Fatty acid synthesis in heart

BIOCHIMICA

360

ET BIOPHYSICA

ACTA

1313.45.5740

FATTY

ACID

SYNTHESIS

E. M. WIT-PEETERS,

IN HEART

H. R. SCHOLTE

AND H. L. ELENBAAS

Laboratory of Biochemistry, B. C. P. Jansen Institute*, University of Amsterdam (The Netherlands) (Received

March 13th,

Igp)

SUMMARY

I. The fatty-acid synthesizing capacity of guinea-pig heart is confined to the mitochondria; no synthesis of fatty acid could be demonstrated in the microsomal or soluble fractions. 2. ATP citrate lyase (EC 4.1.3.8) and acetyl-CoA carboxylase (EC 6.4.1.2), which in rat liver were localized in the non-particulate fraction of the cell, are absent from guinea-pig heart. Evidence is presented that the CoA-dependent oxaloacetate formation from citrate, which in guinea pig heart is a mitochondrial process, is catalysed by citrate synthase. 3, The fatty acid-synthesizing system in guinea-pig heart is firmly bound to the mitochondrial membrane fraction. The low latency of the fatty acid synthesis activity in guinea-pig heart mitochondria compared with that in rat-liver mitochondria, the outer membrane of which does not catalyse fatty acid synthesis, suggests that both the inner and outer membranes of these heart mitochondria possess activity.

INTRODUCTION

In 1960 H~LSMANN* showed that rat- and rabbit-heart mitochondria can synthesize long-chain fatty acids. The mitochondrial system elongates fatty acyl-CoA esters with acetyl-CoA, NADH serving as the hydrogen donor2-B. Although in heart only the mitochondrial system has been well investigated, in liver three enzyme systems that synthesize long-chain fatty acids have been described, residing respectively in the mitochondria, microsomes and the non-particulate fraction. These systems can be distinguished by their different requirements for substrates and hydrogen donors+8. It has been shown that, under the right conditions, these three systems can be measured independently in cell homogenates and subcellular fractionsO. Since heart is, in contrast to liver, predominately an energy-consuming organ and no data are available in the literature on the relative contributions of the different * Postal address:

Plantage

Biochim. Biophys. Acta, 210

Muidergracht

(1970)360-370

12, Amsterdam,

The Netherlands.

FATTY ACID SYNTHESIS

361

IN HEART

cell fractions to fatty acid synthesis in heart, we measured the three systems described for liver in an intracellular-partition study of guinea-pig heart. In addition we determined the activities of ATP citrate lyase (EC 4.1.3.8), which in liver and mammary gland is involved in the extra-mitochondrial production of acetyl-CoAIO-ls, and of acetyl-CoA carboxylase (EC 6.4.1.2), which provides malonyl-CoA for the microsomal and non-particulate fatty acid-synthesizing systems. METHODS

