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Fat metabolism in higher plants
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
162, 158-165 (1974)
Fat Metabolism Properties
of a Soluble
Stearyl-Acyl
in Higher Carrier
Carfhamus J. G. JAWORSKI Department of Biochemislry
and Biophysics,
Protein
Plants Desaturase
from Maturing
finctoriusl* ’ AND P. K. STUMPF University
of California,
Davis,
California
95616
Received November 19, 1973 A soluble extract from maturing safflower seeds (Carthamus tinctorius) synthesized [l%]oleic acid from [%]malonate, or [14C]stearyl-acyl carrier protein. Stearyl-acyl carrier protein was generated from [14C]malonate by the seed extract. The desaturase had only a trace of activity when stearyl-CoA was the substrate. The stearyl-acyl carrier protein desaturase had a specific requirement for ferredoxin which was only partially replaced by flavodoxin. While NADPH was an effective reductant, NADH was ineffective. However, the most effective reductant was a system composed of ferredoxin, grana lamellae, ascorbic acid, dichlorophenolindophenol, and light. No NADPH requirement was observed when this reducing system was employed. Stearylacyl carrier protein desaturase activity was enhanced by dithiothreitol and reduced glutathione, but was partially inhibited by&mercaptoethanol. The desaturase activity was inhibited by 1 mM potassium cyanide but insensitive to carbon monoxide. No lipid micelle requirement could be demonstrated.
Oleic acid synthesis occurs with membrane-bound systems isolated from fungi (1) yeast (2), and liver (3) and each of these systems is specific for stearyl-CoA. In developing castor bean, oleic acid biosynthesis is localized in the proplastid fraction (4). Soluble enzyme systems capable of synthesizing oleic acid have been isolated from Euglena (5, 6), spinach chloroplasts (6), and soybean cotyledons (7, 8). Although the Euglena system has been partially characterized, it is not clear whether stearyl-ACP3 or stearyl-CoA is the substrate (6). Synthesis of oleic and linoleic acids by cell-free extracts of developing safflower seeds (Carthamus tinctorius) occurred maximally in extracts prepared from seeds that 1 This is paper LIX in a series. Paper LVIII is the preceding paper (Arch. Biochem. Biophys. 161, 147-157, 1974). Paper LX is the following paper (Arch. Biochem. Biophys. 161, 166-173, 1974). 2 Suported in part by NIH Grant GM 19213-01. 3 Abbreviations : ACP, acyl carrier protein ; BSTFA, bis(trimethylsilyl)trifluoroacetamide.
were harvested 1418 days after flowering (9). Extracts from these developing seeds were capable of converting oleyl-CoA to linoleyl-CoA (10-12). The oleyl-CoA desaturase was a membrane-bound enzyme (10, 12) and its activity was dependent on NADH and molecular oxygen (12). Furthermore, since this desaturase demonstrated a high specificity for oleyl-CoA (II), the question as to the origin of oleyl-CoA was of interest. A very efficient synthesis of oleylCoA must occur since linoleic acid comprises 75% of the total fatty acids in this oil-rich seed. In this report data will be presented demonstrating oleic acid synthesis by a soluble fraction from extracts of maturing safflower seeds. The substrate is stearylACP. MATERIALS
Materials. NADP, NADPH, glucose-6-phosphate, NADH, ATP, and imidazole were obtained from Sigma; dithiothreitol was from Calbiochem; CoASH was purchased from P-L Biochemical,
158 Copyright All rights
@ 1974 by Academic Press, of reproduction in any form
Inc. reserved.
AND METHODS
IIW. I )I~:Al~:~c~t~ll~~lr~s~~ (111M2) was obtained from What mtn. l’iflef~ll ~~~~uYII~ I1 i-I~~FP-2131’ 011 ( ;:W (:hronl Q, lO’,‘( ICCISS-s o,, c:as C11r0m I’, and 14’.:, horon trifluoridc in methanol were from hpl)lied Science 1,ahoraiories. His(trimethylsilyl)t riflaoro:lc.ct:tltl~~~~, (136TPA) was from Supelco, lnr. j“~‘~(~]m:rlorr:~ t ca (5.5 (‘i, mole) was from New l~~~rgl:~ntl N rlc*le:tr; 11-I’(: lstearatc (48.7 Ci;‘mole), 11.“<‘]ole:~te (5:<.5 (‘i ‘IIIO~P), and sodi~un[:iH]horc)hgtiridc (125 (‘i molp) were from Amersham Srarle. I’rlrifird spinacah fcrredoxin was a generolls gift of I jr. I%oh 13~~c~hanan, U.C. Berkeley, and c,ytoc*hrornc ,‘L,ia, (~ytochrome ra, and flavodoxin were generous gifts of l)r. J. I,e(;all, Centre Nat iollale do la Iterhercte Scientifique, France. ACP was isolated front E. chili 13 by the method of Alhcbrt, bI:ijerlrs, :md \.agelos (13) and purified to the initial achid precipitation.
