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The distribution of palmityl-CoA: Carnitine palmityltransferase in the animal kingdom
Comp. Biochem. Physiol., 1966. Vol. 19, pp. 483 to 487. Pergamon Press Ltd. Printed in Great Britain
SHORT COMMUNICATION THE DISTRIBUTION OF PALMITYL-CoA: CARNITINE PALMITYLTRANSFERASE IN THE ANIMAL KINGDOM* KAARE R. N O R U M and JON BREMER Biological Station of the University of Oslo, Drobak, Norway, and Institute of Clinical Biochemistry, University of Oslo, Rikshospitalet, Oslo, Norway (Received 27 M a y
1966)
A b s t r a c t - - 1 . The distribution of the enzyme carnitine palmityltransferase (EC 2.3.1...) in a variety of animal organisms, representing the main phyla of the animal kingdom, has been studied. 2. The enzyme was found in animals from all the phyla tested. 3. The concentration of the enzyme seems to be correlated to tile metabolic activity of the animal. 4. It is suggested that the distribution and concentration of the carnitine palmityltransferase in animals may reflect their (organ) ability to oxidize fatty acids. INTRODUCTION
CARNITINE(fl-hydroxy-butyrobetaine) has a wide distribution in animals (Fraenkel, 1954). The highest concentrations of the compound are found in mammalian heart and skeletal muscles (Broekhuysen et al., 1965). It is now well established that carnitine plays a major role in the transport of activated fatty acids into the mitochondria, where the fl-oxidation of the fatty acids takes place (Bremer, 1962; Fritz, 1963). The activated fatty acids are most probably carried through the mitochondrial membranes as acylcarnitines. The acylcarnitines are formed according to the reaction: Acyl-CoA + carnitine ~-acylcarnitine + CoA This reaction is catalyzed by two different enzymes. The carnitine acetyltransferase (EC 2.3.1.7) is active in the transfer of short-chain fatty acids (Fritz et al., 1963), and the carnitine palmityltransferase (EC 2.3.1...) catalyzes the transfer of long-chain fatty acids (Norum, 1964). We have recently studied the distribution of carnitine palmityltransferase in human organs and tissues. The concentrations of the enzyme in the different organs and tissues seem to be related to the capacity for fatty-acid oxidation in the organ or tissue (Norum, 1966). * This study was supported by grants from Nicolai Andresen's Fund for biological research. 483
484
KAARE R. NORUM AND JON BREMER
In the present communication we report the distribution of the carnitine palmityltransferase in a variety of animal organisms, representing the main phyla of the animal kingdom. MATERIALS AND METHODS The animals were collected at the Biological Station of the University of Oslo, Drobak, during the summer of 1965. The animals or their organs were frozen and stored at - 2 0 ° C until used. The carnitine palmityltransferase is stable for several months at this temperature. The animal material was, after thawing, cut into small pieces and homogenized in a Potter-Elvehjem homogenizer with a tight-fitting Teflon pestle in 1-4 vol. of 0.25 M sucrose. As the aim of the present investigation was to study the distribution of the carnitine palmityltransferase in a variety of animals, it was considered more important to deal with a great number of organisms, rather than to base each determination on a large number of individual assays. All the figures listed, however, represent the average of at least two tests. The activity of the carnitine palmityltransferase was assayed be means of an isotope-exchange assay as previously described (Norum, 1964). The results are presented as specific activity of the enzyme, i.e. the activity, in arbitrary units (Norum, 1964), per mg of nitrogen. The nitrogen was determined by a microKjeldahl procedure. RESULTS AND DISCUSSION Table 1 shows that the carnitine palmityltransferase was found in most of the animals tested, although in barely detectable amounts in many tissues of the lower species. The tissues of the higher animals had the highest activity of the enzyme. However, there seems to be no strict correlation between the development of the animal and the activity of the carnitine palmityltransferase. More probably there is a correlation between the metabolic activity of the animal or the animal tissue and the activity of the enzyme. It is interesting to note that the activity of the enzyme is relatively high in the female gonads, and that the activity increases with the development and the fertilizability of the eggs. This may reflect the ability of the fertilized egg to utilize stored lipids for its development. We have tested the carnitine palmityltransferase activity in two strains of Escherichia coli, one strain of Bacillus anitratum and one strain of Candida albicans. No enzyme activity could be found in these micro-organisms. The number of micro-organisms tested is too low to permit any conclusion to be drawn. Carnitine has been found in Streptococcus haemolyticus and brewer's yeast (Fraenkel, 1953). Fungi also contain mitochondria and the carnitine palmityltransferase has been shown to be a true mitochondrial enzyme (Norum & Bremer, 1966). One would expect therefore that the varnitine palmityltransferase is only present in microorganisms containing both carnitine and mitochondria.
