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Stimulation by acetoacetate of DPNH oxidation by liver mitochondria
564
PRELIMINARY NOTES
VOL. 36 (1959)
That fatty acid liberation was independent of the shaking rate, was not affected by doubling or halving the amount of substrate, and was the same whether substrate was added directly to the Warburg vessel or homogenized with albumin*, NaC1 or NaHCO~ before addition, argue against attributing the low reactivity of the saturated triglycerides to their solid state, and lessened enzyme-substrate contacts. Also arguing against this explanation was the observation that the hydrolysis of coconut oil (liquid at 37 °) was o-Io % less than a mixture of its constituent triglyceridess (solid at 37°). In addition, a preliminary comparison of the fatty acids enzymically released after 4 h from the triglycerides of human $I 20-400 lipoproteins (in solution though containing tristearin, tripalmitin, triolein, and trilinolein e) indicates that the fatty acids have a higher oleic acid conteht than do the lipoprotein triglycerides remaining after hydrolysis. An explanation for the difference in hydrolysis of the triolein and trilinolein, based on the difference in their physical state, is even less likely since both triglycerides are liquids. Their hydrolysis was also not increased by the same procedures described for the tripalmitin and tristearin. This work was supported in part by the American Heart Association and by the National Heart Institute of the National Institutes of Health. B. SHORE Department o/Physiology, Washington University School o/Medicine, O.M. COLVlN St. Louis, Mo., (U.S.A.) V.G. SHORE 1 B. SHORE, Proc. Soc. Exptl. Biol. Med., 83 (1953) 216. E. KORN, Chemistry o/ Lipides as Related to Atherosclerosis, C. C. T h o m a s , Springfield, Ilk, 1958 , p. 169. 3 D. S. ROBINSON AND J. E. FRENCH, Quart. J. Exptl. Physiol., 42 (1957) 151. 4 L. A. CARLSON AND L. B. WADSTROM, Clin. Chim. Acta, 2 (1957) 9. 5 R. RUYSSEN, ed., The Blood Lipids and the Clearing Factor, I I I r d I n t e r n a t i o n a l Conference on Biochemical P r o b l e m s of Lipids, Brussels, 1956. e V. P. DOLE, Chemistry o[ Lipides as Related to Atherosclerosis, C. C. T h o m a s , Springfield, Ii1., 1958, p. 189. 7 B. SHORE, O. M. COLVIN AND V. G. SHORE, in preparation. s H. J. DEUEL, The Lipids, Vol. I, Interscience, New York, 1951, p. 205. 9 A. NASON AND I. R. LEHMAN, J. Biol. Chem., 222 (1956) 511.
Received August 22nd, 1959 * Erratic d a t a were obtained in t h e W a r b u r g with a t r i g l y c e r i d e - a l b u m i n - e t h a n o l emulsion 9.
Stimulation by acetoacetate of DPN H oxidation by liver mitochondria Various investigators have demonstrated that freshly isolated liver mitochondria oxidize exogenous D P N H at a negligible rate 1-~ which, however, can be stimulated by hypotonic pretreatment of mitochondrial, 3 or addition of cytochrome c1,5,e. These observations have supported the concept, proposed by LEHNINGER ~, of an "internal" and an "external" pathway for the oxidation of D P N H by mitochondria. Abbreviations: D P N , D P N H , oxidized and reduced d i p h o s p h o p y r i d i n e nucleotide; reduced t r i p h o s p h o p y r i d i n e nucleotide; ADP, adenosine diphosphate.