(‘hcmic~allv l)reparcd jl-‘%]stearyl-ACP was a gift of I)r. Inder L ijay, and was prepared by the met.hod of I
spinach c*hl~,roplast l:L~lElliLe prcp:trcYl I)>- lll(’ method of Kat~nangara, ,Jac~~hson, :LII([ St unrpl (18). The c*hloroplast lamellae were washc~~ twicxc with isolation medium c*ont,aining no sot-hit 01 :Irlcl stored at -1OY1 in the isolation mrdi\nn caont:lilling 0.6 M sorhitol. l(cactions containing (~hioroplast lanlellxe were r,ln at 14Y: wit ir 1000 fi 4, of white light. The reac*t,ion was st.oppcd I)y :tddit,iorl of 0.2 ml of 8.0 M KOfI, and the relat ivc, r;cdio:lc, tivity in the individtL:rl fatty acids dcterrrlinc~tl :~s described in thcl [“C]fat tg acid analysis sc~,t ioIl. I)? ,,01‘0 fatty ac’id synthesis was c,:Lrricxtl r~llt Itsing 5 mg protein of saflower heed 0x1 r:i(st ill~ii-hated with 70 pmoles imidazole t)rdY’c,r, pll 7. 1, 0.’ .~~les c’o.4, 0.5 ccn~olrs AN.41)H, 0.5 ~molrs X,2 1)l’, -4.0 pmolcs glll~ose-6.phosph;tte, 2 ,~moles ATP. 1 pmoles dithiothreittrl. 100 /ifs of E. r.rj/i A(‘t’, 50 pg of spinac~h i’erredoxill, arid 1.25 ,~mc~les oI’ /.“V * /malonatt~ (COO,ClUOclam) in it total vol~m~f~ of 0.7 ml for 1 hr ai 14°C’. The reaction was str)pp(ad trh addition of 0.2 ml of X.0 M KOII and the rclat iv{, radioactivit>, in the individrull fatty :ic,ids tlc~tc~r.. mined as descaribrd below. 1)isappearanc.e of thioestcr of :tcyl-(‘(I;\ ;111d stearyl-i\(‘l’ in Ihe prrsenc*r of s;tlllow(~r sc~tl ~5’;. t,ract was monitored t)y illc,\that ing :ul:rc~rtr~)ic~:~ll~ 0.2 nmole of the s\tt)stratc, 5 mg protein of salllowrr seed extract, and 60 ~molcs imidazolr, pI1 i.4, in a total volume of 0.6 ml al l-L”(: for the drsiglls tcstl length of time. Thiorster c,onrent rat ion was q,u~“tit,ated hy the Harrctn :trrd Mooney :tn:rlysis (17 I. NAI)PII oxidase :rcstivity WZLSassayc~l rpc~trc,photometric~ally. Increasing amolmts of s:~lllow~r seed ext,racnt were inctthated with 300pmolrs imid:tzole hrrffer, pH 7.3, 0.5 j~noles of NAI)I’II. and 0.13 pmoles of dic~hlorophrr~olirldophrrrol in :I total volllme of :%ml. I:eaction was mollitc,rc~~l 1)~ t,hr decrease in ahsorhanc~e at 600 nm. of qlr:rryl-.4(‘1’ The I’erredoxin req\tiremrLnt , desatllrase was established t)y t hr mt>thotl 01 hlortcnson (19). Two-milliliter aliqrlots of safflower seed extracBt were treated with v:trious amounts of preeqr~ilibrated I)EAT’. ~crlll~lost~ (19) in a conical centrifuge tithe for 5 min :\I 2°C’. ‘l’hc~ I )EAIGc*ellrdose was pellet,ed by cent rif klging i tI a swinging-hllck(:t elinic~al c*entrifltge and tht> supernat,ant was assayed for de ),DM olrate s)-nthcsis from /2-l”<: ]malonatc in the prcseIIc(l or :it)scrrrf~ of 50 pfs of prlre spinac.h ferredoxin. Suc9wse rlc,rsify gmtlient (.P)/tliS16g/~fjl))i. S:tUlower extracats were fractionated by sIIcrosc drnsit,y gradient centrifugation by the method of Lord, Kagawa, and Becvers (JO) except, that i he srrds were gently homogenized with a mort,ar and pestle.
160
JAWORSKT AND STUMPF
netted to a Nuclear-Chicago I3iospan 4998 proportional tube detector. The glr contained a lo-ft stainless-steel column (0.25 in. o.d.) packed with either 1577, Hi-Eff-2BP on Gas Chrom Q or 10% EGSS-X on Gas Chrom P. All analyses were run isothermally at 182°C with a helium flow rate of 60 ml/min. Under these conditions, methyl stearate and methyl oleate were completely separated. Typical analysis of reaction products involved saponification with 2 N KOH for 30 min at 8O”C, acidification, and Bligh and Dyer extraction (21). Fatty acids were methylated with 147, boron trifluoride in methanol (22). Fatty alcohols were chromatographed as the trimethyl silyl derivat,ive prepared with BSTFA. The identity of [Wloleic acid as the enzyme product was cdnfirmed by cochromatography with authentic oleic acid on glc, on 15y0 AgNOI-silica gel G thin-layer chromatogram, and by reductive ozonolysis and subsequent glc analysis. By these techniques the chain length, degree of unsaturation, and position of the double bond were est,ablished . RESULTS
Conditions of maximum oleic acid synthesis. Initial studies of oleic acid synthesis were carried out by using a complex system which consisted of a ‘de novo fatty acidsynthesizing system as well as a stearyl desaturase, and measuring the amount of the substrate, [2J4C]malonic acid, which was incorporated into [14C]oleic acid,. the final product. Particular attention was paid to conditions which yielded the highest percentage of olcic acid relative to other longchain fatty acids synthesized. The system was very sensitive to temperature. While fatty acid synthesis as well as the synthesis of oleic acid increased as the temperature increased from 14°C to 37”C, the percentage of oleic acid decreased, with a corresponding increase in the relative amounts of palmitic and stearic acids. The lower percentage yield of oleate was not simply related to decreasing oxygen concentrations existing at higher temperatures (23, 24) but was more likely due to a combination of several temperaturesensitive factors influencing the system. Although we have not further investigated the influence of temperature on this system, the temperature which allowed the synthesis of the highest percentage of oleic acid, i.e., 14”C, was chosen as the reaction tcmperature.
It was also noted initially that high acyl carrier protein (ACP) conc.entrations and enzyme extract with low-protein concentrations gave low percentage yields of oleic acid. This effect was completely reversed by supplementing the reaction mixture with purified spinach fcrredoxin. Addition of increasing amounts of ferredoxin to the reaction mixture resulted in maximum stimulation of oleic acid synthesis at 50 pg ferredoxin/0.7 ml (Fig. 1). The requirement for ferredoxin was determined directly tising bhe method of Mortenson (19). By titrating the enzyme extract wit’h DEAE-cellulose, a necessary component for oleic acid synthesis was removed, and this requirement was fulfilled by supplementing the extract with ferredoxin, as shown in Fig. 2. To determine if hhis stimulation was specific for ferredoxin, a number of other electron carriers were examined with both untreated and DEAE-tiellulose-treated enzyme extracts. As shown in Table I, only flavodoxin was partially capable of replacing the ferredoxin in extracts treated with DEAEcellulose, but there was no stimulation of untreated extracts with Aavodoxin. While some electron carriers were inactive, others, namely, cyt ~553,cyt ca, FMN, and FAD, were inhibitory. The stearyl-ACP desaturase has an absolute requirement for oxygen. As illustrated
Ferredoxin
added I(ugj0.7ml)
FIG. 1. Effect of increasing amounts of ferredoxin on oleic acid synthesis in extracts not treated with DEAE-cellulose. Reaction conditions for de ILOVO fatty acid synthesis are described in the text.
DEAE added
(mg/ml)
P‘ic. 2. Ikmonstration of ferredoxin requiremen t, for de /IWO oleic acid synthesis in developing safflower seed extract. Experimental mnditions are described in t,he text.
None E’erredoxin (spinach) Flavodoxin (D. gigs) Cyl c (horse heart ) Cyt 552 (Euglet~a) I’lastoc~yanin (Eugle/~tr) Cyt (‘j51 (Il. gigas) Cyt, (‘3 (D. gigm) FMN FA I )
55 82 57 50 50 45 0 7 22 26
I, Fifty mirrograrns of eac*h electron was added to the assay mixture.
