- Email: [email protected]
Fat metabolism in higher plants
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 189, No. 2, August, pp. 382-391, 1978
Fat Metabolism Characterization JOHN Department
of Plant Acyl-ACP
B. OHLROGGE, of Biochemistry
in Higher
WARD
and Acyl-CoA
E. SHINE,Z
and Biophysics,
Received December
Plants
University
AND
of California,
Hydrolases’ P. K. STUMPF Davis,
California
95616
16, 1977; revised March 3, 1978
Avocado mesocarp extracts contain both acyl-CoA and acyl-acyl carrier protein (ACP) hydrolase activities. These activities have been separated by a sequence of ammonium sulfate fractionations, DEAE-cellulose, and hydroxyapatite column chromatography. Two distinct acyl-CoA hydrolase fractions and one acyl-ACP hydrolase fraction were obtained. The acyl-ACP hydrolase fraction was essentially free of acyl-CoA hydrolase activity, had a pH optimum of 9.5 and a molecular weight of 70-80,000 based on sucrose density gradient centrifugation and gel filtration chromatography. Substrate specificity studies showed that lauroyl-ACP, myristoyl-ACP, palmitoyl-ACP, and stearoyl-ACP were slowly hydrolyzed but oleoyl-ACP was rapidly hydrolyzed to free fatty acid. These results suggest a possible role for acyl-ACP hydrolase as one component of a switching system which allows, indirectly, acyl transfer from ACP to CoA derivatives in plant cells.
Avocado mesocarp extracts contain both acyl-CoA and acyl-acyl carrier protein (ACP)” hydrolase activities (1). While, at present, the role of acyl-CoA hydrolases in plant lipid metabolism is not clear, we have suggested that the acyl-ACP hydrolase, with high specificity for oleoyl-ACP, functions as part of a switching system by converting a primary product of fatty acid synthesis, namely oleoyl-ACP, to free oleic acid which is then in turn transformed to oleoylCoA by an acyl-CoA thiokinase (1). The relevance of this two enzyme switching system which makes possible the transference of acyl moieties from ACP linked reactions to CoA linked reactions, e.g., desaturation of oleoyl-CoA to linoleoyl-CoA (2), transference of acyl-CoAs to suitable acceptors for phospholipid biosynthesis (3) and other activities such as /?-oxidation is evident. Thus the purpose of the present investiga’ This is paper No. 71 in a series. For paper No. 70, see Ref. 30. Supported in part by NSF grant #PCM7601495. ’ Present Address: Kesearrh Department, FritoLay Incorporated, 9(H) North Loop 12, Irving, TX 75060. ” Abbreviations used: ACP, acyl carrier protein; DTT, dithiothreitol. 382
0003-9861/78/1892-0382$02.00/O Copyrighl 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved
tion was to ascertain whether separate enzymes are responsible for catalyzing the hydrolysis of acyl-CoA and acyl-ACP thiolesters and if so to characterize the substrate specificity and properties of these enzyme activities. This paper will, therefore, describe the separation, partial purification and some properties of the acylACP hydrolase (EC 3.1.2.). In addition, data concerning two distinct acyl-CoA hydrolases will also be presented. EXPERIMENTAL
PROCEDURES
Reagents
Nonradioactive acyl-CoAs were obtained from P-L Biochemicals, Inc., Milwaukee, Wisconsin. [ I-“‘C]Lauroyl-CoA (58 Ci/mole) and [1-‘4C]palmitoyl-CoA (58 Ci/mole) were purchased from DHOM Products, North Hollywood, California, while [1-“ClmyristoylCoA (52 Ci/mole), [I-‘“Clstearoyl-CoA (62 Ci/mole), and [I-“CJoleoyl-CoA (55 Ci/mole) were obtained from New England Nuclear, Boston, Massachusetts. Purity of the acyl-CoA solutions was verified by comparing our absorbance ratios with those cited in catalog specifications. [ l-‘4C]Lauric acid (20 Ci/mole), [1-?]myristic acid (45 Ci/mole, [l-‘4C]paImitic acid (58 Ci/mole), [l-Y!]stearic acid (47 Ci/mole), and [l-14C]oleic acid (54 Ci/mole) were obtained from either New England Nuclear or Amersham-Searle and their purity checked
PLANT
ACYL
THIOESTER
before use by thin layer chromatography with appropriate solvent systems. Nucleotides, glucose 6-phosphate, glucose 6-phosphate dehydrogenase, and alkaline phosphatase were obtained from Sigma Co., St. Louis, Missouri. Protein concentration was estimated by the method of Bradford (4) with bovine serum albumin as the standard. Hydrolase
Assay System
Acyl-CoA and acyl-ACP hydrolases were routinely assayed at room temperature (20-23°C) at pH 8.5 in 0.1 M Tricine buffer. The reaction volume for acylCoA hydrolase assays was 0.5 ml in 13 x 100 mm test tubes and for acyl-ACP hydrolase 0.05 ml in 6 x 50 mm test tubes. The reaction was initiated by the addition of substrate and terminated after 5 min for acyl-CoA and IO-15 min for acyl-ACP by adding an equal volume of 1 M acetic acid in isopropanol containing 1 mM oleic acid. Free fatty acids were extracted with petroleum ether saturated with 50%>isopropanol and the radioactivity in the petroleum ether extract counted with 10 ml of PCS scintillation fluid (Amersham-Searle). Thin layer chromatography of the petroleum ether extract indicated that greater than 90% of the radioactivity migrated with free fatty acid. Under the above conditions the reaction proceded linearly with respect to time and amount of enzyme added. One unit of activity is defined as 1 nmol of substrate hydrolyzed per minute. Purification
of Acyl Hydrolases
Mesocarp tissue from four near-ripe avocados (Persea anierica var. Fuerte) was homogenized with a Brinkman Polytron at 4°C in 1 liter of 0.1 M sodium phosphate, pH 7.0, containing 10m4M 2-mercaptobenzothiazole. The homogenate was centrifuged 18,OOOg for 15 min and the supernatant poured through cheesecloth to remove the fat layer and mixed with 200 g acid washed polyvinylpolypyrrolidone (Sigma). This mixture was filtered through Miracloth and then brought to 25% ammonium sulfate saturation by adding solid ammonium sulfate while maintaining the pH at 7.1. After 15 min of stirring at 4’C, the suspension was centrifuged at. 15,000g for 10 min and the supernatant again separated from the fat layer by filtration through cheesecloth. The supernatant was then adjusted to 50% ammonium sulfate saturation with solid ammonium sulfate and after centrifugation, the pellet was resuspended in 50 ml 0.01 M sodium phosphate, pH 7.6, containing 1 mM EDTA and loo-* M 2-mercaptobenzothiazole. The suspension was dialyzed 5 h against 6 liters of the same buffer and then dialyzed 12 h against 5 mM sodium phosphate, pH 7.8, containing 0.5 x IO-’ M 2-mercaptobenzothiazole and 0.5 mM dithiothreitol. The 25-50% ammonium sulfate fraction was chromatographed on DEAE-cellulose (6.5~cm bed height) and eluted with a linear gradient from 5 mM
HYDROLASES
383
sodium phosphate, pH 7.8, plus 0.5 mM DTT to 0.1 M sodium phosphate, pH 7.8, plus 0.2 M KC1 and 0.5 mM DTT. Recovery of acyl-CoA and acyl-ACP hydrolase activities from the column was 56% and 570/o,respectively. Fractions 40-60 from DEAF,-cellulose were concen trated using Millipore immersible molecular separators and then dialyzed against 0.05 M sodium phosphate, pH 6.9 containing 1 mM DTT. This fraction was chromatographed on a 75-ml hydroxyapatite column eluted with a linear gradient from 0.05 M sodium phosphate, pH 6.9, plus 1 mM DTT to 0.3 M sodium phosphate, pH 6.9 plus 1 mM DTT. Recovery of acylACP and acyl-CoA activities from the hydroxyapatite column was 83% and 65% respectively. Molecular
Weight Estimations
Acyl-ACP hydrolase fractions with high activity were centrifuged in 6-23% sucrose density gradients for 19 h at 36,000 rpm in a Beckman SW 56 rotor at 2°C. Bovine alkaline phosphatase was included in each run as an internal molecular weight standard and assayed by the method of Torriani (5). The molecular weight was estimated from the relative sedimentation rates by the method of Martin and Ames (6). Molecular weights were also estimated by gel filtration using Sephacryl S-200 (Pharmacia) and Sepharose 6B in 1.0 x 50 cm and 1.5 x 90 cm columns, respectively, with cytochrome c, carbonic anhydrase, ovalbumin, and aldoiase as standards. Isolation
of Acyl Carrier
Protein
The concentration of acyl carrier protein was estimated by measuring the amount of [“‘C]malonyl-CoA transacylated to ACP-SH at equilibrium by the malonyl-CoA-ACP transacylase method of Alberts et al. (7) under conditions of excess malonyl-CoA relative to ACP-SH. Crude acyl carrier protein was prepared by trichloroacetic acid precipitation of the 100,OOOgsupernatant from an E. coti extract, resuspending the precipitate in phosphate buffer, adjusting to 70% saturation with ammonium sulfate, centrifuging and precipitating the supernatant with trichloroacetic acid. This precipitate was further purified by chromatography on DEAEcellulose and DEAE-Sephadex as described by Majerus et al. (8). Sodium dodecyl sulfate disk gel electrophoresis indicated that the purified fractions were at least 80% homogeneous. Analysis of the purified ACP by the method of Barron and Mooney (9) indicated the presence of approximately 5% acyl-ACP (mainly palmitic and vaccenic). Therefore, the ACP was further purified by binding to activated thiolSepharose 4B (Pharmacia). ACP-SH (1.0 pM) was incubated overnight at room temperature with 100 ml of activated thiol-Sepharose 4B in 0.1 M Tris-HCl pH 8 containing 1 mM EDTA and 0.3 M NaCl. The suspension was then poured into a column which was
384
OHLROGGE.
