/ . Biochem., 81, 911-921 (1977)
Analysis of Reverse Acceptor Control in Mitochondria1
Takeshi TSUJIMOTO Department of Physiology, Wakayama Medical College, 9-bancho, Wakayama, Wakayama 640 Received for publication, July 27, 1976
The reduction of NAD{P) by ADP, a reversal of the normal change, in rat kidney mitochondria incubated in isotonic media containing NaCl and EDTA was studied by dual-wavelength spectrophotometry. Dual-wavelength spectrophotometry and respiratory studies showed that with increasing concentrations of NaCl and EDTA, rapid respiratory rate and marked oxidation of NAD(P) became evident in state 4, respiratory stimulation by ADP was reduced considerably, and the oxidation-reduction pattern of NAD(P) by ADP was converted to a diminished initial oxidation and subsequent reductive-reoxidative changes. Spectroscopic and enzymatic assays confirmed that the NAD(P) reductive change by ADP observed in dualwavelength spectrophotometry appeared to coincide with a remarkable swelling-shrinkage reaction larger in magnitude than the diminished oxidation of NAD by ADP. Under experimental conditions which might involve a significant swelling-shrinkage reaction, the redox kinetics observed by dual-wavelength spectrophotometry do no show the true NAD + : NADH change. The marked swelling-shrinkage was ascribed to an increase of ionic permeability due to the chelation by EDTA of membrane-bound magnesium with loss of potassium and of the integrity of mitochondria. ADP-induced inhibition of respiration andreduction of NAD, which will be designated here as "true reverse acceptor control," was not observed. Instead, the rapid respiratory rate in state 4, the decline of respiratory control, oxidized NAD in state 4, inhibition of further oxidation of NAD in state 3, and the small phosphorylation deficiency observed in the NaCl-EDTA media, suggested that a leaky or loosely coupling state rather than uncoupling led to the apparent reduction.
An insight into the mechanism of ionic selectivity exhibited by biological membranes might lead to a better understanding of the origin of such pheno-
mena as selective ion transport, accumulation or exclusion of ions, or resting membrane potentials and action potentials (1, 2). The selectivities of biological membranes towards Na+ and K+ are particularly important. * The purchase of a Hitachi 356 spectrophotometer was ^ ^ ^ ^ j ^ d i f f e r e n c e s l n t h e actions of the supported in part by a "grant for public school research . , . . . . . ... • mm r ,L w . . . _, two ions on rrutochondnal oxidative phosphorylequipment" in 1969 from the Ministry of Education, . . . . „ . . a t l 0 n and r e I a t e d Science and Culture of Japan. ^actions are well documented, Abbreviations: ME, 0.3 M mannitol and 1 mM EDTA, t h e mechanism is not still clear (3-7). pH 7.4; MT, 0.3 M mannitol and 5 mM Tris-Cl, pH 7.4; T ^ report describes a study on one aspect of EGTA, ethylene glycol bis(j9-aminoethyl ether)-N, N'- Na+ action on mitochondrial membranes, and is a tetraacetate. reinvestigation of a recent report by Weiner and Vol. 81, No. 4, 1977 911
912
T. TSUJIMOTO
Lardy (8), in which they described phenomena similar to the " reverse acceptor control" of Lehninger and Gregg (9), using rat kidney cortex mitochondria incubated in an isotonic solution containing NaCI, EDTA, and Pj: these were pronounced and rapid reduction of pyndine nucleotides associated with a slowing of respiration by ADP. Attention was focused on the reductive change of NAD by ADP. It is well known that the swelling-shrinkage reaction of mitochondria and the oxidation-reduction kinetics of respiratory carriers are linked {10-12), and the swelling reaction was expected to