13 91 15 9 13 10 0 8 -
I
RETENTION
TIME lnilf,
FIG. 3. (;as radiochromstogram of the methylated products of tie IIWO fatt,v acid synt.hesix I).\salllower seed extracts klrldcr anaerobic~ and aerobic conditions. Conditions of t IIP K?.Ivc~hronmtography are described in thr trrt.
carrier
in l:ig. 3, \vhilc no synthesis of olric acid ocrxrwd unacrohically, undor aerobic co11ditions, TO-90 71 of 14C incorporated into fatt,y acids is found in olric acid. The rcmsindw of thr 14C is usually found in palrnitic and stc>aric acids. ASuOstr~a~e specijicity. Inwstigation of the substrut,c spwificity was carrkd out under conditions of optimal okatc~ synthesis. *4s show-n in Tabk II, both chemically and cw zymknlly prepawd stcaryl-AU’s wcrc efl’w-
Suhstrale
(/;
(‘0nvwGon -~
Htesrgl-ACP (chemic.ally prepared) Stearyl-ACl’ (enz;?-micsnlly prcpared) Stearyl Co.4 Stearnte (NH4+ salt) Palmityl CoA I’almitat~ (NH,+ salt)
50 60 <.5 0 0 0
‘L Each reaction mixture contained 0.5 nmoles of sllbst,rate. Inrld~:ti ion conditions are described in the tclxt,.
162
JAWOR.SKI AND STUMPF
qualit> with each preparat’ion showing excellent substrate activity. As a result, all subsequent experiment’s were performed using cneymically prepared stearyl-ACP. The preparation and characterization of this substrate are fully described in the succecding paper (16). The stearyl-ACP desaturase was only slightly active when stearyl-CoA was the substrate and none of the other substrates used were desaturated. The identit’y of oleic acid as the product of the dcsaturation using stearyl-ACP was confirmed as described in the Methods section. As illustrated in Fig. 4, the desaturation reaction was linear for 20 min and essentially complete after 60 min. Since the desaturase was in a crude extract, it was also important to examine the significance of hydrolytic reactions which may have consumed the substrate. The enzymic hydrolysis of the thioester of stearyl-ACP by the safflower extract was determined as well as that of stearyl-CoA and oleyl-CoA. As shown in Fig. 5, there are competing reactions involving the cleavage of thioesters which took place during incubation. The loss of thioester of stearyl-ACP and stearyl-CoA occurred at approximately t,he same rate while the loss of thioester in oleyl-CoA was much more rapid. Presumably, the thioester is either hydrolyzed to yield a free acid or the acyl group was transfcrrcd to lipid acceptors to yield an oxygen ester. m-
0
I’o
bb Time of wubatlon
(mlnutesj
FIG. 4. Time course of stearyl-ACP desaturase reaction. Each assay contained 0.2 nmoles of [‘GJstearyl-ACP.
% thlal ester
20. 0
, 5 IO
20
Time of lncubatlon
30 (minutes)
FIG. 5. Loss of thioesters in an assay mixture as a function of time. Each assay contained 0.2 nmoles of substrate. Assay conditions are described in the text.
Localization of stearyl-ACP desaturase. Stearyl-ACP desaturase was consistently observed in the 105,OOOysupernatant of crude enzyme extracts. To determine if the stearylACP desaturase was exclusively in the soluble fraction, a 2SOq supernatant fraction prepared from developing safflower seeds was separated by sucrose density gradient centrifugation (20). While the cell organelles migrated as discrete bands, both the fatty acid synthetase and stearyl-ACP desaturase activities were totally located in the soluble fraction. Therefore, both the fatty acid synthetase enzymes and the stearyl-ACP dFsaturase occur as soluble systems in the cytoplasm of the cotyledonous cell. NADPH requirement. The desaturase required a reduced pyridine nucleotide for a,ctivity. As shown in Fig. 6, the system had an apparent K, of about 2 X 1OF M for NADPH and was essentially saturated with 10 nmoles in the 0.6.ml reaction mixture. NADH was completely inactive as an electron donor in this system. Since previous workers (6) have implicated NADPH oxidase as linking NADPH to an electron acceptor for the activation of oxygen, the extract was assayed for t’his enzyme activity using dichlorophenolindophenol as the electron acceptor. The amount of extract used in a routine assay could reduce about 2 pmoles of DCIP per minute, which would more then meet the electron requirements of the dcsaturase system.
STEARYLACP
under the usual conditions except that the O2 concemration of the atmosphere was 10 ‘;h instead of the, -“1 % (air), and the rcwt~ion was ca,rricd out, in the dark. Although avian liver stearyl-CoA dcsat~urnsc rquircd lipid micelles for full activity (27), this wquircmerit \vax not obwrved with thcb voluble stcaryl-ACP desaturaw of safflon-or.
NADPH
1 5
F. 0 10
IO 20 NADPH or NADH added (nmolesi
, 25 LAMELLAE
50
I( i:-;
DESA’I’URASI:,
.J
100
j JJ~ chlorophyll/
Fro. 6. Effect of increasing amounts of reduced pyridine mtcleotide or spinach lamellae on stearylACP desaturase activity. Each assay contained 0.2 nmoles of [r4C]stearyl-ACP. NADP+ and glucose-6-phosphate were replaced in the assay by the specified amount, of either NADPH or NADH, or spinach lamellae. Assay conditions are described in the text.
In order to determine whether KADPH was an obligatory component of the desaturase or only a nonspecific source of electrons, a chloroplast lamellae system was employed to photoreduce ferredoxin in the light in the absence of KADPH. As can be seen in Fig. 6, the safflower desaturase was even more active when coupled to phot’ochemically reduced ferredoxin than when coupled to ferredoxin reduced by the NADPH oxidase. In the abssancc of lamellae or ferredoxin, there was no desaturase activity. Washed lamellac at’ a concentration of 50 pg chlorophyll/ml had no drsaturase act’ivity. E$ect of sulfh~cl~~yl compounds. The react,ion mixt’ure required a sulfhydryl compound for maximum activity. As seen in Table III, both dithiothrcitol and reduced glutathione enhanced tho drsaturasc activity. However, thrl des~turnw was inhibited by P-mercapto-
To date, invest,igations on the detiaturation of long-chain fatt,;- acids have 1)~weded slowly by comparison to t!hose concerned with fatty acid synthesis since all the prol)arations obtained from yeast and animal tissue have been m~~mbranr:-bound cmy~IIes. Extracts of the developing safflower seed COIItain a completely solublth as \vell :w
L’!1 :; ,3.!! ti1 .T ; !)
None Dithiothreitol Reduced glutathione P-~lercaptoetharlol
a Each assay contained 0.2 nmole of i’“C’]stearyl-ACP and 1 pmole of the specified sttlfhydryl compotmtl.
Inhibitor
Experiment __~_____--..-~
~-~~~. x2 6X Iii
1
Sane KCN (0.1 m>i) KC?G (1 .o IllhI I
2
lO$( (12 + SO’,; X3 (control)
c+ ll:LIIo!.
