Vol. 6o ITACONIC ACID FORMATION BY A. TERREUS. 2 147

Calam, C. T., Oxford, A. E. & Raistrick, H. (1939).Biochem. J. 33, 1488.

Cavallini, D., Frontali, N. & Toschi, G. (1949). Nature,Loud., 163, 568.

Corzo, R. H. & Tatum, E. L. (1953). Fed. Proc. 12, 470.Fittig, R. & Miller, H. E. (1889). Liebigs Ann. 255, 43.Friedemann, T. E. (1950). Circular No. 16, Dept. Nutrition& Metabolism, Northwestern University Medical School,Chicago.

Kinoshita, K. (1931). Acta Phytochim., Tokyo, 5, 271.Krampitz, L. 0. & Werkman, C. H. (1941). Biochem. J. 35,

595.Krebs, H. A. & Eggleston, L. V. (1944). Biochem. J. 38,426.Lardy, H. A. (1949). Manometric Techniques and Tissue

Metabolism, pp. 163-4, ed. Umbreit, W. W., Burris, R. H.& Stauffer, J. F. Minneapolis: Burgess Publishing Co.

Larsen, H. (1954). Methods in Enzymology, 3, sect. m, ed.Colowick, S. P. & Kaplan, N. 0. New York: AcademicPress.

Larsen, H. & Eimhjellen, K. E. (1955). Biochem. J. 60,135.Malachowski, R. & Maslowski, M. (1928). Ber. dtsch. chem.

Ge8. 61, 2521.Marokwald, W. & Axelrod, S. (1899). Ber. dt8ch. chem. Ge8.

82, 713.Martius, C. & Lynen, F. (1950). Advanc. Enzymol. 10, 167.Michael, A. & Tissot, G. (1892). J. prakt. Chem. 46, 285.Morrison, J. F. (1954). Biochem. J. 56, xxxvi.Morrison, J. F. & Peters, R. A. (1954). Biochem. J. 56,

xxxvi.Nekhorocheff, J. & Wajzer, J. (1953). Bull. Soc. Chim. biol.,

Pari8, 85, 695.Ochoa, S. (1948). J. biol. Chem. 174, 133.Peters, R. A. (1952). Proc. Boy. Soc. B, 139, 143.Potter, V. R. & Heidelberger, C. (1949). Nature, Lond., 164,

180.Robertson, W. van B. (1942). Science, 96, 93.Szent-Gyorgyi, A. (1930). Biochem. J. 24, 1723.Walker, T. K. (1949). Advanc. Enzyrnol. 9, 537.

Biosynthesis of Fatty Acids in Cell-free Preparations2. SYNTHESIS OF FATTY ACIDS FROM ACETATE BY A SOLUBLE ENZYME SYSTEM

PREPARED FROM RAT MAMMARY GLAND

BY G. POPJAK* A ALISA TIETZtThe National In8titute for Medical Research, MiU Hill, London, N. W. 7

(Received 15 November 1954)

We have reported previously the synthesis of short-and long-chain fatty acids from acetate by cell-freesuspensions (homogenates) of mammary gland oflactating rats and sheep (Popj6k & Tietz, 1954a).The following conditions were required for syn-thesis in the preparations of rat mammary gland:(a) aerobic incubation, (b) the co-oxidation of anyof the three keto acids: pyruvate, oxaloacetate orx-oxoglutarate, and (c) the addition of adenosinetriphosphate (ATP), although the last was notstrictly required with pyruvate and a-oxoglutarate.The maximum synthesis of fatty acids from acetateoccurred in the presence of acetate (0-02M), oxalo-acetate (002M) and of ATP (O-O1M) and when thegas phase was air instead of pure 02-

In this article the preparation and some of theproperties of a soluble enzyme system from themammary gland of lactating rats are described. Theresults have already been presented in a preliminaryform (Popjak & Tietz, 1953, 1954b).

MATERIAL AND METHODSPreparation of the soluble enzyme system. Homogenates of

the mammary gland of lactating rats, 10-14 days afterparturition, were prepared first as described previously(Popjak & Tietz, 1954a) and were then fractionated byhigh-speed centrifuging. The homogenate, from whichwhole cells, cell debris, nuclei, etc., have been removed bypreliminary centrifuging at 400g for 10 min., was centri-fuged first at 25 000g for 30 min. and at 00. A clear,transparent pink supernatant (Sp. I) was taken off andfiltered through a small pad of cotton wool to remove a thinfilm of fat from the top. The sediment, designated as'mitochondria', was washed twice by dispersion in fresh,ice-cold buffer and centrifuging at 25 000g for 15 min.A sample of Sp. I was centrifuged further at 2-4° and at104000g for 30 min. in a Spinco preparative centrifuge. Thesupernatant (Sp. II), which in appearance was similar toSp. I, was taken off and filtered as described for Sp. I. Thesediment, a pinkish brown translucent pellet, designated as'microsomes', was washed once with fresh buffer andsedimented at 104000g for 10 min.The 'mitochondria' obtained from 5 ml. of homogenate

were suspended with the aid of a small glass homogenizer in2-5 ml. of buffer or in the same volume of Sp. I, to provide2 ml. for incubation and 0-5 ml. for determination of dryweight. The 'microsomes' obtained from 10 ml. of Sp. Iwere treated in the same way with buffer or Sp. II.

