BIOCHIMICA ET BIOPHYSICAACTA
217
BBA 65149
ON T H E OXIDATION OF ACETATE AND PYRUVATE BY GUINEA-PIG
H E A R T SARCOSOMES
E. JACK DAVIS
Laboratory of Physiological Chemistry, University of A rnslerdam, Amsterdam (The Netherlands)
(Received October i6th, i964)
SUMMARY
I. Sarcosomes isolated from guinea-pig heart have been used in a comparative
study of the mechanism of oxidation of pyruvate and acetate by these particles.
2. Acetate oxidation, without added malate, is very rapid when the concentration of phosphate is low, but is strongly inhibited by higher phosphate concentrations. In the presence of concentrations of phosphate up to 30 raM, acetate is wel.1
oxidized when malate is also present.
3. Acetate oxidation in the presence of 2,4-dinitrophenol is initiated on addition
of ATP. Magnesium inhibits this oxidation and the inhibition is relieved by oligomycin.
4. Pyruvate is rapidly oxidized in the absence of added citric acid cycle intermediates during the first minutes of incubation, the rate rapidly decreasing with time
to near zero (3-1o rain). Evidence is presented which strongly suggests that the supply
of citric acid cycle intermediates is depleted during the oxidation of pyruvate.
5. Carnitine has no effect on the oxidation of acetate or pyruvate in the citric
acid cycle, but stimulates the oxidative decarboxylation of pyruvate, probably as a
result of acetylcarnitine formation from pyruvate.
6. Under the conditions of these experiments, pyruvate oxidation is decreased
about 50% by an equimolar concentration of acetate.
INTRODUCTION
Although there is convincing evidence that short-chain fatty acids are very
rapidly oxidized by the intact heart, and that the utilization of exogenous glucose,
lactate or pyruvate is markedly decreased in their presence1, 2, their rapid oxidation
by isolated heart mitochondria has not been demonstrated (see, e.g. refs. 3-5).
The present communication shows that under the appropriate conditions acetate is rapidly oxidized by isolated guinea-pig sarcosomes, both in the absence and
presence of added malate. Undei the same conditions, however, pyruvate oxidation,
although very rapid at the onset of incubation, rapidly decreases with time when no
exogenous malate is added (cf. refs. 6, 7)- The present results also suggest, in essential
Biochim. Biophys. Acta, 96 (1965) 217-23o
218
E . J . DAVIS
agreement with the conclusions of others, that the oxidation of acetate or of pyruvate
by heart sarcosomes via the citric acid cycle is unaffected by carnitine, but that
carnitine stimulates the oxidative decarboxylation of pyruvate without affecting the
oxidation of the acetyl-CoA formed.
The implications of these observations are discussed in terms of present concepts
concerning the oxidative metabolism of fatty acids and pyruvate.
METHODS
Guinea-pig heart sarcosomes were prepared by a modification of the method
described by CHANCE AND HAGIHARAs for pigeon-heart sarcosomes. Hearts were
minced and homogenized with a glass and Teflon homogenizer in approx. 5 vol. of a
cold medium containing o.25 M sucrose, o.oi M EDTA, o.oI M Tris-HC1 buffer
(pH 7-5) and o.5 mg/ml of Bacillus subtilis alkaline proteinase. The suspension was
immediately diluted with an equal volume of o.25 M sucrose-o.oi M EDTA and
centrifuged at 6oo × g for 3 min. The supernatant fraction was re-centrituged for
IO min at 12 ooo X g. The mitochondrial pellet thus obtained was resuspended in
2o ml of the sucrose-EDTA medium and centrifilged at 8ooo × g for 5 min. The
sarcosomes were finally resuspended in 1.o-1. 5 ml of this medium. All operations were
carried out at o-2 °.
Unlabelled pyruvate was always freshly prepared. Sodium [2-14Cjpyruvate was
stored at --2o ° as a o.2 M solution, and was used for only about 4 weeks after purchase
as a precaution against contamination by breakdown products% a°.
Reaction media for manometric experiments contained 225 mM sucrose, io mM
KCl, IO mM Tris-HC1 buffer (pH 7.5), and I mM EDTA (derived from the mitochondrial suspension), and the additions indicated in the legends to tables and
figures. Flasks with two side-arms were used, with, in the centre well, o.I ml 2 M
KOH and a fluted filter paper, or when 14C-labelled substrates were used, o.i ml
i M hyamine in methanol. The reaction was stopped by the addition of o.o5 ml 2 M
HCIOa from a side-arm. After removal of protein by centrifugation, the HCIO4 was
removed as KCIO4 in the cold. In some experiments oxygen uptake was measured
polarographically with a Gilson Medical Electronics Oxygraph. The reaction temperature was always 25 °.
In manometric experiments that were carried out with 14C-labelled pyruvate,
reactions were stopped with 0.05 ml 2 M HCI04, after which the flasks were allowed
to remain in the shaking apparatus for 30 rain in order to insure that all dissolved
14CO2 was released from the reaction medium and trapped in the hyamine in the centre
well. The hyamine with dissolved C02 was removed from the centre wells and radioactivity was counted in a Nuclear Chicago Model-72o liquid scintillation counter.
Pyruvate was determined spectrophotometrically with lactate dehydrogenase
(EC 1.1.1.27) and NADH. Protein was estimated by the biuret method as described
by CLELAND AND SLATER 11.
