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Thyroxine-enhanced oxidative phosphorylation of rat-liver mitochondria
188
PRELIMINARY NOTES
Thyroxine-enhanced oxidative phosphorylation of rat-liver mitochondria During the past ten years m a n y investigators have reported that thyroxine was capable of uncoupling mitochondrial oxidative phosphorylation. Recently BRONK1 reported that thyroxine-treated mitochondrial "digitonin-particles" yielded elevated P/O ratios. In the present communication we shall report our findings concerning the effect of varying the concentration of L-thyroxine on the P/O ratio of intact rat-liver mitochondria. We have found that lO -4 to IO-s M L-thyroxine uncouples oxidative phosphorylation. Also we have found that lO -5 M L-thyroxine elevates the P/O ratio about 60 % when fl-hydroxybutyrate was the substrate and about IOO % when reduced cytochrome c was the substrate. Liver mitochondria were isolated from normal male Sprague-Dawley rats by a standard procedure 2. The system that was used to study the effect of thyroxine on mitochondrial P/O ratios was almost identical to that described by TAPLEY AND COOPERs. Mitochondria were preincubated in hypotonic sucrose for IO min as an initial step in all experiments. Oxygen consumption was measured by the Warburg apparatus and phosphate esterification was determined by difference. In Fig. I it can be seen that " v e r y low" concentrations of L-thyroxine have essentially no effect on the P/O ratio resulting from fl-hydroxybutyrate oxidation. When the concentration of L-thyroxine was raised above lO -6 M the resulting P/O ratios were increased with a m a x i m u m at IO-~ M. As the concentration was further increased to lO -3 M, uncoupling was found to occur. The observed change in P/O ratios was always due to a change in the utilization of inorganic phosphate, oxidation remaining unchanged. When ascorbic acid-reduced cytochrome c was used as the substrate in essentially p O
o
3.fi
2.C
LC
I
10.7
10"6
I
I
I
10- s 10-4 10-3 i'L-THYROXINE] MOLES/LITER
Fig. I. Effect of L - t h y r o x i n e on rat-livel m i t o c h o n d r i a l P / O ratios. E&ch r e a c t i o n vessel contained, in a total vol. of 3 ml, o.045 # m o l e c y t o c h r o m e c, 3.o # m o l e s d i p h o s p h o p y r i d i n e nucleotide, 15.1 # m o l e s a d e n o s i n e d i p h o s p h a t e . 15,o # m o l e s MgCI 2, 30.0 # m o l e s N a F , 3 7 . 5 / , m o l e s KHzPO4, 4o.0 # m o l e s f l - h y d r o x y b u t y r a t e , I.O ml m i t o c h o n d r i a , s u s p e n d e d in o.i M t r i s ( h y d r o x y m e t h y l ) a m i n o m e t h a n e buffer, p H 7.4 c o n t a i n i n g a n a m o u n t derived f r o m 0. 5 g w e t w e i g h t of liver. ( + ) = Control a n d (o) = w i t h L-thyroxine.
Biochim. Biophys. Acta, 37 (196o) 188-189
189
PRELIMINARY NOTES
the same system it was found that lO -5 M L-thyroxine enhanced the P/O value from a normal value of o.55 to I.IO. Again, the increase was due only to increased inorganic phosphate utilization, oxidation remaining the same for the control and the thyroxinetreated system. It is concluded that within a fairly critical concentration range L-thyroxine causes a "tighter coupling" of the phosphorylative reactions associated with rat-liver mitochondrial oxidative phosphorylation. This increased "energetic efficiency" is coupled to the oxidation of ferrocytochrome c by oxygen. These results indicate that the maximum amount of mitochondrial adenosine triphosphate that can be formed by passage of a pair of electrons from D P N H to oxygen is not three but more likely /our molecules. This study was aided by grants from the American Heart Association, the National Science Foundation (G-II57), the U.S. Public Health Service (H-2Io2C) and the Kentucky Heart Association.
Department o/Biochemistry, University o[ Louisville School o/ Medicine, Louisville, Ky. (U.S.A.)
R. DUNCAN DALLAM R. BROOKS HOWARD
1 j . 1~. BRONK, Biochem. Biophys. Acre, 27 (1958) 667. R. D. DALLAM,Arch. Biochem. Biophys.. 77 (1957) 395. D. F. TAPLEY AND C. COOPER. J. Biol. Chem., 222 (1956) 341.
Received October I7th, 1959 Biochim. Biophys. Acre, 37 (z96°) I88-z89
Activation of iodine utilization in thyroid-gland homogenates by cytochrome c and quinones In a previous report we described the incorporation of zszI [iodide] into iodoproteins by cell-free preparations of sheep-thyroid tissue and the activation of these preparations by ravin cofactors *. The present communication presents the results of further experiments dealing, in particular, with the activation by cytochrome c and various quinones. Cell-free tissue preparations were prepared from sheep-thyroid glands and incubated with zslI- as described previously1. Cytochrome c was added to the incubation mixture in dry crystalline form and quinones in 20 ~1 of ethanol solution. The incubations were terminated by addition of propylthiouracil, and the samples were analyzed by chromatography on filter paper 2. The results shown in Table I indicate that the incorporation of lszI- into protein by the particulate (mitochondrial-microsomal) fraction was augmented by incubating with 0.0005 M FMN or menadione. Cytochrome c had no effect. Interestingly enough, when the particulate material was incubated in the presence of the soluble fraction (IOO,OOO × g supernatant), cytochrome c definitely activated lsZI-utilization; under these conditions FMN was inactive. Thus, the soluble fraction appears to contain a component(s) that is necessary for cytochrome c action and that blocks FMN action. Abbreviation: FMN, r a v i n mononucleotide.
