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Fat metabolism in higher plants
ARCHIVI~X
OF
13IOCHEMISTIIY
AND
BIOPHYRICS
412427
Iti,
Fat Metabolism XLIV. Fatty
Acid Synthesis from
in Higher by a Soluble
Solarium
II;. I’. HUANG2 Department
of Biochemislr~/
and
(1971)
Received
Fatty
Acid Synthetase
tuberosum’ 1’. Ii. STUMl’F
bNI)
Biophysics,
Plants
&ziversit!/ December
of
California,
Davis, California
96616
9, 1970
A highly st,able pot.ato tuber preparat.iott containing the fatty acid syttthetsse ettzymes has been obtained and the cottdit,iotts for t,he de not~o synthesis of fat& acids have been examined. Uttder st,attdard cottditiotts the syst.em formed CR to Ct6 satttrated fatky acids. While 50-70~0 of t,lte newly syttthesixed produ& ocrnrred as free fat.ty acid, t,he remainder accumulated as ACP t.hiolesters (CIA and Ctsj and ss CoA thiolesters (CS , C,U , attd Ct?). The cottcentratiotts of malottyl-CoA, TPNIT, ACP, and pH greatly altered the pat,tertt of fatty acids syni.hesixed. In addition to acetylCoA, propiottyl and but.yryl-CoA served as elrective acceptors to form odd and even long-chain fat,iy acids, respectively. Palmityl thiolest.erase is present in preparations of the synthesizing system although the enzyme can be separated from the syttthetase enzymes. IDs specificit.y is limited lo the C~S and Ct8 fatt.y a.cyl CoA derivutives.
Fatt.y acid synthetases have been characterized from a number of sources,notably yeast (l), avian liver (2, 3), mammalian liver (4), adipose tissue (5), Escherichia coli (6-S), and higher plant,s (9, 10). The fatt,y acid synthetases from yeast., (pigeon) liver, (rat) liver, and Mycobacterium phlei are isolated as tightly bound complexes. However, the fatty acid-synt,hesizing syst,em from E. coli consists of at least, six soluble enzymes and a low molecular, heat-stable protein known as acyl carrier prot,ein. This latter protein contains 4’-phosphopantetheine as a prosthetic group and functions as a cofactor in fat,ty acid biosynthesis (11, 13). The end products of the yeast fat,ty acid synthetase complex are fatty acyl CoA derivatives (1). However, the end products 1 This investigation was supported by NSF Grant, GB-19733X. 2 Present address: National Institute of Arthrit is and Metabolic Diseases, National Inst.it,utes of Health, Public Health Service, Bet,hesda, Maryland. 412
of avian liver (3, 13), mammalian liver (4), and adipose tissue (5, 14) fatty acid synthetase complexes are free fatty acids whereas in E. coli, fatty acyl ACP derivat.ives have been shown t.o be the reaction products (15). As yet there is no detailed study on the react.ion products of plant, fatty acid synthet.ases. The compositions of fat.ty acid synthesized by the yeast fatty acid synthetaxe complex have been shown to be determined by the malonyl-CoA/acetyl-CoA concent&ion ratio. The studies of both rabbit, mammary gland and rat liver fat,t,y acid synt,hetase complexes suggested that t.he pat.tern of fat.ty acids synthesized is determined by malonyl-CoA concenbrtltion (17). A number of st.udies on fatt.y acid synthesis have utilized organisms and tissues of high lipid contents, such as adipose tissue, liver, and fatty seeds. These organisms and tissues have obvious advant,ages in yielding highly acbive synthetases. However, with the exception of work in the pea system,
FATTY
ACII)
SYSTHETASE
there is very little information available about the fatty acid-synthesizing systems from t,he low lipid-containing plant tissues. This paper reports on the properties of a soluble fat,tv acid synthetase from a low lipid-contairiing t.issue, namely the potato tuber. MATERIALS Potato tubers (Solarium tuberosum) of White Rose variety were purchased from local markets. Dithiothreit,ol and TPNH were obtained from Calbiochem; chromatographically pure CoA from P-L Biochemicals, Milwaukee; Sephadex G-lOO120 and DPNH from Sigma Chemical Company; malonic acid 1,3-14C, decanoic-1-‘4C, lauric-l-l%, myristyl-1-‘4C CoA, stearyl-l-l% CoA, from New England Nuclear Corporation; palmityl-1-‘4C CoA from Dhom Products, Ltd.; polyclar AT from General Aniline and Film Corporation, Dye Stuff and Chemicals Division; 2-mercaptobenzothiazole (MERCAP) from K and K Laboratories, Inc; chromat,ography media (ITLC-SC; type, 20 X 20 cm) from Gelman Instrument Company; l)EAl<-cellulose (DE-52) from Whatman. METHODS E. coli ACP was isolat,ed according t.o the method of Simoni et al. (18). Malonyl-1,3-14C CoA was prepared according to the method of Trams and Brady (34) modified by Dr. Vageos, Washington University. Acetyl-CoA and propionyl CoA were made according t,o the method of Simon and Shemin (19). Butyry-CoA, crotonyl CoA, isobnt.yryl CoA, Valery1 CoA, and caproyl Co.4 were prepared according to the same method except, t.he reactions were run in 50yc acet.one in order to aid in the solubility of various anhydrides. I)ecanoyl-l-l% CoA and lauryl-l-l% CoA were synthesized by t.he mixed anhydride method of Goldman and Vagelos (20). Fatty acyl thioesters of CoA were determined by t.he absorption at 232 nm (21) and the t.hiol determinat,ion by Ellman’s method (22) after cleavage with neut,ral hydroxylamine. Preparation of fatty acid synlhelase. White pot,at,o tubers (Solarium tuberosum, var. White Rose), purchased from local markets, were peeled and cut int.0 small chunks. For each 300 g of potato, t.he following components were added: 30 g of acid-washed polyclar AT, 7.5 g of acidwashed Norit A, and 60 ml of 0.5 M potassium phosphate buffer, pH 7.2, with 0.05 M mercaptoet.hanol and 0.6 rnM of 2-mercaptobenzothiazole at 4”. The mixt,ure was homogenized in a Waring Blendor for 2 min at top speed. The slurry was squeezed through two layers of cheesecloth, and
OF
Solonctrrl
/uberos,o)~
413
the filtrate was centrifuged at 48,OOOg for 26 min in a Sorvall IX-2B centrifuge. The pooled supernatant fluid was 230 ml at, pH 6.8. A 0.05 vol of 1 Y MnCl2 (11.5 ml) was added slowly to the crude ext.ract with constant. stirring. The pH was maintained at 7.0 by adding 6 N KOH. The turbid solution was then centrifuged at 48,000g for 20 miu. To 236 ml of M&l:! supernatant fluid 57.2 g of solid ammonium sulfate were added gradually to @lo sat.uration. The solution was cent.rifuged at 48,000g for 20 min, and t,he precipitate was discarded. Ammonium sulfate (34.6 g) was added to the supernatant fluid (210 ml) to 65% saturation. The result.ing precipitate was collected by centrifugation and the supernatant fluid was discarded. The precipit,ate was dissolved in 0.01 M potassium phosphate buffer (pH 7.0) with 0.1 mM EDTA to a final protein concentration of 20 mgi ml. The solution was t,hen frozen in l-ml aliquots and stored at -15” under N) until ready for use. Preparation of palmilyl CoA thioleslerase. The 4&65yc ammonium sulfate precipitate was used as the start.ing material. The concentrated protein solution (12 ml containing 917 mg protein) was fractionated on a Sephadex G-100 column (4.1 X 110 cm) equilibrated wit.h 0.01 M potassium phosphate buffer (pII 7.0) containing 1 mM mercaptoethanol. Fract.ions of 8.5 ml were collect,ed and assayed for palmit,yl-CoA t.hiolesterase activity. Effluent fractions of high activit.y from Sephadex column were pooled (450 mg of prot.ein) and put onto a DEAE-cellulose (Whatman DE-52) column (2.5 X 24 cm) which had been equilibrated with 0.01 ?M pot.assium phosphate buffer (pH 7.0) with 1 my mercaptoethanol. After washing with t.he same buffer, the column was eluted with a linear gradient made from 500 ml of 0.01 M potassium phosphate (pH 7.0) with 0.07 M pot.assium chloride and 500 ml of 0.25 M potassium chloride in the same buffer. Fractions of 5 ml were collected and assayed for palmityl-CoA thiolesteraae aetivity. Assay of jaf1.y acid synthetase. The standard reaction mixture contained potassium phosphat,e buffer, pH 7.0, 5Ormoles; purified E. coli ACP, 70 pg; acetyl-CoA, 0.07 pmole; DPNH, 0.2 pmole; TPNH, 0.2 pmole; malonyl-1,3-14C CoA, 0.032 amole (2@0,000 cpm); and 1 t.o 2 mg of potato fatty acid synthetaae in a total volume of 0.5 ml. The incubations were carried out at 37” for 30 min and stopped by t,he addition of 0.2 ml of 40% KOH. After 30 min of hydrolysis at 80”, the reaction mixtures were acidified with 0.25 ml of concentrated HCl. After addit,ion of 2 ml of water, the reaction mixtures were extracted wit.h 16 ml of CHClyjCIIaOH (7.5:5.5). Separation into two phases were acheived by t.he addition of another 2 ml of water. The chloroform layer was washed
414
HUANG
AYI) _
with 5 ml of solution containing 0.5% malonir acid and 0.5% acetic acid in 1 N HCl sat.urated with sodium chloride and dried over anhydrous NaW. An aliquot was then dried in a scinit,llation vial and counted in a Packard liquid scintillation counter by the usual procedures. Assay of palntilyl CoA thiolesterase. The assay procedure for palmityl CoA thiolesterase was baaed on t,he enzyme-cat,alymed release of radioactive fatty acid from its thioest.er of CoA. The assay mixture contained 10 pmoles of Tris chloride (pH 8.0), 0.025amole of palmityl-1-W CoA (125,000 cpm), and 3-10 pg of thiolest.erase in a t.otal volume of 0.1 ml. The reaction was carried out in a 10 X 75-mm test tube and incubated at 37” for 1 min. After 1 min of incubation the reaction was terminat.ed by the addition of 0.01 ml of glacial acetic acid at 2”. Ten-microliter aliquots of the reaction mixture were put onto a 1.4 X 6.0-cm strip of Gelman chromatography media. The reaction product., free fat,ty acid, was separated from it,s substrat,e, acyl-CoA by placing the strip into a small jar containing a solvent system of water-saturat,ed ether/formic acid (7: 1). Separation was accomplished in 4 min. The reaction product moved to the solvent front and the substrat,e stayed at the origin (23). Free fatt.y acid produced in t.he reaction could be measured by cutting the strip 1.7 cm below the top, and the radioactivit,y was counted in a scintillation counter. Analysis of reaction producl. The products of fatty acid synt,hetase reaction were analyzed by an Aerograph Model A-90 P2 Nuclear Chicago Biospan Model 4998 radiomonitoring system after met.hylat,ion with diazomethane. Various chain1engt.h fatty acids were separated on a 5 ft X 0.25 in. colurm~ packed wit.h 12% diethylene glycol succinate (DEGS) on Anakrom P (60-70 mesh). Separation was carried out by temperature programming from 120-200” with standard fat,t,y acids (from CsYOto C19:0) in each injection. The percentage distribution of various radioact,ive fatty acids were calculated from t,he integrator which registered the total radioactivity of each component. Protein concentrat.ion was determined by the Lowry procedure according to Miller (24). RESULTS
Properties of fatty mid synthetase. Potato tubers contain phenolic compounds which complex either reversibly with proteins by hydrogen bonding or irreversibly by oxidation to quinones followed by covalent condensation of t.he quinones wth the react.ive groups of proteins (2~). The crude extract. was, therefore, relatively unst,able. However, by employing the Polyclar AT-Xorit
,YTUW’F *
A - mercaptoet.hanol - 2 - mercaptobenzo - t hiazide system, t,he partially purified synt.hetase system was stable for mont.hs at -115” under Na. Under standard iwsay condit:ions 0.32 mpmoles of malonyl-CoA was incorporated per minute per milligram protein into cle ~IOVO synthesized fatty acids. Repeated freezing and thawing caused considerable loss in activity. When the prot.ein solut,ion was cenkifuged at, 198,000~/ for 5 hr over 70 %i of t.he synt.hetasc activity remained in t.he supernatant fluid. Unlike the fatty acid synthetaw complex from yeast and mammalian tissue, potato fat.ty acid synthetase was similar to t,he E. coli system and ot.her higher plant fatky acid-synthesizing systems (9). Requirenwnts
Jar
fatty
acid
syrrthesis.
