Fat metabolism in higher plants

ARCHIVES OF BIOCHEMISTRY Vol. 190, No. 1, September,

AND BIOPHYSICS pp. 210-220,1978

Fat Metabolism The Elongation

of Saturated

and Unsaturated Acyl-CoAs Isolated Spinach Chloroplasts’

W. A. VANCE Department

in Higher Plants

of Biochemistry

AND

System

from

P. K. STUMPF

and Biophysics,

University

December

5, 1977; revised

Received

by a Stromal

of California, April

Davis,

California

95616

25, 1978

A soluble enzyme system from the stroma of chloroplasts isolated from Spinacease oleraceae elongated various long chain acyl-CoAs using acetyl-CoA as a two-carbon donor. Partial purification of the system was achieved by ammonium sulfate fractionation and molecular sieve chromatography. The elongation system required NADPH and NADH for the reduction steps. Several nucleoside triphosphates markedly stimulated elongation. Inhibition occurred with several thiol binding reagents and with free CoA. The possible significance of elongation via acyl-CoAs in chloroplasts is discussed.

Although early proposals for the mechanism of fatty acid synthesis suggested a simple reversal of the P-oxidation sequence for fatty acid degradation, the requirement for both malonyl-CoA and acyl carrier protein in a wide variety of tissues argued conclusively for a distinct and separate pathway for fatty acid synthesis. In recent years, however, evidence is accumulating which suggests a reversal of the P-oxidation sequence as a mechanism for limited or specialized fatty acid synthesis. Synthesis is “limited” in the sense that it occurs primarily as an elongation of a previously existing fatty acid. The term “elongation” is used in this paper to describe this mode of synthesis. Malonyl-CoA independent elongation of fatty acids has been observed in mammalian mitochondrial and microsomal systems by Seubert et al. (1, 2) and by Wakil (3, 4). Their systems closely resembled the reversal of P-oxidation with the exception that the final reaction was catalyzed by an enoyl-CoA reductase:NADPH (EC 1.3.1.8) in place of an acyl-CoA:FAD dehydrogenase. Participation of this reaction in the elongation sequence permitted a theoretical net ‘Supported in part by NSF Grant GM Chis is paper LXX1 in a series. Paper LXX in Arch. Biochem. Biophys. 173,427 (1976).

19213-06. appeared

210

0003-9861/78/1901-0210$02.09/O Copyright All rights

0 1978 by Academic Press, Inc. of reproduction in any form reserved.

AGo’ of -10.35 k&mole (5) for the overall process. An elongation system for pahnityl-ACP2 has been described (6) which is responsible for the synthesis of stearyl-ACP (the highly specific substrate for stearyl-ACP desaturase). These elongation and desaturase systems are found in a variety of higher plants (7).

The existence of an additional fatty acid elongation system in chloroplasts was indicated from the observation in this laboratory that hexadecatrienoic acid was elongated with [1-14C]acetate to [1-14C]a-linolenic acid in the presence of cyanide or avidin (7). Previously the synthesis of CXlinolenic acid had been thought to occur solely by the sequential desaturation of stearate to oleic to linoleic acids (or their appropriate derivatives) and thence to CXlinolenic acid (8). The relatively large amounts of hexadecatrienoic acid in Brassica napis (9) and spinach (10) prompted Heyes and Shoreland (11) to suggest that it may be a precursor of a-linolenic acid. This suggestion was tested by Jacobson et *Abbreviations used: ACP, acyl carrier protein; Hepes, 4-(2-hydroxyethyh-I-piperazineethanesulfonic acid; Tricine, N-tris(hydroxymethyl)methylglycine; POPP, 1,4-bis[2-(5-phenyloxazoylllbenzene; PPO, 2,5diphenyloxazole.

ACYL-CoA

ELONGATION

BY

CHLOROPLAST

(7) when they observed that this precursor could indeed be elongated to a-linolenic acid. Elongation of hexadecatrienoic acid was examined using an enzyme system prepared from the 106,500g supernatant obtained from disrupted spinach chloroplasts (7). The elongation of cis-7,10,13-hexadecatrienoic acid (16:3) by [1-14C]acetate was found to be enhanced by the addition of NADP’, NADH, ATP, Mg’, CoASH and an NADPH generating system. This preliminary evidence indicated the existence of a pathway which utilized acylCoAs with cofactors similar to what might be expected for reversal of P-oxidation even though evidence for P-oxidation of fatty acids has not been shown to occur in chloroplasts. This paper will describe this pathway with respect to substrate specificity, required cofactors, and inhibitors that were determined with an ammonium sulfate fraction from the stroma of chloroplasts isolated from spinach leaves.

al.

MATERIALS

AND

METHODS

Reagents. NADPH (tetrasodium salt), NADP+ (sodium salt), NADH (sodium salt), glucose-g-phosphate dehydrogenase (EC 1.1.1.49), glucose g-phosphate (monosodium salt), avidin, Z-ascorbic acid, ATP (disodium salt), ADP (sodium salt), AMP (free acid) 3’:5’cyclic AMP (sodium salt), GTP (sodium salt), and Lcysteine (free base) were from Sigma Chemical Co., St. Louis, MO. UTP (trisodium salt), CTP (d&odium salt), Tricine, and Hepes were from Calbiochem, La Jolla, CA. Coenzyme A was from P-L BiochemicaIs, Inc., Milwaukee, Wise. cis-3,6-Dodecadienoic acid (12:2), cis-5,8-tetradecanoic acid (14:2), and cis-7,10hexadecadienoic acid (162) were the generous gift of Professor H. W. Sprecher, Ohio State University. cis7,10,13-Hexadecatrienoic acid (16:3) was extracted from chloroplast membranes according to the method of Bligh and Dyer (12) and purified as the methyl ester by argentation chromatography (13) and gas-liquid chromatography. PCS (phase combining solution for liquid scintillation spectrometry of aqueous samples) was from Amersham/Searle, Arlington Heights, Ill., and 15% HI-EFF-2BP on Gas-Chrom Q @O/l00 mesh) and 10% EGSS-X on Gas-Chrom P (100/120 mesh) were from Applied Science Laboratories, State College, Pa. Triton X-100, hydroxide of Hyamine-10X, dimethyl POPOP, and PPO were from Packard Instrument Co., Downers Grove, Ill. EDTA, 2 mercaptoethanol, sorbitol, MgClz .6HzO, ethylchloroformate, triethylamine, tetrahydrofuran, and perchloric acid

