterial wall must be clarified. We have undertaken an investigation of the fatty acid synthesizing systems in aorta in an effort to evaluate their contribution to arterial fatty acid metabolism. This paper describes the capacity of subcellular fractions of human aorta for fatty acid synthesis and reports the isolation and partial purification of fatty acid synthetase from chicken aorta and the characterization of its enzymic and immunological properties. Materials and Methods Chemicals
The 14C-labeled acyl-CoA derivatives were obtained from New England Nuclear. Unlabeled acyl-CoA derivatives were purchased from P.L. Biochemicals. All other chemicals were reagent grade and were obtained from local commercial sources. Collection
0 f tissue
Segments of human aorta were removed at postmortem examinations within 2 h of death and were collected into ice-cold normal saline. Aortas from freshly killed frying chickens were collected in the same way. Preparation
of subcellular
fractions
All manipulations were performed at 0-4°C. Adhering fat and connective tissue was removed and the intima-media portions of the aortas were rinsed in 0.05 M potassium phosphate, pH 7.4, containing 0.25 M sucrose, 10 n-&f 2-mercaptoethanol and 1 mM EDTA (homogenizing medium). The aortas were homogenized for 15 set at setting 10 with a Polytron ST-20 (Brinkman Instruments) in 5 volumes (v/w) of homogenizing medium. The homogenate was fractionated by differential centrifugation. After an initial centrifugation for 6 min at 800 X g to remove the bulk of the cell debris and collagen, the supernatant was centrifuged for 12 min at 7,700 X g. The resulting mitochondrial pellet was resuspended in homogenizing buffer and recentrifuged for 10 min at 6,000 X g and resuspended in a small volume of the same buffer. The post-mitochondrial
105
supernatant was centrifuged for 60 min at 105,000 X g and the microsomal pellet was resuspended in a small volume of the same buffer and used without further washing. Enzyme
assays
Fatty acid synthetase activity was measured routinely by incubating appropriate amounts of enzyme with 30 nmoles of acetyl-CoA and 56 nmoles of NADPH in the presence of 60 mM potassium phosphate, pH 7.0, 4 mM dithiothreitol. After a 5-min pre-incubation at room temperature (25” C), the reaction was started by adding 45 nmoles of malonyl-CoA bringing the final volume to 0.5 ml. The rate of NADPH oxidation was measured at 340 nm in an Aminco DW-2 spectrophotometer. A unit of enzyme activity is defined as the amount catalyzing the oxidation of 1 pmole of NADPH per min under these conditions. Fatty acid synthetase activity in the high speed supematant was also measured by a radioactive assay. The same incubation mixture was used except that [ 1,3-14C]malonyl-CoA (2 pCi/pmole) was substituted for malonyl-CoA, and amount of NADPH was increased to 500 nmoles. Incubations were performed for 20 min at room temperature and terminated by the addition of 0.2 ml of 5 M HCl. Radioactivity incorporated into fatty acids was determined by extracting the acidified mixture with redistilled pentane and counting an aliquot in toluene based liquid scintillation medium. Fatty acid elongation activity in mitochondria and microsomes was measured by the rate of incorporation of [l-“C] acetyl-CoA and [1,3-14C]malonylCoA respectively into endogenous fatty acids. The reaction mixture contained in a final volume of 0.5 ml, 25 pmoles potassium phosphate, pH 7.0, 2.5 pmoles MgC12, 5 pmoles ATP, and 0.5 mg protein. Incubations with microsomes contained in addition, 50 nmoles [1,3-14C]malonyl-CoA (2 pC!i/mole) and 2.5 pmoles NADPH, while mitochondrial incubations contained 50 nmoles [ 1-14C] acetyl-CoA (2 /&i/pmole) and 2.5 pmoles NADH. Reactions were performed at 37°C for 20 min and were terminated by the addition of 0.2 ml 10 N KOH. The mixture was saponified at 95°C for 20 min, cooled and acidified, and the fatty acids were extracted with pentane. After washing the extract, an aliquot was taken to dryness in a scintillation vial and the radioactivity was determined using toluene based scintillation medium. Preparation
of [‘4C]acetyl-labeled
fatty acid synthetase
Chicken aorta synthetase was dialyzed against 0.1 M sodium phosphate, pH 7.0, 1 mM EDTA, 1% 2-mercaptoethanol. Dialyzed synthetase (200 1-11,130 pg) was incubated for 2 min at room temperature with 37 nmoles of [ l-14C]acetyl-CoA in a final volume of 0.2 ml. The reaction was terminated by the addition of 2.5 pmoles of N-ethylmaleimide. To determine the radioactivity bound to the synthetase, the protein was precipitated by the addition of 1.0 ml of cold 5% aqueous perchloric acid. The precipitate was collected on a Millipore filter (25 mm, 0.45 pm) and washed with 30 ml of cold 5% aqueous perchloric acid. The filters were dissolved in scintillation vials with 0.1 ml 0.5 N NaOH and 1.0 ml methanol and counted in a dioxane based scintillation medium.
106 Sodium
dodecylsulphate
disc electrophoresis
The 14C-labeled synthetase was denatured by the addition of 20 ~1 of a solution containing 10% SDS and 100 mM iodacetamide to 200 ~1 of the enzyme solution. After incubation for 2 h at room temperature the samples were applied in duplicate (100 ~1) to SDS gels and electrophoresis was performed as described by Stoops et al. [lo]. The gels were stained with Coomassie blue, destained, photographed, densitometer scans were made, and finally cut into 2 mm sections. The sections were heated overnight at 70°C with 1.0 ml of 0.2 M NaOH in sealed scintillation vials. The extracts were acidified and radioactivity was determined using Triton X-100 : toluene scintillation medium. Identification of products products The radiolabeled
of fatty acid synthesis and elongation were identified as the fatty acid methyl esters. Esterification was performed by reaction with an ethereal solution of diazomethane. Separation and identification of the methyl esters was achieved on a 15% ethyleneglycolsuccinate column in a Varian Series ‘2700 gas chromatograph. Mass was detected using a flame ionization detector and radioactivity was monitored continuously with a Nuclear Chicago counting system. Separation of fatty acid methyl esters on the basis of unsaturation was performed using silver nitrate impregnated silica gel thin layer chromatography as described by Jones et al. [ 111. Decarboxylation of radiolabeled fatty acids was carried out by the method of Brady et al. [ 121.