Under the standard assay condition, as showy in Fig. 1, fatty acids synthesized bv the pot,at,o system increased linearly \I-ith respect to t,ime for 30 min. The enzyme system showed an absolute requirement for E. wEi ACP (Fig. 8) and TI’NH (Fig. 3). Becauseof the existence of malonylCoA decarboxylase in the enzyme preparation, acetyl CoA was not absolutely required (Fig. 4). However, :t clear de-
2awo
Iaow
l
v
Y
I
t
I
I
I
I
20
40
60
so
100
120
TIME (min.) FIN. 1. Time course of fatty acid synt,hesis under standard assay conditions with 2 mg of protein. The enzyme activity is expressed as total radioactivity (count,s per minute) incorporated int,o fatt.y acid.
FATTY
ACID
40
SYNTHETASE
so
100
OF Solanccm
160
200
240
415.
Ircbermum
280
320
JG AU FIG.
2. Requirement
of E. coti ACP for fatty acid synthesis.
were acidified to pH 2.0 then followed by pentane ext.ract,ion. About, 50-700/u of the react,ion products were free acisd. The rest. of the pent,ane-insoluble reaction products could be recovered by furt,her pent.ane extraction aft.er acidificat.ion of t.he alkaline hydrolyzate. The alkali hydrolyzable port.ion was considered to be thioester-bound fatty acid. In order t,o characterize the properties of thioester-bound fatty acids, the reaction
40
80
120
160
203
243
280
mp mole TPNH
FIG. 3. Requirement synthesis.
of TPNH
for fatt.y acid
pendence of acet.yl CoA as a primer was observed. Both DPXH and dit,hiothreitol stimulated the reaction. Addition of 0.2 rmmole of DPXH or 5 pmoles of dithiothreitol t,o the react,ion mixture increased the syntbesizing capacity about, 10%. Thus DPSH is not an absolute component. in I t.he reduction steps of fatty acid synthesis. 20 40 60 80 100 The effect of various sulfhydryvl reagents mp mde acetyl COP, on fatty acid synthesis is shown in I+?g. 5. A?halysis of the reaction proclucts. After FIG. 4. Requirement. of acetyl-CoA acid synt.hesis. 30 min of incubation, the reaction mixtures
120
140
for fatty
416
IIUANG
AND STUMPF
mixtures were separated on a standardized Sephadex G-100 column. As shown in Fig. 6, there are three radioactive peaks corresponding to an apparent molecular weight, of 63,000, 8-9,000, and l-2,000 from the standard curve. Each of the radioact.ive peaks was pooled and acidified after alkaline
20
40
60
80
1M
hydrolysis. Peak 1 contained relatively low pentane-ext.ractable radioactivity which would suggest polar components. .Further investigations were not carried out, on this fraction. Pentane extra&ion of peak 2 yielded myristic acid, palmitic acid, and unknown components, presumably hydroxy acids. Hecause peak 2 corresponds t,o approximat.ely NOO-9000 mol wt, these components may be bound t.o h’. coli ACP which has a molecular weight of about 8700. Peak 3 contains the highest amount, of radioactivit,> wiith a molecular weight around 1000-2000. Pentane extract,ion of the acidified alkaline hydrolyzate yielded octanoic, decanoic, and lauric acids. Ether extraction of the same fraction gave malonate. Obviously peak 3 cons&s of acyl CoA esters. The synthesized fatty acids were separated by GLC and degraded by the permanganate oxidation of Harris and *James (26). The resu1t.s are shown in Table I. The con&ant ratio of radioactivity to mass
120
CONC. X IO%
FIG. 5. Effect of sulfhydryl acid syntheaie.
reagents on fatty
I
40
60
80
100
120
140
TUBE NUMBER FIQ. 6. Separation of the reaction product by Sephadex G-100 column fractionation. Separations were carried out, in a calibrated Sephadex G-100 column (1.6 X 120 cm) at 3-4’. Blue dextran-2000 wa8 added to the reaction mixture just before putting the sample into the column. After separation, a O.l-ml aliquot wea put into scintillation vial with 10 ml of Bray’s solution. The radioactivit,y was determined by a Packard liquid-scintillation counter. -a--, ODzao; +, radioactivity.
FATTY
ACID
SYXTHETASE
OF SoZun,rnz ~rtberosror~
TABLE I Miss Rxrros OF ~~YItISTITI ?LND P..\LMIT.\TV _ .- ..p--.---Parent compound KMllO4 _-.-.. .- ..---... Oxidation product C,S CV ..~.. __ ...3.18 Cl8 3.18 Cl:, 3.0 1.36 Cl, 1.45 2.99 Cl3 3.1 1.14 Cl2 -.R Ratios were calculated using the peak area of radioactive trace and mass trace.
117
16;O
ILUIIOXTI~ITY:
K n
60
6.6
70
PH 7. Effect of incubation pH on the fatty acid synthetase activity. The pH values indicated in the figures are t,he actual reaction pH’s measured by the pH meter. FIG.
indicated myrktate
t,he de )iovo synt,hesis of bot.h and palmitate. pH optimum of fatty acid synthesis. The pH optimum of potato fatty acid synthetase is approximately pH 7.0 in phosphate buffer aa shown in Fig. 7. Pattern of fatty acids syn.thesizedby potato fatty acid synthetase.Figure 8 shows t,he
typical pattern of radioact.ive fatty acids produced under the standard assay condition. Each of the individual fatty acid methylesters was collect,ed from gasliquid chromatography and identified further by comparing with standard fatty acid methylesters. Octanoic acid, decanoic acid, lauric acid, myristic acid, palmitic acid, and trace amount, of stearic acid were
FIG. 8. Analysis of the reaction product methyl est.ers in a 5 ft X 0.25 in. 1)EGS column. Separation was carried out by temperature programming from 120-200”.
synthesized from malonyl-CoA. Ko unsaturat.ed fat,ty acid was detectable. Unlike other fatty acid synt,hetases from higher plants, which synthesize mainly palmitat,e and st.earate, the potat,o fat,ty acid synthetase synthesizes fatt,y acids of varying chain length (from Cs t.o Cl,). Because of t,he versatility of t.his system in synthesizing varying fatt,y acids, the effects of varying the concentrat,ions of malonyl-CoA, acetyl-CoA, TPNH, and ACY on t.he final pr0duct.s were examined. As shown in Fig. 9, the effect of malonylCoA concentration on t.he chain-length of the reaction pr0duct.s is obvious. At low malonyl-CoA concentration, t,he reaction products were widely diskibuted among various chain-length fatty acids, whereas, at) high malonyl-CoA concentrat,ion, palmitnte was the predominant fatty acid.
418
HUANG
AND STUMPF
60-B 50-
II
40 3020 10 l-l
l-l
6O-c
60-C so-
50 -
40 30 20 r 10 I-l
l-l
CS
=I0
CS Cl2
Cl4
cl6
FIG. 9. Effect of malonyl-CoA concentration on the fatt,y acid composition in the reaction prodoct. The reactions were carried out under st,andard assay conditions with various amounts of malonyl-CoA. The malonyl-CoA concentrations used in the experiments ae shown in Fig. 9A, B, and C were 4.9, 24.5, and 65.2 mpmoles, respectively.
When the malonyl-CoA concentration was further decreased to 3.26 /IM, little if any myristate or palmitate accumulated. Octanoate, decanoate, and laurate became the predominant products. The shift in the patt,ern of fat.t:y acid with respect to malonyl-CoA concentration agreed with the results obt.ained from yeast., rabbit mammary gland, and rat liver, ail multienzyme complexes. When malonyl-CoA concentration was kept constant with variable amounts of scetyl-CoA in the assay system, the results are shown in Fig. 10. The fatty acid compositions of the reaction product kept
Cl0
Cl2
Cl4
cl6
FIQ. 10. Wfect of acetyl-CoA concentration on t,he compositions of fatty acid synthesized. The acetyl-CoA concentrations used in the experiments aa shown in Fig. lOA, B, and C were 4.8, 23.8, and 142.5 mpmoles, respectively.
fairly constant, over a wide range of acetylCoA concentrations. Although the malonylCoA/acetyl-CoA concentration ratios vary from 6.75 to 0.22, no significant changes were observed. Thus t,he absolute rather than the relative concent.rat,ion of malonylCoA is critical. The effect. of TPNH concentration on fatty acid composition of the reaction product is shown in Fig. 11. The results indicat,ed that at lower TP3H concentration t>he palmitate synthesis was less favorable, and at higher TPXH concentration my&ate and palmitate became t,he predominant products. Figure 12 shows the striking effect of ACP concentration on the fatty acid composition of the final products. -4t low ACP
FATTY
60
B
ACID
SYNTHETASE
41.9
tuberosum
n
50 Q
OF Xolanum
60
B
7
40 50 40 30 20
v
10
=I0 CS Cl2 cl4 cl6 FIG. 11. Effect of TPNH concentration on the composition of fattt,y acid synthesized. The TPNH concentrations used in the experiments as shown in Fig. llA, B, and C were 4.0, 40.0, and 120.0 mpmoles .
concentration (Fig. 12A), palm&ate was the major component which accounted for almost 80% of the t.ot,al product. However, at higher ACP concentration (Fig. 12C) various chain-length fatty acids were equally distributed. Myristate and palmitate synthesis were highly reduced under high ACP concentration. As shown in Fig. 13, the change in the reaction mixture pH also greatly perturbs the composition of reaction products. Palm&ate was sharply decreased from 52% of total fatty acid synthesized to only 270 when the pH was changed from 6.3 to 5.3. At higher pH value, shorter chain-length fatty acids (C,-CIZ) became the major products. These results clearly indicate the com-
n
l-l
n
%
'10
'12
'14
'16
FIG. 12. Effect of ACP concent,ration
on the composition of fatt,y acid synthesized. The ACP concentrations used in the experiments as shown in Fig. 12a, B, and C were 6.9, 55.2, and 256pg, respectively.
plexky of conditions which participate in the det,ermination of chain length of de uovo synthesized fatty acids. It is quite possible that superimposed on the enzymes involved in synthesizing the fatty acids are ancillary enzymes, such as malonyl-CoA decarboxylase, which would regulate the level of malonyl-CoA and long-chain acyl t,hioesterases which would alter in activity as a fun&ion of pH. Sulfhydryl reagents can either activat,e or inactivat,e the total synthesis of fat,ty acids. They also affect t,he composition of the reaction product t.o a different extent. As shown in Fig. 14, three sulfhydryl reagents at two concentration levels were
4'20
HUANG
AND STUMPF
60 60 i?