Sodium pyruvate, a-oxoglutarate and lactate dehydrogenase were obtained
from C. F. Boehringer und Soehne; NAD+, NADH, ADP and ATP from Sigma
Chemical Co. ; sodium acetate, L-glutamate and 2,4-dinitrophenol from British Drug
Houses; sodium [2-14Clpyruvate from Radiochemical Centre; and crystalline bacterial alkaline proteinase from Nagase & Co., Ltd., Osaka (Japan).
Biochim. Biophys. Acta, 96 (1965) 217 230
OXIDATION OF ACETATE AND PYRUVATE BY SARCOSOMES
219
Hexokinase (EC 2.7.1.1 ) was prepared as described by DARROW AND COLOWICK12, omitting the final crystallization step.
RESULTS
The oxidation of acetate
The oxidation of pyruvate, glutamate or acetate by the sarcosomes in the
presence of ADP was not stimulated by addition of Mg2+, cytochrome c, NAD +,
NADP + or CoA. The respiratory-control ratio E(respiratory rate after addition of
ADP) :(respiratory rate after exhaustion of ADP)l on the addition of ADP was 8-12
for glutamate (Fig. I) or pyruvate when no Mg2. was added. Addition of 5 mM MgC12
decreased the ratio to 2-3. Acetate oxidation was similarly affected by the addition
of ADP, giving a control ratio of 5-9 in the absence of added Mgi+ (see Fig. I).
t
T
T
ADP~~~
-~rr
1
I
pato0m
Fig. I. O x i d a t i o n of a c e t a t e a n d g l u t a m a t e in a b s e n c e a n d presence of Mg *+. T r a c i n g s of Gilson
Medical E l e c t r o n i c s O x y g r a p h records. R e a c t i o n m e d i a c o n t a i n e d in a d d i t i o n to t h e basic c o m p o n e n t s : 5 m M p o t a s s i u m p h o s p h a t e buffer (pH 7.4), 1-o4 m g s a r c o s o m a l p r o t e i n a n d t h e addit i o n s as i n d i c a t e d in a t o t a l v o l u m e of 2.0 ml. C o n c e n t r a t i o n s : acetate, io m M ; g l u t a m a t e ,
i o m M ; MgC12, 5 r a M ; a n d A D P , a p p r o x , i . o / , m o l e (first addition) a n d 5 . o # m o l e s (second
addition). A b b r e v i a t i o n s : Acet, a c e t a t e ; Glu, g l u t a m a t e .
Acetate was oxidized rapidly and at a constant rate, both in the presence and
absence of added malate, if mitochondria were prepared as described in METHODS
(Table I). If the concentration of EDTA in the isolation medium was I mM or less,
however, the results were quite variable, but in general acetate was oxidized very
poorly or not at all under these conditions. Acetate oxidation proceeded at about
equal rates in the presence of phosphate and phosphate acceptor, or in the presence
of 2,4-dinitrophenol and ATP. In the absence of dinitrophenol, added ATP was not
necessary to drive the reaction. VAN DEN BERGH1~ has observed that the oxidation
of short-chain fatty acids by rat-liver mitochondria is strongly inhibited by relatively
low concentrations of phosphate when no intermediate of the citric acid cycle is
added. Table I shows that the same is trlle for acetate oxidation by heart sarcosomes,
Biochim. Biophys. Acta, 96 (1965) 217-23o
220
E.J.
TABLE
I
RATE
ACETATE
OF
OXIDATION,
AS COMPARED
TO THAT
OF OTHER
SUBSTRATES
MEASURED
DAVIS
POLARO-
GRAPHICALLY
R e a c t i o n m e d i u m c o n t a i n e d 2 0 0 m M s u c r o s e , i o m M KC1, i o m M T r i s - H C 1 ( p H 7-5), I m M E D T A ,
o.5-1. 4 mg sarcosomal
protein and the additions
listed. Reaction
volume,
2.0 ml. The values
represent the means of 5 or more determinations,
with the range in brackets.
Additions
Substrate
.
.
.
.
.
.
.
.
.
.
.
.
.
Respiratorycontrolratio
Respiratory rate
(mt~atoms/mg protein[
min)
Phos- Malate DNP* A T P ADP** MgCl 2
phate (mM) (#M) (raM)
(raM)
(mM)
Acetate
(io mM)
Glutamate
(io mM)
5
--
--
--
+
--
6.5 15.4-1o.o)
270 (212-3o4)
5
5
0.5
--
---
--
+
+
-5
6.7 (5.42.0 (i.6
264 1216-292)
276 (2oo-314)
5
5
5
--o.5
5°
5°
5°
5
5
---
----
----
272 12o4-31o)
260 (208-294)
--
0.5
5°
5
--
--
--
120
5
3°
3°
--o-5
50
5°
51)
5
5
5
----
5
---
----
i6 (
o- 52)
48 ( 22- 84)
254 (I98-288)
--
--
--
+
--
8.2 (6.6-12.5)
296 (244
320)
---
+
--
7-9 16.6-11.8)
288 (244
304)
5
5
Pyruvate
0.5
5
--
---
5°
5°
5
--
--
--
+
9.8)
3.1)
o
---
---
3 1 ° (254-352)
90 (72-124)
--
--
280
3°
31o
294
-
O-acetyl-nLcarnitine
(io raM)
5
0-5
--
5
5
---
5°
5°
2,4_dinitrophenol.