C’yaGle irrhihitici/r. As show-n in the Table IV, potassium cynnidt:, which had been prepared fresh and neutralized, was strongly inhibitory at a wnccntration of 1 .O m&r but only slightl:. at 0.1 m&r. Carbon monoxide, hon.w(r, had no t#cct, on desaturase activity-. Thea latter c,spcriment was carried out
(, (‘onversion - ~-~ -
Thiol compound
lOs’, co
+
lo’;
02 +
soy;
:(4 S?
:3 I
u Each assay contained 0.2 nmoles of strarlyACP. Carbon monoxide assay was carried 011t in t,he dark.
164
JAWORSKI
systems which are also capable of synthesizing oleic acid have been prepared from Euglena gracilis (5, 6) spinach chloroplasts (5, 6), and soybean cotyledons (7, 8). The most extensively characterized system was prepared from Euglena by Nagai and Bloch (6). The developing nonphotosynthetic safflower seed system is similar to the Euglena system in that (1) both employ NADPH effectively as an electron donor and an NADPH oxidase is readily demonstrated in the extract, (2) both have a ferredoxin requirement, and (3) both are cyanide sensitive, but carbon monoxide insensitive. Thus, the electron-generating and carrier systems for the two enzymes appear to be similar. Unlike the Euglena system (6), however, the safflower desaturase appears to be specific for stearyl-ACP. Only a negligible activity could be demonstrated with stearyl-CoA as the substrate. It was of interest that cyanide markedly inhibited the soluble desaturase system. A cyanide-sensitive factor has been observed in the avian stearyl-CoA desaturase system (25-27) and the cyanide-sensitive site has been placed between cyt-bg and the desaturase (26). It seems likely that in the safflower system the cyanide-sensitive site is located after ferredoxin and at or before the desaturase site since both ferredoxin and NADPH oxidase are insensitive to cyanide. No phospholipid requirement has been observed in the safflower system in contrast to the avian system which requires lipid micelles for optimal activity (26, 27). In the crude extracts the rate-limiting step for desaturation involved ferredoxin since in these extracts conditions which reduced the rate of dcsaturation relative to fatty acid synthesis, e.g., high ACP concentration or low protein concentration, could be reversed by supplementing the reaction mixture with ferredoxin. Furthermore, when NADPH + NADPH oxidase was replaced with a chloroplast lamellae system, desaturase activity was increased still further. The complete replacement of NADPH by a grana system which reduced ferredoxin implies that NADPH is not an obligatory reductant but only one of a number of possible reductants available to the system in the cell. In photo-
AND
STUMPF
synthetic organisms such as Euglena, an important source of electrons for reducing ferredoxin is quite possibly Hz0 via photosystems I and II with NADPH as an ancillary source. Both the safflower and the Euglena systems specifically required ferredoxin. Other electron carriers assayed were either inactive or inhibitory in that they probably diverted the electron flow away from the desaturase system. It should also be recognized that ferredoxin may not be the natural electron carrier but is simply capable of efficiently replacing the carrier which was removed by the DEAE-cellulose procedure of Mortenson (19). With the availability of large amounts of tissue, purification of the enzymes involved in desaturation will be undertaken to extend and clarify these observations. REFERENCES 1. BAKER, N., AND LYNEN, F. (1971) Eur. J. Biothem. 19, 20&210. 2. BLOOMFIELD, D. K., AND BLOCH, K. (1960) J. Biol. Chem. 236, 337-345. 3. M.~RsH, J. B., AND JAMES, A. T. (1962) Riochim. Biophys. Acta 60, 320-328. 4. ZILKEY, B. F., AND QNVIN, 1). T. (1972) Cwt. J. Bot. 60, 323-326. 5. N~c.41, J., AND BLOCH, K. (1965) J. Biol. Chun. 246, PC3702-3703. 6. NAGAI, J., AND BLOCH, K. (1968) J. Biol. Chem. 243, 46264633. 7. INKPEN, J. A., AND QUACKENBUSH, F. W. (1969) Lipids 4, 539-543. 8. RINNE, R. W. (1969) Plant Physiol. 44, 89-94. 9. MCM~HON, V., AND STUMPF, P.K. (1966) Plant Physiol. 41, 14&156. 10. MCMAHON, V., AND STUMPF, P. K. (1964) Biochim. Biophys. Acta 84, 359-361. 11. VIJAY, I. K., .IND STUMPF, P. K. (1971) J. Biol. Chem. 246, 29162917. 12. VIJAY, I. K., AND STUMPF, P. K. (1972) J. Biol. Chem. 247, 360-366. 13. ALHERTS, A. W., MAJYRUS, P. W., AND VAGELOS, P. R. (1969) Methods Enzymol. 14,43-50. 14. GALLIARD, T., AND STUMPF, P. K. (1968) i?~ Biochemical Preparations (Lands, W. E. M., ed.), Vol. 12, p. 66, Wiley, New York. 15. BIRGE, C. H., SILBERT, D. F., AND VAGELOS, P. R. (1967) Biochem. Biophys. Res. Cow mm. 29, 808-814. 16. JAWORSKI, J., AND STUMPF, P. K. (1974) Arch. Biochem. Biophys. 162, 166-173. 17. BARRON, E. J., AND MOONEY, L. A. (1968) Anal. Chem. 40. 1742-1744.
STlGAI:YI,-ACP 18.
K\NN.ING.UI.\, STUMPF,
I)fCSATIJf:.~Sf’
C. C;., JXOUSON, B. S., .\m P. K. (1973) Plant Physiol. 62, 156-
161. 19.
I,. I’,. (1964) Biochim.
~~ORTENSON,
BiqJh~/.S.
Acta 81, 473-478. 20. LORD, J. M., K.\o.\w.\,
T.,
BEEVF:R~, H.
.iND
(1972) J’roc. .\‘a/. Acad. Sci. C:SA 69, 21292432. 21.
BLIGH,
15. (i.,
Hiochem. 2".
MOKRISON,
I,ipitl
.\NI)
1)Y~ll,
w.
J.
(1959)
Ph!/siol. 37, 911-917. W. IX., .\ND SMITH, 1,. Rrs. 6, 600-608.
M.
?a//.
.I.
(1964) J.
