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[114] Energy-linked transport of Ca++, phosphate, and adenine nucleotides
[114]
ENERGY-LINKED TRANSPORT
745
[114] E n e r g y - L i n k e d T r a n s p o r t of C a +*, P h o s p h a t e , and Adenine Nucleotides B y ERNESTO CARAFOLI1 a n d ALBERT L. LEHNINGER
Mitochondria isolated from a variety of tissues can accumulate certain ions from the suspending medium in a process linked to electron transport; among these are the cations Ca ++, Sr ÷÷, Mn ÷÷, and Mg +÷ and the anions phosphate, sulfate, and acetate. When accumulation of divalent cations occurs, it replaces oxidative phosphorylation; these processes are therefore alternative and each is stoichiometric with electron transport. During uptake of Ca ++, large amounts of ADP or ATP may also be accumulated. The accumulation of Ca *+ and phosphate, the most intensively studied ions, can be observed under two different sets of conditions. In the first, termed massive loading, mitochondria are exposed to relatively high concentrations of Ca +÷ (,~3 mM) and they accumulate amounts of Ca ÷+ up to 2.5 micromoles per milligram of mitochondrial protein; phosphate is required in this case and it is also accumulated, la,2 Although such massive accumulation of Ca ++ and phosphate is stoichiometric with electron transport, the excess Ca ++ in the medium causes loss of acceptor control and of capacity for oxidative phosphorylation. In the second condition, termed limited loading, mitochondria are presented with small amounts of divalent cations (-~0.1 mM, or ~100 micromoles Ca *+ per milligram of protein); phosphate is not required, but if it is present, it is also accumulated. Under these conditions there is no damage to oxidative phosphorylation or respiratory control. The dynamics and stoichiometry of ion uptake are best studied in limited-loading experiments? -5 Massive Loading with Ca +÷ Massive accumulation of Ca ++ and P~ can be supported either by electron transport or by ATP hydrolysis,la,2 When it is supported by oxidation of a respiratory substrate, ATP is also required in the medium for the mitochondria to retain the accumulated calcium phosphate. In the ATP-driven system, no respiratory substrate is needed, but high 1This manuscript was prepared while the author was at the Department of Physiological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland. 1, F. D. Vasington and J. V. Murphy, J. Biol. Chem. 237, 2670 (1962). C. S. Rossi and A. L. Lehninger, Biochem. Z. 338, 698 (1963). s C. S. Rossi and A. L. Lehninger, J. Biol. Chem. 230, 3971 (1964). J. B. Chappell, M. Cohn, and G. D. Greville, in "Energy-Linked Functions of Mitochondria" (B. Chance, ed.), p. 253. Academic Press, New York, 1963. 5B. Chance, J. Biol. Chem. 240, 2729 (1965).
746
ENERGY-LINKED REACTIONS
[114]
concentrations of A T P ( ~ 1 5 mM) are required. Reagents and Reaction Media. The components are given on the basis of a 3.0 ml total volume; the systems m a y be scaled down or up as required. RESPIRATION-SUPPORTED SYSTEM
Stock solutions
0.1 M Tris-chloride, pH 7.2 0.1 M Na-succinate or DL-fl-hydroxybutyrate, pH 7.2 0.1 M MgC12 0.4 M Na-K phosphate buffer, pH 7.2 0.8 M NaC1 0.15 M Na-ATP, pH 7.2 0.03 M 45CaC1~(0,05 ~C per ~mole) Mitochondrial suspension (protein, 10 mg/ml in 0.25 M sucrose) H20 Total volume:
Volume (ml)
Final concentration (mM)
0.3 0.3
10 10
0.3 0.3 0.3 0.06 0.3 0.3
10 4.0 80 3.0 3.0 1.0 mg protein per ml
0.84 3.0
ATP-suPPORTED SYSTEM. Same as above, with respiratory substrate replaced by water and the final A T P concentration raised to 15 mM. Procedure. Mitochondria are added last to start the reaction, which is usually carried out for 10 minutes with shaking in air at 30 ° in beakers or large centrifuge tubes. The tubes are then rapidly cooled in cracked ice, and centrifuged in the cold at 20,000 g for 4 minutes to separate the "loaded" mitochondria from the suspending medium.
