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Two systems for the activation of fatty acids in rat-liver mitochondria
BIOCHIMICA ET BIOPHYSICA ACTA
442 PRELIMINARY
NOTE
BBA 51024
Two systems for the activation of fatty acids in rat-liver
mitochondria
It is well known that fatty acids undergo an enzymatic activation with formation of thiol esters of coenzyme A prior to their oxidation in mitochondria. The fatty acid-activating enzymes (acid: CoAligase (AMP), EC 6.2.1.2. and EC 6.2.1.3) catalyze the following reaction : R * COOH + ATP + CoA -+R.CO+CoA+AMP+PPI
(I)
The activation of fatty acid by this reaction in intact mitochondria is inhibited by z,+dinitrophenol, independently of whether the ATP is generated in the mitochondrialp* or added externally. In the latter case the inhibition can be overcome by addition of oligomycin, which inhibits the dinitrophenol-induced ATPase activity of the mitochondriaa, leaving the added ATP available for activation of the fatty acid (Fig. I, Expt. A).
With Pi
Fig. I. Palmitate oxidation,as measuredwith the Clark“oxygen electrode”. The reaction medium contained2 pmoles palmitic acid, 1.5 mM ADP, 15 mM KCI, 5 mM MgCl,, 2 mM EDTA, 50 mM Tris-HCl buffer, 25 mM sucrose and 12 mg mitochondrial protein. In Expt. A 30 mM potassium phosphate buffer was present initially. The reaction volume was 2 ml, the temperature the pH 7.5 Dinitrophenol was added to a concentration of 0.1 mM, ATP to 5 mM.
25’ and
In this communication evidence is presented that a second pathway for activation of fatty acids exists in rat-liver mitochondria. This conclusion arose from the finding that dinitrophenol is not inhibitory to fatty acid oxidation when inorganic phosphate is absent from the reaction medium. As can be seen in Expt. B of Fig. I, removal of inorganic phosphate from the medium resulted in a strongly decreased rate of respiration. The respiration was greatly stimulated by dinitrophenol to a rate equal to that obtained with phosphate in Expt. A. However, in the presence of dinitrophenol, the respiration was strongly inhibited by the subsequent addition of phosphate. A 50% inhibition was found at 2-3 mM phosphate. Biochim.
Bio$hys.
Acta, g8 (1965)
442-444
PRELIMINARYNOTE
443
It is clear, therefore, that two separate systems exist in isolated mitochondria for the activation of fatty acids. One system requires phosphate and is inhibited by dinitrophenol, the other is insensitive to dinitrophenol but is inhibited by phosphate. In Table I the two systems are compared. Whereas the dinitrophenol-sensitive oxidation reaction is only 4o-50% inhibited by interruption of the Krebs cycle, the dinitrophenol-insensitive process is completely dependent on the operation of the Krebs cycle. In the latter system addition of malate can overcome the inhibition by malonate, but not that by arsenite. This shows that the dinitrophenol-insensitive system is dependent on that part of the Krebs cycle lying between the arsenite- and malonate-sensitive sites. In the last three lines of Table I it can be seen that it is indeed the a-oxoglutarate oxidation step of the Krebs cycle which supplies the energy for the dinitrophenol-insensitive fatty acid oxidation. TABLE I COMPARISON OPTHEDINITROPHENOL-SENSITIVE
AND
-INSENSITIVE
SYSTEMS
FOR FATTY
ACID
OXI-
DATION