60 I
62
40
:
30
B
% pti 7.35
20 10 l-l 60
rl
’ pH 6.30
50 40 30
l-l c,
%I
c,
c,,
c14
‘16
FIG. 13. Effect of incubat,ion pH on the composition of fatty acid synt.hesized. The buffers used in t,hese experiment.5 as shown in Fig. 13A, B, and C were phosphat,e buffer, pH 6.4-pH 7.3.
compared to the control wit.hout. adding sulfhydryl reagent. In general, in t,he absence of -SH reagent, palmitate and ~~ristatc were t,he major products. Addldlon of -SH reagent decreased palmitate formation. C;lutat.hione (Fig. 14~) was more effective than dithiothreitol and mercaptoethanol.
Cl0
52
Cl4
cl6
FIG. 14. Effect of sulfhydryl reagents on the composition of fatty acid synthesized. Various concentration levels of glut.at.hione (A), dithiothreitol (B), and mercaptoethanol (C) were used as indicated. 0, control experiment without sulfhydryl reagent; q , reaction mixture contains 1 mM of respective sulfhydryl reagent; n , reaction mixture contains 10 mM of respect.ive sulfhydryl reagent.
CoA were less effect,ive. The low rat.e of fatty acid synthesis with caproyl-CoA as primer could be relat.ed t.o possible low activit,y of acyl-CoA-AC1 transferase towwd caproyl-CoA. The relatively low activity of crotonyl-Cob as t,he primer is similar to that of rat brain enzyme (27). The products of synthesis were extracted, Effect of various acgl CoA as th.e “primmer” and analyzed as described of jattg acirr! synthesis. When fatty acid methylated, synthetase assays were carried out under before. Odd-chain (derived from propionylCoA and valeryl-CoA) and branched-chain standard assay conditions with various fatty acids short-chain acyl Cohs as primers, the rate (derived from lsobutyryl-CoA) of fatty acid synthesis was markedly af- were easily identified by gas-liquid chromatography. The coexistence of even-chain fected. As shown in Fig. 15, butyryl-CoA and propionyl-CoA were bet.ter acceptors and odd-chain fatty acids in the propionylthan acet,yl-CoA. However, valeryl-CoA, CoA and valeryl-CoA experiments suggest.ed crotonyl-Cob, isobutyl-CoA, and caproylthe formation of acetyl--CoA from malonyl-
FATTY
I
ACID
SYSTHETASE
I
,
I
20
40
60
mpmole
431
OF Solanrtna fztberosum
80
I
I
I
I
100
120
140
160
Acyl CaA
FIG. 15. Effect of various acyl-CoA’s OILthe rate of fatty acid synt,hesis. The reactions were carried out under standard assay conditions with various amolmts of nonradioactive short-chain acyl-&.4’s as primers.
CoA by the malonyl-Co-4 decarboxylase reaction. The coexist.ence of even-chain and branched-chain fatty acids was also observed in isobut,yryl-CoA experiments. The competition of various acyl-CoAs w&h acetyl-CoA as t,he primer for fatt) acid synthesis can be seen from the results obtained in the propionyl-Co-4 experiments (Fig. 1G). The results showed that at higher propionyl-CoA concentrations there was no det.ectable even-chain fatty acids in the reaction product. As primers, valeryl-CoA and isobut,yr\rlCo-4 did not stimulate synthesis above t,he background level of synthesis in their absence. However, the ratio of odd-chain fatty acids and branched-chain fatty acids to even-chain fatt,y acids did proportionally increase as the concent,ration of valerylCoA and isobut,yryl-CoA increased. Isolation of thioleskmse. Fatt,y acidsynthesizing systems of potato t.uber spt.hesiaed both free acids and fatty acyl thiolesters. The formation of free acids related to the presence of a very active
t,hiolesterase in fatty acid synthetase prcparations. When the potato fatty acid synt,hetase (40-G % ammonium sulfate fraction) was fractionated on a Sephadex G-100 column (Fig. 17) and then followed by DEAE-cellulose column chromatography, the t.hiolesterase could be separated int.0 three enzyme peaks (Fig. 18). The results indicate that these thiolesterases are multiple forms with similar molecular weight. P,roperties of thiolesterase. Under the standard assay condition using palmit.yl1-14C CoA as the subst:ratc t.he enzyme shows a time-dependent palmit,-+CoA hydrolysis up to 1 min (l:ig. 19). The rate of hydrolysis is also dependent on the prot,ein concentration (Fig. 20). Since t.wo major enzyme forms were not well separated from DEAE-cellulose column, only fractions 70 and 90 were used for comparison. So detail study on peak 1 (fractions 50-60) was carried out. Under standard assay conditions the partially purified pot,ato thiolcsterase had a specific activity of 200 mpmoles of palmityl-Coh hydrolyzed per minute
422
HUANG
AND
c, c, $0 51 Cl2 Cl3 cu Cl5 Cl6 Cl7 16. The reaction product with propionylCoA a~ primer. The result shown in Fig. 16A is the control experiment in the absence of propionylCoA. The results with 14 wmoles and 35 mpmoles of propionyl-CoA are shown in Fig. 16B and Fig. 16C, respectively. FIG.
STUMPF
per milligram protein whereas the highly purified E. coli enzyme had a corresponding specific activity of 3700 (30). The pH optimum for palmityl-CoA hydrolysis of both fractions is very similar (Fig. 21). The control experiments in the absence of enzyme have no detectable hydrolysis in 1 min of assay over the pH range under test (from pH 5.5 to pH 8.7). Incubation of both enzymes with 2 rrrm of diisopropylfluorophosphate for 40 min at 25” had no effect on their hydrolytic activity. These two enzymes were also very resistant to sulfhydryl inhibitors. With 1 maI mill&molar concentration of Y-ethylmaleimide or p-chloromercuribenzoate, t.he enzyme lost only 20% of the activit.y. Kane of the sulfhydryl reagents (dithiothreitol, glutathione, and mercaptoethanol) activat,ed or stimulated the hydrolyt,ic activity. Chain-length specijicity
of thiolesterase.
Both thiolest,erases were t,ested for their substrate specificity for various acyl-CoAs. As shown in Fig. 22, both thiolesterases show exactly the same substrate specificit.y
TUBE NUMBER FIG. 17. Sephadex G-100 column chromatography profile of ammonium sulfate fraction. The assay W&B carried out under standard assay conditions with 21 amoles of palmityl1-W CoA (260,000 cpm) and 10 ~1 of enzyme from each fraction. The enzyme activity is expressed aa counts per minute of palmityl-1-W CoA hydrolyzed per 10 4 of reaction mixture during 1 min of incubation at 27”. -•-, protein concentration profile; +, palmityl CoA thiolesteraze activity.
FATTY
ACID
20
SYSTHETASE
40
60
OF Solarium luberosum
80
loo
lx:
123
140
TUBE NUMBER FIG. 18. DEAE-cellulose chromatography of Sephadex G-100 fract,ion. The condition of the separation was described in Methods. The assay was carried out under st.andard assay conditions with 21 qmoles of palmityl-1-W CoA (260,000 cpm) and 10 ~1 of enzyme from eachfraction.Enzyme act,ivit,y is expressed as count,s per minute of palmityl-1-W CoA hydrolyzed per 10~1 of reaction mixture during 1 min incubation at 37”. --a--, prot.ein coucentration profile; +-palmityl CoA thiolesterase activity.
for thioesters of long-chain fatty acids. At low acyl-CoA concentrations, the initial rates of hydrolysis of palmityl-, stearyl-, and oleyl-CoA were almost the same. At higher levels of acyl-CoA concentrations, t.he rate of hydrolysis of oleyl- and stearyl-CoA were much higher than palmityl-CoA. Myristyl-CoA and decanoylCoA were hydrolyzed at very low rates. Both potato thiolesterases were, therefore, specific for long-chain fatty acyl thiolesters. DISCUSSION
The fatty acid-synthesizing system isolated from potat. tuber is similar t.o the soluble synthetase of avocado mesocarp (9). When assayed with 14C-malonyl-CoA, the potato synthetase system required TPNH, E. coli ACP, and acetyl-CoA. The fatty acid-synthesizing activity of t,he potato tuber synthetase varied depending on the season and time of storage of t,he tissue. In general, the fat.ty acid synthetase
was very active as compared t.o the fatty acid synthetase isolated from high fat tissue. It is interesting t.hat the fatty acid synthetase itself was not the limiting factor for fatty acid synthesis in potato tuber-a high starch-low lipid storage Gssue. Unlike the fatty acid synthetase isolated from ot,her tissue, the potato system synthesized fatty acid from Cs : o up to Cls: ,, but formed very small amounts of Cle: ,,. The occurrence of various chain-length fatty acids in the reaction product was not due to t.he ut,ilization of E. coli ACP. Similar results were obtained with both avocado and chloroplast ACP’s. Earlier experiments in which 1J4Cacetate was incubated with intact potato tuber slices (28) clearly showed that the distribution of 14C was 18 % in palmit,ic acid, 30 % in stearic acid, and 51% in oleic acid. Xo 1°C count.s were associat.ed either with polyunsaturated fatty acids or with fatt.y acids of chain lengths less than C&.
414
HUANG
I
I
0.5
10
V
1 15
AKD
STUMPF
I 20
TIME Ctdrd
PH
FIG. 19. Time course of enzymatic hydrolysis of palmityl-1-W CoA. The assay was carried out under si-andard assay conditions with 25 mpmoles of palmi tyl-1-W CoA (125,000 cpm) and 5.5 pg of paritally purified enzyme from DEAF-cellulose chromatography. The enzyme activity is expressed as counts per minut,e of palmityl-1-W CoA hydrolyzed per 10 ~1 of reaction mixture during 1 min of incubation at 37”.
21. Effect on pH on palmit.yl-CoA thiolester&se activity. The reactions were carried out under standard assay conditions with 5 pg of protein from fraction “70” and 4% of protein from fraction “90” as shown in Fig. 18.---, fraction 70; -----, fraction 90. n , Tris buffer; 0, phosphate FIG.
apoo6000t ISE -G I-” 40002 2poo/
L/L2D
41)
60
pg PROTEIN 20. Protein concentration versus enzyme activity curve. The reactions were carried out. under standard assay conditions with 25 mgmolee of palmit,yl-1-W CoA (125,000 cpm) and partially purified palmityl-CoA thiolest,erase from DEAEcellulose column chromatography. The enzyme activity is expressed as counts per minute of palmityl-1-W CoA hydrolyzed per lO+d of reaction mixture during 1 min of incubation at 37”.
