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[87] Energy-coupling in nonphosphorylating submitochondrial particles
[87]
ENERGY-COUPLING IN UNCOUPLED PARTICLES
543
mercuric acetate 50~o at 10-8 M, and both are specifically inhibited by ADP but by no other nucleoside diphosphates. On the basis of these results it is tentatively concluded that both reactions are catalyzed by the same enzyme. The extraction and purification procedure described here has been found to give reproducible results providing the mitochondria are not damaged by exposure to hypotonie sucrose solutions or to salt solution of ionic strength in excess of 0.01. Attempts to utilize hypotonic media, strong salt solutions, or freezing and thawing resulted in the extraction of large amounts of adenylate kinase activity which introduces complications for assay of the specific ATP-ADP exchange enzyme activity.
[87] E n e r g y - C o u p l i n g in Nonphosphorylating Submitochondrial Particles
By
CHUAN-PU
L E E 1 a n d LARS E R N S T E R
Demonstration of Energy-Coupling The energy-linked pyridine nucleotide transhydrogenase reactionla (cf. this volume [113]) can be used as a tool for the demonstration 2 of energy-coupling in so-called "nonphosphorylating"submitochondrial electron transport particles. The approach is based on the concept that the energy-linked transhydrogenase reaction: NADH
+ N A D P + + I ~ X --* N A D + + N A D P H
-{-I + X
(I)
involves the utilization of a nonphosphorylated high-energy compound, denoted I ~ X, which is an intermediate of the respiratory chain-linked oxidative phosphorylation system: respiration
I+X , I,-,X I~XWADPWP~I+X+ATP
(2) (3)
(where I and X are hypothetie energy-transfer carriers). In phosphorylating submitochondrial particles, the energy-linked transhydrogenase reaction can be driven by either the respiratory chain, Reaction (2), or ATP, reversal of Reaction (3), at approximately equal capacities. In contrast, "nonphosphorylating" particles can derive energy for driving the energy-linked transhydrogenase reaction much less efficiently from 1See footnote 1, page 33. ~"L. Danielson and L. Ernster, Biochem. Biophys. Res. Commun. 10, 91 (1963); in "Energy-Linked Functions of Mitochondria" (B. Chance, ed.), p. 157. Academic Press, New York, 1963; Biochem. Z. 338, 188 (1963). C. P. Lee, G. F. Azzone, and L. Ernster, Nature t01, 152 (1964).
544
COVPLING FACTORS
[87]
ATP than from the respiratory chain. This is consistent with the conclusion that these particles still possess the capacity for generating I ~ X by respiratory energy-coupling (Reaction 2), although their phosphorylating capacity, i.e., their ability to utilize I ~ X for converting ADP and P~ into ATP (Reaction 3), is weakened or lost. It is also found 2 that in such particles oligomycin markedly stimulates, and may even be obligatory for, the respiratory chain-driven transhydrogenase reaction. Illustrative data are summarized in the table. The data shown in this table compare two types of phosphorylating particles with three types of "nonphosphorylating" particles. "Heavy" beef-heart mitochondria, prepared according to LSw and Vallin, ~ and stored in 250 m M sucrose suspension at --10 ° for at least 3 days, were used as the starting material for the various preparations. The general procedure for the preparation of submitochondrial particles was as follows: The frozen mitochondrial suspension was thawed, and diluted with 250 mM sucrose to contain about 20-30 mg of protein per milliliter. Depending on the type of preparation to be investigated, various reagents were added as will be specified below. The suspension was then saturated with N2 and subjected to sonic oscillation for 2 minutes at the maximal output in a 20-kc Raytheon sonicator cooled with running tap water (4-8°). The suspension was diluted with an equal volume of 250 mM sucrose and centrifuged at 12,000 g for 10 minutes. The supernatant fraction was decanted and centrifuged at 105,000 g for 40 minutes. The sediment was washed by homogenization with 10 volumes of 250 mM sucrose