I li.i
23. H.\RHIS, I’., .\NU .J.uII+, A. T. (196!)i /<;oc,/IPY)/. J 112, 325-330. 24. H.\ERIS, P., .~ND J.GXICS, A. T. (19691 /Z~ocl~iv/. Niophys. Acta 18’7, 13-18. 25. W.\IXI,, I;. J. (1964) i/r IVfetaholisn~ and f’hysiological Significance of Lipids (I ):tw~oll, 1:. M. C., and I
OF
BIOCHEMISTRY
AND
BIOPHYSICS
162, 158-165 (1974)
Fat Metabolism Properties
of a Soluble
Stearyl-Acyl
in Higher Carrier
Carfhamus J. G. JAWORSKI Department of Biochemislry
and Biophysics,
Protein
Plants Desaturase
from Maturing
finctoriusl* ’ AND P. K. STUMPF University
of California,
Davis,
California
95616
Received November 19, 1973 A soluble extract from maturing safflower seeds (Carthamus tinctorius) synthesized [l%]oleic acid from [%]malonate, or [14C]stearyl-acyl carrier protein. Stearyl-acyl carrier protein was generated from [14C]malonate by the seed extract. The desaturase had only a trace of activity when stearyl-CoA was the substrate. The stearyl-acyl carrier protein desaturase had a specific requirement for ferredoxin which was only partially replaced by flavodoxin. While NADPH was an effective reductant, NADH was ineffective. However, the most effective reductant was a system composed of ferredoxin, grana lamellae, ascorbic acid, dichlorophenolindophenol, and light. No NADPH requirement was observed when this reducing system was employed. Stearylacyl carrier protein desaturase activity was enhanced by dithiothreitol and reduced glutathione, but was partially inhibited by&mercaptoethanol. The desaturase activity was inhibited by 1 mM potassium cyanide but insensitive to carbon monoxide. No lipid micelle requirement could be demonstrated.
Oleic acid synthesis occurs with membrane-bound systems isolated from fungi (1) yeast (2), and liver (3) and each of these systems is specific for stearyl-CoA. In developing castor bean, oleic acid biosynthesis is localized in the proplastid fraction (4). Soluble enzyme systems capable of synthesizing oleic acid have been isolated from Euglena (5, 6), spinach chloroplasts (6), and soybean cotyledons (7, 8). Although the Euglena system has been partially characterized, it is not clear whether stearyl-ACP3 or stearyl-CoA is the substrate (6). Synthesis of oleic and linoleic acids by cell-free extracts of developing safflower seeds (Carthamus tinctorius) occurred maximally in extracts prepared from seeds that 1 This is paper LIX in a series. Paper LVIII is the preceding paper (Arch. Biochem. Biophys. 161, 147-157, 1974). Paper LX is the following paper (Arch. Biochem. Biophys. 161, 166-173, 1974). 2 Suported in part by NIH Grant GM 19213-01. 3 Abbreviations : ACP, acyl carrier protein ; BSTFA, bis(trimethylsilyl)trifluoroacetamide.
were harvested 1418 days after flowering (9). Extracts from these developing seeds were capable of converting oleyl-CoA to linoleyl-CoA (10-12). The oleyl-CoA desaturase was a membrane-bound enzyme (10, 12) and its activity was dependent on NADH and molecular oxygen (12). Furthermore, since this desaturase demonstrated a high specificity for oleyl-CoA (II), the question as to the origin of oleyl-CoA was of interest. A very efficient synthesis of oleylCoA must occur since linoleic acid comprises 75% of the total fatty acids in this oil-rich seed. In this report data will be presented demonstrating oleic acid synthesis by a soluble fraction from extracts of maturing safflower seeds. The substrate is stearylACP. MATERIALS
Materials. NADP, NADPH, glucose-6-phosphate, NADH, ATP, and imidazole were obtained from Sigma; dithiothreitol was from Calbiochem; CoASH was purchased from P-L Biochemical,
158 Copyright All rights
@ 1974 by Academic Press, of reproduction in any form
Inc. reserved.
AND METHODS
IIW. I )I~:Al~:~c~t~ll~~lr~s~~ (111M2) was obtained from What mtn. l’iflef~ll ~~~~uYII~ I1 i-I~~FP-2131’ 011 ( ;:W (:hronl Q, lO’,‘( ICCISS-s o,, c:as C11r0m I’, and 14’.:, horon trifluoridc in methanol were from hpl)lied Science 1,ahoraiories. His(trimethylsilyl)t riflaoro:lc.ct:tltl~~~~, (136TPA) was from Supelco, lnr. j“~‘~(~]m:rlorr:~ t ca (5.5 (‘i, mole) was from New l~~~rgl:~ntl N rlc*le:tr; 11-I’(: lstearatc (48.7 Ci;‘mole), 11.“<‘]ole:~te (5:<.5 (‘i ‘IIIO~P), and sodi~un[:iH]horc)hgtiridc (125 (‘i molp) were from Amersham Srarle. I’rlrifird spinacah fcrredoxin was a generolls gift of I jr. I%oh 13~~c~hanan, U.C. Berkeley, and c,ytoc*hrornc ,‘L,ia, (~ytochrome ra, and flavodoxin were generous gifts of l)r. J. I,e(;all, Centre Nat iollale do la Iterhercte Scientifique, France. ACP was isolated front E. chili 13 by the method of Alhcbrt, bI:ijerlrs, :md \.agelos (13) and purified to the initial achid precipitation.