Oxygen uptake was measured with differential manometers. The reaction medium contained 3 mM octanoate, 30 mM glucose, 15 mM KCI, 5 mM MgCl,, z mM EDTA, 50 mM Tris-HCl buffer, 50 mM sucrose, 4.6 mg mitochondrial protein and either 30 mM potassium phosphate buffer, I mM ADP and ISO Cori units of hexokinase or 0.1 mM a,4-dinitrophenol. Other additions were IO mM L-malate, IO mM a-oxoglutarate, IO mM malonate and I mM anenite. The reaction volume was I ml, the temperature 25’, the gas phase air and the pH 7.5. Substrate
Additions
Respiratory
rate (pl O,/mg protein/h)
Plus phosphate, dinitro#henol Octanoate Octanoate
+ malate
Octanoate a-oxoglutarate Octanoate + a-oxoglutarate
None Malonate Arsenite None Malonate Arsenite Malonate Malonate Malonate
62 40 37
no
No phosphate. dinitro$heuol
plus
55 I 2
62 5I 9 I I4 49
$ 40
As early as 1945, LEHNINGER* described the exceptional ability of a-oxoglutarate to activate fatty acid oxidation in the absence of added adenine nucleotides. Recently, ROSSI AND GIBSON’ (see also ref. 6) have demonstrated the presence in liver mitochondria of a GTP-specific fatty acid-activating enzyme, which catalyses the reaction R - COOH + GTP + CoA --+R~CO~CoA+GDP+Pr It seems very likely that this enzyme is involved in the dinitrophenol-insensitive fatty acid oxidation. The finding that orthophosphate is a product in Eqn. 2 may well explain the inhibitory effect of phosphate on the dinitrophenol-insensitive fatty acid oxidation. It was found, however, that Reaction I is unaffected by the addition of inorganic pyrophosphate. Low concentrations of arsenate, which is known to uncouple a-oxoglutaratelinked substrate-level phosphorylation’, strongly inhibited the dinitrophenol-insensitive fatty acid oxidation, Biochim.
Biophys.
Acta, 98 (1963)
442-444
PRELIMINARYNOTE
444
The dinitrophenol-insensitive fatty acid-activating system is active with all even-numbered saturated fatty acids ranging from acetic to stearic acid. It is also active with all odd-numbered acids tested (C,, CI, C,* and C,,). Of the unsaturated fatty acids only oleic acid was tested. At concentrations of oleic acid which completely uncouple oxidative phosphorylationa, its oxidation is unaffected by addition of dinitrophenol, strongly stimulated by removal of inorganic phosphate and completely inhibited by addition of malonate, indicating that the energy for the activation of oleic acid is also supplied by a-oxoglutarate oxidation. A detailed’account of these investigations will be presented later. The author is grateful to Dr. A. L. LEHNINGERfor his hospitality, advice and encouragement and to Miss Y. DOORNBOSfor technical assistance. This work was started during the tenure of a U.S. Public Health Service International Postdoctoral Fellowship. S. G. VANDEN BERGH
De$artment of Physiological Chemistry, The Johns Hopkins School of Medicine, Baltimore, Md. (U.S.A.), and Laboratory of Biochemistry * University of Amsterdam, Amsterdam (The Netherlands) REFERENCES
R. J. CROSS,J. V. TAGGART, G. A. Covo AND D. E. GREEN, J. Biol. Chem., 177 (1949) 655. E. P. KENNEDY AND A. L. LEHNINGER, J. Biol. Chem., Igo (1951) 361. H. A. LARDY, D. JOHNSON AND W. C. MCMURRAY, Awl. Biochem. Biofihys., 78 (1958) 587. A. L. LEHNINGER, J. Biol. Chem., 161 (1945) 437. C. R. ROSSI AND D. M. GIBSON, J. Biol. Chem., qg (1964) 1694. C. R. ROSSI AND M. SACCHETTO,Expwientia. 15 (1959) 64. D. R. SANADI, D. M. GIBSON, P. AYENGAR AND L. OUELLET, Biochim. Biophys. Acta, 13 (1954) 146. 8 P. BORST, J. A. Loos, E. J. V. J. CHRISTAND E .C. SLATER,Biochim.Biophys. Acta, 62 (1962) 509. I 2 3 4 5 6 7
Keceived January 6th, 1965
* Formerly : Laboratory dam (The Netherlands).
of Physiological
Chemistry.
Postal address Biochim.
: J . D. Meijerplein 3. Amster-
Biophys.