and centrifuged at 105,000 g for 40 minutes. The particles were finally suspended in 250 mM sucrose to give a protein concentration of about 20 mg/ml. A recovery of 20-30% of the mitochondrial protein was usually obtained. In the case of the phosphorylating particles studied, the sonicating medium consisted of either 5 mM MnC12, 10 mM MgS04 and 1 mM sodium succinate, pH 7.5 (preparation described by Smith and Hansen4), or 15 mM MgS04 and 1 m M ATP, pH 7.5 (ETPH preparation of Linnane and Ziegler5 as modified by LSw and VallinS). "Nonphosphorylating" particles were prepared in a sonicating medium consisting of either 2 mM EDTA, pH 7.5 ("modified" E T P H of Linnane and ZieglerS), or 20 m M NH40H (A-particles of Conover et al2). A third type of "nonphosphorylating" particle preparation was made by sonication of the 8H. L5w and I. Vallin, Biochlm. Biophys. Acta 69, 361 (1963). ~A. L. Smith and M. ttansen, Biochem. Biophys. Res. Commun. 8, 33 (1962); Biochim. Biophys. Acta 81, 214 (1964). SA. W. Linnane and D. M. Ziegler, Biochlm. Biophys. Acta 29, 630 (1958). *T. E. Conover, R. L. Prairie, and E. Racker, J. Biol. Chem. 238, 2831 (1963).
I87]
ENERGY-COUPLII~G IN UNCOUPLED
•
v
PARTICLES
545
[
.~ °~
o ,,~
,--, o
.-+
~
o!
.! ~o
'~
•~
~;~'~
~
~
~
0
~-~
.~
~'~
~
z~
+~
~
~ ~~ ~g
~.~.~ o
9..!
•
÷ +~ - ~+ ~ ~ N
0
.4
5
~
~
Z
r~
÷~.~ ~
546
COUPLING FACTORS
[87]
mitochondria in a medium containing 2 mM EDTA and 2 mM sodiumpotassium phosphate, pit 7.5, and subsequent treatment of the particles so obtained with urea. One milliliter of particle suspension in 250 mM sucrose, containing 20 mg of protein per milliliter, was mixed with 1 ml of 4 M urea and incubated for 6 minutes at 20 °. Five milliliters of cold distilled water was then added, and the incubation was continued for another 6 minutes at 0 °. The reaction mixture was diluted with 5 ml 250 mM sucrose and centrifuged at 105,000 g for 40 minutes. The sediment was washed by homogenization with 10 volumes of 250 mM sucrose and recentrifuged at 105,000 g for 40 minutes. The particles were finally suspended in 250 mM sucrose. The table indicates the phosphorylating efficiency of the various types of particles as measured with suceinate as the substrate, as well as their energy-linked transhydrogenase activities with either suceinate or ATP as the sucrose of energy, the former both in the absence and in the presence of oligomyein. The transhydrogenase reaction was assayed spectrophotometrieally in the presence of oxidized glutathione and glutathione reductase as described in this volume [113]. Results similar to those shown in the table with the urea-treated EDTA particles have been reported by Haas 7 using a Keilin-Hartree heart muscle preparation. Stimulation of Oxidative Phosphorylation and Its Reversal in "Nonphosphorylating" Particles b y Oligomycin The demonstration 8 of this effect of oligomyein has emerged from ~he observations that oligomyein stimulates the respiratory chain-driven pyridine nucleotides transhydrogenase reaction in "nonphosphorylating" particles 2 (cf. the table) and that the amount of oligomyein required for maximal stimulation is considerably smaller than that required for inhibition of the ATPase activity of the same particles2 Typical results obtained with EDTA particles are shown in Fig. 1. Forward oxidative phosphorylation is assayed with N A D H as substrate, and the reverse process is assayed as ATP-supported reduction of NAD ÷ by succinate. Maximal stimulation of both reactions occurs with an amount of oligomycin ranging between 0.2 and 0.3 ~g per milligram of protein. Larger amounts of oligomycin are inhibitory. Mg ÷÷, which is obligatory for both the forward and reverse reactions, in high concentrations diminishes the effect of oligomycin on the forward reaction but not on the reverse. 7D. W. Haas, Biochim. Biophys. Acta 89, 543 (1964). s C. P. Lee and L. Ernster, Biochem. Biophys. Res. Commun. 18, 523 (1965); Symp. Regulation Metabolic Processes in Mitochondria, Bari, 1965 Vol. 7, p. 218. B.B.A. Library, Elsevier, Amsterdam, 1966.