** o . 5 - i # m o l e w a s
--
5
(90-210)
+
--
--
---
7.2 (6.O-lO.2)
---
(220-33o)
( o- 64)
(248-340)
(236-324)
ist
5th
ist
5th
min
min
rain
rain
274 (21o 320)
280 1216-31o )
*
5/zmoles
added
when
respiratory-control
ratio
was
determined;
otherwise
were added.
the addition of malate relieving the inhibition. As mentioned earlier, added Mg ~+
did not have any stimulating effect on the oxidation of pyruvate. Added Mg 2+
occasionally inhibited acetate oxidation in the presence of phosphate and phosphate
acceptor, and always in the presence of 2,4-dinitrophenol and ATP.
Fig. 2 shows the effects of phosphate, Mg z+ and malate on acetate oxidation in
presence of 2,4-dinitrophenol and ATP. Although, when the phosphate concentration is kept low, malate does not stimulate acetate oxidation (Table I), it somehow protects the acetate-oxidizing system against the phosphate effect. In experiments using an oxygen polarograph, optimal rates of acetate oxidation in the
presence of 2,4-dinitrophenol and ATP were not obtained unless a small amount
1o.1- 5 mM) of phosphate was added. This effect is most probably due to the fact that
phosphate becomes limiting for the substrate-linked phosphorylation step, a-oxoglutarate -+ succinate, which is not uncoupled by 2,4-dinitrophenoP 4,15. No correBiochim. Biophys. Acta,
96 (1965) 217-23 o
OXIDATION OF ACETATE AND PYRUVATE BY SARCOSOMES
221
80
.9 60
~4o
6
3
20
4
0
6
0
12
18
24
6
12
30
0
Incubation time (rain)
18
24
30
36
F i g . 2. T h e e f f e c t s o f p h o s p h a t e , M g 2+ a n d m a l a t e o n a c e t a t e o x i d a t i o n i n t h e p r e s e n c e o f 2, 4d i n i t r o p h e n o l a n d A T P . R e a c t i o n s w e r e c a r r i e d o u t in ~ V a r b u r g v e s s e l s c o n t a i n i n g i n a d d i t i o n
to the basic medium, io mM ATP, io mM acetate, 5 °/~M 2,4-dinitrophenol and 0.80 mg sarcos o m a l p r o t e i n p l u s t h e f o l l o w i n g : f l a s k s 2 a n d 4, 5 m M p o t a s s i u m p h o s p h a t e ( p H 7-4); f l a s k s 6
a n d 8, 0. 5 m M m a l a t e ; f l a s k s 5 - 8 , 3 ° m M p o t a s s i u m p h o s p h a t e ; f l a s k s 3, 4, 7 a n d 8, 5 m M MgC12.
R e a c t i o n v o l u m e w a s i . o m l . A f t e r a 4 - r a i n p e r i o d o f e q u i l i b r a t i o n , 2 , 4 - d i n i t r o p h e n o l (5 ° ~ M ) w a s
a d d e d f r o m a s i d e - a r m , a n d o x y g e n u p t a k e w a s m e a s u r e d f o r 24 r a i n .
sponding phosphate dependence was observed in manometric experiments, possibly
due to the fact that phosphate concentration did not become limiting during the course
of incubation, since the mitochondrial concentration was considerably higher.
Table II shows that, when measured in the presence of Mg~+, ATP and 2,4-dinitrophenol, acetate is much more rapidly oxidized in the presence of oligomycin than
in its absence. It therefore appears that an oligomycin-sensitive1~ Mg2+-activated
mitochondrial ATPase largely prevents acetate activation in the absence of oligomycin.
TABLE
II
OF OLIGOMYCIN ON THE OXIDATION
PHENOL ( D N P ) AND A T P
THE EFFECT
OF A C E T A T E IN P R E S E N C E
OF
M g 2+, 2,4-DINITRO-
Data were taken from an experiment measuring oxygen uptake polarographically. Incubation
m e d i a c o n t a i n e d 2 0 5 m M s u c r o s e , IO m M KC1, IO m M T r i s - H C l ( p H 7.5), 5 m M p o t a s s i u m
p h o s p h a t e ( p H 7.5), 0 - 2 5 m M E D T A , IO m M s o d i u m a c e t a t e , 0.9 m g m i t o c h o n d r i a l p r o t e i n a n d
t h e a d d i t i o n s l i s t e d . R e a c t i o n v o l u m e w a s 2.o m] a n d t e m p e r a t u r e w a s 25 °.
DNP A T P A D P MgClz Oligo- Respiratory rate
(#M) (raM) (raM) (mM) mycin (mualoms O/mg
(pg) protein/rnin)
-5°
5°
5°
5°
-5
5
5
5
[
. .
---~
-.
-.
5
5
5
-2
2
ISl rain
4th rain
240
228
65
I32
t36
-224
3°
132
13o
Pyruvate oxidation
In the absence of added malate or other citric acid cycle intermediates, pyruvate
is initially oxidized very rapidly, the rate decreasing progressively with time (Fig. 3)Biochim. Biophys. Acta, 9 6 (1965) 2 1 7 - 2 3 o
222
E.J.
