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The relationship between the temperature and the effect of 2,4 dinitrophenol on the swelling of isolated mitochondria
THE
RELATIONSHIP EFFECT
BETWEEN
QF ISOLATED 6. DI Istituto
THE TEMPERATURE
OF 2,4 DINITROPHENOL
di Patologia
gene&e
AND THE
ON THE S~~E~L~N~
MITOCHONDRIA1 SABAT02 dell’ Universita
di Milano,
Milan,
Italy
Received December 8. 1958
N a previous work 111it was observed that, as already noted by other authors f2-4, G-81, 2,4 dinitrophenol (DNP) inhibits the swelling of mitoehondria incubated a% temperatures between 23°C and 28°C in the absence of oxidizable substrates. Subsequent experiments showed that at 37°C DNP no longer protected the mitochondria from swelling. The present communication is concerned with a detailed analysis of the influence of the temperature on the inhibition by DNP of the swelling of mitochondria. The particles were incubated a% different temperatures both in phosphate buffer pH 7.2-Mg++ medium and in tris(hydroxymethyl)aminomethane (TRIS) buffer pH 7.2-sucrose medium. Neither of the incubation media adopted in this work contained oxidizable substrate, so that no oxidation or phosphorylation of any magnitude was occurring in the mitochondria. Experimental.-Adult albino rats of both sexes, weighing 200 to 250 g, were used throughout these experiments. The rats received a standard diet until 12-14 hours prior to the experiment, when they were killed by decapitation. The livers were quickly removed and cooled for three minutes in partially frozen 0.25 M sucrose. The procedure for isolating the mitochondria was very similar to that employed by Fonnesu and Davies [4], always washing the mitochondria twice. The technique used for measuring the mitochondrial swelling was as follows: 5 ml: of mitochondria suspended in 0.25 M sucrose (giving at 520 rnp an optical density of approximately 0.400 when diluted 30 times with cold 0.25 M sucrose) were pipetted into 100 ml conical flasks containing 10 ml of incubation medium. Soon after the addition of the mitochondria, a sample (1 ml) was removed, diluted with 9 volumes of 0.25 M sucrose kept a%the incubation temperatures (cf. Figs. 1-4) and the optical density determined in a Beckmann spectrophotometer mod. DU in cuvettes of 10 mm light path.. ‘The value of this first reading was taken as the zero time or initial optical density. The open flasks were shaken horizontally in a bath at different temperatures; at various interval of time, 1 ml samples were removed, diluted and read in the spectrophotometer, as indicated above. Results.-Fig. I shows that mitochondria incubated at different temperatm-es for 30 minutes in TRIS buffer-sucrose medium containing DNP lW3 M, IO-” M and 1 Aided by a grant from the Consiglio Nazionale delle Ricerche, Centro per la Patologia CelMare “P. Rondoni”, Milan0 (Italy). 2 Present address: Graduate Department of Biochemistry, Brandeis University, Waltham 54, Massachusetts, U.S.A. Experimental
Cell Research 16
G. Di Sabato
442
lO-5 M are greatly protected against swelling at temperatures ranging between 28°C and 37°C. The protective effect decreases above this temperature. After 60 minutes of incubation the greatest protection has been found at 28°C. At higher temperatures the protection from swelling by DNP decreases rapidly and disappears above 37°C (Fig. 2). The protection by DNP is of the same degree for the three molarities of DNP used.
q E :: m x d d
0.40
0.40
0.35
0.35
0.30
a E
0.30
l-2 m 0.25 x
0.25
0.20
0.15
0.10
0.15
I
I
o”
loo
TEMPERATURE
6
I
I
I
2o”
3o”
4o”
OF INCUBATION
Fig. 1.
0.10
I
o”
I
he
TEMPERATURE
2100
-
3o”
4100
OF INCUBATION
Fig. 2.
Fig. l.-Effect of DNP on the swelling of isolated rat liver mitochondria after 30 minutes of incubation at different temperatures in TRIS buffer-sucrose medium. The composition of the incubation medium (after the addition of the mitochondria) was as follows: 0.02 M TRIS buffer pH 7.2; 0.03 M sucrose; DNP Na pH 7.2 at the molarities indicated below. In the control flask DNP was substituted with water. Symbols: +, Control; 0, DNP 1OW 1M; V, DNP lo-” M, 0, DNP 1O-5 1M. For the other experimental conditions, see text. Fig. 2.-Effect of DNP on the swelling of isolated rat liver mitochondria after 60 minutes of incubation at different temperatures in TRIS buffer-sucrose medium. Medium composition and symbols as in Fig. 1. For the other experimental conditions, see text.
