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The effect of acetate oxidation on the endogenous adenine nucleotides of rat heart mitochondria
BIOCHEMICAL
Vol. 79, No. 4, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
THE EFFECT OF ACETATE OXIDATION ON THE ENECGENOUSADENINE NUCLEOTIDES OF RAT HE4RIMI'IKHONDRIA E. Jack Davis and W. I. A. Davis-van Thienen Indiana University School of Medicine Department of Biochemistry Indianapolis, Indiana 46202 U.S.A. Received
October
28,
1977 SUMMARY
When citrate cycle substrates or glutamate are oxidized by rat heart mitochondria, AMP ccmprises only a few percent of the endogenous pool of adenine nucleotides. however, when acetate is oxidized, greater than half or about 30% of the total mitochondrial pool of adenine nucleotides is converted to AMP in the ADP-stimulated or resting state, respectively. Supporting substrates which form GTP as a result of their oxidation partially, but not canpletely, reverse the accumulation of AMP which results from acetate metabolism.
Recent evidence f&n a number of laboratories nificant
amount of acetate
to be oxidized
ess is analogous to the production oxidation
regard, giving
in peripheral
as free acetate
tissues.
in preference
other short-chain
fatty
large extent within tate activation adenylate
kinase
enous nucleotides
vt
(E.C. 2.7.4.31, of isolated
sion has been shawn to elevate skeletal Copyright AI1 rights
followed
by
end product of its hepatic
metabolism,
in its concentration rapidly
in plasma (5).
oxidize
to some other substMtes (7,8).
matrix.
(9).
the total
for further
of acetate
heart mitochondria AMP level
(6,7),
(along with at least
to a
Since AMP formed due to ace-
is not available the effect
acetate
Acetate
is unique in that it is activated
the mitochondrial
in this
Such a proc-
in this
heart mitochondria
acids)
in certain
Ethanol is a unique substrate
is the principal
Cardiac muscle and isolated
in other organs.
of ketone bodies by the liver,
rise to a pronounced elevation
apparently
suggests that a sig-
may be produced during normal metabolism
organs of the body, subsequently
their
(l-4)
oxidation
was examined.
processing
by
on the endogAcetate perfu-
in perfused heart
(10) and
muscles (11).
0 1977 by Academic of reproduction in any
Press, Inc. form reserved.
1155 ISSN
0006-291
X
BIOCHEMICAL
Vol. 79, No. 4, 1977
The present investigation acetate either respiration,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
shows that when rat heart mitochondria oxidizing
in metabolic State 3 (121, or when ADP is limiting
mitochondrial
AMP acmtes,
the total
mitochondrial
nucleotides.
lized -via
the substrate-linked
for maximum
to account for as much as half of Supporting substrates which are metabo-
phosphorylation
step of succinyl-CoA synthetase
diminished the accumulation of AMP, but did not lower its concentration
to that
found with substrates other than acetate.
Rat heart mitochondria were prepared as previously described (6). The basic incubation medim contained 225 mMsucrose, 10 mMKCl, 10 mMTris HC$4 (pH 7.41, 5 ti potassim phosphate fqH 7.4) and 0.5 ti ATP and 0.5 uC1 (8- ClATP, or 0.5 mMADPplus 0.5 uCi (8- CJADP. Approtitely 0.5 mMMTA (derived from the mitochondrial suspension) was also present. Incubationslbere initiated C-ADP (withwith mitocho@ria and carried out for 6 min., in t@ presence of C-ATP and 0.5 or 1.0 mM out added Mg 1, or for 2 min. in the presence of State 4 (12) was reached beR&12 * In the former case, controlled respiratory fos the reactions were terminated. Respiratory control ratios without added Mg were 8-15 with substrates other than acetate, and 3-6 with acetate plus s&ate. In the latter case, MgCl was used to stimulate respiration through the magnesium-stimulated ATPase p&sent in heart mitochondrial preparations (13). Reactions were terminated by rapid separation of the mitochoridria through silicone oil, followed by column separation of nucleotides (14,15). Respiration was measuredpolarographically. RESlLTSANDDISCUSSION Table I shows the distribution after
of adenine nucleotides obtained in State 4
a period of ADP-stimulated respiration.
high mitochondrial
ATP/ADP ratio,
Glutamate maintained a relatively
and a very low concentration
with acetate as supporting substrate,
the AMP concentration
creased over that with glutamate, representing
of AMP. However,
was 20-fold in-
a 30%decrease in the endogenous
pool of ATP and ADP. Tables II and III
are results of experiments in which respiration
stimulated by MgC12in the presence of ATP. a law AMP concentration, fective
in this respect.