TPNH,
VOL 36 (1959)
PRELIMINARY NOTES
565
W e h a v e o b s e r v e d t h a t c a t a l y t i c quantities of either a c e t o a c e t a t e or f l - h y d r o x y b u t y r a t e s t i m u l a t e the o x i d a t i o n of D P N H b y m i t o c h o n d r i a w i t h c o n c o m i t a n t phosphate uptake, presumably via the DPN-linked D-fl-hydroxybutyrate dehydrogenase. These results suggest a n o t h e r p a t h w a y for the o x i d a t i o n of e x t e r n a l D P N H which does n o t d e p e n d u p o n h y p o t o n i c t r e a t m e n t of m i t o c h o n d r i a or the presence of exogenous c y t o c h r o m e c. R a t liver m i t o c h o n d r i a isolated in 2.5 % p o l y v i n y l p y r r o l i d o n e - o . 2 5 M sucrose 7 were e m p l o y e d in most of t h e e x p e r i m e n t s described because p r e l i m i n a r y spectrop h o t o m e t r i c studies i n d i c a t e d less d a i l y v a r i a t i o n in the r a t e of D P N H o x i d a t i o n w i t h fresh m i t o c h o n d r i a l p r e p a r a t i o n s isolated with this m e d i u m t h a n with 0.25 M sucrose s. However, c o m p a r a b l e results h a v e been o b s e r v e d with m i t o c h o n d r i a p r e p a r e d in 0.25 M sucrose. TABLE I ~FFECT
OF ACETOACETATE
AND fl-HYDROXYBUTYRATE
ON
DPNH
OXIDATION
Incubation medium (3.0 ml) contained: o.oi M phosphate (pH 7.4), o.ooo33 M ADP, o.oi66 M glucose, i5o K.M. units hexokinase, o.oI M tris(hydroxymethyl)aminomethane (pH 7.4), 0.0o6 M MgC1v o.o2 M KF, o.Io 214 KC1, and mitochondria (i.5-2.o mg N). 8/~moles DPNH added after 8-min preincubation. Reaction time, 35 min at 30 °. 0 2 uptake measured manometfically and phosphate colorimetrically9. Endogenous respiration (Li #atoms) and phosphorylation have been subtracted from these values. Additions
Oxygen Uptake I,atoms
Phosphate Uptake itmoles
P/O
None
I .o
O. I
O. I
0.3/tmole acetoacetate i .o/~mole acetoacetate 3.o #moles acetoacetate 0.6/~mole DL-fl-hydroxybutyrate 2.o ttmoles DL-fl-hydroxybutyrate
3.6 5.3 5.9 3.7 5.4
7.0 i t .o 12.4 8.o 13. I
1.9 2. I 2. I 2.2 2.4
Expt.
I
None
I ,O
O
O
6.o/~moles amytal I.o #mole acetoacetate I.o #mole acetoacetate + 6.0 #moles amytal i.o #mole acetoacetate + 5 #g antimycin A
o.4 4.0 0.7 0. 3
o
o
6.8
1.7
O
O
O
o
As shown in T a b l e I, the r a t e of m i t o c h o n d r i a l o x i d a t i o n of exogenous D P N H was v e r y slow with negligible p h o s p h a t e u p t a k e . I n m a n y e x p e r i m e n t s with D P N H alone 0 3 u p t a k e c o m m e n c e d o n l y after the first I O - I 5 rain, suggesting t h a t a swelling of t h e m i t o c h o n d r i a occurred during t h e incubation, p e r m i t t i n g access of the D P N H t o the " i n t e r n a l " p a t h w a y . A d d i t i o n of either a c e t o a c e t a t e or f l - h y d r o x y b u t y r a t e s t i m u l a t e d b o t h respiration a n d the coupled p h o s p h o r y l a t i o n ; m a x i m a l s t i m u l a t i o n occurred w i t h 3 / , m o l e s acetoacetate, f l - H y d r o x y b u t y r a t e was as effective as acetoa c e t a t e in s t i m u l a t i n g the o x i d a t i o n of exogenous D P N H . I n these e x p e r i m e n t s the racemic m i x t u r e of I)L-fl-hydroxybutyrate was e m p l o y e d a n d it is a s s u m e d t h a t only the I) form was active. The increased respiration o b s e r v e d could n o t be a c c o u n t e d for b y the o x i d a t i o n of f l - h y d r o x y b u t y r a t e alone. W i t h either a c e t o a c e t a t e or f l - h y d r o x y b u t y r a t e , P / O ratios of 1.5-2.I were generally o b s e r v e d a l t h o u g h values as high as 2. 7 have been measured. The o x i d a t i o n a n d coupled p h o s p h o r y l a t i o n were