DNP shows a protective effect also on the mitochondria incubated in phosphate medium. After 30 minutes of incubation the protective effect reaches a buffer-Mg* maximum at a temperature ranging between 18°C and 28”C, then diminishes rapidly and disappears above 35°C (Fig. 3). After 60 minutes of incubation the protection reaches a maximum at 18°C; at 28°C or higher temperatures there is no protection (Fig. 4). The experiments carried out in phosphate buffer-Mg++ medium show that the protection of the mitochondria increases with the molarity of DNP. This could be due to an antagonism between phosphate and DNP. Indeed, incubating mitochondria at 28°C in TRIS buffer-sucrose medium in the presence of orthophosphate 0.001 M or 0.01 M and in the absence of Mg H, it has been found that the protective effect Experimental
Cell Research 16
DNP and mifochondrial
swelling
443
of DNP (1O-3 nia and IO-* M) is greater at the lower (0.001 M) than at the higher (0.01 A8) phosphate concentration. Some experiments have been carried out incubating mitochondria at different temperatures in TRIS buffer-sucrose medium in the presence of thyroxine PO-* M, 1OW M, and 1O-6 M. In our working conditions, thyroxine does not show any effect on the mitochondrial swelling at 18”C, while the swelling is enhanced at higher temperatures (28X, 37”C, 42°C). The swelling by thyroxine, when it occurs, is more evident in the first IO-20 minutes of incubation.
0.35
q E :: W x d d
0.30
0.25
0.20
0.15
O.lO-/ Q0
TEMPERATURE
OF INCUBATION
Fig. 3.
loo
2o”
TEMPERATURE
30°
4o”
OF iNCUBATlON, Fig.
4.
Fig. 3.-Effect of DNP on the swelling of isolated rat liver mitochondria after 30 minutes of incubation at different temperatures in phosphate buffer-Mg ++ medium. The composition of the ,incubation medium (after the addition of the mitochondria) was as follows: 0.005 M MgSD,; 0.01 M KH,PO,-K,HPO, buffer pH 7.2; 0.08 M sucrose; 0.02 Af NaCI; DNP Na pH 7.2 at the molarities indicated in Fig. 1. In the control flask DNP was substituted with water. Symbols as in Fig. I. For the other experimental conditions, see text. Fig. 4.-Effect of DNP on the swelling of isolated rat liver mitochondria after 60 minutes of incubation at different temperatures in phosphate buffer-I@ ++ medium. Medium composition as in Fig. 3. Symbols as in Fig. 1. For the other experimental conditions, see text.
Discussion.-The above results show clearly that the protective effect of DNP decreases with the increase of the temperature. Furthermore, if the incubation time is prolonged to 60 minutes, the protective effect of DNP disappears between 37°C and 42°C. The protective effect of DNP is dependent on the temperature either in TRIS buffer-sucrose medium or in phosphate buffer-Mg +&medium; in the last medium, however, the effect is less evident than in TRIS buffer-sucrose medium, probably because of an antagonism between phosphate and DNP. The protection of the swelling of the mitochondria exhibited by DNP confirm:~ Experimental
Cell Research 16
T. Huh
444
and Alexandra von der Decken
that the high energy phosphate bonds are not essential for the maintainance of the mitochondrial morphology. Since the test system used in the above experiments did not include oxidizable substrates, no oxidation or phosphorylation was occurring in the mitochondria. It is also to be noted that the concentrations of DNP used in these experiments are known to stimulate ATPase activity. The protective effect of DNP may be explained on the basis of the known affinity of DNP toward amino acids [5]. The drug could bind to the mitochondrial proteins and inhibit in this way the mitochondrial swelling. Prolonging the incubation time or increasing the temperature, these bonds would split off or would not be formed at all, so that the protective effect of DNP would be reduced or abolished. This is only a hypothesis which needs further experimental support. REFERENCES 1. DI SABATO, G. and FONNESU, A., In press. 2. EMMELOT, P. and Bos, C. J., ExpptI. Cell Research 12, 191 (1957). 3. FONNESU, A. and DAVIES, R. E., Biochem. J. 61, vi (1955).