SHINE,
eluted first with 150 ml of the above buffer and second with 150 ml of the above buffer containing 50 mM cysteine (freshly prepared at pH 8.0). Fractions containing acyl carrier protein eluted by cysteine were pooled and concentrated by acid precipitation. Preparation
of Acyl-ACPs
Spinach stroma method. [‘?]Stearoyl-ACP was prepared by a modification of the method of Jaworski and Stumpf (10). Chloroplasts from the inner leaves of spinach prepared by the method of Kannangara et al. (11) were ruptured in a French press and centrifuged 100,OOOgfor 90 min. The supernatant was used as a source of fatty acid synthetase and was incubated with 35 pM [1,3-?I!]malonyl-CoA, 0.23 mM NADH, 0.21 rnM NADPH, 0.58 mM ATP, 0.56 mM glucose-6PO,, 1 unit glucose-6-POr dehydrogenase, 0.15 mM MgCl%, 0.08 mM MnCL, and 3-4 pM crude acyl carrier protein (approx. 10% pure). After incubation at 30°C for 10 min the reaction was stopped by adjusting the solution to 5% trichloroacetic acid. This mixture was centrifuged and the pellet resuspended in 0.1 M potassium phosphate buffer and the pH adjusted to 6.8 while solid ammonium sulfate was added to 70% saturation. This mixture was stirred at 4°C for 30 min and centrifuged 12,OOOgfor 15 min. The pellet was discarded and the supernatant protein was precipitated with 5% trichloroacetic acid and the pellet obtained after centrifugation was redissolved in 0.1 M potassium phosphate. After removing any undissolved protein by centrifugation, the supernatant protein was again precipitated and redissolved in a small volume of 0.01 M potassium phosphate, pH 7.0. Aliquots (0.1 ml) of this solution were then stored at -2O’C after adding just enough 1 M acetic acid to precipitate the protein. Prior to use in assaying acyl-ACP hydrolase activity just enough 0.1 M NaOH was added to redissolve the protein. This procedure gave approximately 50% conversion of [‘“Clmalonyl-CoA to [?]acyl-ACP. Unreacted ACP-SH was not removed from these preparations prior to use. The fatty acid composition of the acyl-ACP prepared by this method was determined by gas-liquid chromatography to be 81% stearic acid and 19% pahnitic acid. [‘4C]Palmitoyl-ACP (44 Ci/mole) was prepared similarly except [1-‘“Clacetate was the substrate and the spinach stroma fatty acid synthetase was pretreated at 37’C for 30 min which largely eliminated the elongation of palmitoyl-ACP to stearoyl-ACP (12). Gas chromatographic analysis of the product indicated 15% stearic and 15% lauric and myristic in addition to palmitoyl-ACP. [‘?]Oleoyl-ACP was prepared by enzymatic desaturation of the [‘4C]stearoyl-ACP prepared above. The reaction mixture contained in 10 ml: 0.4 PCi stearoylACP prepared above, 0.02 M sodium ascorbate, 0.07 M potassium phosphate, pH 6.0,40 X 19’units catalase,
AND
STUMPF
1 mM DDT, 900 pg ferredoxin, 10 I umts of partially purified stearoyl-ACP desaturase from Carthamus tinctorius (free of acyl-ACP hydrolase) and a photochemical reducing system consisting of 50 WM dichlorophenolindophenol and 50 pg/ml spinach chloroplast lamellae (10). This mixture was incubated at 24°C for 30 min in the light. [‘4C]Oleoyl-ACP (0.06 PCi) was iso!ated from the reaction mixture by the same method as described above for stearoyl-ACP and gas-liquid chromatography of this preparation indicated a fatty acid composition of 60% 18:1-ACP, 25% 16:0-ACP and 159 18:0ACP. Escherichia coli acyl-ACP synthetase method. ACP-SH was acylated with lauric, myristic and palmitic acids by the method of Ray and Cronan (13) using E. coli strain TR3 (generously provided by Allen Spencer of J. Cronan’s laboratory) as the source of enzyme. Endogeneous acyl-ACP-free ACP-SH was employed. The reaction mixture was incubated for 2 h at 37°C and terminated by addition of trichloroacetic acid. The acyl-ACP products were purified as described above for the preparation of crude ACP and then were separated from unreacted ACP-SH by passage through activated thiol-Sepharose and concentrated and desalted by ultrafiltration on Amicon UM02 membranes. Unreacted free fatty acid was separated from associated acyl-ACP by extraction with petroleum ether after acidifying with acetic acid. The acyl-ACP solution was then lyophilized and stored at -20°C until use. The conversions of lauric, myristic and palmitic acids to acyl-ACP by this procedure were approximately 10% 15% and lo%, respectively. The palmitoyl-ACP synthesized by this method gave rates of hydrolysis essentially identical to those observed with palmitoyl-ACP synthesized by the spinach stroma method described above. Preparation
of Coconut and Jojoba Extracts
Acetone powder extracts were prepared from developing coconut (Cocos nucifera) endosperm and developing jojoba (Simmondsia chinensis) cotyledons by blending the tissue 3 times in 10 vol of acetone at -20°C. The extract was then filtered and rinsed with diethyl ether and allowed to dry. The extracts (0.2 g) were resuspended in 5 ml of 0.1 M sodium phosphate, pH 7.5, plus 1 mM DTT before assaying for acyl-ACP hydrolase activity. RESULTS
Purification of Acyl-ACP Hydrolase The crude extract from avocado mesocarp contained approximately equal units of acyl-ACP and acyl-CoA hydrolase activity. Preliminary ammonium sulfate fractionations of avocado extracts indicated that of the total hydrolase activity re-
PLANT
ACYL
THIOESTER
covered in various fractions, 92% of the acyl-ACP hydrolase activity and 73% of the acyl-CoA hydrolase activity were found in the 25-50% ammonium sulfate fraction. This fraction was, therefore, applied to a DEAE-cellulose column and eluted with increasing concentrations of phosphate buffer and KC1 (Fig. 1). Two major peaks of acyl-CoA hydrolase activity and one peak of acyl-ACP activity were eluted from the column under these conditions. In Fig. 1 the acyl-ACP hydrolase peak appears to elute coincident to the second acyl-CoA hydrolase peak; however, in some runs, under similar conditions, the acyl-ACP hydrolase activity eluted just prior to the acylCoA hydrolase activity suggesting that both activities were not associated with the same protein. The first and second peaks of acyl-CoA hydrolase activity to elute from the DEAE-cellulose column will be referred to as acyl-CoA hydrolase I (CoA I) and acyl-CoA hydrolase II (CoA II), respectively. Acyl-CoA hydrolase I and acyl-CoA hydrolase II represented approximately 20% and 60% respectively of the total acylCoA hydrolase activity to elute from the DEAE-cellulose column. Fractions 40-60 from the DEAE-cellulose column were concentrated, dialyzed and further purified by hydroxyapatite chromatography which yielded a clear sep-
HYDROLASES
385
aration of two hydrolase peaks; the first contained acyl-CoA hydrolase II and the second acyl-ACP hydrolase (Fig. 2). A 360fold purification of acyl-ACP hydrolase activity had been achieved at this state. After hydroxyapatite chromatography the acylACP hydrolase activity was unstable. An approximately 30% decrease in activity was observed during 12 h storage at 4°C. This loss in activity was largely arrested after pooling and concentrating the active fractions. However, several attempts to purify further and characterize the hydroxyapatite fractions by gel filtration using either polyacrylamide or polydextran media led to loss of >95% of the activity. The purification attained through the hydroxyapatite stage is summarized in Table I. After the hydroxyapatite chromatography step the purified acyl-ACP hydrolase cleaved oleoyl-ACP at a rate approximately lo-fold greater than oleoyl-CoA. The actual specificity of the acyl-ACP hydrolase for acyl carrier protein thioesters may be even greater since there was a slight tailing of the acyl-CoA hydrolase activity into the acyl-ACP hydrolase peak. Molecular Weight of Acyl-ACP Hydrolase Although gel filtration of the more purified fractions was unsuccessful, gel filtration of the 25-50% ammonium sulfate frac-
FIG. 1. DEAE-cellulose chromatography of the 25-50% ammonium sulfate fraction from avocado mesocarp. Approximately 250 mg of protein was applied to a 6.5 X 3 cm column and eluted with a 2-liter linear gradient from 5 mM sodium phosphate, pH 7.8, plus 0.5 mM DTT to 0.1 M sodium phosphate, pH 7.8 plus 0.2 M KC1 and 0.5 mM DTT. The flow rate was 2-3 ml/min and 20-25 ml fractions were collected. B----M, Acyl-ACP hydrolase activity (nmoles/min/ml enzyme); A--k, acyl-CoA hydrolase activity (nmoles/min/ml enzyme); W, protein concentration (pg/ml).