566
PRELIMINARY NOTES
VOL. 36 (1959)
inhibited by amytal and antimycin A, indicating that DPNH, in the presence of acetoacetate, is oxidized via the phosphorylating "internal" pathway which is sensitive to these respiratory inhibitors 5. 2,4-Dinitrophenol (5" lO-4 M) completely uncoupled the stimulated phosphorylation and slightly inhibited respiration. With mitochondria prepared in 0.25 M sucrose, the rates of oxidation by molecular oxygen of fi-hydroxybutyrate, succinate, DPNH, and D P N H + acetoacetate were in the relative ratio of I.O, 1.3, o.I and 0.6. Catalytic quantities (I/~mole) of the trlcarboxylic acid cycle intermediates and pyrnvate did not stimulate either D P N H oxidation or the coupled phosphorylation. Some of these substrates, but not acetoacetate or fl-hydroxybutyrate, did stimulate the endogenous respiration with concomitant phosphate uptake. These results rule out the possibility that acetoacetate or fl-hydroxybutyrate have a non-specific effect on D P N H oxidation. Addition of coenzyme A had no effect on oxidation of D P N H in the absence or presence or acetoacetate, indicating that the acetoacetate stimulation does not function through the L-fl-hydroxybutyryl-CoA-dehydrogenaseTM. The oxidation of T P N H by mitochondria prepared in 0.25 M sucrose was unaffected by acetoacetate, but the presence of catalytic quantities (I.O/zmole) of both acetoacetate and DPN stimulated oxygen uptake and phosphorylation. These observations suggest the participation of both pyridine nucleotide transhydrogenase n and the acetoacetate-stimulated pathway in the oxidation of TPNH. These results demonstrate a pathway for the transfer of reducing equivalents from extramitochondrial D P N H to intramitochondrial DPN which is apparently catalyzed by DPN-linked D-fi-hydroxybutyrate dehydrogenase. In this system acetoacetate and D-fl-hydroxybutyrate function as a shuttle between the intra- and extramitochondrial DPNH, permitting the oxidation of exogenous D P N H without either an alteration in the permeability of the mitochondria or the intervention of external cytochrome c. A full report of these observations will be presented for publication shortly. This investigation was supported by the Cancer Chemotherapy National Service Center, National Cancer Institute, under the National Institutes of Health Contract Number SA-43-ph-I886.
Merck Institute/or Therapeutic Research, Rahway, N.J. (U.S.A.)
THOMAS M. DEVLIN BERNICE H. BEDELL
1 A. L. LEHNI•GER, J. Biol. Chem., 19o (1951) 345. A. L. LEHNINGER, Harvey Lectures, 49 (1953-54) 176. 3 B. CHANCE AND G. R. WILLIAMS, J. Biol. Chem., 217 (1955) 4o9. 4 M. E. PULLMAN AND E. RACKER, Science, 123 (1956) 11o5. 5 L. ERNSTER, O. JALLING, H. L~W AND O. LINDB~RG, Exp. Cell Res., Suppl., 3 (I955) 124. e G. MALEY, J. Biol. Chem., 224 (1957) lO29. A. NOVlKOFF, in Mitochondria and Other Cytoplasmic Inclusions, A c a d e m i c Press, Inc., N e w York, 1957, p. 92. 8 G. H. HOGEBOOM, W. C. SCHNEIDER AND G. E. PALLADE, J. Biol. Chem., 172 (1948) 619. 9 0 . H. LOWRY AND J. A. LOPEZ, J. Biol. Chem., 162 (1946) 421. 10 A. L. LEHNINGER AND G. D. GREVlLLE, Biochim. Biophys. Acta, 12 (1953) 188. 11 A. M. STEIN, N. O. KAPLAN AND M. CIOTTI, J. Biol. Chem., 234 (1959) 979.