4. __
ibid. 64, 769 (1956). F., Biochem. J. 39, 507 (1945). D. F., J. Biol. Chem. 222, 325 (1956).
5. SANGER, 6. TAPLEY, 7. TAPLEY, 8. WITTER,
THE
D. F., COOPER, C. and LEHNINGER, A. L., Biochim. et Biophys. Ada R. F. and COTTONE, M. A., Biochim. ef Biophys. Ada 22, 364 (1956).
TRANSFER
OF SOLUBLE
NUCLEIC T. HULTIN The Wenner-Gren
POLYNUCLEOTIDES
ACID OF RAT LIVER and ALEXANDRA
Institute for Experimental
TO THE
RIBO-
MICROSOMES
VON Biology,
18, 597 (1955).
DER DECKEN
University
of Stockholm,
Sweden
ReceivedDecember19, 1958
AN energy-dependent transfer of labeled polynucleotides to microsomal nucleoprotein particles by a cell-free rat liver system was described in a previous communication [l]. A similar observation has been briefly mentioned also by Hoagland [a]. A few additional characteristics of the transfer reaction will be given in this report. Experimental.-Rats weighing about 200 g were used. As in the previous experiments they were treated in vivo with [3zP] phosphate (2mc) or [14C] adenine (0.1 mc) for about 15 hours. Soluble liver or kidney fractions were prepared, and they were in most cases precipitated at pH 5 by the addition of acetic acid. After washing, the precipitates were dissolved in 0.1 M tris buffer, pH 7.8. Polynucleotides (“s-RNA”)l ’ Abbreviations:
ATP,
GTP,
CTP,
UTP,
adenosine,
guanosine, cytidine, uridine triphosphates; tris(hydroxymethyl)aminomethane;
PEP, phosphoenolpuruvic acid; RNA, ribonucleic acid; Tris, TCA,
trichloroacetic
Experimental
acid.
Cell Research 16
RELATIONSHIP EFFECT
BETWEEN
QF ISOLATED 6. DI Istituto
THE TEMPERATURE
OF 2,4 DINITROPHENOL
di Patologia
gene&e
AND THE
ON THE S~~E~L~N~
MITOCHONDRIA1 SABAT02 dell’ Universita
di Milano,
Milan,
Italy
Received December 8. 1958
N a previous work 111it was observed that, as already noted by other authors f2-4, G-81, 2,4 dinitrophenol (DNP) inhibits the swelling of mitoehondria incubated a% temperatures between 23°C and 28°C in the absence of oxidizable substrates. Subsequent experiments showed that at 37°C DNP no longer protected the mitochondria from swelling. The present communication is concerned with a detailed analysis of the influence of the temperature on the inhibition by DNP of the swelling of mitochondria. The particles were incubated a% different temperatures both in phosphate buffer pH 7.2-Mg++ medium and in tris(hydroxymethyl)aminomethane (TRIS) buffer pH 7.2-sucrose medium. Neither of the incubation media adopted in this work contained oxidizable substrate, so that no oxidation or phosphorylation of any magnitude was occurring in the mitochondria. Experimental.-Adult albino rats of both sexes, weighing 200 to 250 g, were used throughout these experiments. The rats received a standard diet until 12-14 hours prior to the experiment, when they were killed by decapitation. The livers were quickly removed and cooled for three minutes in partially frozen 0.25 M sucrose. The procedure for isolating the mitochondria was very similar to that employed by Fonnesu and Davies [4], always washing the mitochondria twice. The technique used for measuring the mitochondrial swelling was as follows: 5 ml: of mitochondria suspended in 0.25 M sucrose (giving at 520 rnp an optical density of approximately 0.400 when diluted 30 times with cold 0.25 M sucrose) were pipetted into 100 ml conical flasks containing 10 ml of incubation medium. Soon after the addition of the mitochondria, a sample (1 ml) was removed, diluted with 9 volumes of 0.25 M sucrose kept a%the incubation temperatures (cf. Figs. 1-4) and the optical density determined in a Beckmann spectrophotometer mod. DU in cuvettes of 10 mm light path.. ‘The value of this first reading was taken as the zero time or initial optical density. The open flasks were shaken horizontally in a bath at different temperatures; at various interval of time, 1 ml samples were removed, diluted and read in the spectrophotometer, as indicated above. Results.-Fig. I shows that mitochondria incubated at different temperatm-es for 30 minutes in TRIS buffer-sucrose medium containing DNP lW3 M, IO-” M and 1 Aided by a grant from the Consiglio Nazionale delle Ricerche, Centro per la Patologia CelMare “P. Rondoni”, Milan0 (Italy). 2 Present address: Graduate Department of Biochemistry, Brandeis University, Waltham 54, Massachusetts, U.S.A. Experimental
Cell Research 16
G. Di Sabato
442
lO-5 M are greatly protected against swelling at temperatures ranging between 28°C and 37°C. The protective effect decreases above this temperature. After 60 minutes of incubation the greatest protection has been found at 28°C. At higher temperatures the protection from swelling by DNP decreases rapidly and disappears above 37°C (Fig. 2). The protection by DNP is of the same degree for the three molarities of DNP used.
