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Carnitine-coenzyme A transacetylase in mitochondria from various organs
SHORT COMMUNICATIONS
205
sc 53oo4
Carnitine-coenzyme A transacetylase in mitochondria from various organs The group transfer of acetyl from CoASH to carnitine, as first described by FRIEDMAN AND FRAENKEL 1 in tissue extracts prompted the characterization of the carnitine transacetylase (acetyl-CoA:carnitine 0-acetyltransferase, EC 2.3.1.7) catalysing the following reaction: acetylcarnitine + C o A S H - + carnitine + CoASAc
(i)
Thus a participation of carnitine in metabolism by forming another type of"activated" acetic acid was directly demonstrated. On the other hand carnitine had been shown to be a cofactor in fatty acid oxidationS, 8. The association of the carnitine transacetylase with fatty acid oxidation was substantiated by the finding that the carnitine esters of fatty acids are highly efficient substrates for various mitochondria 4. An obligatory requirement for carnitine was found for the oxidation of fatty acids by mitochondria from skeletal muscles 4, and the flight muscles 5 of the locust. It can be assumed that the carnitine esters are oxidized only after a transacylation to the CoASH derivatives of the fatty acids. In order to establish further the role of carnitine in fatty acid oxidation, and its probable specificity to certain organs, the activity of carnitine transacetylase in mitochondria from different organs was examined. A combined assay on carnitine transacetylase based on the following reactions was worked out: acetylcarnitine + CoASH
carnitine ~ransacetylase > carnitine + CoASAc
CoASAc + oxaloacetate malate + D P N +
citrate s y n t h a s e ~ citrate + CoASH
malate dehydrogenase ~ oxaloacetate + D P N H + H +
(I) (2) (3)
Thus, by the combined addition of citrate synthase (EC 4.1.3.7) and malate dehydrogenase (EC 1.1.1.37) as well as of CoASH, D P N H is formed in equivalent amounts to the acetylcarnitine transacetylated in Reaction (i). The test mixture for the estimation of transacetylase activity had the following composition : 0.2 M Tris buffer (pH 8.o), 0. 5 mM EDTA, 5 mM DPN, 50 mM L-malate, malate dehydrogenase (20 units) (Boehringer), citrate synthase (0.003 ml, specific activity, 31.2), 0.3 mM CoASH, 2 mM acetylcarnitine. The absorption changes due to the formation of D P N H were recorded at 366 m#. The mitochondria were prepared according to the procedure described elsewhere6,L Transacetylase was extracted from the mitochondria with o.I M phosphate buffer (pH 7.2) after disruption with the ultraturrax desintegrator (Janke and Kunkel). The enzyme was extracted thus easily into the supernatant. Repeated extraction 8 of both the heart muscle of the rat (Table I) and of the flight muscle of the locust has demonstrated that the transacetylase activity is localized exclusively in the "soluble" fraction of the mitochondria. The measurement of the transacetylase activity shows that, only after the addition of acetylcarnitine to the extract, containing all additions, D P N H is generated at a linear rate, which Biochim. Biophys. Acta, 84 (1964) 205-207
206
SHORT COMMUNICATIONS TABLE
I
REPEATED EXTRACTION OF THE HEART MUSCLE OF THE RAT
Treatment with
Isotonic sucrose I Isotonic sucrose I I o . i M p h o s p h a t e buffer Disintegration in o . I M phosphate buffer
Enzyme activity in
#moles/h per g fresh weight
supernatant ( i o o ooo × g) supernatant (ioo ooo × g) supernatant ( i o o ooo × g)
24
supernatant (IOO ooo × g) resuspended s e d i m e n t
625 7
6 [
reflects the activity of the carnitine transacetylase. On a further addition of butyryl-, decanoyl-, or palmitylcarnitine the reaction is largely inhibited. This is interpreted as demonstrating that these acylcarnitines can effectively compete with acetylcarnitine for the transacetylase reaction. It m a y be concluded therefore that the same enzyme assayed for the transfer of the acetyl group from carnitine to CoASH can also transfer acyl groups of medium and long carbon chains. The activity of the carnitine transacetylase, measured in mitochondria of various TABLE
II
THE ACTIVITY OF CARNITINE TRANSACETYLASE WITH ACETYLCARNITINE AS SUBSTRATE IN MITOCHONDRIA
Organ
#moles/h per g
of protein
Rat:
Liver Kidney Skeletal muscle H e a r t muscle
57 850 79oo 65oo
Pigeon:
Breast muscle Heart muscle
1385o 555 °
Locust: Flight muscle Bee: Flight muscle*
515 ° o
* Measured after repeated extraction in the various extracts using the treatments described in Table I.