DAVIS
I
1~latorn0
Fig. 3. O x i d a t i o n of p y r u v a t e in t h e a b s e n c e a n d presence of m a l a t e . T r a c i n g s of Gilson Medical
E l e c t r o n i c s O x y g r a p h records. R e a c t i o n m e d i a c o n t a i n e d in a d d i t i o n to t h e basic c o m p o n e n t s ,
20 m M glucose, 5 m M p o t a s s i u m p h o s p h a t e buffer (pH 7.4), 5 m M MgC12, i m M A T P , 0.88 m g
s a r c o s o m a l p r o t e i n a n d t h e a d d i t i o n s as i n d i c a t e d in a total v o l u m e of 2.0 ml. C o n c e n t r a t i o n s :
p y r u v a t e (Pyr), io m M ; h e x o k i n a s e (HK), 15o units1"; m a l a t e (Mal), 0.5 mM.
Malate prevented the decline or restored the oxidation after it had declined nearly to
zero. Similar results were obtained when a-oxoglutarate was added instead of malate.
There results are quite similar to those reported by SLATER et al. 6,7 with rat and
rabbit-heart sarcosomes.
Fig. 4 shows that, when added as sole exogenous substrate, glutamate was
rapidly and linearly oxidized. However, when glutamate was added to a mitochondrial
suspension after preincubation with pyruvate, glutamate was not oxidized until malate
was added. Indeed, when pyruvate and glutamate were added simultaneously, the
pattern of oxygen uptake resembled that of pyruvate alone. These results are also
similar to those reported by SLATER et al.6, ~.
ONP
Ip!torn
~'-~
I rnin
GItl
Fig. 4. O x i d a t i o n of g l u t a m a t e before a n d after p r e - i n c u b a t i o n of h e a r t s a r c o s o m e s w i t h p y r u v a t e .
T r a c i n g s of Gilson Medical Electronics O x y g r a p h records. R e a c t i o n m e d i a c o n t a i n e d , in a d d i t i o n
to t h e basic c o m p o n e n t s , 5 m M p o t a s s i u m p h o s p h a t e buffer (pH 7-4), 1-3o m g s a r c o s o m a l p r o t e i n
a n d t h e a d d i t i o n s as i n d i c a t e d : p y r u v a t e (Pyr), io raM; g l u t a m a t e (Glu), io m M ; m a l a t e (Mal),
o. 5 m.%; a n d 2,4-dinitrophenol (DNP), o.o 5 mM. R e a c t i o n v o l u m e , 2 ml.
Biochim. Biophys. Acta, 96 (1965) 217-23o
223
O X I D A T I O N OF A C E T A T E A N D P Y R U V A T E BY SARCOSOMES
The possibility that the inhibition of pyruvate oxidation was due to the
accumulation of an intermediate of the Krebs cycle was tested by measuring the r a t o
between the 02 uptake and pyruvate disappearance. As shown in Table I I I pyruvate
disappearance results in oxygen uptake which is not significantly different from that
expected if all the pyruvate removed from the medium is completely oxidized.
TABLE
II i
THE STOICHEIOMETRY OF PYRUVATE OXIDATION IN THE PRESENCE AND ABSENCE OF ACETATE
E x p t s . I a n d 2. i n c u b a t i o n s w e r e c a r r i e d o u t w i t h t h e s t a n d a r d r e a c t i o n m i x t u r e p l u s t h e foll o w i n g : 25 m M p o t a s s i u m p h o s p h a t e b u f f e r ( p H 7.4), i m M A T P , 4 ° m M g l u c o s e , 2 m M MgC12,
a n d 0.87 m g ( E x p t . i) o r 1.o 5 m g ( E x p t . 2) m i t o c h o n d r i a l p r o t e i n . R e a c t i o n s w e r e i n i t i a t e d b y
t h e a d d i t i o n o f 15o u n i t s x6 h e x o k i n a s e f r o m o n e s i d e - a r m , a n d s t o p p e d 45 m i n l a t e r o n t h e a d d i t i o n o f 0.05 m l 2 M H C 1 0 4 f r o m a s e c o n d s i d e - a r m . T h e r e a c t i o n v o l u m e w a s i . o m l . E x p t . 3.
E a c h flask c o n t a i n e d t h e s t a n d a r d reaction m i x t u r e plus: 5 m M p o t a s s i u m p h o s p h a t e buffer
( p H 7.4), 15 m M A T P , a n d 1.2o m g m i t o c h o n d r i a l p r o t e i n , in a f i n a l v o l u m e o f i . o m l . R e a c t i o n s
w e r e i n i t i a t e d b y t h e a d d i t i o n o f 0.0.5/*mole 2 , 4 - d i n i t r o p h e n o l f r o m s i d e - a r m i, a n d s t o p p e d
w i t h o.o5 m l 2 M H C 1 0 , 4 ° m i n l a t e r f r o m s i d e - a r m 2. E a c h r e s u l t in E x p t s . 2 a n d 3 is t h e m e a n
of t w o s e p a r a t e d e t e r m i n a t i o n s .
Expt. Flask
I
I
2"
3
4
5
6
Pyruvate Acetate
(mM)
(mm)
Malate
(raM)
-APyruvate - A O
(/*moles) (/*atoms)
-AO/
-- APyruvate
2
2
6
6
6
6
-----6
-0.5
-0. 5
0.5
0.5
0.98
1.95
o.51
2.02
2.04
0.89
4.8
9.1
2.3
9.3
9.4
9.9
4.9
4.7
4.5
4 .6
4 .6
II.I
I
2
3
4
5
4
-4
.