q E :: m x d d
0.40
0.40
0.35
0.35
0.30
a E
0.30
l-2 m 0.25 x
0.25
0.20
0.15
0.10
0.15
I
I
o”
loo
TEMPERATURE
6
I
I
I
2o”
3o”
4o”
OF INCUBATION
Fig. 1.
0.10
I
o”
I
he
TEMPERATURE
2100
-
3o”
4100
OF INCUBATION
Fig. 2.
Fig. l.-Effect of DNP on the swelling of isolated rat liver mitochondria after 30 minutes of incubation at different temperatures in TRIS buffer-sucrose medium. The composition of the incubation medium (after the addition of the mitochondria) was as follows: 0.02 M TRIS buffer pH 7.2; 0.03 M sucrose; DNP Na pH 7.2 at the molarities indicated below. In the control flask DNP was substituted with water. Symbols: +, Control; 0, DNP 1OW 1M; V, DNP lo-” M, 0, DNP 1O-5 1M. For the other experimental conditions, see text. Fig. 2.-Effect of DNP on the swelling of isolated rat liver mitochondria after 60 minutes of incubation at different temperatures in TRIS buffer-sucrose medium. Medium composition and symbols as in Fig. 1. For the other experimental conditions, see text.
DNP shows a protective effect also on the mitochondria incubated in phosphate medium. After 30 minutes of incubation the protective effect reaches a buffer-Mg* maximum at a temperature ranging between 18°C and 28”C, then diminishes rapidly and disappears above 35°C (Fig. 3). After 60 minutes of incubation the protection reaches a maximum at 18°C; at 28°C or higher temperatures there is no protection (Fig. 4). The experiments carried out in phosphate buffer-Mg++ medium show that the protection of the mitochondria increases with the molarity of DNP. This could be due to an antagonism between phosphate and DNP. Indeed, incubating mitochondria at 28°C in TRIS buffer-sucrose medium in the presence of orthophosphate 0.001 M or 0.01 M and in the absence of Mg H, it has been found that the protective effect Experimental
Cell Research 16
DNP and mifochondrial
swelling
443
of DNP (1O-3 nia and IO-* M) is greater at the lower (0.001 M) than at the higher (0.01 A8) phosphate concentration. Some experiments have been carried out incubating mitochondria at different temperatures in TRIS buffer-sucrose medium in the presence of thyroxine PO-* M, 1OW M, and 1O-6 M. In our working conditions, thyroxine does not show any effect on the mitochondrial swelling at 18”C, while the swelling is enhanced at higher temperatures (28X, 37”C, 42°C). The swelling by thyroxine, when it occurs, is more evident in the first IO-20 minutes of incubation.
0.35
q E :: W x d d
0.30
0.25
0.20
0.15
O.lO-/ Q0
TEMPERATURE
OF INCUBATION
Fig. 3.
loo
2o”
TEMPERATURE
30°
4o”
OF iNCUBATlON, Fig.
4.