organs, is summarized in Table II. The transacetylase activity shows marked differences between the mitochondria from various organs. High activity is found in the mitochondria from the muscles of the rat and the pigeon, low activity in the mitochondria from kidney, and particularly from liver, of the rat. This is in accordance with the requirement of the carnitine esters for fatty acid oxidation 4: Only mitochondria from kidney, and in particular from liver, are able to oxidize short-chain, as well as Biochim. Biophys. Acta, 84 (1964) 2 o 5 - 2 o 7
207
SHORT COMMUNICATIONS
long-chain, free fatty acids without the addition of carnitine. Mitochondria from heart muscle and in particular from rat-skeletal and pigeon-breast muscle are largely or absolutely (in the latter cases) dependent on the supply of carnitine. Particularly striking are the differences in the transacetylase activity between the muscles of two insects which are known for their ability to burn fatty acids (locust) or only carbohydrates (bee) in flight. No trace of a transacetylase activity could be detected in extracts of the flight muscle of the bee. The exclusive localization of the carnitine transacetylase in the mitochondria is in contrast to its suggested roleg, 1° in the transfer of "active acetyl" from the intrato the extra-mitochondrial space in the cell. However, the assumption that the transacetylase is related to the role of carnitine in fatty acid oxidation is further supported. Thus the enzyme appears to be specifically active also for medium- and longchain fatty acids although tested here for convenience with the aeetylcarnitine ester. In this context a comparison of various tissues is of special interest. The transacetylase activity varies considerably between the mitochondria from tissues which have an equally high capacity for fatty acid oxidation, for example between skeletal muscle and liver of rat. Thus the carnitine transacetylase is not an obligatory enzyme of the fatty acid oxidation system. The transacetylase activity reflects qualities in the mitochondria which are specific to the tissue and which are superimposed on the basic metabolic pattern. Assuming that carnitine may flmction in the transport of fatty acids in the mitochondria, the distribution of the transacetylase may reflect certain differences in the organization of basic metabolic pathways for example between the mitochondria of liver and of muscle which are also suggested on other grounds. We are very indebted to Mr. C. BODE for providing the carnitine esters of fatty acids and to Dr. EGGERER for his generous gift of citrate synthase. One of the authors (A. M. TH. B.) wants to express his thanks to the Alexander von Humboldt-Stiffung for having granted him a stipend. This work was supported by grants from the Deutsche Forschungsgemeinschaft.
Zoological Institute, N ij megen (Netherlands) Physiologisch-Chemisches Institut, Marburg /Lahn (Germany)
A. M. TH. BEENAKKERS M. KLINGENBERG
1 S. FRIEDMAN AND G. FRAENKEL, Arch. Biochem. Biophys., 59 (1955) 491. J. B. FRITZ, Am. J. Physiol., 197 (1959) 297. 3 j . B. FRITZ AND B. McEwEN, Science, 129 (1959) 334. 4 C. BODE AND M. KLINGENBERG, Bioehim. Biophys. Aeta, in the press. 5 A. M. TH. BEENAKKERS, in preparation. e M. KLINGENBERG, W. SLENCZKA AND E. RITT, Bioehem. Z., 332 (1959) 47v M. KLINGENBERG AND P. SCHOLLMEYER, Biochem. Z., 333 (196o) 335. 8 D. PETTE, R. W. BROSEMER AND W. VOGELL, Biochem. Z., s u b m i t t e d for publication. 9 j . BREMER, J. Biol. Chem., 237 (1962) 2228. 10 j. BREMER, J. Biol. Chem., 237 (1962) 3628.