Ada,
g8 (1965) 442-444
442 PRELIMINARY
NOTE
BBA 51024
Two systems for the activation of fatty acids in rat-liver
mitochondria
It is well known that fatty acids undergo an enzymatic activation with formation of thiol esters of coenzyme A prior to their oxidation in mitochondria. The fatty acid-activating enzymes (acid: CoAligase (AMP), EC 6.2.1.2. and EC 6.2.1.3) catalyze the following reaction : R * COOH + ATP + CoA -+R.CO+CoA+AMP+PPI
(I)
The activation of fatty acid by this reaction in intact mitochondria is inhibited by z,+dinitrophenol, independently of whether the ATP is generated in the mitochondrialp* or added externally. In the latter case the inhibition can be overcome by addition of oligomycin, which inhibits the dinitrophenol-induced ATPase activity of the mitochondriaa, leaving the added ATP available for activation of the fatty acid (Fig. I, Expt. A).
With Pi
Fig. I. Palmitate oxidation,as measuredwith the Clark“oxygen electrode”. The reaction medium contained2 pmoles palmitic acid, 1.5 mM ADP, 15 mM KCI, 5 mM MgCl,, 2 mM EDTA, 50 mM Tris-HCl buffer, 25 mM sucrose and 12 mg mitochondrial protein. In Expt. A 30 mM potassium phosphate buffer was present initially. The reaction volume was 2 ml, the temperature the pH 7.5 Dinitrophenol was added to a concentration of 0.1 mM, ATP to 5 mM.
25’ and
In this communication evidence is presented that a second pathway for activation of fatty acids exists in rat-liver mitochondria. This conclusion arose from the finding that dinitrophenol is not inhibitory to fatty acid oxidation when inorganic phosphate is absent from the reaction medium. As can be seen in Expt. B of Fig. I, removal of inorganic phosphate from the medium resulted in a strongly decreased rate of respiration. The respiration was greatly stimulated by dinitrophenol to a rate equal to that obtained with phosphate in Expt. A. However, in the presence of dinitrophenol, the respiration was strongly inhibited by the subsequent addition of phosphate. A 50% inhibition was found at 2-3 mM phosphate. Biochim.
Bio$hys.
Acta, g8 (1965)
442-444
PRELIMINARYNOTE
443
It is clear, therefore, that two separate systems exist in isolated mitochondria for the activation of fatty acids. One system requires phosphate and is inhibited by dinitrophenol, the other is insensitive to dinitrophenol but is inhibited by phosphate. In Table I the two systems are compared. Whereas the dinitrophenol-sensitive oxidation reaction is only 4o-50% inhibited by interruption of the Krebs cycle, the dinitrophenol-insensitive process is completely dependent on the operation of the Krebs cycle. In the latter system addition of malate can overcome the inhibition by malonate, but not that by arsenite. This shows that the dinitrophenol-insensitive system is dependent on that part of the Krebs cycle lying between the arsenite- and malonate-sensitive sites. In the last three lines of Table I it can be seen that it is indeed the a-oxoglutarate oxidation step of the Krebs cycle which supplies the energy for the dinitrophenol-insensitive fatty acid oxidation. TABLE I COMPARISON OPTHEDINITROPHENOL-SENSITIVE
AND
-INSENSITIVE
SYSTEMS
FOR FATTY
ACID
OXI-
DATION
Oxygen uptake was measured with differential manometers. The reaction medium contained 3 mM octanoate, 30 mM glucose, 15 mM KCI, 5 mM MgCl,, z mM EDTA, 50 mM Tris-HCl buffer, 50 mM sucrose, 4.6 mg mitochondrial protein and either 30 mM potassium phosphate buffer, I mM ADP and ISO Cori units of hexokinase or 0.1 mM a,4-dinitrophenol. Other additions were IO mM L-malate, IO mM a-oxoglutarate, IO mM malonate and I mM anenite. The reaction volume was I ml, the temperature 25’, the gas phase air and the pH 7.5. Substrate