Immunological
procedures
Goat antiserum against highly purified chicken liver fatty acid synthetase was partially purified by DEAE-cellulose chromatography. Ouchterlony double diffusion analysis was performed as described by Clausen [13] using commercially available agar plates (Hyland). Equivalence point determinations were performed as described by Kabat and Mayer [ 141. Radioactivity
and protein
determination
Radioactivity was determined using a Nuclear Chicago Mark II liquid scintillation system. Addition of a 14C toluene internal standard indicated a counting efficiency of 86--89%. Protein was measured by the method of Lowry et al. [15]. Results Fatty acid synthesis
in human aorta
Active lipid synthesis from [2-14C]acetate has been shown by Chobanian and Manzur [6] in normal intima and fatty streaks of human arterial segments. We extended our studies to determine the contribution towards fatty acid synthesis of the various subcellular fractions, and the effect of arteriosclerosis on the activities. Table 1 shows the incorporation of 14C-labeled acetyl CoA or malonyl CoA into fatty acids by mitochondria, microsomes and the supematant fraction of normal human aorta. The activit.ies of both de nouo synthesis and elongation
107 TABLE
I
RELATIVE CONTRIBUTIONS HUMAN AORTA
OF SUBCELLULAR
FRACTIONS
TO FATTY
ACID
SYNTHESIS
IN
Values are from duplicate measurements on fractions from necroscopy code 195. 20 year-old-male. Fatty acid synthesis was measured using either [l-14Clacetyl CoA or [1,3-14Clmalonyl CoA as described in Materials and Methods. Fatty acid production was calculated on the basis of an elongation mechanism for the particulate fractions and on de nouo synthesis of palmitic acid by the high speed supernatant. Fraction
Yield (mg protein/g wet weight aorta)
Fatty acid synthesis (nmoles fatty acid/g aorta/h)
High speed supernatant Microsomes Mitochondria
18.4 4.4 3.2
4.23 7.81 1.37
are lower than detected in chicken aorta, agreeing with the studies of Rao and Rao [16] who found that lipid synthesis in chick aorta was higher than in any other species. Nevertheless the results show that fatty acid biosynthesis in human aorta can contribute to the accumulation of aortic lipids. Chobanian [6] reported that lipid synthesis in the fatty streak region was greater than either normal intima or advanced atheromata. We determined the fatty acid synthetase activity in samples of human aorta with various degrees of fibrous-arteriosclerotic involvement. Table 2 lists the results obtained and it is apparent that fatty acid synthetase activity is lower in all lipid-atherosclerotic segments and even in adjacent areas from the same aorta. On the other hand segments with fibrous-arteriosclerotic lesions had near normal activities suggesting that either the fibrous plaques were the result of a different process or that they represented an earlier stage in the development of the atheromatous lesions. No significant difference in fatty acid synthetase activity could be found in this study between segments of thoracic or abdominal origin.
TABLE
2
EFFECT
OF ATHEROSCLEROSIS
ON FATTY
ACID SYNTHETASE
ACTIVITY
IN HUMAN
AORTAS
Necroscopy code
Age and sex
Type of lesion
Protein in HSS (mg/g wet weight of aorta)
acid synthetase Fatty (nmol/h/mg protein a)
195 195
20, M
none none
15.4 14.5
0.28 0.29
60 60
60. M
lipid lipid
19.5 17.5
0.06 0.06
fibrous lipid none
21.7 21.6 20.0
0.11 0.06 0.06
51 51 51
61. M
42
69, M
fibrous
18.6
0.26
196
70, F
lipid
13.8
0.13
202 202
88, F
lipid none
26.8 23.4
0.06 0.09
a BY [1,3-14Clmalonyl
CoA incorporation
(see Table 1).
108 TABLE
3
FATTY
ACID SYNTHESIS
BY SUBCELLULAR
Fatty acid synthesis was measured calculated assuming a mechanism production by the soluble fraction.
FRACTIONS
OF CHICKEN
as indicated under Materials and Methods. of elongation for the particulate fractions, Values are mean f SE of the mean.
AORTA Fatty acid production and de nouo palmitic
Fraction
Yield (mg protein/g wet weight aorta)
Fatty acid synthesis (nmoles fattty acid/g aorta/h)
High speed supernatant Microsomes Mitochondria
14.1 * 1.1 4.1 * 0.2 3.0 t 0.4
12.69 19.57 20.48
was acid
f 1.13 + 0.77 + 1.57
Fatty acid synthesis in chicken aorta Chicken aorta was selected for further studies on the nature of the fatty acid synthetase in aorta. Table 3 shows the fatty acid synthetase activity of the subcellular fractions and the yields of each obtained routinely. Conditions of activity determination were such that proportionality with time and added protein was observed. Assay requirements were generally similar to those reported in other tissues [17] and found in human aorta. Microsomal elongation of fatty acids required malonyl-CoA, ATP and NADPH, the requirement for NADPH being only partially replaced by NADH. In mitochondria, elongation is dependent on acetyl-CoA, ATP and NADH. NADPH does not substitute for NADH. Analysis of the products of elongation by microsomes indicated that 50% of the radioactivity was incorporated into long chain polyunsaturated fatty acids, 30% in mono- and dieneoic-acids, and 20% in saturated fatty acids of which C l&O compromised 80%. The mitochondrial system also incorporated radioactivity into a variety of saturated and unsaturated long chain fatty acids. In this case Cl8 was the shortest chain length detectable. The products of the soluble fatty acid synthesizing system in the crude extract were identified as being palmitic and stearic acids in the proportions 60% and 40% respectively. Decarboxylation analysis of product fatty acids supported the assumption that elon-
TABLE
4
ISOLATION Fraction
HSS b 1 2 3 4 5 6 7
OF FATTY
ACID SYNTHETASE
Protein
FROM
(mg)
Activity a (nmoI/min)
1960 1862 1281 102 20 8 4 2.5
3057 2646 878 534 165 199 203
a BY NADH oxidation. b High speed supernatant from 140 ammonium sulphate precipitation.
AORTA
Specific activity (nmoI/mg/min) 0.20 1.64 2.1 8.6 27 21 50 81
392
g chicken
CHICKEN
aorta
segments,
8 to 10 preparations
were combined
after
109
gation was the mode of synthesis in mitochondria and microsomes soluble fraction (crude extract) a de nouo mechanism was indicated
while in the [Ml.
Protein purification The results of the procedure are summarized in Table 4. Unless otherwise specified, all buffers contained 10% glycerol, 1 mM dithiothreitol and 1 n-J4 EDTA. The high speed supematant was fractionated by ammonium sulphate precipitation. The portion precipitating between 30% and 50% saturation was collected and redissolved in 0.05 M potassium phosphate, pH 7.4. The enzyme solution was stored at -80” C after freezing in liquid N2 (Fraction 1). When sufficient material had been accumulated (8-10 preparations) the stored solutions were thawed in ice-water, combined, and dialyzed against 0.05 M potassium phosphate, pH 7.4. Calcium phosphate gel (20 mg/ml) was added to the solution in the proportion of 1 mg gel/mg protein. The mixture was immediately centrifuged and the supematant retained (Fraction 2). After adjusting the ionic strength of the supematant to 0.03 M potassium phosphate, it was applied to a DEAEcellulose column 4 X 42 cm which had been equilibrated with 0.01 M potassium phosphate, pH 7.4. The column was washed with the same buffer and synthetase activity was eluted with a linear gradient of 500 ml each of 0.01 M and 0.25 M potassium phosphate pH 7.4. Fractions containing activity were pooled and an equal volume of saturated ammonium sulphate, pH 7.4 was added to precipitate the active protein. The precipitate was collected by centrifugation as before and redissolved in a minimum volume of 0.25 M potassium phosphate, pH 7.4 (Fraction 3). The solution (2 ml) was applied to a 2.5 X 30 cm column of Sepharose-6B (Pharmacia) equilibrated with 0.25 M potassium phosphate, pH 7.4. The enzyme was eluted with the same buffer, activity being eluted with a K,, of 0.14. The pooled activity was precipitated from 50% saturated ammonium sulphate as before, taken up in a small volume of 0.01 M potassium phosphate, pH 7.4, 10 mM dithiothreitol and dialyzed against the same buffer (Fraction 4). The dialyzed solution was applied to a 1.2 X 4.5 cm column of DEAE-Biogel (Bio-Rad) equilibrated with the 0.01 M potassium phosphate, pH 7.4, 10 m&I dithiothreitol. The column was washed with the same buffer and the enzyme was eluted with a linear gradient of 20 ml each of 0.01 M potassium phosphate, pH 7.4 and 0.20 M potassium phosphate, pH 7.4, both containing 10 mM dithiothreitol. Fractions containing activity were pooled and ammonium sulphate precipitation was performed as before (Fraction 5). This step was essential for the success of subsequent purification steps. The precipitated activity was redissolved in ml of 0.25 M potassium phosphate buffer, pH 7.0, 10 mM dithiothreitol and chromatographed on a 1.2 X 47 cm column of Sepharose-6B with the same buffer. Active fractions were pooled and ammonium sulphate precipitation was performed (Fraction 6). The enzyme was redissolved in 0.01 M potassium phosphate, pH 7.0, 10 m&Z dithiothreitol and dialyzed against the same buffer. The dialyzed solution was applied to a final 0.5 X 6 cm column of DEAE-Biogel and after washing with 0.01 M potassium phosphate, pH 7.0 activity was eluted by the stepwise
110
addition of 5 ml each of 0.05 M, phate, pH 7.0, all containing 10 highest activity were pooled and in 0.5 M potassium phosphate pH
0.10 M, 0.15 M and 0.20 M potassium phosmJ4 dithiothreitol. The tubes containing the the activity was precipitated and redissolved 7.0, 10 mM dithiothreitol (Fraction 7).