occur in media containing NaCI and EDTA. It has been shown that the reductive change of NAD(P) by ADP observed by dual-wavelength spectrophotometry is an apparent phenomenon due to the influence of a remarkable swelling-shrinkage reaction. Reduced oxidation of NAD by ADP accentuates the phenomenon. Loss of respiratory control was found in NaCl-EDTA media, but real reduction of NAD and slowing of respiration by ADP were not observed. Characteristic states of respiration and phosphorylation in NaCl-EDTA media are discussed in relation to the mechanism of sodium action. MATERIALS AND METHODS Male Wistar rats (200-300 g) starved overnight were stunned and exsanguinated. Kidneys were quickly removed and placed in ice-cold ME medium (0.3 M mannitol, 1 mM EDTA, pH 7.4). Decapsulation with a paper towel was carried out, followed by mid-sagittal section, removal of papilla and medulla (inner white and outer red), mincing, and washing. After squeezing in a press, a 10% homogenate in ME medium was prepared with a Potter-Elvehjem homogenizer. The homogenate was centrifuged at 700 xg for 6 min, and the supernatant was centrifuged for lOmin at 5,000xg. Care was taken to wipe off lipid adhering to the centrifuge tube, and to rinse out the fluffy layer with MT medium (0.3 M mannitol, 5 mM Tris-Cl, pH 7.4) after each centrifugation. The pellets were washed twice by resuspension in MT medium with a Nylon brush, with centrifugation for 10 min at 5,500 x g. The preparation was suspended in MT medium. Protein con-
centration was estimated by the biuret method (13). Oxygen consumption and dual-wavelength kinetics or swelling-shrinkage reaction (absorbance change at a single wavelength) were simultaneously recorded with a combination of a Clark-type membrane electrode and Hitachi 356 double-beam dual-wavelength spectrophotometer (14). The reaction vessel, made by the author, was a readily disassembled air-tight cell, composed of a rectangular quartz cell (light path, 15 mm) and a cap made from polyacrylate: the latter was equipped with a hole for adding reagents (inner diameter, 0.5 mm; length, 20 mm; with a side arm for suction), a stirring rod (obliquely inserted, DC motor driven), and an O, electrode (vertically inserted, made by the author). The cell and cap were held together by a rubber seal, and fixed into a thermostatic compartment with a bolt and nut. Total volume was kept to 5.2 ml by suction from the side arm. The pH and oxygen consumption were simultaneously recorded with a combination of a Clarktype electrode (Yellow Springs Instrument) and a Radiometer 26c pH meter equipped with a GK 2321 combined electrode and a bucking voltage source (15). The reaction vessel, made by the author, was a readily disassembled air-tight cell, composed of a cap made polycarbonate rod and a cylindrical glass tube: the latter was fixed to a thermostatic vessel; the Oa electrode was inserted into the side with a magnetic stirrer on the bottom. The cap was inserted into the cylindrical glass tube, which was equipped with a hole for adding reagents (inner diameter, 2.5 mm; height, 5 cm; with a side arm for suction 1 cm beneath the top) and an H + electrode (vertically inserted). O-ring seals were used between all parts. Total volume was kept to 6 ml by suction from the side arm. Estimation of ATP synthesis was carried out according to Nishimura et al. (16). Buffering capacity was assumed based on the internal HC1 standard method. A value of 0.89 was used for n in the following equation. ADP- 3
IT
ADP-*
Pi"1 +
ATP' 4
+ IT1 + «H = IT Pr
+H.0
ATP-»
Enzymatic assays of NADH : NAD+ ratio (17) or ATP/ADP/AMP concentrations (18) were done with extracted samples using enzymes from Boehrin/. Biochem.