--
-4
4
2.08
-0.96
--
lO.6
11.3
II.O
0.2
0.3
5.1
-11. 4
--
0.5
0.5
0. 5
.
0.5
1
2
3
4
5
5
---
-5
5
5
0-5
0.5
0.5
--
2.55
r.63
----
12,4
12.3
12.2
I2.I
4.9
7 .6
---
.
.
.
.
.
--
" F o r F l a s k 2, p y r u v a t e c o n c e n t r a t i o n b e c a m e l i m i t i n g . I n r e m a i n i n g f l a s k s ( E x p t . i)
containing malate, phosphate concentration became limiting at the end of incubation. For
f l a s k s w i t h o u t m a l a t e , p y r u v a t e o x i d a t i o n w a s v e r y s l o w a f t e r t h e first 15 m i n o f i n c u b a t i o n .
In another set of experiments, pyruvate or acetate oxidation was measured in
the presence of malonate and small amounts of malate. The sum reactions expected
are
pyruvate
+ m a l a t e + 2 0 2 --> s u e c i n a t e + 3 COs
and
acetate + malate +
I1[2 O , - + s u c c i n a t e + 2 CO S
respectively. Table IV shows that, in these experiments too, the - - A O / - - J s u b s t r a t e
ratios are very near to those expected, with no indication for accumulation of intermediates or for reactions outside the citric acid cycle.
In the experiment shown in Fig. 5, mitochondria were incubated with pyruvate
until the rate of oxygen uptake had nearly ceased. Varying amounts of malate were
Biochim. Biophys. Acta,
96 (1965) 2 1 7 - 2 3 o
224
E . j . DAVIS
TABLE IV
STOICFIEIOMETRY
OF
PYRUVATE
AND
ACETATE
OXIDATION
IN
THE
PRESENCE
OF
MALONATE
AND
MALATE
Each flask contained, in addition to the basic m e d i u m : 5 mM potassiuul p h o s p h a t e buffer (pH 7.4),
I m2vI ATP, 2 mM MgCI 2, 4 ° mM glucose, 15 mM sodium malonate, hexokinasc (side-arm l,
15o units); m a l a t e (side-arm 2, i.o or 1.4 ffmoles) and I . I 5 mg mitochondrial protein in a total
v o l u m e of t.o ml. After a 3-min period of t e m p e r a t u r e equilibration, hexokinase was tipped in.
Then, after an initial small b u r s t of oxygen u p t a k e had subsided, malate was added from sidea r m 2, and O 2 u p t a k e recorded until a s h a r p cut-off was observed 15 to 25 min after addition o f
malate. The exact point of cut-off was obtained by extrapolation. Each value is the m e a n o f
two separate determinations.
Substrate
Malate
(ffmoles)
£10
(#atoms)
(theoretical)
-- zJO
(/*atoms)
(observed)
Pyruvate
(IO mM)
I.O
1. 4
4.0
5.6
3.83
5.34
Acetate
(io mM)
i.o
1. 4
3.0
4.2
2.8 i
4.1o
then added, and the oxygen uptake was recorded for periods up to 15o min. It is of
especial interest that pyruvate oxidation is initially stimulated by all concentrations
of malate which were added, but that the rate of oxygen uptake gradually approaches
the control rate (no malate added) when very low concentrations of malate are added.
I t should also be noted that the increment of increased oxygen uptake is far in excess
of that expected for the oxidation of all the malate present.
o
5
2~z
d
o~
C_'~
C
~T- 2
0
12
24
36
48
60
72
Time (min) a f t e r addition rnotote
84
96
108
120
Fig. 5. The effect of low concentrations of matate on p y r u v a t e oxidation in the presence of 2, 4dinitrophenol. I n c u b a t i o n s were carried o u t in \ V a r b u r g flasks containing the s t a n d a r d incubation m e d i u m plus the following: 5 mM p o t a s s i u m p h o s p h a t e buffer (pH 7.4); i mM A T P ; IO mM
s o d i u m p y r u v a t e and o.56 mg mitochondrial protein in a total volume of i.o ml. Reactions were
initiated b y the addition of o.o 5 mM 2,4-dinitrophenol f r o m a side-arm. Pre-incubations w i t h o u t
m a l a t e were carried o u t for 35 min, followed b y the addition of malate as follows: (I) 2o/2M;
(2) 6o #M; and (3) 20o ffM. I n c u b a t i o n s were s u b s e q u e n t l y carried o u t for an additional 12o min.
E a c h p o i n t represents t h a t increment of 02 u p t a k e per 6-min interval due to the addition of
malate. The total oxygen c o n s u m p t i o n , ill the absence of added malate, during the second interval
was 12. 5 M1, of which io ffl occurred during the first 42 rain of this interval.