were reagent sources.

STROMAL grade

211

SYSTEM and

obtained

from

commercial

Substrates. [l-‘4C]Acetyl-CoA (58 Ci/mol) and [1,3“C]malonyl-CoA (54 Ci/mol) were obtained from Schwarz/Mann. Acetyl-CoA, malonyl-CoA, decanoylCoA, dodecanoyl-CoA, tetradecanoyl-CoA, hexadecanoyl-CoA, and octadecanoyl-Cob were obtained from P-L Biochemicals. cis-9-Hexadecenoyl-CoA, c&+3,6dodecadienoyl-CoA, cis-5,8-tetradecadienoyl-CoA, cis-7,10-hexadecadienoyl-CoA, and cis-7,10,13-hexa decatrienoyl-CoA were prepared essentially according to the method described by Young and Lynen (14) for acylation of CoASH by a carboxylate-formate mixed anhydride. Centrifugation and washing of the product precipitated by 0.8% perchloric acid was as described by Goldman and Vagelos (15). Free fatty acids were removed from the pellet with acetone and diethyl ether as described by Seubert (16). Purity of product was determined by calculating absorption ratios measured spectrophotometrically at 232,250, and 280 nm divided by the value determined at 260 mn. These values were compared to the spectral constants cited for commercial sources of saturated and unsaturated acyl-CoAs (17). Preparation of spinach stromal enzyme system. Fresh spinach purchased from the local market was washed thoroughly and the young centrally located leaves were removed and chilled overnight (in a plastic bag). The leaves were deveined and chopped with scissors and mixed with an equal volume of chilled isolation medium (0.6 M sorbitol, 0.1 M Tricine, 5 mM cysteine, 1 mu EDTA, 0.1% (w/v) Z-ascorbic acid titrated to pH 7.9 with 8 N KOH at 0°C). Homogenization was accomplished by fit employing a Waring blender at 70% maximum voltage for 15-20 s then a Brinkman Polytron at 60% maximum voltage for up to 30 s. The homogenate was filtered through one layer of nylon net 28 )nn mesh and two layers of Miracloth, then centrifuged in a Sorvall RC-2B refrigerated centrifuge at 3,500g for 45 s at 0°C in a Sorvall SS-34 rotor. The supernatant was rapidly poured off and the chloroplasts were pooled and diluted to a suitable viscosity for disruption in a French pressure cell at 15,006 psi. The resulting pressate was centrifuged at 106,600g for 90 min in a Beckman model L centrifuge fitted with a type 40 rotor. The supernatant containing the soluble enzymes of the stroma, was fractionated by a series of precipitations with a saturated solution of ammonium sulfate (enzyme grade from Schwarz/Mann) containing 5 mu mercaptoethanol and 1 mM EDTA titrated to pH 7.9 at O’C (a 1:lO dilution gave a pH of 7.5). The precipitate at 35 to 75% saturation was pelleted by centrifugation at 48,600g for 10 min and was dissolved in buffer containing 50 mu Hepes and 10 mM mercaptoethanol, pH 7.5, at 0°C. Reprecipitation of the proteins was performed with increasing amounts of saturated ammonium sulfate solution and the following fractions were obtained: The 35-40% fraction had a specific activity of 34 pmol

212 of product per mg of protein per fraction, 88; the 50-60% fraction, fraction was devoid of activity. were stored in the resuspension (20%, v/v) at -5°C.