IO
FIG.
m
30
40
Acyl CcA X 10%
22. Chain-length specificity of thioleeterese. The reactions were carried out. under standard assay conditions with 5 pg of protein from fraction 70 ss shown in Fig. 18. The enzyme activity is expressed se m&moles of substrate hydrolyzed during 1-min incubation at 37”. FIG.
FATTY
ACID
SYNTHJZTASE
Thus, the partially purified synthetase would appear to represent only part of the total complement of enzymes responsible for the synthesis and further alteration of fatty acids in the intact tissue, namely, the enzymes for the conversion of CZ --+ Cls. Other evidence from t,his laborat,ory (29) clearly suggests that there are at least three discret.e systems involved in plant tissues for the construction of the hydrocarbon chain; (a) the Cz -+ CM system, (b) Cl6 -+ Cls syst,em, and (c) the CM + c20 . - . system. Of further interest, in addition to the free fatty acids produced in the react,ion mixture, considerable amounts of thiolesterbound fatty acids were formed. Octanoic, decanoic, and lauric acids were found as CoA esters, and myristic and palmitic acid and some unknown minor components, presumably the intermediates of fatty acid synthesis, were also found as ACP thiolesters. The formation of CoA t.hiolesters suggests t,hat a transferase transfers the acyl component of acyl ACP to CoA to form acyl-CoA. This result is similar to that observed by the yeast fatty acid synthet,ase complex (1). The absence of myristyland palmityl-CoA thiolesters can be explained by the observation t,hat these thiolesters are readily hydrolyzed by thiolesterases which favor the hydrolysis of long-chain fatty acyl CoAs (Fig. 22). The existence of myristyl and pahnityl ACP derivatives may relate to the relative low of thiolesterases toward fat.ty activity acyl ACP derivatives (30, 31). Detailed studies on fatty acid compositions under various assay conditions provided further knowledge on the chainlength specificity at the termination of fatty acid biosynthesis. The effect of malonyl-CoA concentration on the pattern of fatty acids synthesized by fatty acid synthebase complexes from yeast (16), rat liver, and rabbit mammary gland (17) have been well documented. The results obt,ained from potato fatty acid synthetase agreed with those obtained from yeast and mammalian tissue, namely, t.hat at. low malonyl-CoA concentrations shorter-
OF Solunum tuberosum
425
chain fatty acids were the major product (Fig. 9) and at high malonyl-CoA concentrations longer-chain fatty acids (C,, : ,,, CM : O) became predominant, (Fig. 9). Variat,ion of acetyl-CoA concentrations did not cause significant. change in t,he pattern of fatty acid synthesized (Fig. lo), even though the malonyl-CoA/acet,ylCoA ratio changes from 6.75 to 0.22. In contrast,, in the yeast fatty acid synthetase complex, the malonyl-CoA/acetyl-CoA concentrat,ion rat,io is considered as a factor influencing the chain length of products. In the yeast fatty acid synthet,ase complex t,here are 3 moles of 4’-phosphopantetheine per mole of synthetase complex which is composed of three functional synthetase units. The acyl moiety of both acet,yl-CoA and malonyl-CoA is capable of binding to the t,hiol group of 4’-phosphopant.etheine of fat,t.y acid synthet,ase complex (32, 33). The competition between acetyl-CoA and malonyl-CoA for this acyl-binding site (the sit.e of the chain-elongation react.ion) could account, for the observed shift in the chain length of synthesized fatty acids. In the pot,ato fatty acid synthetase t,hc &ketoacyl ACP synthetase catalyzes the following reaction (33) : R-CO-S-ACP + HS-E of R-CO-S-E + ACP-SH RCO-S-E + HOOCCHt CO-S-ACP e RCOCHe CO-S-ACP + CO* + HS-E
At, saturating ACP concentration t)he competition bet.ween acetyl-CoA and malonyl-CoA for ACP is not important. However, t.he format,ion of enzyme-bound acetyl-thiolester is import.ant in the initiat.ion of the condensation reaction. Presumably, the concent,ration of enzymcbound acetyl-thiolester is maintained at a relat.ively const,ant level over a wide range of acetyl-CoA concentration and therefore, the actual malonyl-CoA/acetyl-CoA ratio does not influence the fatty acid pattern in the final product,. Nevertheless, t,he malonyl-CoA concentration it.self is import,ant in governing the final product. The effect of TPNH concentration on t.he pattern of fatty acid synt.hesized is similar to that of malonyl-CoA; lower
426
HUANG
AND
STUMPF
TPNH concentrations favor shorter-chain 3. Hsn, R. Y., WASSON, G., AND PORTER, J. W., J. Biol. C’hem. 240, 3736 (1965). and higher TPNH concentrations favor 4. BURTON, D. N., HA.\NIK, A. G., AND PORTER, longer-chain fatty acids synthesis. At low J. W., Arch. Rio&em. Biophys. 126, 141 TPNH concentration there were no de(1968). tectable keto- and unsaturated fatty acid 5. MARTIN, D. B., HORNINQ, M. G., AND VACIEMS, which would suggest t.hat rate of reduction P. It., J. Biol. Chem. 236,663 (1961). is faster than the condensation reaction. 6. LEN.LARZ, W. J., LIQHT, R. J., .\ND BLOCH, K., No accumulation of triacetic acid was obPTOC. Nat. A&l. Sci. U.S.A. 46,640 (1962): served. 7. GOLDMAN, P., ALBERTS, A. W., AND VAQEMS, The effect of ACP concentration on the P. R., J. Biol. Chem. 256, 1255 (1963). 8. WAKIL, S. J., PUGH, E. L., AND SAUER, F., pattern of fatty acid synthesized by potato PTOC. Arat. Acad. Sci. U.S.A. 52, 106 (1964). synthetase was different from those wit,h 9. OVERATR, P., AND STUMPF, P. K., J. Biol. malonyl-CoA and TPNH. At low ACP Chem. 239,4103 (1964). concentration, t.he concentrations of acet,yl10. BROOKS, J. L., AND STIJMPF, P. K., AT&. ACP, malonyl-ACP, &9 well m fatt.y acylBiochem. Biophye. 116,108 (1966). ACP would be relatively low and the prob11. PUQH, E. L., AND WAKIL, S. J., J. Biol. Chem. ability of t,ermination of chain growth by 240,4727 (1965). transfer of CoA derivatives and then sub- 12. MA.JERUS, P. W., ALBERTS, A. W., AND sequent hydrolysis to form free acid would VAQELOS, P. It., J. Biol. Chem. 240, 4723 be less. The reaction product, is then mainly (1965). P. H. W., BOCK, 13. YANG, P. C., BUTTERWORTH, determined by t.hermodynamic determinants R. M., AND PORTER, J. W., J. Biol. Chem. which probably favor G: ,I and CIS : o syn242.3501 (1967). thesis. On the contrary, at, high ACP con14. L.%RRABEE, A. R., MCDANIEL, E. G., BACEERcentration the competit,ion between acetylMAN, H. A., AND VAQELOS, P. R., PTOC. Nat. ACP and other acyl-ACPs for condensaAcad. Sci. U.S.A. 54, 267 (1965). tion enzyme would be greater. In addition, 15. PUGH, E. L., SAUER, F., WAITE, M., TOOMEY, the increase in fatt,y acyl-ACP concentraR. E., AND WAKIL, S. J., J. Biol. Chem. 241, tions would favor t,hc transferase reaction 2635 (1966). which would transfer fatt.y acyl-ACP de- 16. SUMPER, M., DESTERHELT, D., RIEPERTINQER, C., .4ND LYNEN, F., EUT. J. Biochem. 10,377 rivatives to fatty acyl-CoAs and with sub(1969). sequent hydrolysis to free acids. CAEEY, E. M., DILS, R., AND HANSEN, H. J. M., 17. The effect of pH on the reaction product Biochem. J . 117,633 (1970). is more complicated, because it may involve R. D., GRIDDLE, R. S., AND STUMPF, several react,ions which are undefined in 18. SIYONI, P. K., J. Biol. Chem. 242,573 (1967). the potato tuber. 19. SIMON, E. J., AND SHEMIN, D., J. Amer. Chem. The isolat.ion of t,hiolesterase from potato Sot. 76, 2520 (1953). tuber suggests that at, least. two isozymes of 20. GOLDMAN, P., AND VAQEMS, P. R., J. Biol. similar molecular weight exist,. Unlike t,he Chem. 236.2620 (1961). thiolesterases from B. c&i (30, 31), both 21. STADTMAN, E. R., in “Methods in Ensymology” (S. Colowick and N. 0. Kaplan, eds), potato thiolesterases have similar properties. Vol. 3, p. 931. Academic Press, New York The thiolesterases in potato tuber are very (1957). active and have relatively higher V,., 22. ELLMAN, G. L., Arch. Biochem. Biophys. 62, than h’. coli enzymes. ACKNOWLEDGMENTS
We wish to acknowledge the assietance of Mrs. Barbara Clover in this investigation. REFERENCES F., Fed. Proc. 20,941 (1961). 2. BREBSLER, R., AND WAKIL, S. J., J. Biol. Chem. 836, 1643 (1961).
1. LYNEN,
70 (1959). 23. HuANQ, K. P., Anal B&hem. 27, 98 (1970). 24. MILLER, G. L., Anal. Chem. 91,964 (1959). 25. LOOMIS, W. D., in “Methods in Enzymology” (J. M. Lamenstein, ea.), Vol. 13, p. 555. Academic Press, New York (1969). 26. HARRIS, R. V., AND J-ES, A. T., Biochim. Biophys. Acla 166,456 (1965). 27. BRADY, R. O., J. Biol. Chem. 236,3099 (1960).
FATTY 23. WILLEMOT,
C., AND
STUMPF,
46,679 (1967). 29. HARWOOD, J., AND
ACID
SYNTHETASE
P. K., Can.J. Bot.
STUMPF,
P. K.,
Arch.
Biochem.Biophys. lpB, 281 (1971). 30. BARNES, E. M., JR., AND WAKIL, S. J., .I. Biol. Chem. 243, 2966 (1968). 31. BARNES, E. M., JR., SWINDELL, A. C., AND WAKIL, S. J., J. Biol. Chem. 246,3122 (1970). 32. LYNEN, F., OMTERHELT, D., SCHWEIZER, E.,
OF SoEanum tuberosum AND
WILLECKE,
mentalization Metabolism,” York (1968). 33. GREENSPAN, VIGELOS,
427
K., “Cellular Compartand Control of Fatty Acid p. 7. Academic Prezs, New
$1. D., ALBERTS, A. W., P. R., J. Biol. Chem.%,
AND
6477
(1969).