Fractionation of guinea-& heart For each experiment two hearts were homogenized in 0.25 M sucrose, filtered through a Nylon or Perlon net and partioned into subcellular fractions essentially according to HULSMANS”with some modifications. All steps were carried out at o-5”. The homogenate was centrifuged for IO min at 200 x ga,. The pellet was twice resuspended and centrifuged for IO min at IIO xg,,. The final pellet was resuspended to yield N or nuclear fraction. The E or cytoplasmic extract fraction was the combined supematants from the three centrifugations.The sum of the values obtained separately for enzymic activity and protein content of the N and E fractions was assumed to be representative of the whole tissue. Recoveries in the N, M, L, P and S fractions (see below) were based on the sum of the N and E activities. Three particulate fractions were isolated from the E fraction by successive centrifugations for IO min at 2200 x gaV (M or heavy mitochondrial fraction), IO min at 13500 x gav (L or light mitochondrial fraction), 60 min at 125000 xgav (P or microsomal fraction). The final fraction (S or cytosol fraction) was the supematant after centrifugation for 60 min at 125000 xgav. The M and L pellets were washed twice, and the combined supematants were used to isolate the succeeding fraction. The P pellet was not washed. In some experiments, the M and L fraction were isolated together (M+L) by centrifuging the E fraction for ro min at 135ooxg,,. The (M+L) fraction was washed only once. Fractionation of rat liver The fractionation of rat liver was carried out essentially according to the method of DE DUVE et aLla, exactly as the normal partition described by DONALDSON et aLg. Fractionation of guinea-jig heart mitochondria Guinea-pig heart mitochondria were prepared essentially according to HOLTON et a1.10. Guinea-pig heart was homogenized in 0.25 M sucrose and centrifuged for 3 min at 450 xgav. The pellet was washed once and again centrifuged for 3 min at 450 xgsV. From the combined supematants the mitochondria were isolated by centrifugation for IO min at 3000 x ga,. The mitochondrial pellet was resuspended in 0.25 M sucrose and centrifuged for IO min at II ooo xg &“.After washing once more the mitochondria were fractionated into an intermembrane fraction, a membrane fraction and a matrix fraction as follows. The mitochondria were incubated for 15 min at 0” in 40 mM potassium phosphate buffer (pH 7.2) in order to release the enzymes of the intermembrane space 20*21. This concentration of phosphate is lower than the IOO mM used by PETTE~Oand KLINGENBERGAND PFAFF~~,as it was shown that more creatine kinase, the marker enzyme for the intermembrane space, was released with the lower concentration. After taking a sample of the phosphate-treated mitochondria, to which Biochim. Biophys. Acta, 210 (1970)

36c-370

362

E. M. WIT-PEETERS

et al.

sucrose was added to an end concentration of 0.25 M, the mitochondrial suspension was centrifuged for IO min at ISOOO xg itV, yielding the intermembrane fraction as a supernatant (Fraction I). The pellet was resuspended in 0.25 M sucrose and I mM glutathione (volume, 2 ml) and sonicated for three periods of I min in a M.S.E. sonic disintegrator (21 kcycles/sec; z yrn from peak to peak). The sonicate was then centrifuged for I h at 160000 xgav yielding the membrane fraction (Fraction II) as the pellet and the matrix fraction (Fraction III) as the final supernatant. Assays The subfractions were stored in I- or z-ml portions at about --zg’ and thawed just before testing. Prior to testing I mM glutathione was added and the fractions were subjected to ultrasonic vibration in the cold for respectively one or two periods of I min in a sonic disintegrator (21 kcycles/sec; 2 pm from peak to peak). No glutathione was added to the fractions in which the activities of 6-phosphogluconate dehydrogenase, lactate dehydrogenase, acetyl-CoA carboxylase, ATP citrate lyase, and the microsomal and non-particulate fatty acid-synthesizing system were tested; nor were these fractions sonicated. Malate dehydrogenase (EC I.I.I.~~), cytochrome c oxidase (EC r.g.3.1), rotenone-insensitive NADPH-cytochrome c reductase, acid phosphatase (EC 3.1.3.2), glucase-6-phosphatase (EC 3.1.3.9.), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), lactate dehydrogenase (EC 1.1.1.27), propionyl-CoA carboxylase (EC 6.4.1.3), malonylCoA decarboxylase (EC 4.1.1.9) and creatine kinase (EC 27.32) were tested as described in ref. 22 (see also ref. 23), 24, 25, 18, 18, 26, 27, 28, 28 and 29, respectively. Fatty acid synthesis was tested as described by DONALDSONet aL9 in a standard incubation medium containing go mM KCl, 50 mM potassium phosphate buffer (pH 7.4), I mM EDTA, 5 mM M&l, and 5 mM KCN to which were added IO mM ATP, 5 mM NADH and 0.3 mM [I-r4C]acetyl-CoA (Medium I or “mitochondrial” medium), or IO mM ATP, 5 mM NADPH and 0.3 mM [r(3)-14C]malonyl-CoA (Medium II or “microsomal” medium) or 5 mM NADPH, 0.3 mM [I(3)-14C]malonyl-CoA and 0.042 mM unlabeled acetyl-CoA (Medium III or “non-particulate” medium). ATP citrate lyase (EC 4.1.3.8) was determined essentially according to HOWANITZ AND LEVY~~. The CoA-dependent oxaloacetate formation from citrate was measured spectrophotometrically with NADH and malate dehydrogenase. The incubation medium contained IO mM Tris-HCl buffer (pH 7.3), 2.6 mM MgCl,, zo mM potassium citrate, 0.3 mM KCN, 1.5 ,uM rotenone, IO mM mercaptoethanol, 3.33 mM ATP, 0.15 mM NADH, 0.5 pg malate dehydrogenase (Boehringer) and 0.125 mM coenzyme A (Boehringer). Acetyl-CoA carboxylase (EC 6.4.1.2) was tested by measuring the acetyl-CoAdependent r4C0, fixation after preincubation of the enzyme preparation with citrate (see ref. 31).The assay was carried out in liquid-scintillation vessels in a metabolic shaker. During the preincubation (I h) the medium contained 60 mM Tris, brought to pH 7.0 with H,PO,, zo mM MgC12, 8 mM potassium citrate and j mM z-mercaptoethanol. The incubation was carried out in 60 mM Tris, brought to pH 7.0 with H,PO,, IO mM MgCl,, 4 mM potassium citrate, 2.5 mM a-mercaptoethanol, 40 mM KH14C0,, 3.6 mM ATP (dipotassium salt) and 0.42 mM acetyl-CoA (total volume, 0.5 ml). After incubating for 5 min in case of rat liver and for IO min in case of guinea-pig heart, the reaction was stopped with 0.05 ml 2.5 M H,SO,. With longer incubations the Boichim. Biophys. Acta.