Received November ISt, 1963 Biochim. Biophys. Aaa, 84 (1964) 205-207
205
sc 53oo4
Carnitine-coenzyme A transacetylase in mitochondria from various organs The group transfer of acetyl from CoASH to carnitine, as first described by FRIEDMAN AND FRAENKEL 1 in tissue extracts prompted the characterization of the carnitine transacetylase (acetyl-CoA:carnitine 0-acetyltransferase, EC 2.3.1.7) catalysing the following reaction: acetylcarnitine + C o A S H - + carnitine + CoASAc
(i)
Thus a participation of carnitine in metabolism by forming another type of"activated" acetic acid was directly demonstrated. On the other hand carnitine had been shown to be a cofactor in fatty acid oxidationS, 8. The association of the carnitine transacetylase with fatty acid oxidation was substantiated by the finding that the carnitine esters of fatty acids are highly efficient substrates for various mitochondria 4. An obligatory requirement for carnitine was found for the oxidation of fatty acids by mitochondria from skeletal muscles 4, and the flight muscles 5 of the locust. It can be assumed that the carnitine esters are oxidized only after a transacylation to the CoASH derivatives of the fatty acids. In order to establish further the role of carnitine in fatty acid oxidation, and its probable specificity to certain organs, the activity of carnitine transacetylase in mitochondria from different organs was examined. A combined assay on carnitine transacetylase based on the following reactions was worked out: acetylcarnitine + CoASH
carnitine ~ransacetylase > carnitine + CoASAc
CoASAc + oxaloacetate malate + D P N +
citrate s y n t h a s e ~ citrate + CoASH
malate dehydrogenase ~ oxaloacetate + D P N H + H +
(I) (2) (3)
Thus, by the combined addition of citrate synthase (EC 4.1.3.7) and malate dehydrogenase (EC 1.1.1.37) as well as of CoASH, D P N H is formed in equivalent amounts to the acetylcarnitine transacetylated in Reaction (i). The test mixture for the estimation of transacetylase activity had the following composition : 0.2 M Tris buffer (pH 8.o), 0. 5 mM EDTA, 5 mM DPN, 50 mM L-malate, malate dehydrogenase (20 units) (Boehringer), citrate synthase (0.003 ml, specific activity, 31.2), 0.3 mM CoASH, 2 mM acetylcarnitine. The absorption changes due to the formation of D P N H were recorded at 366 m#. The mitochondria were prepared according to the procedure described elsewhere6,L Transacetylase was extracted from the mitochondria with o.I M phosphate buffer (pH 7.2) after disruption with the ultraturrax desintegrator (Janke and Kunkel). The enzyme was extracted thus easily into the supernatant. Repeated extraction 8 of both the heart muscle of the rat (Table I) and of the flight muscle of the locust has demonstrated that the transacetylase activity is localized exclusively in the "soluble" fraction of the mitochondria. The measurement of the transacetylase activity shows that, only after the addition of acetylcarnitine to the extract, containing all additions, D P N H is generated at a linear rate, which Biochim. Biophys. Acta, 84 (1964) 205-207
206
SHORT COMMUNICATIONS TABLE
I
REPEATED EXTRACTION OF THE HEART MUSCLE OF THE RAT
Treatment with
Isotonic sucrose I Isotonic sucrose I I o . i M p h o s p h a t e buffer Disintegration in o . I M phosphate buffer
Enzyme activity in
#moles/h per g fresh weight
supernatant ( i o o ooo × g) supernatant (ioo ooo × g) supernatant ( i o o ooo × g)
24
supernatant (IOO ooo × g) resuspended s e d i m e n t
625 7
6 [
reflects the activity of the carnitine transacetylase. On a further addition of butyryl-, decanoyl-, or palmitylcarnitine the reaction is largely inhibited. This is interpreted as demonstrating that these acylcarnitines can effectively compete with acetylcarnitine for the transacetylase reaction. It m a y be concluded therefore that the same enzyme assayed for the transfer of the acetyl group from carnitine to CoASH can also transfer acyl groups of medium and long carbon chains. The activity of the carnitine transacetylase, measured in mitochondria of various TABLE
II
THE ACTIVITY OF CARNITINE TRANSACETYLASE WITH ACETYLCARNITINE AS SUBSTRATE IN MITOCHONDRIA
Organ
#moles/h per g
of protein
Rat:
Liver Kidney Skeletal muscle H e a r t muscle
57 850 79oo 65oo
Pigeon:
Breast muscle Heart muscle
1385o 555 °
Locust: Flight muscle Bee: Flight muscle*
515 ° o
* Measured after repeated extraction in the various extracts using the treatments described in Table I.