34. Ta.u,xs, E. Cr., :IND BR.~DT, 1%.O., J. Amer. Chem.Sot. 33, 2972 (1960).
OF
13IOCHEMISTIIY
AND
BIOPHYRICS
412427
Iti,
Fat Metabolism XLIV. Fatty
Acid Synthesis from
in Higher by a Soluble
Solarium
II;. I’. HUANG2 Department
of Biochemislr~/
and
(1971)
Received
Fatty
Acid Synthetase
tuberosum’ 1’. Ii. STUMl’F
bNI)
Biophysics,
Plants
&ziversit!/ December
of
California,
Davis, California
96616
9, 1970
A highly st,able pot.ato tuber preparat.iott containing the fatty acid syttthetsse ettzymes has been obtained and the cottdit,iotts for t,he de not~o synthesis of fat& acids have been examined. Uttder st,attdard cottditiotts the syst.em formed CR to Ct6 satttrated fatky acids. While 50-70~0 of t,lte newly syttthesixed produ& ocrnrred as free fat.ty acid, t,he remainder accumulated as ACP t.hiolesters (CIA and Ctsj and ss CoA thiolesters (CS , C,U , attd Ct?). The cottcentratiotts of malottyl-CoA, TPNIT, ACP, and pH greatly altered the pat,tertt of fatty acids syni.hesixed. In addition to acetylCoA, propiottyl and but.yryl-CoA served as elrective acceptors to form odd and even long-chain fat,iy acids, respectively. Palmityl thiolest.erase is present in preparations of the synthesizing system although the enzyme can be separated from the syttthetase enzymes. IDs specificit.y is limited lo the C~S and Ct8 fatt.y a.cyl CoA derivutives.
Fatt.y acid synthetases have been characterized from a number of sources,notably yeast (l), avian liver (2, 3), mammalian liver (4), adipose tissue (5), Escherichia coli (6-S), and higher plant,s (9, 10). The fatt,y acid synthetases from yeast., (pigeon) liver, (rat) liver, and Mycobacterium phlei are isolated as tightly bound complexes. However, the fatty acid-synt,hesizing syst,em from E. coli consists of at least, six soluble enzymes and a low molecular, heat-stable protein known as acyl carrier prot,ein. This latter protein contains 4’-phosphopantetheine as a prosthetic group and functions as a cofactor in fat,ty acid biosynthesis (11, 13). The end products of the yeast fat,ty acid synthetase complex are fatty acyl CoA derivatives (1). However, the end products 1 This investigation was supported by NSF Grant, GB-19733X. 2 Present address: National Institute of Arthrit is and Metabolic Diseases, National Inst.it,utes of Health, Public Health Service, Bet,hesda, Maryland. 412
of avian liver (3, 13), mammalian liver (4), and adipose tissue (5, 14) fatty acid synthetase complexes are free fatty acids whereas in E. coli, fatty acyl ACP derivat.ives have been shown t.o be the reaction products (15). As yet there is no detailed study on the react.ion products of plant, fatty acid synthet.ases. The compositions of fat.ty acid synthesized by the yeast fatty acid synthetaxe complex have been shown to be determined by the malonyl-CoA/acetyl-CoA concent&ion ratio. The studies of both rabbit, mammary gland and rat liver fat,t,y acid synt,hetase complexes suggested that t.he pat.tern of fat.ty acids synthesized is determined by malonyl-CoA concenbrtltion (17). A number of st.udies on fatt.y acid synthesis have utilized organisms and tissues of high lipid contents, such as adipose tissue, liver, and fatty seeds. These organisms and tissues have obvious advant,ages in yielding highly acbive synthetases. However, with the exception of work in the pea system,
FATTY
ACII)
SYSTHETASE
there is very little information available about the fatty acid-synthesizing systems from t,he low lipid-containing plant tissues. This paper reports on the properties of a soluble fat,tv acid synthetase from a low lipid-contairiing t.issue, namely the potato tuber. MATERIALS Potato tubers (Solarium tuberosum) of White Rose variety were purchased from local markets. Dithiothreit,ol and TPNH were obtained from Calbiochem; chromatographically pure CoA from P-L Biochemicals, Milwaukee; Sephadex G-lOO120 and DPNH from Sigma Chemical Company; malonic acid 1,3-14C, decanoic-1-‘4C, lauric-l-l%, myristyl-1-‘4C CoA, stearyl-l-l% CoA, from New England Nuclear Corporation; palmityl-1-‘4C CoA from Dhom Products, Ltd.; polyclar AT from General Aniline and Film Corporation, Dye Stuff and Chemicals Division; 2-mercaptobenzothiazole (MERCAP) from K and K Laboratories, Inc; chromat,ography media (ITLC-SC; type, 20 X 20 cm) from Gelman Instrument Company; l)EAl<-cellulose (DE-52) from Whatman. METHODS E. coli ACP was isolat,ed according t.o the method of Simoni et al. (18). Malonyl-1,3-14C CoA was prepared according to the method of Trams and Brady (34) modified by Dr. Vageos, Washington University. Acetyl-CoA and propionyl CoA were made according t,o the method of Simon and Shemin (19). Butyry-CoA, crotonyl CoA, isobnt.yryl CoA, Valery1 CoA, and caproyl Co.4 were prepared according to the same method except, t.he reactions were run in 50yc acet.one in order to aid in the solubility of various anhydrides. I)ecanoyl-l-l% CoA and lauryl-l-l% CoA were synthesized by t.he mixed anhydride method of Goldman and Vagelos (20). Fatty acyl thioesters of CoA were determined by t.he absorption at 232 nm (21) and the t.hiol determinat,ion by Ellman’s method (22) after cleavage with neut,ral hydroxylamine. Preparation of fatty acid synlhelase. White pot,at,o tubers (Solarium tuberosum, var. White Rose), purchased from local markets, were peeled and cut int.0 small chunks. For each 300 g of potato, t.he following components were added: 30 g of acid-washed polyclar AT, 7.5 g of acidwashed Norit A, and 60 ml of 0.5 M potassium phosphate buffer, pH 7.2, with 0.05 M mercaptoet.hanol and 0.6 rnM of 2-mercaptobenzothiazole at 4”. The mixt,ure was homogenized in a Waring Blendor for 2 min at top speed. The slurry was squeezed through two layers of cheesecloth, and
OF
Solonctrrl
/uberos,o)~
413
the filtrate was centrifuged at 48,OOOg for 26 min in a Sorvall IX-2B centrifuge. The pooled supernatant fluid was 230 ml at, pH 6.8. A 0.05 vol of 1 Y MnCl2 (11.5 ml) was added slowly to the crude ext.ract with constant. stirring. The pH was maintained at 7.0 by adding 6 N KOH. The turbid solution was then centrifuged at 48,000g for 20 miu. To 236 ml of M&l:! supernatant fluid 57.2 g of solid ammonium sulfate were added gradually to @lo sat.uration. The solution was cent.rifuged at 48,000g for 20 min, and t,he precipitate was discarded. Ammonium sulfate (34.6 g) was added to the supernatant fluid (210 ml) to 65% saturation. The result.ing precipitate was collected by centrifugation and the supernatant fluid was discarded. The precipit,ate was dissolved in 0.01 M potassium phosphate buffer (pH 7.0) with 0.1 mM EDTA to a final protein concentration of 20 mgi ml. The solution was t,hen frozen in l-ml aliquots and stored at -15” under N) until ready for use. Preparation of palmilyl CoA thioleslerase. The 4&65yc ammonium sulfate precipitate was used as the start.ing material. The concentrated protein solution (12 ml containing 917 mg protein) was fractionated on a Sephadex G-100 column (4.1 X 110 cm) equilibrated wit.h 0.01 M potassium phosphate buffer (pII 7.0) containing 1 mM mercaptoethanol. Fract.ions of 8.5 ml were collect,ed and assayed for palmit,yl-CoA t.hiolesterase activity. Effluent fractions of high activit.y from Sephadex column were pooled (450 mg of prot.ein) and put onto a DEAE-cellulose (Whatman DE-52) column (2.5 X 24 cm) which had been equilibrated with 0.01 ?M pot.assium phosphate buffer (pH 7.0) with 1 my mercaptoethanol. After washing with t.he same buffer, the column was eluted with a linear gradient made from 500 ml of 0.01 M potassium phosphate (pH 7.0) with 0.07 M pot.assium chloride and 500 ml of 0.25 M potassium chloride in the same buffer. Fractions of 5 ml were collected and assayed for palmityl-CoA thiolesteraae aetivity. Assay of jaf1.y acid synthetase. The standard reaction mixture contained potassium phosphat,e buffer, pH 7.0, 5Ormoles; purified E. coli ACP, 70 pg; acetyl-CoA, 0.07 pmole; DPNH, 0.2 pmole; TPNH, 0.2 pmole; malonyl-1,3-14C CoA, 0.032 amole (2@0,000 cpm); and 1 t.o 2 mg of potato fatty acid synthetaae in a total volume of 0.5 ml. The incubations were carried out at 37” for 30 min and stopped by t,he addition of 0.2 ml of 40% KOH. After 30 min of hydrolysis at 80”, the reaction mixtures were acidified with 0.25 ml of concentrated HCl. After addit,ion of 2 ml of water, the reaction mixtures were extracted wit.h 16 ml of CHClyjCIIaOH (7.5:5.5). Separation into two phases were acheived by t.he addition of another 2 ml of water. The chloroform layer was washed
414
HUANG
AYI) _
with 5 ml of solution containing 0.5% malonir acid and 0.5% acetic acid in 1 N HCl sat.urated with sodium chloride and dried over anhydrous NaW. An aliquot was then dried in a scinit,llation vial and counted in a Packard liquid scintillation counter by the usual procedures. Assay of palntilyl CoA thiolesterase. The assay procedure for palmityl CoA thiolesterase was baaed on t,he enzyme-cat,alymed release of radioactive fatty acid from its thioest.er of CoA. The assay mixture contained 10 pmoles of Tris chloride (pH 8.0), 0.025amole of palmityl-1-W CoA (125,000 cpm), and 3-10 pg of thiolest.erase in a t.otal volume of 0.1 ml. The reaction was carried out in a 10 X 75-mm test tube and incubated at 37” for 1 min. After 1 min of incubation the reaction was terminat.ed by the addition of 0.01 ml of glacial acetic acid at 2”. Ten-microliter aliquots of the reaction mixture were put onto a 1.4 X 6.0-cm strip of Gelman chromatography media. The reaction product., free fat,ty acid, was separated from it,s substrat,e, acyl-CoA by placing the strip into a small jar containing a solvent system of water-saturat,ed ether/formic acid (7: 1). Separation was accomplished in 4 min. The reaction product moved to the solvent front and the substrat,e stayed at the origin (23). Free fatt.y acid produced in t.he reaction could be measured by cutting the strip 1.7 cm below the top, and the radioactivit,y was counted in a scintillation counter. Analysis of reaction producl. The products of fatty acid synt,hetase reaction were analyzed by an Aerograph Model A-90 P2 Nuclear Chicago Biospan Model 4998 radiomonitoring system after met.hylat,ion with diazomethane. Various chain1engt.h fatty acids were separated on a 5 ft X 0.25 in. colurm~ packed wit.h 12% diethylene glycol succinate (DEGS) on Anakrom P (60-70 mesh). Separation was carried out by temperature programming from 120-200” with standard fat,t,y acids (from CsYOto C19:0) in each injection. The percentage distribution of various radioact,ive fatty acids were calculated from t,he integrator which registered the total radioactivity of each component. Protein concentrat.ion was determined by the Lowry procedure according to Miller (24). RESULTS
Properties of fatty mid synthetase. Potato tubers contain phenolic compounds which complex either reversibly with proteins by hydrogen bonding or irreversibly by oxidation to quinones followed by covalent condensation of t.he quinones wth the react.ive groups of proteins (2~). The crude extract. was, therefore, relatively unst,able. However, by employing the Polyclar AT-Xorit
,YTUW’F *
A - mercaptoet.hanol - 2 - mercaptobenzo - t hiazide system, t,he partially purified synt.hetase system was stable for mont.hs at -115” under Na. Under standard iwsay condit:ions 0.32 mpmoles of malonyl-CoA was incorporated per minute per milligram protein into cle ~IOVO synthesized fatty acids. Repeated freezing and thawing caused considerable loss in activity. When the prot.ein solut,ion was cenkifuged at, 198,000~/ for 5 hr over 70 %i of t.he synt.hetasc activity remained in t.he supernatant fluid. Unlike the fatty acid synthetaw complex from yeast and mammalian tissue, potato fat.ty acid synthetase was similar to t,he E. coli system and ot.her higher plant fatky acid-synthesizing systems (9). Requirenwnts
Jar
fatty
acid
syrrthesis.