dent frwnlow
ATP inboth
&xoglutarate
oxidation
with pyruvate or acetyl -carnitine
was maintained
being almost as ef-
Malate alone was unable to support respiration intra-
and extramitochondrial
absence of an acceptor of oxaloacetate.
spaces), owingtothe
On the other hand, acetate oxidation
caused an accumulation of more than half of the total
1156
(evi-
mitochondrial
adenine
Vol. 79, No. 4, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
THE EFFECTOF ACETATEOXIDATIONONMYITOCHONDRIALADENINENUC~IDESIN STATE4 AFl'ERA PERIODOF ADP-Sl?IMULATED RESPIRATION Substrate Added Glutamate + Malate Acetate + Malate
AMP (% of total
ADP endogenous
ATP
ATP/ADP (external>
1.5 f 0.5
14.1 + 2.6
84.4 f 3.8
35 2 3
30.2 i: 3.7
11.2 2 1.9
57.9 +- 3.5
30 + 5
The basic mediumcontained in addition 0.5 mMADP, 0.5 Wi (8-14C)ADP, 0.9 mg mitochondrial. pyotein/ml, and where indicated, 10 mMglutamate, 10 mMacetate and 0.5 &i m&&e. Incubation time, 2 min.; Temperature, 30°. Values are means+ S.E.M. of 3 or 4 determinations.
TABLEII THE EFFECTSOF VARIOUSSUBSTRATES ONTHEADENINENUCLEOTIDES OF RAT HEART MITCCHONDRIARESPIRING IN PRESENCE OFMgC12ANDATP Substrate Malate
AMP (% of total
ATP
ATP/ADP Lzxternal)
Zgenousl
9.1
75.5
15.4
0.2
55.5
29.0
15.5
8.5
a-oxoglutarate
3.2
30.5
66.3
19.5
a-oxoglutarate + acetate
25.6
25.9
48.5
23.3
glutamate + acetate
20.6
45.1
34.3
5.2
acetate + mlate + arsenite
19.9
76.5
3.6
0.4
pyruvate + malate
6.8
54.8
38.4
10.0
acetyl-camitine +mlate
8.5
58.0
39.5
3.5
Acetate + Malate
The basic mediumcontained in addition 0.5 mMATP, 0.5 UCi (8-14C> ATP, 0.5 mM MgCl , 0.9 mg mitochondrial protein/ml and other additions as indicated: glut&ate, a-oxoglutarate, acetate, and py-ruvate, 10 mM; (-)acetyl carnitine, 2 r&l; malate, 0.5 mM; and sodiumarsenite, 2 mM. Incubation time, 6 min. Values are the mans of 2 to 4 detenninations.
1157
Vol. 79, No. 4, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE III RATES OF ADP-SUPPURTED AND MgCl -li'JWCED RESPIRATION OF HEART MI'IOCHONDRIA IN PREkNCE OF VARIOUS SUBSTRATES Substrate
Glutamate,
malate
Glutamate, arsenite
malate,
Glutamate,
acetate
Glutamate, arsenite
acetate,
Acetate,
malate
Acetate, arsenite
malate,
a-oxoglutamte,
acetate
a-oxoglutarate,
acetate,
State 3 Respiration
Respiration in Presence of MgC12
natoms/min/mg protein
--o.smM
248
92
194
108
--
---
225
81
162
24
--
V-B
90
81
84
16
--
--
195
92
200
12
-^
me-
arsenite
1.omt-l
Respiration was measured polamgraphically, being stimulated with either ADP orMgC12 after an initial 2 min. preincubationperiod. Dataare fromone mitochondrial PrepaMtion and are typical of others. Incubations contained the basic medium, 0.5 mM ATP and the additions as indicated.
as AMP. Glutamate
nucleotides through their x7ssulting
pathway of oxidation,
frmacetate
An additional which muld
or a-oxoglutarate, only partially
fomtion
experiment
was attempted
last
these conditions, reason for this that mitochondrial
line
the rise
in AMP
and Table III).
is not clear,
ATP is extremely
phosphorylation,
AMP when acetate
Hmever,acetatewas
even though an acetyl-acceptor observation
with the use of arsenite,
of GTP via substrate-linked
might be expected to give rise to even higher (Table II,
prevented
oxidation. control
prevent
which both give rise to GIP
is present
notoxidizedunder
was provided
by malate.
but is perhaps explained
low under these conditions
1158
and
The
by the fact
(Table II),
Vol. 79, No. 4, 1977
BIOCHEMICAL
thereby becoming limiting
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
for acetate
activation.