210

(1970)

360-370

363

FATTY ACID SYNTHESIS IN HEART

enzyme activity is no longer linear with the incubation time. This is probably due to the decarboxylation of the product, malonyl-CoA, by malonyl-CoA decarboxylase (see ref. 28). After z h the vessels were gassed with I?, and 1z.5 ml scintiIlation liquid was added (6.67 mf toluene containing the ffuors, 3.33 ml Triton x-100 and 2.5 mi ethanoF2). After standing at 4” the fluid was mixed and counted. All enzyme activities are expressed in units of ymoles substrate metabolized per min. Protein was determined according to CLELAND AND SLATER~~ except in the intramitochondrial partition studies (Table VII, Fig. 4), where the method of LOWRY et aLs4 was used. Cytochrome c was prepared from beef heart35, and acetyl-CoA, propionyl-Cob and malonyl-CoA were synthesized by the thiophenol method36. RESULTS

~~t~uc~l~~la4~~~~~~~~~0~ Q~~~ty acid ~y~th~~~sin g~~~e~-~~g heart The requirements for the mitochondrial fatty acid-synthesizing system of guinea-pig heart have been studied in an earlier papers; the mitochond~al system elongates fatty acids with acetyl-CoA as condensing unit, NADH as hydrogen donor and ATP to activate endogenous fatty acids. Table I and Fig. I show that the intraTABLE

X

INTRACELLULAR

NADH

IN

DISTRIBUTION

GUINEA-PIG

OF THE

FATTY

ACID-SYNTHESIZING

SYSTEM

USING

ATP

ACETYL-CO&

AND

HEART

As markerenzymeswere usedcytochromec oxidasefor the mitochondria,rotenone-insensitive NADPHcytochrome c reductase for the microsomes and acid phosphatase for the lysosomes. E is cytoplasmic extract, N is the nuclear fraction, M is the heavy mitochondrial fraction, L is the light mitochondrial fraction, l? is the microsomal fraction and S is the particle-free supernatant. Absolute

Percentage values

values E+N

Protein (mg) Cytochrome c oxidase (units) Rotenon~-i~~ns~tive NADPHcytochrome c reductase (munits) Acid phosphatase (munits) Fatty acid synthesis* (munits) Incubation

medium

Recovery

E+N

N

I73 92.5

100

41.2

15.0

100

3z.9

35.2

25.0

754 786

100

32.5 34.’