REVERSE ACCEPTOR CONTROL ger with a Beckman DU spectroscope (with a Gilford optical density converter) or a Hitachi 356 spectrophotometer. Mitochondrial space was assayed according to the procedure of Hunter and Brierley (19). Double labeling using ["H] water and [14C] mannitol or ["C] dextran was performed. After incubation, the mitochondrial pellet was recovered centrifugally, and extracted with 0.5 N HC1O4. The extract (0.2 ml) was mixed with 10 ml of Packard Instagel, and counted on an Aloka liquid scintillation spectrometer (LSC-651) equipped with a data processor (ACM-100). The labeled reagent was obtained from the Radiochemical Centre, Amersham. Metal content (Ca, Mg, and K) was assayed with a Nippon Jarrell-Ash atomic absorption spectrophotometer using a 0.1 N HC1 extract of the centrifugally recovered mitochondrial pellet. All chemicals used were of the highest purity available. Precautions such as the deionization of mannitol or adenine nucleotide with ion-exchange resin, and neutralization of reagents with Tris were taken to avoid cation contamination. All incubations were performed at 25°C, unless otherwise specified. RESULTS Mitochondria prepared in ME media and suspended in MT media showed well coupled respiration and usual kinetics of NAD(P) oxido-reduction. However, as shown in Fig. 1, state 4 respiration (as defined by Chance and Williams, 20) became faster and NAD(P) shifted to a more oxidized level when mannitol in the medium was replaced with NaQ and the total osmolarity was kept at 0.3. These findings are in accord with those of Weiner and Lardy (8), but separate additions of substrate and Pi in this study were carried out to clarify the situation. On increasing the concentration of NaQ, initial reduction of NAD(P) by glutamate-malate and subsequent reoxidative change by Pi (at 5 ITIM NaQ) became oxidative change followed by small reductive changes (at concentrations higher than 50 mM NaQ). The NAD(P) redox level shifted to a considerably more oxidized state. The usual sequence of oxidative-reductive change on ADP addition was not converted solely to a reductiveoxidative change, but the initial oxidative change always remained, even though it was markedly Vol. 81, No. 4, 1977
913
5min Fig. 1. Effect of NaCl on NAD(P) oxido-reduction and respiration. The media contained 5 mM Tris-Cl, 0.5 mM EDTA and various concentrations of NaCl: a, 5; b, 16; c, 50; d, 150 (mM). Total osmolarity was adjusted to 0.3 with mannitol. Total volume, 5.2 ml. Ph, 7.4. NAD(P) oxidoreduction (solid line) and respiration (dashed line) following additions of kidney mitochondria (1.0 mg protein/ml), glutamate-malate (3 mM), Pi (5 HIM) and ADP (108 ftu) are shown. Increase of ^M0-»74 representing NAD(P) reduction is indicated as downward deflection. Traces between 5 to lOmin after addition of glutamate-malate are deleted for the sake of brevity. Numerals beside the O, trace indicate the respiratory rate (natoms O/min/mg protein). suppressed by the increasing reductive tendency with increasing concentration of NaQ. State 3 respiration was significantly low in 0.15 M NaQ, but slowing of respiration by ADP (reverse acceptor control, Ref. 8) was not observed even when the loss of respiratory control was marked (in such cases, however, significant synthesis of ATP was observed, as described later). Replacing mannitol with K Q caused only a slight oxidative shift of NAD(P), slight reduction by ADP, and a slight decrease of respiratory control. Redox kinetics in the 0.15 M K Q media were similar to those shown in 16 mM NaQ+268mM mannitol (cf. Fig. 1). EDTA Requirement for the Appearance of NAD(P) Reductive Change by ADP in Dual-wave-
914
T. TSUJIMOTO
^MII /^\* a
/
l p i lADP
• '
1
( B)
-.
~
in
a
\ h
/
y
^ ; V,
~T
w
i
^
1 "
5 A 3 co -Log [EDTA or EGTA,MJ
i
EDTA " "^
1"°
5 4 3 -LogCEDTAorEGTA.M]
Fig. 3. Effect of EDTA or EGTA on swelling in mannitol media. The ordinate indicates the reciprocal of initial swelling rate at 650 nm in 0.25 osmolar manX nitol containing 5 mM Tris-Cl, 3.2 mM Na succmate, X X 4.8 ftu rotenone, EDTA or EGTA as indicated on the abscissa, and kidney mitochondria (0.93 mg protein/ml), :e 5min at pH 7.4, 22°C. Swelling was initiated by the addition Fig. 2. Effect of EDTA on NADfP) oxido-reduction of Na acetate (27 3 mM). Left figure (A); mitochondria and respiration in NaCl media. The media contained prepared by homogenization in media containing 1 mM 0.15 M NaCl, 5 mM Tns-Cl and various concentrations of EDTA and washed twice with EDTA-free mannitol were used. Right figure: (B) mitochondria prepared by EDTA. a, 0; b, 15; c, 29; d, 59; e, 472 (//M). NADfP) oxido (upward deflection)-reduction (downward deflec- homogenization in media containing 1 mM EGTA and tion) and respiration (dashed line) following addition of washed twice with EGTA-free mannitol were used. reagents at the same concentrations as in Fig. 1 are EDTA effect: solid line. EGTA effect- dashed line. shown.