The data are taken to suggest that an elevated is the case after nucleotide
ethanol
ingestion,
may perturb
pool in cardiac muscle,
via nucleoside
concentration,
the intramitcchondrial
thereby lowering
the total
This would presmably
ADP and perhaps the ATP/ADP ratio. being activated
acetate
(and AMP being produced) more rapidly
as adenine
pool of ATP and
result
fmn acetate
than AMP can be recycled
monophosphate kinase.
Supported by USPHS Grants AA00289 and AM13939, and the Grace M. Showalter Trust. REFERENCES Davis, E. J. (1968) Biochim. Biophys. Acta, 162, l-10. Knowles, S. E., Jamett, I. G., Filsell, 0. H. and Ballard, F. J. (1974) Biochem. J., 142, 401-411. 3. Bernson, F. S.. (1976) Eur. J. B&hem., 67, 403-410. 4. Seufert, C. D., Graf, M., Janson, G., Kuhn, A. and Soling, H. D. (1974) Biochem. Biophys. Res. Cumnm., 57, 901-909. 5. Forsander, 0. A. and Raiha (196OrJ. Biol. Chem., 235, 34-36. 6. Davis, E. J. (1965) Biochim. Biophys. Acta, E, 217-230. 7. Davis, E. J. and Quastel, J. H. (1964) Can. J. Biochem., 4& 1605-1621. 8. Williamson, J. R. (1965) J. Biol. Chem., 240, 2308-2321. 9. Aas, M. (1971) Biochim. Biophys. Acta, 231, 32-47. 10. Fandle, P. J., England, P. J. and Denton, R. M. (1970) Biochem. J., 117, 677-695. Spydevold, 0., Davis, E. J. and Ekmer, J. (1976) Eur. J. Biochem., 11, Il. 155-165. 12. Chance, B. and Williams, G. R. (1956) Adv. &zyml. Relat. Areas Mol Biol., 2, 65-134. 13. Chao, D. L-S. and Davis, E. J. (1972) Biochemistry, 11, 1943-1952. 14. Heldt, H. W., IQinge.nberg, M. and Milovancev, M. (19n) Eur. J. Biochem., 0, 434-440. 15. Davis, E. J. and Lmeng, L. (1975) J. Biol. Chem., 250, 2275-2282. 1.
2.
1159
Vol. 79, No. 4, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
THE EFFECT OF ACETATE OXIDATION ON THE ENECGENOUSADENINE NUCLEOTIDES OF RAT HE4RIMI'IKHONDRIA E. Jack Davis and W. I. A. Davis-van Thienen Indiana University School of Medicine Department of Biochemistry Indianapolis, Indiana 46202 U.S.A. Received
October
28,
1977 SUMMARY
When citrate cycle substrates or glutamate are oxidized by rat heart mitochondria, AMP ccmprises only a few percent of the endogenous pool of adenine nucleotides. however, when acetate is oxidized, greater than half or about 30% of the total mitochondrial pool of adenine nucleotides is converted to AMP in the ADP-stimulated or resting state, respectively. Supporting substrates which form GTP as a result of their oxidation partially, but not canpletely, reverse the accumulation of AMP which results from acetate metabolism.
Recent evidence f&n a number of laboratories nificant
amount of acetate
to be oxidized
ess is analogous to the production oxidation
regard, giving
in peripheral
as free acetate
tissues.
in preference
other short-chain
fatty
large extent within tate activation adenylate
kinase
enous nucleotides
vt
(E.C. 2.7.4.31, of isolated
sion has been shawn to elevate skeletal Copyright AI1 rights
followed
by
end product of its hepatic
metabolism,
in its concentration rapidly
in plasma (5).
oxidize
to some other substMtes (7,8).
matrix.
(9).
the total
for further
of acetate
heart mitochondria AMP level
(6,7),
(along with at least
to a
Since AMP formed due to ace-
is not available the effect
acetate
Acetate
is unique in that it is activated
the mitochondrial
in this
Such a proc-
in this
heart mitochondria
acids)
in certain
Ethanol is a unique substrate
is the principal
Cardiac muscle and isolated
in other organs.
of ketone bodies by the liver,
rise to a pronounced elevation
apparently
suggests that a sig-
may be produced during normal metabolism
organs of the body, subsequently
their
(l-4)
oxidation
was examined.
processing
by
on the endogAcetate perfu-
in perfused heart
(10) and
muscles (11).
0 1977 by Academic of reproduction in any
Press, Inc. form reserved.