Biochim. Biophys. Acta, 96 (1965) 217-23 o
OXIDATION OF ACETATE AND PYRUVATE BY SARCOSOMES
225
Competition between acetate and pyruvate for oxidation in the presence of malate
The locus of the inhibition by acetate of the oxidation of pyruvate in the
intact heart appears to be at the level of the oxidative decarboxylation of pyruvate
rather than its subsequent fate in the citric acid cyclO, 2. It was therefore of interest
to study the competition between acetate and pyruvate in intact mitochondria which
are capable of oxidizing both substrates. It seemed unlikely that phosphate, ADP or
intermediates of the citric acid cycle are limiting factors for oxidation in situ, so that
"State 3" conditions were used throughout in an attempt to simulate conditions
which are believed to exist in the intact heart. Substrates were therefore oxidized in
the presence of malate and (I) phosphate and phosphate acceptor, or (2) 2,4-dinitrophenol and ATP. Table I I I shows that in no case was the rate of oxygen uptake in the
presence of both substrates significantly greater than that of pyruvate alone. It was
pointed out earlier that in some preparations, acetate was poorly oxidized in the
presence of phosphate acceptor when Mg 2+ was also present. The data in Table I I I
(Expts. I and 2) are therefore representative only of those experiments in which acetate alone was rapidly oxidized. Since acetate inhibited pyruvate disappearance by
3o-6o~o (depending on the conditions), it would appear that the two substrates
compete equally for oxidation in the citric acid cycle.
The effects of carnitine and acetyl-carnitine on the oxidation of acetate and pyruvate
Since the original observation by FRITZ18,19 that carnitine stimulates the
oxidation of long-chain f a t t y acids in liver slices and homogenates, a number of
publications have appeared in an attempt to explain the role of carnitine in fatty acid
metabolism. F•ITZ et al. 2° found that low concentrations of carnitine stimulated the
oxidation of long-chain f a t t y acids by heart homogenates, with little or no effect on
the oxidation of short-chain f a t t y acids. BREMEIO observed, however, that in mito-
hfitOAlc~ AC,
'NP
ATP
lpatom
0
\
\
1 mfn
Fig. 6. T h e o x i d a t i o n of a c e t a t e a n d O-acetyl-DL-carnitine in t h e presence of 2,4-dinitrophenol.
T r a c i n g s of Gilson Medical Electronics O x y g r a p h records. R e a c t i o n m e d i a c o n t a i n e d in a d d i t i o n
to t h e basic c o m p o n e n t s , 5 m M p o t a s s i u m p h o s p h a t e buffer (pH 7.4), io m M A T P (second t r a c i n g
only), io m M a c e t a t e (third t r a c i n g only) a n d t h e a d d i t i o n s as i n d i c a t e d : m i t o c h o n d r i a (Mito),
1.o8 m g m i t o c h o n d r i a l p r o t e i n ; O-acetyl-DL-carnitine (AC), io m M ; a c e t a t e (Acet), io raM; DLc a r n i t i n e (C), IO raM; a n d 2,4-dinitrophenol (DNP), 0.05 raM. Volume, 2.0 m | .
Biochim. Biophys. Acta, 96 (1965) 217-23o
226
E.J. DAVIS
chondrial preparations which did not oxidize acetate, acetylcarnitine was rapidly
oxidized, and that the oxidation of pyruvate was stimulated by carnitine. It was
therefore of interest to examine the effects of carnitine on the utilization of acetate
and pyruvate in heart sarcosomes which oxidize free acetate.
Fig. 6 demonstrates the fact that acetate, in the presence of 2,4-dinitrophenol
and ATP, is oxidized at about the same rate as acetylcarnitine. Added ATP is not
required for acetylcarnitine oxidation. A similar result is obtained when acetate or
acetylcarnitine is oxidized in the presence of phosphate acceptor. Neither carnitine
nor acetylcarnitine significantly affected the rate of oxygen consumption by heart
sarcosomes in the presence of acetate.
When pyruvate oxidation was studied, however, carnitine had the following
effects: (I) in the absence of malate, carnitine increased the oxygen uptake due to
pyruvate without affecting its oxidation in the citric acid cycle. In Expt. i of Table
V, the increased oxygen uptake in the presence of carnitine (2.50 m i n u s 1.38 ~ 1.12)
corresponded with an increased pyruvate disappearance (I.6I m i n u s 0.54 = I.O7) of
one molecule of pyruvate per atom of oxygen consumption. In the presence of malate,
carnitine increased the rate of pyruvate disappearance without any significant effect
on the rate of oxygen uptake.
DISCUSSION
To the author's knowledge, there are no reports in the literature that heart
sarcosomes can oxidize acetate or other short-chain fatty acids at a rate compatible
with that known to occur in the intact organ, although PLAUT AND PLAUT21 were able
to obtain low but significant rates of acetate oxidation. However, since pyruvate is
known to be rapidly oxidized by heart mitochondria in the presence of catalytic
amounts of Krebs-cycle intermediates it would be expected that acetate would be
oxidized as well, unless the acetate-activating enzyme (acetyl-CoA synthetase;
acetate:CoA ligase (AMP), EC 6.2.1.1) were inactivated or removed from the Initochondria during isolation.
The results presented in the present paper demonstrate that guinea-pig heart
sarcosomes, under the conditions described, are capable of oxidizing acetate at a very
rapid rate without the addition of any co-factor or intermediate of the citric-acid
cycle. The inhibitory effect of phosphate is not understood*. The fact that the addition
of co-factors (even of Mg 2+) had no stimulatory influence on the oxidation of any of
the various substrates studied indicates comparatively little loss of mitochondrial
enzymes or co-enzymes during preparation. This result is in contrast to a number of
published reports that there is an absolute requirement for Mg2+ by heart mitochondria oxidizing pyruvate 21-2s. Indeed, REISS AND HELLERMAN23 pointed out a
requirement for added CoA and cytochrome c as well.