12). EDTA, a prerequisite for the appearance of length Spectrophotometry—The results in Fig. 1 NAD(P) reduction, has been claimed to cause an were obtained in the presence of 0.5 mM EDTA. increase of mitochondrial permeability to monoAs shown in Fig. 2, however, as little as 30 /JM valenf cations (21-23). EDTA induced reoxidation of NAD(P) following ^Figure 3 shows the effects of EDTA (solid line) reduction with substrate, reductive change after and EGTA (dashed line) on the swelling of mitooxidative change with Pi, acceleration of state 4 chondria (represented by the reciprocal of initial respiration, and reductive change on adding ADP. swelling rate) using either 1 mM EDTA (Fig. 3A) The presence of less than 29 /JM EDTA caused a or EGTA (Fig. 3B) preparations. EDTA accelergradual inhibition of state 4 respiration with marked ated the swelling except at around 10 fiM, but reoxidation of NAD(P), marked oxidation with Pi, EGTA inhibited the swelling. The concentration and loss of respiratory stimulation on adding ADP. of EDTA which inhibited the swelling of EDTA Without EDTA, a highly damped oscillatory change preparation (Fig. 3A) was lower than in the EGTA of NAD(P) and gradual inhibition of respiration preparation (Fig. 3B), and the concentration of were seen. On the other hand, EGTA at similar EGTA which maximally inhibited the swelling of concentrations did not induce loss of respiratory EDTA preparation (Fig. 3A) was slightly higher control, and could not substitute for EDTA. than in the EGTA preparation (Fig. 3B), indicating Influence of Swelling-Shrinkage Phenomena on that EDTA remained in the preparation even after Dual-Wavelength Spectrophotometry—Possible ef- two washings with EDTA-free mannitol: a separate fects of the mitochondrial swelling-shrinkage reac- experiment in our laboratory indicated some tion following the transition between different states residual binding (0.7 ng EDTA/mg protein) (24). of metabolism on the spectrophotometry of reFigure 4 shows the absorbance change at 520 spiratory pigments have been well documented (11, nm of a mitochondrial suspension in NaCl media J. Biochem.
mption
REVERSE ACCEPTOR CONTROL GlutMaK
nsuo
/ X,
^
915
/
Glut-Mal|Pi lADP
/
r^ a
1 O
\A
^—
Pit
d
V V
Analysis of Reverse Acceptor Control in Mitochondria1
Takeshi TSUJIMOTO Department of Physiology, Wakayama Medical College, 9-bancho, Wakayama, Wakayama 640 Received for publication, July 27, 1976
The reduction of NAD{P) by ADP, a reversal of the normal change, in rat kidney mitochondria incubated in isotonic media containing NaCl and EDTA was studied by dual-wavelength spectrophotometry. Dual-wavelength spectrophotometry and respiratory studies showed that with increasing concentrations of NaCl and EDTA, rapid respiratory rate and marked oxidation of NAD(P) became evident in state 4, respiratory stimulation by ADP was reduced considerably, and the oxidation-reduction pattern of NAD(P) by ADP was converted to a diminished initial oxidation and subsequent reductive-reoxidative changes. Spectroscopic and enzymatic assays confirmed that the NAD(P) reductive change by ADP observed in dualwavelength spectrophotometry appeared to coincide with a remarkable swelling-shrinkage reaction larger in magnitude than the diminished oxidation of NAD by ADP. Under experimental conditions which might involve a significant swelling-shrinkage reaction, the redox kinetics observed by dual-wavelength spectrophotometry do no show the true NAD + : NADH change. The marked swelling-shrinkage was ascribed to an increase of ionic permeability due to the chelation by EDTA of membrane-bound magnesium with loss of potassium and of the integrity of mitochondria. ADP-induced inhibition of respiration andreduction of NAD, which will be designated here as "true reverse acceptor control," was not observed. Instead, the rapid respiratory rate in state 4, the decline of respiratory control, oxidized NAD in state 4, inhibition of further oxidation of NAD in state 3, and the small phosphorylation deficiency observed in the NaCl-EDTA media, suggested that a leaky or loosely coupling state rather than uncoupling led to the apparent reduction.