Requirements of the fatty acid syn thetase reaction The reaction was dependent on the presence of both acetyl- and malonylCoA. When acetyl-CoA was omitted from the assay system, the rate of malonyl-CoA dependent oxidation of NADPH was negligible compared to the rate observed in the presence of acetyl-CoA. Acetyl-CoA could not be replaced by butyryl-CoA as a primer, optimum levels of which gave rates only 7.5% of the control values. The aorta synthetase also showed a high degree of specificity towards NADPH as the hydrogen donor, the rates obtained with NADH being less than 10% of those in the presence of NADPH. Dithiothreitol was found to be an essential component of the assay system for maximum activity and was required throughout the purification and during storage for maintenance of activity. The activity of the aorta synthetase was inhibited by N-ethylmaleimide and iodoacetamide, further implying the involvement of sulphydryl groups in enzyme activity. Although not required for maximum activity in the assay system, EDTA was included in all buffers to protect the enzyme from heavy metal inhibition. Identification of products The 14C-labeled products from incubation of the purified enzyme with [1,3-14C]malonyl-CoA (see Methods) were able to be extracted from acid solu-
!O
I
0
IO
5 TIME
15 ( minutes
1
4
20
25
I
Fig. 1. Gas chromatograms of the radioactive products of purified aorta fatty acid synthetase. Authentic palmitic and stearic acids were added to the reaction products. Trace A represents the response of the mass detector, trace B the response of the radioactivity detector.
111
tion with pentane without prior treatment with alkali. The products were therefore free fatty acids and not acyl-CoA derivatives. Gas liquid radiochromatography of the products was carried out to identify the fatty acids synthesized. Almost all the radioactivity of the methylated material was coincident with authentic methylated carrier palmitic acid (Fig. 1). These results demonstrate that the major product of fatty acid synthetase is free palmitic acid. Enzymatic properties of chicken aorta synthetase The synthetase has a single, broad optimum of activity at pH 7.0 to 7.5 (Fig. 2). Figure 3 shows the increase in reaction rates measured at pH 7.0 as a function of increasing amounts of substrate. From double reciprocal plots (see insets) the apparent ‘K, values for NADPH, acetyl-CoA and malonyl-CoA are The reaction rate is 8.9 X 10” M, 25 X 10% M and 60 X 10” M, respectively. proportional to the amount of enzyme added and is linear for over 5 min under the assay conditions described. Molecular weight estimation SDS gel electrophoresis of [ 14C]acetyl-labeled aorta fatty acid synthetase gave the results shown in Fig. 4. Four major bands were seen, with the slowest migrating band corresponding to purified chicken liver synthetase. It was estimated that the aorta synthetase was approximately 10% pure in this preparation. That this band was in fact the aorta synthetase was demonstrated by the
30
v 20
IO
6.0
6.5
1.0
7.5
8.0
8.5
9.0
PH Fig. 2. Effect of pH on aorta fatty acid synthetase activity. The spectrophotometric used. The reaction rate (v) is expressed as nmol NADPH oxidized/min/ml.
assay system was
112
I MALONYL-co A I” M
IACm-CoAlpM
INADPHI”M
Fig. 3. Effect of substrate concentration on the activity of aorta fatty acid synthetase. Assay conditions were as described in Methods except that the malonyl-CoA, acetyl-CoA. and NADPH concentrations were varied as indicated. The reaction rate (v) is expressed as nmol NADPH oxidized/min/ml.
finding of all the 14C-label from acetyl and malonyl associated with this band. Assays of the binding capacity of the aorta synthetase preparation indicated that more than 70% of the possible protein-bound counts were recovered from the gel in the band. No radioactivity was detected in any other position. From a calibration curve of log (molecular weight) against relative mobility, the molecular weight of the aorta synthetase in SDS was estimated to be 220,000. Under non-dissociating conditions (see Protein Purification) the aorta synthe-
Fig. 4. Sodium dodecylsulphatc disc electrophoresis [I4 Clacetyl-labeled aorta fatty acid synthetase; (A) highly purified chicken liver syntbetase run under identical conditions; (B) aorta synthetase. The radioactivity associated with 2 mm slices tif gel (B) is shown above. Direction of migration was from left to right.
113
Fig. 5. Doilble diffusion precipitin reactions in agar of aorta (A). and liver (L). fatty acid synthetase. The center well coctained goat antiserum prepared against chicken liver fatty acid synthetase. The precipitin lines were stained with 0.5% Amido Black.
ENZYME ADDED I milliunits
I
Fig. 6. Immunochemical equivalence of aorta and liver fatty acid synthetase. Varying amounts of purified aorta (A) or liver (0) synthetase were incubated at 4’C with 20 ~1 of immune gammaglobulin fraction from goat serum. Fatty acid synthetase activity was measured in the supernatant solution after centrifugation of the incubation.
114
tase activity chromatographed on Sepharose-6B with a K,, of 0.12, a value similar to that obtained for the liver synthetase and corresponding to a molecular weight of about 500,000.
Immunological pi-operties The results of double diffusion analysis of the aorta synthetase and liver synthetase against antiserum to the chicken synthetase are shown in Fig. 5. The smooth continuum of the precipitin lines and the absence of spur formation indicates the cross reaction of homologous antigens, supporting the similarity between the synthetases from aorta and liver. Furthermore, identical equivalence points (Fig. 6) for the synthetases from both tissues were obtained on addition of increasing amounts of enzyme to a constant amount of antiserum against the liver enzyme. Discussion Arterial wall lipid metabolism has been the subject of renewed interest in the past decade, progress in this subject has been reviewed recently by Geer and his co-workers [19]. Whereat [20] has reviewed in detail the status of fatty acid biosynthesis in aorta. De nouo synthesis of saturated fatty acids by aorta has been reported in human, monkey and pigeon [4-6]. Although the system responsible has not been fully characterized, Howard showed that malonyl-CoA was the preferred substrate for de nouo fatty acid synthesis in high-speed supernatant fraction of monkey aorta [9]. The present results confirm that aorta is capable of both de nouo synthesis and elongation of fatty acids. We have purified the fatty acid synthesizing activity some 800-fold from the soluble fraction of aorta homogenates and have established that the enzyme closely resembles the fatty acid synthetase isolated from chicken liver. Essentially similar procedures for the purification were used, and although comparable specific activities were not reached this may be ascribed to the technical difficulties encountered with this tissue. The concentration of the enzyme (per mg soluble protein) is 200-fold lower in aorta than in liver. Furthermore the contaminating proteins remaining with the purified synthetase appear to exist, in the native state, as an aggregate since they can only be separated from the synthetase under dissociating conditions. The molecular weight of the aorta synthetase as determined by SDS electrophoresis (220,000) is identical to that of the liver enzyme and under non-dissociating conditions the two enzymes are eluted from Sepharose-6B at the same K,, (0.12, equivalent MW 450,000). These results support a model, developed from extensive studies with the liver enzyme [lo], in which the fatty acid synthetase is a dimer of MW 450,000 which dissociates in SDS into two polypeptides of MW 220,000. Further evidence of the close similarity of the two enzymes is provided by the antigenic homology shown on double diffusion analysis and equivalence point determination. The product of the synthesis is palmitic acid in both cases, and the assay requirements for the two enzymes are almost identical. The stability of the enzymes is remarkably similar, both requiring phosphate ion, dithiothreitol and glycerol for maintenance of activity.