17.6 6.9

IO.0

100

28.2

100

37.1

23.1

22.2

-

*

-.-.

--

M

L

S

(%I

5.5

31.4 4.9

100.3 102.5

26.3 17.4

24.5 25.1

111.7

5.4

0.0

P 4.7

8.0

25.7

-

110.9 91.3

I. rotenonc-insensitive

NAWH-cytochrome cytochrome c oxidase rcductase D

’ 0

. 20

X

. 40

’ 60

* ’ 60 100

’

*

.

*

*

1

c acid phwphataee

fatty acid synthesis

-

-

total protein

Fig. I. The intracellular distribution of the fatty acid-synthesizing and NADH in guinea-pig heart. See also Table I. Biochim.

system, using acetyl-CoA,

Biophys. Acta,

210

(1970)

ATP

360-370

E. M. WIT-PEETERS et al.

364

cellular distribution of this enzyme system resembles that of the marker enzyme for the mitochondria, cytochrome c oxidase. In rat liver there exist, apart from the mitochondrial system, two other fatty acid-synthesizing systems. The microsomal system elongates endogenous fatty acids with malonyl-CoA, and requires NADH or NADPH and ATP (see ref. 7) ; the nonparticulate system synthesizes fatty acids de EOTJO from malonyl-CoA, NADPH and acetyl-CoA (see ref. 8). In subcellular fractions of rat liver these three systems can be measured independently of each other using incubation Medium I for the mitochondrial, Medium II for the microsomaf, and Medium III for the non-particulate systems (see METNODS for the incubation media}. It can be seen in Table II that only the mitochondrial system is active in guinea-pig heart. The slow fatty acid synthesis in all fractions with Medium II, with T.4BLE FATTY

II ACID

INCUBATION

SYNTHESIS

IN

SUBCELLULAR

FRACTIONS

OF

GUINEA-PIG

HEART

UNDER

DIFFERENT

CONDITIONS

Guinea-pig heart was fractionated into subcellular fractions as described in METHODS. E is the cytoplasmic extract, N is the nuclear fraction, M +L is the mitochondrial fraction, P is the microsomal fraction and S the particle-free supernatant. In addition to the components of the standard medium, incubation medium I contained 0.3 mM [I-K]acetyl-CoA, 5 mM NADH and IO mM ATP; incubation medium II 0.3 mM [r(3)-r*C]malonyl-CoA, 5 mM NADPH and IO mM ATP; incubation medium III 0.3 mM ~x(3)-1*C]malonyl-~o_~, 5 m&f NADPH and o.o@ mM acetyl-CoA. .___ ___~.___ C&2 fvac&?n pm&s of wbstrate incorporaled -~per min ___~ per mg protein Medium III Medium I ^_ Medium II N E M+L P S

61 103 164 128 6

0

4 6 21 8 2

.-.

3 I o 2

~--

(as with Medium I) the highest specific activity in fraction M+L, can be explained by decarboxylation of malonyl-CoA by malonyl-CoA decarboxylase, a mitochondrial by the mitochondrial system. Although the enzymeZ8, and subsequent incorporation mitochondrial system works preferentially with NADH, it shows also some activity with NADPH6. Intracellular

localization of ATP

citrate lyase and acetyl-CoA carboxylase ilz rat liver

Table III and Fig. z show the carboxylase in subcellular fractions follow that of the marker enzyme 6-phosphogluconate dehydrogenase. CoA carboxylase are both localized Activity of ATP

distribution of ATP citrate lyase and acetyl-CoA of rat liver. Both distribution patterns closely for the non-particulate fraction of the liver cell, We conclude that ATP citrate Iyase and acetylexchrsively in the cytosol of rat liver.

