Vol.

THE JOURNAL OF B~OLOCICAL CHEM~~Y 250, No. 21, Issue of November 10, pp. 8361-8375, Printed m U.S.A.

1975

The Steady State Concentrations Coenzyme A Thioester, Citrate, Tricarboxylate Cycle Oxidations
RICHARD G. HANSFORD AND ROGER N. JOHNSON

of Coenzyme A-SH and and Isocitrate during in Rabbit Heart Mitochondria

(Received for publication, May 2, 1975)

From the Gerontology Research Center, National Institute of Child Health and Human National Institutes of Health, Baltimore City Hospitals, Baltimore, Maryland 21224

Development,

The steady state mitochondrial content of coenzyme A-SH (CoA), acetyl-CoA, succinyl-CoA, and long chain acyl-CoA has been determined during the oxidation of palmitoylcarnitine by rabbit heart mitochondria. Variation of the substrate concentration during ADP-stimulated (state 3) respiration varies the mitochondrial content of long chain acyl-CoA and the rate of 0, uptake, and permits the conclusion that the K, of p oxidation for intramitochondrial long chain acyl-CoA is approximately 1 nmol/mg of mitochondrial protein. At near saturating concentrations of palmitoylcarnitine, plus Lmalate, the addition of ADP causes a decrease in acetyl-CoA, an increase in CoA and succinyl-CoA, and no clear change in long chain acyl-CoA content. These changes reverse upon the depletion of ADP (state 3 - 4 transition). Similar changes in CoA, acetyl-CoA, and succinyl-CoA are seen during state 4 - 3 + 4 transitions with pyruvate plus L-malate and octanoate plus L-malate as substrates. These results suggest a limitation of flux by citrate synthase during the controlled oxidation of these three substrates. The ratio acetyl-CoA/succinyl-CoA was determined not only during state 3 and state 4 oxidation of palmitoylcarnitine plus L-malate and pyruvate plus L-malate, but also during intermediate respiratory states (state 3%) generated by adding glucose and varying amounts of hexokinase. These intermediate states are characterized by a high succinyl-CoA content, relative to either state 3 or state 4, and a suboptimal flux through citrate synthase, estimated either by pyruvate disappearance or by 0, uptake. Despite this, no correlation was found between acety-CoA/succinyl-CoA ratio and flux through citrate synthase with the possible exception of the region between 72 and 100% of the state 3 rate with acetylcarnitine as substrate. Experiments involving the state 4 + 3 + 4 transition with P-oxoglutarate plus L-malate as substrate showed a high succinyl-CoA content in state 4, and inclusion of 2-oxoglutarate in an experiment with pyruvate plus L-malate and varying activities of hexokinase gave decreased acetyl-CoA/succinyl-CoA ratios at low citrate synthase flux. However, increased citrate synthase flux achieved with increased hexokinase was not associated with a rise in this ratio. Thus it seemed more probable that under all of the conditions studied citrate synthase is regulated by the availability of its other substrate, oxalacetate. This prompted the estimation of mitochondrial NAD+ and NADH in states 3 and 4 as a determinant of oxalacetate concentration, and the estimation of intramitochondrial citrate concentration, as a determinant of oxalacetate binding to citrate synthase. Mitochondrial citrate content was found to rise during state 3 + 4 transitions, but not to decrease during state 4 + 3 transitions. Its contribution to citrate synthase regulation under these experimental conditions is thus equivocal. The mitochondrial isocitrate content, however, decreased during the state 4 + 3 transition and this is consistent with an activation of isocitrate dehydrogenase. This was true with either pyruvate plus L-malate or palmitoylcarnitine plus L-malate as substrate. Experiments with uncoupling agents and oligomycin allowed the demonstration that a decreased NAD(P)H/NAD(P) + ratio is more important than a decreased ATP/ADP ratio in the activation of isocitrate dehydrogenase. An evaluation of the possible contribution of NAD-isocitrate dehydrogenase (EC 1.1.1.41) and NADP-isocitrate dehydrogenase (EC 1.1.1.42) is made. The steady state concentrations of CoA, CoA thioesters, citrate, and isocitrate are compared with values obtained for perfused heart, with glucose or glucose plus palmitate as substrate.