An insight into the mechanism of ionic selectivity exhibited by biological membranes might lead to a better understanding of the origin of such pheno-
mena as selective ion transport, accumulation or exclusion of ions, or resting membrane potentials and action potentials (1, 2). The selectivities of biological membranes towards Na+ and K+ are particularly important. * The purchase of a Hitachi 356 spectrophotometer was ^ ^ ^ ^ j ^ d i f f e r e n c e s l n t h e actions of the supported in part by a "grant for public school research . , . . . . . ... • mm r ,L w . . . _, two ions on rrutochondnal oxidative phosphorylequipment" in 1969 from the Ministry of Education, . . . . „ . . a t l 0 n and r e I a t e d Science and Culture of Japan. ^actions are well documented, Abbreviations: ME, 0.3 M mannitol and 1 mM EDTA, t h e mechanism is not still clear (3-7). pH 7.4; MT, 0.3 M mannitol and 5 mM Tris-Cl, pH 7.4; T ^ report describes a study on one aspect of EGTA, ethylene glycol bis(j9-aminoethyl ether)-N, N'- Na+ action on mitochondrial membranes, and is a tetraacetate. reinvestigation of a recent report by Weiner and Vol. 81, No. 4, 1977 911
912
T. TSUJIMOTO
Lardy (8), in which they described phenomena similar to the " reverse acceptor control" of Lehninger and Gregg (9), using rat kidney cortex mitochondria incubated in an isotonic solution containing NaCI, EDTA, and Pj: these were pronounced and rapid reduction of pyndine nucleotides associated with a slowing of respiration by ADP. Attention was focused on the reductive change of NAD by ADP. It is well known that the swelling-shrinkage reaction of mitochondria and the oxidation-reduction kinetics of respiratory carriers are linked {10-12), and the swelling reaction was expected to occur in media containing NaCI and EDTA. It has been shown that the reductive change of NAD(P) by ADP observed by dual-wavelength spectrophotometry is an apparent phenomenon due to the influence of a remarkable swelling-shrinkage reaction. Reduced oxidation of NAD by ADP accentuates the phenomenon. Loss of respiratory control was found in NaCl-EDTA media, but real reduction of NAD and slowing of respiration by ADP were not observed. Characteristic states of respiration and phosphorylation in NaCl-EDTA media are discussed in relation to the mechanism of sodium action. MATERIALS AND METHODS Male Wistar rats (200-300 g) starved overnight were stunned and exsanguinated. Kidneys were quickly removed and placed in ice-cold ME medium (0.3 M mannitol, 1 mM EDTA, pH 7.4). Decapsulation with a paper towel was carried out, followed by mid-sagittal section, removal of papilla and medulla (inner white and outer red), mincing, and washing. After squeezing in a press, a 10% homogenate in ME medium was prepared with a Potter-Elvehjem homogenizer. The homogenate was centrifuged at 700 xg for 6 min, and the supernatant was centrifuged for lOmin at 5,000xg. Care was taken to wipe off lipid adhering to the centrifuge tube, and to rinse out the fluffy layer with MT medium (0.3 M mannitol, 5 mM Tris-Cl, pH 7.4) after each centrifugation. The pellets were washed twice by resuspension in MT medium with a Nylon brush, with centrifugation for 10 min at 5,500 x g. The preparation was suspended in MT medium. Protein con-
centration was estimated by the biuret method (13). Oxygen consumption and dual-wavelength kinetics or swelling-shrinkage reaction (absorbance change at a single wavelength) were simultaneously recorded with a combination of a Clark-type membrane electrode and Hitachi 356 double-beam dual-wavelength spectrophotometer (14). The reaction vessel, made by the author, was a readily disassembled air-tight cell, composed of a rectangular quartz cell (light path, 15 mm) and a cap made from polyacrylate: the latter was equipped with a hole for adding reagents (inner diameter, 0.5 mm; length, 20 mm; with a side arm for suction), a stirring rod (obliquely inserted, DC motor driven), and an O, electrode (vertically inserted, made by the author). The cell and cap were held together by a rubber seal, and fixed into a thermostatic compartment with a bolt and nut. Total volume was kept to 5.2 ml by suction from the side arm. The pH and oxygen consumption were simultaneously recorded with a combination of a Clarktype electrode (Yellow Springs Instrument) and a Radiometer 26c pH meter equipped with a GK 2321 combined electrode and a bucking voltage source (15). The reaction vessel, made by the author, was a readily disassembled air-tight cell, composed of a cap made polycarbonate rod and a cylindrical glass tube: the latter was fixed to a thermostatic vessel; the Oa electrode was inserted into the side with a magnetic stirrer on the bottom. The cap was inserted into the cylindrical glass tube, which was equipped with a hole for adding reagents (inner diameter, 2.5 mm; height, 5 cm; with a side arm for suction 1 cm beneath the top) and an H + electrode (vertically inserted). O-ring seals were used between all parts. Total volume was kept to 6 ml by suction from the side arm. Estimation of ATP synthesis was carried out according to Nishimura et al. (16). Buffering capacity was assumed based on the internal HC1 standard method. A value of 0.89 was used for n in the following equation. ADP- 3
IT
ADP-*
Pi"1 +
ATP' 4
+ IT1 + «H = IT Pr
+H.0
ATP-»
Enzymatic assays of NADH : NAD+ ratio (17) or ATP/ADP/AMP concentrations (18) were done with extracted samples using enzymes from Boehrin/. Biochem.