After it was found that all the enzymic activity requiredfor fatty acid synthesis was present in Sp. II, the procedurewas shortened. Homogenates were prepared in eitherphosphate buffer (0-154m-KCI, 100 parts; 0-154m-MgCl2,

* Present address: M.R.C. Experimental Radiopath-ology Research Unit, Hammersmith Hospital, DucaneRoad, London, W. 12.

t Holder of the Glaxo Laboratories and Friends of theHebrew University of Jerusalem Scholarship while workingat the National Institute for Medical Research.Paper 1 of this series: PopjAk & Tietz (1954a). Present

address: Department of Biochemistry, Hadassah MedicalSchool, Jerusalem, Israel.

10-2

G. POPJAK AND A. TIETZ10 parts; 0-Im potassium phosphate buffer, pH 7 4, 35parts) or in THAM buffer (0-154M-KCI, 9 parts; and 0-5Maminotrishydroxymethylmethane buffer, pH 7.5, 1 part),and centrifuged immediately at 104000g for 1 hr. Most ofthe fat which separated on the surface was scooped off andthe rest removed by filtering the supernatant through asmall pad of cotton wool. The filtered supernatant providedthe soluble mammary-gland enzyme system (MGE). Theprecipitate was discarded.

Since there was no observable difference between theenzymic activities of MGE's prepared in phosphate or inTHAM buffer, no distinction will be made between the two

I955[14C]acetate and the methods of 14C counting and calcula-tions were described in our previous communication(PopjAk & Tietz, 1954a).

RESULTS

A comparison of the various fractions obtained fromthe centrifugal fractionation of mammary-glandhomogenates for their ability to synthesize fattyacids from acetate is shown in Table 1. The condi-tions of these experiments were those found optimal

Table 1. Fatty acid synthesis from [carboxy-14C] acetate by fractions of rat nammmary-gland homogenatesEach flask contained: K acetate, 60umoles (5jAc 14C); K oxaloacetate, 0-02M; ATP, O-O1M, and 2 ml. of the indicated

preparation; final volume, 3 ml. Fatty acid synthesis expressed as umoles x 10-3 acetate incorporated/100 mg. dry weight(corrected for salt and fat content). Preparation no.

Fraction incubatedFull homogenateSp. IMitochondriaMitochondria + Sp. ISp. IIMicrosomesMicrosomes + Sp. II

1126-95

35-206-203-85

11421-2

32-4, 27-88-48, 9-30

12-90, 14-10

1168-9

90-0

48-50-0

25-6

11712-175-010-34.54

18-50-012-4

1192-59-1

41-5

1203-6

17-3

62-1

types of preparation. However, when the MGE was pre-pared in THAM buffer, 50,umoles of potassium phosphatebuffer, pH 7-4, and 30pmoles of MgCl2 were added later tothe incubation mixture (total volume, 3 ml.).The synthetic activity of the various batches of MGE

varied a good deal, although not as much as the activity ofthe homogenates (cf. PopjaLk & Tietz, 1954a). These varia-tions are not surprising in view of the crude nature of theenzyme preparations and since the synthesis of fatty acidsdepends probably on a number of enzymes and coenzymes.The synthetic activity of different batches of enzymesunder various conditions may easily be compared since theresults are expressed throughout this paper as ,umoles(or 1 x 103 pmoles) of acetate incorporated into fatty acidsper 100 mg. salt- and fat-free dry weight of the enzymepreparations.

Incubations. Aerobic and anaerobic incubations weremade in Warburg vessels as described previously for homo-genates (Popjflk & Tietz, 1954a). In the anaerobic incuba-tions ATP and [14C]acetate were tipped in from the side armafter the enzyme preparations had been incubated under Nsfor 10 min. Commercial N. and N3 purified by passingthrough an ammoniacal cuprous chloride solution wereused.

Extraction offatty acids. This was done as described pre-viously for homogenates (Popja6k & Tietz, 1954a) exceptthat an ethanol-ether (3: 1, v/v) extract of the mammary-gland fat, obtained in the course of the centrifuging, wasadded at the end ofthe incubation and before saponificationto the preparations of Sp. I, Sp. II and MGE on account ofthe very low fat content of these. The amount of fat addedwas equivalent to 10 mg. of mixed fatty acids.

[carboxy-"C]Acetate (obtained from the RadiochemicalCentre, Amersham, Bucks) was used as precursor of fattyacids. The purification of fatty acids from contaminating

for fatty acid synthesis by the homogenates. It canbe seen from Table 1 that both supernatants, Sp. Iand Sp. II, were more active (on a dry weight basis)than the full homogenates from which they wereobtained. The mitochondrial fraction showed onlya very slight synthetic activity. Moreover, when itwas combined with Sp. I, fatty acid synthesis by themixture was depressed to the originally lower levelofthe full homogenates. The microsomes completelylacked the ability to synthesize fatty acids and,in the few experiments tested (nos. 116 and117), seemed to depress slightly the activity ofSp. II.The soluble enzyme preparations (MGE), with

the exception ofone experiment (no. 142), were also

Table 2. Comparison of fatty acid synthesis from[carboxy-14C]acetate by full homogenate andsoluble mammary gland enzyme system (MGE)

Experimental details as in Table 1. Results expressedas umoles x 10-3 acetate incorporated into fatty acids/100 mg. dry weight.

