- Email: [email protected]
Effect of mercurials on NADH oxidation and energy-linked hydrogen ion gradient in submitochondrial particles
ARCHIVES
OF
Effect
BIOCHEMISTRY
AND
of Mercurials
Hydrogen
BIOPHYSICS
on NADH
Ion Gradient
C. K. RAMAKRISHNA Retina
Foundation,
Department
Received
137, 388-391 (1970)
October
Oxidation
and
Energy-Linked
in Submitochondrial KURUP
of Bioenergetics
D. RAO SANADP
AND
Research,
31, 1969; accepted
Particles’
Boston, Massachusetts
January
02114
21, 1970
Earlier work has shown that Ihe sensitivity of NADH oxidation in submitochondrial particles to inhibition by mercurial increases, if the particles are ‘preconditioned’ by allowing them to oxidize a small amount of NADH before exposure to the inhibitor. This inhibition is relieved by the addition of ethylenediamine tetraacetate (EDTA). The effect of EDTA and mersalyl on the decolorization of membrane-bound bromthymol blue, when it is energized by NADH oxidation, has been measured. Under conditions where EDTA overcame the inhibition of oxidation by mersalyl, it did not affect the bromthymol blue response. The involvement of an -SH in the energy-driven establishment of a proton gradient thus becomes apparent.
Tyler et al. (1, 2) observed that the NADH oxidase activity of bovine heart submitochondrial particles became highly sensitive to inhibition by organic mercurials (90% by 15 pM mersalyl) when the particles were “preconditioned” or “sensitized” by permitting them to oxidize a small amount of NADH to completion before exposure to the inhibitor. Under comparable conditions, the activity of the untreated particles was inhibited only 20%. This was essentially confirmed by Mersmann et al. (3) who, however, observed significant inhibition (50 %) even without preconditioning. We report here that in the presence of EDTA the preconditioning effect leading to high sensitivity to mercurial is masked. As a result, the sensitivity of energy-linked reactions to mercurials becomes apparent. 1 This is Part XVIII of the series, Studies on Oxidative Phosphorylation. This work was supported by grants from the Public Health Service (I-ROI-GM, 13641 and GRS 5-Sol-FR-05527), the Life Insurance Medical Research Fund (GB 67-20) and the American Heart Association, Inc. (67-49). 2 Present address: Indian Institute of Science, Department of Biochemistry, Bangalore, India. 3 Also at Department of Biological Chemistry, Harvard Medical School.
Energy-linked reactions driven by coupling to ascorbate oxidation are also sensitive to mercurials while this oxidation is fully resistant (4, 5). EXPERIMENTAL
PROCEDURES
The experiments reported in Fig. 1 were carried out with phosphorylating submitochondrial particles prepared from heavy-layer beef heart mitochondria as described by Linnane and Ziegler (6). These particles were designated ETPH4 by the above authors, and the same terminology will be employed here. The particles used for t,he experiments in Fig. 2 were made by sonic disruption at pH 8.8 in 0.4-0.6 mM EDTA (7) and will be referred to as ammonia-EDTA particles. For the experiments in Fig. 1, oxygen uptake was determined polarographically with the Clark oxygen electrode supplied by Yellow Springs Instrument Company, Ohio. The experiments in Fig. 2 were monitored for oxygen with a vibrating platinum electrode in an open cuvette. The change in color of membrane-bound bromthymol blue was measured in an Aminco-Chance dual-wavelength spectrophotometer using the 61&700-rnr wavelength pair (5,s). The maximum deflect,ion in these experiments is taken as a function of the steady 4 The abbreviations used are: ETPH, phosphorylating electron transport particle prepared from heavy-layer beef heart mitochondria.