547
ENERGY-COUPLING IN UNCOUPLED PARTICLES
[87]
Oxidative
phosphorylation (NADH "02) I .0~
2 mM MgSO4
0
a
0.5
±025~cg olig./mg prot.
0.5
p---- Q_________-----_-0
0.4
0.8 1 .2 1 .6 pg oligomycin/mg protein
5
10 MgSO4 ,MM
15
Reversal of oxid . phosphorylation (Succ.--NAD+ )
.d 60
10 MM MgS04
2 v n *) " E 0 E 40
+0.25Ng olig ./mg prot .
40
of ¢w z v 20 0 E 'E
60
201
0.4
0.8 1.2 N.g oligomycin/mg protein
I
f
_
5
10 MgS04' MM
15
Fia . 1 . Effects of oligomycin and Mg - on oxidative phosphorylation and ATPsupported succinate-linked NAD+ reduction . [From C. P. Lee and L. Ernster, Symp . Regulation Metabolic Processes in Mitochondria, Bari, 1965 Vol . 7, p . 218. B.B.A . Library, Elsevier, Amsterdam, 1966 .] Oxidative phosphorylation was assayed in a reaction mixture consisting of 180 mM sucrose, 50 mM Tris-acetate buffer, pH 7.5, 2 mM ADP, 15 mM glucose, 75 Kunitz-MacDonald units of yeast hexokinase, 3 mM "P, (12 X 108 cpm/micromole), particles (prepared in the presence of 2 mM EDTA, pH 8.6) containing 0.6 mg protein, and 1 mM NADH as the substrate . Other additions as indicated : panel A, 2 mM MgS04 and varying amounts of oligomycin ; panel B, 025 #g oligomycin per milligram of protein and varying concentrations of MgSO, . Final volume, 3 .1 ml ; temperature, 30° . O Z consumption was measured with a Clark oxygen electrode, and the esterification of P, was determined by the isotope distribution method [O . Lindberg and L . Ernster, Methods Biochem . Anal . 3, 1 (1955)] . The reaction mixture for the ATPsupported reduction of NAD+ by succinate consisted of 180 mM sucrose, 50 mM Tris-acetate buffer, pH 7 .5, 1 .6 mM KCN, 0.2 mM NAD*, 5 mM succinate, particles (prepared in the presence of 2 mM EDTA, pH 8 .7) containing 0 .6 mg protein ; 3 mM ATP was added to start the reaction . Other additions as indicated : panel C, 10 mM MgSO4 and varying amounts of oligomycin ; panel D, 0 .25 jug oligomycin per milligram of protein and varying concentrations of MgSO.. Final volume, 3 ml ; temperature 30° .