386
OHLROGGE,
SHINE,
AND
..-. ! ! ! ’ ! i !i.
.-?
.I
,A*,?I I* ‘: #t \; ;. , .w . .-;:t,
/ ! i ! i ! i ! i I .;. i’ !I i k;**., !i i i is’ i
,‘f
l
STUMPF
\
‘\. \ -0.~
. .
. . I\\.,
d
a
QIQ20¶040~6070U)9a
iraction
ltumbrr
FIG. 2. Hydroxyapatite chromatography of DEAE fractions 40-60. After concentration and dialysis, fractions 40-60 from the DEAE column (Fig. 1) were applied to a 10 x 3 cm hydroxyapatite column and eluted with a 600 ml linear gradient from 0.05 M sodium phosphate, pH 6.9, plus 1 mM DTT to 0.3 M sodium phosphate, pH 6.9 plus 1 mM DTT. The flow rate was 1 mf/min and 6 ml fractions were collected. W, Acyl-ACP hydrolase activity (nmoles/min/ml enzyme); W, acyl-CoA hydrolase activity (nmoles/min/ml enzyme). TABLE I PARTIAL PURIFICATION OF ACYL-ACP HYDROLASE~ Total Procedure Specific Yield Puriticaunits activity tion (units/ mo g mesomg protein) carp) Crude extrct 25-50s (NH,),SO, DEAE-cellulose Hydroxyapatite
80 43
0.42 0.53
100 53
1.0 1.3
24
7.30
34
17.0
21
150.00
28
360.0
n Unit = 1 nmol of l&O-ACP hydrolyzed
per min.
tion did yield essentially 100% recovery of acyl-ACP hydrolase activity (Fig. 3). Calculation of the apparent molecular weight from these data gave a value of 70-80,000. The molecular weight of the acyl-ACP hydrolase was also estimated by sucrose density gradients of the protein at three stages of purification; 25-50s ammonium sulfate fraction, DEAE-cellulose purified and hydroxyapatite purified. Each stage of purification resulted in calculation of an apparent molecular weight between 70-75,000. Molecular Weights of Acyl-CoA Hydrolases Gel filtration of the first acyl-CoA hydrolase peak to elute from DEAE-cellulose gave an apparent molecular weight of 200,000.
Gel filtration of fractions 40-60 from the DEAE-cellulose column gave an apparent molecular weight of 70-80,000 for acyl-CoA hydrolase II. pH Activity
Profiles
Each hydrolase could be distinguished by a different pH-activity profile (Fig. 4). The acyl-ACP hydrolase had a pH optimum of 9.5 (Fig. 4a) while the acyl-CoA hydrolase II displayed a pH optimum at 8.4 to 8.6. The activity of acyl-CoA hydrolase I appeared to increase over the entire pH range of 5.0 to 10.5 although with the phosphate and tricine buffers decreases occurred at the upper end of their buffering capacity (perhaps suggesting an inhibition of this enzyme by anions). Measurements of activity above pH 10.5 were difficult due to nonenzymatic hydrolysis of substrate. Thermal
Stability
Acyl-CoA hydrolase II and acyl-ACP hydrolase could be distinguished by marked differences in their thermal stability. AcylCoA hydrolase II lost approximately 75% of its activity after 20 minutes incubation at 50°C whereas the acyl-ACP hydrolase lost no activity by this treatment (Fig. 5). Sensitivity of Hydrolases to Agents Neither acyl-CoA hydrolase I CoA hydrolase II was inhibited by of the products of the reaction,
Various or acyladdition namely,
PLANT
ACYL
THIOESTER
HYDROLASES
387
FIG. 3. Gel filtration of acyl-ACP hydrolase activity. The 2540% ammonium sulfate fraction (0.5 ml) was applied to a 1.0 x 50 cm column of Sephacryl S-200 (Pharmacia). The column yas eluted at room temperature with 0.1 M Tricine, pH 7.5, plus 0.5 mu DTT at a flow rate of 0.08 ml/min. Cytochrome c (0.2 mg) was included with the sample as an internal molecular weight standard and is responsible for the final peak in protein concentration. In the insert the K., calculated for the standards, aldolase (A), ovalbumin (W), and cytochrome c (0) are plotted versus their molecular weights. The Kav calculated for the acyl-ACP hydrolase is indicated by the arrow. W AcylACP hydrolase activity (nmoles/min/ml enzyme). O---4 Protein concentration (pg protein/ml).
CoASH or free fatty acid (Table II). In contrast, the acyl-ACP hydrolase was inhibited by its product, free ACP. Each hydrolase could be further distinguished by its sensitivity to the various active site reagents listed in Table II. These data lend furt,her support that each hydrolase activity is associated with a separate protein rather than to introconvertable forms of the same protein. The sensitivity of the acyl-ACP hydrolase to the sulfhydryl reagents (N-ethylmaleimide, iodoacetaand 5,5’-dithiobis-(2-nitrobenzoic mide acid) but not to the active serine reagent, phenylmethylsulfonylfluoride suggests that this hydrolase may be a sulfhydryl type esterase. Acyl-CoA hydrolase I was remarkably resistant to reagents to which acylCoA hydrolase II was reactive. Inhibition by ACP-SH The inhibition of the acyl-ACP hydrolase noted in Table II was further investigated. Since (as described under Experimental Procedures) our preparations of acyl-ACP contained low levels of free ACP-SH it was necessary in inhibition studies to use acylACP preparations containing no free ACP. In addition, we observed that our preparations of ACP-SH from E. coli were contam-
inated with approximately 5% acyl-ACP. Therefore, both acyl-ACP and ACP-SH were further purified by binding to activated thiol sepharose. While acyl-ACP does not bind to the column and is washed out, ACP-SH is eluted with cysteine. With increasing concentrations of ACPSH, we did not observe straight line Dixon plots suggesting that ACP-SH is not inhibiting in a simple competitive manner with substrate. Insufficient amounts of substrate free of ACP-SH prevented a more detailed kinetic analysis of the ACP-SH inhibition. Substrate Specificity The specificity of the acyl-ACP hydrolase with acyl-ACP substrates having different acyl chains was evaluated. The reaction rate increased with increasing chain length from 12 to 18 carbons (Fig. 6a). In addition, as had been observed previously with crude extracts of spinach, avocado and safflower (l), the purified acyl-ACP hydrolase displayed a marked selectivity for oleoyl-ACP relative to the saturated acylACPs. Insufficient quantities of the acylACP substrates prevented an accurate determination of K, values for the different substrates. However, we estimate that the K,‘s for palmitoyl, stearoyl, and oleoyl-
388
OHLROGGE,
04 ’ 5
I
9
SHINE,
II
PH FIG. 4. pH Activity profiles: Assays were with 2 stearyl-ACP or stearyl-CoA. Buffers used were succinate (A), phosphate (O), Tricine (B), and glycine (A); each adjusted to the pH with NaOH or HCl. Nonenzymatic hydrolysis at the higher pH’s has been subtracted. (a) Acyl-ACP hydrolase activity from DEAE-cellulose fractions 40-60 (Fig. 1). (b) Acyl-CoA hydrolase I activity from DEAE-cellulose fraction 3 (Fig. 1). (c) Acyl-CoA hydrolase II activity from DEAE-cellulose fractions 40-60 (Fig. 1). PM
ACP are below 0.2 PM and thus the relative rates of hydrolysis shown in Fig. 6a at a concentration of 1 PM are approximate V,,, comparisons. When the acyl-ACP hydrolase was incubated with a combination of 1 FM oleoyl and 1 pM stearoyl-ACP the rate of hydrolysis was approximately an average of the two hydrolysis rates rather than a sum. Thus, it is unlikely that these two substrates react at separate enzyme active sites. We also investigated the substrate specificity of hydrolysis of acyl-ACP substrates by two plant systems which differed mark-
AND
STUMPF
edly in the chain length of fatty acids synthesized in these tissues. We chose coconut (Cocos nuciferu) which produces predominantly 12 and 14 carbon fatty acids and the developing jojoba seed (Simmondsia chinensis) which synthesizes 20 and 22 carbon fatty acids. Figure 6, b and c, indicates that the specificity of acyl-ACP hydrolysis by extracts from these two tissues is very similar to that observed with avocado. Thus, from these data we cannot attribute the difference in fatty acid chain length synthesized by these plants to different acyl-ACP hydrolase specificities. In contrast to the acyl-ACP hydrolase, the two acyl-CoA hydrolases of avocado had a broader specificity and did not show as great a preference for the oleoyl derivative. Compariative reaction rates with the various substrates are shown in Fig. 6, d and e. It is well known that long chain acylCoAs form micelles at critical concentrations (usually at the 2-5 PM level), and adsorb strongly to proteins and at the airwater interphase (14). Thus, the concentrations cited in Fig. 6 represent only the amounts added to the reaction mixture and not the actual concentration in the bulk phase. For these reasons substrate versus velocity plots did not allow accurate K,,, determinations. However, in general, the relative order of reactivity of the acyl-CoA substrates shown in Fig. 6 did not change
-. 20
x..erer-.-A 0
20
&OS
at 50?“c
FIG. 5. Thermal stability. Aliquots of fractions 40-60 from the DEAE-cellulose purification (Fig. 1) were heated at 50°C for the times indicated and then assayed with either 2 PM l&O-ACP (u) or 2 PM l&O-CoA (A-,--A).