385
&IERCURIAL
AND
ENERGY-LINKED
state concentration of energized carriers. The experimental conditions have been described in the legends t,o the figures. RESULTS
The effect of mersalyl on the NADH oxidase activity of ETPH is shown in Fig. 1. In agreement with the observations
pH GR,SDIENT
389
of Tyler et al. (a), mersalyl (10 PM) almost completely inhibited NADH oxidation when the particles were “preconditioned” (compare tracings 1 and 2). The inhibition in the first cycle of NADH oxidation (tracing 6) was less but increased with time. The tracings 2-S show the effect of EDTA on the inhibition. It is seen that EDTA, when
FIG. 1. Influence of mersalyl on the NADH oxidase activity of heart mitochondrial particles. The reaction system contained 50 DIM phosphate buffer, pH 7.4, and 0.5 mg of ETPH (protein) in 1.5 ml. Further additions were made as indicated. Oxygen uptake was measured polarographically at 30” using a Clark oxygen electrode. The transient oxygen uptake after the addition of Hg in Traces -l and 6 may be due to the oxidation of unknown endogenous substrates facilitated by the uncoupling caused by Hg. It is not seen in all experiments and may depend on the prior exposure of the particles to slightly varying experimental conditions.
390
KURUP
AND
added before mersalyl (tracing 5) gave complete protection to the particles. It was effective even when added soon after mersalyl (tracings 3 and 4) but less effective if the addition of EDTA is delayed (tracings 2 and 3). In this respect EDTA differed from ferricyanide which was able to protect the “preconditioned” particles from the inhibitory action of mersalyl only when added before the mercurial but was almost totally ineffective when added after the mercurial (data are not shown), in agreement with the observations of Tyler et al. (2). Incubation of the untreated particles with mersalyl for 1 min caused considerable (about 50%) inhibition of NADH oxidase activity as seen in tracing 6. However, addition of EDTA before mersalyl (tracing 7) again afforded protection. Ferricyanide, under these conditions, was ineffective in protecting the oxidase from inhibition by mersalyl (compare tracings 6 and 8). Conceivably, the ferricyanide gets reduced immediately after the first addition of NADH to ferrocyanide, which is inactive as a protective agent (2). The decolorization of membrane-bound bromothymol blue, which is dependent on substrate oxidation in mitochondrial particles (8)) is a sensitive index of the energized state in the oxidative phosphorylation reactions. The inhibition of the color change, produced during the oxidation of ascorbate-N, N, N’, N’-tetramethyl-p-phenylenediamine by mersalyl has been reported earlier (4, 5). The cytochrome c oxidase involved in this system is insensitive to mersalyl; consequently the effect of the inhibitor has been ascribed to uncoupling. Since the coupling efficiency as estimated by P/O or the bromthymol blue response with ascorbate-phenylenediamine in mitochondrial particles is quite low, it was of interest to examine the effect of mersalyl with NADH as the substrate which shows superior efficiency in both systems. The effect of mersalyl on NADH oxidation and associated bromthymol blue color change is shown in Fig. 2. These experiments were carried out in Tris-chloride buffer using ammonia-EDTA particles, but ETPH also gave similar results. Ad-
SANADI NADH
&a-700~~
NADH
(180) FIG. 2. The influence of mersalyl
on bromthymol blue response and NADH oxidase activity. The reaction system contained 20 mM Tris-chloride buffer, pH 7.4, 0.20 M sucrose, 6.7 pM bromthymol blue, and 1 mg ammonia-EDTA particle protein in a total reaction volume of 6 ml. Mersalyl (10 PM) and EDTA (0.6 mM) were added as shown. The response was measured by the absorbance decrease at 618-706 rnp on the addition of 0.1 mM NADH. The values in parentheses given under the curves are the rates of oxygen uptake (natoms X mini X mg-i protein) measured simultaneously using a vibrating platinum electrode.