Determination o] the Uptake o] Ca +* and InorgaNic Phosphate Reagents Sucrose, 0.25 M Sodium lauryl sulfate, 0.2% Trichloroacetic acid 8%
Procedure. After sedimentation of the mitochondria, the supernatant reaction media are saved for analysis. The sedimented mitochondria are washed twice with 5.0-ml portions of cold 0.25 M sucrose. Prior to the last centrifugation, the suspensions of washed mitochondria are divided into two equal portions. After centrifugation, one pellet is extracted with 5.0 ml of cold trichloroacetic acid and aliquots of the clarified extract are used for P~ determination by the method of Gomori2 The other pellet is G. Gomori, ]. Lab. Clin. Med. 27, 955 (1942).
[114]
ENERGY-LINKED TRANSPORT
747
dissolved in 5.0 ml sodium lauryl sulfate. Aliquots (0.3 ml) of the sodium lauryl sulfate extracts of the pellets, and of the original clarified reaction media, are plated on aluminum planchets (2.0 cm diameter), evaporated to dryness to yield "thin" samples, and counted in a gas-flow Geiger tube. The distribution of radioactivity between washed pellets and the incubation medium is used to calculate the uptake of Ca ++. The preexisting intramitochondrial Ca ++ (10 millimicromoles per milligram of protein) is very low compared to the large amounts of Ca ++ taken up (up to 2500 millimicromoles Ca ++ per milligram of protein); the Ca ++ uptake data therefore need not be corrected for exchange. Since P~ uptake is stoichiometric with that of Ca ++, measurement of accumulated P~ in the mitochondrial trichloroacetic acid extract will suffice as a simpler means of following accumulation of Ca ++ and phosphate.
Properties of the Massive Loading Reaction For each pair of electrons passing through each energy-conserving site of the respiratory chain, about 1.67 molecules of Ca ++ and 1.0 molecule of Pi are accumulated; Ca ++ and Pi are thus accumulated in a molar ratio of 1.67, which is that of hydroxylapatite.2 The ATP-supported system is less efficient. Mg ++ is essential in the incubation medium, and some of it is accumulated with the Ca ++ and phosphate. 7 Antimycin A and cyanide block Ca ++ uptake in systems supported by electron transport, whereas oligomycin has no effect. Oligomycin, on the other hand, greatly inhibits the ATP-supported system, which is otherwise only slightly inhibited by antimycin A or cyanide. Ion uptake in either type of system is inhibited by true uncoupling agents such as 2,4-dinitrophenol.la, o Massive uptake of s~Sr++ can be studied under the same conditions described above;S the uptake of ssSr++ is determined by counting aliquots of the clarified reaction medium or of the trichloroacetic acid extracts of the washed mitochondria in a scintillation counter.
Uptake of A T P or ADP during Massive Uptake of Ca++and P~ Accumulation of ATP and ADP may be measured in the same medium used for study of the uptake of Ca ++ and Pi. In this case, ATP8-14C (0.05-0.1/~C) is employed, and the Ca ++ added is unlabeled. AMP of the medium is not accumulated under these conditions2 7E. Carafoli, C. S. Rossi, and A. L. Lehninger, J. Biol. Chem. 239, 2055 (1964). E. Carafoli, S. Weiland, and A. L. Lehninger, Biochim. Biophys. Acta, 97, 88 (1965). E. Carafoli, C. S. Rossi, and A. L. Lehninger, J. Biol. Chem. 2,40, 2254 (1965).
748
ENERGY-LINKED REACTIONS
[114]
Procedure. At the end of the incubation, the mitochondria are centrifuged and washed twice with 5.0-ml portions of 0.25 M sucrose. Before the last centrifugation, the suspensions are divided into two aliquots. One of the resulting pellets is dissolved in 0.2% sodium lauryl sulfate; aliquots of the solutions are plated and counted in a low-background gas flow counter. The other pellet is extracted with 6% cold perchloric acid. Aliquots of the perehloric acid extract may be applied to paper, and the labeled ATP and ADP separated and identified by paper electrophoresis2
Limited Loading with Ca ++ In the limited loading system,~ the mitochondria are usually incubated in cuvettes of the Clark oxygen polarograph linked to a strip chart recorder. 1° A known amount of Ca ++ is added to mitochondria respiring in state 4, and the rate and amount of extra oxygen uptake is recorded, up to the point when oxygen uptake returns to the initial resting rate. Ca ++ uptake is then measured in an aliquot of the medium.11 Reagents and Reaction M e d i u m . This is equilibrated with air.