Under the standard assay condition, as showy in Fig. 1, fatty acids synthesized bv the pot,at,o system increased linearly \I-ith respect to t,ime for 30 min. The enzyme system showed an absolute requirement for E. wEi ACP (Fig. 8) and TI’NH (Fig. 3). Becauseof the existence of malonylCoA decarboxylase in the enzyme preparation, acetyl CoA was not absolutely required (Fig. 4). However, :t clear de-
2awo
Iaow
l
v
Y
I
t
I
I
I
I
20
40
60
so
100
120
TIME (min.) FIN. 1. Time course of fatty acid synt,hesis under standard assay conditions with 2 mg of protein. The enzyme activity is expressed as total radioactivity (count,s per minute) incorporated int,o fatt.y acid.
FATTY
ACID
40
SYNTHETASE
so
100
OF Solanccm
160
200
240
415.
Ircbermum
280
320
JG AU FIG.
2. Requirement
of E. coti ACP for fatty acid synthesis.
were acidified to pH 2.0 then followed by pentane ext.ract,ion. About, 50-700/u of the react,ion products were free acisd. The rest. of the pent,ane-insoluble reaction products could be recovered by furt,her pent.ane extraction aft.er acidificat.ion of t.he alkaline hydrolyzate. The alkali hydrolyzable port.ion was considered to be thioester-bound fatty acid. In order t,o characterize the properties of thioester-bound fatty acids, the reaction
40
80
120
160
203
243
280
mp mole TPNH
FIG. 3. Requirement synthesis.
of TPNH
for fatt.y acid
pendence of acet.yl CoA as a primer was observed. Both DPXH and dit,hiothreitol stimulated the reaction. Addition of 0.2 rmmole of DPXH or 5 pmoles of dithiothreitol t,o the react,ion mixture increased the syntbesizing capacity about, 10%. Thus DPSH is not an absolute component. in I t.he reduction steps of fatty acid synthesis. 20 40 60 80 100 The effect of various sulfhydryvl reagents mp mde acetyl COP, on fatty acid synthesis is shown in I+?g. 5. A?halysis of the reaction proclucts. After FIG. 4. Requirement. of acetyl-CoA acid synt.hesis. 30 min of incubation, the reaction mixtures
120
140
for fatty
416
IIUANG
AND STUMPF
mixtures were separated on a standardized Sephadex G-100 column. As shown in Fig. 6, there are three radioactive peaks corresponding to an apparent molecular weight, of 63,000, 8-9,000, and l-2,000 from the standard curve. Each of the radioact.ive peaks was pooled and acidified after alkaline
20
40
60
80
1M
hydrolysis. Peak 1 contained relatively low pentane-ext.ractable radioactivity which would suggest polar components. .Further investigations were not carried out, on this fraction. Pentane extra&ion of peak 2 yielded myristic acid, palmitic acid, and unknown components, presumably hydroxy acids. Hecause peak 2 corresponds t,o approximat.ely NOO-9000 mol wt, these components may be bound t.o h’. coli ACP which has a molecular weight of about 8700. Peak 3 contains the highest amount, of radioactivit,> wiith a molecular weight around 1000-2000. Pentane extract,ion of the acidified alkaline hydrolyzate yielded octanoic, decanoic, and lauric acids. Ether extraction of the same fraction gave malonate. Obviously peak 3 cons&s of acyl CoA esters. The synthesized fatty acids were separated by GLC and degraded by the permanganate oxidation of Harris and *James (26). The resu1t.s are shown in Table I. The con&ant ratio of radioactivity to mass
120
CONC. X IO%
FIG. 5. Effect of sulfhydryl acid syntheaie.
reagents on fatty
I
40
60
80
100
120
140
TUBE NUMBER FIQ. 6. Separation of the reaction product by Sephadex G-100 column fractionation. Separations were carried out, in a calibrated Sephadex G-100 column (1.6 X 120 cm) at 3-4’. Blue dextran-2000 wa8 added to the reaction mixture just before putting the sample into the column. After separation, a O.l-ml aliquot wea put into scintillation vial with 10 ml of Bray’s solution. The radioactivit,y was determined by a Packard liquid-scintillation counter. -a--, ODzao; +, radioactivity.
FATTY
ACID
SYXTHETASE
OF SoZun,rnz ~rtberosror~
TABLE I Miss Rxrros OF ~~YItISTITI ?LND P..\LMIT.\TV _ .- ..p--.---Parent compound KMllO4 _-.-.. .- ..---... Oxidation product C,S CV ..~.. __ ...3.18 Cl8 3.18 Cl:, 3.0 1.36 Cl, 1.45 2.99 Cl3 3.1 1.14 Cl2 -.R Ratios were calculated using the peak area of radioactive trace and mass trace.
117
16;O
ILUIIOXTI~ITY:
K n
60
6.6
70
PH 7. Effect of incubation pH on the fatty acid synthetase activity. The pH values indicated in the figures are t,he actual reaction pH’s measured by the pH meter. FIG.
indicated myrktate
t,he de )iovo synt,hesis of bot.h and palmitate. pH optimum of fatty acid synthesis. The pH optimum of potato fatty acid synthetase is approximately pH 7.0 in phosphate buffer aa shown in Fig. 7. Pattern of fatty acids syn.thesizedby potato fatty acid synthetase.Figure 8 shows t,he
typical pattern of radioact.ive fatty acids produced under the standard assay condition. Each of the individual fatty acid methylesters was collect,ed from gasliquid chromatography and identified further by comparing with standard fatty acid methylesters. Octanoic acid, decanoic acid, lauric acid, myristic acid, palmitic acid, and trace amount, of stearic acid were
FIG. 8. Analysis of the reaction product methyl est.ers in a 5 ft X 0.25 in. 1)EGS column. Separation was carried out by temperature programming from 120-200”.
synthesized from malonyl-CoA. Ko unsaturat.ed fat,ty acid was detectable. Unlike other fatty acid synt,hetases from higher plants, which synthesize mainly palmitat,e and st.earate, the potat,o fat,ty acid synthetase synthesizes fatt,y acids of varying chain length (from Cs t.o Cl,). Because of t,he versatility of t.his system in synthesizing varying fatt,y acids, the effects of varying the concentrat,ions of malonyl-CoA, acetyl-CoA, TPNH, and ACY on t.he final pr0duct.s were examined. As shown in Fig. 9, the effect of malonylCoA concentration on t.he chain-length of the reaction pr0duct.s is obvious. At low malonyl-CoA concentration, t,he reaction products were widely diskibuted among various chain-length fatty acids, whereas, at) high malonyl-CoA concentrat,ion, palmitnte was the predominant fatty acid.
418
HUANG
AND STUMPF
60-B 50-
II
40 3020 10 l-l
l-l
6O-c
60-C so-
50 -
40 30 20 r 10 I-l
l-l
CS
=I0
CS Cl2
Cl4
cl6
FIG. 9. Effect of malonyl-CoA concentration on the fatt,y acid composition in the reaction prodoct. The reactions were carried out under st,andard assay conditions with various amounts of malonyl-CoA. The malonyl-CoA concentrations used in the experiments ae shown in Fig. 9A, B, and C were 4.9, 24.5, and 65.2 mpmoles, respectively.
When the malonyl-CoA concentration was further decreased to 3.26 /IM, little if any myristate or palmitate accumulated. Octanoate, decanoate, and laurate became the predominant products. The shift in the patt,ern of fat.t:y acid with respect to malonyl-CoA concentration agreed with the results obt.ained from yeast., rabbit mammary gland, and rat liver, ail multienzyme complexes. When malonyl-CoA concentration was kept constant with variable amounts of scetyl-CoA in the assay system, the results are shown in Fig. 10. The fatty acid compositions of the reaction product kept
Cl0
Cl2
Cl4
cl6
FIQ. 10. Wfect of acetyl-CoA concentration on t,he compositions of fatty acid synthesized. The acetyl-CoA concentrations used in the experiments aa shown in Fig. lOA, B, and C were 4.8, 23.8, and 142.5 mpmoles, respectively.
fairly constant, over a wide range of acetylCoA concentrations. Although the malonylCoA/acetyl-CoA concentration ratios vary from 6.75 to 0.22, no significant changes were observed. Thus t,he absolute rather than the relative concent.rat,ion of malonylCoA is critical. The effect. of TPNH concentration on fatty acid composition of the reaction product is shown in Fig. 11. The results indicat,ed that at lower TP3H concentration t>he palmitate synthesis was less favorable, and at higher TPXH concentration my&ate and palmitate became t,he predominant products. Figure 12 shows the striking effect of ACP concentration on the fatty acid composition of the final products. -4t low ACP
FATTY
60
B
ACID
SYNTHETASE
41.9
tuberosum
n
50 Q
OF Xolanum
60
B
7
40 50 40 30 20
v
10
=I0 CS Cl2 cl4 cl6 FIG. 11. Effect of TPNH concentration on the composition of fattt,y acid synthesized. The TPNH concentrations used in the experiments as shown in Fig. llA, B, and C were 4.0, 40.0, and 120.0 mpmoles .
concentration (Fig. 12A), palm&ate was the major component which accounted for almost 80% of the t.ot,al product. However, at higher ACP concentration (Fig. 12C) various chain-length fatty acids were equally distributed. Myristate and palmitate synthesis were highly reduced under high ACP concentration. As shown in Fig. 13, the change in the reaction mixture pH also greatly perturbs the composition of reaction products. Palm&ate was sharply decreased from 52% of total fatty acid synthesized to only 270 when the pH was changed from 6.3 to 5.3. At higher pH value, shorter chain-length fatty acids (C,-CIZ) became the major products. These results clearly indicate the com-
n
l-l
n
%
'10
'12
'14
'16
FIG. 12. Effect of ACP concent,ration
on the composition of fatt,y acid synthesized. The ACP concentrations used in the experiments as shown in Fig. 12a, B, and C were 6.9, 55.2, and 256pg, respectively.
plexky of conditions which participate in the det,ermination of chain length of de uovo synthesized fatty acids. It is quite possible that superimposed on the enzymes involved in synthesizing the fatty acids are ancillary enzymes, such as malonyl-CoA decarboxylase, which would regulate the level of malonyl-CoA and long-chain acyl t,hioesterases which would alter in activity as a fun&ion of pH. Sulfhydryl reagents can either activat,e or inactivat,e the total synthesis of fat,ty acids. They also affect t,he composition of the reaction product t.o a different extent. As shown in Fig. 14, three sulfhydryl reagents at two concentration levels were
4'20
HUANG
AND STUMPF
60 60 i?