dition of mersalyl before the first addition of NADH inhibited the first cycle of response only slightly (compare tracings A and B). The oxidation of NADH added a second time as well as the associated color change were inhibited strongly. (The rates of oxygen uptake determined simultaneously are given in parentheses under the curves.) When mersalyl was added to the “preconditioned” particles and incubated for 6
MERCURIAL
AND
ENERGY-LINKED
min (for maximum effect) oxygen uptake and bromthymol blue response were inhibited (tracing C). However, addition of EDTA before the second addition of NADH prevented the inhibition of the oxidase activity almost completely in agreement with Fig. 1, curve 4, but did not restore the energy-driven bromthymol blue response (tracing D). DISCUSSION
The enhanced sensitivity of the preconditioned system to mercurials was postulated by Tyler et al. (2) to be due to the conversion of a disulfide group present in the untreated particles to the mercurialligands.” Ferricyasensitive “iron-sulfur nide protected the particles, presumably, by oxidizing these and re-forming the disulfide. It is not known by what mechanism EDTA is able to reverse this inhibition even when added after the preconditioned particles are exposed to the mercurial. It has been observed (9) that in cytochrome oxidase the number of sulfhydryl groups accessible to nitroprusside was doubled in the presence of EDTA. A similar increase in the sulfhydryl groups accessible to the mercurial would explain how EDTA could retard the the reaction between the mercurial and the functionally active sulfhydryl group, but reversal of established inhibition (Fig. 1, tracing 3) would be harder to explain on this basis. Another possibility is that EDTA makes the functional sulfhydryl less accessible or less reactive to the mercurial by inducing conformational changes in the protein. The persistent inhibition of the energydriven bromthymol blue response even under conditions where the inhibition of oxidation is relieved by EDTA (Fig. 2, curve D) clearly distinguishes the more sensitive -SH on the energy-transfer pathway from the SH associated with NADH oxidation. The former must function at an early step in energy conservation preceding the site of oligomycin inhibition since the bromthymol blue response driven by oxidative energy is not inhibited by oligomycin (5, 8). Similar sensitivity to mersalyl _.has been observed in the bromthymol blue
pH GRADIENT
391
response with ascorbate-tetramethyl-pphenylenediamine as substrate (5), this oxidation being completely insensitive to mersalyl. These results are consistent with the previous demonstration of uncoupling of phosphorylation without affecting significantly the oxidation rate by arsenite in the presence of 2,3-dimercaptopropanol or by Cd2+ (10-12). The compounds also inhibited ATP-P1 exchange (13, 14) and the energy-driven nicotinamide nucleotide transhydrogenase (5). ACKNOWLEDGMENTS The assistance of Miriam Gilman in this work and of Richard Waitkus in the preparation of the particles is gratefully acknowledged. REFERENCES 1. TYLER, D. D., BUTOW, It. A., GONZE, J., AND ESTABROOK, R. W., Biochem. Biophys. Res. Commun. 19, 551 (1965). 2. TYLER, D. D., GONZE, J., ESTABROOK, R. W., AND BUTO~, R. A., in “Non-heme Iron Proteins” (A. San Pietro, ed.), p. 447. Antioch Press, Yellow Springs, Ohio (1965). 3. MERSMANN, H., LUSTY, J., AND SINGER, T. P., Biochem. Biophys. Res. Commun. 26, 43 (1966). 4. SAIYADI, D. R., LAM, K. W., AND KUKUP, C. K. It., hoc. Nat. Acad. Sci. U.S.A. 61, 277 (1968). 5. KURUP, C. K. R., AND SANADI, D. R., Biochemistry 7,4483 (1968). 6. LINNANE, A. W., AND ZIEOLER, D. M., Biochim. Biophys. Ada 29, 630 (1958). 7. LAM, K. W., WARSHAW, J. B., AND SANADI, D. R., Arch. Biochem. Biophys. 119, 477 (1967). 8. CHANCE, B., AND MEL.~, L., J. Biol. Chem. 242, 830 (1967). 9. KIRSCHBAUM, J., AND WAINIO, W. W., Biochim. Biophys. Ada 118, 643 (1966). 10. FLUHARTY, A. L., AND SANADI, D. It., J. Biol. Chem. 236, 2772 (1961). 11. FLUHARTY, A. L., AND SANADI, D. R., Biochemistry 1, 276 (1962). 12. FONYO, A., AND BEssM~N, S., Biochem. Biophys. Res. Commun. 24, 61 (1966). 13. COOPER, C., AND LEHNINGER, A. L., J. Biol. Chem. 219, 519 (1956). 14. BOYER, P. D., BIEBER, L. L., MITCHELL, R. A., AND SZABOLCSI, G., J. Biol. Chem. 241, 5384 (1966).