PLANT
ACYL
THIOESTER TABLE
INHIBITION
OF ACYL
THIOESTER
HYDROLASES II
HYDROLASES
BY DIFFERENT
Concentration
Reagent
Iodoacetamide acid)
0.90
1.0 rnM
5.0mM 0.3mM
PhenyImethyIsulfonylfluoride
0.8 mM 10 PM 50 PM 1.0 /.iM 5.0 PM 3.0 PM 15.0 pLM 1% 5% 0.001% 0.005%
CoASH acid
ACP-SH Isopropanol Tergitol
0.96
1.0 rnM
55.Dithiobis-(2-nitrobenzoic
Patmitic
1.0 rnM
5.0mM 5.0mM
15-S-9
” The first four inhibitors for hydrolase activity.
were preincubated
REAGENTS”
Rate relative Acyl-CoA hydrolase I
N-Ethylmaleimide
389
1.0
0.92 1.0 1.0 1.0 0.96 1.1 1.1 1.0 1.0 1.4 0.90 1.1 1.4 0.73 0.44
to control
Acyl-CoA hydrolase II 0.32 0.23 0.65 0.48 0.74 0.48 0.71 0.60 1.0 1.0 I.0 1.0 0.77 0.67 0.90 0.37 0.90 0.42
with enzyme for 1 h and then diluted lo-20.fold
over the concentration range of 0.1 to 10 PM. Hydrolysis of either acyl-CoA or acylACP was not inhibited by addition of phospholipids or triglycerides. Thus it is unlikely that the activity we are reporting is due to a non-specific phospholipid or triglyceride lipase. DISCUSSION
The chain length of fatty acids synthesized by various fatty acid synthetases has been shown to be a function of the concentration and activity of a wide variety of substrates, cofactors and enzymes (15-22). Thus it is unlikely that the function of controlling fatty acid chain length can be attributed to a single enzyme activity. In several organisms acyl hydrolases have been proposed as controlling elements in determining the chain length of fatty acids synthesized (15-17). Flick and Bloch (15) reported that addition of an acyl thioester hydrolase to a fatty acid synthetase from Mycobacterium phlei roughly doubled the proportion of short chain acids synthesized. Similarly, Knudsen et al. (16) have recently shown that addition of a purified medium
Acyl-ACP hydrolase 0.60 0.15 0.26 0.80 1.0 1.0 1.0 1.0 1.0 1.0 0.80 0.50 1.0 0.73 1.1 1.0 before assaying
chain acyl hydrolase isolated from rabbit mammary gland to the mammary fatty acid synthetase resulted in a decrease in chain length of the fatty acids produced. In addition, Agradi et al. (17) showed that removal by trypsin cleavage of thioesterase from a multienzyme fatty acid synthetase led to an increase in fatty acid chain length. In contrast, Cronan et al. (18) have proposed that, for E. coli, acyl transfer into phospholipid is a major determinant of fatty acid chain length although previous investigations of the /I-ketoacyl-ACP synthetase indicated that this enzyme’s specificity is also consistent with the pattern of fatty acids synthesized (19). Sumper et al. (21) proposed a model for the yeast fatty acid synthetase which described the pattern of fatty acids synthesized as a function of the relative rates of the condensation reaction versus acyl transfer to coenzyme A. Since the principal fatty acids in most plants are the Cl8 unsaturated acids (221, a mechanism must be at hand to guarantee the biosynthesis of these acids. We would like to suggest that the predominance of these Cl8 fatty acids (oleic, linoieic, and
390
OHLROGGE,
SHINE,
AND
STUMPF
80. cdl I g
4
Ml
.i
C. lojoba
-II
12:o 14% l&O IBE0 l&l
f 2 aa .E z 22
0
12:0 14:0 16:0 18:O 183 E. 8 CoAII
4
'
Lll
12:O l4:O 16:O 18:O 18:l
FIG. 6. Substrate specificity of acyl thioester hydrolases. (A-C) Relative hydrolase activity with acyl-ACP substrates. Substrates were prepared as described under Experimental Procedures and assayed at a concentration of 1 pM with the DEAE-cellulose purified avocado enzyme or with acetone powder from coconut and jojoba. (D) Specificity of acyl-CoA hydrolase I from avocado. Substrates at a concentration of 1 pM were assayed with enzyme from fraction 3 of DEAE-cellulose (Fig. 1). (E) Specificity of acyl-CoA hydrolase II from avocado. Substrates at a concentration of 1 PM were assayed with enzyme from fractions 40-60 of DEAE-cellulose (Fig. 1).
linolenic) is the result of the coordinated action of a series of enzyme systems all employing ACP as the thioester moiety. Thus, the synthesis of palmitoyl-ACP from acetyl-ACP and malonyl-ACP, the specific conversion of palmitoyl-ACP to stearoylACP by the palmitoyl-ACP elongase (12), the highly specific desaturation of stearolyACP to oleoyl-ACP by stearoyl-ACP desaturase (10) and the specific hydrolysis of oleoyl-ACP to free oleic acid by the acylACP hydrolase guarantee the formation of oleic acid which then can be converted to oleoyl-CoA for further modifications (2). Thus, chain termination of fatty acid biosynthesis in plants may result from the specificity of each of the above enzymes which act in concert to assure the predominance of 18 carbon fatty acids in most plants. Intriguing is the observation that this oleate specific acyl-ACP hydrolase also occurs in plants which differ markedly in
the chain length of the major fatty acids synthesized (Fig. 6). We are currently investigating what other mechanisms of chain termination might account for these different fatty acid compositions. ACKNOWLEDGMENTS We wish to thank Don Andrews for excellent technical assistance, Ms. Barbara Clover for help with the preparation of this paper and Dr. Tom McKeon for purified stearoyl-ACP desaturase. REFERENCES 1. SHINE, W. E., MANCHA, M., AND STUMPF, P. K. (1976) Arch. Biochem. Biophys. 172, 110-116. 2. VIJAY, I. K., AND STUMPF, P. K. (1971). J. Biol. Chem.
246,2910-2917.
3. SHINE, W. E., MANCHA, M., AND STUMPF, P. K. (1976) Arch. Biochem. Biophys. 173, 472-479. 4. BRADFORD, M. M. (1976) Anal. Biochem. 72, 248-254. 5. TORRIANI, A. (1966) in Procedures
Research
(Cantoni,
in Nucleic Acid G. L., and Davies, D. R.,
PLANT eds.),
pp. 224-234,
Harper
& Row,
ACYL New
6. MARTIN, R. G., AND AMMES, B. N. (1961) Chem. 236,1372-1379.
7.
8.
9. 10. 11.
12.
13.
THIOESTER York.
R. E., AND CLELAND,
W. W. (1969)
them. Biophys. Res. Commun. 34,555-559.
P. K., AND BLOCH, K. (1974) J. Biol. Chem.
249, 1031-1036. 16. KNUDSEN,
Nat. Acad. Sci. 73,4374-4378. 14. BARDEN,
15. FLICK,
J. Biol.
ALBERTS, A. W., MAJERLJS, P. W., TALAMO, B., 3, AND VAGELOS, P. R. (1964) Biochemistry 1563-1571. MAJERUS, P. W., ALBF.RTS, A. W., AND VAGELOS, P. R. (1969) in Methods of Enzymology (Lowenster, J. M., ed.), Vol. 14, pp. 43-50, Academic Press, New York. BAHRON, E. J., AND MOONEY, L. A. (1968) Anal. Chem. 40, 1742-1744. JAWORSKI, J. G., AND STUMPF, P. K. (1974) Arch. Biochem. Biophys. 162, 166-173. KANNANGAHA, C. G., JACOBSON, B. S., AND Plant Physiol. 52, STUMPF, P. K. (1973) 156-161. JAWORSKI, J. G., GOLDSCHMIDT, E. E., AND STUMPF, I’. K. (1974). Arc/z. Biochem. Biophys. 163, 769-776. RAY, T. K., AND CRONAN, J. E., JR. (1976) Proc.
Bio-
391
HYDROLASES
J., CLARK,
S., AND
DILS,
R.
(1976)
Biochem. J. 160,683-691. 17. AGRADI,
E., LIBERTINI,
Biochem.
E., AND SMITH,
S. (1976).
Biophys.
Res. Commun. 68, 894-900. 18. CRONAN, J. E., JR., WEISBERG, L. J., AND ALLEN, R. G. (1975) J. Biol. Chem. 250,5835-5840. 19. GREENSPAN, M. D., BIRGE, C. H., POWELL, G., HANCOCK,
W. S., AND VAGELOS,
P. R. (1970)
Science 170, 1203-1204. 20. BARDEN, R. E., AND CLELAND, W. W. (1969) Biol. Chem. 244, 3677-3684.