+ *i *li.t.t O.21L DyRL.hLVOi

Preparationno.131134135

141142143

Full homogenate7-5

22-531-1

18-2107-0

8-2

MGE72-547-3

(a) 56-0(b) 62-0

165-094.459-0

148

.v aTJTy &U1CW SynToiosz

BIOSYNTHESIS OF FATTY ACIDS. 2

Table 3. The effect of short and long homogenization onfatty acid 8ynthesi8 by full hormogenate and MGE

Experimental details as in Table 1. Results expressed as jAtmoles x 10-8 acetate incorporated into fatty acids/100 mg.dry weight.

Mode of homogenization

1 min. in a loose homogenizer*f

Full homogenate187*076*018*8

1 min. in loose homogenizer* +1 min. in tight homogenizert

MGE Full homogenate75-0

130*5 11-473-0 4.9

MGE325-0135*092*0

* Difference between diameter of tube of homogenizer and that of the pestle, 0 4 mm.t Difference between the diameter of tube and that of the pestle, 0-2 mm.

Table 4. The effect offreezing and thawing on fatty acid synthei8 by the MGE

Each flask contained: K acetate, 60,umoles (5 .sC 14C); ATP, 001M; 2 ml. of MGE; further additions were as follows:K oxaloacetate, 0-02M; K a-oxoglutarate, 0*02m and K malonate 0*05M; final volume, 3 ml. Results expressed as jumolesacetate incorporated into fatty acids/100 mg. dry weight.

Preparationno.

135136139140

No. ofdays keptfrozen

6242

Fatty acid synthesis

Additionsx-Oxoglutaratem-OxoglutarateOxaloacetateOxaloacetate

141 2 Oxaloacetate152 2 a-Oxoglutarate 1

5 + malonate |162 27 m-Oxoglutarate + malonate

Fresh MGE0-1680-0690*1350-092

0*1650-0770*760

Frozen MGE0*0530*0640-182

(a) 0-214(b) 0*233

0*1&7I0-1450-1460-281

more active than the homogenates in synthesizingfatty acids from acetate (Table 2). It is of interestto note that, while prolonged homogenizing im-paired very considerably the synthetic activity ofhomogenates, there was no impairment in theactivity of the MGE (Table 3). A poorly activehomogenate could still yield a highly active MGE.The change produced in the homogenates by pro-

longed grinding leading to a decreased syntheticactivity is unknown at present.The MGE retained its enzymic activities after

being kept frozen in solid C02 for several days(Table 4). Surprisingly, the synthetic activity ofthe preparations was frequently greater after a

few (2-5) days' storage in solid C02 than whenfreshly tested. This change is attributed to the pre-

cipitation of some inactive protein, which almostinvariably occurred on thawing. Further experi-ments, which will be described below, were carriedout with either fresh or stored preparations as

convenient. It should be pointed out that experi-ments made with any one batch ofMGE are enteredin the tables under the same number, but these werenot necessarily carried out on the same day; this isthe explanation for the apparent variation in thesynthetic activity of the same batch ofMGE underotherwise identical conditions.

The rate offatty acid &ynthei8 by the MGE

The effect of varying times of incubation on theamounts of acetate incorporated into fatty acids isillustrated in Fig. 1. These experiments were set up

100

an--l 7V

" 80

c 70c

E 60

.E 50x

E 40'40

30._

u 20L1

10

*AA a,

-A

20 40 60 80 100120 140 160 180 200Time (min.)

Fig. 1. Rate of fatty acid synthesis by soluble mammarygland enzyme system (MGE).

Preparationno.138132140

Vol. 60 149

I

F

G. POPJAK AND A. TIETZin such a way that 2 ml. of MGE were mixed with1 ml. ofadditions in Warburg vessels and incubationwas started immediately. The first vessel wasremoved after 10 min. (the time interval usuallyallowed for equilibration before O uptake measure-ments were started). Further vessels were removed10, 20, 30 min., 1, 2 and 3 hr. later. It can be seenfrom Fig. 1 that 50% of maximum incorporationwas reached about 15 mini. after the incubation hadbeen started and maximum incorporation of [14C]-acetate into fatty acids was attained in 1 hr.; oncethe maximum was reached no further changes in thespecific activity of the fatty acids occurred. Incu-bation for 70 min. was therefore used.

Type offatty acids synthesizedThe fatty acids recovered from preparation no.