citrate lyase and acetyl-CoA carboxylase in guinea-pig

heart

It is clear from Table IV and Fig. 3 that in guinea-pig heart both the acetylCoA carboxylation and the CoA-dependent oxaloacetate formation from citrate are mitochondrial processes; their distribution patterns resemble that of cytochrome c oxidase and closely foIlow that of propionyl-CoA carboxylase, an enzyme which in rat liver28 and guinea-pig heart (see below) is a mitochondrial matrix enzyme. The slight activity of both enzymic reactions in the S fraction can be explained by leakage Bs’ockim. Biophys.

Acta,

210

(rg7o)

360-370

3%

FATTY ACID SYNTHESIS IN HEART TABLE

III

THE INTRACELLULAR

DISTRIBUTION

OF

ATP

CITRATE

LYASE

AND

ACETYL-COA

CARBOXYLASE

IN RAT LIVER

As marker enzymes were used cytochrome c oxidase for the mitochondria, acid phosphatase for the lysosomes, glucose&phosphatase for the microsomes and 6-phosphogluconate dehydrogenase for the nonparticulate fraction of the cell. E is cytoplasmic extract, N is the nuclear fraction, M the heavy mitochondrial fraction, L the light mitochondrial fraction, P the microsomal fraction and S the particle-free supernatant. Percentage

Absolute

Reco-

values

values

Protein (mg) Cytochrome c oxidase (units) Acid phosphatase (units) Glucose-6-phosphatase (units) 6Phosphogluconate dehydrogenase (units) Acetyl-CoA carboxylase (units) ATP citrate lyase (units)

E+N

IQ-N

N

1763 322 87.4 69.5

100 100 100 100 100 100

21.6 3.40 3.78

100

very

.-___

M

L

P

S

26.1

I7.5

25.1

so.7

10.4 18.5

23.2 16.0

4.= 3.4 42.5 6.8

25.1

50.2

10.4 11.3 9.1

4.5 4.2 3.8

0.3 0.6 0.4

12.3 2.0 10.3

68.1 60.6 81.6

1.9 15.8 59.2

r?J 0.0 0.6 0.7

97.9 106.2 92.5 101.2 95.6 78.7 105.2

acid phaephatase

cytochrome

c oxidaee

0 20 40 60 80 100

I”

% total protein

Fig. 2. The intracellular distribution See also Table III.

of ATP citrate lyase and acetyl-CoA

carboxylase

in rat liver.

of matrix enzymes out of the mitochon~ia as a result of the isolation procedureas. It is clear that guinea-pig heart cytosol contains neither acetyl-CoA carboxylase nor ATP citrate lyase. Table V shows that the acetyl-CoA carboxylation in each subcellular fraction of guinea-pig heart is I-Z O/oof the propionyl-CoA carboxylase activity in that fraction and is probably due to the non-specificity of propionylCoA carboxylase37. Table VI shows that the CoA-dependent oxaloacetate formation from citrate by intact guinea-pig heart mitochondria is latent ; the mitochondria must be disrupted, for example by sonication, to show activity. Since all the substrates for this reaction readily penetrate the outer mitochondrial membrane 58,the activity must be localized on the inner side of the mitochondrial inner membrane. ATP, an absolute requirement for the non-particulate ATP citrate lyase in rat liver (line 3) is not required for the mitocbond~al reaction in heart (line 2). The possibility that the mitochondria contain sufficient endogenous ATP to maintain the reaction is excluded by the fact that the Biochim.Biophys.

Ada,

210

(1970)

360-370

E. M. WIT-PEETERS

366 TABLE

et d.