(‘hcmic~allv l)reparcd jl-‘%]stearyl-ACP was a gift of I)r. Inder L ijay, and was prepared by the met.hod of I
spinach c*hl~,roplast l:L~lElliLe prcp:trcYl I)>- lll(’ method of Kat~nangara, ,Jac~~hson, :LII([ St unrpl (18). The c*hloroplast lamellae were washc~~ twicxc with isolation medium c*ont,aining no sot-hit 01 :Irlcl stored at -1OY1 in the isolation mrdi\nn caont:lilling 0.6 M sorhitol. l(cactions containing (~hioroplast lanlellxe were r,ln at 14Y: wit ir 1000 fi 4, of white light. The reac*t,ion was st.oppcd I)y :tddit,iorl of 0.2 ml of 8.0 M KOfI, and the relat ivc, r;cdio:lc, tivity in the individtL:rl fatty acids dcterrrlinc~tl :~s described in thcl [“C]fat tg acid analysis sc~,t ioIl. I)? ,,01‘0 fatty ac’id synthesis was c,:Lrricxtl r~llt Itsing 5 mg protein of saflower heed 0x1 r:i(st ill~ii-hated with 70 pmoles imidazole t)rdY’c,r, pll 7. 1, 0.’ .~~les c’o.4, 0.5 ccn~olrs AN.41)H, 0.5 ~molrs X,2 1)l’, -4.0 pmolcs glll~ose-6.phosph;tte, 2 ,~moles ATP. 1 pmoles dithiothreittrl. 100 /ifs of E. r.rj/i A(‘t’, 50 pg of spinac~h i’erredoxill, arid 1.25 ,~mc~les oI’ /.“V * /malonatt~ (COO,ClUOclam) in it total vol~m~f~ of 0.7 ml for 1 hr ai 14°C’. The reaction was str)pp(ad trh addition of 0.2 ml of X.0 M KOII and the rclat iv{, radioactivit>, in the individrull fatty :ic,ids tlc~tc~r.. mined as descaribrd below. 1)isappearanc.e of thioestcr of :tcyl-(‘(I;\ ;111d stearyl-i\(‘l’ in Ihe prrsenc*r of s;tlllow(~r sc~tl ~5’;. t,ract was monitored t)y illc,\that ing :ul:rc~rtr~)ic~:~ll~ 0.2 nmole of the s\tt)stratc, 5 mg protein of salllowrr seed extract, and 60 ~molcs imidazolr, pI1 i.4, in a total volume of 0.6 ml al l-L”(: for the drsiglls tcstl length of time. Thiorster c,onrent rat ion was q,u~“tit,ated hy the Harrctn :trrd Mooney :tn:rlysis (17 I. NAI)PII oxidase :rcstivity WZLSassayc~l rpc~trc,photometric~ally. Increasing amolmts of s:~lllow~r seed ext,racnt were inctthated with 300pmolrs imid:tzole hrrffer, pH 7.3, 0.5 j~noles of NAI)I’II. and 0.13 pmoles of dic~hlorophrr~olirldophrrrol in :I total volllme of :%ml. I:eaction was mollitc,rc~~l 1)~ t,hr decrease in ahsorhanc~e at 600 nm. of qlr:rryl-.4(‘1’ The I’erredoxin req\tiremrLnt , desatllrase was established t)y t hr mt>thotl 01 hlortcnson (19). Two-milliliter aliqrlots of safflower seed extracBt were treated with v:trious amounts of preeqr~ilibrated I)EAT’. ~crlll~lost~ (19) in a conical centrifuge tithe for 5 min :\I 2°C’. ‘l’hc~ I )EAIGc*ellrdose was pellet,ed by cent rif klging i tI a swinging-hllck(:t elinic~al c*entrifltge and tht> supernat,ant was assayed for de ),DM olrate s)-nthcsis from /2-l”<: ]malonatc in the prcseIIc(l or :it)scrrrf~ of 50 pfs of prlre spinac.h ferredoxin. Suc9wse rlc,rsify gmtlient (.P)/tliS16g/~fjl))i. S:tUlower extracats were fractionated by sIIcrosc drnsit,y gradient centrifugation by the method of Lord, Kagawa, and Becvers (JO) except, that i he srrds were gently homogenized with a mort,ar and pestle.
160
JAWORSKT AND STUMPF
netted to a Nuclear-Chicago I3iospan 4998 proportional tube detector. The glr contained a lo-ft stainless-steel column (0.25 in. o.d.) packed with either 1577, Hi-Eff-2BP on Gas Chrom Q or 10% EGSS-X on Gas Chrom P. All analyses were run isothermally at 182°C with a helium flow rate of 60 ml/min. Under these conditions, methyl stearate and methyl oleate were completely separated. Typical analysis of reaction products involved saponification with 2 N KOH for 30 min at 8O”C, acidification, and Bligh and Dyer extraction (21). Fatty acids were methylated with 147, boron trifluoride in methanol (22). Fatty alcohols were chromatographed as the trimethyl silyl derivat,ive prepared with BSTFA. The identity of [Wloleic acid as the enzyme product was cdnfirmed by cochromatography with authentic oleic acid on glc, on 15y0 AgNOI-silica gel G thin-layer chromatogram, and by reductive ozonolysis and subsequent glc analysis. By these techniques the chain length, degree of unsaturation, and position of the double bond were est,ablished . RESULTS
Conditions of maximum oleic acid synthesis. Initial studies of oleic acid synthesis were carried out by using a complex system which consisted of a ‘de novo fatty acidsynthesizing system as well as a stearyl desaturase, and measuring the amount of the substrate, [2J4C]malonic acid, which was incorporated into [14C]oleic acid,. the final product. Particular attention was paid to conditions which yielded the highest percentage of olcic acid relative to other longchain fatty acids synthesized. The system was very sensitive to temperature. While fatty acid synthesis as well as the synthesis of oleic acid increased as the temperature increased from 14°C to 37”C, the percentage of oleic acid decreased, with a corresponding increase in the relative amounts of palmitic and stearic acids. The lower percentage yield of oleate was not simply related to decreasing oxygen concentrations existing at higher temperatures (23, 24) but was more likely due to a combination of several temperaturesensitive factors influencing the system. Although we have not further investigated the influence of temperature on this system, the temperature which allowed the synthesis of the highest percentage of oleic acid, i.e., 14”C, was chosen as the reaction tcmperature.
It was also noted initially that high acyl carrier protein (ACP) conc.entrations and enzyme extract with low-protein concentrations gave low percentage yields of oleic acid. This effect was completely reversed by supplementing the reaction mixture with purified spinach fcrredoxin. Addition of increasing amounts of ferredoxin to the reaction mixture resulted in maximum stimulation of oleic acid synthesis at 50 pg ferredoxin/0.7 ml (Fig. 1). The requirement for ferredoxin was determined directly tising bhe method of Mortenson (19). By titrating the enzyme extract wit’h DEAE-cellulose, a necessary component for oleic acid synthesis was removed, and this requirement was fulfilled by supplementing the extract with ferredoxin, as shown in Fig. 2. To determine if hhis stimulation was specific for ferredoxin, a number of other electron carriers were examined with both untreated and DEAE-tiellulose-treated enzyme extracts. As shown in Table I, only flavodoxin was partially capable of replacing the ferredoxin in extracts treated with DEAEcellulose, but there was no stimulation of untreated extracts with Aavodoxin. While some electron carriers were inactive, others, namely, cyt ~553,cyt ca, FMN, and FAD, were inhibitory. The stearyl-ACP desaturase has an absolute requirement for oxygen. As illustrated
Ferredoxin
added I(ugj0.7ml)
FIG. 1. Effect of increasing amounts of ferredoxin on oleic acid synthesis in extracts not treated with DEAE-cellulose. Reaction conditions for de ILOVO fatty acid synthesis are described in the text.
DEAE added
(mg/ml)
P‘ic. 2. Ikmonstration of ferredoxin requiremen t, for de /IWO oleic acid synthesis in developing safflower seed extract. Experimental mnditions are described in t,he text.
None E’erredoxin (spinach) Flavodoxin (D. gigs) Cyl c (horse heart ) Cyt 552 (Euglet~a) I’lastoc~yanin (Eugle/~tr) Cyt (‘j51 (Il. gigas) Cyt, (‘3 (D. gigm) FMN FA I )
55 82 57 50 50 45 0 7 22 26
I, Fifty mirrograrns of eac*h electron was added to the assay mixture.