OF
Effect
BIOCHEMISTRY
AND
of Mercurials
Hydrogen
BIOPHYSICS
on NADH
Ion Gradient
C. K. RAMAKRISHNA Retina
Foundation,
Department
Received
137, 388-391 (1970)
October
Oxidation
and
Energy-Linked
in Submitochondrial KURUP
of Bioenergetics
D. RAO SANADP
AND
Research,
31, 1969; accepted
Particles’
Boston, Massachusetts
January
02114
21, 1970
Earlier work has shown that Ihe sensitivity of NADH oxidation in submitochondrial particles to inhibition by mercurial increases, if the particles are ‘preconditioned’ by allowing them to oxidize a small amount of NADH before exposure to the inhibitor. This inhibition is relieved by the addition of ethylenediamine tetraacetate (EDTA). The effect of EDTA and mersalyl on the decolorization of membrane-bound bromthymol blue, when it is energized by NADH oxidation, has been measured. Under conditions where EDTA overcame the inhibition of oxidation by mersalyl, it did not affect the bromthymol blue response. The involvement of an -SH in the energy-driven establishment of a proton gradient thus becomes apparent.
Tyler et al. (1, 2) observed that the NADH oxidase activity of bovine heart submitochondrial particles became highly sensitive to inhibition by organic mercurials (90% by 15 pM mersalyl) when the particles were “preconditioned” or “sensitized” by permitting them to oxidize a small amount of NADH to completion before exposure to the inhibitor. Under comparable conditions, the activity of the untreated particles was inhibited only 20%. This was essentially confirmed by Mersmann et al. (3) who, however, observed significant inhibition (50 %) even without preconditioning. We report here that in the presence of EDTA the preconditioning effect leading to high sensitivity to mercurial is masked. As a result, the sensitivity of energy-linked reactions to mercurials becomes apparent. 1 This is Part XVIII of the series, Studies on Oxidative Phosphorylation. This work was supported by grants from the Public Health Service (I-ROI-GM, 13641 and GRS 5-Sol-FR-05527), the Life Insurance Medical Research Fund (GB 67-20) and the American Heart Association, Inc. (67-49). 2 Present address: Indian Institute of Science, Department of Biochemistry, Bangalore, India. 3 Also at Department of Biological Chemistry, Harvard Medical School.
Energy-linked reactions driven by coupling to ascorbate oxidation are also sensitive to mercurials while this oxidation is fully resistant (4, 5). EXPERIMENTAL
PROCEDURES
The experiments reported in Fig. 1 were carried out with phosphorylating submitochondrial particles prepared from heavy-layer beef heart mitochondria as described by Linnane and Ziegler (6). These particles were designated ETPH4 by the above authors, and the same terminology will be employed here. The particles used for t,he experiments in Fig. 2 were made by sonic disruption at pH 8.8 in 0.4-0.6 mM EDTA (7) and will be referred to as ammonia-EDTA particles. For the experiments in Fig. 1, oxygen uptake was determined polarographically with the Clark oxygen electrode supplied by Yellow Springs Instrument Company, Ohio. The experiments in Fig. 2 were monitored for oxygen with a vibrating platinum electrode in an open cuvette. The change in color of membrane-bound bromthymol blue was measured in an Aminco-Chance dual-wavelength spectrophotometer using the 61&700-rnr wavelength pair (5,s). The maximum deflect,ion in these experiments is taken as a function of the steady 4 The abbreviations used are: ETPH, phosphorylating electron transport particle prepared from heavy-layer beef heart mitochondria.