60 I
62
40
:
30
B
% pti 7.35
20 10 l-l 60
rl
’ pH 6.30
50 40 30
l-l c,
%I
c,
c,,
c14
‘16
FIG. 13. Effect of incubat,ion pH on the composition of fatty acid synt.hesized. The buffers used in t,hese experiment.5 as shown in Fig. 13A, B, and C were phosphat,e buffer, pH 6.4-pH 7.3.
compared to the control wit.hout. adding sulfhydryl reagent. In general, in t,he absence of -SH reagent, palmitate and ~~ristatc were t,he major products. Addldlon of -SH reagent decreased palmitate formation. C;lutat.hione (Fig. 14~) was more effective than dithiothreitol and mercaptoethanol.
Cl0
52
Cl4
cl6
FIG. 14. Effect of sulfhydryl reagents on the composition of fatty acid synthesized. Various concentration levels of glut.at.hione (A), dithiothreitol (B), and mercaptoethanol (C) were used as indicated. 0, control experiment without sulfhydryl reagent; q , reaction mixture contains 1 mM of respective sulfhydryl reagent; n , reaction mixture contains 10 mM of respect.ive sulfhydryl reagent.
CoA were less effect,ive. The low rat.e of fatty acid synthesis with caproyl-CoA as primer could be relat.ed t.o possible low activit,y of acyl-CoA-AC1 transferase towwd caproyl-CoA. The relatively low activity of crotonyl-Cob as t,he primer is similar to that of rat brain enzyme (27). The products of synthesis were extracted, Effect of various acgl CoA as th.e “primmer” and analyzed as described of jattg acirr! synthesis. When fatty acid methylated, synthetase assays were carried out under before. Odd-chain (derived from propionylCoA and valeryl-CoA) and branched-chain standard assay conditions with various fatty acids short-chain acyl Cohs as primers, the rate (derived from lsobutyryl-CoA) of fatty acid synthesis was markedly af- were easily identified by gas-liquid chromatography. The coexistence of even-chain fected. As shown in Fig. 15, butyryl-CoA and propionyl-CoA were bet.ter acceptors and odd-chain fatty acids in the propionylthan acet,yl-CoA. However, valeryl-CoA, CoA and valeryl-CoA experiments suggest.ed crotonyl-Cob, isobutyl-CoA, and caproylthe formation of acetyl--CoA from malonyl-
FATTY
I
ACID
SYSTHETASE
I
,
I
20
40
60
mpmole
431
OF Solanrtna fztberosum
80
I
I
I
I
100
120
140
160
Acyl CaA
FIG. 15. Effect of various acyl-CoA’s OILthe rate of fatty acid synt,hesis. The reactions were carried out under standard assay conditions with various amolmts of nonradioactive short-chain acyl-&.4’s as primers.
CoA by the malonyl-Co-4 decarboxylase reaction. The coexist.ence of even-chain and branched-chain fatty acids was also observed in isobut,yryl-CoA experiments. The competition of various acyl-CoAs w&h acetyl-CoA as t,he primer for fatt) acid synthesis can be seen from the results obtained in the propionyl-Co-4 experiments (Fig. 1G). The results showed that at higher propionyl-CoA concentrations there was no det.ectable even-chain fatty acids in the reaction product. As primers, valeryl-CoA and isobut,yr\rlCo-4 did not stimulate synthesis above t,he background level of synthesis in their absence. However, the ratio of odd-chain fatty acids and branched-chain fatty acids to even-chain fatt,y acids did proportionally increase as the concent,ration of valerylCoA and isobut,yryl-CoA increased. Isolation of thioleskmse. Fatt,y acidsynthesizing systems of potato t.uber spt.hesiaed both free acids and fatty acyl thiolesters. The formation of free acids related to the presence of a very active
t,hiolesterase in fatty acid synthetase prcparations. When the potato fatty acid synt,hetase (40-G % ammonium sulfate fraction) was fractionated on a Sephadex G-100 column (Fig. 17) and then followed by DEAE-cellulose column chromatography, the t.hiolesterase could be separated int.0 three enzyme peaks (Fig. 18). The results indicate that these thiolesterases are multiple forms with similar molecular weight. P,roperties of thiolesterase. Under the standard assay condition using palmit.yl1-14C CoA as the subst:ratc t.he enzyme shows a time-dependent palmit,-+CoA hydrolysis up to 1 min (l:ig. 19). The rate of hydrolysis is also dependent on the prot,ein concentration (Fig. 20). Since t.wo major enzyme forms were not well separated from DEAE-cellulose column, only fractions 70 and 90 were used for comparison. So detail study on peak 1 (fractions 50-60) was carried out. Under standard assay conditions the partially purified pot,ato thiolcsterase had a specific activity of 200 mpmoles of palmityl-Coh hydrolyzed per minute
422
HUANG
AND
c, c, $0 51 Cl2 Cl3 cu Cl5 Cl6 Cl7 16. The reaction product with propionylCoA a~ primer. The result shown in Fig. 16A is the control experiment in the absence of propionylCoA. The results with 14 wmoles and 35 mpmoles of propionyl-CoA are shown in Fig. 16B and Fig. 16C, respectively. FIG.
STUMPF
per milligram protein whereas the highly purified E. coli enzyme had a corresponding specific activity of 3700 (30). The pH optimum for palmityl-CoA hydrolysis of both fractions is very similar (Fig. 21). The control experiments in the absence of enzyme have no detectable hydrolysis in 1 min of assay over the pH range under test (from pH 5.5 to pH 8.7). Incubation of both enzymes with 2 rrrm of diisopropylfluorophosphate for 40 min at 25” had no effect on their hydrolytic activity. These two enzymes were also very resistant to sulfhydryl inhibitors. With 1 maI mill&molar concentration of Y-ethylmaleimide or p-chloromercuribenzoate, t.he enzyme lost only 20% of the activit.y. Kane of the sulfhydryl reagents (dithiothreitol, glutathione, and mercaptoethanol) activat,ed or stimulated the hydrolyt,ic activity. Chain-length specijicity
of thiolesterase.
Both thiolest,erases were t,ested for their substrate specificity for various acyl-CoAs. As shown in Fig. 22, both thiolesterases show exactly the same substrate specificit.y
TUBE NUMBER FIG. 17. Sephadex G-100 column chromatography profile of ammonium sulfate fraction. The assay W&B carried out under standard assay conditions with 21 amoles of palmityl1-W CoA (260,000 cpm) and 10 ~1 of enzyme from each fraction. The enzyme activity is expressed aa counts per minute of palmityl-1-W CoA hydrolyzed per 10 4 of reaction mixture during 1 min of incubation at 27”. -•-, protein concentration profile; +, palmityl CoA thiolesteraze activity.
FATTY
ACID
20
SYSTHETASE
40
60
OF Solarium luberosum
80
loo
lx:
123
140
TUBE NUMBER FIG. 18. DEAE-cellulose chromatography of Sephadex G-100 fract,ion. The condition of the separation was described in Methods. The assay was carried out under st.andard assay conditions with 21 qmoles of palmityl-1-W CoA (260,000 cpm) and 10 ~1 of enzyme from eachfraction.Enzyme act,ivit,y is expressed as count,s per minute of palmityl-1-W CoA hydrolyzed per 10~1 of reaction mixture during 1 min incubation at 37”. --a--, prot.ein coucentration profile; +-palmityl CoA thiolesterase activity.
for thioesters of long-chain fatty acids. At low acyl-CoA concentrations, the initial rates of hydrolysis of palmityl-, stearyl-, and oleyl-CoA were almost the same. At higher levels of acyl-CoA concentrations, t.he rate of hydrolysis of oleyl- and stearyl-CoA were much higher than palmityl-CoA. Myristyl-CoA and decanoylCoA were hydrolyzed at very low rates. Both potato thiolesterases were, therefore, specific for long-chain fatty acyl thiolesters. DISCUSSION
The fatty acid-synthesizing system isolated from potat. tuber is similar t.o the soluble synthetase of avocado mesocarp (9). When assayed with 14C-malonyl-CoA, the potato synthetase system required TPNH, E. coli ACP, and acetyl-CoA. The fatty acid-synthesizing activity of t,he potato tuber synthetase varied depending on the season and time of storage of t,he tissue. In general, the fat.ty acid synthetase
was very active as compared t.o the fatty acid synthetase isolated from high fat tissue. It is interesting t.hat the fatty acid synthetase itself was not the limiting factor for fatty acid synthesis in potato tuber-a high starch-low lipid storage Gssue. Unlike the fatty acid synthetase isolated from ot,her tissue, the potato system synthesized fatty acid from Cs : o up to Cls: ,, but formed very small amounts of Cle: ,,. The occurrence of various chain-length fatty acids in the reaction product was not due to t.he ut,ilization of E. coli ACP. Similar results were obtained with both avocado and chloroplast ACP’s. Earlier experiments in which 1J4Cacetate was incubated with intact potato tuber slices (28) clearly showed that the distribution of 14C was 18 % in palmit,ic acid, 30 % in stearic acid, and 51% in oleic acid. Xo 1°C count.s were associat.ed either with polyunsaturated fatty acids or with fatt.y acids of chain lengths less than C&.
414
HUANG
I
I
0.5
10
V
1 15
AKD
STUMPF
I 20
TIME Ctdrd
PH
FIG. 19. Time course of enzymatic hydrolysis of palmityl-1-W CoA. The assay was carried out under si-andard assay conditions with 25 mpmoles of palmi tyl-1-W CoA (125,000 cpm) and 5.5 pg of paritally purified enzyme from DEAF-cellulose chromatography. The enzyme activity is expressed as counts per minut,e of palmityl-1-W CoA hydrolyzed per 10 ~1 of reaction mixture during 1 min of incubation at 37”.
21. Effect on pH on palmit.yl-CoA thiolester&se activity. The reactions were carried out under standard assay conditions with 5 pg of protein from fraction “70” and 4% of protein from fraction “90” as shown in Fig. 18.---, fraction 70; -----, fraction 90. n , Tris buffer; 0, phosphate FIG.
apoo6000t ISE -G I-” 40002 2poo/
L/L2D
41)
60
pg PROTEIN 20. Protein concentration versus enzyme activity curve. The reactions were carried out. under standard assay conditions with 25 mgmolee of palmit,yl-1-W CoA (125,000 cpm) and partially purified palmityl-CoA thiolest,erase from DEAEcellulose column chromatography. The enzyme activity is expressed as counts per minute of palmityl-1-W CoA hydrolyzed per lO+d of reaction mixture during 1 min of incubation at 37”.