548
COUPLING FACTORS
[87]
For obtaining the effects of oligomycin as described above, it is essential to maintain the pH of the EDTA-containing sonicating medium within the range 8.5-9.0. At pH values below 8.5, the particles may retain a substantial capacity for both oxidative phosphorylation and ATP-supported succinate-linked NAD ÷ reduction even when assayed in the absence of oligomycin; at pH values above 9.0, the stimulating effect of oligomycin becomes less pronounced, especially as the stimulation of the forward oxidative phosphorylation is concerned. Particles prepared in the presence of ammonia/, 9 as well as the Keilin-Hartree heart muscle preparation/° show a response to oligomycin similar to that of EDTA particles. Results analogous to those shown in Fig. 1 are obtained with succinate or with ascorbate + TMPD (or PMS) rather than NADH as the substrafe for oxidative phosphorylation, and with ascorbate + TMPD rather than succinate as the hydrogen donor for the ATP-supported NAD ÷ reduction2 With NADH as substrate, in the absence of added Mg ++, oligomycin inhibits the respiration of EDTA particles by up to 80%, and the inhibition is relieved by phosphorylation-uncoupling concentrations of 2,4-dinitrophenol or dicoumarol. Oligomycin also stimulates the P~-ATP exchange activity of EDTA particles by 4 to 5-fold.11 The optimal oligomycin concentration for this effect is the same as for the maximal stimulation of oxdiative phosphorylation and its reversal. The effect of Mg +÷is similar to that on the reversal of oxidative phosphorylation. The optimal range of oligomycin concentration for all the above effects may vary with the batch of oligomycin used. A practical detail of great importance in performing titrations with oligomycin is to wash the reaction chambers very thoroughly with both water and ethanol between the single assays in order to remove adhering traces of oligomycin. Oligomycins A, B, C, and D (rutamycin) act in an essentially similar fashion; aurovertin does not replace oligomycin in any of the stimulating effects described above, and even abolishes these effects of oligomycin.TM Possible mechanisms involved in the above effects of oligomycin have been discussed by Lee and Ernster. 8 Fessenden and Racker 9 have studied the relationship between the phosphorylation stimulating effects of oligomycin and of soluble "coupling factors." Dj. Fessenden and iE. Racker, J. Biol. Chem. 241, 2483 (1966). 1oK. van Dam and H. F. ter Welle, Syrup. Regulation of Metabolic Proeesse, in Mitochond,ia, Bari, 1965 Vol. 7, p. 237. B.B.A. Library, Elsevier, Amsterdam, 1966. C. P. Lee and L. Ernster, unpublished results, 1965. 120. Lindberg and L. Ernster, Methods Biochem. Anal. 3, 1 (1955).
ENERGY-COUPLING IN UNCOUPLED PARTICLES
543
mercuric acetate 50~o at 10-8 M, and both are specifically inhibited by ADP but by no other nucleoside diphosphates. On the basis of these results it is tentatively concluded that both reactions are catalyzed by the same enzyme. The extraction and purification procedure described here has been found to give reproducible results providing the mitochondria are not damaged by exposure to hypotonie sucrose solutions or to salt solution of ionic strength in excess of 0.01. Attempts to utilize hypotonic media, strong salt solutions, or freezing and thawing resulted in the extraction of large amounts of adenylate kinase activity which introduces complications for assay of the specific ATP-ADP exchange enzyme activity.
[87] E n e r g y - C o u p l i n g in Nonphosphorylating Submitochondrial Particles
By
CHUAN-PU
L E E 1 a n d LARS E R N S T E R
Demonstration of Energy-Coupling The energy-linked pyridine nucleotide transhydrogenase reactionla (cf. this volume [113]) can be used as a tool for the demonstration 2 of energy-coupling in so-called "nonphosphorylating"submitochondrial electron transport particles. The approach is based on the concept that the energy-linked transhydrogenase reaction: NADH
+ N A D P + + I ~ X --* N A D + + N A D P H
-{-I + X
(I)
involves the utilization of a nonphosphorylated high-energy compound, denoted I ~ X, which is an intermediate of the respiratory chain-linked oxidative phosphorylation system: respiration
I+X , I,-,X I~XWADPWP~I+X+ATP
(2) (3)
(where I and X are hypothetie energy-transfer carriers). In phosphorylating submitochondrial particles, the energy-linked transhydrogenase reaction can be driven by either the respiratory chain, Reaction (2), or ATP, reversal of Reaction (3), at approximately equal capacities. In contrast, "nonphosphorylating" particles can derive energy for driving the energy-linked transhydrogenase reaction much less efficiently from 1See footnote 1, page 33. ~"L. Danielson and L. Ernster, Biochem. Biophys. Res. Commun. 10, 91 (1963); in "Energy-Linked Functions of Mitochondria" (B. Chance, ed.), p. 157. Academic Press, New York, 1963; Biochem. Z. 338, 188 (1963). C. P. Lee, G. F. Azzone, and L. Ernster, Nature t01, 152 (1964).