140 after incubation with [carboxy-"4C]acetate in thepresence of oxaloacetate and ATP were resolvedinto two groups by reversed-phase chromatography(Howard & Martin, 1950). The first group containedall the acids which were removed from the columnwith 45% (v/v) aqueous acetone; i.e. the first bandwhich contains butyric, caproic (hexanoic), andcaprylic (octanoic) acids, plus a second band ofcapric (decanoic) acid. On changing the solvent to75% (v/v) aqueous acetone all long-chain fattyacids with a chain length between C12 and C1lemerged in one band.Both acid groups were radioactive: 5-34 x 10- HaC

(75-8%) were recovered in the first fraction and1-70 x 10-31 c (24-2 %) in the second fraction. Since

an unknown mixture of fatty acids was addedbefore saponification, the true specific activity ofeach fraction in the preparation could not be calcu-lated. The results, however, indicate that the MGEsynthesizes both short- and long-chain fatty acidsfrom acetate, with a preference for short-chainacids.

The effect ofATP, pyruvate and some intermediate8 ofthe citric acid cycle onfatty acid syntheswi by the MGE

In the experiments described so far the experi-mental conditions were those found optimal for thefull homogenates. It was found, however, thatwhereas in the full homogenate the addition ofpyruvate or oxaloacetate or a-oxoglutarate and ofATP was required to activate fatty acid synthesisfrom acetate, in the soluble preparation ATP alonewas sufficient for activation. An absolute require-ment for ATP was demonstrated; only traces of[14C]acetate were incorporated into fatty acidsunless ATP was added to the incubation mixture.In contrast to observations with the full homo-genate, even oc-oxoglutarate could stimulate fattyacid synthesis only in the presence of ATP. Whenincreasing amounts of ATP were added (in thepresence of acetate plus oxaloacetate) the amountsof acetate incorporated into fatty acids also in-creased, a concentration of OO1OM being optimum(Table 5).The effects of pyruvate and of some inter-

mediates of the citric acid cycle (added in thepresence of ATP) were further studied. Table 6

Table 5. The effect of varying concentrations of ATP onfatty acid synthesis by the MGE

Each flask contained: K acetate, 0-02m (5 &c 140); K oxaloacetate, 0-02m; MGE, 2 ml. (prepared in phosphate buffer);ATP was added as indicated; final volume, 3 ml. Fatty acid synthesis expressed as ,umoles x 10-3 acetate incorporated/100 mg. dry weight.

Final concentration of ATP addedPreparation

no.120142

None4.74.45

0-001M 0-002M 0-OOOM5.9 13-25

10-7 16-9 26-2

0-01M 0-02M62-159.5 60*0

Table 6. The effect of pyruvate and of some intermediate8 of the citric acid cycle onfatty acidsynthesis by the MGE

Each flask contained: K acetate, 60,umoles (5ic 140); ATP, 0010m; THAM buffer, pH 7-6, 0-025m; MGE (in phosphatebuffer), 2 ml.; total volume, 3 ml. Additions were as indicated. Fatty acid synthesis expressed as l&moles acetateincorporated into fatty aoids/100 mg. dry weight.

K pyruvate0-0320-0380-027

0-014

Additions, final conon., 0-02M

K oxaloacetate0-0470-0590-0260-1820-2240-0470 0730-014

*K oc-oxoglutarate0-1180-1680-0650-0860*5050-0310-0520-163

K succinate0-0410 0700-044

0 0450-0520 070

Preparationno.184135136139140143145146

None0-0260-0580 0350-0220-0310-0140-0180*045

150 I955

BIOSYNTHESIS OF FATTY ACIDS. 2

shows that oxaloacetate stimulated fatty acidsynthesis in most of the preparations although itseemed to inhibit others. ca-Oxoglutarate had amarked stimulating effect in all preparations, aug-menting the incorporation of [14C]acetate into fattyacids 2 to 5 times. In one preparation (no. 140) a15-fold increase was observed. Succinate seemed tostimulate fatty acid synthesis also, while citrate,fumarate and malate (not shown in the table) werewithout effect. Pyruvate, in contrast to its be-haviour in the full homogenate, did not stimulatefatty acid synthesis by the MGE; in two experi-ments (nos. 135 and 136) a slight inhibitory effectwas observed.

Oxygen conr&umption by the MGE

As already described for the full homogenates(Popjak & Tietz, 1954a) only a very low endogenous02 uptake was observed. ATP, however, verymarkedly stimulated the 02-consumption. The

.-

b

-o

4,E

0

E

10 to

9 ~~~~~~~~~~/9

8

7Af-V

6 ,oS ~~~ /~

4/ ~~~~~/5

4/

/.0 N}X4~~/Al~~~~~~~~~t

.-.....o -0

...... .. ......N ATP

20 40 60 80 100 120 140 160 180Time (min.)

Fig. 2. The effect of varying concentrations of ATP in thepresence of acetate (0-02M) and oxaloacetate (0-02m) on

oxygen uptake by MGE.

dependence of the 02 uptake on ATP added (in thepresence of acetate plus oxaloacetate) can be seen

from Fig. 2. With the lower concentrations ofATP(0001M-0-005M) the curves flattened out after an

interval and became parallel with the curve ob-tained in the absence ofATP. This sudden inflexionof the curves suggests that ATP was used up duringthe preceding periods. Although with O-1OM ATP

the initial rate of 02 uptake was lower than with0-005M ATP, the 02 uptake continued uncheckedfor 3 hr. The addition of substrates alone (acetate,pyruvate, oxaloacetate or a-oxoglutarate) hadno effect on the 02 uptake unless ATP was alsoadded. Under this condition, oxaloacetate in-variably very markedly stimulated the 02 uptake.m-Oxoglutarate and pyruvate had a more moderateeffect. A typical experiment is illustrated by Fig. 3.