13 91 15 9 13 10 0 8 -
I
RETENTION
TIME lnilf,
FIG. 3. (;as radiochromstogram of the methylated products of tie IIWO fatt,v acid synt.hesix I).\salllower seed extracts klrldcr anaerobic~ and aerobic conditions. Conditions of t IIP K?.Ivc~hronmtography are described in thr trrt.
carrier
in l:ig. 3, \vhilc no synthesis of olric acid ocrxrwd unacrohically, undor aerobic co11ditions, TO-90 71 of 14C incorporated into fatt,y acids is found in olric acid. The rcmsindw of thr 14C is usually found in palrnitic and stc>aric acids. ASuOstr~a~e specijicity. Inwstigation of the substrut,c spwificity was carrkd out under conditions of optimal okatc~ synthesis. *4s show-n in Tabk II, both chemically and cw zymknlly prepawd stcaryl-AU’s wcrc efl’w-
Suhstrale
(/;
(‘0nvwGon -~
Htesrgl-ACP (chemic.ally prepared) Stearyl-ACl’ (enz;?-micsnlly prcpared) Stearyl Co.4 Stearnte (NH4+ salt) Palmityl CoA I’almitat~ (NH,+ salt)
50 60 <.5 0 0 0
‘L Each reaction mixture contained 0.5 nmoles of sllbst,rate. Inrld~:ti ion conditions are described in the tclxt,.
162
JAWOR.SKI AND STUMPF
qualit> with each preparat’ion showing excellent substrate activity. As a result, all subsequent experiment’s were performed using cneymically prepared stearyl-ACP. The preparation and characterization of this substrate are fully described in the succecding paper (16). The stearyl-ACP desaturase was only slightly active when stearyl-CoA was the substrate and none of the other substrates used were desaturated. The identit’y of oleic acid as the product of the dcsaturation using stearyl-ACP was confirmed as described in the Methods section. As illustrated in Fig. 4, the desaturation reaction was linear for 20 min and essentially complete after 60 min. Since the desaturase was in a crude extract, it was also important to examine the significance of hydrolytic reactions which may have consumed the substrate. The enzymic hydrolysis of the thioester of stearyl-ACP by the safflower extract was determined as well as that of stearyl-CoA and oleyl-CoA. As shown in Fig. 5, there are competing reactions involving the cleavage of thioesters which took place during incubation. The loss of thioester of stearyl-ACP and stearyl-CoA occurred at approximately t,he same rate while the loss of thioester in oleyl-CoA was much more rapid. Presumably, the thioester is either hydrolyzed to yield a free acid or the acyl group was transfcrrcd to lipid acceptors to yield an oxygen ester. m-
0
I’o
bb Time of wubatlon
(mlnutesj
FIG. 4. Time course of stearyl-ACP desaturase reaction. Each assay contained 0.2 nmoles of [‘GJstearyl-ACP.
% thlal ester
20. 0
, 5 IO
20
Time of lncubatlon
30 (minutes)
FIG. 5. Loss of thioesters in an assay mixture as a function of time. Each assay contained 0.2 nmoles of substrate. Assay conditions are described in the text.
Localization of stearyl-ACP desaturase. Stearyl-ACP desaturase was consistently observed in the 105,OOOysupernatant of crude enzyme extracts. To determine if the stearylACP desaturase was exclusively in the soluble fraction, a 2SOq supernatant fraction prepared from developing safflower seeds was separated by sucrose density gradient centrifugation (20). While the cell organelles migrated as discrete bands, both the fatty acid synthetase and stearyl-ACP desaturase activities were totally located in the soluble fraction. Therefore, both the fatty acid synthetase enzymes and the stearyl-ACP dFsaturase occur as soluble systems in the cytoplasm of the cotyledonous cell. NADPH requirement. The desaturase required a reduced pyridine nucleotide for a,ctivity. As shown in Fig. 6, the system had an apparent K, of about 2 X 1OF M for NADPH and was essentially saturated with 10 nmoles in the 0.6.ml reaction mixture. NADH was completely inactive as an electron donor in this system. Since previous workers (6) have implicated NADPH oxidase as linking NADPH to an electron acceptor for the activation of oxygen, the extract was assayed for t’his enzyme activity using dichlorophenolindophenol as the electron acceptor. The amount of extract used in a routine assay could reduce about 2 pmoles of DCIP per minute, which would more then meet the electron requirements of the dcsaturase system.
STEARYLACP
under the usual conditions except that the O2 concemration of the atmosphere was 10 ‘;h instead of the, -“1 % (air), and the rcwt~ion was ca,rricd out, in the dark. Although avian liver stearyl-CoA dcsat~urnsc rquircd lipid micelles for full activity (27), this wquircmerit \vax not obwrved with thcb voluble stcaryl-ACP desaturaw of safflon-or.
NADPH
1 5
F. 0 10
IO 20 NADPH or NADH added (nmolesi
, 25 LAMELLAE
50
I( i:-;
DESA’I’URASI:,
.J
100
j JJ~ chlorophyll/
Fro. 6. Effect of increasing amounts of reduced pyridine mtcleotide or spinach lamellae on stearylACP desaturase activity. Each assay contained 0.2 nmoles of [r4C]stearyl-ACP. NADP+ and glucose-6-phosphate were replaced in the assay by the specified amount, of either NADPH or NADH, or spinach lamellae. Assay conditions are described in the text.
In order to determine whether KADPH was an obligatory component of the desaturase or only a nonspecific source of electrons, a chloroplast lamellae system was employed to photoreduce ferredoxin in the light in the absence of KADPH. As can be seen in Fig. 6, the safflower desaturase was even more active when coupled to phot’ochemically reduced ferredoxin than when coupled to ferredoxin reduced by the NADPH oxidase. In the abssancc of lamellae or ferredoxin, there was no desaturase activity. Washed lamellac at’ a concentration of 50 pg chlorophyll/ml had no drsaturase act’ivity. E$ect of sulfh~cl~~yl compounds. The react,ion mixt’ure required a sulfhydryl compound for maximum activity. As seen in Table III, both dithiothrcitol and reduced glutathione enhanced tho drsaturasc activity. However, thrl des~turnw was inhibited by P-mercapto-
To date, invest,igations on the detiaturation of long-chain fatt,;- acids have 1)~weded slowly by comparison to t!hose concerned with fatty acid synthesis since all the prol)arations obtained from yeast and animal tissue have been m~~mbranr:-bound cmy~IIes. Extracts of the developing safflower seed COIItain a completely solublth as \vell :w
L’!1 :; ,3.!! ti1 .T ; !)
None Dithiothreitol Reduced glutathione P-~lercaptoetharlol
a Each assay contained 0.2 nmole of i’“C’]stearyl-ACP and 1 pmole of the specified sttlfhydryl compotmtl.
Inhibitor
Experiment __~_____--..-~
~-~~~. x2 6X Iii
1
Sane KCN (0.1 m>i) KC?G (1 .o IllhI I
2
lO$( (12 + SO’,; X3 (control)
c+ ll:LIIo!.