VANCE

AND

30 min; the 40-50% 57; and the 60-70% Individual fractions buffer plus glycerol

Assays and analyses ofproducts. Elongation

activity was assayed with the following amounts of reactants in 0.5 ml final volume in a 13 X 100 mm screw cap culture tube: long chain acyl-CoA, 10 nmol; [l-“Clacetyl-CoA, 17.2 nmol; ATP, 250 nmol; Mg’+, 250 nmol; NADPH, 125 nmol; NADH, 125 nmol; avidin, 0.25 unit; Hepes buffer at pH 7.5, 17.5 pmol; mercaptoethanol, 2.5 pmol; and enzyme protein, 1-2 mg. Optimal incubation conditions were determined to be 30°C for 30 min, pH 7.5. Reactions were terminated by the addition of 0.2 ml 8 N KOH and saponified by heating at 80°C for 30 min. To improve extraction of product acids 100 nmol of palmitic acid were added prior to saponification. Extraction of the acids was performed by first adding 0.3 ml 10 N HCl, then 2 vol of petroleum ether and mixing vigorously. Three extractions were performed and the petroleum ether was pooled and evaporated under a stream of nitrogen. The free acids were converted to methyl esters by the addition of diazomethane dissolved in diethyl ether and 2-3 drops of anhydrous methanol. The methyl esters were redissolved in toluene and an aliquot removed (usually 10%) and counted in a Beckman model LS-230 liquid scintillation spectrometer. The scintillant was composed of 0.6% POP and 0.05% POPOP in toluene. The 14C counts/min (cpm) were converted to disintegrations/min by determining counting efficiency after correcting for quenching by the external standard ratio method. Product acids were identified by comparison with authentic standards (Supelco Co.) after gas-liquid chromatography analysis on 15% HI-EFFSBP. A 0.47 x 150 cm stainless steel column was used in a Varian model 920 gas chromatograph equipped with a thermal conductivity detector and coupled to a Nuclear Chicago Biospan 4998 radioactivity detector. Carrier gas was helium at a flow rate of 60 ml/min. Propane at a flow rate of 60 ml/min was added to the effluent from the thermoconductivity cell producing a final effluent flow rate of 120 ml/min in the “‘C detector chamber. Detector responses (mass and radioactivity) were recorded by a Leeds and Northrop dual channel chart recorder set at 1 inch/IO min. Counts per minute were integrated by a Nuclear Chicago model 8770A scalar and recorded on an Esterline Angus chart recorder set at 1 inch/l0 min. Proof of structure of a specific fatty acid was performed by fust determining the chain length of the catalytically reduced product. The position of the first olelinic bond from the carboxyl end was determined by reductive ozonolytic cleavage of unsaturated acids and gas-liquid chromatography analysis of the fragments according to the method of Stein and Nicolaides (18). Elongation was differentiated from de nouo syn-

STUMPF thesis by chemical a-oxidation (19) of the saturated (or catalytically reduced) free fatty acids. RESULTS

General observations. Both the location of elongation activity in the chloroplast stroma and insensitivity to inhibition by avidin observed by Jacobson et al. (7, 20) were confirmed. By precipitating twice with ammonium sulfate and desalting on BioGel P-10, a preparation was obtained for which the minimum cofactor requirements for elongation of hexadecatrienoic acid with [1-14C]acetate were defined (Table I). The requirement for CoASH and not ACP provided additional evidence to distinguish this elongation from de nova synthesis or palmityl-ACP elongation (6, 21). The observed requirement for ATP and Mg2+ could be assigned to the conversion of hexadecatrienoate and [ 1-14C]acetate to their CoA derivatives by acyl-CoA synthetase(s). The requirement for a reduced pyridine nucleotide(s) would be assigned to the two reduction steps in the elongation sequence. Two-carbon donor. The experimental results obtained using [l-‘4C]acetyl-CoA in the presence of avidin (Fig. 1) were strong presumptive evidence that malonyl-CoA was not participating in the elongation reactions. Nevertheless, when [1,3-14C]maloTABLE COFACTOR Cofactors

REQUIREMENT

of elongation system

Complete - CoASH - ACP - ATP - Mg’+ - NADP+ - NADH

I FOR ELONGATION[I-“C]Linolenate formed (pmol/mg protein) 1008

5 960 0 16

23 26

R Each reaction mixture contained the following concentrations of reactants in 0.5 ml final volume: 163 (free fatty acid), 50 pM; [l-‘4C]acetate (58 Ci/mol), 172 PM (5 @i); ATP, 2 mhf; Mg’+, 0.4 mu; NADP+ or NADH, 0.4 mM; glucose B-phosphate, 4 mu; glucoseB-phosphate dehydrogenase (Yeast), 0.15 unit; Hepes buffer at pH 8, 0.1 M; and enzyme protein (30-508 ammonium sulfate cut), 3 mg. Reactions were incubated for 3 h at 23’C under an atmosphere of nitrogen. Termination, extraction, and analysis of fatty acids was as described under Materials and Methods.

ACYL-CoA

ELONGATION

BY

CHLOROPLAST

!D

FIG. 1. Determination of the preferred two-carbon donor in elongation in lauroyl-CoA. Half-milliliter reaction mixtures contained 35 mru Hepes buffer (pH 7.5), 5 mu mercaptoethanol, 0.25 unit of avidin, 20 pM lauroyl-CoA, 0.5 mM ATP, 0.5 mu MgClx, 0.25 mru NADPH. 0.25 mru NADH, [I-“Clacetyl-CoA (58 Ci/mol) at the concentrations indicated, [1,3-“C]malonyl-CoA (54 Ci/mol) at the concentrations indicated, and 1.2 mg of protein from the 40-50% ammonium sulfate fraction. Reactions were incubated at 30°C under an atmosphere of nitrogen for 30 min then terminated by the addition of 0.2 ml of 8 N KOH. Extraction and analysis of product fatty acids was as described under Materials and Methods.

nyl-CoA was examined as a possible substrate, it was incorporated at about half the rate observed when [l-14C]acetyl-CoA was used. These results were analyzed with respect to the stability of malonyl-CoA; that is, the enzymic decarboxylation of malonylCoA to acetyl-CoA in the presence and absence of lauroyl CoA. The decarboxylation of [1,3-‘4C]malonyl-CoA was measured by trapping evolved 14C02 on filter paper dipped in hydroxide of Hyamine-10X. When the amount of 14C02 was quantitated for each point of the malonyl-CoA concentration curve, it was found that 14C02 was equal to or slightly less than the [ 1-14C]myristate produced from the elongation of lauroyl-CoA. Thus the release of 14C02 from [ 1,3-14C]malonyl-CoA does not precede but is probably the result of a condensation reaction. In the absence of lauroyl-CoA, malonyl-CoA is not decarboxylated to acetyl-CoA. Presumably either malonyl-CoA participates at a lower rate in the condensation reaction or there are two distinct condensing enzymes, employing either acetyl-CoA or malonyl-CoA.