Fig. 3.-Effect of DNP on the swelling of isolated rat liver mitochondria after 30 minutes of incubation at different temperatures in phosphate buffer-Mg ++ medium. The composition of the ,incubation medium (after the addition of the mitochondria) was as follows: 0.005 M MgSD,; 0.01 M KH,PO,-K,HPO, buffer pH 7.2; 0.08 M sucrose; 0.02 Af NaCI; DNP Na pH 7.2 at the molarities indicated in Fig. 1. In the control flask DNP was substituted with water. Symbols as in Fig. I. For the other experimental conditions, see text. Fig. 4.-Effect of DNP on the swelling of isolated rat liver mitochondria after 60 minutes of incubation at different temperatures in phosphate buffer-I@ ++ medium. Medium composition as in Fig. 3. Symbols as in Fig. 1. For the other experimental conditions, see text.
Discussion.-The above results show clearly that the protective effect of DNP decreases with the increase of the temperature. Furthermore, if the incubation time is prolonged to 60 minutes, the protective effect of DNP disappears between 37°C and 42°C. The protective effect of DNP is dependent on the temperature either in TRIS buffer-sucrose medium or in phosphate buffer-Mg +&medium; in the last medium, however, the effect is less evident than in TRIS buffer-sucrose medium, probably because of an antagonism between phosphate and DNP. The protection of the swelling of the mitochondria exhibited by DNP confirm:~ Experimental
Cell Research 16
T. Huh
444
and Alexandra von der Decken
that the high energy phosphate bonds are not essential for the maintainance of the mitochondrial morphology. Since the test system used in the above experiments did not include oxidizable substrates, no oxidation or phosphorylation was occurring in the mitochondria. It is also to be noted that the concentrations of DNP used in these experiments are known to stimulate ATPase activity. The protective effect of DNP may be explained on the basis of the known affinity of DNP toward amino acids [5]. The drug could bind to the mitochondrial proteins and inhibit in this way the mitochondrial swelling. Prolonging the incubation time or increasing the temperature, these bonds would split off or would not be formed at all, so that the protective effect of DNP would be reduced or abolished. This is only a hypothesis which needs further experimental support. REFERENCES 1. DI SABATO, G. and FONNESU, A., In press. 2. EMMELOT, P. and Bos, C. J., ExpptI. Cell Research 12, 191 (1957). 3. FONNESU, A. and DAVIES, R. E., Biochem. J. 61, vi (1955).
4. __
ibid. 64, 769 (1956). F., Biochem. J. 39, 507 (1945). D. F., J. Biol. Chem. 222, 325 (1956).
5. SANGER, 6. TAPLEY, 7. TAPLEY, 8. WITTER,
THE
D. F., COOPER, C. and LEHNINGER, A. L., Biochim. et Biophys. Ada R. F. and COTTONE, M. A., Biochim. ef Biophys. Ada 22, 364 (1956).
TRANSFER
OF SOLUBLE
NUCLEIC T. HULTIN The Wenner-Gren
POLYNUCLEOTIDES
ACID OF RAT LIVER and ALEXANDRA
Institute for Experimental
TO THE
RIBO-
MICROSOMES
VON Biology,
18, 597 (1955).
DER DECKEN
University
of Stockholm,
Sweden
ReceivedDecember19, 1958
AN energy-dependent transfer of labeled polynucleotides to microsomal nucleoprotein particles by a cell-free rat liver system was described in a previous communication [l]. A similar observation has been briefly mentioned also by Hoagland [a]. A few additional characteristics of the transfer reaction will be given in this report. Experimental.-Rats weighing about 200 g were used. As in the previous experiments they were treated in vivo with [3zP] phosphate (2mc) or [14C] adenine (0.1 mc) for about 15 hours. Soluble liver or kidney fractions were prepared, and they were in most cases precipitated at pH 5 by the addition of acetic acid. After washing, the precipitates were dissolved in 0.1 M tris buffer, pH 7.8. Polynucleotides (“s-RNA”)l ’ Abbreviations:
ATP,
GTP,
CTP,
UTP,
adenosine,
guanosine, cytidine, uridine triphosphates; tris(hydroxymethyl)aminomethane;
PEP, phosphoenolpuruvic acid; RNA, ribonucleic acid; Tris, TCA,
trichloroacetic
Experimental
acid.
Cell Research 16