IO
FIG.
m
30
40
Acyl CcA X 10%
22. Chain-length specificity of thioleeterese. The reactions were carried out. under standard assay conditions with 5 pg of protein from fraction 70 ss shown in Fig. 18. The enzyme activity is expressed se m&moles of substrate hydrolyzed during 1-min incubation at 37”. FIG.
FATTY
ACID
SYNTHJZTASE
Thus, the partially purified synthetase would appear to represent only part of the total complement of enzymes responsible for the synthesis and further alteration of fatty acids in the intact tissue, namely, the enzymes for the conversion of CZ --+ Cls. Other evidence from t,his laborat,ory (29) clearly suggests that there are at least three discret.e systems involved in plant tissues for the construction of the hydrocarbon chain; (a) the Cz -+ CM system, (b) Cl6 -+ Cls syst,em, and (c) the CM + c20 . - . system. Of further interest, in addition to the free fatty acids produced in the react,ion mixture, considerable amounts of thiolesterbound fatty acids were formed. Octanoic, decanoic, and lauric acids were found as CoA esters, and myristic and palmitic acid and some unknown minor components, presumably the intermediates of fatty acid synthesis, were also found as ACP thiolesters. The formation of CoA t.hiolesters suggests t,hat a transferase transfers the acyl component of acyl ACP to CoA to form acyl-CoA. This result is similar to that observed by the yeast fatty acid synthet,ase complex (1). The absence of myristyland palmityl-CoA thiolesters can be explained by the observation t,hat these thiolesters are readily hydrolyzed by thiolesterases which favor the hydrolysis of long-chain fatty acyl CoAs (Fig. 22). The existence of myristyl and pahnityl ACP derivatives may relate to the relative low of thiolesterases toward fat.ty activity acyl ACP derivatives (30, 31). Detailed studies on fatty acid compositions under various assay conditions provided further knowledge on the chainlength specificity at the termination of fatty acid biosynthesis. The effect of malonyl-CoA concentration on the pattern of fatty acids synthesized by fatty acid synthebase complexes from yeast (16), rat liver, and rabbit mammary gland (17) have been well documented. The results obt,ained from potato fatty acid synthetase agreed with those obtained from yeast and mammalian tissue, namely, t.hat at. low malonyl-CoA concentrations shorter-
OF Solunum tuberosum
425
chain fatty acids were the major product (Fig. 9) and at high malonyl-CoA concentrations longer-chain fatty acids (C,, : ,,, CM : O) became predominant, (Fig. 9). Variat,ion of acetyl-CoA concentrations did not cause significant. change in t,he pattern of fatty acid synthesized (Fig. lo), even though the malonyl-CoA/acet,ylCoA ratio changes from 6.75 to 0.22. In contrast,, in the yeast fatty acid synthetase complex, the malonyl-CoA/acetyl-CoA concentrat,ion rat,io is considered as a factor influencing the chain length of products. In the yeast fatty acid synthet,ase complex t,here are 3 moles of 4’-phosphopantetheine per mole of synthetase complex which is composed of three functional synthetase units. The acyl moiety of both acet,yl-CoA and malonyl-CoA is capable of binding to the t,hiol group of 4’-phosphopant.etheine of fat,t.y acid synthet,ase complex (32, 33). The competition between acetyl-CoA and malonyl-CoA for this acyl-binding site (the sit.e of the chain-elongation react.ion) could account, for the observed shift in the chain length of synthesized fatty acids. In the pot,ato fatty acid synthetase t,hc &ketoacyl ACP synthetase catalyzes the following reaction (33) : R-CO-S-ACP + HS-E of R-CO-S-E + ACP-SH RCO-S-E + HOOCCHt CO-S-ACP e RCOCHe CO-S-ACP + CO* + HS-E
At, saturating ACP concentration t)he competition bet.ween acetyl-CoA and malonyl-CoA for ACP is not important. However, t.he format,ion of enzyme-bound acetyl-thiolester is import.ant in the initiat.ion of the condensation reaction. Presumably, the concent,ration of enzymcbound acetyl-thiolester is maintained at a relat.ively const,ant level over a wide range of acetyl-CoA concentration and therefore, the actual malonyl-CoA/acetyl-CoA ratio does not influence the fatty acid pattern in the final product,. Nevertheless, t,he malonyl-CoA concentration it.self is import,ant in governing the final product. The effect of TPNH concentration on t.he pattern of fatty acid synt.hesized is similar to that of malonyl-CoA; lower
426
HUANG
AND
STUMPF
TPNH concentrations favor shorter-chain 3. Hsn, R. Y., WASSON, G., AND PORTER, J. W., J. Biol. C’hem. 240, 3736 (1965). and higher TPNH concentrations favor 4. BURTON, D. N., HA.\NIK, A. G., AND PORTER, longer-chain fatty acids synthesis. At low J. W., Arch. Rio&em. Biophys. 126, 141 TPNH concentration there were no de(1968). tectable keto- and unsaturated fatty acid 5. MARTIN, D. B., HORNINQ, M. G., AND VACIEMS, which would suggest t.hat rate of reduction P. It., J. Biol. Chem. 236,663 (1961). is faster than the condensation reaction. 6. LEN.LARZ, W. J., LIQHT, R. J., .\ND BLOCH, K., No accumulation of triacetic acid was obPTOC. Nat. A&l. Sci. U.S.A. 46,640 (1962): served. 7. GOLDMAN, P., ALBERTS, A. W., AND VAQEMS, The effect of ACP concentration on the P. R., J. Biol. Chem. 256, 1255 (1963). 8. WAKIL, S. J., PUGH, E. L., AND SAUER, F., pattern of fatty acid synthesized by potato PTOC. Arat. Acad. Sci. U.S.A. 52, 106 (1964). synthetase was different from those wit,h 9. OVERATR, P., AND STUMPF, P. K., J. Biol. malonyl-CoA and TPNH. At low ACP Chem. 239,4103 (1964). concentration, t.he concentrations of acet,yl10. BROOKS, J. L., AND STIJMPF, P. K., AT&. ACP, malonyl-ACP, &9 well m fatt.y acylBiochem. Biophye. 116,108 (1966). ACP would be relatively low and the prob11. PUQH, E. L., AND WAKIL, S. J., J. Biol. Chem. ability of t,ermination of chain growth by 240,4727 (1965). transfer of CoA derivatives and then sub- 12. MA.JERUS, P. W., ALBERTS, A. W., AND sequent hydrolysis to form free acid would VAQELOS, P. It., J. Biol. Chem. 240, 4723 be less. The reaction product, is then mainly (1965). P. H. W., BOCK, 13. YANG, P. C., BUTTERWORTH, determined by t.hermodynamic determinants R. M., AND PORTER, J. W., J. Biol. Chem. which probably favor G: ,I and CIS : o syn242.3501 (1967). thesis. On the contrary, at, high ACP con14. L.%RRABEE, A. R., MCDANIEL, E. G., BACEERcentration the competit,ion between acetylMAN, H. A., AND VAQELOS, P. R., PTOC. Nat. ACP and other acyl-ACPs for condensaAcad. Sci. U.S.A. 54, 267 (1965). tion enzyme would be greater. In addition, 15. PUGH, E. L., SAUER, F., WAITE, M., TOOMEY, the increase in fatt,y acyl-ACP concentraR. E., AND WAKIL, S. J., J. Biol. Chem. 241, tions would favor t,hc transferase reaction 2635 (1966). which would transfer fatt.y acyl-ACP de- 16. SUMPER, M., DESTERHELT, D., RIEPERTINQER, C., .4ND LYNEN, F., EUT. J. Biochem. 10,377 rivatives to fatty acyl-CoAs and with sub(1969). sequent hydrolysis to free acids. CAEEY, E. M., DILS, R., AND HANSEN, H. J. M., 17. The effect of pH on the reaction product Biochem. J . 117,633 (1970). is more complicated, because it may involve R. D., GRIDDLE, R. S., AND STUMPF, several react,ions which are undefined in 18. SIYONI, P. K., J. Biol. Chem. 242,573 (1967). the potato tuber. 19. SIMON, E. J., AND SHEMIN, D., J. Amer. Chem. The isolat.ion of t,hiolesterase from potato Sot. 76, 2520 (1953). tuber suggests that at, least. two isozymes of 20. GOLDMAN, P., AND VAQEMS, P. R., J. Biol. similar molecular weight exist,. Unlike t,he Chem. 236.2620 (1961). thiolesterases from B. c&i (30, 31), both 21. STADTMAN, E. R., in “Methods in Ensymology” (S. Colowick and N. 0. Kaplan, eds), potato thiolesterases have similar properties. Vol. 3, p. 931. Academic Press, New York The thiolesterases in potato tuber are very (1957). active and have relatively higher V,., 22. ELLMAN, G. L., Arch. Biochem. Biophys. 62, than h’. coli enzymes. ACKNOWLEDGMENTS
We wish to acknowledge the assietance of Mrs. Barbara Clover in this investigation. REFERENCES F., Fed. Proc. 20,941 (1961). 2. BREBSLER, R., AND WAKIL, S. J., J. Biol. Chem. 836, 1643 (1961).
1. LYNEN,
70 (1959). 23. HuANQ, K. P., Anal B&hem. 27, 98 (1970). 24. MILLER, G. L., Anal. Chem. 91,964 (1959). 25. LOOMIS, W. D., in “Methods in Enzymology” (J. M. Lamenstein, ea.), Vol. 13, p. 555. Academic Press, New York (1969). 26. HARRIS, R. V., AND J-ES, A. T., Biochim. Biophys. Acla 166,456 (1965). 27. BRADY, R. O., J. Biol. Chem. 236,3099 (1960).
FATTY 23. WILLEMOT,
C., AND
STUMPF,
46,679 (1967). 29. HARWOOD, J., AND
ACID
SYNTHETASE
P. K., Can.J. Bot.
STUMPF,
P. K.,
Arch.
Biochem.Biophys. lpB, 281 (1971). 30. BARNES, E. M., JR., AND WAKIL, S. J., .I. Biol. Chem. 243, 2966 (1968). 31. BARNES, E. M., JR., SWINDELL, A. C., AND WAKIL, S. J., J. Biol. Chem. 246,3122 (1970). 32. LYNEN, F., OMTERHELT, D., SCHWEIZER, E.,
OF SoEanum tuberosum AND
WILLECKE,
mentalization Metabolism,” York (1968). 33. GREENSPAN, VIGELOS,
427
K., “Cellular Compartand Control of Fatty Acid p. 7. Academic Prezs, New
$1. D., ALBERTS, A. W., P. R., J. Biol. Chem.%,
AND
6477
(1969).
34. Ta.u,xs, E. Cr., :IND BR.~DT, 1%.O., J. Amer. Chem.Sot. 33, 2972 (1960).