Reagent 0.1 M Tri~-chloride, pH 7.2 0.8 M NaC1 0.1 M Na-suecinate, pH 7.2 H~O Total volume:
Volume (ml)
Final concentration (mM)
0.19 0.19 0.09 1.33 1.80
10 80 10
Procedure. After thermoequilibration of the above medium at 25 ° in the polarograph cuvette, 0.10 ml of mitochondrial suspension (5.0 mg protein) is added and the course of the state 4 oxygen uptake is followed. The contents of the euvette are continuously stirred by a magnetic bar. Sixty seconds after the addition of the mitoehondria, 45CaCl.~ (200-600 millimicromoles) is added with a mierosyringe (6.7-20 #l). When the respiration has returned to the resting rate, a 1.0-ml aliquot of the suspension is removed with a 1.0-ml syringe equipped with a needle. The needle is quickly replaced with a Swinny adapter containing a Millipore filter (0.8 ~ pore size) and 0.3-0.4 ml of clear filtrate are collected by exerting moderate pressure on the plunger of the syringe.TM The entire
10W. W. Kielley and J. R. Bronk, J. Biol. Chem. 230, 521 (1958). 1~E. Carafoli, Biochim. Biophys. Acta 97, 107 (1965). ~tt. Rasmussen, A. Waldorf, D. D. Dziewiatow~ki, and It. F. DeLuca, Biochim. Biophys. Acta 75, 250 (1963).
[l14a]
FATTY ACID ACTIVATION
749
operation requires approximately 10 seconds. An aliquot of the clear filtrate (0.1-0.2 ml) is plated and counted for 4~Ca++. The Ca÷+:0 accumulation ratio is the ratio of millimicromoles Ca +* accumulated to millimicroatoms of extra oxygen uptake stimulated by the addition of Ca ++, as recorded by the polarograph trace. Its value will depend on the respiratory substrate; it is approximately 5-6 for fl-hydroxybutyrate, 3.6-4.0 for sueeinate. 2 The baseline state 4 respiration is also capable of coupling to Ca ÷+ accumulation, but the efficiency is low. The Ca÷÷:0 activation ratio is the ratio (millimicromoles Ca ++ added): (millimicroatoms extra oxygen taken up); it is usually slightly larger than the Ca +÷:0 accumulation ratio. Properties of the Reaction. Usually not more than 1% of the added Ca *÷ is left in the supernatant 30 seconds after the respiration has returned to the resting rate. Mg ÷÷ ions and ATP are not necessary for simple uptake of Ca ÷÷ in the absence of P,. When P~ is added, it is also accumulated if the system contains 1.0 mM ATP plus 10 mM MgC12, or 1.0/~g of oligomycin? Limited uptake of Sr ÷÷11 and Mn ÷÷4 can be studied under the same conditions, by employing 85Sr+÷ or 54Mn*÷. With these cations, respiration returns to the resting rate in the presence of inorganic phosphate; ATP is not required.
[ 1 14a] F a t t y A c i d A c t i v a t i o n a n d O x i d a t i o n b y M i t o c h o n d r i a
By SIMON G. VAN DEN"BERGH In animal cells fatty acid activation occurs both in the mitochondria and in the microsomes, but the mitochondria are the only site of fatty acid oxidation. Fatty acids are different from other mitochondrial respiratory substrates in three important respects: (a) they need activation with formation of thiol esters of coenzyme A prior to their oxidation; (b) they are potent uncoupling agents; and (c) they are powerful mitochondrial swelling agents. These properties all have specific consequences and require special precautions. Although fatty acid breakdown has been studied in mitochondria isolated from a variety of animal and plant tissues, we shall in this chapter mainly refer to rat liver mitochondria and only in the last paragraph consider some of the properties of fatty acid breakdown in other types of mitochondria.