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The oxidation of fatty acids supplies a major portion of the energy requirements of mammalian heart (see Neely et al. (1) for a review). One method of studying the control of this

process is to intermediates when the flux

study the steady state concentrations of key and investigate the way in which they change through the pathway is changed (see Rolleston

8361

8362 (2) for a review). This approach has been applied to CoA, acyl-CoA derivatives, and tricarboxylate cycle intermediates in whole heart (3) but there are at present no data obtained with isolated heart mitochondria, oxidizing fatty acids. Such data may be useful in evaluating results obtained with the whole heart which will reflect contributions from both mitochondria and cytosol. This may be especially true for CoA and CoA thioesters, in that these are divided into two intracellular pools by the impermeability of the mitochondrial membrane. This study presents determinations of mitochondrial CoA, acetyl-CoA, succinyl-Coil, and long chain acyl-CoA, and documents the changes that occur as the concentration of the substrate, palmitoylcarnitine, is varied and as the mitochondrial energy state is varied by the increased availability of ADP. Such studies focused attention on the control of citrate synthase (EC 4.1.3.7), and the results of these experiments and others involving pyruvate and acetylcarnitine as substrates have been examined to see whether they fit a model of control by the ratio acety-CoA/succinyl-CoA advanced by LaNoue et al. (4). In addition, studies have been made of the intramitochondrial content of citrate and isocitrate and the response to mitochondrial energy state. These experiments allow the identification of isocitrate dehydrogenase as a control site, which may be important in the context of the control of citrate synthase, because citrate inhibits citrate synthase competitively with respect to oxalacetate (5). It is shown that the oxidation-reduction state of nicotinamide nucleotide is more important than the ATP/ADP ratio in determining flux through the isocitrate dehydrogenase step.
EXPERIMENTAL PROCEDURE

Williamson and Corkey (8) and then assaying CoA by the cycling technique (7. 9). Sampling, extracting. neutralization, and assay procedures for citrate, isocitrate, and 2.oxoglutarate were as described previously (10) with the mitochondrial content of these compounds being determined by the subtraction of a supernatant content, obtained after microcentrifugation. from the total content of the incubation medium containing mitochondria. The extraction and assay of mitochondrial NAD +, NADP+, NADH, and NADPH were as described by Williamson and Corkey (8). LVhere duplicate experiments were performed they were matched closely with respect to time of sampling and, when tricarboxylate cycle intermediates were assayed, with respect to protein concentration. The values presented are means and Students t test was applied to establish the significance of differences between groups of data; where limits of error are given, the standard error of the mean is shown. with the number of samples in parentheses. In all extraction experiments, 0, uptake was monitored continuously, using a Clark-type 0, electrode. Where rates of 0, uptake are quoted, these were obtained in parallel incubations involving air-saturated medium and a completely closed chamber of volume 2 ml. The recordings of intramitochondrial NAD(P)H were obtained in parallel incubations. using a Farrand fluorimeter, as described previously (11). ATP and ADP were measured in extracts as described by Williamson and Corkey (8) using either the fluorimeter or a Beckman-Gilford spectrophotometer, as appropriate. Pyruvate in the extracts was measured spectrophotometrically with lactate dehgdrogenase. Extraction and Estimation of Isocitrate Dehydrogevmses and Nicotinamide Nucleotide Transhydrogenase-Two methods were used to remove the latency of NAD-&citrate dehydrogenase. In one procedure, 1 ml of mitochondrial suspension containing 15 to 20 mg of protein was diluted with 2 ml of 50 mM KP,, pH 7.2, containing 2 mM ADP, 1 mu EDTA, and 10 rn~ dithiothreitol. The suspension was sonicated using a Branson sonifier, setting 2, in bursts (3 x 20 s). 40 s

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elapsing between each burst. Cooling during sonication was effected by

an ice/water mixture. Suhmitochondrial particles were sedimented at 100,000 x g for 45 min and the supernatant fraction was used for the assay of NAD-isocitrate dehydrogenase. The assa!. medium comprised 50 tnM KP,, pH 7.2, 10 rn~ MgCI,. 0.5 pg of rotenone/ml. 1 kg of oligomycin/ml, and NAD+. Isocitrate was added as a mixture of citrate and m-isocitrate containing a 25.fold molar excess of citrate over threo-n,-isocitrate to start the reaction, and NAD reduction was followed spectrophotometrically at 340 nm. In the second procedure, mitochondria were added directly to a medium containing 50 trIM KP,, 10 mM M&l,, 1 mM dithiothreitol, 0.5 pg of rotenone/ml. 1 py of oligomycin/ml, 0.05t (v/v) Triton X-100,