1155 ISSN
0006-291
X
BIOCHEMICAL
Vol. 79, No. 4, 1977
The present investigation acetate either respiration,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
shows that when rat heart mitochondria oxidizing
in metabolic State 3 (121, or when ADP is limiting
mitochondrial
AMP acmtes,
the total
mitochondrial
nucleotides.
lized -via
the substrate-linked
for maximum
to account for as much as half of Supporting substrates which are metabo-
phosphorylation
step of succinyl-CoA synthetase
diminished the accumulation of AMP, but did not lower its concentration
to that
found with substrates other than acetate.
Rat heart mitochondria were prepared as previously described (6). The basic incubation medim contained 225 mMsucrose, 10 mMKCl, 10 mMTris HC$4 (pH 7.41, 5 ti potassim phosphate fqH 7.4) and 0.5 ti ATP and 0.5 uC1 (8- ClATP, or 0.5 mMADPplus 0.5 uCi (8- CJADP. Approtitely 0.5 mMMTA (derived from the mitochondrial suspension) was also present. Incubationslbere initiated C-ADP (withwith mitocho@ria and carried out for 6 min., in t@ presence of C-ATP and 0.5 or 1.0 mM out added Mg 1, or for 2 min. in the presence of State 4 (12) was reached beR&12 * In the former case, controlled respiratory fos the reactions were terminated. Respiratory control ratios without added Mg were 8-15 with substrates other than acetate, and 3-6 with acetate plus s&ate. In the latter case, MgCl was used to stimulate respiration through the magnesium-stimulated ATPase p&sent in heart mitochondrial preparations (13). Reactions were terminated by rapid separation of the mitochoridria through silicone oil, followed by column separation of nucleotides (14,15). Respiration was measuredpolarographically. RESlLTSANDDISCUSSION Table I shows the distribution after
of adenine nucleotides obtained in State 4
a period of ADP-stimulated respiration.
high mitochondrial
ATP/ADP ratio,
Glutamate maintained a relatively
and a very low concentration
with acetate as supporting substrate,
the AMP concentration
creased over that with glutamate, representing
of AMP. However,
was 20-fold in-
a 30%decrease in the endogenous
pool of ATP and ADP. Tables II and III
are results of experiments in which respiration
stimulated by MgC12in the presence of ATP. a law AMP concentration, fective
in this respect.
dent frwnlow
ATP inboth
&xoglutarate
oxidation
with pyruvate or acetyl -carnitine
was maintained
being almost as ef-
Malate alone was unable to support respiration intra-
and extramitochondrial
absence of an acceptor of oxaloacetate.
spaces), owingtothe
On the other hand, acetate oxidation
caused an accumulation of more than half of the total
1156
(evi-
mitochondrial
adenine
Vol. 79, No. 4, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
THE EFFECTOF ACETATEOXIDATIONONMYITOCHONDRIALADENINENUC~IDESIN STATE4 AFl'ERA PERIODOF ADP-Sl?IMULATED RESPIRATION Substrate Added Glutamate + Malate Acetate + Malate
AMP (% of total
ADP endogenous
ATP
ATP/ADP (external>
1.5 f 0.5
14.1 + 2.6
84.4 f 3.8
35 2 3
30.2 i: 3.7
11.2 2 1.9
57.9 +- 3.5
30 + 5
The basic mediumcontained in addition 0.5 mMADP, 0.5 Wi (8-14C)ADP, 0.9 mg mitochondrial. pyotein/ml, and where indicated, 10 mMglutamate, 10 mMacetate and 0.5 &i m&&e. Incubation time, 2 min.; Temperature, 30°. Values are means+ S.E.M. of 3 or 4 determinations.
TABLEII THE EFFECTSOF VARIOUSSUBSTRATES ONTHEADENINENUCLEOTIDES OF RAT HEART MITCCHONDRIARESPIRING IN PRESENCE OFMgC12ANDATP Substrate Malate
AMP (% of total
ATP
ATP/ADP Lzxternal)
Zgenousl
9.1
75.5
15.4
0.2
55.5
29.0
15.5
8.5
a-oxoglutarate
3.2
30.5
66.3
19.5
a-oxoglutarate + acetate
25.6
25.9
48.5
23.3
glutamate + acetate
20.6
45.1
34.3
5.2
acetate + mlate + arsenite
19.9
76.5
3.6
0.4
pyruvate + malate
6.8
54.8
38.4
10.0
acetyl-camitine +mlate
8.5
58.0
39.5
3.5
Acetate + Malate
The basic mediumcontained in addition 0.5 mMATP, 0.5 UCi (8-14C> ATP, 0.5 mM MgCl , 0.9 mg mitochondrial protein/ml and other additions as indicated: glut&ate, a-oxoglutarate, acetate, and py-ruvate, 10 mM; (-)acetyl carnitine, 2 r&l; malate, 0.5 mM; and sodiumarsenite, 2 mM. Incubation time, 6 min. Values are the mans of 2 to 4 detenninations.