Biochim. Biophys. Acre, 37 (196o) 189-191
PRELIMINARY NOTES
Thyroxine-enhanced oxidative phosphorylation of rat-liver mitochondria During the past ten years m a n y investigators have reported that thyroxine was capable of uncoupling mitochondrial oxidative phosphorylation. Recently BRONK1 reported that thyroxine-treated mitochondrial "digitonin-particles" yielded elevated P/O ratios. In the present communication we shall report our findings concerning the effect of varying the concentration of L-thyroxine on the P/O ratio of intact rat-liver mitochondria. We have found that lO -4 to IO-s M L-thyroxine uncouples oxidative phosphorylation. Also we have found that lO -5 M L-thyroxine elevates the P/O ratio about 60 % when fl-hydroxybutyrate was the substrate and about IOO % when reduced cytochrome c was the substrate. Liver mitochondria were isolated from normal male Sprague-Dawley rats by a standard procedure 2. The system that was used to study the effect of thyroxine on mitochondrial P/O ratios was almost identical to that described by TAPLEY AND COOPERs. Mitochondria were preincubated in hypotonic sucrose for IO min as an initial step in all experiments. Oxygen consumption was measured by the Warburg apparatus and phosphate esterification was determined by difference. In Fig. I it can be seen that " v e r y low" concentrations of L-thyroxine have essentially no effect on the P/O ratio resulting from fl-hydroxybutyrate oxidation. When the concentration of L-thyroxine was raised above lO -6 M the resulting P/O ratios were increased with a m a x i m u m at IO-~ M. As the concentration was further increased to lO -3 M, uncoupling was found to occur. The observed change in P/O ratios was always due to a change in the utilization of inorganic phosphate, oxidation remaining unchanged. When ascorbic acid-reduced cytochrome c was used as the substrate in essentially p O
o
3.fi
2.C
LC
I
10.7
10"6
I
I
I
10- s 10-4 10-3 i'L-THYROXINE] MOLES/LITER
Fig. I. Effect of L - t h y r o x i n e on rat-livel m i t o c h o n d r i a l P / O ratios. E&ch r e a c t i o n vessel contained, in a total vol. of 3 ml, o.045 # m o l e c y t o c h r o m e c, 3.o # m o l e s d i p h o s p h o p y r i d i n e nucleotide, 15.1 # m o l e s a d e n o s i n e d i p h o s p h a t e . 15,o # m o l e s MgCI 2, 30.0 # m o l e s N a F , 3 7 . 5 / , m o l e s KHzPO4, 4o.0 # m o l e s f l - h y d r o x y b u t y r a t e , I.O ml m i t o c h o n d r i a , s u s p e n d e d in o.i M t r i s ( h y d r o x y m e t h y l ) a m i n o m e t h a n e buffer, p H 7.4 c o n t a i n i n g a n a m o u n t derived f r o m 0. 5 g w e t w e i g h t of liver. ( + ) = Control a n d (o) = w i t h L-thyroxine.
Biochim. Biophys. Acta, 37 (196o) 188-189
189
PRELIMINARY NOTES
the same system it was found that lO -5 M L-thyroxine enhanced the P/O value from a normal value of o.55 to I.IO. Again, the increase was due only to increased inorganic phosphate utilization, oxidation remaining the same for the control and the thyroxinetreated system. It is concluded that within a fairly critical concentration range L-thyroxine causes a "tighter coupling" of the phosphorylative reactions associated with rat-liver mitochondrial oxidative phosphorylation. This increased "energetic efficiency" is coupled to the oxidation of ferrocytochrome c by oxygen. These results indicate that the maximum amount of mitochondrial adenosine triphosphate that can be formed by passage of a pair of electrons from D P N H to oxygen is not three but more likely /our molecules. This study was aided by grants from the American Heart Association, the National Science Foundation (G-II57), the U.S. Public Health Service (H-2Io2C) and the Kentucky Heart Association.
Department o/Biochemistry, University o[ Louisville School o/ Medicine, Louisville, Ky. (U.S.A.)
R. DUNCAN DALLAM R. BROOKS HOWARD
1 j . 1~. BRONK, Biochem. Biophys. Acre, 27 (1958) 667. R. D. DALLAM,Arch. Biochem. Biophys.. 77 (1957) 395. D. F. TAPLEY AND C. COOPER. J. Biol. Chem., 222 (1956) 341.
Received October I7th, 1959 Biochim. Biophys. Acre, 37 (z96°) I88-z89
Activation of iodine utilization in thyroid-gland homogenates by cytochrome c and quinones In a previous report we described the incorporation of zszI [iodide] into iodoproteins by cell-free preparations of sheep-thyroid tissue and the activation of these preparations by ravin cofactors *. The present communication presents the results of further experiments dealing, in particular, with the activation by cytochrome c and various quinones. Cell-free tissue preparations were prepared from sheep-thyroid glands and incubated with zslI- as described previously1. Cytochrome c was added to the incubation mixture in dry crystalline form and quinones in 20 ~1 of ethanol solution. The incubations were terminated by addition of propylthiouracil, and the samples were analyzed by chromatography on filter paper 2. The results shown in Table I indicate that the incorporation of lszI- into protein by the particulate (mitochondrial-microsomal) fraction was augmented by incubating with 0.0005 M FMN or menadione. Cytochrome c had no effect. Interestingly enough, when the particulate material was incubated in the presence of the soluble fraction (IOO,OOO × g supernatant), cytochrome c definitely activated lsZI-utilization; under these conditions FMN was inactive. Thus, the soluble fraction appears to contain a component(s) that is necessary for cytochrome c action and that blocks FMN action. Abbreviation: FMN, r a v i n mononucleotide.
Biochim. Biophys. Acre, 37 (196o) 189-191