IV

THE INTRACELLULARDISTRII~UTIONOF THE COA-DEPENDENT OX.~LOACETATEFORMATIONFROM CITRATE, ACETYL-CO-4 CARBOXYLATIONAND PROPIONYL-COA CARBOXYLASE IN GUINEA-PIG HEART As marker enzymes were used cytochrome c oxidase for the mitochondria, rotenone-insensitive NADPHcytochrome c reductase for the microsomes and lactate dehydrogenase for the non-particulate fraction of the cell. E is the cytoplasmic extract, N the nuclear fraction, M+L the mitochondrial fraction, P the microsomal fraction, and S the particle-free supernatant. Absolute values

Percentage values

N+E

___~ Protein (mg) Cytochrome c oxidase (units) Rotenone-insensitive NADPH-cytochrome c reductase (munits) Lactate dehydrogenase (units) CoA-dependent oxaloacetate formation from citrate (munits) Acetyl-CoA carboxylation (munits) Propionyl-CoA carboxylase (munits)

TABLE THE IN

205.9 163.2

100 IO0

565 483 142.7 13.05 915

26.2 15.2

25.2 78.4

6.7 8.9

42.6 0.0

100.7 102.5

IO0

‘7.5 2.0

29.2 2.2

21.2 I.4

25.6 93.1

93.5 98.7

100 100 IO0

26.8 26.5 21.3

38.6 36.4 47.2

8.5 3.0 2.3

6.2 6.6 9.6

x0. I 72.5 80.4 -

100

V

SPECIFIC

ACTIVITIES

SUBCELLULAR

OF

THE

FRACTIONS

For E, N, M+L,

OF

ACETYL-COA GUINEA-PIG

CARBOXYLATION

AND

PROPIONYL-COA

P and S see legend to Table IV.

Cell fvaction

Acetyl-CoA carboxylation (munits/mg protein)

Propionyl-CoA carboxylase (munits/mg protein)

N E M+L P S

0.078 0.063 0.092 0.024

4.74 8.35 I.52

0.010

1.00

3.62

propioryl-CoA carboxylase

5 -

CARBOXYLASE

HEART

CoA-dependent formation from

acetyl-CoA carboztyiation

cytochromec

0 A A citrate

rotenoneinsewitive

oxidase

NADPH-cytochrome

c

lactate

dehydrogenase

redxtas~

0

20

40

% t otal

60

80

100

protein

Fig. 3. The intracellular distribution citrate, the acetyl-CoA carboxylation Table VI. Biochim. Biophys. Acta,

210

of the CoA-dependent oxaloacetate (OAA) formation from and propionyl-CoA carboxylase in guinea-pig heart. See also

(1970) 36c-370

367

FATTY ACID SYNTHESIS IN HEART TABLE

VL

THE EFFECT OF SONICATION, NATANT

OF

GUINEA-PIG

ADDITION AND OF THE ADDITION OF A PARTICLE-FREE SUPERCOA-DEPENDENT OXALOACETATE FORMATION FROM CITRATE BY HEART MITOCHONDRIA

RAT

The standard

LIVER

OF

ON

incubation

ATP

THE

medium for the ATP

citrate Iyase assay was used with or without

-_

ATP.

Formation of oxaloacetate (munits)

0.29 mg intact guinea-pig

heart mitochondria

0.29 mg sonicated guinea-pig heart mitochond~a 0.42 mg rat liver supernatant 0.29 mg sonicated guinea-pig heart mitochondria+ 0.42 mg rat liver supernatant

-ATP

+ATP

0.00 1.14 0.00

0.00 I.I9 2.04

1.24

3.17

ATP citrate lyase activity of the liver cytosol remains dependent on ATP addition even in the presence of mitochondria (line 4). The localization of the CoA-dependent oxaloacetate formation from citrate inside the inner mitochondrial membrane and its independence of ATP suggest that this reaction is catalysed by citrate synthase (EC 4.1.3.7). TABLE