544
COVPLING FACTORS
[87]
ATP than from the respiratory chain. This is consistent with the conclusion that these particles still possess the capacity for generating I ~ X by respiratory energy-coupling (Reaction 2), although their phosphorylating capacity, i.e., their ability to utilize I ~ X for converting ADP and P~ into ATP (Reaction 3), is weakened or lost. It is also found 2 that in such particles oligomycin markedly stimulates, and may even be obligatory for, the respiratory chain-driven transhydrogenase reaction. Illustrative data are summarized in the table. The data shown in this table compare two types of phosphorylating particles with three types of "nonphosphorylating" particles. "Heavy" beef-heart mitochondria, prepared according to LSw and Vallin, ~ and stored in 250 m M sucrose suspension at --10 ° for at least 3 days, were used as the starting material for the various preparations. The general procedure for the preparation of submitochondrial particles was as follows: The frozen mitochondrial suspension was thawed, and diluted with 250 mM sucrose to contain about 20-30 mg of protein per milliliter. Depending on the type of preparation to be investigated, various reagents were added as will be specified below. The suspension was then saturated with N2 and subjected to sonic oscillation for 2 minutes at the maximal output in a 20-kc Raytheon sonicator cooled with running tap water (4-8°). The suspension was diluted with an equal volume of 250 mM sucrose and centrifuged at 12,000 g for 10 minutes. The supernatant fraction was decanted and centrifuged at 105,000 g for 40 minutes. The sediment was washed by homogenization with 10 volumes of 250 mM sucrose and centrifuged at 105,000 g for 40 minutes. The particles were finally suspended in 250 mM sucrose to give a protein concentration of about 20 mg/ml. A recovery of 20-30% of the mitochondrial protein was usually obtained. In the case of the phosphorylating particles studied, the sonicating medium consisted of either 5 mM MnC12, 10 mM MgS04 and 1 mM sodium succinate, pH 7.5 (preparation described by Smith and Hansen4), or 15 mM MgS04 and 1 m M ATP, pH 7.5 (ETPH preparation of Linnane and Ziegler5 as modified by LSw and VallinS). "Nonphosphorylating" particles were prepared in a sonicating medium consisting of either 2 mM EDTA, pH 7.5 ("modified" E T P H of Linnane and ZieglerS), or 20 m M NH40H (A-particles of Conover et al2). A third type of "nonphosphorylating" particle preparation was made by sonication of the 8H. L5w and I. Vallin, Biochlm. Biophys. Acta 69, 361 (1963). ~A. L. Smith and M. ttansen, Biochem. Biophys. Res. Commun. 8, 33 (1962); Biochim. Biophys. Acta 81, 214 (1964). SA. W. Linnane and D. M. Ziegler, Biochlm. Biophys. Acta 29, 630 (1958). *T. E. Conover, R. L. Prairie, and E. Racker, J. Biol. Chem. 238, 2831 (1963).
I87]
ENERGY-COUPLII~G IN UNCOUPLED
•
v
PARTICLES
545
[
.~ °~
o ,,~
,--, o
.-+
~
o!
.! ~o
'~
•~
~;~'~
~
~
~
0
~-~
.~
~'~
~
z~
+~
~
~ ~~ ~g
~.~.~ o
9..!