Additions
Respiratory
rate (pl O,/mg protein/h)
Plus phosphate, dinitro#henol Octanoate Octanoate
+ malate
Octanoate a-oxoglutarate Octanoate + a-oxoglutarate
None Malonate Arsenite None Malonate Arsenite Malonate Malonate Malonate
62 40 37
no
No phosphate. dinitro$heuol
plus
55 I 2
62 5I 9 I I4 49
$ 40
As early as 1945, LEHNINGER* described the exceptional ability of a-oxoglutarate to activate fatty acid oxidation in the absence of added adenine nucleotides. Recently, ROSSI AND GIBSON’ (see also ref. 6) have demonstrated the presence in liver mitochondria of a GTP-specific fatty acid-activating enzyme, which catalyses the reaction R - COOH + GTP + CoA --+R~CO~CoA+GDP+Pr It seems very likely that this enzyme is involved in the dinitrophenol-insensitive fatty acid oxidation. The finding that orthophosphate is a product in Eqn. 2 may well explain the inhibitory effect of phosphate on the dinitrophenol-insensitive fatty acid oxidation. It was found, however, that Reaction I is unaffected by the addition of inorganic pyrophosphate. Low concentrations of arsenate, which is known to uncouple a-oxoglutaratelinked substrate-level phosphorylation’, strongly inhibited the dinitrophenol-insensitive fatty acid oxidation, Biochim.
Biophys.
Acta, 98 (1963)
442-444
PRELIMINARYNOTE
444
The dinitrophenol-insensitive fatty acid-activating system is active with all even-numbered saturated fatty acids ranging from acetic to stearic acid. It is also active with all odd-numbered acids tested (C,, CI, C,* and C,,). Of the unsaturated fatty acids only oleic acid was tested. At concentrations of oleic acid which completely uncouple oxidative phosphorylationa, its oxidation is unaffected by addition of dinitrophenol, strongly stimulated by removal of inorganic phosphate and completely inhibited by addition of malonate, indicating that the energy for the activation of oleic acid is also supplied by a-oxoglutarate oxidation. A detailed’account of these investigations will be presented later. The author is grateful to Dr. A. L. LEHNINGERfor his hospitality, advice and encouragement and to Miss Y. DOORNBOSfor technical assistance. This work was started during the tenure of a U.S. Public Health Service International Postdoctoral Fellowship. S. G. VANDEN BERGH
De$artment of Physiological Chemistry, The Johns Hopkins School of Medicine, Baltimore, Md. (U.S.A.), and Laboratory of Biochemistry * University of Amsterdam, Amsterdam (The Netherlands) REFERENCES
R. J. CROSS,J. V. TAGGART, G. A. Covo AND D. E. GREEN, J. Biol. Chem., 177 (1949) 655. E. P. KENNEDY AND A. L. LEHNINGER, J. Biol. Chem., Igo (1951) 361. H. A. LARDY, D. JOHNSON AND W. C. MCMURRAY, Awl. Biochem. Biofihys., 78 (1958) 587. A. L. LEHNINGER, J. Biol. Chem., 161 (1945) 437. C. R. ROSSI AND D. M. GIBSON, J. Biol. Chem., qg (1964) 1694. C. R. ROSSI AND M. SACCHETTO,Expwientia. 15 (1959) 64. D. R. SANADI, D. M. GIBSON, P. AYENGAR AND L. OUELLET, Biochim. Biophys. Acta, 13 (1954) 146. 8 P. BORST, J. A. Loos, E. J. V. J. CHRISTAND E .C. SLATER,Biochim.Biophys. Acta, 62 (1962) 509. I 2 3 4 5 6 7
Keceived January 6th, 1965
* Formerly : Laboratory dam (The Netherlands).
of Physiological
Chemistry.
Postal address Biochim.
: J . D. Meijerplein 3. Amster-
Biophys.
Ada,
g8 (1965) 442-444