VII

INTRAMITOCHONDRIAL

THE HEART

DISTRIBUTION

OF

THE

FATTY

ACID-SYNTHESIZING

SYSTEX

IN

GUINEA-PIG

XlTOCHONDRIA

Cytochrome c oxidase was used as marker for the inner membrane, creatine kinase for intermembrane Fraction I is the IO min, space and malate dehydrogenase for matrix space. Mit. = mitochondria. 18000 xgav supernatant after phosphate treatment, Fraction 2 is the 60 min, 160000 xfiav pellet after sonication, and Fraction 3 the 60 min, 160000 x g BV supernatant (see METHODS).

Protein (mg) Cytochrome G oxidase (units) Creatine kinase (units) Malate dehydrogenase (units) Fatty acid synthesis (munits) Propionyl-CoA

carboxylase

* After treatment

(munits)

Recovery (%I)

Absolute ualzkes

Percentage vu&es

&fit. *

MiZ. *

Fr. I

Fr. 2

Fr. 3

36.58 84.10 51.46 89.48 5.55

IO0 100 100 IO0 IO0

7.2 0.5 78.8 4,’ 0.0

7o,9 107.2 12.1 19.4 98.2

26.6 2.6

104.7 IIO.3

4.3 79.2 4.5

95.2 102.7 102.7

IO0

I.7

3.4

98.8

103.9

370

with phosphate.

Intramitochondrial localizatioti of the fatty acid-synthesizing system in guinea-pig heart mitocho&ia Guinea-pig heart mitochondria were fractionatid into an inter-membrane fraction (Fr. r), a membrane fraction (Fr. 2) and a matrix fraction (Fr. 3) (see METHODS) ; no differentiation was made between inner and outer membrane. The distribution pattern of fatty acid synthesis closely follows that of cytochrome c oxidase, the marker enzyme for the inner membrane (Table VII and Fig. 4). However, since the inner and outer membranes were not separated, this experiment is unable to distinguish between a localization in either membrane, or in both. In an earlier paper? it has been shown that in rat liver the mitochondrial fatty acid-synthesizing system is confined to the inner membrane, no activity in the outer membrane being demonstrated. The observation, shown in Table VIII, that fatty acid synthesis is highly latent in intact rat-liver mitochondria (the activity is stimuBiochim. Bdo#ys.

Acta, 2x0 (1970) 360-370

368

E. M. WIT-PEETERS

et al.

1 PWkMyLCoA carboxylase

1

3

2

-II

malate dehydmgenase 3

cytochrome E axidase

fatty acid synth&s

creatine kinase 2 1

0

l..‘.

1

3 .

h I.

2 *

I.

r-l I ._I,

+-I

3

Ill I

I

*

1)

20 40 60 80 100 X total protein

Fig. 4. The intramitochondrial heart. See also Table VII.

TABLE THE

af the fatty acid-synthesizing

system in guinea-pig

VIII

EFFECT

OF FATTY

distribution

OF SONICATION

ACID

SYNTHESIS

OF GUINEA-PIG AND

HEART

MALONYL-C0.k

AND

RAT-LIVER

MITOCHONDRIA

ON TAE

ACTIVITY

DECARBOXYLASE

Malonyl-CoA decarboxylase was determined as in ref. 28, but 40 mM potassium phosphate buffer (pH 7.0) was replaced by 250 mM sucrose and 4 mM potassium phosphate buffer (pH 7.0) in order to maintain isotonicity. ---~ -___.-.-Spec~$c activity (munitslmg protein) Stimulation factor Intact rn~t~~ho~d~~a Sonic~>-~~r% Gknea-pig heart Fatty acid synthesis* Malonyl-CoA decarboxylase Rat livw Fatty acid synthesis* Malonyl-CoA decarboxylase * Measured in incubation

0.032 0.23

0.096 2.35

0.020

0.281

1.02

7.05

medium I (see

3.0 10.2

14.0 6.9

METHODS).