•
÷ +~ - ~+ ~ ~ N
0
.4
5
~
~
Z
r~
÷~.~ ~
546
COUPLING FACTORS
[87]
mitochondria in a medium containing 2 mM EDTA and 2 mM sodiumpotassium phosphate, pit 7.5, and subsequent treatment of the particles so obtained with urea. One milliliter of particle suspension in 250 mM sucrose, containing 20 mg of protein per milliliter, was mixed with 1 ml of 4 M urea and incubated for 6 minutes at 20 °. Five milliliters of cold distilled water was then added, and the incubation was continued for another 6 minutes at 0 °. The reaction mixture was diluted with 5 ml 250 mM sucrose and centrifuged at 105,000 g for 40 minutes. The sediment was washed by homogenization with 10 volumes of 250 mM sucrose and recentrifuged at 105,000 g for 40 minutes. The particles were finally suspended in 250 mM sucrose. The table indicates the phosphorylating efficiency of the various types of particles as measured with suceinate as the substrate, as well as their energy-linked transhydrogenase activities with either suceinate or ATP as the sucrose of energy, the former both in the absence and in the presence of oligomyein. The transhydrogenase reaction was assayed spectrophotometrieally in the presence of oxidized glutathione and glutathione reductase as described in this volume [113]. Results similar to those shown in the table with the urea-treated EDTA particles have been reported by Haas 7 using a Keilin-Hartree heart muscle preparation. Stimulation of Oxidative Phosphorylation and Its Reversal in "Nonphosphorylating" Particles b y Oligomycin The demonstration 8 of this effect of oligomyein has emerged from ~he observations that oligomyein stimulates the respiratory chain-driven pyridine nucleotides transhydrogenase reaction in "nonphosphorylating" particles 2 (cf. the table) and that the amount of oligomyein required for maximal stimulation is considerably smaller than that required for inhibition of the ATPase activity of the same particles2 Typical results obtained with EDTA particles are shown in Fig. 1. Forward oxidative phosphorylation is assayed with N A D H as substrate, and the reverse process is assayed as ATP-supported reduction of NAD ÷ by succinate. Maximal stimulation of both reactions occurs with an amount of oligomycin ranging between 0.2 and 0.3 ~g per milligram of protein. Larger amounts of oligomycin are inhibitory. Mg ÷÷, which is obligatory for both the forward and reverse reactions, in high concentrations diminishes the effect of oligomycin on the forward reaction but not on the reverse. 7D. W. Haas, Biochim. Biophys. Acta 89, 543 (1964). s C. P. Lee and L. Ernster, Biochem. Biophys. Res. Commun. 18, 523 (1965); Symp. Regulation Metabolic Processes in Mitochondria, Bari, 1965 Vol. 7, p. 218. B.B.A. Library, Elsevier, Amsterdam, 1966.
547
ENERGY-COUPLING IN UNCOUPLED PARTICLES
[87]
Oxidative
phosphorylation (NADH "02) I .0~
2 mM MgSO4
0
a
0.5
±025~cg olig./mg prot.
0.5
p---- Q_________-----_-0
0.4
0.8 1 .2 1 .6 pg oligomycin/mg protein
5
10 MgSO4 ,MM
15
Reversal of oxid . phosphorylation (Succ.--NAD+ )
.d 60
10 MM MgS04
2 v n *) " E 0 E 40
+0.25Ng olig ./mg prot .