115
The regulation of saturated fatty acid biosynthesis in liver and brain has been reviewed recently by Volpe and Vagelos [ 211. Immunochemical techniques have been used to study the content, synthesis, and degradation of fatty acid synthetase in these tissues in various nutritional states and during development [ 221. We intend to use the antigenic similarity discovered in this study to apply these sensitive techniques to the study of the effect of hormones, nutritional state, development, and atherosclerosis on the aorta’ fatty acid synthetase. These studies will give insight into the regulation of fatty acid synthesis in aorta and the reasons for the apparent increase in lipids associated with atherosclerotic involvement. References 1 Howard. Jr.. CF., Lipogenesis from glucose-2-l 4 C and acetate-l-’ 4 C in aorta, J. Lipid Res., 12 (1971) 725-730. 2 Howard, Jr., C.F., Aortic Iipogenesis during aerobic and hypoxic incubation. Atherosclerosis, 15 (1972) 359-369. 3 Lofland, Jr., H.B.. Mousy, D.M., Hoffman, C.W. and Clarkson, T.B.. Lipid metabolism in pigeon aorta during atherogenesis, J. Lipid Res., 6 (1965) 112-118. 4 St. Clair. R.W.. Lofland, Jr.. H.B. and Clarkson, T.B., Composition and synthesis of fatty acids in atherosclerotic aortas of the pigeon, J. Lipid Res., 9 (1968) 739-747. 5 LofIand, Jr., H.B., St. Cl&, R.W., Clarkson, T.B., Bullock. B.C., and Lehner, N.D.M.. Atherosclerosis in Cebus monkeys. Part 2 (Arterial metabolism), Exp. Molec. Path., 9 (1968) 57-70. 6 Chobanian, A.V. and Manzur, F.. Metabolism of lipid in the human fatty streak lesion, J. Lipid Res., 13 (1972) 201-206. 7 Whereat, A.F.. Fatty acid synthesis in cell-free system from rabbit aorta, J. Lipid Res., 7 (1966) 671677. 8 FiIipovic. I. and Buddecke, E., Increased fatty acid synthesis of arterial tissue in hypoxia, Europ. J. Biochem., 20 (1971) 587-592. 9 Howard, Jr., C.F., De now synthesis and elongation of fatty acids by subcellular fractions of monkey aorta, J. Lipid Res., 9 (1968) 254-261. 10 Stoops, J.K., Arslanian, M.J., Oh, Y.H.. Aune, K.C., Vanaman, T.C. and WakiI, S.J., Presence of two polypeptide chains comprising fatty acid synthetase, Proc. Nat. Acad. Sci. (Wash.), 72 (1975) 19401944. 11 Jones, P.D., Holloway. P.W., Peluffo. R.O. and WakiI. S.J., A requirement for lipids by the microsomal stearyl CoA desaturase, J. Biol. Chem., 244 (1969) 744-754. 12 Brady, R.O., Bradley, R.M. and Trams. E.G., Biosynthesis of fatty acids, Part 1 (Studies with enzymes obtained from liver). J. Biol. Chem., 235 (1960) 3093-3098. 13 Clausen, J., In: T.S. Work and E. Work (Eds.), Laboratory Techniques in Biochemistry and Molecular Biology, North-Holland. Amsterdam, 1972, pp. 399-572. 14 Kabat. E.A. and Mayer. M.M., Experimental Immunochemistry. 2nd edition, C.C. Thomas, Springfield, Ill., 1961. 15 Lowry. O.H., Rosebrough, N.J.. Farr, A.L., and Randall, R.J., Protein measurements with the FoIin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 16 Rae, A.R. and Rae. B.S.N.. Incorporation of [1-14C]acetate into the lipids of aortas of different species, J. Atheroscler. Res. 8 (1968) 59-67. 17 Seubert, W. and Podack, E.R.. Mechanisms and physiological roles of fatty acid chain elongation in microsomes and mitochondria, Molec. and Cellular Biochem., 1 (1973) 2940. 18 WakiI. S.J.. Lipid Metabolism, Academic Press, New York, N.Y., 1970. pp. 148. 19 Geer, J.C., Panganamala, R.V.. Newman, H.A.I. and CornwaII. D.G.. In: R.W. Wissler and J.C. Geer (Eds.), Pathogenesis of Atherosclerosis. American Association of Pathology and Bacteriology, 1971, pp. 200-213. 20 Whereat. A.F., In: R. Paoletti and D. Kritchevsky (Eds.), Advances in Lipid Research. Academic Press, New York. N.Y., 1971, pp. 119-159. 21 Volpe, J.J. and Vagelos. P.R. In: E.E. SneII (Ed.), Annual Review of Biochemistry, Annual Reviews Inc., Palo Alto, Cal., 42 (1973) 21-60. 22 Gibson, D.M., Lyons, R.T.. Scott. D.F. and Muto, Y.. In: G. Weber (Ed.), Advances in Enzyme Regulation, Vol. 10. Pergamon Press, New York, N.Y., 1972, pp. 187-204.
Afherosclerosis, 26 (1977) 103-115 @ Elsevier/North-Holland Biomedical Press
FATTY
ACID SYNTHESIS
ISOLATION
OF FATTY
IN AORTA
ACID SYNTHETASE
ALAN C. WILSON, MAHDI MURTADHA Marrs McLean Department 77030 (U.S.A.)
FROM CHICKEN AORTA
and SALIH J. WAKIL
of Biochemistry,
Baylor
College
of Medicine,
Houston,
Texas
(Received 14 May, 1976) (Accepted 4 August, 1976)
Summary
Fatty acid synthesis by subcellular fractions of human aorta was studied by measuring the incorporation of either radioactive acetyl-CoA or malonyl-CoA into long chain fatty acids. The high speed supematant fraction contained fatty acid synthetase and was capable of de nom fatty acid synthesis. The fatty acid synthetase from chicken aorta was purified 800-fold from the high speed supematant and was judged to be 10% pure at this level. Its molecular weight was estimated to be 450,000 on the basis of agarose gel filtration chromatography, while under dissociating conditions a molecular weight of 220,000 was obtained on sodium dodecyl sulphate disc gel electrophoresis. Fatty acid synthesis was dependent on acetyl-CoA, malonyl-CoA and NADPH. The major product was free palmitic acid. In enzymatic and physical characteristics the chicken aorta fatty acid synthetase strongly resembles the synthetase isolated from chicken liver. The two enzymes cross-react immunochemically and this homology provides the possibility of studying the synthesis and degradation of the aorta synthetase during the development of atherosclerosis. ~~ _____ Key words:
Aorta Human
___~~__
Chicken
-Fatty
acid
chain
elongation
-Fatty
acid
synthetase
-
_.___
Introduction
The development of atherosclerosis is characterized by the focal accumulation of lipids in the vessel walls. The origin of this lipid may be either from the This
work
was
supported
by
United
States
Public
Health
Service
grant
HL-17269-01
(1)
P-3
l-A-3.