385
&IERCURIAL
AND
ENERGY-LINKED
state concentration of energized carriers. The experimental conditions have been described in the legends t,o the figures. RESULTS
The effect of mersalyl on the NADH oxidase activity of ETPH is shown in Fig. 1. In agreement with the observations
pH GR,SDIENT
389
of Tyler et al. (a), mersalyl (10 PM) almost completely inhibited NADH oxidation when the particles were “preconditioned” (compare tracings 1 and 2). The inhibition in the first cycle of NADH oxidation (tracing 6) was less but increased with time. The tracings 2-S show the effect of EDTA on the inhibition. It is seen that EDTA, when
FIG. 1. Influence of mersalyl on the NADH oxidase activity of heart mitochondrial particles. The reaction system contained 50 DIM phosphate buffer, pH 7.4, and 0.5 mg of ETPH (protein) in 1.5 ml. Further additions were made as indicated. Oxygen uptake was measured polarographically at 30” using a Clark oxygen electrode. The transient oxygen uptake after the addition of Hg in Traces -l and 6 may be due to the oxidation of unknown endogenous substrates facilitated by the uncoupling caused by Hg. It is not seen in all experiments and may depend on the prior exposure of the particles to slightly varying experimental conditions.
390
KURUP
AND
added before mersalyl (tracing 5) gave complete protection to the particles. It was effective even when added soon after mersalyl (tracings 3 and 4) but less effective if the addition of EDTA is delayed (tracings 2 and 3). In this respect EDTA differed from ferricyanide which was able to protect the “preconditioned” particles from the inhibitory action of mersalyl only when added before the mercurial but was almost totally ineffective when added after the mercurial (data are not shown), in agreement with the observations of Tyler et al. (2). Incubation of the untreated particles with mersalyl for 1 min caused considerable (about 50%) inhibition of NADH oxidase activity as seen in tracing 6. However, addition of EDTA before mersalyl (tracing 7) again afforded protection. Ferricyanide, under these conditions, was ineffective in protecting the oxidase from inhibition by mersalyl (compare tracings 6 and 8). Conceivably, the ferricyanide gets reduced immediately after the first addition of NADH to ferrocyanide, which is inactive as a protective agent (2). The decolorization of membrane-bound bromothymol blue, which is dependent on substrate oxidation in mitochondrial particles (8)) is a sensitive index of the energized state in the oxidative phosphorylation reactions. The inhibition of the color change, produced during the oxidation of ascorbate-N, N, N’, N’-tetramethyl-p-phenylenediamine by mersalyl has been reported earlier (4, 5). The cytochrome c oxidase involved in this system is insensitive to mersalyl; consequently the effect of the inhibitor has been ascribed to uncoupling. Since the coupling efficiency as estimated by P/O or the bromthymol blue response with ascorbate-phenylenediamine in mitochondrial particles is quite low, it was of interest to examine the effect of mersalyl with NADH as the substrate which shows superior efficiency in both systems. The effect of mersalyl on NADH oxidation and associated bromthymol blue color change is shown in Fig. 2. These experiments were carried out in Tris-chloride buffer using ammonia-EDTA particles, but ETPH also gave similar results. Ad-
SANADI NADH
&a-700~~
NADH
(180) FIG. 2. The influence of mersalyl
on bromthymol blue response and NADH oxidase activity. The reaction system contained 20 mM Tris-chloride buffer, pH 7.4, 0.20 M sucrose, 6.7 pM bromthymol blue, and 1 mg ammonia-EDTA particle protein in a total reaction volume of 6 ml. Mersalyl (10 PM) and EDTA (0.6 mM) were added as shown. The response was measured by the absorbance decrease at 618-706 rnp on the addition of 0.1 mM NADH. The values in parentheses given under the curves are the rates of oxygen uptake (natoms X mini X mg-i protein) measured simultaneously using a vibrating platinum electrode.