REVERSE ACCEPTOR CONTROL ger with a Beckman DU spectroscope (with a Gilford optical density converter) or a Hitachi 356 spectrophotometer. Mitochondrial space was assayed according to the procedure of Hunter and Brierley (19). Double labeling using ["H] water and [14C] mannitol or ["C] dextran was performed. After incubation, the mitochondrial pellet was recovered centrifugally, and extracted with 0.5 N HC1O4. The extract (0.2 ml) was mixed with 10 ml of Packard Instagel, and counted on an Aloka liquid scintillation spectrometer (LSC-651) equipped with a data processor (ACM-100). The labeled reagent was obtained from the Radiochemical Centre, Amersham. Metal content (Ca, Mg, and K) was assayed with a Nippon Jarrell-Ash atomic absorption spectrophotometer using a 0.1 N HC1 extract of the centrifugally recovered mitochondrial pellet. All chemicals used were of the highest purity available. Precautions such as the deionization of mannitol or adenine nucleotide with ion-exchange resin, and neutralization of reagents with Tris were taken to avoid cation contamination. All incubations were performed at 25°C, unless otherwise specified. RESULTS Mitochondria prepared in ME media and suspended in MT media showed well coupled respiration and usual kinetics of NAD(P) oxido-reduction. However, as shown in Fig. 1, state 4 respiration (as defined by Chance and Williams, 20) became faster and NAD(P) shifted to a more oxidized level when mannitol in the medium was replaced with NaQ and the total osmolarity was kept at 0.3. These findings are in accord with those of Weiner and Lardy (8), but separate additions of substrate and Pi in this study were carried out to clarify the situation. On increasing the concentration of NaQ, initial reduction of NAD(P) by glutamate-malate and subsequent reoxidative change by Pi (at 5 ITIM NaQ) became oxidative change followed by small reductive changes (at concentrations higher than 50 mM NaQ). The NAD(P) redox level shifted to a considerably more oxidized state. The usual sequence of oxidative-reductive change on ADP addition was not converted solely to a reductiveoxidative change, but the initial oxidative change always remained, even though it was markedly Vol. 81, No. 4, 1977
913
5min Fig. 1. Effect of NaCl on NAD(P) oxido-reduction and respiration. The media contained 5 mM Tris-Cl, 0.5 mM EDTA and various concentrations of NaCl: a, 5; b, 16; c, 50; d, 150 (mM). Total osmolarity was adjusted to 0.3 with mannitol. Total volume, 5.2 ml. Ph, 7.4. NAD(P) oxidoreduction (solid line) and respiration (dashed line) following additions of kidney mitochondria (1.0 mg protein/ml), glutamate-malate (3 mM), Pi (5 HIM) and ADP (108 ftu) are shown. Increase of ^M0-»74 representing NAD(P) reduction is indicated as downward deflection. Traces between 5 to lOmin after addition of glutamate-malate are deleted for the sake of brevity. Numerals beside the O, trace indicate the respiratory rate (natoms O/min/mg protein). suppressed by the increasing reductive tendency with increasing concentration of NaQ. State 3 respiration was significantly low in 0.15 M NaQ, but slowing of respiration by ADP (reverse acceptor control, Ref. 8) was not observed even when the loss of respiratory control was marked (in such cases, however, significant synthesis of ATP was observed, as described later). Replacing mannitol with K Q caused only a slight oxidative shift of NAD(P), slight reduction by ADP, and a slight decrease of respiratory control. Redox kinetics in the 0.15 M K Q media were similar to those shown in 16 mM NaQ+268mM mannitol (cf. Fig. 1). EDTA Requirement for the Appearance of NAD(P) Reductive Change by ADP in Dual-wave-
914
T. TSUJIMOTO
^MII /^\* a
/
l p i lADP
• '
1
( B)
-.