40
of ¢w z v 20 0 E 'E
60
201
0.4
0.8 1.2 N.g oligomycin/mg protein
I
f
_
5
10 MgS04' MM
15
Fia . 1 . Effects of oligomycin and Mg - on oxidative phosphorylation and ATPsupported succinate-linked NAD+ reduction . [From C. P. Lee and L. Ernster, Symp . Regulation Metabolic Processes in Mitochondria, Bari, 1965 Vol . 7, p . 218. B.B.A . Library, Elsevier, Amsterdam, 1966 .] Oxidative phosphorylation was assayed in a reaction mixture consisting of 180 mM sucrose, 50 mM Tris-acetate buffer, pH 7.5, 2 mM ADP, 15 mM glucose, 75 Kunitz-MacDonald units of yeast hexokinase, 3 mM "P, (12 X 108 cpm/micromole), particles (prepared in the presence of 2 mM EDTA, pH 8.6) containing 0.6 mg protein, and 1 mM NADH as the substrate . Other additions as indicated : panel A, 2 mM MgS04 and varying amounts of oligomycin ; panel B, 025 #g oligomycin per milligram of protein and varying concentrations of MgSO, . Final volume, 3 .1 ml ; temperature, 30° . O Z consumption was measured with a Clark oxygen electrode, and the esterification of P, was determined by the isotope distribution method [O . Lindberg and L . Ernster, Methods Biochem . Anal . 3, 1 (1955)] . The reaction mixture for the ATPsupported reduction of NAD+ by succinate consisted of 180 mM sucrose, 50 mM Tris-acetate buffer, pH 7 .5, 1 .6 mM KCN, 0.2 mM NAD*, 5 mM succinate, particles (prepared in the presence of 2 mM EDTA, pH 8 .7) containing 0 .6 mg protein ; 3 mM ATP was added to start the reaction . Other additions as indicated : panel C, 10 mM MgSO4 and varying amounts of oligomycin ; panel D, 0 .25 jug oligomycin per milligram of protein and varying concentrations of MgSO.. Final volume, 3 ml ; temperature 30° .
548
COUPLING FACTORS
[87]
For obtaining the effects of oligomycin as described above, it is essential to maintain the pH of the EDTA-containing sonicating medium within the range 8.5-9.0. At pH values below 8.5, the particles may retain a substantial capacity for both oxidative phosphorylation and ATP-supported succinate-linked NAD ÷ reduction even when assayed in the absence of oligomycin; at pH values above 9.0, the stimulating effect of oligomycin becomes less pronounced, especially as the stimulation of the forward oxidative phosphorylation is concerned. Particles prepared in the presence of ammonia/, 9 as well as the Keilin-Hartree heart muscle preparation/° show a response to oligomycin similar to that of EDTA particles. Results analogous to those shown in Fig. 1 are obtained with succinate or with ascorbate + TMPD (or PMS) rather than NADH as the substrafe for oxidative phosphorylation, and with ascorbate + TMPD rather than succinate as the hydrogen donor for the ATP-supported NAD ÷ reduction2 With NADH as substrate, in the absence of added Mg ++, oligomycin inhibits the respiration of EDTA particles by up to 80%, and the inhibition is relieved by phosphorylation-uncoupling concentrations of 2,4-dinitrophenol or dicoumarol. Oligomycin also stimulates the P~-ATP exchange activity of EDTA particles by 4 to 5-fold.11 The optimal oligomycin concentration for this effect is the same as for the maximal stimulation of oxdiative phosphorylation and its reversal. The effect of Mg +÷is similar to that on the reversal of oxidative phosphorylation. The optimal range of oligomycin concentration for all the above effects may vary with the batch of oligomycin used. A practical detail of great importance in performing titrations with oligomycin is to wash the reaction chambers very thoroughly with both water and ethanol between the single assays in order to remove adhering traces of oligomycin. Oligomycins A, B, C, and D (rutamycin) act in an essentially similar fashion; aurovertin does not replace oligomycin in any of the stimulating effects described above, and even abolishes these effects of oligomycin.TM Possible mechanisms involved in the above effects of oligomycin have been discussed by Lee and Ernster. 8 Fessenden and Racker 9 have studied the relationship between the phosphorylation stimulating effects of oligomycin and of soluble "coupling factors." Dj. Fessenden and iE. Racker, J. Biol. Chem. 241, 2483 (1966). 1oK. van Dam and H. F. ter Welle, Syrup. Regulation of Metabolic Proeesse, in Mitochond,ia, Bari, 1965 Vol. 7, p. 237. B.B.A. Library, Elsevier, Amsterdam, 1966. C. P. Lee and L. Ernster, unpublished results, 1965. 120. Lindberg and L. Ernster, Methods Biochem. Anal. 3, 1 (1955).