dition of mersalyl before the first addition of NADH inhibited the first cycle of response only slightly (compare tracings A and B). The oxidation of NADH added a second time as well as the associated color change were inhibited strongly. (The rates of oxygen uptake determined simultaneously are given in parentheses under the curves.) When mersalyl was added to the “preconditioned” particles and incubated for 6
MERCURIAL
AND
ENERGY-LINKED
min (for maximum effect) oxygen uptake and bromthymol blue response were inhibited (tracing C). However, addition of EDTA before the second addition of NADH prevented the inhibition of the oxidase activity almost completely in agreement with Fig. 1, curve 4, but did not restore the energy-driven bromthymol blue response (tracing D). DISCUSSION
The enhanced sensitivity of the preconditioned system to mercurials was postulated by Tyler et al. (2) to be due to the conversion of a disulfide group present in the untreated particles to the mercurialligands.” Ferricyasensitive “iron-sulfur nide protected the particles, presumably, by oxidizing these and re-forming the disulfide. It is not known by what mechanism EDTA is able to reverse this inhibition even when added after the preconditioned particles are exposed to the mercurial. It has been observed (9) that in cytochrome oxidase the number of sulfhydryl groups accessible to nitroprusside was doubled in the presence of EDTA. A similar increase in the sulfhydryl groups accessible to the mercurial would explain how EDTA could retard the the reaction between the mercurial and the functionally active sulfhydryl group, but reversal of established inhibition (Fig. 1, tracing 3) would be harder to explain on this basis. Another possibility is that EDTA makes the functional sulfhydryl less accessible or less reactive to the mercurial by inducing conformational changes in the protein. The persistent inhibition of the energydriven bromthymol blue response even under conditions where the inhibition of oxidation is relieved by EDTA (Fig. 2, curve D) clearly distinguishes the more sensitive -SH on the energy-transfer pathway from the SH associated with NADH oxidation. The former must function at an early step in energy conservation preceding the site of oligomycin inhibition since the bromthymol blue response driven by oxidative energy is not inhibited by oligomycin (5, 8). Similar sensitivity to mersalyl _.has been observed in the bromthymol blue
pH GRADIENT
391
response with ascorbate-tetramethyl-pphenylenediamine as substrate (5), this oxidation being completely insensitive to mersalyl. These results are consistent with the previous demonstration of uncoupling of phosphorylation without affecting significantly the oxidation rate by arsenite in the presence of 2,3-dimercaptopropanol or by Cd2+ (10-12). The compounds also inhibited ATP-P1 exchange (13, 14) and the energy-driven nicotinamide nucleotide transhydrogenase (5). ACKNOWLEDGMENTS The assistance of Miriam Gilman in this work and of Richard Waitkus in the preparation of the particles is gratefully acknowledged. REFERENCES 1. TYLER, D. D., BUTOW, It. A., GONZE, J., AND ESTABROOK, R. W., Biochem. Biophys. Res. Commun. 19, 551 (1965). 2. TYLER, D. D., GONZE, J., ESTABROOK, R. W., AND BUTO~, R. A., in “Non-heme Iron Proteins” (A. San Pietro, ed.), p. 447. Antioch Press, Yellow Springs, Ohio (1965). 3. MERSMANN, H., LUSTY, J., AND SINGER, T. P., Biochem. Biophys. Res. Commun. 26, 43 (1966). 4. SAIYADI, D. R., LAM, K. W., AND KUKUP, C. K. It., hoc. Nat. Acad. Sci. U.S.A. 61, 277 (1968). 5. KURUP, C. K. R., AND SANADI, D. R., Biochemistry 7,4483 (1968). 6. LINNANE, A. W., AND ZIEOLER, D. M., Biochim. Biophys. Ada 29, 630 (1958). 7. LAM, K. W., WARSHAW, J. B., AND SANADI, D. R., Arch. Biochem. Biophys. 119, 477 (1967). 8. CHANCE, B., AND MEL.~, L., J. Biol. Chem. 242, 830 (1967). 9. KIRSCHBAUM, J., AND WAINIO, W. W., Biochim. Biophys. Ada 118, 643 (1966). 10. FLUHARTY, A. L., AND SANADI, D. It., J. Biol. Chem. 236, 2772 (1961). 11. FLUHARTY, A. L., AND SANADI, D. R., Biochemistry 1, 276 (1962). 12. FONYO, A., AND BEssM~N, S., Biochem. Biophys. Res. Commun. 24, 61 (1966). 13. COOPER, C., AND LEHNINGER, A. L., J. Biol. Chem. 219, 519 (1956). 14. BOYER, P. D., BIEBER, L. L., MITCHELL, R. A., AND SZABOLCSI, G., J. Biol. Chem. 241, 5384 (1966).