STROMAL

SYSTEM

213

Double reciprocal plots of substrate versus product for acetyl-CoA gave an apparent K,,, of 6.5-7.0 x 10e5 M and a V,,, of approximately 11.5 pm01 min-’ mg-’ using enzyme from the 40-50s ammonium sulfate fraction and lauroyl-CoA as acceptor. Acyl-CoA acceptors. The results in Fig. 2 summarize the specificity of acyl-CoAs as substrates for the elongation reaction. Lauroyl-CoA was the optimal substrate for elongation. Saturated acyl-CoAs with longer chain lengths had progressively less activity as substrate. Unsaturated acylCoAs had less activity than the corresponding saturated acyl-CoAs. Serious limitations are imposed on the interpretation of results obtained where the concentration of a long chain acyl-CoA was at or below its critical micellar concentration (CMC). Because of the formation of a surface excess at the air-water interface, the concentration of acyl-CoA becomes a function of the surface to volume ratio of the reaction mixture (22). In addition, the nonspecific adsorption of acyl-CoA to protein impairs the calculation of the free acylCoa concentration. These factors made it difficult to measure an apparent K,,, for each acyl-CoA examined. Indeed the shapes of the curves for 16:3-CoA and 12:0CoA elongation in the experiments shown in Fig. 3 imply that a micelle can serve as substrate (since the CMC for acyl-CoAs is approximately 3 PM). This may not be the case when the concentration values are corrected for surface excess and nonspecific adsorption. For example, above 12 PM 16:3CoA, the V,,, is constant and not hyperbolic, producing a bimodal double reciprocal plot. This is characteristic of those enzymes which employ monomers but not micelles as substrates (Class II kinetics by the terminology of Gatt et al. (23)). Pyridine nucleotide specificity. The requirement for a source of reducing equivalents was further examined with respect to specificity. The results of a series of concentration studies are presented in Fig. 4. It was observed that NADPH and NADH together gave better results than either individual nucleotide at equivalent total concentrations. Thus, there may not be strict specificity for a reduced pyridine nucleotide by either reduction reaction since either

214

VANCE

AND

STUMPF

\

16 Carbon

.-z 4 I

; ,D

Oirmoic

Acyl-CoA’s .

A. Nunbo in sryl

-

l

product

\

01 carbon atoms group of substrate

6.

Numbar substrats

of olsfin bonds .scyI-CDL’S

in

FIG. 2. Acyl-CoA acceptors in elongation. Half-milliliter reaction mixtures contained 35 mM Hepes buffer (pH 7.5), 5 mu mercaptoethanol, 0.25 unit of avidin, 0.5 mM ATP, 0.5 mu MgCb, 0.25 mu NADPH, 0.25 mM NADH, 34.5 pM [1-Wlacetyl-CoA (58 Ci/mol), 20 pM acyl-CoAs in frame A and 10 pM acyl-CoAs in frame B, and 1 mg of protein from the 40-5056 ammonium sulfate fraction. Reactions were incubated at 30°C under an atmosphere of nitrogen then terminated by the addition of 0.2 ml of 8 N KOH. Extraction and analysis of fatty acids was as described under Materials and Methods.

‘.

/AoPN+NAON

;

NADPII

16:3-toA

-

l6:3-Cal

I 0

J 0

10

26 Substrate

30 llcyl-CoA

40

50 IUMI

FIG. 3. Comparative kinetics of elongation of lauroyl-CoA and l&3-CoA (saturated vs. polyunsaturated). Half-millilter reaction mixtures contained 35 rnre Hepes buffer (pH 7.9), 3.5 mre mercaptoethanol, 0.5 unit of avidin, 0.5 mu ATP, 0.5 mu MgCh, 0.25 mu NADPH, 0.25 mM NADH, 34.5 p [1-‘%]acetylCoA (58 Ci/mol), acyl-CoA at the concentrations indicated, and 2 mg of protein from a 30-509 ammonium sulfate fraction. Reactions were incubated at 30°C under an atmosphere of 0.2 ml of 8 N KOH. Extraction and analysis of fatty acids was as described under Materials and Methods.

I I25 IInduced

250 pyridinr

500 nuclsntide

IUMI

FIG. 4. Determination of the preferred reduced pyridine nucleotide. Half-milliliter reaction mixtures contained 35 rnhr Hepes buffer (pH 7.5), 5 mM mercaptoethanol, 0.5 unit of avidin, 0.5 II~M ATP, 0.5 mM MgClx, reduced pyridine nucleotides at the concentrations indicated (NADPH + NADH is l:l), 34.5 PM [l‘%]acetyl-CoA (58 Ci/mol), 20 PM lauroyl-CoA, and 2 mg of protein from a 30-5056 ammonium sulfate fraction. Reaction mixtures were incubated at 30°C under an atmosphere of nitrogen for 30 min then terminated by the addition of 0.2 ml 8 N KOH. Extraction and analysis of fatty acids was as described under Materials and Methods.