ENERGY-LINKED TRANSPORT
745
[114] E n e r g y - L i n k e d T r a n s p o r t of C a +*, P h o s p h a t e , and Adenine Nucleotides B y ERNESTO CARAFOLI1 a n d ALBERT L. LEHNINGER
Mitochondria isolated from a variety of tissues can accumulate certain ions from the suspending medium in a process linked to electron transport; among these are the cations Ca ++, Sr ÷÷, Mn ÷÷, and Mg +÷ and the anions phosphate, sulfate, and acetate. When accumulation of divalent cations occurs, it replaces oxidative phosphorylation; these processes are therefore alternative and each is stoichiometric with electron transport. During uptake of Ca ++, large amounts of ADP or ATP may also be accumulated. The accumulation of Ca *+ and phosphate, the most intensively studied ions, can be observed under two different sets of conditions. In the first, termed massive loading, mitochondria are exposed to relatively high concentrations of Ca +÷ (,~3 mM) and they accumulate amounts of Ca ÷+ up to 2.5 micromoles per milligram of mitochondrial protein; phosphate is required in this case and it is also accumulated, la,2 Although such massive accumulation of Ca ++ and phosphate is stoichiometric with electron transport, the excess Ca ++ in the medium causes loss of acceptor control and of capacity for oxidative phosphorylation. In the second condition, termed limited loading, mitochondria are presented with small amounts of divalent cations (-~0.1 mM, or ~100 micromoles Ca *+ per milligram of protein); phosphate is not required, but if it is present, it is also accumulated. Under these conditions there is no damage to oxidative phosphorylation or respiratory control. The dynamics and stoichiometry of ion uptake are best studied in limited-loading experiments? -5 Massive Loading with Ca +÷ Massive accumulation of Ca ++ and P~ can be supported either by electron transport or by ATP hydrolysis,la,2 When it is supported by oxidation of a respiratory substrate, ATP is also required in the medium for the mitochondria to retain the accumulated calcium phosphate. In the ATP-driven system, no respiratory substrate is needed, but high 1This manuscript was prepared while the author was at the Department of Physiological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland. 1, F. D. Vasington and J. V. Murphy, J. Biol. Chem. 237, 2670 (1962). C. S. Rossi and A. L. Lehninger, Biochem. Z. 338, 698 (1963). s C. S. Rossi and A. L. Lehninger, J. Biol. Chem. 230, 3971 (1964). J. B. Chappell, M. Cohn, and G. D. Greville, in "Energy-Linked Functions of Mitochondria" (B. Chance, ed.), p. 253. Academic Press, New York, 1963. 5B. Chance, J. Biol. Chem. 240, 2729 (1965).
746
ENERGY-LINKED REACTIONS
[114]
concentrations of A T P ( ~ 1 5 mM) are required. Reagents and Reaction Media. The components are given on the basis of a 3.0 ml total volume; the systems m a y be scaled down or up as required. RESPIRATION-SUPPORTED SYSTEM
Stock solutions
0.1 M Tris-chloride, pH 7.2 0.1 M Na-succinate or DL-fl-hydroxybutyrate, pH 7.2 0.1 M MgC12 0.4 M Na-K phosphate buffer, pH 7.2 0.8 M NaC1 0.15 M Na-ATP, pH 7.2 0.03 M 45CaC1~(0,05 ~C per ~mole) Mitochondrial suspension (protein, 10 mg/ml in 0.25 M sucrose) H20 Total volume:
Volume (ml)
Final concentration (mM)
0.3 0.3
10 10
0.3 0.3 0.3 0.06 0.3 0.3
10 4.0 80 3.0 3.0 1.0 mg protein per ml
0.84 3.0
ATP-suPPORTED SYSTEM. Same as above, with respiratory substrate replaced by water and the final A T P concentration raised to 15 mM. Procedure. Mitochondria are added last to start the reaction, which is usually carried out for 10 minutes with shaking in air at 30 ° in beakers or large centrifuge tubes. The tubes are then rapidly cooled in cracked ice, and centrifuged in the cold at 20,000 g for 4 minutes to separate the "loaded" mitochondria from the suspending medium.