Preparation of Mitochondria-Mitochondria were prepared from the heart of one New Zealand white rabbit by a slight modification of the method of Chance and Hagihara (6). The heart was chopped with scissors and then passed through a tissue press with holes of l-mm diameter. The heart muscle was then incubated with 7.5 mg of Nagarse (from Enzyme Development Corp., New York) in a total volume of 40 ml of 0.25 M sucrose/l0 tnM N-tris(hydroxymethyl)methyl-P-aminoethanesulfonate (TES)/l mM ethyleneglycol bis(&aminoethyl ether). N,N-tetraacetic acid (EGTA) pH 7.2, at 0. Incubation was carried out in a Dounce homogenizer, with a Teflon pestle for a total of 12 min. Homogenization was carried out by hand, as soon as mechanically possible, with a total of five complete passes. The homogenate was then centrifuged at 10,000 x g for 6 min and the entire pellet was resuspended in 60 ml of preparation medium. The suspension was centrifuged at 350 x g for 10 rain and the supernatant layer was decanted. The latter was then centrifuged at 10,000 x g for 6 min to yield mitochondrial pellets. These were resuspended and pooled to give a total volume of 30 ml. The final centrifugation was for 7 min at 7,500 x g to permit the removal of an upper fluffy iayer from the pellet. The mitochondria were resuspended in 2 ml of preparation medium, to give a suspension of approximately 20 mg of protein/ml. For some experiments the mitochondrial suspension was depleted of endog.enous substrate by adding it to 15 ml of 0,.saturated basal medium (see below) to which ADP had been added to 0.5 mM, glucose to 5 mM, M&l, to 2.5 mM, hexokinase to 10 units/ml, and L-malate to 0.1 ITIM. After 7 min at 25, the diluted mitochondrial suspension was cooled and centrifuged at 7500 x g for 7 min. The pellet was resuspended as described previously. Incubatiorl of Mitochondria and Extraction and Assay of Intermediates-All incubations used a basal medium comprising 0.12 M KCl, 20 mM KTES, pH 7.2, and 10 mM KP,, pH 7.2. Other additions are detailed in the figure legends. All experiments were carried out at 25. The sampling, extraction, neutralization, and assay procedures for CoA, acetyl-CoA, and succiny-CoA were exactly as described previously (7). Long chain acy-CoA was determined in the washed pellets of HClO,-insoluble material by hydrolyzing the thioester as described by In this paper, CoA is used to denote coenzyme A-SH

and KAD+. The final protein concentration

was about 0.2 mg/ml and

the pH was 7.2. The reaction was begun by addition of a citrate--isocitrate mixture and followed at 340 nm as described above. For the assay of SADP-isocitrate dehgdrogenase, mitochondria were added to the medium containing Triton X-100 used for the SADlinked enzyme, NADP+, replacing NAD+. All other conditions were as described for the NAD-linked enzyme. NADPH-NAD + transhydrogenase was assayed in submitochondrial particles isolated by the method of Hansen and Smith (12) as described hy Lee and Ernster (13). The assay procedure (14) used a trapping system of 3 mM pyruvate and an excess activity of lactate dehydrogenase (0.7 units/ml final concentration). The reaction was initiated by addition of NAD+ to 0.4 mu. The NADPH concentration was 0.1 ITIM. Under these conditions the assay was linear with added particle protein. Transhydrogenase activity was calculated with respect to mitochondrial protein hy estimating the cytochrome a content of submitochondrial particles and intact mitochondria, using the difference in absorbance between 605 and 630 nm of air-oxidized, dithionite-reduced cytochrome. Reagents and Enzymes-Reagents were of the highest purity available commercially. The uncoupling agent FCCP was from Pierce Chemical and oligomycin was from Sigma Chemical Co. All enzymes used were from Boehringer-Mannheim Corp., New York.
RESULTS AND DISCUSSION

The Steady State Mitochondrial Thioester during Oxidation of

Contents of Palmitoylcarnitine

CoA and
and

CoA Oc-

The abbreviation used is: FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone.