ACYL-CoA

ELONGATION

BY

CHLOROPLAST

nucleotide results in production of [l-14C]myristate from lauroyl-CoA and [14C]acetyl-CoA. This is interesting from the standpoint that neither P-oxidation nor the enzyme studied by Seubert et al. (2) demonstrated overlapping specificity. In order to determine if there was a preference in the final reduction reaction for one or the other pyridine nucleotide experiments similar to those of Hinsch et al. (24) were employed. By taking advantage of the specificity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) from baker’s yeast and Leuconostoc mesenteroides (25), ,&[“H]NADPH (26) and [3H]NADH can be generated from n-[1-3H]glucose-6-phosphate and the corresponding pyridine nucleotide. The isotope effect observed with glucose-6-phosphate dehydrogenase (24) was not strictly accounted for in these experiments; however, the reactions which generated the reduced pyridine nucleotides in situ were allowed to remain at equilibrium for 10 min after the maximum absorbance at 340 nm was observed. The substrate 2,3-trans-myristoyl-CoA was also generated in situ by the 40-50% ammonium sulfate enzyme from added DL-P-OH-myristoyl-CoA. The final reduction reaction using a [3H]-labeled pyridine nucleotide would thus produce [“Hlmyristoyl-CoA labeled in the C3 position. Product identity was verified by gas-liquid chromatography analysis using ,&OH-methyl myristate and methyl myristate as standards. The results of the experiment summarized in Table II confirm the data of Fig. 4 that with /I-OH-myristoyl-CoA as substrate, NADP3H was about twice as effective as NAD3H in forming [3-3H]myristoylCoA; moreover equal amounts of NADP3H and NADH or NADPH and NAD3H resulted in a reduction by approximately onehalf in the extent of 3H incorporation into the product. The results would suggest that both NADPH and NADH can serve as reductants in the conversion of 2,3-transmyristoyl-CoA to myristoyl-CoA. Whether or not an isotope effect was involved in the reduction of 2,3-trans-myristoyl-CoA was not determined. A spectrophotometric measurement of the decrease in absorbance at 340 nm of the

STROMAL

215

SYSTEM TABLE

II

3H INCORPORATION FROM PYRIDINE NUCLEOTIDES WITH /3-HYDROXYMYRISTOYL-COA AS SUBSTRATE” NADPH NAD’H NADH [3-“HINADP’H My&ate (am) (pm) (am) (pm) (pmol/ w) 250 250

250

250 250

250 -

1060 467 474 249

a Half-milliliter reaction mixtures contained 50 mM Hepes buffer (pH 7.5), 3.5 mu mercaptoethanol, 1 mM ATP, 5 mru MgCL, 0.25 mu n-[l-3H]glucose (99.5 Ci/ mol), 2 units of hexokinase, either 0.25 mM NADP’ and 1.5 units of glucose-6-phosphate dehydrogenase from baker’s yeast or 0.25 mu NAD’ and 1.5 units of glucose-6-phosphate dehydrogenase from Leuconostot mesenteroides, 0.25 mM reduced pyridine nucleotide where indicated, 20 PM /3-OH-myristoyl-CoA. Reaction mixtures were allowed to generate reduced pyridine nucleotide until the absorbance at 340 nm stopped increasing. Reactions were initiated by the addition of 0.65 mg of protein from a 40-50% ammomum sulfate fraction. Incubations were performed at 30°C under an atmosphere of nitrogen for 30 min then terminated by the addition of 0.2 ml of 8 N KOH. Extraction and analysis of fatty acids was as described under Materials and Methods. Two reaction mixtures contained 2 mu acetyl-CoA in order to favor further elongation.

reaction mixtures described in Table II (utilizing unlabeled glucose) was made. The rates of reduction were several fold higher than those observed in the radio assay but confirmed that NADPH is the preferred substrate. Thus both types of experiments demonstrated that while 2,3-trans-enoylCoA reductase did not have strict pyridine nucleotide specificity there was a preference for NADPH. Nucleoside phosphate involvement. It was expected that when both acyl-CoA and acetyl-CoA were the substrates for elongation, the requirement for ATP would be obviated. While Seubert et al. (2) and Wakil (3) did not detect any nucleoside triphosphate requirement for their systems, in the chloroplast system a 4-fold enhancement occurred in the presence of ATP and Mg’+. The results of an ATP:Mg2+ (1:l) concentration study, summarized in Fig. 5, demonstrates this enhancement effect. One possible explanation for this stimulation may be that activation by covalent

216

VANCE

AND

STUMPF TABLE ACTIVATION

Initial concentration ATP/ Mg*+ (PM) before dilution 250 0 256 0 0

FIG. 5. Activation of elongation by ATP/Mg’+. Half-milliliter reaction mixtures contained 35 mu Hepes buffer (pH 7.9), 3.5 mu mercaptoethanol, 0.5 unit of avidin, ATP and Me (1:l) at the concentrations indicated, 0.25 mu NADPH, 0.25 mu NADH, 34.5 pM [l-‘4C]acetyl-CoA (58 Ci/mol), 24 PM pahnitoyl-CoA, and 1.5 mg of protein from the 40~50% ammonium sulfate fraction. Reactions were incubated at 30°C under an atmosphere of nitrogen for 30 min then terminated by the addition of 0.2 ml of 8 N KOH. Extraction and analysis of fatty acids was as described under Materials and Methods.

modification (e.g., phosphorylation or adenylylation) or very tight binding by ATP was occurring. Incubation of the enzymes in the presence of high concentration of ATP followed by a 50-fold dilution resulted in decreasing elongation activity as shown in Table III. These data would therefore argue against a phosphorylation or adenylation of the enzyme protein by ATP. Apparently the continued presence of a high concentration of ATP is required for stimulation. The specificity of the enhancement effect as a function of the nucleoside phosphate is summarized in Table IV. Interestingly, several nucleoside triphosphates as well as ADP were able to stimulate elongation. Since an active nucleoside diphosphate kinase has been demonstrated by Mazelis (27) for spinach chloroplasts it is possible that ADP is being converted to AMP and ATP. AMP as well as cyclic AMP had no effect. Since pyrophosphate bonds of nucleosides are involved in the synthesis of acylCoAs (28) the possibility existed that ATP was regenerating the substrates for elon-

III

OF ELONGATION ATP/Mg’+”