THE D I S T R I B U T I O N OF CARNITINE PALMITYLTRANSFERASE T A B L E 1 - - D I S T R I B U T I O N OF CARNITINE PALMITYLTRANSFERASE I N ANIMALS AND ANIMAL TISSUES
Animal tested Porifera Halichondria panicea Coelenterata Scyphozoa Aurelia aurita Actinozoa Metridium senile Platyhelminthes App. Nemertini Ignot. sp. Annelida Chaetopoda Nerds sp. Arthropoda Crustacea Homarus vulgaris Homarus vulgaris Homarus vulgaris Homarus vulgaris Carcinus maenas Carcinus maenas Insecta Apis mellifica Mollusca Gastropoda Buccinum undatum Buccinum undatum Buccinum undatum Bivalvia(Pelecypoda) Myt~se~ Myt~se~ Mytilus edulis Modiolus modiolus Echinodermata Asteroidea Asterias rubens Echinoidea Psammechinus miliaris Psammechinus miliaris Psammechinus miliaris
Tissue used
Specific activity of carnitine palmityltransferase (arbitrary units)
Whole animal
0"18
Whole animal
0-5
Whole animal
Trace
Whole animal
0"28
Whole animal
Trace
Heart Muscle Liver (hepato-pancreas) Gonad Muscle Gonad ? Whole animal
Liver (hepato-pancreas) Foot Whole animal (without liver and foot)
5"26 0"1 7"45 1 "03 0"81 8"3 11"9
0 0"12 0"23
Whole animal Liver (hepato-pancreas) Adductor muscle Gonad $
0-21
Internal organs
0"1
Eggs, non-fertile Eggs, fertile Sperms, fertile
0-7 3"5 0"27
0 0 2"04
485
486
KAARE R. NORUM AND JON BRE~VIEE TABLE 1--(Continued)
Animal tested Echinodermata (cont.) Holothuroidea Stichopus tremulus Stichopus tremulus Stichopus tremulus Chordata Tunicata Ascidia mentula Ascidia mentula Cyclostomata Myxine glutinosa Myxine glutinosa Myxine glutinosa Myxine glutinosa Myxine glutinosa Pisces Gadus morrhua Gadus morrhua Gadus morrhua Gadus morrhua Gadus morrhua Gadus morrhua Gadus morrhua Amphibia Bufo vulgaris Bufo vulgaris Mammalia Rattus norvegicus Rattus norvegicus
Tissue used
Specific activity of carnitine palmityltransferase (arbitrary units)
Gonad ~ Intestine Muscle
Trace 0 Trace
Intestine Whole animal (without intestine)
Trace
Muscle Heart Liver Intestine Eggs, non-fertile
0 5'8 18"5 3"6 0'21
Muscle Heart Liver Kidney Spleen Intestine Gonad ?
2"5 19"6 33-1 11 '3 4-8 0-77 6-9
Heart Liver
42'0 60-0
Heart Liver
72-0 52-0
0
I n human organs the highest concentrations of carnitine palmityltransferase are found in those organs which have the highest rate of fatty-acid oxidation (Norum, 1966). It may therefore tentatively be suggested that the distribution of the earnitine palmityltransferase in animals reflects their (organ) ability to oxidize fatty acids. REFERENCES BREMER J. (1962) Carnitine in intermediary metabolism. The metabolism of fatty acid esters of carnitine by mitochondria, ft. biol. Chem. 237, 3628-3632. BROEmtUYSEN J., ROZENBLUMC., GrtXSLAIN M. & DELTOUR G. (1965) Distribution of carnitine in the rat. In Recent Research on Carnitine, pp. 23-25. M.I.T. Press, Cambridge, Mass.
THE
DISTRIBUTION
OF
CARNITINE
PALMITYLTRANSFERASE
487
FRAENKEL G. (1953) Studies on the distribution of vitamin BT (carnitine). Biol. Bull., Woods Hole 104, 359-371. FRAENKEL G. (1954) T h e distribution of vitamin B T (carnitine) throughout the animal kingdom. Archs Biochem. Biophys. 50, 486-495. FRITZ I. B. (1963) Carnitine and its role in the fatty acid metabolism. Advances in Lipid Research, Vol. 1, pp. 285-334. Academic Press, New York. FRITZ I. B., SCHULTZ S. K. • SRERE P. A. (1963) Properties of partially purified carnitine acetyltransferase. J. biol. Chem. 238, 2509-2517. NORUM K. R. (1964) Palmityl-CoA: carnitine palmityltransferase. Purification from calf liver mitochondria and some properties of the enzyme. Biochirn. biophys. Acta 89, 95108. NORUM K. R. (1966) T h e organ and subcellular distribution of palmityl-CoA: carnitine palmityltransferase in man. Acta physiol. Scand. 66, 172-181. NORUM K. R. & BREMER J. (1966) T h e subcellular distribution of acyl-CoA: camitine acyltransferases in rat liver. Submitted for publication.