104
plasma lipoproteins or from endogenous synthesis in the arterial wall. Increased fatty acid synthesis has been reported to occur in atherosclerotic aortas from rabbit, pigeon, monkey, and humans [l-6]. Whereat [7] isolated subcellular fractions from rabbit aorta and, measuring [l-14C]acetate incorporation, identified mitochondria as the major site of fatty acid synthesis. Filipovic and his co-workers [8] using calf arterial segments, found increased incorporation of [ l-‘4C]acetate into triglyceride fatty acids in hypoxia and proposed that mitochondrial fatty acid chain elongation was the predominant process. Howard [9] investigated fatty acid synthesis by subcellular fractions of monkey aorta and concluded that aorta is able to carry out both de nouo synthesis and elongation of fatty acids. Aortic high-speed supernatant extracts synthesized palmitic and stearic acids completely from malonyl-CoA. Both mitochondria and microsomes elongated fatty acids with acetyl-CoA, microsomes also elongated fatty acids with malonyl-CoA. In order to understand the reasons for the apparent increased synthesis of fatty acids, the mechanisms regulating fatty acid synthesis in the arterial wall must be clarified. We have undertaken an investigation of the fatty acid synthesizing systems in aorta in an effort to evaluate their contribution to arterial fatty acid metabolism. This paper describes the capacity of subcellular fractions of human aorta for fatty acid synthesis and reports the isolation and partial purification of fatty acid synthetase from chicken aorta and the characterization of its enzymic and immunological properties. Materials and Methods Chemicals
The 14C-labeled acyl-CoA derivatives were obtained from New England Nuclear. Unlabeled acyl-CoA derivatives were purchased from P.L. Biochemicals. All other chemicals were reagent grade and were obtained from local commercial sources. Collection
0 f tissue
Segments of human aorta were removed at postmortem examinations within 2 h of death and were collected into ice-cold normal saline. Aortas from freshly killed frying chickens were collected in the same way. Preparation
of subcellular
fractions
All manipulations were performed at 0-4°C. Adhering fat and connective tissue was removed and the intima-media portions of the aortas were rinsed in 0.05 M potassium phosphate, pH 7.4, containing 0.25 M sucrose, 10 n-&f 2-mercaptoethanol and 1 mM EDTA (homogenizing medium). The aortas were homogenized for 15 set at setting 10 with a Polytron ST-20 (Brinkman Instruments) in 5 volumes (v/w) of homogenizing medium. The homogenate was fractionated by differential centrifugation. After an initial centrifugation for 6 min at 800 X g to remove the bulk of the cell debris and collagen, the supernatant was centrifuged for 12 min at 7,700 X g. The resulting mitochondrial pellet was resuspended in homogenizing buffer and recentrifuged for 10 min at 6,000 X g and resuspended in a small volume of the same buffer. The post-mitochondrial
105
supernatant was centrifuged for 60 min at 105,000 X g and the microsomal pellet was resuspended in a small volume of the same buffer and used without further washing. Enzyme
assays
Fatty acid synthetase activity was measured routinely by incubating appropriate amounts of enzyme with 30 nmoles of acetyl-CoA and 56 nmoles of NADPH in the presence of 60 mM potassium phosphate, pH 7.0, 4 mM dithiothreitol. After a 5-min pre-incubation at room temperature (25” C), the reaction was started by adding 45 nmoles of malonyl-CoA bringing the final volume to 0.5 ml. The rate of NADPH oxidation was measured at 340 nm in an Aminco DW-2 spectrophotometer. A unit of enzyme activity is defined as the amount catalyzing the oxidation of 1 pmole of NADPH per min under these conditions. Fatty acid synthetase activity in the high speed supematant was also measured by a radioactive assay. The same incubation mixture was used except that [ 1,3-14C]malonyl-CoA (2 pCi/pmole) was substituted for malonyl-CoA, and amount of NADPH was increased to 500 nmoles. Incubations were performed for 20 min at room temperature and terminated by the addition of 0.2 ml of 5 M HCl. Radioactivity incorporated into fatty acids was determined by extracting the acidified mixture with redistilled pentane and counting an aliquot in toluene based liquid scintillation medium. Fatty acid elongation activity in mitochondria and microsomes was measured by the rate of incorporation of [l-“C] acetyl-CoA and [1,3-14C]malonylCoA respectively into endogenous fatty acids. The reaction mixture contained in a final volume of 0.5 ml, 25 pmoles potassium phosphate, pH 7.0, 2.5 pmoles MgC12, 5 pmoles ATP, and 0.5 mg protein. Incubations with microsomes contained in addition, 50 nmoles [1,3-14C]malonyl-CoA (2 pC!i/mole) and 2.5 pmoles NADPH, while mitochondrial incubations contained 50 nmoles [ 1-14C] acetyl-CoA (2 /&i/pmole) and 2.5 pmoles NADH. Reactions were performed at 37°C for 20 min and were terminated by the addition of 0.2 ml 10 N KOH. The mixture was saponified at 95°C for 20 min, cooled and acidified, and the fatty acids were extracted with pentane. After washing the extract, an aliquot was taken to dryness in a scintillation vial and the radioactivity was determined using toluene based scintillation medium. Preparation
of [‘4C]acetyl-labeled
fatty acid synthetase
Chicken aorta synthetase was dialyzed against 0.1 M sodium phosphate, pH 7.0, 1 mM EDTA, 1% 2-mercaptoethanol. Dialyzed synthetase (200 1-11,130 pg) was incubated for 2 min at room temperature with 37 nmoles of [ l-14C]acetyl-CoA in a final volume of 0.2 ml. The reaction was terminated by the addition of 2.5 pmoles of N-ethylmaleimide. To determine the radioactivity bound to the synthetase, the protein was precipitated by the addition of 1.0 ml of cold 5% aqueous perchloric acid. The precipitate was collected on a Millipore filter (25 mm, 0.45 pm) and washed with 30 ml of cold 5% aqueous perchloric acid. The filters were dissolved in scintillation vials with 0.1 ml 0.5 N NaOH and 1.0 ml methanol and counted in a dioxane based scintillation medium.
106 Sodium
dodecylsulphate
disc electrophoresis
The 14C-labeled synthetase was denatured by the addition of 20 ~1 of a solution containing 10% SDS and 100 mM iodacetamide to 200 ~1 of the enzyme solution. After incubation for 2 h at room temperature the samples were applied in duplicate (100 ~1) to SDS gels and electrophoresis was performed as described by Stoops et al. [lo]. The gels were stained with Coomassie blue, destained, photographed, densitometer scans were made, and finally cut into 2 mm sections. The sections were heated overnight at 70°C with 1.0 ml of 0.2 M NaOH in sealed scintillation vials. The extracts were acidified and radioactivity was determined using Triton X-100 : toluene scintillation medium. Identification of products products The radiolabeled
of fatty acid synthesis and elongation were identified as the fatty acid methyl esters. Esterification was performed by reaction with an ethereal solution of diazomethane. Separation and identification of the methyl esters was achieved on a 15% ethyleneglycolsuccinate column in a Varian Series ‘2700 gas chromatograph. Mass was detected using a flame ionization detector and radioactivity was monitored continuously with a Nuclear Chicago counting system. Separation of fatty acid methyl esters on the basis of unsaturation was performed using silver nitrate impregnated silica gel thin layer chromatography as described by Jones et al. [ 111. Decarboxylation of radiolabeled fatty acids was carried out by the method of Brady et al. [ 121.