Another indication of the relative "intactness" of these mitochondria and
possibly an explanation for their unique ability to oxidize acetate is their high degree
* Note added in pro@ VAN DEN BERG87 has recently provided the explanation for inhibition by phosphate of fatty acid utilization by liver mitochondria in the presence of dinitrophenol.
His data, however, do not appear to explain the anomalous effect of phosphate reported ill the
present communication. (Received January 5th, 1965).
Biochim, Biophys. Aeta, 96 (1965) 217-23o
OXIDATION OF ACETATE AND PYRUVATE BY SARCOSOMES
227
of respiratory control when incubated in the absence of added magnesium. There is
loss of most of that control on the addition of Mg 2÷. Corresponding to the loss of
respiratory control in the presence of Mg2~ is a loss of ability to oxidize acetate in
some preparations, while in others Mg 2+ has no effect. However, when sarcosomes
are incubated with acetate in the presence of 2,4-dinitrophenol and ATP, Mg z+ invariably prevents acetate oxidation. I t is clear from the experiments of SEATER AND
HOETON24 that E D T A does not necessarily bind sarcosomal Mg 2+, so it is not surprising that there is no Mg 2+ requirement by s.~rcosomes which have not lost their
Mg~+ during isolation.
In contrast to acetate, pyruvate is not oxidized by the sarcosomes tbr any
length of time in the absence of Krebs-cycle intermediate. In this respect and in the
inhibition by pyruvate of glutamate oxidation the preparation used agreed with
those studied b y SEATER et al.6, ~, who suggested that pyruvate caused depletion of
the sarcosomes from their oxaloacetate due to an acetyl-CoA-catalysed decarboxylation. This explanation is supported by the experiment shown in Fig. 5, in which,
when a very low concentration of malate is added, malate or one of its oxidation
products is depleted during the oxidation of the pyruvate. This result is particularly
TABLE V
THE
EFFECTS
ADDED
OF CARNITINE
ON THE
OXIDATION
OF PYRUVATE
IN THE
PRESENCE
AND
ABSENCE
OF
MALATE
E x p t . I. S t a n d a r d i n c u b a t i o n m i x t u r e plus t h e following: 25 mM p o t a s s i u m p h o s p h a t e buffer
(pH 7.4), i mM ATP, 2 mM MgC12, 4 ° mM glucose, 3 / , m o l e s s o d i u m [2-14C]pyruvate (87o ooo
d i s i n t e g r a t i o n s / m i n ) , a n d 1.36 m g m i t o c h o n d r i a l p r o t e i n in a t o t a l v o l u m e of I.O ml. R e a c t i o n s
were i n i t i a t e d b y t h e a d d i t i o n of h e x o k i n a s e (15o units) from s i d e - a r m i, a n d s t o p p e d a f t e r
60 m i n b y t h e a d d i t i o n of 0.05 ml 2 M HC104 from t h e second s i de -a rm. E x p t . 2. The i n c u b a t i o n
m i x t u r e was t h e s a m e as in E x p t . i, w i t h t h e followi ng e x c e p t i o n s : 5 / z m o l e s s o d i u m [2-14C~p y r u v a t e (1.45. i o e d i s i n t e g r a t i o n s / m i n ) , o.88 m g m i t o c h o n d r i a l p r o t e i n , a n d i . o / z m o l e m a l a t e
per flask, a n d DL-carnitine as i n d i c a t e d . I n c u b a t i o n peri od w a s 20 min. E a c h r e s u l t is t h e m e a n
of t w o s e p a r a t e d e t e r m i n a t i o n s .
Expt. I
Flask Pyruvate
(ltmoles)
Carnitine
(mM)
-- AO
(/,atoms)
z]Pyruvate
([zmoles)
1"C02
(disintegrations]
rain)
i
2
3
4
o
o
io
io
0.35
1.38
0.44
2.50
o
0.54
o
1.6t
-63 200
-55 5 ° o
Expt.
o
3
o
3
2
Flask DL-Carnitine --zlPyruvate Rate of oxygen consumption x4COz
(raM)
(llmoles)
(disintegrations]
(5-Io rain) (z5-2o rain) rain)
(mllatoms/mg proteinlmin )
i
2
3
4
-o.i
I.O
io
1.3o
1.37
1.85
3.09
300
292
29o
274
264
272
27o
272
23
19
21
18
ooo
8oo
35 °
300
Biochim. Biophys. Acta, 96 (1965) 217-23o
228
E.J. DAVIS
striking in view of the fact that both acetate and glutamate are oxidized optimally
at constant rates in the absence of added malate.
Moreover, tile experiment described in Table V, Expt. I, gives good support to
the conclusion that pyruvate oxidation declines owing to lack of acetyl acceptor (i.e.
oxaloacetate) for the acetyl-CoA, since carnitine increases pyruvate oxidation without
affecting CO= production from C-2. Under these conditions, oxidation of pyruvate is
described b y the reactions
pyruvate + CoA + 1/20 2
-+ acetyl-CoA q- CO~
acetyl-CoA + carnitine
~ acetyl-carnitine + CoA
sum: pyruvate + carnitine + 1/2 02 ~ acetyl-carnitine + CO2
(I)
+
(2)
(3)
I f the acetyl-CoA derived from pyruvate is more readily available than acetylCoA from acetate for the enzymes catalysing the decarboxylation of oxaloacetate
(via malonyl-CoA formation6, v) then it might be expected that pyruvate is a more
efficient precursor than acetate for long-chain fatty acids in mitochondria25, 26, since
malonyl-CoA formation is known to be rate-limiting for f a t t y acid synthesis in heart
sarsocomes 25. Preliminary experiments have shown that, under conditions in which
both substrates are rapidly oxidized during the first minutes of incubation, pyruvate
is much more rapidly incorporated into long-chain f a t t y acids than is acetate 27.