OF PALMITOYL-COA

Final concentration ATP/ Mg*+ (PM) after dilution 5 5 250 250 0

BY

yclk (pmol/mg) 77 73 203 199 60

B Half-milliliter reaction mixtures contained 35 mM Hepes buffer (pH 7.9), 3.5 mru mercaptoethanol, 0.5 unit of avidin, ATP at the concentrations indicated, MgClz at concentrations equivalent to ATP concentrations, 0.25 mu NADPH, 0.25 mM NADH, 34.5 mM [1-“Clacetyl-CoA (58 Ci/mmol), 24 pM palmityl-CoA, and 1.5 mg of protein from a 30-50% ammonium sulfate fraction. Concentrated enzyme protein was preincubated for 5 min with 0.25 mM ATP/Me and then added to the reaction mixture, resulting in a 50fold dilution. Final reaction mixtures were incubated at 30°C under an atmosphere of nitrogen for 30 min then terminated by the addition of 0.2 ml of 8 N KOH. Extraction and analysis of fatty acids was as described under Materials and Methods.

TABLE NUCLEOTIDE Nucleotide/metal

IV

SPECIFICITY FOR ELONGATION ACTIVATION” Concentration

b-f) None Mg2+ ATP ATP/Mg’+ ADP/Mg’+ AMP/Mg’+ UTP/Mg’+ CTP/Mg’ GTP/Mg’+ cAMP/ATP/Mg”

0.5 0.5 0.5/0.5 0.5/0.5 0.5/0.5 0.5/0.5 0.5/0.5 0.5/0.5 0.25/0.5/0.5

ri-wlLinoleate (nmol/me) 100 118 155 363 327 85 327 279 152 344

a Half-milliliter reaction mixtures contained 35 n-nu Hepes buffer (pH 7.9), 3.5 mru mercaptoethanol, 0.5 unit of avidin, nucleoside phosphates at the concentrations indicated, Mg*+ at the concentrations indicated, 0.25 mM NADPH, 0.25 mM NADH, 34.5 go [l“C]acetyl-CoA (58 Ci/mol), 24 PM palmitolinoleylCoA (16:2CoA), and 1 mg of protein from a 40-50% ammonium sulfate fraction. Reaction mixtures were incubated at 23°C under an atmosphere of nitrogen for 30 min then terminated by the addition of 0.2 ml 8 N KOH. Extraction and analysis of fatty acids was as described under Materials and Methods.

ACYL-CoA

ELONGATION

BY

CHLOROPLAST

gation. Measurement of thioesterase activity indicated that hydrolysis of substrate was not sufficient to require resynthesis by an ATP/CoA thiokinase reaction to account for the 4-fold enhancement. Inhibitors. It was found during enzyme purification that the presence of 1 to 10 rnru mercaptoethanol enhanced enzymatic activity. The involvement of an active thiol group was examined by assaying elongation activity in the presence of known sulfhydryl inhibitors (Table V). The strong inhibition observed with iodoacetamide indicated that a reduced sulfhydryl group may be participating in one of the reactions. Possible activation was reexamined with additional thiol reducing agents at increasing concentrations as shown in Fig. 6. The results of the inhibition and activation experiments strongly suggest the presence of an active thiol group in at least one of the elongation enzymes. Of interest, increasing concentrations of thiol compounds consistently gave activation at low levels and marked inhibition at higher levels. CoA was equally effective as an inhibitor of the elongation reaction. Figure 7 documents this effect in the presence of 5 mu mercaptoethanol. In the absence of mercaptoethanol, as little as 5 PM CoASH almost totally inhibited elongation (Fig. 6). Whether this effect is of physiologTABLE INHIBITOR Inhibitor

;;m;-

V STUDIES~ [l-‘4c]Lino-

leate (pmol)

(mid

Percentage inhibition

None Iodoacetamide 5,5’-Dithiobis(2ni trobenxoic N-Ethylmaleimide

-

161 34 97

-

0.5 0.5 0.5

88

46

79 40

acid)

a Half-milliliter reaction mixtures contained 35 mru Hepes buffer (pH 7.5), 0.5 unit of avidin, 0.5 nuu ATP, 0.5 mru MgClx, 0.25 mu NADPH, 0.25 mru NADH, 34.5 pM [1-“‘Clacetyl-CoA (58 Ci/mol), 24 )JM palmitolinoleyl-CoA (16:2-CoA), and 2 mg of protein from a 40-50% ammonium sulfate fraction. Reactions were incubated at 23°C under an atmosphere of nitrogen for 30 min then terminated by the addition of 0.2 ml 8 N KOH. Extraction and analysis of fatty acids was as described under Materials and Methods.

STROMAL

217

SYSTEM

Dithiothreitol

0

5

10 Thiol

15

20

tmM1

FIG. 6. Effect of thiol reducing agents on elongation activity. Half-milliliter reaction mixtures contained 50 nnu Hepes buffer (pH 7.5), thiol reducing agents at the concentrations indicated, 0.5 unit of avidin, 0.5 mu ATP, 0.5 mu MgClz, 0.25 mu NADPH, 0.25 mM NADH, 34.5 PM [l-‘4C]acetyl-CoA (58 Ci/mol), 20 PM lauroyl-CoA, and 1 mg of protein from the 40-508 ammonium sulfate was done in the absence of mercaptoethanol. The enzyme was stored in 20% glycerol/buffer (less mercaptoethanol) overnight at -5°C and assayed the following day. Reactions were incubated at 30°C under an atmosphere of nitrogen for 30 min then terminated by the addition of 0.2 ml 8 N KOH. Extraction and analysis of the fatty acids was as described under Materials and Methods.

ical importance tion.