J.
21. SUMPER, M., OESTERHELT, D., RIEPERTINGER, C., AND LYNEN, F. (1969) Eur. J. Biochem. 10,
377-387. 22.
HUANG,
K. P., AND STUMPF, P. K. (1971) 143,412-427.
Arch.
Biochem. Biophys.
23. HITCHCOCK, C., AND NICHOLAS, B. W. (1971) Plant York.
Lipid
Biochemistry,
Academic
in Press, New
30. SHINE, W. E., MANCHA, M., AND STUMPF, P. K. (1976) Arch. Biochem. Biophys. 173, 742-749.
Fat Metabolism Characterization JOHN Department
of Plant Acyl-ACP
B. OHLROGGE, of Biochemistry
in Higher
WARD
and Acyl-CoA
E. SHINE,Z
and Biophysics,
Received December
Plants
University
AND
of California,
Hydrolases’ P. K. STUMPF Davis,
California
95616
16, 1977; revised March 3, 1978
Avocado mesocarp extracts contain both acyl-CoA and acyl-acyl carrier protein (ACP) hydrolase activities. These activities have been separated by a sequence of ammonium sulfate fractionations, DEAE-cellulose, and hydroxyapatite column chromatography. Two distinct acyl-CoA hydrolase fractions and one acyl-ACP hydrolase fraction were obtained. The acyl-ACP hydrolase fraction was essentially free of acyl-CoA hydrolase activity, had a pH optimum of 9.5 and a molecular weight of 70-80,000 based on sucrose density gradient centrifugation and gel filtration chromatography. Substrate specificity studies showed that lauroyl-ACP, myristoyl-ACP, palmitoyl-ACP, and stearoyl-ACP were slowly hydrolyzed but oleoyl-ACP was rapidly hydrolyzed to free fatty acid. These results suggest a possible role for acyl-ACP hydrolase as one component of a switching system which allows, indirectly, acyl transfer from ACP to CoA derivatives in plant cells.
Avocado mesocarp extracts contain both acyl-CoA and acyl-acyl carrier protein (ACP)” hydrolase activities (1). While, at present, the role of acyl-CoA hydrolases in plant lipid metabolism is not clear, we have suggested that the acyl-ACP hydrolase, with high specificity for oleoyl-ACP, functions as part of a switching system by converting a primary product of fatty acid synthesis, namely oleoyl-ACP, to free oleic acid which is then in turn transformed to oleoylCoA by an acyl-CoA thiokinase (1). The relevance of this two enzyme switching system which makes possible the transference of acyl moieties from ACP linked reactions to CoA linked reactions, e.g., desaturation of oleoyl-CoA to linoleoyl-CoA (2), transference of acyl-CoAs to suitable acceptors for phospholipid biosynthesis (3) and other activities such as /?-oxidation is evident. Thus the purpose of the present investiga’ This is paper No. 71 in a series. For paper No. 70, see Ref. 30. Supported in part by NSF grant #PCM7601495. ’ Present Address: Kesearrh Department, FritoLay Incorporated, 9(H) North Loop 12, Irving, TX 75060. ” Abbreviations used: ACP, acyl carrier protein; DTT, dithiothreitol. 382
0003-9861/78/1892-0382$02.00/O Copyrighl 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved
tion was to ascertain whether separate enzymes are responsible for catalyzing the hydrolysis of acyl-CoA and acyl-ACP thiolesters and if so to characterize the substrate specificity and properties of these enzyme activities. This paper will, therefore, describe the separation, partial purification and some properties of the acylACP hydrolase (EC 3.1.2.). In addition, data concerning two distinct acyl-CoA hydrolases will also be presented. EXPERIMENTAL
PROCEDURES
Reagents
Nonradioactive acyl-CoAs were obtained from P-L Biochemicals, Inc., Milwaukee, Wisconsin. [ I-“‘C]Lauroyl-CoA (58 Ci/mole) and [1-‘4C]palmitoyl-CoA (58 Ci/mole) were purchased from DHOM Products, North Hollywood, California, while [1-“ClmyristoylCoA (52 Ci/mole), [I-‘“Clstearoyl-CoA (62 Ci/mole), and [I-“CJoleoyl-CoA (55 Ci/mole) were obtained from New England Nuclear, Boston, Massachusetts. Purity of the acyl-CoA solutions was verified by comparing our absorbance ratios with those cited in catalog specifications. [ l-‘4C]Lauric acid (20 Ci/mole), [1-?]myristic acid (45 Ci/mole, [l-‘4C]paImitic acid (58 Ci/mole), [l-Y!]stearic acid (47 Ci/mole), and [l-14C]oleic acid (54 Ci/mole) were obtained from either New England Nuclear or Amersham-Searle and their purity checked
PLANT
ACYL
THIOESTER
before use by thin layer chromatography with appropriate solvent systems. Nucleotides, glucose 6-phosphate, glucose 6-phosphate dehydrogenase, and alkaline phosphatase were obtained from Sigma Co., St. Louis, Missouri. Protein concentration was estimated by the method of Bradford (4) with bovine serum albumin as the standard. Hydrolase
Assay System
Acyl-CoA and acyl-ACP hydrolases were routinely assayed at room temperature (20-23°C) at pH 8.5 in 0.1 M Tricine buffer. The reaction volume for acylCoA hydrolase assays was 0.5 ml in 13 x 100 mm test tubes and for acyl-ACP hydrolase 0.05 ml in 6 x 50 mm test tubes. The reaction was initiated by the addition of substrate and terminated after 5 min for acyl-CoA and IO-15 min for acyl-ACP by adding an equal volume of 1 M acetic acid in isopropanol containing 1 mM oleic acid. Free fatty acids were extracted with petroleum ether saturated with 50%>isopropanol and the radioactivity in the petroleum ether extract counted with 10 ml of PCS scintillation fluid (Amersham-Searle). Thin layer chromatography of the petroleum ether extract indicated that greater than 90% of the radioactivity migrated with free fatty acid. Under the above conditions the reaction proceded linearly with respect to time and amount of enzyme added. One unit of activity is defined as 1 nmol of substrate hydrolyzed per minute. Purification
of Acyl Hydrolases
Mesocarp tissue from four near-ripe avocados (Persea anierica var. Fuerte) was homogenized with a Brinkman Polytron at 4°C in 1 liter of 0.1 M sodium phosphate, pH 7.0, containing 10m4M 2-mercaptobenzothiazole. The homogenate was centrifuged 18,OOOg for 15 min and the supernatant poured through cheesecloth to remove the fat layer and mixed with 200 g acid washed polyvinylpolypyrrolidone (Sigma). This mixture was filtered through Miracloth and then brought to 25% ammonium sulfate saturation by adding solid ammonium sulfate while maintaining the pH at 7.1. After 15 min of stirring at 4’C, the suspension was centrifuged at. 15,000g for 10 min and the supernatant again separated from the fat layer by filtration through cheesecloth. The supernatant was then adjusted to 50% ammonium sulfate saturation with solid ammonium sulfate and after centrifugation, the pellet was resuspended in 50 ml 0.01 M sodium phosphate, pH 7.6, containing 1 mM EDTA and loo-* M 2-mercaptobenzothiazole. The suspension was dialyzed 5 h against 6 liters of the same buffer and then dialyzed 12 h against 5 mM sodium phosphate, pH 7.8, containing 0.5 x IO-’ M 2-mercaptobenzothiazole and 0.5 mM dithiothreitol. The 25-50% ammonium sulfate fraction was chromatographed on DEAE-cellulose (6.5~cm bed height) and eluted with a linear gradient from 5 mM
HYDROLASES
383
sodium phosphate, pH 7.8, plus 0.5 mM DTT to 0.1 M sodium phosphate, pH 7.8, plus 0.2 M KC1 and 0.5 mM DTT. Recovery of acyl-CoA and acyl-ACP hydrolase activities from the column was 56% and 570/o,respectively. Fractions 40-60 from DEAF,-cellulose were concen trated using Millipore immersible molecular separators and then dialyzed against 0.05 M sodium phosphate, pH 6.9 containing 1 mM DTT. This fraction was chromatographed on a 75-ml hydroxyapatite column eluted with a linear gradient from 0.05 M sodium phosphate, pH 6.9, plus 1 mM DTT to 0.3 M sodium phosphate, pH 6.9 plus 1 mM DTT. Recovery of acylACP and acyl-CoA activities from the hydroxyapatite column was 83% and 65% respectively. Molecular
Weight Estimations
Acyl-ACP hydrolase fractions with high activity were centrifuged in 6-23% sucrose density gradients for 19 h at 36,000 rpm in a Beckman SW 56 rotor at 2°C. Bovine alkaline phosphatase was included in each run as an internal molecular weight standard and assayed by the method of Torriani (5). The molecular weight was estimated from the relative sedimentation rates by the method of Martin and Ames (6). Molecular weights were also estimated by gel filtration using Sephacryl S-200 (Pharmacia) and Sepharose 6B in 1.0 x 50 cm and 1.5 x 90 cm columns, respectively, with cytochrome c, carbonic anhydrase, ovalbumin, and aldoiase as standards. Isolation
of Acyl Carrier
Protein
The concentration of acyl carrier protein was estimated by measuring the amount of [“‘C]malonyl-CoA transacylated to ACP-SH at equilibrium by the malonyl-CoA-ACP transacylase method of Alberts et al. (7) under conditions of excess malonyl-CoA relative to ACP-SH. Crude acyl carrier protein was prepared by trichloroacetic acid precipitation of the 100,OOOgsupernatant from an E. coti extract, resuspending the precipitate in phosphate buffer, adjusting to 70% saturation with ammonium sulfate, centrifuging and precipitating the supernatant with trichloroacetic acid. This precipitate was further purified by chromatography on DEAEcellulose and DEAE-Sephadex as described by Majerus et al. (8). Sodium dodecyl sulfate disk gel electrophoresis indicated that the purified fractions were at least 80% homogeneous. Analysis of the purified ACP by the method of Barron and Mooney (9) indicated the presence of approximately 5% acyl-ACP (mainly palmitic and vaccenic). Therefore, the ACP was further purified by binding to activated thiolSepharose 4B (Pharmacia). ACP-SH (1.0 pM) was incubated overnight at room temperature with 100 ml of activated thiol-Sepharose 4B in 0.1 M Tris-HCl pH 8 containing 1 mM EDTA and 0.3 M NaCl. The suspension was then poured into a column which was
384
OHLROGGE.