~
in
a
\ h
/
y
^ ; V,
~T
w
i
^
1 "
5 A 3 co -Log [EDTA or EGTA,MJ
i
EDTA " "^
1"°
5 4 3 -LogCEDTAorEGTA.M]
Fig. 3. Effect of EDTA or EGTA on swelling in mannitol media. The ordinate indicates the reciprocal of initial swelling rate at 650 nm in 0.25 osmolar manX nitol containing 5 mM Tris-Cl, 3.2 mM Na succmate, X X 4.8 ftu rotenone, EDTA or EGTA as indicated on the abscissa, and kidney mitochondria (0.93 mg protein/ml), :e 5min at pH 7.4, 22°C. Swelling was initiated by the addition Fig. 2. Effect of EDTA on NADfP) oxido-reduction of Na acetate (27 3 mM). Left figure (A); mitochondria and respiration in NaCl media. The media contained prepared by homogenization in media containing 1 mM 0.15 M NaCl, 5 mM Tns-Cl and various concentrations of EDTA and washed twice with EDTA-free mannitol were used. Right figure: (B) mitochondria prepared by EDTA. a, 0; b, 15; c, 29; d, 59; e, 472 (//M). NADfP) oxido (upward deflection)-reduction (downward deflec- homogenization in media containing 1 mM EGTA and tion) and respiration (dashed line) following addition of washed twice with EGTA-free mannitol were used. reagents at the same concentrations as in Fig. 1 are EDTA effect: solid line. EGTA effect- dashed line. shown.
12). EDTA, a prerequisite for the appearance of length Spectrophotometry—The results in Fig. 1 NAD(P) reduction, has been claimed to cause an were obtained in the presence of 0.5 mM EDTA. increase of mitochondrial permeability to monoAs shown in Fig. 2, however, as little as 30 /JM valenf cations (21-23). EDTA induced reoxidation of NAD(P) following ^Figure 3 shows the effects of EDTA (solid line) reduction with substrate, reductive change after and EGTA (dashed line) on the swelling of mitooxidative change with Pi, acceleration of state 4 chondria (represented by the reciprocal of initial respiration, and reductive change on adding ADP. swelling rate) using either 1 mM EDTA (Fig. 3A) The presence of less than 29 /JM EDTA caused a or EGTA (Fig. 3B) preparations. EDTA accelergradual inhibition of state 4 respiration with marked ated the swelling except at around 10 fiM, but reoxidation of NAD(P), marked oxidation with Pi, EGTA inhibited the swelling. The concentration and loss of respiratory stimulation on adding ADP. of EDTA which inhibited the swelling of EDTA Without EDTA, a highly damped oscillatory change preparation (Fig. 3A) was lower than in the EGTA of NAD(P) and gradual inhibition of respiration preparation (Fig. 3B), and the concentration of were seen. On the other hand, EGTA at similar EGTA which maximally inhibited the swelling of concentrations did not induce loss of respiratory EDTA preparation (Fig. 3A) was slightly higher control, and could not substitute for EDTA. than in the EGTA preparation (Fig. 3B), indicating Influence of Swelling-Shrinkage Phenomena on that EDTA remained in the preparation even after Dual-Wavelength Spectrophotometry—Possible ef- two washings with EDTA-free mannitol: a separate fects of the mitochondrial swelling-shrinkage reac- experiment in our laboratory indicated some tion following the transition between different states residual binding (0.7 ng EDTA/mg protein) (24). of metabolism on the spectrophotometry of reFigure 4 shows the absorbance change at 520 spiratory pigments have been well documented (11, nm of a mitochondrial suspension in NaCl media J. Biochem.
mption
REVERSE ACCEPTOR CONTROL GlutMaK
nsuo
/ X,
^
915
/
Glut-Mal|Pi lADP
/
r^ a
1 O
\A
^—
Pit
d
V V