requires

further

investiga-

Molecular sieve chromatography. Since at least four reactions are required for elongation, it was of interest to determine whether or not the enzymes for these reactions were associated in a large complex or were separate entities. Fractionation of the 40-50s ammonium sulfate cut on Sepharose 6B which has an exclusion limit of 4 x lo6 daltons and an effective fractionation range of 1.5 x lo6 to lo5 daltons produced the elution profile shown in Fig. 8. The major protein peak was assayed for elongation with an assay utilizing /?-OH-myristoyl-CoA and NADP3H to measure activities which could presumably involve (a) the oxidation-reduction of the /?-hydroxyl function to the /3-keto function of P-OH-myristoyl-CoA, (b) the dehydration of P-OHmyristoyl-CoA to 2,3-trans-tetradecenoylCoA, and (c) the reduction of the double bond system to form myristoyl-CoA. The insertion of 3H into /?-OH-myristate and

VANCE

218

AND STUMPF

Protein

0

50

100

150

200

FIG. 7. Inhibition by reduced coenzyme A. Halfmilliliter reaction mixtures contained 35 mM Hepes buffer (pH 7.5), 5 mM mercaptoethanol, 0.5 unit of avidin, 0.5 mM ATP, 0.5 mM MgClz, 0.25 mu NADPH, 0.25 mru NADH, 34.5 pM [l-‘4C]acetyl-CoA (58 Ci/mol), 24 PM palmitoyl-CoA, CoASH at the concentrations indicated, and 1.6 mg of protein from a 30-50% ammonium sulfate fraction. Reactions were incubated at 30°C under an atmosphere of nitrogen for 30 min then terminated by the addition of 0.2 ml 8 N KOH. Extraction and analysis of fatty acids was as described under Materials and Methods.

trans-2,3-myristate may be attributed to the reversibility of a P-OH-acyl-CoA reductase-type reaction. The origin of 3H in myristate would be from the action of an enoylCoA reductase (sufficient dehydratase activity would be required to produce the substrate for reduction). In Fig. 8 the low dehydratase activity in fractions 55 through 63 permitted the accumulation of P-OHmyristate (labeled and unlabeled) so that the operation of the /?-keto reductase (pOH-acyl-CoA reductase) could be detected. On the other hand, the increase in dehydratase activity observed in fractions 64-68 is in part the result of decreasing P-OH-acylCoA reductase activity and in part to low enoyl-CoA reductase activity. It should be borne in mind that labeled tetradecenoylCoA should arise only from labeled P-OHmyristoyl-CoA (the reverse reaction of enoyl-CoA reductase should be stereospecific for the hydrogens inserted or removed). Thus the apparent decline of dehydratase activity observed after fraction 68 would be explained by increase of the enoyl-CoA re-

Fraction

No.

8. Relative amounts of intermediate and product acids produced from P-OH-myristoyl-CoA and NADP3H. Half-milliliter reaction mixtures contained 50 mM Hepes and 5 mM KzHP04 buffer (pH 7.9), 5 mru mercaptoethanol, 1 mru ATP, 5 mM MgCL, 0.5 mM NADP+, 0.25 mM n-[l-*H]glucose, 1.5 units of glucose-6-phosphate dehydrogenase (from baker’s yeast, 2 units of hexokinase, 20 pM P-OH-myristoylCoA, and protein contained in a 0.25~ml aliquot from each 1.5~ml fraction. Reactions were incubated at 30°C under an atmosphere of nitrogen for 30 min then terminated by the addition of 0.2 ml 8 N KOH. Extraction and analysis of fatty acids was as described under methods. Proportional amounts of each acid were assigned based on integrating the counts/min for each peak on the gas-liquid chromatography trace divided by the total counta/min for the run after correction for background. The shift in the primary acid synthesized was most dramatic between fractions 64 and 70. Only the major portion of the protein peak was assayed for activity. FIG.

ductase activity. Whether or not dehydratase activity was indeed declining cannot be determined from these experiments but it is important to note that dehydratase activity must be present to provide substrate for the enoyl-CoA reductase whose activity is rapidly increasing in the late fraction. We conclude from these experiments that under the conditions of fractionation and enzymatic assay that the enzymatic activity can be successfully separated. If all the activities coincided under

ACYL-CoA

ELONGATION

BY

CHLOROPLAST

the umbrella of the protein peak then it could indicate either a multifunctional enzyme or a multienzyme complex. If there was no coincidence of activities, the results would suggest separate enzyme systems. Figure 8 clearly indicates that there was no coincidence of activity and thus presumably the enzymes were discrete entities. DISCUSSION

Previous investigators (29) have documented a biosynthetic pathway for fatty acid synthesis in spinach chloroplasts which was ACP dependent. Jaworski et al. (6) described an elongation system for palmitoyl-ACP to stearoyl-ACP with properties distinct from the de nouo system. In addition, Jacobson et al. (7, 20) have provided evidence for the elongation of hexadecatrienoic acid to a-linolenoic acid utilizing acetate. This paper defines the latter pathway as coenzyme A dependent and characterizes its substrate specificity. The system is of interest in that acetyl-CoA is the CZ donor, both saturated and unsaturated acyl-CoAs serve as primer units with lauroyl-CoA the optimum substrate, and ATP markedly activates the system whereas from CoASH is an effective inhibitor. It is difficult to assess directly the physiological importance of this non-ACP dependent elongation system in the chloroplast. It has been consistently observed that free pahnitic added to spinach leaf slices is not elongated to stearic acid. However, in viva and in vitro studies clearly suggest that 16:3-CoA is elongated to 18:3CoA in spinach preparations. Based on data in this paper, about 1% of the acyl-CoA is elongated to the next higher homolog under optimal in vitro conditions in 30 min. Since both the level of ATP and free CoA have profound effects on the rates of elongation under in vitro conditions, it will be difficult to evaluate the activity of this elongation system in the intact chloroplast. A direct test of the capacity of isolated chloroplasts to elongate added acyl-CoAs is difficult to design since the outer envelopes of chloroplasts are impermeable to these substrates. Preliminary data would suggest that the elongation enzymes are catalyzing reactions