SHORT COMMUNICATION THE DISTRIBUTION OF PALMITYL-CoA: CARNITINE PALMITYLTRANSFERASE IN THE ANIMAL KINGDOM* KAARE R. N O R U M and JON BREMER Biological Station of the University of Oslo, Drobak, Norway, and Institute of Clinical Biochemistry, University of Oslo, Rikshospitalet, Oslo, Norway (Received 27 M a y
1966)
A b s t r a c t - - 1 . The distribution of the enzyme carnitine palmityltransferase (EC 2.3.1...) in a variety of animal organisms, representing the main phyla of the animal kingdom, has been studied. 2. The enzyme was found in animals from all the phyla tested. 3. The concentration of the enzyme seems to be correlated to tile metabolic activity of the animal. 4. It is suggested that the distribution and concentration of the carnitine palmityltransferase in animals may reflect their (organ) ability to oxidize fatty acids. INTRODUCTION
CARNITINE(fl-hydroxy-butyrobetaine) has a wide distribution in animals (Fraenkel, 1954). The highest concentrations of the compound are found in mammalian heart and skeletal muscles (Broekhuysen et al., 1965). It is now well established that carnitine plays a major role in the transport of activated fatty acids into the mitochondria, where the fl-oxidation of the fatty acids takes place (Bremer, 1962; Fritz, 1963). The activated fatty acids are most probably carried through the mitochondrial membranes as acylcarnitines. The acylcarnitines are formed according to the reaction: Acyl-CoA + carnitine ~-acylcarnitine + CoA This reaction is catalyzed by two different enzymes. The carnitine acetyltransferase (EC 2.3.1.7) is active in the transfer of short-chain fatty acids (Fritz et al., 1963), and the carnitine palmityltransferase (EC 2.3.1...) catalyzes the transfer of long-chain fatty acids (Norum, 1964). We have recently studied the distribution of carnitine palmityltransferase in human organs and tissues. The concentrations of the enzyme in the different organs and tissues seem to be related to the capacity for fatty-acid oxidation in the organ or tissue (Norum, 1966). * This study was supported by grants from Nicolai Andresen's Fund for biological research. 483
484
KAARE R. NORUM AND JON BREMER
In the present communication we report the distribution of the carnitine palmityltransferase in a variety of animal organisms, representing the main phyla of the animal kingdom. MATERIALS AND METHODS The animals were collected at the Biological Station of the University of Oslo, Drobak, during the summer of 1965. The animals or their organs were frozen and stored at - 2 0 ° C until used. The carnitine palmityltransferase is stable for several months at this temperature. The animal material was, after thawing, cut into small pieces and homogenized in a Potter-Elvehjem homogenizer with a tight-fitting Teflon pestle in 1-4 vol. of 0.25 M sucrose. As the aim of the present investigation was to study the distribution of the carnitine palmityltransferase in a variety of animals, it was considered more important to deal with a great number of organisms, rather than to base each determination on a large number of individual assays. All the figures listed, however, represent the average of at least two tests. The activity of the carnitine palmityltransferase was assayed be means of an isotope-exchange assay as previously described (Norum, 1964). The results are presented as specific activity of the enzyme, i.e. the activity, in arbitrary units (Norum, 1964), per mg of nitrogen. The nitrogen was determined by a microKjeldahl procedure. RESULTS AND DISCUSSION Table 1 shows that the carnitine palmityltransferase was found in most of the animals tested, although in barely detectable amounts in many tissues of the lower species. The tissues of the higher animals had the highest activity of the enzyme. However, there seems to be no strict correlation between the development of the animal and the activity of the carnitine palmityltransferase. More probably there is a correlation between the metabolic activity of the animal or the animal tissue and the activity of the enzyme. It is interesting to note that the activity of the enzyme is relatively high in the female gonads, and that the activity increases with the development and the fertilizability of the eggs. This may reflect the ability of the fertilized egg to utilize stored lipids for its development. We have tested the carnitine palmityltransferase activity in two strains of Escherichia coli, one strain of Bacillus anitratum and one strain of Candida albicans. No enzyme activity could be found in these micro-organisms. The number of micro-organisms tested is too low to permit any conclusion to be drawn. Carnitine has been found in Streptococcus haemolyticus and brewer's yeast (Fraenkel, 1953). Fungi also contain mitochondria and the carnitine palmityltransferase has been shown to be a true mitochondrial enzyme (Norum & Bremer, 1966). One would expect therefore that the varnitine palmityltransferase is only present in microorganisms containing both carnitine and mitochondria.