8363
.* (Al ADP

0.6-

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2 MINUTES

CoA and CoA thioester during state 4 - 3 + 4 FIG. 1. Mitochondrial transitions with palmitoylcarnitine and L-malate as substrate. At zero suspension (protein 18.4 mg/mll were time, 1.5 ml of mitochondrial added to 20 ml of a basal medium containing 12 @M palmitoyl-L-carnitine and 0.5 mM r--malate. The medium was saturated with air. At the point indicated, 12 pmol of ADP were added. Samples (2 ml) were tanoate-Fig. 1 presents the results of an experiment in which mitochondrial CoA and CoA thioester were estimated during the oxidation of palmitoylcarnitine plus 0.5 mM L-malate. It is seen that during the initial phase of controlled respiration (state 4, according to Chance, and Williams (15)) acetyl-CoA content is high, whereas that of CoA is exceedingly low. On

withdrawn, described indicated electrode l - - -0. long chain

deproteinized. and assayed fcr CoA and CoA thioesters as under Experimental Procedure. The points 01 sampling by symbols in A correspond with the numbers on the oxygen trace shown in B. O---O, CoA; O-0, acetyl-CoA; succiny-CoA; W-a, HClO,-insoluble CoA (referred to as acyl-CoA in the text).

activation of respiration by ADP (state 3) there is a marked fall in acety-CoA content, with a concomitant increase in CoA content. In addition, succiny-CoA content increases slightly. This latter change is defined with more exactness in a later section. On re-entry into state 4, the changes reverse, with the absolute amounts measured being somewhat higher, owing to a decline in long chain acy-CoA content with a decline in the concentration of the substrate. palmitoylcarnitine. Although in this experiment the long chain acyl-CoA content appeared to rise during the state 4 + 3 transition, the opposite behavior in other experiments (Fig. 5 and not shown) indicates that there is no clear response to changes in flux. The absence of any large change suggests that increased carnitine palmitoyltransferase activity (EC 2.3.1.a) which would be expected in state 3 owing to the rise in CoA content must be matched by an increase in the activity of p oxidation in this state, the latter presumably due to decreased concentration of NADH and FADH,. The quantitative contribution of long chain acyl:CoA to the mitochondrial pool of CoA and thioester is greater than that in perfused heart even at high palmitate concentrations (31, suggesting that the ratio of the concentration of long chain acy-CoA to CoA is higher in the mitochondria than in the

cytosol. The low concentrations of CoA and acetyl-CoA in mitochondria oxidizing palmitoylcarnitine would be difficult to measure using conventional stoichiometric assays, but are amenable to the kinetic assay described by Allred and Guy (9). The modification of this assay to include succinyl-CoA synthetase (7) permits the measurement of succiny-CoA at equally low concentrations. The clear decrease in acetyl-CoA content on activating respiration which is seen in Fig. 1 implicates the tricarboxylate cycle. and specifically citrate synthase, as being rate limiting during the controlled (state 4) oxidation of palmitoylcarnitine by isolated heart mitochondria. This is in accord with a conclusion reached by Oram et al. (3) on the control of the oxidation of high concentrations of palmitate by perfused rat heart. There is no real conflict with the conclusion reached by Pande (16) that the activity of /3 oxidation limits the rate of oxidation of the palmitoyl group by rat heart the earlier study (16), which mitochondria. This is because compared the rate of palmitoyl group disappearance in the presence and absence of a functioning tricarboxylate cycle, exclusively used conditions which maximally activate oxidative phosphorylation (ADP) or electron transfer (uncoupling agent). By contrast, the present study is concerned with those factors which regulate the oxidation of palmitoyl groups and pyruvate when the rate of oxidative phosphorylation is a minimum (state 4, Fig. 1) or is less than maximal (state 3?2, see below). It is noteworthy that Garland et al. (17) found changes precisely opposite to those reported here during the state 4 + 3 transition, in experiments involving the oxidation of

8364 palmitoylcarnitine by rat liver mitochondria. The large increase in acety-CoA content, which they reported, suggests an increase in the activity of p oxidation relative to citrate synthase, upon the activation of respiration. This emerges as a major difference between heart and liver mitochondria. The presence of a high content of acetyl-CoA during state 4 respiration in the present work (Fig. 1) and the irreversible nature of the citrate synthase reaction raise the question of the control of this enzyme, and this is discussed in a later section. It has been shown that no decrease in acety-CoA content occurs on increasing the pressure development of rat hearts oxidizing octanoate (3), contrary to the decrease observed with palmitate. For this reason an experiment was carried out similar in.design to that shown in Fig. 1, but using 0.25 mM octanoate plus 0.5 mM L-malate as substrate (Fig. 2). This was found to be an optimal concentration of octanoate in the absence of serum albumin, giving a State 3 rate of oxygen consumption of 0.275 lg.atoms of 0 Jmin/mg of protein. It is seen that the decrease in acetyl-CoA content with the activation of respiration was marked, and indeed greater than that obtained with palmitoylcarnitine (Fig. 1). The discrepancy between this result and that obtained with perfused hearts (3) is unexplained, but may reflect species difference.
Steady State Thioester during Concentrations-There Mitochondrial Oxidation Contents
of Pyruuate, of CoA and CoA Present at Varying