Immunological
procedures
Goat antiserum against highly purified chicken liver fatty acid synthetase was partially purified by DEAE-cellulose chromatography. Ouchterlony double diffusion analysis was performed as described by Clausen [13] using commercially available agar plates (Hyland). Equivalence point determinations were performed as described by Kabat and Mayer [ 141. Radioactivity
and protein
determination
Radioactivity was determined using a Nuclear Chicago Mark II liquid scintillation system. Addition of a 14C toluene internal standard indicated a counting efficiency of 86--89%. Protein was measured by the method of Lowry et al. [15]. Results Fatty acid synthesis
in human aorta
Active lipid synthesis from [2-14C]acetate has been shown by Chobanian and Manzur [6] in normal intima and fatty streaks of human arterial segments. We extended our studies to determine the contribution towards fatty acid synthesis of the various subcellular fractions, and the effect of arteriosclerosis on the activities. Table 1 shows the incorporation of 14C-labeled acetyl CoA or malonyl CoA into fatty acids by mitochondria, microsomes and the supematant fraction of normal human aorta. The activit.ies of both de nouo synthesis and elongation
107 TABLE
I
RELATIVE CONTRIBUTIONS HUMAN AORTA
OF SUBCELLULAR
FRACTIONS
TO FATTY
ACID
SYNTHESIS
IN
Values are from duplicate measurements on fractions from necroscopy code 195. 20 year-old-male. Fatty acid synthesis was measured using either [l-14Clacetyl CoA or [1,3-14Clmalonyl CoA as described in Materials and Methods. Fatty acid production was calculated on the basis of an elongation mechanism for the particulate fractions and on de nouo synthesis of palmitic acid by the high speed supernatant. Fraction
Yield (mg protein/g wet weight aorta)
Fatty acid synthesis (nmoles fatty acid/g aorta/h)
High speed supernatant Microsomes Mitochondria
18.4 4.4 3.2
4.23 7.81 1.37
are lower than detected in chicken aorta, agreeing with the studies of Rao and Rao [16] who found that lipid synthesis in chick aorta was higher than in any other species. Nevertheless the results show that fatty acid biosynthesis in human aorta can contribute to the accumulation of aortic lipids. Chobanian [6] reported that lipid synthesis in the fatty streak region was greater than either normal intima or advanced atheromata. We determined the fatty acid synthetase activity in samples of human aorta with various degrees of fibrous-arteriosclerotic involvement. Table 2 lists the results obtained and it is apparent that fatty acid synthetase activity is lower in all lipid-atherosclerotic segments and even in adjacent areas from the same aorta. On the other hand segments with fibrous-arteriosclerotic lesions had near normal activities suggesting that either the fibrous plaques were the result of a different process or that they represented an earlier stage in the development of the atheromatous lesions. No significant difference in fatty acid synthetase activity could be found in this study between segments of thoracic or abdominal origin.
TABLE
2
EFFECT
OF ATHEROSCLEROSIS
ON FATTY
ACID SYNTHETASE
ACTIVITY
IN HUMAN
AORTAS
Necroscopy code
Age and sex
Type of lesion
Protein in HSS (mg/g wet weight of aorta)
acid synthetase Fatty (nmol/h/mg protein a)
195 195
20, M
none none
15.4 14.5
0.28 0.29
60 60
60. M
lipid lipid
19.5 17.5
0.06 0.06
fibrous lipid none
21.7 21.6 20.0
0.11 0.06 0.06
51 51 51
61. M
42
69, M
fibrous
18.6
0.26
196
70, F
lipid
13.8
0.13
202 202
88, F
lipid none
26.8 23.4
0.06 0.09
a BY [1,3-14Clmalonyl
CoA incorporation
(see Table 1).
108 TABLE
3
FATTY
ACID SYNTHESIS
BY SUBCELLULAR
Fatty acid synthesis was measured calculated assuming a mechanism production by the soluble fraction.
FRACTIONS
OF CHICKEN
as indicated under Materials and Methods. of elongation for the particulate fractions, Values are mean f SE of the mean.
AORTA Fatty acid production and de nouo palmitic
Fraction
Yield (mg protein/g wet weight aorta)
Fatty acid synthesis (nmoles fattty acid/g aorta/h)
High speed supernatant Microsomes Mitochondria
14.1 * 1.1 4.1 * 0.2 3.0 t 0.4
12.69 19.57 20.48
was acid
f 1.13 + 0.77 + 1.57
Fatty acid synthesis in chicken aorta Chicken aorta was selected for further studies on the nature of the fatty acid synthetase in aorta. Table 3 shows the fatty acid synthetase activity of the subcellular fractions and the yields of each obtained routinely. Conditions of activity determination were such that proportionality with time and added protein was observed. Assay requirements were generally similar to those reported in other tissues [17] and found in human aorta. Microsomal elongation of fatty acids required malonyl-CoA, ATP and NADPH, the requirement for NADPH being only partially replaced by NADH. In mitochondria, elongation is dependent on acetyl-CoA, ATP and NADH. NADPH does not substitute for NADH. Analysis of the products of elongation by microsomes indicated that 50% of the radioactivity was incorporated into long chain polyunsaturated fatty acids, 30% in mono- and dieneoic-acids, and 20% in saturated fatty acids of which C l&O compromised 80%. The mitochondrial system also incorporated radioactivity into a variety of saturated and unsaturated long chain fatty acids. In this case Cl8 was the shortest chain length detectable. The products of the soluble fatty acid synthesizing system in the crude extract were identified as being palmitic and stearic acids in the proportions 60% and 40% respectively. Decarboxylation analysis of product fatty acids supported the assumption that elon-
TABLE
4
ISOLATION Fraction
HSS b 1 2 3 4 5 6 7
OF FATTY
ACID SYNTHETASE
Protein
FROM
(mg)
Activity a (nmoI/min)
1960 1862 1281 102 20 8 4 2.5
3057 2646 878 534 165 199 203
a BY NADH oxidation. b High speed supernatant from 140 ammonium sulphate precipitation.
AORTA
Specific activity (nmoI/mg/min) 0.20 1.64 2.1 8.6 27 21 50 81
392
g chicken
CHICKEN
aorta
segments,
8 to 10 preparations
were combined
after
109
gation was the mode of synthesis in mitochondria and microsomes soluble fraction (crude extract) a de nouo mechanism was indicated
while in the [Ml.