C’yaGle irrhihitici/r. As show-n in the Table IV, potassium cynnidt:, which had been prepared fresh and neutralized, was strongly inhibitory at a wnccntration of 1 .O m&r but only slightl:. at 0.1 m&r. Carbon monoxide, hon.w(r, had no t#cct, on desaturase activity-. Thea latter c,spcriment was carried out
(, (‘onversion - ~-~ -
Thiol compound
lOs’, co
+
lo’;
02 +
soy;
:(4 S?
:3 I
u Each assay contained 0.2 nmoles of strarlyACP. Carbon monoxide assay was carried 011t in t,he dark.
164
JAWORSKI
systems which are also capable of synthesizing oleic acid have been prepared from Euglena gracilis (5, 6) spinach chloroplasts (5, 6), and soybean cotyledons (7, 8). The most extensively characterized system was prepared from Euglena by Nagai and Bloch (6). The developing nonphotosynthetic safflower seed system is similar to the Euglena system in that (1) both employ NADPH effectively as an electron donor and an NADPH oxidase is readily demonstrated in the extract, (2) both have a ferredoxin requirement, and (3) both are cyanide sensitive, but carbon monoxide insensitive. Thus, the electron-generating and carrier systems for the two enzymes appear to be similar. Unlike the Euglena system (6), however, the safflower desaturase appears to be specific for stearyl-ACP. Only a negligible activity could be demonstrated with stearyl-CoA as the substrate. It was of interest that cyanide markedly inhibited the soluble desaturase system. A cyanide-sensitive factor has been observed in the avian stearyl-CoA desaturase system (25-27) and the cyanide-sensitive site has been placed between cyt-bg and the desaturase (26). It seems likely that in the safflower system the cyanide-sensitive site is located after ferredoxin and at or before the desaturase site since both ferredoxin and NADPH oxidase are insensitive to cyanide. No phospholipid requirement has been observed in the safflower system in contrast to the avian system which requires lipid micelles for optimal activity (26, 27). In the crude extracts the rate-limiting step for desaturation involved ferredoxin since in these extracts conditions which reduced the rate of dcsaturation relative to fatty acid synthesis, e.g., high ACP concentration or low protein concentration, could be reversed by supplementing the reaction mixture with ferredoxin. Furthermore, when NADPH + NADPH oxidase was replaced with a chloroplast lamellae system, desaturase activity was increased still further. The complete replacement of NADPH by a grana system which reduced ferredoxin implies that NADPH is not an obligatory reductant but only one of a number of possible reductants available to the system in the cell. In photo-
AND
STUMPF
synthetic organisms such as Euglena, an important source of electrons for reducing ferredoxin is quite possibly Hz0 via photosystems I and II with NADPH as an ancillary source. Both the safflower and the Euglena systems specifically required ferredoxin. Other electron carriers assayed were either inactive or inhibitory in that they probably diverted the electron flow away from the desaturase system. It should also be recognized that ferredoxin may not be the natural electron carrier but is simply capable of efficiently replacing the carrier which was removed by the DEAE-cellulose procedure of Mortenson (19). With the availability of large amounts of tissue, purification of the enzymes involved in desaturation will be undertaken to extend and clarify these observations. REFERENCES 1. BAKER, N., AND LYNEN, F. (1971) Eur. J. Biothem. 19, 20&210. 2. BLOOMFIELD, D. K., AND BLOCH, K. (1960) J. Biol. Chem. 236, 337-345. 3. M.~RsH, J. B., AND JAMES, A. T. (1962) Riochim. Biophys. Acta 60, 320-328. 4. ZILKEY, B. F., AND QNVIN, 1). T. (1972) Cwt. J. Bot. 60, 323-326. 5. N~c.41, J., AND BLOCH, K. (1965) J. Biol. Chun. 246, PC3702-3703. 6. NAGAI, J., AND BLOCH, K. (1968) J. Biol. Chem. 243, 46264633. 7. INKPEN, J. A., AND QUACKENBUSH, F. W. (1969) Lipids 4, 539-543. 8. RINNE, R. W. (1969) Plant Physiol. 44, 89-94. 9. MCM~HON, V., AND STUMPF, P.K. (1966) Plant Physiol. 41, 14&156. 10. MCMAHON, V., AND STUMPF, P. K. (1964) Biochim. Biophys. Acta 84, 359-361. 11. VIJAY, I. K., .IND STUMPF, P. K. (1971) J. Biol. Chem. 246, 29162917. 12. VIJAY, I. K., AND STUMPF, P. K. (1972) J. Biol. Chem. 247, 360-366. 13. ALHERTS, A. W., MAJYRUS, P. W., AND VAGELOS, P. R. (1969) Methods Enzymol. 14,43-50. 14. GALLIARD, T., AND STUMPF, P. K. (1968) i?~ Biochemical Preparations (Lands, W. E. M., ed.), Vol. 12, p. 66, Wiley, New York. 15. BIRGE, C. H., SILBERT, D. F., AND VAGELOS, P. R. (1967) Biochem. Biophys. Res. Cow mm. 29, 808-814. 16. JAWORSKI, J., AND STUMPF, P. K. (1974) Arch. Biochem. Biophys. 162, 166-173. 17. BARRON, E. J., AND MOONEY, L. A. (1968) Anal. Chem. 40. 1742-1744.
STlGAI:YI,-ACP 18.
K\NN.ING.UI.\, STUMPF,
I)fCSATIJf:.~Sf’
C. C;., JXOUSON, B. S., .\m P. K. (1973) Plant Physiol. 62, 156-
161. 19.
I,. I’,. (1964) Biochim.
~~ORTENSON,
BiqJh~/.S.
Acta 81, 473-478. 20. LORD, J. M., K.\o.\w.\,
T.,
BEEVF:R~, H.
.iND
(1972) J’roc. .\‘a/. Acad. Sci. C:SA 69, 21292432. 21.
BLIGH,
15. (i.,
Hiochem. 2".
MOKRISON,
I,ipitl
.\NI)
1)Y~ll,
w.
J.
(1959)
Ph!/siol. 37, 911-917. W. IX., .\ND SMITH, 1,. Rrs. 6, 600-608.
M.
?a//.
.I.
(1964) J.
I li.i
23. H.\RHIS, I’., .\NU .J.uII+, A. T. (196!)i /<;oc,/IPY)/. J 112, 325-330. 24. H.\ERIS, P., .~ND J.GXICS, A. T. (19691 /Z~ocl~iv/. Niophys. Acta 18’7, 13-18. 25. W.\IXI,, I;. J. (1964) i/r IVfetaholisn~ and f’hysiological Significance of Lipids (I ):tw~oll, 1:. M. C., and I