4o

E

8,

o0'

a

I 80 100 120 140 160 1.80Time (min.)

Fig. 3. The effects of pyruvate (0'02M), oxaloacetate(0-02M) and of ox-oxoglutarate (0-02M) on the oxygenuptake by MGE in the presence of acetate (0-02m) plusATP (0-01m). 0, Acetate+ATP+oxaloacetate; A,acetate +ATP + c-oxoglutarate; 0, acetate +ATP -pyruvate; x, acetate+ATP.

Acetate and other intermediates of the citric acidcycle: citrate, succinate, fumarate and malate werewithout effect.

The effect of inhibitor8 onfatty acid 8yntheis8

In order to get some insight into the enzymicsystems involved in the biosynthesis of fatty acidsby the MGE, an attempt was made to inhibit fattyacid synthesis with various compounds known toinhibit some vital process. The following substanceswere chosen: HgCl2 (1 x 10-4M), arsenate (0-O0M),2:4-dinitrophenol (2 x 10-4M), cyanide (001M),azide (0-01M), fluoride (0.05M) and malonate(0.05M). As can be seen from the results shown inTable 7, mercury and arsenate almost completelyinhibited the incorporation of acetate into fatty

151Vol. 6o

G. POPJAK AND A. TIETZ I955Table 7. The effect of inhibitors on fatty acid 8ynthesi8 and on oxygen uptake by the MGE

Each flask contained: K acetate, 60!moles (5,uc 14C); K oxaloacetate, 0-02M; ATP, 0-01m; MGE, 2 ml. (prepared inphosphate buffer); final volume, 3 ml. Fatty acid synthesis expressed as umoles x 10- acetate incorporated/100 mg.dry weight.

Preparation no.

Substance addedand concentration

None2:4-Dinitrophenol, 2 x 10-4MKF, 0-05mNa3AsO4, 0-05MNaN8, 0-01MKCN, 0-O1MHgCl,2 1 x 10-4MK malonate, 0-05M

141

Fatty acid- Qo° synthesis2-61 186-54-46 118-02-11 169-01-95 14-43-15 184-0

57-62-48 9-82-43 328-0

acids. Of the two substances, only arsenate reducedthe 02 uptake. Cyanide caused a 30-70% inhibition,while azide was without effect. 2:4-Dinitrophenolcaused a 30% inhibition of fatty acid synthesis anda 40% inhibition in 02 uptake. Fluoride was

without effect. Malonate, however, markedlystimulated fatty acid synthesis, without affectingthe 02 uptake. These effects of malonate were mostsurprising and further investigations on thisfinding were undertaken.

The effect of malonate on fatty acid8ynthesi by the MGE

It can be seen from Table 8 that malonatemarkedly stimulated fatty acid synthesis when onlyacetate and ATP were added. However, no effectwas observed in the absence of ATP (cf. Table 9).Comparing the stimulating effect of malonate or ofox-oxoglutarate, malonate seemed to have an even

stronger effect than a-oxoglutarate, especially whenadded at a concentration of 0-05M instead of0-02M.

We further compared the effect of malonate on

fatty acid synthesis in the presence of oxaloacetateor of oc-oxoglutarate. When malonate was addedtogether with either of the two substrates, thestimulating effect was higher, particularly with

Table 9. Comparison of the effect of oxaloacetate,a-oxoglutarate and of malonate on fatty acidsynthesiw by the MGE

Each flask contained: K acetate, 60,umoles (5,uc 14C);THAM buffer, pH 7-6, 0-025M; MGE, 2 ml. (prepared inphosphate buffer); final concentration of additions: Koxaloacetate and K m-oxoglutarate, 0-02M; K malonate,0-05M; ATP, 0-O1M. Results expressed as Zmoles x 10-8acetate incorporated into fatty acids/100 mg. dry weight.

AdditionsNoneMalonateOxaloacetateOxaloacetate + malonatea-Oxoglutaratea-Oxoglutarate + malonate

Fatty acid synthesis

Without ATP With ATP- 18-212-6 118-0- 72-710-9 241-0

51-711-3 517-0

Table 8. The effect of nalonate and of o-oxoglutarateonfatty acid synthes8 by the MGE

Each flask contained: K acetate, 60,umoles (5L'c 14C);ATP, 0-O1M; THAM buffer, pH 7-6, 0-025M; MGE, 2 ml.(prepared in phosphate buffer); additions were as indi-cated. Fatty acid synthesis expressed as jAmoles x 10-8acetate incorporated/100 mg. dry weight.

Additions, final concn. 0-02M

Preparationno.

140145

KNone a-oxoglutarate14-4 31-018-2 51-7

146 45-0153 8-0

162-520-2

* 0-05M.