Although competition between pyruvate and acetate for oxidation by heart
sarcosomes is demonstrated b y the results shown in Table II, the "preference" for
acetate or other fatty acids which obtains in the perfused heart and in the intact
animal is not s e e n . GARLAND AND RANDLE 2s have shown, in an a t t e m p t to explain
f a t t y acid inhibition of pyruvate oxidation, that the level of acetyl-CoA in perfused
rat hearts is markedly increased in the presence of f a t t y acids, and that partially
purified pyruvate dehydrogenase is inhibited by acetyl-CoA. In support of these
results PEARSON AND TUBBS29 observed a similar effect of acetate,/~-hydroxybutyrate
and butyrate on the level of acetylcarnitine in perfnsed hearts (an equilibrium
would be expected to exist between acetyl-CoA and acetylcarnitine in the presence
of carnitine acetyltransferase (acetyl-CoA:carnitine O-acetyltransferase, EC 2.3.1.7}
which is known to be present in hearta°). The inability to demonstrate a very marked
inhibition of pyruvate utilization by acetate in heart sarcosomes m a y be due to a
partial loss or inactivation of the acetateactivating enzyme during preparation of
the sarcosomes. Although acetate activation is apparently not limiting for its subsequent oxidation, it is quite conceivable that the rate of acetyl-CoA formation in
these sarcosomes is much lower than that obtained in the intact cell. Another consideration for the comparison of the competition between pyruvate and f a t t y acids
in the isolated heart and in heart sareosomes is the likelihood that most of the
pyruvate added to perfused heart preparations is quickly converted to lactate,
so that in effect, a low concentration of pyruvate is competing with a relatively
higher concentration of fatty acid. I f this be the case, the preference for fatty acids
for oxidation m a y be only apparent.
Carnitine increases the rate of oxidation of long-chain f a t t y acids by preparations from a number of tissues18,19. Enzymes have been shown to exist in heart which
catalyse the reversible acetylation and acylation of carnitine from acetyl-CoA 31 and
palmityl-CoA, respectively 32,33. BREMER has shown that 0-acetyl-DL-carnitine 4 and
Biochim. Biophys. Acla, 96 (1965) 217-23o
OXIDATION OF ACETATE AND PYRUVATE BY SARCOSOMES
229
0-palmitoyl-DL-carnitine 31 are rapidly oxidized b y mitochondria from various tissues
which do not oxidize free acetate or palmitate. FRITZ AND YUE 33 have observed that
carnitine markedly stimulates acetyl-CoA oxidation b y heart sarcosomes but does
not significantly increase the oxidation of free acetate. CHAPPELL34 found a requirement for carnitine for the oxidation of palmitoyl-CoA or of palmitate in presence of
CoA + ATP by liver mitochondria. The above authors generally agree that the
formation of acyl-carnitine is a step in the transfer of f a t t y acids to the intramitochondrial site of oxidation.
I t is noteworthy that carnitine was reported to have little or no effect on the
oxidation of short-chain f a t t y acids by heart homogenates TM or sarcosomes 35, while
oxidation of long-chain f a t t y acids was stimulated several fold. BREMER4 found,
however, that pyruvate oxidation was stimulated by carnitine. The carnitine stimulation of pyruvate oxidation was partly accounted for by an increased formation of
acetylcarnitine. The present data, in qualitative agreement with BREMER, demonstrate a stoicheiometric relationship (I :I) between increased pyruvate disappearance
and oxygen consumption, while acetate oxidation is unaffected by carnitine.
HfJLSMANN et al. 36 have recently shown that carnitine stimulates the oxidation
of a-oxoglutarate (in the presence of malonate and acetoacetate or pyruvate), and
have suggested that the stimulatory effect of carnitine is brought about by a release
of CoA (acetylcarnitine formation) for the synthesis of succinyl-CoA, since the supply
of CoA is probably limiting under their conditions. I t seems unlikely, in view of the
present data, that the same situation obtains when the breakdown oi acetyl-CoA is
not made limiting b y the presence of malonate or b y the absence of phosphate acceptor. The results here presented are therefore consistent with the thesis of BREMER31
a n d of FRITZ AND YUE 32 that carnitine is active as a general acyl carrier in the intracellular transport of f a t t y acids. Acetate or pyruvate oxidation, according to this
mechanism, would not be stimulated b y carnitine since both substrates are freely
permeable to the cellular and mitochondrial membranes. Neither does acetyl-CoA
formed within the mitochondrion from acetate or pyruvate require the intervention
of carnitine for transport of active acetyl to its locus for oxidation.
ACKNOWLEDGEMENTS
The author is indebted to Professor E. C. SLATER for m a n y helpful discussions
during the course of the investigation. This investigation was supported in part by a
U.S. Public Health Service Fellowship no. 5-72-HE-I9, 854-o2 from the National
Heart Institute.
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