SHINE,
eluted first with 150 ml of the above buffer and second with 150 ml of the above buffer containing 50 mM cysteine (freshly prepared at pH 8.0). Fractions containing acyl carrier protein eluted by cysteine were pooled and concentrated by acid precipitation. Preparation
of Acyl-ACPs
Spinach stroma method. [‘?]Stearoyl-ACP was prepared by a modification of the method of Jaworski and Stumpf (10). Chloroplasts from the inner leaves of spinach prepared by the method of Kannangara et al. (11) were ruptured in a French press and centrifuged 100,OOOgfor 90 min. The supernatant was used as a source of fatty acid synthetase and was incubated with 35 pM [1,3-?I!]malonyl-CoA, 0.23 mM NADH, 0.21 rnM NADPH, 0.58 mM ATP, 0.56 mM glucose-6PO,, 1 unit glucose-6-POr dehydrogenase, 0.15 mM MgCl%, 0.08 mM MnCL, and 3-4 pM crude acyl carrier protein (approx. 10% pure). After incubation at 30°C for 10 min the reaction was stopped by adjusting the solution to 5% trichloroacetic acid. This mixture was centrifuged and the pellet resuspended in 0.1 M potassium phosphate buffer and the pH adjusted to 6.8 while solid ammonium sulfate was added to 70% saturation. This mixture was stirred at 4°C for 30 min and centrifuged 12,OOOgfor 15 min. The pellet was discarded and the supernatant protein was precipitated with 5% trichloroacetic acid and the pellet obtained after centrifugation was redissolved in 0.1 M potassium phosphate. After removing any undissolved protein by centrifugation, the supernatant protein was again precipitated and redissolved in a small volume of 0.01 M potassium phosphate, pH 7.0. Aliquots (0.1 ml) of this solution were then stored at -2O’C after adding just enough 1 M acetic acid to precipitate the protein. Prior to use in assaying acyl-ACP hydrolase activity just enough 0.1 M NaOH was added to redissolve the protein. This procedure gave approximately 50% conversion of [‘“Clmalonyl-CoA to [?]acyl-ACP. Unreacted ACP-SH was not removed from these preparations prior to use. The fatty acid composition of the acyl-ACP prepared by this method was determined by gas-liquid chromatography to be 81% stearic acid and 19% pahnitic acid. [‘4C]Palmitoyl-ACP (44 Ci/mole) was prepared similarly except [1-‘“Clacetate was the substrate and the spinach stroma fatty acid synthetase was pretreated at 37’C for 30 min which largely eliminated the elongation of palmitoyl-ACP to stearoyl-ACP (12). Gas chromatographic analysis of the product indicated 15% stearic and 15% lauric and myristic in addition to palmitoyl-ACP. [‘?]Oleoyl-ACP was prepared by enzymatic desaturation of the [‘4C]stearoyl-ACP prepared above. The reaction mixture contained in 10 ml: 0.4 PCi stearoylACP prepared above, 0.02 M sodium ascorbate, 0.07 M potassium phosphate, pH 6.0,40 X 19’units catalase,
AND
STUMPF
1 mM DDT, 900 pg ferredoxin, 10 I umts of partially purified stearoyl-ACP desaturase from Carthamus tinctorius (free of acyl-ACP hydrolase) and a photochemical reducing system consisting of 50 WM dichlorophenolindophenol and 50 pg/ml spinach chloroplast lamellae (10). This mixture was incubated at 24°C for 30 min in the light. [‘4C]Oleoyl-ACP (0.06 PCi) was iso!ated from the reaction mixture by the same method as described above for stearoyl-ACP and gas-liquid chromatography of this preparation indicated a fatty acid composition of 60% 18:1-ACP, 25% 16:0-ACP and 159 18:0ACP. Escherichia coli acyl-ACP synthetase method. ACP-SH was acylated with lauric, myristic and palmitic acids by the method of Ray and Cronan (13) using E. coli strain TR3 (generously provided by Allen Spencer of J. Cronan’s laboratory) as the source of enzyme. Endogeneous acyl-ACP-free ACP-SH was employed. The reaction mixture was incubated for 2 h at 37°C and terminated by addition of trichloroacetic acid. The acyl-ACP products were purified as described above for the preparation of crude ACP and then were separated from unreacted ACP-SH by passage through activated thiol-Sepharose and concentrated and desalted by ultrafiltration on Amicon UM02 membranes. Unreacted free fatty acid was separated from associated acyl-ACP by extraction with petroleum ether after acidifying with acetic acid. The acyl-ACP solution was then lyophilized and stored at -20°C until use. The conversions of lauric, myristic and palmitic acids to acyl-ACP by this procedure were approximately 10% 15% and lo%, respectively. The palmitoyl-ACP synthesized by this method gave rates of hydrolysis essentially identical to those observed with palmitoyl-ACP synthesized by the spinach stroma method described above. Preparation
of Coconut and Jojoba Extracts
Acetone powder extracts were prepared from developing coconut (Cocos nucifera) endosperm and developing jojoba (Simmondsia chinensis) cotyledons by blending the tissue 3 times in 10 vol of acetone at -20°C. The extract was then filtered and rinsed with diethyl ether and allowed to dry. The extracts (0.2 g) were resuspended in 5 ml of 0.1 M sodium phosphate, pH 7.5, plus 1 mM DTT before assaying for acyl-ACP hydrolase activity. RESULTS
Purification of Acyl-ACP Hydrolase The crude extract from avocado mesocarp contained approximately equal units of acyl-ACP and acyl-CoA hydrolase activity. Preliminary ammonium sulfate fractionations of avocado extracts indicated that of the total hydrolase activity re-
PLANT
ACYL
THIOESTER
covered in various fractions, 92% of the acyl-ACP hydrolase activity and 73% of the acyl-CoA hydrolase activity were found in the 25-50% ammonium sulfate fraction. This fraction was, therefore, applied to a DEAE-cellulose column and eluted with increasing concentrations of phosphate buffer and KC1 (Fig. 1). Two major peaks of acyl-CoA hydrolase activity and one peak of acyl-ACP activity were eluted from the column under these conditions. In Fig. 1 the acyl-ACP hydrolase peak appears to elute coincident to the second acyl-CoA hydrolase peak; however, in some runs, under similar conditions, the acyl-ACP hydrolase activity eluted just prior to the acylCoA hydrolase activity suggesting that both activities were not associated with the same protein. The first and second peaks of acyl-CoA hydrolase activity to elute from the DEAE-cellulose column will be referred to as acyl-CoA hydrolase I (CoA I) and acyl-CoA hydrolase II (CoA II), respectively. Acyl-CoA hydrolase I and acyl-CoA hydrolase II represented approximately 20% and 60% respectively of the total acylCoA hydrolase activity to elute from the DEAE-cellulose column. Fractions 40-60 from the DEAE-cellulose column were concentrated, dialyzed and further purified by hydroxyapatite chromatography which yielded a clear sep-
HYDROLASES
385
aration of two hydrolase peaks; the first contained acyl-CoA hydrolase II and the second acyl-ACP hydrolase (Fig. 2). A 360fold purification of acyl-ACP hydrolase activity had been achieved at this state. After hydroxyapatite chromatography the acylACP hydrolase activity was unstable. An approximately 30% decrease in activity was observed during 12 h storage at 4°C. This loss in activity was largely arrested after pooling and concentrating the active fractions. However, several attempts to purify further and characterize the hydroxyapatite fractions by gel filtration using either polyacrylamide or polydextran media led to loss of >95% of the activity. The purification attained through the hydroxyapatite stage is summarized in Table I. After the hydroxyapatite chromatography step the purified acyl-ACP hydrolase cleaved oleoyl-ACP at a rate approximately lo-fold greater than oleoyl-CoA. The actual specificity of the acyl-ACP hydrolase for acyl carrier protein thioesters may be even greater since there was a slight tailing of the acyl-CoA hydrolase activity into the acyl-ACP hydrolase peak. Molecular Weight of Acyl-ACP Hydrolase Although gel filtration of the more purified fractions was unsuccessful, gel filtration of the 25-50% ammonium sulfate frac-
FIG. 1. DEAE-cellulose chromatography of the 25-50% ammonium sulfate fraction from avocado mesocarp. Approximately 250 mg of protein was applied to a 6.5 X 3 cm column and eluted with a 2-liter linear gradient from 5 mM sodium phosphate, pH 7.8, plus 0.5 mM DTT to 0.1 M sodium phosphate, pH 7.8 plus 0.2 M KC1 and 0.5 mM DTT. The flow rate was 2-3 ml/min and 20-25 ml fractions were collected. B----M, Acyl-ACP hydrolase activity (nmoles/min/ml enzyme); A--k, acyl-CoA hydrolase activity (nmoles/min/ml enzyme); W, protein concentration (pg/ml).