K malonate93-033.9118.0*67-5106.0*

Table 10. The effect of malonate in the pre8ence ofo-oxoglutarate and of ATP on fatty acid synthe8isby the MGE

Each flask contained: K acetate, 60I&moles (5,uo 14C);ATP, 0-L1M; K phosphate buffer, pH 7 4, 0-015M; MgCl8,0-O1M; MGE, 2 ml. (prepared in THAM buffer); finalconcentration of additions: K oc-oxoglutarate, 0-02M;K malonate, 0-05M. Fatty acid synthesis expressed as

,umoles acetate incorporated/100 mg. dry weight.

Preparationno.

154156157

Additions

ax-OxoglutarateNone oc-Oxoglutarate + malonate0-053 0-427 1-5600-020 0-115 1-2000X010 0-018 0-144

142

Fatty acidsynthesis

35-224-653.411-7

25-89.7

214-0

- Q022-411-432-011-69

1-932-30

152

BIOSYNTHESIS OF FATTY ACIDS. 2

x-oxoglutarate, than would be expected if theeffects of the two substances in combination wereadditive (Tables 9 and 10). While x-oxoglutaratecaused a threefold increase, malonate a sixfoldincrease, a-oxoglutarate and malonate when addedtogether augmented fatty acid synthesis 30 times.

The effect of anaerobic incubation onfatty acid synthes8w

As can be seen from the results shown in Table 1 1,aerobic incubation is not essential for fatty acidsynthesis in the presence of ATP, x-oxoglutarate

effect of AMP, ADP and of ATP was compared,fluoride (0-05M) was added to inhibit adenosinetri-phosphatase, myokinase and pyrophosphatase (ifpresent in the MGE). Table 12 shows that AMPcould not effect fatty acid synthesis while ADP wasat least as effective as ATP. All three compounds,however, stimdilated 02-consumption. In thisexperiment and in all preceding experiments com-mercial samples of AMP, ADP and of ATP ofunknown purity were used. For further compari-sons the commercial preparations were purifiedchromatographically on a Dowex-1 ion-exchange

Table 11. The effect of anaerobic incubation on fatty acid 8ynthe8i8from [14C]acetate by the MGE

Each flask contained: K acetate, 60 &moles (5,mc 14C); ATP, 0-01M; MGE, 2 ml.; final concentration of additions:K oxaloacetate, 0-02m; K a-oxoglutarate, 0-02m; K malonate, 0-05m; final volume, 3 ml. Fatty acid synthesis expressedas ,Amoles acetate incorporated/100 mg. dry weight.

AdditionsOxaloacetateac-Oxoglutarate + malonate

162 ac-Oxoglutarate +malonate163 a-Oxoglutarate + malonate164 m-Oxoglutarate +malonate

* Purified N2.

and malonate. Except in one experiment (no. 162)incubation under N2 did not inhibit fatty acidsynthesis; in two experiments (nos. 158 and 164)even a slight stimulation was observed. With oxalo-acetate plus ATP as sparkers, however, fatty acidsynthesis was inhibited by anaerobic incubation(prep. no. 140), just as was found for the full homo-genates (PopjAk & Tietz, 1954a).

The effect of adenylic acid, adenosine dipho8phateand ofATP on fatty acid 8ynthesi8 by the MGESince an absolute requirement of ATP for fatty

acid synthesis was demonstrated (cf. Table 5) it wasof interest to determine whether muscle adenylicacid (AMP) or adenosine diphosphate (ADP) couldreplace ATP. In all experiments in which the

Table 12. The effects ofAMP, ADP and ofATP onfatty acid synthesi8 by the MGE (preparationno. 147)

Each flask contained: K acetate, 60ILmoles (5jLC 14C);K oc-oxoglutarate, 0-02M; MgCl2, 0 01M; KF, 0-05M;MGE, 2ml. (prepared in THAM buffer); final volume,3 ml. Fatty acid synthesis expressed as ,umoles acetateincorporated/100 mg. dry weight.

Fatty acidsynthesis0*0040-0080-0610*040

Fatty acid synthesis

In air In N20-246 0-0290-146 0.371*

0-2561*910 0-4690-281 0-2020-142 0-200

Table 13. Compari8on of the effect of ADP and ofATP on fatty acid synthe8is by the MGE

Conditions as in Table 12, plus 0-05M K malonate.Fatty acid synthesis expressed as umoles acetate in-corporated/100 mg. dry weight of the preparation.

AdditionsNoneCommercial ATP (001M)Purified ATP (00012M)Purified ATP (0.006M)Purified ADP (00012M)Purified ADP (0.006M)

Preparation no.

158 1620-0070.150 1.3200-0220-1030-017 0-1250-125 1-225

column as described by Cohn & Carter (1950). Theresults obtained (Table 13) were identical with thosefound when the commercial preparations were used.