Received August 3Ist, 1959
PRELIMINARY NOTES
VOL. 36 (1959)
That fatty acid liberation was independent of the shaking rate, was not affected by doubling or halving the amount of substrate, and was the same whether substrate was added directly to the Warburg vessel or homogenized with albumin*, NaC1 or NaHCO~ before addition, argue against attributing the low reactivity of the saturated triglycerides to their solid state, and lessened enzyme-substrate contacts. Also arguing against this explanation was the observation that the hydrolysis of coconut oil (liquid at 37 °) was o-Io % less than a mixture of its constituent triglyceridess (solid at 37°). In addition, a preliminary comparison of the fatty acids enzymically released after 4 h from the triglycerides of human $I 20-400 lipoproteins (in solution though containing tristearin, tripalmitin, triolein, and trilinolein e) indicates that the fatty acids have a higher oleic acid conteht than do the lipoprotein triglycerides remaining after hydrolysis. An explanation for the difference in hydrolysis of the triolein and trilinolein, based on the difference in their physical state, is even less likely since both triglycerides are liquids. Their hydrolysis was also not increased by the same procedures described for the tripalmitin and tristearin. This work was supported in part by the American Heart Association and by the National Heart Institute of the National Institutes of Health. B. SHORE Department o/Physiology, Washington University School o/Medicine, O.M. COLVlN St. Louis, Mo., (U.S.A.) V.G. SHORE 1 B. SHORE, Proc. Soc. Exptl. Biol. Med., 83 (1953) 216. E. KORN, Chemistry o/ Lipides as Related to Atherosclerosis, C. C. T h o m a s , Springfield, Ilk, 1958 , p. 169. 3 D. S. ROBINSON AND J. E. FRENCH, Quart. J. Exptl. Physiol., 42 (1957) 151. 4 L. A. CARLSON AND L. B. WADSTROM, Clin. Chim. Acta, 2 (1957) 9. 5 R. RUYSSEN, ed., The Blood Lipids and the Clearing Factor, I I I r d I n t e r n a t i o n a l Conference on Biochemical P r o b l e m s of Lipids, Brussels, 1956. e V. P. DOLE, Chemistry o[ Lipides as Related to Atherosclerosis, C. C. T h o m a s , Springfield, Ii1., 1958, p. 189. 7 B. SHORE, O. M. COLVIN AND V. G. SHORE, in preparation. s H. J. DEUEL, The Lipids, Vol. I, Interscience, New York, 1951, p. 205. 9 A. NASON AND I. R. LEHMAN, J. Biol. Chem., 222 (1956) 511.
Received August 22nd, 1959 * Erratic d a t a were obtained in t h e W a r b u r g with a t r i g l y c e r i d e - a l b u m i n - e t h a n o l emulsion 9.
Stimulation by acetoacetate of DPN H oxidation by liver mitochondria Various investigators have demonstrated that freshly isolated liver mitochondria oxidize exogenous D P N H at a negligible rate 1-~ which, however, can be stimulated by hypotonic pretreatment of mitochondrial, 3 or addition of cytochrome c1,5,e. These observations have supported the concept, proposed by LEHNINGER ~, of an "internal" and an "external" pathway for the oxidation of D P N H by mitochondria. Abbreviations: D P N , D P N H , oxidized and reduced d i p h o s p h o p y r i d i n e nucleotide; reduced t r i p h o s p h o p y r i d i n e nucleotide; ADP, adenosine diphosphate.