386
OHLROGGE,
SHINE,
AND
..-. ! ! ! ’ ! i !i.
.-?
.I
,A*,?I I* ‘: #t \; ;. , .w . .-;:t,
/ ! i ! i ! i ! i I .;. i’ !I i k;**., !i i i is’ i
,‘f
l
STUMPF
\
‘\. \ -0.~
. .
. . I\\.,
d
a
QIQ20¶040~6070U)9a
iraction
ltumbrr
FIG. 2. Hydroxyapatite chromatography of DEAE fractions 40-60. After concentration and dialysis, fractions 40-60 from the DEAE column (Fig. 1) were applied to a 10 x 3 cm hydroxyapatite column and eluted with a 600 ml linear gradient from 0.05 M sodium phosphate, pH 6.9, plus 1 mM DTT to 0.3 M sodium phosphate, pH 6.9 plus 1 mM DTT. The flow rate was 1 mf/min and 6 ml fractions were collected. W, Acyl-ACP hydrolase activity (nmoles/min/ml enzyme); W, acyl-CoA hydrolase activity (nmoles/min/ml enzyme). TABLE I PARTIAL PURIFICATION OF ACYL-ACP HYDROLASE~ Total Procedure Specific Yield Puriticaunits activity tion (units/ mo g mesomg protein) carp) Crude extrct 25-50s (NH,),SO, DEAE-cellulose Hydroxyapatite
80 43
0.42 0.53
100 53
1.0 1.3
24
7.30
34
17.0
21
150.00
28
360.0
n Unit = 1 nmol of l&O-ACP hydrolyzed
per min.
tion did yield essentially 100% recovery of acyl-ACP hydrolase activity (Fig. 3). Calculation of the apparent molecular weight from these data gave a value of 70-80,000. The molecular weight of the acyl-ACP hydrolase was also estimated by sucrose density gradients of the protein at three stages of purification; 25-50s ammonium sulfate fraction, DEAE-cellulose purified and hydroxyapatite purified. Each stage of purification resulted in calculation of an apparent molecular weight between 70-75,000. Molecular Weights of Acyl-CoA Hydrolases Gel filtration of the first acyl-CoA hydrolase peak to elute from DEAE-cellulose gave an apparent molecular weight of 200,000.
Gel filtration of fractions 40-60 from the DEAE-cellulose column gave an apparent molecular weight of 70-80,000 for acyl-CoA hydrolase II. pH Activity
Profiles
Each hydrolase could be distinguished by a different pH-activity profile (Fig. 4). The acyl-ACP hydrolase had a pH optimum of 9.5 (Fig. 4a) while the acyl-CoA hydrolase II displayed a pH optimum at 8.4 to 8.6. The activity of acyl-CoA hydrolase I appeared to increase over the entire pH range of 5.0 to 10.5 although with the phosphate and tricine buffers decreases occurred at the upper end of their buffering capacity (perhaps suggesting an inhibition of this enzyme by anions). Measurements of activity above pH 10.5 were difficult due to nonenzymatic hydrolysis of substrate. Thermal
Stability
Acyl-CoA hydrolase II and acyl-ACP hydrolase could be distinguished by marked differences in their thermal stability. AcylCoA hydrolase II lost approximately 75% of its activity after 20 minutes incubation at 50°C whereas the acyl-ACP hydrolase lost no activity by this treatment (Fig. 5). Sensitivity of Hydrolases to Agents Neither acyl-CoA hydrolase I CoA hydrolase II was inhibited by of the products of the reaction,
Various or acyladdition namely,
PLANT
ACYL
THIOESTER
HYDROLASES
387
FIG. 3. Gel filtration of acyl-ACP hydrolase activity. The 2540% ammonium sulfate fraction (0.5 ml) was applied to a 1.0 x 50 cm column of Sephacryl S-200 (Pharmacia). The column yas eluted at room temperature with 0.1 M Tricine, pH 7.5, plus 0.5 mu DTT at a flow rate of 0.08 ml/min. Cytochrome c (0.2 mg) was included with the sample as an internal molecular weight standard and is responsible for the final peak in protein concentration. In the insert the K., calculated for the standards, aldolase (A), ovalbumin (W), and cytochrome c (0) are plotted versus their molecular weights. The Kav calculated for the acyl-ACP hydrolase is indicated by the arrow. W AcylACP hydrolase activity (nmoles/min/ml enzyme). O---4 Protein concentration (pg protein/ml).
CoASH or free fatty acid (Table II). In contrast, the acyl-ACP hydrolase was inhibited by its product, free ACP. Each hydrolase could be further distinguished by its sensitivity to the various active site reagents listed in Table II. These data lend furt,her support that each hydrolase activity is associated with a separate protein rather than to introconvertable forms of the same protein. The sensitivity of the acyl-ACP hydrolase to the sulfhydryl reagents (N-ethylmaleimide, iodoacetaand 5,5’-dithiobis-(2-nitrobenzoic mide acid) but not to the active serine reagent, phenylmethylsulfonylfluoride suggests that this hydrolase may be a sulfhydryl type esterase. Acyl-CoA hydrolase I was remarkably resistant to reagents to which acylCoA hydrolase II was reactive. Inhibition by ACP-SH The inhibition of the acyl-ACP hydrolase noted in Table II was further investigated. Since (as described under Experimental Procedures) our preparations of acyl-ACP contained low levels of free ACP-SH it was necessary in inhibition studies to use acylACP preparations containing no free ACP. In addition, we observed that our preparations of ACP-SH from E. coli were contam-
inated with approximately 5% acyl-ACP. Therefore, both acyl-ACP and ACP-SH were further purified by binding to activated thiol sepharose. While acyl-ACP does not bind to the column and is washed out, ACP-SH is eluted with cysteine. With increasing concentrations of ACPSH, we did not observe straight line Dixon plots suggesting that ACP-SH is not inhibiting in a simple competitive manner with substrate. Insufficient amounts of substrate free of ACP-SH prevented a more detailed kinetic analysis of the ACP-SH inhibition. Substrate Specificity The specificity of the acyl-ACP hydrolase with acyl-ACP substrates having different acyl chains was evaluated. The reaction rate increased with increasing chain length from 12 to 18 carbons (Fig. 6a). In addition, as had been observed previously with crude extracts of spinach, avocado and safflower (l), the purified acyl-ACP hydrolase displayed a marked selectivity for oleoyl-ACP relative to the saturated acylACPs. Insufficient quantities of the acylACP substrates prevented an accurate determination of K, values for the different substrates. However, we estimate that the K,‘s for palmitoyl, stearoyl, and oleoyl-
388
OHLROGGE,
04 ’ 5
I
9
SHINE,
II
PH FIG. 4. pH Activity profiles: Assays were with 2 stearyl-ACP or stearyl-CoA. Buffers used were succinate (A), phosphate (O), Tricine (B), and glycine (A); each adjusted to the pH with NaOH or HCl. Nonenzymatic hydrolysis at the higher pH’s has been subtracted. (a) Acyl-ACP hydrolase activity from DEAE-cellulose fractions 40-60 (Fig. 1). (b) Acyl-CoA hydrolase I activity from DEAE-cellulose fraction 3 (Fig. 1). (c) Acyl-CoA hydrolase II activity from DEAE-cellulose fractions 40-60 (Fig. 1). PM
ACP are below 0.2 PM and thus the relative rates of hydrolysis shown in Fig. 6a at a concentration of 1 PM are approximate V,,, comparisons. When the acyl-ACP hydrolase was incubated with a combination of 1 FM oleoyl and 1 pM stearoyl-ACP the rate of hydrolysis was approximately an average of the two hydrolysis rates rather than a sum. Thus, it is unlikely that these two substrates react at separate enzyme active sites. We also investigated the substrate specificity of hydrolysis of acyl-ACP substrates by two plant systems which differed mark-
AND
STUMPF
edly in the chain length of fatty acids synthesized in these tissues. We chose coconut (Cocos nuciferu) which produces predominantly 12 and 14 carbon fatty acids and the developing jojoba seed (Simmondsia chinensis) which synthesizes 20 and 22 carbon fatty acids. Figure 6, b and c, indicates that the specificity of acyl-ACP hydrolysis by extracts from these two tissues is very similar to that observed with avocado. Thus, from these data we cannot attribute the difference in fatty acid chain length synthesized by these plants to different acyl-ACP hydrolase specificities. In contrast to the acyl-ACP hydrolase, the two acyl-CoA hydrolases of avocado had a broader specificity and did not show as great a preference for the oleoyl derivative. Compariative reaction rates with the various substrates are shown in Fig. 6, d and e. It is well known that long chain acylCoAs form micelles at critical concentrations (usually at the 2-5 PM level), and adsorb strongly to proteins and at the airwater interphase (14). Thus, the concentrations cited in Fig. 6 represent only the amounts added to the reaction mixture and not the actual concentration in the bulk phase. For these reasons substrate versus velocity plots did not allow accurate K,,, determinations. However, in general, the relative order of reactivity of the acyl-CoA substrates shown in Fig. 6 did not change
-. 20
x..erer-.-A 0
20
&OS
at 50?“c
FIG. 5. Thermal stability. Aliquots of fractions 40-60 from the DEAE-cellulose purification (Fig. 1) were heated at 50°C for the times indicated and then assayed with either 2 PM l&O-ACP (u) or 2 PM l&O-CoA (A-,--A).