Protein purification The results of the procedure are summarized in Table 4. Unless otherwise specified, all buffers contained 10% glycerol, 1 mM dithiothreitol and 1 n-J4 EDTA. The high speed supematant was fractionated by ammonium sulphate precipitation. The portion precipitating between 30% and 50% saturation was collected and redissolved in 0.05 M potassium phosphate, pH 7.4. The enzyme solution was stored at -80” C after freezing in liquid N2 (Fraction 1). When sufficient material had been accumulated (8-10 preparations) the stored solutions were thawed in ice-water, combined, and dialyzed against 0.05 M potassium phosphate, pH 7.4. Calcium phosphate gel (20 mg/ml) was added to the solution in the proportion of 1 mg gel/mg protein. The mixture was immediately centrifuged and the supematant retained (Fraction 2). After adjusting the ionic strength of the supematant to 0.03 M potassium phosphate, it was applied to a DEAEcellulose column 4 X 42 cm which had been equilibrated with 0.01 M potassium phosphate, pH 7.4. The column was washed with the same buffer and synthetase activity was eluted with a linear gradient of 500 ml each of 0.01 M and 0.25 M potassium phosphate pH 7.4. Fractions containing activity were pooled and an equal volume of saturated ammonium sulphate, pH 7.4 was added to precipitate the active protein. The precipitate was collected by centrifugation as before and redissolved in a minimum volume of 0.25 M potassium phosphate, pH 7.4 (Fraction 3). The solution (2 ml) was applied to a 2.5 X 30 cm column of Sepharose-6B (Pharmacia) equilibrated with 0.25 M potassium phosphate, pH 7.4. The enzyme was eluted with the same buffer, activity being eluted with a K,, of 0.14. The pooled activity was precipitated from 50% saturated ammonium sulphate as before, taken up in a small volume of 0.01 M potassium phosphate, pH 7.4, 10 mM dithiothreitol and dialyzed against the same buffer (Fraction 4). The dialyzed solution was applied to a 1.2 X 4.5 cm column of DEAE-Biogel (Bio-Rad) equilibrated with the 0.01 M potassium phosphate, pH 7.4, 10 m&I dithiothreitol. The column was washed with the same buffer and the enzyme was eluted with a linear gradient of 20 ml each of 0.01 M potassium phosphate, pH 7.4 and 0.20 M potassium phosphate, pH 7.4, both containing 10 mM dithiothreitol. Fractions containing activity were pooled and ammonium sulphate precipitation was performed as before (Fraction 5). This step was essential for the success of subsequent purification steps. The precipitated activity was redissolved in ml of 0.25 M potassium phosphate buffer, pH 7.0, 10 mM dithiothreitol and chromatographed on a 1.2 X 47 cm column of Sepharose-6B with the same buffer. Active fractions were pooled and ammonium sulphate precipitation was performed (Fraction 6). The enzyme was redissolved in 0.01 M potassium phosphate, pH 7.0, 10 m&Z dithiothreitol and dialyzed against the same buffer. The dialyzed solution was applied to a final 0.5 X 6 cm column of DEAE-Biogel and after washing with 0.01 M potassium phosphate, pH 7.0 activity was eluted by the stepwise
110
addition of 5 ml each of 0.05 M, phate, pH 7.0, all containing 10 highest activity were pooled and in 0.5 M potassium phosphate pH
0.10 M, 0.15 M and 0.20 M potassium phosmJ4 dithiothreitol. The tubes containing the the activity was precipitated and redissolved 7.0, 10 mM dithiothreitol (Fraction 7).
Requirements of the fatty acid syn thetase reaction The reaction was dependent on the presence of both acetyl- and malonylCoA. When acetyl-CoA was omitted from the assay system, the rate of malonyl-CoA dependent oxidation of NADPH was negligible compared to the rate observed in the presence of acetyl-CoA. Acetyl-CoA could not be replaced by butyryl-CoA as a primer, optimum levels of which gave rates only 7.5% of the control values. The aorta synthetase also showed a high degree of specificity towards NADPH as the hydrogen donor, the rates obtained with NADH being less than 10% of those in the presence of NADPH. Dithiothreitol was found to be an essential component of the assay system for maximum activity and was required throughout the purification and during storage for maintenance of activity. The activity of the aorta synthetase was inhibited by N-ethylmaleimide and iodoacetamide, further implying the involvement of sulphydryl groups in enzyme activity. Although not required for maximum activity in the assay system, EDTA was included in all buffers to protect the enzyme from heavy metal inhibition. Identification of products The 14C-labeled products from incubation of the purified enzyme with [1,3-14C]malonyl-CoA (see Methods) were able to be extracted from acid solu-
!O
I
0
IO
5 TIME
15 ( minutes
1
4
20
25
I
Fig. 1. Gas chromatograms of the radioactive products of purified aorta fatty acid synthetase. Authentic palmitic and stearic acids were added to the reaction products. Trace A represents the response of the mass detector, trace B the response of the radioactivity detector.
111
tion with pentane without prior treatment with alkali. The products were therefore free fatty acids and not acyl-CoA derivatives. Gas liquid radiochromatography of the products was carried out to identify the fatty acids synthesized. Almost all the radioactivity of the methylated material was coincident with authentic methylated carrier palmitic acid (Fig. 1). These results demonstrate that the major product of fatty acid synthetase is free palmitic acid. Enzymatic properties of chicken aorta synthetase The synthetase has a single, broad optimum of activity at pH 7.0 to 7.5 (Fig. 2). Figure 3 shows the increase in reaction rates measured at pH 7.0 as a function of increasing amounts of substrate. From double reciprocal plots (see insets) the apparent ‘K, values for NADPH, acetyl-CoA and malonyl-CoA are The reaction rate is 8.9 X 10” M, 25 X 10% M and 60 X 10” M, respectively. proportional to the amount of enzyme added and is linear for over 5 min under the assay conditions described. Molecular weight estimation SDS gel electrophoresis of [ 14C]acetyl-labeled aorta fatty acid synthetase gave the results shown in Fig. 4. Four major bands were seen, with the slowest migrating band corresponding to purified chicken liver synthetase. It was estimated that the aorta synthetase was approximately 10% pure in this preparation. That this band was in fact the aorta synthetase was demonstrated by the
30
v 20
IO
6.0
6.5
1.0
7.5
8.0
8.5
9.0
PH Fig. 2. Effect of pH on aorta fatty acid synthetase activity. The spectrophotometric used. The reaction rate (v) is expressed as nmol NADPH oxidized/min/ml.
assay system was
112
I MALONYL-co A I” M
IACm-CoAlpM
INADPHI”M
Fig. 3. Effect of substrate concentration on the activity of aorta fatty acid synthetase. Assay conditions were as described in Methods except that the malonyl-CoA, acetyl-CoA. and NADPH concentrations were varied as indicated. The reaction rate (v) is expressed as nmol NADPH oxidized/min/ml.
finding of all the 14C-label from acetyl and malonyl associated with this band. Assays of the binding capacity of the aorta synthetase preparation indicated that more than 70% of the possible protein-bound counts were recovered from the gel in the band. No radioactivity was detected in any other position. From a calibration curve of log (molecular weight) against relative mobility, the molecular weight of the aorta synthetase in SDS was estimated to be 220,000. Under non-dissociating conditions (see Protein Purification) the aorta synthe-
Fig. 4. Sodium dodecylsulphatc disc electrophoresis [I4 Clacetyl-labeled aorta fatty acid synthetase; (A) highly purified chicken liver syntbetase run under identical conditions; (B) aorta synthetase. The radioactivity associated with 2 mm slices tif gel (B) is shown above. Direction of migration was from left to right.
113
Fig. 5. Doilble diffusion precipitin reactions in agar of aorta (A). and liver (L). fatty acid synthetase. The center well coctained goat antiserum prepared against chicken liver fatty acid synthetase. The precipitin lines were stained with 0.5% Amido Black.
ENZYME ADDED I milliunits
I
Fig. 6. Immunochemical equivalence of aorta and liver fatty acid synthetase. Varying amounts of purified aorta (A) or liver (0) synthetase were incubated at 4’C with 20 ~1 of immune gammaglobulin fraction from goat serum. Fatty acid synthetase activity was measured in the supernatant solution after centrifugation of the incubation.