organs, is summarized in Table II. The transacetylase activity shows marked differences between the mitochondria from various organs. High activity is found in the mitochondria from the muscles of the rat and the pigeon, low activity in the mitochondria from kidney, and particularly from liver, of the rat. This is in accordance with the requirement of the carnitine esters for fatty acid oxidation 4: Only mitochondria from kidney, and in particular from liver, are able to oxidize short-chain, as well as Biochim. Biophys. Acta, 84 (1964) 2 o 5 - 2 o 7
207
SHORT COMMUNICATIONS
long-chain, free fatty acids without the addition of carnitine. Mitochondria from heart muscle and in particular from rat-skeletal and pigeon-breast muscle are largely or absolutely (in the latter cases) dependent on the supply of carnitine. Particularly striking are the differences in the transacetylase activity between the muscles of two insects which are known for their ability to burn fatty acids (locust) or only carbohydrates (bee) in flight. No trace of a transacetylase activity could be detected in extracts of the flight muscle of the bee. The exclusive localization of the carnitine transacetylase in the mitochondria is in contrast to its suggested roleg, 1° in the transfer of "active acetyl" from the intrato the extra-mitochondrial space in the cell. However, the assumption that the transacetylase is related to the role of carnitine in fatty acid oxidation is further supported. Thus the enzyme appears to be specifically active also for medium- and longchain fatty acids although tested here for convenience with the aeetylcarnitine ester. In this context a comparison of various tissues is of special interest. The transacetylase activity varies considerably between the mitochondria from tissues which have an equally high capacity for fatty acid oxidation, for example between skeletal muscle and liver of rat. Thus the carnitine transacetylase is not an obligatory enzyme of the fatty acid oxidation system. The transacetylase activity reflects qualities in the mitochondria which are specific to the tissue and which are superimposed on the basic metabolic pattern. Assuming that carnitine may flmction in the transport of fatty acids in the mitochondria, the distribution of the transacetylase may reflect certain differences in the organization of basic metabolic pathways for example between the mitochondria of liver and of muscle which are also suggested on other grounds. We are very indebted to Mr. C. BODE for providing the carnitine esters of fatty acids and to Dr. EGGERER for his generous gift of citrate synthase. One of the authors (A. M. TH. B.) wants to express his thanks to the Alexander von Humboldt-Stiffung for having granted him a stipend. This work was supported by grants from the Deutsche Forschungsgemeinschaft.
Zoological Institute, N ij megen (Netherlands) Physiologisch-Chemisches Institut, Marburg /Lahn (Germany)
A. M. TH. BEENAKKERS M. KLINGENBERG
1 S. FRIEDMAN AND G. FRAENKEL, Arch. Biochem. Biophys., 59 (1955) 491. J. B. FRITZ, Am. J. Physiol., 197 (1959) 297. 3 j . B. FRITZ AND B. McEwEN, Science, 129 (1959) 334. 4 C. BODE AND M. KLINGENBERG, Bioehim. Biophys. Aeta, in the press. 5 A. M. TH. BEENAKKERS, in preparation. e M. KLINGENBERG, W. SLENCZKA AND E. RITT, Bioehem. Z., 332 (1959) 47v M. KLINGENBERG AND P. SCHOLLMEYER, Biochem. Z., 333 (196o) 335. 8 D. PETTE, R. W. BROSEMER AND W. VOGELL, Biochem. Z., s u b m i t t e d for publication. 9 j . BREMER, J. Biol. Chem., 237 (1962) 2228. 10 j. BREMER, J. Biol. Chem., 237 (1962) 3628.
Received November ISt, 1963 Biochim. Biophys. Aaa, 84 (1964) 205-207