TPNH,
VOL 36 (1959)
PRELIMINARY NOTES
565
W e h a v e o b s e r v e d t h a t c a t a l y t i c quantities of either a c e t o a c e t a t e or f l - h y d r o x y b u t y r a t e s t i m u l a t e the o x i d a t i o n of D P N H b y m i t o c h o n d r i a w i t h c o n c o m i t a n t phosphate uptake, presumably via the DPN-linked D-fl-hydroxybutyrate dehydrogenase. These results suggest a n o t h e r p a t h w a y for the o x i d a t i o n of e x t e r n a l D P N H which does n o t d e p e n d u p o n h y p o t o n i c t r e a t m e n t of m i t o c h o n d r i a or the presence of exogenous c y t o c h r o m e c. R a t liver m i t o c h o n d r i a isolated in 2.5 % p o l y v i n y l p y r r o l i d o n e - o . 2 5 M sucrose 7 were e m p l o y e d in most of t h e e x p e r i m e n t s described because p r e l i m i n a r y spectrop h o t o m e t r i c studies i n d i c a t e d less d a i l y v a r i a t i o n in the r a t e of D P N H o x i d a t i o n w i t h fresh m i t o c h o n d r i a l p r e p a r a t i o n s isolated with this m e d i u m t h a n with 0.25 M sucrose s. However, c o m p a r a b l e results h a v e been o b s e r v e d with m i t o c h o n d r i a p r e p a r e d in 0.25 M sucrose. TABLE I ~FFECT
OF ACETOACETATE
AND fl-HYDROXYBUTYRATE
ON
DPNH
OXIDATION
Incubation medium (3.0 ml) contained: o.oi M phosphate (pH 7.4), o.ooo33 M ADP, o.oi66 M glucose, i5o K.M. units hexokinase, o.oI M tris(hydroxymethyl)aminomethane (pH 7.4), 0.0o6 M MgC1v o.o2 M KF, o.Io 214 KC1, and mitochondria (i.5-2.o mg N). 8/~moles DPNH added after 8-min preincubation. Reaction time, 35 min at 30 °. 0 2 uptake measured manometfically and phosphate colorimetrically9. Endogenous respiration (Li #atoms) and phosphorylation have been subtracted from these values. Additions
Oxygen Uptake I,atoms
Phosphate Uptake itmoles
P/O
None
I .o
O. I
O. I
0.3/tmole acetoacetate i .o/~mole acetoacetate 3.o #moles acetoacetate 0.6/~mole DL-fl-hydroxybutyrate 2.o ttmoles DL-fl-hydroxybutyrate
3.6 5.3 5.9 3.7 5.4
7.0 i t .o 12.4 8.o 13. I
1.9 2. I 2. I 2.2 2.4
Expt.
I
None
I ,O
O
O
6.o/~moles amytal I.o #mole acetoacetate I.o #mole acetoacetate + 6.0 #moles amytal i.o #mole acetoacetate + 5 #g antimycin A
o.4 4.0 0.7 0. 3
o
o
6.8
1.7
O
O
O
o
As shown in T a b l e I, the r a t e of m i t o c h o n d r i a l o x i d a t i o n of exogenous D P N H was v e r y slow with negligible p h o s p h a t e u p t a k e . I n m a n y e x p e r i m e n t s with D P N H alone 0 3 u p t a k e c o m m e n c e d o n l y after the first I O - I 5 rain, suggesting t h a t a swelling of t h e m i t o c h o n d r i a occurred during t h e incubation, p e r m i t t i n g access of the D P N H t o the " i n t e r n a l " p a t h w a y . A d d i t i o n of either a c e t o a c e t a t e or f l - h y d r o x y b u t y r a t e s t i m u l a t e d b o t h respiration a n d the coupled p h o s p h o r y l a t i o n ; m a x i m a l s t i m u l a t i o n occurred w i t h 3 / , m o l e s acetoacetate, f l - H y d r o x y b u t y r a t e was as effective as acetoa c e t a t e in s t i m u l a t i n g the o x i d a t i o n of exogenous D P N H . I n these e x p e r i m e n t s the racemic m i x t u r e of I)L-fl-hydroxybutyrate was e m p l o y e d a n d it is a s s u m e d t h a t only the I) form was active. The increased respiration o b s e r v e d could n o t be a c c o u n t e d for b y the o x i d a t i o n of f l - h y d r o x y b u t y r a t e alone. W i t h either a c e t o a c e t a t e or f l - h y d r o x y b u t y r a t e , P / O ratios of 1.5-2.I were generally o b s e r v e d a l t h o u g h values as high as 2. 7 have been measured. The o x i d a t i o n a n d coupled p h o s p h o r y l a t i o n were