THE D I S T R I B U T I O N OF CARNITINE PALMITYLTRANSFERASE T A B L E 1 - - D I S T R I B U T I O N OF CARNITINE PALMITYLTRANSFERASE I N ANIMALS AND ANIMAL TISSUES
Animal tested Porifera Halichondria panicea Coelenterata Scyphozoa Aurelia aurita Actinozoa Metridium senile Platyhelminthes App. Nemertini Ignot. sp. Annelida Chaetopoda Nerds sp. Arthropoda Crustacea Homarus vulgaris Homarus vulgaris Homarus vulgaris Homarus vulgaris Carcinus maenas Carcinus maenas Insecta Apis mellifica Mollusca Gastropoda Buccinum undatum Buccinum undatum Buccinum undatum Bivalvia(Pelecypoda) Myt~se~ Myt~se~ Mytilus edulis Modiolus modiolus Echinodermata Asteroidea Asterias rubens Echinoidea Psammechinus miliaris Psammechinus miliaris Psammechinus miliaris
Tissue used
Specific activity of carnitine palmityltransferase (arbitrary units)
Whole animal
0"18
Whole animal
0-5
Whole animal
Trace
Whole animal
0"28
Whole animal
Trace
Heart Muscle Liver (hepato-pancreas) Gonad Muscle Gonad ? Whole animal
Liver (hepato-pancreas) Foot Whole animal (without liver and foot)
5"26 0"1 7"45 1 "03 0"81 8"3 11"9
0 0"12 0"23
Whole animal Liver (hepato-pancreas) Adductor muscle Gonad $
0-21
Internal organs
0"1
Eggs, non-fertile Eggs, fertile Sperms, fertile
0-7 3"5 0"27
0 0 2"04
485
486
KAARE R. NORUM AND JON BRE~VIEE TABLE 1--(Continued)
Animal tested Echinodermata (cont.) Holothuroidea Stichopus tremulus Stichopus tremulus Stichopus tremulus Chordata Tunicata Ascidia mentula Ascidia mentula Cyclostomata Myxine glutinosa Myxine glutinosa Myxine glutinosa Myxine glutinosa Myxine glutinosa Pisces Gadus morrhua Gadus morrhua Gadus morrhua Gadus morrhua Gadus morrhua Gadus morrhua Gadus morrhua Amphibia Bufo vulgaris Bufo vulgaris Mammalia Rattus norvegicus Rattus norvegicus
Tissue used
Specific activity of carnitine palmityltransferase (arbitrary units)
Gonad ~ Intestine Muscle
Trace 0 Trace
Intestine Whole animal (without intestine)
Trace
Muscle Heart Liver Intestine Eggs, non-fertile
0 5'8 18"5 3"6 0'21
Muscle Heart Liver Kidney Spleen Intestine Gonad ?
2"5 19"6 33-1 11 '3 4-8 0-77 6-9
Heart Liver
42'0 60-0
Heart Liver
72-0 52-0
0
I n human organs the highest concentrations of carnitine palmityltransferase are found in those organs which have the highest rate of fatty-acid oxidation (Norum, 1966). It may therefore tentatively be suggested that the distribution of the earnitine palmityltransferase in animals reflects their (organ) ability to oxidize fatty acids. REFERENCES BREMER J. (1962) Carnitine in intermediary metabolism. The metabolism of fatty acid esters of carnitine by mitochondria, ft. biol. Chem. 237, 3628-3632. BROEmtUYSEN J., ROZENBLUMC., GrtXSLAIN M. & DELTOUR G. (1965) Distribution of carnitine in the rat. In Recent Research on Carnitine, pp. 23-25. M.I.T. Press, Cambridge, Mass.
THE
DISTRIBUTION
OF
CARNITINE
PALMITYLTRANSFERASE
487
FRAENKEL G. (1953) Studies on the distribution of vitamin BT (carnitine). Biol. Bull., Woods Hole 104, 359-371. FRAENKEL G. (1954) T h e distribution of vitamin B T (carnitine) throughout the animal kingdom. Archs Biochem. Biophys. 50, 486-495. FRITZ I. B. (1963) Carnitine and its role in the fatty acid metabolism. Advances in Lipid Research, Vol. 1, pp. 285-334. Academic Press, New York. FRITZ I. B., SCHULTZ S. K. • SRERE P. A. (1963) Properties of partially purified carnitine acetyltransferase. J. biol. Chem. 238, 2509-2517. NORUM K. R. (1964) Palmityl-CoA: carnitine palmityltransferase. Purification from calf liver mitochondria and some properties of the enzyme. Biochirn. biophys. Acta 89, 95108. NORUM K. R. (1966) T h e organ and subcellular distribution of palmityl-CoA: carnitine palmityltransferase in man. Acta physiol. Scand. 66, 172-181. NORUM K. R. & BREMER J. (1966) T h e subcellular distribution of acyl-CoA: camitine acyltransferases in rat liver. Submitted for publication.