qtimes by sonication of the mitochondria), is in agreement with this conclusion. However, in guinea-pig heart mitochondria one third of the total fatty acid synthesis was found with intact mitochondria (Table VIII). This argues for a possible double location of mitochondrial fatty acid synthesis in guinea-pig heart, either in the outer and in the inner membranes, or on the two sides of the inner membrane. The possibility that the low latency of the fatty acid-synthesizing activity in guinea-pig heart mitochondria is due to mechanical damage of the mitochondria is made unlikely by the high latency of malonyl-CoA decarboxylase activity in these mitochondria {Table VIII).

lated

DISCUSSION

It is widely recognized that the liver cell contains at least three separate enzyme systems to synthesize long-chain fatty acids, located in different parts of the cell. E&china.Biophys.

Ada,

210

(rgyoj

36c-370

FATTY ACID SYNTHESISIN HEART

369

Recently their relative contribution to total fatty acid synthesis in liver has been measured8. In heart, however, only the mitochondrial system has well been studiedl-6 and the possible existence of other systems remained uncertain. The recent demonstration by CHRISTYand DAHLEN AND PORTERS that the mitochondrial system can only elongate fatty acids, and does not synthesize fatty acids de ~ovo, made it important to investigate the possible presence of other systems. From the experiments reported in this paper it is clear that a non-particulate system, that synthesizes fatty acids de nova, and a microsomal elongation system, comparable with those of liver, are absent from guinea-pig heart although it cannot be excluded that heart contains another unknown system. The absence in heart of a de nova system implies that the fatty acids for elongation by the mitochondria are supplied by the blood. In rat liver ATP citrate lyase and acetyl-CoA carboxylase, which are both involved in the production of malonyl-CoA for extramitochondrial fatty acid synthesis, are localized in the non-particulate fraction of the cell. These results are in agreement with those of SRERE 39for ATP citrate lyase and of SMITHet ~1.~~for acetylCoA carboxylase. The data reported in this paper show that both enzymes are absent from heart. The localization of the acetyl-CoA carboxylation reaction in the mitochondria of guinea-pig heart and the low activity of the acetyl-CoA carboxylation in comparison with that of propionyl-CoA in all subcellular fractions (Table V) suggest that this reaction is catalysed by propionyl-CoA carboxylase, in agreement with H~~LSMANN~~. Our conclusion that the CoA-dependent oxaloacetate formation from citrate measured in guinea-pig heart is not catalysed by ATP citrate lyase but by citrate synthase is based on the localization of this reaction in mitochondria, and its latency and ATP independence. This conclusion is in disagreement with that of SRERE~~who found ATP citrate lyase activity in rat, rabbit, chicken and pig heart. However, it seems possible that the method used by SRERE~~to extract the enzyme, namely treatment of the tissue with KCl-ethanol, damages the mitochondria in such a way that matrix enzymes, including citrate synthase, are extracted from the mitochondria. Our results are in agreement with those of GARLANDANDRANDI.E~~,who could not demonstrate an ATP-dependent formation of oxaloacetate from citrate by rat heart. The relative impermeability of heart mitochondria for citrate%44 is also understandable in the light of the absence from the heart cytosol of ATP citrate lyase, acetyl-CoA carboxylase and a system for synthesizing fatty acids. ACKNOWLEDGEMENTS We are indebted to Professor E. C. SLATER for his valuable advice and interest. We wish to thank Professor W. E. DONALDSON for collaboration in some of the experiments and Mr. H. L. A. PIJST for skilful technical assistence. The present investigations have been carried out under the auspices of the Netherlands Foundation for Chemical Research (S.O.N.) and with financial aid from the Netherlands Organization for the Advancement of Pure Scientific Research (Z.W.O.).

B&him.

Biophys.

Ada,

210 (1970)

360-370

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Biophys. Acta, 210 (1970) 360-370