566
PRELIMINARY NOTES
VOL. 36 (1959)
inhibited by amytal and antimycin A, indicating that DPNH, in the presence of acetoacetate, is oxidized via the phosphorylating "internal" pathway which is sensitive to these respiratory inhibitors 5. 2,4-Dinitrophenol (5" lO-4 M) completely uncoupled the stimulated phosphorylation and slightly inhibited respiration. With mitochondria prepared in 0.25 M sucrose, the rates of oxidation by molecular oxygen of fi-hydroxybutyrate, succinate, DPNH, and D P N H + acetoacetate were in the relative ratio of I.O, 1.3, o.I and 0.6. Catalytic quantities (I/~mole) of the trlcarboxylic acid cycle intermediates and pyrnvate did not stimulate either D P N H oxidation or the coupled phosphorylation. Some of these substrates, but not acetoacetate or fl-hydroxybutyrate, did stimulate the endogenous respiration with concomitant phosphate uptake. These results rule out the possibility that acetoacetate or fl-hydroxybutyrate have a non-specific effect on D P N H oxidation. Addition of coenzyme A had no effect on oxidation of D P N H in the absence or presence or acetoacetate, indicating that the acetoacetate stimulation does not function through the L-fl-hydroxybutyryl-CoA-dehydrogenaseTM. The oxidation of T P N H by mitochondria prepared in 0.25 M sucrose was unaffected by acetoacetate, but the presence of catalytic quantities (I.O/zmole) of both acetoacetate and DPN stimulated oxygen uptake and phosphorylation. These observations suggest the participation of both pyridine nucleotide transhydrogenase n and the acetoacetate-stimulated pathway in the oxidation of TPNH. These results demonstrate a pathway for the transfer of reducing equivalents from extramitochondrial D P N H to intramitochondrial DPN which is apparently catalyzed by DPN-linked D-fi-hydroxybutyrate dehydrogenase. In this system acetoacetate and D-fl-hydroxybutyrate function as a shuttle between the intra- and extramitochondrial DPNH, permitting the oxidation of exogenous D P N H without either an alteration in the permeability of the mitochondria or the intervention of external cytochrome c. A full report of these observations will be presented for publication shortly. This investigation was supported by the Cancer Chemotherapy National Service Center, National Cancer Institute, under the National Institutes of Health Contract Number SA-43-ph-I886.
Merck Institute/or Therapeutic Research, Rahway, N.J. (U.S.A.)
THOMAS M. DEVLIN BERNICE H. BEDELL
1 A. L. LEHNI•GER, J. Biol. Chem., 19o (1951) 345. A. L. LEHNINGER, Harvey Lectures, 49 (1953-54) 176. 3 B. CHANCE AND G. R. WILLIAMS, J. Biol. Chem., 217 (1955) 4o9. 4 M. E. PULLMAN AND E. RACKER, Science, 123 (1956) 11o5. 5 L. ERNSTER, O. JALLING, H. L~W AND O. LINDB~RG, Exp. Cell Res., Suppl., 3 (I955) 124. e G. MALEY, J. Biol. Chem., 224 (1957) lO29. A. NOVlKOFF, in Mitochondria and Other Cytoplasmic Inclusions, A c a d e m i c Press, Inc., N e w York, 1957, p. 92. 8 G. H. HOGEBOOM, W. C. SCHNEIDER AND G. E. PALLADE, J. Biol. Chem., 172 (1948) 619. 9 0 . H. LOWRY AND J. A. LOPEZ, J. Biol. Chem., 162 (1946) 421. 10 A. L. LEHNINGER AND G. D. GREVlLLE, Biochim. Biophys. Acta, 12 (1953) 188. 11 A. M. STEIN, N. O. KAPLAN AND M. CIOTTI, J. Biol. Chem., 234 (1959) 979.
Received August 3Ist, 1959