Determination o] the Uptake o] Ca +* and InorgaNic Phosphate Reagents Sucrose, 0.25 M Sodium lauryl sulfate, 0.2% Trichloroacetic acid 8%
Procedure. After sedimentation of the mitochondria, the supernatant reaction media are saved for analysis. The sedimented mitochondria are washed twice with 5.0-ml portions of cold 0.25 M sucrose. Prior to the last centrifugation, the suspensions of washed mitochondria are divided into two equal portions. After centrifugation, one pellet is extracted with 5.0 ml of cold trichloroacetic acid and aliquots of the clarified extract are used for P~ determination by the method of Gomori2 The other pellet is G. Gomori, ]. Lab. Clin. Med. 27, 955 (1942).
[114]
ENERGY-LINKED TRANSPORT
747
dissolved in 5.0 ml sodium lauryl sulfate. Aliquots (0.3 ml) of the sodium lauryl sulfate extracts of the pellets, and of the original clarified reaction media, are plated on aluminum planchets (2.0 cm diameter), evaporated to dryness to yield "thin" samples, and counted in a gas-flow Geiger tube. The distribution of radioactivity between washed pellets and the incubation medium is used to calculate the uptake of Ca ++. The preexisting intramitochondrial Ca ++ (10 millimicromoles per milligram of protein) is very low compared to the large amounts of Ca ++ taken up (up to 2500 millimicromoles Ca ++ per milligram of protein); the Ca ++ uptake data therefore need not be corrected for exchange. Since P~ uptake is stoichiometric with that of Ca ++, measurement of accumulated P~ in the mitochondrial trichloroacetic acid extract will suffice as a simpler means of following accumulation of Ca ++ and phosphate.
Properties of the Massive Loading Reaction For each pair of electrons passing through each energy-conserving site of the respiratory chain, about 1.67 molecules of Ca ++ and 1.0 molecule of Pi are accumulated; Ca ++ and Pi are thus accumulated in a molar ratio of 1.67, which is that of hydroxylapatite.2 The ATP-supported system is less efficient. Mg ++ is essential in the incubation medium, and some of it is accumulated with the Ca ++ and phosphate. 7 Antimycin A and cyanide block Ca ++ uptake in systems supported by electron transport, whereas oligomycin has no effect. Oligomycin, on the other hand, greatly inhibits the ATP-supported system, which is otherwise only slightly inhibited by antimycin A or cyanide. Ion uptake in either type of system is inhibited by true uncoupling agents such as 2,4-dinitrophenol.la, o Massive uptake of s~Sr++ can be studied under the same conditions described above;S the uptake of ssSr++ is determined by counting aliquots of the clarified reaction medium or of the trichloroacetic acid extracts of the washed mitochondria in a scintillation counter.
Uptake of A T P or ADP during Massive Uptake of Ca++and P~ Accumulation of ATP and ADP may be measured in the same medium used for study of the uptake of Ca ++ and Pi. In this case, ATP8-14C (0.05-0.1/~C) is employed, and the Ca ++ added is unlabeled. AMP of the medium is not accumulated under these conditions2 7E. Carafoli, C. S. Rossi, and A. L. Lehninger, J. Biol. Chem. 239, 2055 (1964). E. Carafoli, S. Weiland, and A. L. Lehninger, Biochim. Biophys. Acta, 97, 88 (1965). E. Carafoli, C. S. Rossi, and A. L. Lehninger, J. Biol. Chem. 2,40, 2254 (1965).
748
ENERGY-LINKED REACTIONS
[114]
Procedure. At the end of the incubation, the mitochondria are centrifuged and washed twice with 5.0-ml portions of 0.25 M sucrose. Before the last centrifugation, the suspensions are divided into two aliquots. One of the resulting pellets is dissolved in 0.2% sodium lauryl sulfate; aliquots of the solutions are plated and counted in a low-background gas flow counter. The other pellet is extracted with 6% cold perchloric acid. Aliquots of the perehloric acid extract may be applied to paper, and the labeled ATP and ADP separated and identified by paper electrophoresis2
Limited Loading with Ca ++ In the limited loading system,~ the mitochondria are usually incubated in cuvettes of the Clark oxygen polarograph linked to a strip chart recorder. 1° A known amount of Ca ++ is added to mitochondria respiring in state 4, and the rate and amount of extra oxygen uptake is recorded, up to the point when oxygen uptake returns to the initial resting rate. Ca ++ uptake is then measured in an aliquot of the medium.11 Reagents and Reaction M e d i u m . This is equilibrated with air.