DISCUSSION

The distribution of fatty acid-synthesizing enzymesin the mammary gland cells seems to differ fromthat in liver. Brady & Gurin (1952) and Dituri &Gurin (1953) demonstrated that, in pigeon and ratliver, enzymes of both the mitochondria and thecytoplasmic phase were required for fatty acidsynthesis from acetate. In the mammary gland,however, the cytoplasmic fraction alone couldeffect fatty acid synthesis from acetate, althoughthe possibility that some enzymes were extracted

Preparationno.140158

AdditionsNoneAMP (0 01m)ADP (0 01m)ATP (001M)

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G. POPJAK AND A. TIETZ

from the mitochondria in the course of the prepara-tion and were thus contained in the MGE cannot beexcluded. It is worth emphasizing, however, thatthe addition of the 'mitochondrial' fraction to thesoluble preparations inhibited fatty acid synthesis.Recently Kornberg & Pricer (1953a, b) demon-stratedthatinguinea-pigliver preparations enzymesactivating fatty acids with coenzyme A werepresent both in the mitochondrial and solublefractions. We have detected similar enzymes in theMGE (Tietz & Popjak, 1955). There is nowample evidence available to show that biochemicalreactions involving fatty acids require the previousactivation of the acids with coenzymeA (for reviewsee Green, 1954). One of the reactions leading tosuch activation is represented by equation (1)(Mahler, Wakil & Bock, 1953; Kornberg & Pricer,1953a):

Fatty acid +ATP + HS-CoA= fatty acyl-S-CoA+AMP + pyrophosphate (1)

the prototype reaction being the activation ofacetate (Jones, Lipmann, Hilz & Lynen, 1953;Beinert et al. 1953; Hele, 1954). The absoluterequirement for ATP in our MGE preparations is inharmony with this concept. The fact, however, thatATP could be replaced by equimolar amounts ofADP (but not by AMP) suggests the presence ofeither powerful myokinase activity in our pre-parations, or of some other reaction leading to theformation of ATP from ADP. It might be men-

tioned here that the fatty acid-synthesizing solublepreparations of Dituri & Gurin (1953) differ fromthe MGE in that the addition ofATP is not requiredand maximum stimulation of the synthesis isobtained in their preparation with citrate, which hasno effect in the MGE.Measurements of oxygen uptake by the MGE

indicated that oxaloacetate, oc-oxoglutarate andalso pyruvate were oxidized by the preparations ifATP was added. The presence of pyruvic oxidasewas also inferred from the formation of acetyl-coenzyme A from pyruvate in the MGE (Tietz &PopjAk, 1955). Since neither the succinic oxidasenor cytochrome c oxidase systems were detected inthe soluble preparations, it is suggested that therelatively high oxygen uptakes were catalysed byflavine-enzymes.The fact that in the full homogenate fatty acid

synthesis can be linked with the oxidation of a-

oxoglutarate or pyruvate, even in the absence ofadded ATP, suggests that the generation of energy-rich phosphate bonds is coupled to the oxidation ofthese substrates, whereas in the MGE oxidativephosphorylation does not occur, hence ATP isrequired. Moore & Nelson (1952) and Terner(1954a, b) have demonstrated oxidative phos-

phorylation in homogenates of mammary gland.A lack of apparent stimulation of fatty acid syn-thesis from [14C]acetate by pyruvate in MGE pre-parations may be attributed to the dilution of the[14C]acetyl-coenzyme A, formed directly by theacetate activating enzyme, with unlabelled acetyl-coenzyme A derived from the oxidative decarboxy-lation of pyruvate (see above).The stimulating effect of a-oxoglutarate on fatty

acid synthesis by the MGE can be most readilyexplained by the generation of reduced diphospho-pyridine nucleotide (DPNH) through reaction (2)(Kaufman, Gilvarg, Cori & Ochoa, 1953; Sanadi &Littlefield, 1953; Hift, Quellet, Littlefield & Sanadi,1953), thus furnishing a hydrogen donor for thereduction of the intermediary products of synthesis.The lack of inhibition of fatty acid synthesis in theMGE by anaerobic conditions in the presence ofa-oxoglutarate plus ATP, and the stimulatingeffect of added DPN on the synthesis (Tietz &Popjak, 1955) are in harmony with this conclusion.

a-oxoglutaricdehydrogenase

a-Oxoglutarate +HS-CoA +DPNsuccinyl-S-CoA + CO2 +DPNH+ H+. (2)

No satisfactory explanation can as yet be offeredfor the stimulating effects ofsuccinate, oxaloacetateand of malonate on fatty acid synthesis. Furtherwork is planned to elucidate this question.The mechanism ofaction ofsome ofthe inhibitors,

e.g. Hg2+ and arsenate, will be analysed in thefollowing communication (Tietz & Popjak, 1955).

SUMMARY

1. A soluble enzyme system, synthesizing short-and long-chain fatty acids from acetate, has beenprepared by high-speed centrifugal fractionation ofhomogenates ofmammary gland of lactating rats.

2. Neither the 'mitochondrial' nor 'microsomal'fractions of the mammary-gland homogenates wererequired for fatty acid synthesis. The ability of thesoluble supernatant to synthesize fatty acids wasusually greater than that of the full homogenate.

3. In the soluble enzyme preparations ATP alonewas sufficient to activate synthesis from acetate andthe requirement for ATP was absolute. ADP couldeffectively replace ATP.