PLANT
ACYL
THIOESTER TABLE
INHIBITION
OF ACYL
THIOESTER
HYDROLASES II
HYDROLASES
BY DIFFERENT
Concentration
Reagent
Iodoacetamide acid)
0.90
1.0 rnM
5.0mM 0.3mM
PhenyImethyIsulfonylfluoride
0.8 mM 10 PM 50 PM 1.0 /.iM 5.0 PM 3.0 PM 15.0 pLM 1% 5% 0.001% 0.005%
CoASH acid
ACP-SH Isopropanol Tergitol
0.96
1.0 rnM
55.Dithiobis-(2-nitrobenzoic
Patmitic
1.0 rnM
5.0mM 5.0mM
15-S-9
” The first four inhibitors for hydrolase activity.
were preincubated
REAGENTS”
Rate relative Acyl-CoA hydrolase I
N-Ethylmaleimide
389
1.0
0.92 1.0 1.0 1.0 0.96 1.1 1.1 1.0 1.0 1.4 0.90 1.1 1.4 0.73 0.44
to control
Acyl-CoA hydrolase II 0.32 0.23 0.65 0.48 0.74 0.48 0.71 0.60 1.0 1.0 I.0 1.0 0.77 0.67 0.90 0.37 0.90 0.42
with enzyme for 1 h and then diluted lo-20.fold
over the concentration range of 0.1 to 10 PM. Hydrolysis of either acyl-CoA or acylACP was not inhibited by addition of phospholipids or triglycerides. Thus it is unlikely that the activity we are reporting is due to a non-specific phospholipid or triglyceride lipase. DISCUSSION
The chain length of fatty acids synthesized by various fatty acid synthetases has been shown to be a function of the concentration and activity of a wide variety of substrates, cofactors and enzymes (15-22). Thus it is unlikely that the function of controlling fatty acid chain length can be attributed to a single enzyme activity. In several organisms acyl hydrolases have been proposed as controlling elements in determining the chain length of fatty acids synthesized (15-17). Flick and Bloch (15) reported that addition of an acyl thioester hydrolase to a fatty acid synthetase from Mycobacterium phlei roughly doubled the proportion of short chain acids synthesized. Similarly, Knudsen et al. (16) have recently shown that addition of a purified medium
Acyl-ACP hydrolase 0.60 0.15 0.26 0.80 1.0 1.0 1.0 1.0 1.0 1.0 0.80 0.50 1.0 0.73 1.1 1.0 before assaying
chain acyl hydrolase isolated from rabbit mammary gland to the mammary fatty acid synthetase resulted in a decrease in chain length of the fatty acids produced. In addition, Agradi et al. (17) showed that removal by trypsin cleavage of thioesterase from a multienzyme fatty acid synthetase led to an increase in fatty acid chain length. In contrast, Cronan et al. (18) have proposed that, for E. coli, acyl transfer into phospholipid is a major determinant of fatty acid chain length although previous investigations of the /I-ketoacyl-ACP synthetase indicated that this enzyme’s specificity is also consistent with the pattern of fatty acids synthesized (19). Sumper et al. (21) proposed a model for the yeast fatty acid synthetase which described the pattern of fatty acids synthesized as a function of the relative rates of the condensation reaction versus acyl transfer to coenzyme A. Since the principal fatty acids in most plants are the Cl8 unsaturated acids (221, a mechanism must be at hand to guarantee the biosynthesis of these acids. We would like to suggest that the predominance of these Cl8 fatty acids (oleic, linoieic, and
390
OHLROGGE,
SHINE,
AND
STUMPF
80. cdl I g
4
Ml
.i
C. lojoba
-II
12:o 14% l&O IBE0 l&l
f 2 aa .E z 22
0
12:0 14:0 16:0 18:O 183 E. 8 CoAII
4
'
Lll
12:O l4:O 16:O 18:O 18:l
FIG. 6. Substrate specificity of acyl thioester hydrolases. (A-C) Relative hydrolase activity with acyl-ACP substrates. Substrates were prepared as described under Experimental Procedures and assayed at a concentration of 1 pM with the DEAE-cellulose purified avocado enzyme or with acetone powder from coconut and jojoba. (D) Specificity of acyl-CoA hydrolase I from avocado. Substrates at a concentration of 1 pM were assayed with enzyme from fraction 3 of DEAE-cellulose (Fig. 1). (E) Specificity of acyl-CoA hydrolase II from avocado. Substrates at a concentration of 1 PM were assayed with enzyme from fractions 40-60 of DEAE-cellulose (Fig. 1).
linolenic) is the result of the coordinated action of a series of enzyme systems all employing ACP as the thioester moiety. Thus, the synthesis of palmitoyl-ACP from acetyl-ACP and malonyl-ACP, the specific conversion of palmitoyl-ACP to stearoylACP by the palmitoyl-ACP elongase (12), the highly specific desaturation of stearolyACP to oleoyl-ACP by stearoyl-ACP desaturase (10) and the specific hydrolysis of oleoyl-ACP to free oleic acid by the acylACP hydrolase guarantee the formation of oleic acid which then can be converted to oleoyl-CoA for further modifications (2). Thus, chain termination of fatty acid biosynthesis in plants may result from the specificity of each of the above enzymes which act in concert to assure the predominance of 18 carbon fatty acids in most plants. Intriguing is the observation that this oleate specific acyl-ACP hydrolase also occurs in plants which differ markedly in
the chain length of the major fatty acids synthesized (Fig. 6). We are currently investigating what other mechanisms of chain termination might account for these different fatty acid compositions. ACKNOWLEDGMENTS We wish to thank Don Andrews for excellent technical assistance, Ms. Barbara Clover for help with the preparation of this paper and Dr. Tom McKeon for purified stearoyl-ACP desaturase. REFERENCES 1. SHINE, W. E., MANCHA, M., AND STUMPF, P. K. (1976) Arch. Biochem. Biophys. 172, 110-116. 2. VIJAY, I. K., AND STUMPF, P. K. (1971). J. Biol. Chem.
246,2910-2917.
3. SHINE, W. E., MANCHA, M., AND STUMPF, P. K. (1976) Arch. Biochem. Biophys. 173, 472-479. 4. BRADFORD, M. M. (1976) Anal. Biochem. 72, 248-254. 5. TORRIANI, A. (1966) in Procedures
Research
(Cantoni,
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Harper
& Row,
ACYL New
6. MARTIN, R. G., AND AMMES, B. N. (1961) Chem. 236,1372-1379.
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THIOESTER York.
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ALBERTS, A. W., MAJERLJS, P. W., TALAMO, B., 3, AND VAGELOS, P. R. (1964) Biochemistry 1563-1571. MAJERUS, P. W., ALBF.RTS, A. W., AND VAGELOS, P. R. (1969) in Methods of Enzymology (Lowenster, J. M., ed.), Vol. 14, pp. 43-50, Academic Press, New York. BAHRON, E. J., AND MOONEY, L. A. (1968) Anal. Chem. 40, 1742-1744. JAWORSKI, J. G., AND STUMPF, P. K. (1974) Arch. Biochem. Biophys. 162, 166-173. KANNANGAHA, C. G., JACOBSON, B. S., AND Plant Physiol. 52, STUMPF, P. K. (1973) 156-161. JAWORSKI, J. G., GOLDSCHMIDT, E. E., AND STUMPF, I’. K. (1974). Arc/z. Biochem. Biophys. 163, 769-776. RAY, T. K., AND CRONAN, J. E., JR. (1976) Proc.
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DILS,
R.
(1976)
Biochem. J. 160,683-691. 17. AGRADI,
E., LIBERTINI,
Biochem.
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21. SUMPER, M., OESTERHELT, D., RIEPERTINGER, C., AND LYNEN, F. (1969) Eur. J. Biochem. 10,
377-387. 22.
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K. P., AND STUMPF, P. K. (1971) 143,412-427.
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23. HITCHCOCK, C., AND NICHOLAS, B. W. (1971) Plant York.
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