Reagent 0.1 M Tri~-chloride, pH 7.2 0.8 M NaC1 0.1 M Na-suecinate, pH 7.2 H~O Total volume:
Volume (ml)
Final concentration (mM)
0.19 0.19 0.09 1.33 1.80
10 80 10
Procedure. After thermoequilibration of the above medium at 25 ° in the polarograph cuvette, 0.10 ml of mitochondrial suspension (5.0 mg protein) is added and the course of the state 4 oxygen uptake is followed. The contents of the euvette are continuously stirred by a magnetic bar. Sixty seconds after the addition of the mitoehondria, 45CaCl.~ (200-600 millimicromoles) is added with a mierosyringe (6.7-20 #l). When the respiration has returned to the resting rate, a 1.0-ml aliquot of the suspension is removed with a 1.0-ml syringe equipped with a needle. The needle is quickly replaced with a Swinny adapter containing a Millipore filter (0.8 ~ pore size) and 0.3-0.4 ml of clear filtrate are collected by exerting moderate pressure on the plunger of the syringe.TM The entire
10W. W. Kielley and J. R. Bronk, J. Biol. Chem. 230, 521 (1958). 1~E. Carafoli, Biochim. Biophys. Acta 97, 107 (1965). ~tt. Rasmussen, A. Waldorf, D. D. Dziewiatow~ki, and It. F. DeLuca, Biochim. Biophys. Acta 75, 250 (1963).
[l14a]
FATTY ACID ACTIVATION
749
operation requires approximately 10 seconds. An aliquot of the clear filtrate (0.1-0.2 ml) is plated and counted for 4~Ca++. The Ca÷+:0 accumulation ratio is the ratio of millimicromoles Ca +* accumulated to millimicroatoms of extra oxygen uptake stimulated by the addition of Ca ++, as recorded by the polarograph trace. Its value will depend on the respiratory substrate; it is approximately 5-6 for fl-hydroxybutyrate, 3.6-4.0 for sueeinate. 2 The baseline state 4 respiration is also capable of coupling to Ca ÷+ accumulation, but the efficiency is low. The Ca÷÷:0 activation ratio is the ratio (millimicromoles Ca ++ added): (millimicroatoms extra oxygen taken up); it is usually slightly larger than the Ca +÷:0 accumulation ratio. Properties of the Reaction. Usually not more than 1% of the added Ca *÷ is left in the supernatant 30 seconds after the respiration has returned to the resting rate. Mg ÷÷ ions and ATP are not necessary for simple uptake of Ca ÷÷ in the absence of P,. When P~ is added, it is also accumulated if the system contains 1.0 mM ATP plus 10 mM MgC12, or 1.0/~g of oligomycin? Limited uptake of Sr ÷÷11 and Mn ÷÷4 can be studied under the same conditions, by employing 85Sr+÷ or 54Mn*÷. With these cations, respiration returns to the resting rate in the presence of inorganic phosphate; ATP is not required.
[ 1 14a] F a t t y A c i d A c t i v a t i o n a n d O x i d a t i o n b y M i t o c h o n d r i a
By SIMON G. VAN DEN"BERGH In animal cells fatty acid activation occurs both in the mitochondria and in the microsomes, but the mitochondria are the only site of fatty acid oxidation. Fatty acids are different from other mitochondrial respiratory substrates in three important respects: (a) they need activation with formation of thiol esters of coenzyme A prior to their oxidation; (b) they are potent uncoupling agents; and (c) they are powerful mitochondrial swelling agents. These properties all have specific consequences and require special precautions. Although fatty acid breakdown has been studied in mitochondria isolated from a variety of animal and plant tissues, we shall in this chapter mainly refer to rat liver mitochondria and only in the last paragraph consider some of the properties of fatty acid breakdown in other types of mitochondria.