4. Fatty acid synthesis was markedly stimulatedin the presence of ATP by oxaloacetate, a-oxo-glutarate and to a lesser extent by succinate. Aparticularly strong stimulation was caused bymalonate; a combination of a-oxoglutarate withmalonate elicited on occasion as much as a 30-foldincrease in the amount of [14C]acetate incorporatedinto fatty acids.

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Vol. 6o BIOSYNTHESIS OF FATTY ACIDS. 2 1555. Anaerobic incubation caused an inhibition of

synthesis only in the presence of oxaloacetate; noinhibition was observed anaerobically in thepresence of a-oxoglutarate plus malonate.

6. Hg2+ and arsenate almost completely abolishedfatty acid synthesis; cyanide and 2:4-dinitrophenolcaused a moderate (30-70%) inhibition, whileazide and fluoride were without effect.

7. The implication of the results is discussed.We wish to thank Miss Mary O'Donnell for her technical

assistance and Mr J. Orr in the Biophysics Division forcarrying out all the ultracentrifugal operations. MissMaureen Clevely drew the diagrams.

REFERENCESBeinert, H., Green, D. E., Hele, P., Hift, H., von Korff,

R. W. & Ramakrishnan, C. V. (1953). J. biol. Chem. 203,35.

Brady, R. 0. & Gurin, S. (1952). J. biol. Chem. 199, 421.Cohn, W. E. & Carter, C. E. (1950). J. Amer. chem. Soc. 72,

4273.Dituri, F. & Gurin, S. (1953). Arch. Biochem. Biophy8. 43,

231.Green, D. E. (1954). Biol. Rev. 29, 330.

Hele, P. (1954). J. biol. Chem. 206, 671.Hift, H., Quellet, L., Littlefield, J. W. & Sanadi, D. R.

(1953). J. biol. Chem. 204, 565.Howard, G. A. & Martin, A. J. P. (1950). Bioclem. J. 40,

532.Jones, M. E., Lipmann, F., Hilz, H. & Lynen, F. (1953).

J. Amer. chem. Soc. 75, 3285.Kaufman, S., Gilvarg, C., Cori, 0. & Ochoa, S. (1953).

J. biol. Chem. 208, 869.Kornberg, A. & Pricer, W. E. jun. (1953a). J. biol. Chem.

204, 329.Kornberg, A. & Pricer, W. E. jun. (1953b). J. biol. Chem.

204, 345.Mahler, H. R., Wakil, S. J. & Bock, R. M. (1953). J. biol.

Chem. 204, 453.Moore, R. 0. & Nelson, W. L. (1952). Arch. Biochem.

Biophye. 38, 178.Popj4k, G. & Tietz, A. (1953). Biochim. biophy8. Acta, 11,

587.Popj6ik, G. & Tietz, A. (1954a). Biochem. J. 56, 46.PopjAk, G. & Tietz, A. (1954b). Biochem. J. 58, xxii.Sanadi, D. R. & Littlefield, J. W. (1953). J. biol. Chem. 201,

103.Terner, C. (1954 a). Biochem. J. 56, 471.Terner, C. (1954b). Biochem. J. 57, v.Tietz, A. & Popjik, G. (1955). Biochem. J. 60, 155.

Biosynthesis of Fatty Acids in Cell-free Preparations3. COENZYME A DEPENDENT REACTIONS IN A SOLUBLE ENZYME SYSTEM

OF MAMMARY GLAND

BY ALISA TIETZ*The National In8titute for Medical Re8earch, MiU Hill, London

AND G. POPJAKM.R.C. Experimental Radiopathology Re8earch Unit, Hammeramith Ho8pital, Ducane Road, London

(Received 15 November 1954)

In the preceding paper the biosynthesis of short-and long-chain fatty acids from acetate in a solubleenzyme system (MGE) prepared from themammarygland of lactating rats was described, and favour-able conditions for the synthesis were defined(Popjtak & Tietz, 1955). The discovery of coenzymeA (CoA) and of its participation in acetylationreactions by Lipmann and his colleagues (seeLipmann, 1948-9) resulted finally in the identifica-tion and isolation of acetyl-CoA as the active formof acetate by Lynen, Reichert & Rueff (1951). Thesediscoveries and the subsequent extensive studies of,B-oxidation of fatty acids led to the realization that

fatty acids are metabolized as CoA-derivatives(Lynen & Ochoa, 1953; Green, 1954). It became ofinterest therefore to establish whether or not fattyacid synthesis from acetate in the MGE likewiseinvolved the intermediate formation of acetyl-CoAand of higher acyl-CoA homologues. The mainobject of the work reported here was the study ofthis question. The results have already been pre-sented in a preliminary form (Popjtk & Tietz,1954b).

MATERIAL AND METHODS

The preparation of the soluble enzyme system (MGE) fromthe mammary gland of lactating rats has been described inthe preceding paper (PopjAk & Tietz, 1955).

Preparation and asway of coenzyme A. These were carriedout by the method of Kaplan & Lipmann (1948). The crudeCoA preparations were made from rat liver; they usually

* Holder of the Glaxo Laboratories and Friends of theHebrew University of Jerusalem Scholarship while workingat the National Institute for Medical Research. Presentaddress: Department of Biochemistry, Hadassah MedicalSchool, Jerusalem, Israel.