STROMAL

219

SYSTEM

very similar to those first reported by Seubert et al. (2) in animal mitochondria and appear to be discrete proteins since fractionation of the stroma protein on Sepharose 6B clearly indicated overlapping but discrete peaks of activity. Evidence is now accumulating that in a variety of tissues long chain acyl-CoAs are further elongated. For example, in the developing cotyledons of Simmondsia chinensis (Link) oleoyl-CoA appears to be elongated to eicosenoyl-CoA and thence to docosenoyl-CoA. These substrates are then reduced to their corresponding alcohols and both alcohols and acids combine to form C4o-44 waxes (30). It is conceivable that a variant of the stroma system described here is functioning in the Simmondsia system. Whatever the role of this elongation system in the chloroplast, this investigation suggests that an acyl-CoA-acetyl-CoA system is functioning under in vitro conditions. In the intact cell the system may be under rigid control by critical levels of ATP and free CoA and only functional when acylCoAs are available for elongation and when ATP levels are elevated and CoA levels are markedly reduced such as when fatty acids had been converted to their acyl-CoAs by ligase activity. REFERENCES 1. SEUBERT,

W., GREULL,

G., AND LYNEN,

F. (1957)

Angew. Chem. 69,359-361. 2. SEUBERT, W., LAMBERTS, I., KRAMER, R., AND OHLY, B. (1968) Biochim. Biophys. Acta 164, 498-517. 3. WAKIL, S. J. (1961) J. L@pid Res. 2, l-23. 4. HARLAN, W. R., AND WAKIL, S. J. (1963) J. Biol.

Chen. 238,3216-3223. 5. PODACK,

E. R., AND SEUBERT,

W. (1972)

Biochim.

Biophys. Acta 280.235-247. 6. JAWORSKI, STUMPF,

J. G., GOLDSCHMIDT, E. E., AND P. K. (1974) Arch. Biochem. Biophys.

Res. Commun. s&487-493. 7. JACOBSON, STUMPF,

B. S., KANNANGARA, C. G., AND P. K. (1973) Biochem. Biophys. Res. Commun. 51,487-493. 8. HARRIS, R. V., AND JAMES, A. T. (1965) Biochim. Biophys. Acta 106,456-464. F. B. (1945) Nature 156,269-270. 9. MORELAND, 10. ALLEN, C. F., GOOD, P., DAVIS, H. F., CHISIM, P., AND FOWLER, S. D. (1966) J. Amer. Oil Chem. Sot. 43,223-231.

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11. HEYES,

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Biochem. Physiol. Pharmacol. 37,911-916. 13. MORRIS, L. J. (1966) J. Lipid Res. 7, 717-732. 14. YOUNG, D. L., AND LYNEN, F. (1969) J. Biol. Chem. 244,377-383. 15. GOLDMAN, P., AND VAGELOS, P. R. (1961) J. Biol. Chem. 236,2620-2623. 16. SEUBERT, W. (1960) in Biochemical Preparations (Lardy, H. A., ed.), Vol. 7, pp. 80-83, John Wiley & Sons, New York. 17. PABST LABORATORIES (1974) Biochemicals Reference Guide 101.8. 18. STEIN, R. A., AND NICOLAIDES, N. (1962) J. Lipid

Res. 3,476-478. 19. HARRIS, R. V., HARRIS, P., AND JAMES, A. T. (1965) Biochim. Biophys. Actu 106,465-473. 20. JACOBSON, B. S., KANNANGARA, C. G., AND STUMPF, P. K. (1973) Biochem. Biophys. Res. Commun. 52,1190-1198. 21. NAGAI, J., AND BLOCH, K. (1967) J. Biol. Chem. 242,357-362. 22. BARDEN, R. E., AND CLELAND, W. W. (1969) Bio-

STUMPF

them. Biophys. Res. Commun. 34,555-559. 23. GATT, S., AND BARENHOLZ, Y. (1973) in Annual Review of Biochemistry (SneII, E. E., Boyer, P. D., Meister, A., and Richardson, C. C., eds.), Vol. 42, pp. 690, Annual Reviews Inc., Palo Alto, Cdif. 24. HINSCH, W., KLAGES, C., AND SEUBERT, W. (1976)

Eur. J. Biochem. 64,45-55. 25. NOLTMAN, E. A., AND KUBY, S. A. (1963) in The Enzymes (Boyer, P. D., Lardy, H., and Myrback, K., eds.), Ed. 2, Vol. 7, pp. 223-242, Academic Press, New York. 26. STERN, B. K., AND VENNESLAND, B. (1960) J.

Biol. Chem. 235,205-208. 27. MAZELIS, M. (1956) Plant Physiol. 31,37-43. 28. GREEN, D. E., AND ALLMANN, D. W. (1968) in Metabolic Pathways (Greenberg, D. M., ed.), Vol. 2, Ch. 8, pp. l-37, Academic Press, New York. 29. KANNANGARA, C. G., JACOBSON, B. S., AND STUMPF, P. K. (1973) Plant Physiol. 52, 156-161. 30. OHLROCGE, J. B., POLLARD, M. R., AND STUMPF, P. K. (1978) Lipids 13,203-210.