1157
Vol. 79, No. 4, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE III RATES OF ADP-SUPPURTED AND MgCl -li'JWCED RESPIRATION OF HEART MI'IOCHONDRIA IN PREkNCE OF VARIOUS SUBSTRATES Substrate
Glutamate,
malate
Glutamate, arsenite
malate,
Glutamate,
acetate
Glutamate, arsenite
acetate,
Acetate,
malate
Acetate, arsenite
malate,
a-oxoglutamte,
acetate
a-oxoglutarate,
acetate,
State 3 Respiration
Respiration in Presence of MgC12
natoms/min/mg protein
--o.smM
248
92
194
108
--
---
225
81
162
24
--
V-B
90
81
84
16
--
--
195
92
200
12
-^
me-
arsenite
1.omt-l
Respiration was measured polamgraphically, being stimulated with either ADP orMgC12 after an initial 2 min. preincubationperiod. Dataare fromone mitochondrial PrepaMtion and are typical of others. Incubations contained the basic medium, 0.5 mM ATP and the additions as indicated.
as AMP. Glutamate
nucleotides through their x7ssulting
pathway of oxidation,
frmacetate
An additional which muld
or a-oxoglutarate, only partially
fomtion
experiment
was attempted
last
these conditions, reason for this that mitochondrial
line
the rise
in AMP
and Table III).
is not clear,
ATP is extremely
phosphorylation,
AMP when acetate
Hmever,acetatewas
even though an acetyl-acceptor observation
with the use of arsenite,
of GTP via substrate-linked
might be expected to give rise to even higher (Table II,
prevented
oxidation. control
prevent
which both give rise to GIP
is present
notoxidizedunder
was provided
by malate.
but is perhaps explained
low under these conditions
1158
and
The
by the fact
(Table II),
Vol. 79, No. 4, 1977
BIOCHEMICAL
thereby becoming limiting
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
for acetate
activation.
The data are taken to suggest that an elevated is the case after nucleotide
ethanol
ingestion,
may perturb
pool in cardiac muscle,
via nucleoside
concentration,
the intramitcchondrial
thereby lowering
the total
This would presmably
ADP and perhaps the ATP/ADP ratio. being activated
acetate
(and AMP being produced) more rapidly
as adenine
pool of ATP and
result
fmn acetate
than AMP can be recycled
monophosphate kinase.
Supported by USPHS Grants AA00289 and AM13939, and the Grace M. Showalter Trust. REFERENCES Davis, E. J. (1968) Biochim. Biophys. Acta, 162, l-10. Knowles, S. E., Jamett, I. G., Filsell, 0. H. and Ballard, F. J. (1974) Biochem. J., 142, 401-411. 3. Bernson, F. S.. (1976) Eur. J. B&hem., 67, 403-410. 4. Seufert, C. D., Graf, M., Janson, G., Kuhn, A. and Soling, H. D. (1974) Biochem. Biophys. Res. Cumnm., 57, 901-909. 5. Forsander, 0. A. and Raiha (196OrJ. Biol. Chem., 235, 34-36. 6. Davis, E. J. (1965) Biochim. Biophys. Acta, E, 217-230. 7. Davis, E. J. and Quastel, J. H. (1964) Can. J. Biochem., 4& 1605-1621. 8. Williamson, J. R. (1965) J. Biol. Chem., 240, 2308-2321. 9. Aas, M. (1971) Biochim. Biophys. Acta, 231, 32-47. 10. Fandle, P. J., England, P. J. and Denton, R. M. (1970) Biochem. J., 117, 677-695. Spydevold, 0., Davis, E. J. and Ekmer, J. (1976) Eur. J. Biochem., 11, Il. 155-165. 12. Chance, B. and Williams, G. R. (1956) Adv. &zyml. Relat. Areas Mol Biol., 2, 65-134. 13. Chao, D. L-S. and Davis, E. J. (1972) Biochemistry, 11, 1943-1952. 14. Heldt, H. W., IQinge.nberg, M. and Milovancev, M. (19n) Eur. J. Biochem., 0, 434-440. 15. Davis, E. J. and Lmeng, L. (1975) J. Biol. Chem., 250, 2275-2282. 1.
2.
1159