114
tase activity chromatographed on Sepharose-6B with a K,, of 0.12, a value similar to that obtained for the liver synthetase and corresponding to a molecular weight of about 500,000.
Immunological pi-operties The results of double diffusion analysis of the aorta synthetase and liver synthetase against antiserum to the chicken synthetase are shown in Fig. 5. The smooth continuum of the precipitin lines and the absence of spur formation indicates the cross reaction of homologous antigens, supporting the similarity between the synthetases from aorta and liver. Furthermore, identical equivalence points (Fig. 6) for the synthetases from both tissues were obtained on addition of increasing amounts of enzyme to a constant amount of antiserum against the liver enzyme. Discussion Arterial wall lipid metabolism has been the subject of renewed interest in the past decade, progress in this subject has been reviewed recently by Geer and his co-workers [19]. Whereat [20] has reviewed in detail the status of fatty acid biosynthesis in aorta. De nouo synthesis of saturated fatty acids by aorta has been reported in human, monkey and pigeon [4-6]. Although the system responsible has not been fully characterized, Howard showed that malonyl-CoA was the preferred substrate for de nouo fatty acid synthesis in high-speed supernatant fraction of monkey aorta [9]. The present results confirm that aorta is capable of both de nouo synthesis and elongation of fatty acids. We have purified the fatty acid synthesizing activity some 800-fold from the soluble fraction of aorta homogenates and have established that the enzyme closely resembles the fatty acid synthetase isolated from chicken liver. Essentially similar procedures for the purification were used, and although comparable specific activities were not reached this may be ascribed to the technical difficulties encountered with this tissue. The concentration of the enzyme (per mg soluble protein) is 200-fold lower in aorta than in liver. Furthermore the contaminating proteins remaining with the purified synthetase appear to exist, in the native state, as an aggregate since they can only be separated from the synthetase under dissociating conditions. The molecular weight of the aorta synthetase as determined by SDS electrophoresis (220,000) is identical to that of the liver enzyme and under non-dissociating conditions the two enzymes are eluted from Sepharose-6B at the same K,, (0.12, equivalent MW 450,000). These results support a model, developed from extensive studies with the liver enzyme [lo], in which the fatty acid synthetase is a dimer of MW 450,000 which dissociates in SDS into two polypeptides of MW 220,000. Further evidence of the close similarity of the two enzymes is provided by the antigenic homology shown on double diffusion analysis and equivalence point determination. The product of the synthesis is palmitic acid in both cases, and the assay requirements for the two enzymes are almost identical. The stability of the enzymes is remarkably similar, both requiring phosphate ion, dithiothreitol and glycerol for maintenance of activity.
115
The regulation of saturated fatty acid biosynthesis in liver and brain has been reviewed recently by Volpe and Vagelos [ 211. Immunochemical techniques have been used to study the content, synthesis, and degradation of fatty acid synthetase in these tissues in various nutritional states and during development [ 221. We intend to use the antigenic similarity discovered in this study to apply these sensitive techniques to the study of the effect of hormones, nutritional state, development, and atherosclerosis on the aorta’ fatty acid synthetase. These studies will give insight into the regulation of fatty acid synthesis in aorta and the reasons for the apparent increase in lipids associated with atherosclerotic involvement. References 1 Howard. Jr.. CF., Lipogenesis from glucose-2-l 4 C and acetate-l-’ 4 C in aorta, J. Lipid Res., 12 (1971) 725-730. 2 Howard, Jr., C.F., Aortic Iipogenesis during aerobic and hypoxic incubation. Atherosclerosis, 15 (1972) 359-369. 3 Lofland, Jr., H.B.. Mousy, D.M., Hoffman, C.W. and Clarkson, T.B.. Lipid metabolism in pigeon aorta during atherogenesis, J. Lipid Res., 6 (1965) 112-118. 4 St. Clair. R.W.. Lofland, Jr.. H.B. and Clarkson, T.B., Composition and synthesis of fatty acids in atherosclerotic aortas of the pigeon, J. Lipid Res., 9 (1968) 739-747. 5 LofIand, Jr., H.B., St. Cl&, R.W., Clarkson, T.B., Bullock. B.C., and Lehner, N.D.M.. Atherosclerosis in Cebus monkeys. Part 2 (Arterial metabolism), Exp. Molec. Path., 9 (1968) 57-70. 6 Chobanian, A.V. and Manzur, F.. Metabolism of lipid in the human fatty streak lesion, J. Lipid Res., 13 (1972) 201-206. 7 Whereat, A.F.. Fatty acid synthesis in cell-free system from rabbit aorta, J. Lipid Res., 7 (1966) 671677. 8 FiIipovic. I. and Buddecke, E., Increased fatty acid synthesis of arterial tissue in hypoxia, Europ. J. Biochem., 20 (1971) 587-592. 9 Howard, Jr., C.F., De now synthesis and elongation of fatty acids by subcellular fractions of monkey aorta, J. Lipid Res., 9 (1968) 254-261. 10 Stoops, J.K., Arslanian, M.J., Oh, Y.H.. Aune, K.C., Vanaman, T.C. and WakiI, S.J., Presence of two polypeptide chains comprising fatty acid synthetase, Proc. Nat. Acad. Sci. (Wash.), 72 (1975) 19401944. 11 Jones, P.D., Holloway. P.W., Peluffo. R.O. and WakiI. S.J., A requirement for lipids by the microsomal stearyl CoA desaturase, J. Biol. Chem., 244 (1969) 744-754. 12 Brady, R.O., Bradley, R.M. and Trams. E.G., Biosynthesis of fatty acids, Part 1 (Studies with enzymes obtained from liver). J. Biol. Chem., 235 (1960) 3093-3098. 13 Clausen, J., In: T.S. Work and E. Work (Eds.), Laboratory Techniques in Biochemistry and Molecular Biology, North-Holland. Amsterdam, 1972, pp. 399-572. 14 Kabat. E.A. and Mayer. M.M., Experimental Immunochemistry. 2nd edition, C.C. Thomas, Springfield, Ill., 1961. 15 Lowry. O.H., Rosebrough, N.J.. Farr, A.L., and Randall, R.J., Protein measurements with the FoIin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 16 Rae, A.R. and Rae. B.S.N.. Incorporation of [1-14C]acetate into the lipids of aortas of different species, J. Atheroscler. Res. 8 (1968) 59-67. 17 Seubert, W. and Podack, E.R.. Mechanisms and physiological roles of fatty acid chain elongation in microsomes and mitochondria, Molec. and Cellular Biochem., 1 (1973) 2940. 18 WakiI. S.J.. Lipid Metabolism, Academic Press, New York, N.Y., 1970. pp. 148. 19 Geer, J.C., Panganamala, R.V.. Newman, H.A.I. and CornwaII. D.G.. In: R.W. Wissler and J.C. Geer (Eds.), Pathogenesis of Atherosclerosis. American Association of Pathology and Bacteriology, 1971, pp. 200-213. 20 Whereat. A.F., In: R. Paoletti and D. Kritchevsky (Eds.), Advances in Lipid Research. Academic Press, New York. N.Y., 1971, pp. 119-159. 21 Volpe, J.J. and Vagelos. P.R. In: E.E. SneII (Ed.), Annual Review of Biochemistry, Annual Reviews Inc., Palo Alto, Cal., 42 (1973) 21-60. 22 Gibson, D.M., Lyons, R.T.. Scott. D.F. and Muto, Y.. In: G. Weber (Ed.), Advances in Enzyme Regulation, Vol. 10. Pergamon Press, New York, N.Y., 1972, pp. 187-204.