3). It is seen that the state 4 + 3 transition is associated with changes similar to those described for Fig. 1, and that the acetyl-CoA content clearly falls. The CoA/acety-CoA ratio is somewhat higher in state 4 than that shown in Fig. 1, suggesting that pyruvate dehydrogenase is less active in state 4 than is p oxidation as a mechanism of generating acetyl groups. Repetition of the experiment at a lower pyruvate concentration (0.25 mM) gave the results shown in Fig. 4. It is noted that the pattern of changes seen in response to the activation of respiration is very similar to that seen at 2.5 mM pyruvate, but that the CoA/acetyl-CoA ratios are higher in both state 3 and state 4, consistent with a decreased pyruvate dehydrogenase activity at the lower substrate concentration. There is a clear rise in succinyl-CoA content on activating respiration, as is
ADP I STATE 3-4

06

05

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is some ambiguity in the literature about the mitochondrial content of CoA and acety-CoA during controlled (state 4) and active (state 3) pyruvate plus L-malate oxidation. Thus, acetyl-CoA content was found to be the same in states 3 and 4 (18) and this was confirmed for most incubation times in a later study (4). However, the latter work did demonstrate an elevated CoA content in state 3 and another study involving rabbit heart mitochondria demonstrated a fall in acetyl-CoA content on uncoupling, but did not present data for state 3 (19). Because of this ambiguity, an experiment was performed of the design shown in Fig. 1, but with 2.5 mM pyruvate plus 0.5 mM L-malate as substrate (Fig.

OL MINUTES

FIG. 2. Mitochondrial CoA and CoA thioester during state 1- 3 - 4 transitions with octanoate plus L-malate as substrate. The reaction was initiated by the addition of 0.9 7 ml of mitochondrial suspension (protein = 17.7 mg/ml) to 20 ml of a basal medium containing 0.25 mM octanoate and 0.5 mM L-malate. At the point indicated, 40 pmol of ADP were added. The medium was saturated with 0,. O--O. CoA; O---O, acetyl-CoA; l - - -0, succiny-CoA.

(Ai

q lo -I

ADP

OXYGEN I ~(APPROX pg-ATOM/ML)

0.1

0 MINUTES

2 MINUTES

5.

FIG. 3. Mitochondrial CoA and CoA thioester during state 1+ 3 - 4 transitions with a high concentration of pyruvate plus r.-malate as substrate. One milliliter of mitochondrial suspension (13 mg of protein) was added at zero time to 20 ml of a basal medium containing 2.5 rnrvr pyruvate and 0.5 rnM L-malate. The medium was saturated

with air. ADP (13 pmol) was added at the point indicated. Panel A presents the mitochondrial CoA and thioester content, panel R, an oxygen electrode trace from the same experiment. O---O, CoA; 0-O acetyl-CoA; l - - -0, succinyl-CoA.

8365

(A)O./
0.6z Li L 05 \ I)

ADP 1

OXYGEN (APPROX 0.1 MS-ATOM/ML) 4 L, 1 6 : I

I OO

I 1

I 2

I 3 MINUTES

, 4

I 5

I 6

3 MINUTES

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FIG.

substrate. containing l - -0.

4. Mitochondrial CoA and CoA thioester during state 4 + 3 - 4 transitions with a low concentration of pyruvate plus r.-malate as The reaction was initiated by the addition of 1.4 ml ofmitochondrial suspension (protein 24.4 mg/ml) to 20 ml of a basal medium 0.25 mM pyruvate and 0.j mM I,-malate. At the point indicated, 12 Mmol of ADP were added. 0-O. CoA: O---O. acetyl-CoA:

succinyl-CoA.
TABLE Pattern of acylation of mitochondrial pyruuate, generated by lactate I CoA during dehydrogenase oxidation reaction of

found in mitochondria from blowfly flight muscle (7). Such a change is noted as a response to increased work load in the perfused rat heart (1) but has not been shown with isolated heart mitochondria. Thus, LaNoue et al. (4) show rather similar succinyl-CoA contents in states 3 and 4 with acetylcarnitine plus malate as substrate and show a slight fall in succinyl-CoA content on addition of uncoupling agent (20). There are no values in the literature for succiny-CoA content of heart mitochondria oxidizing either pyruvate, palmitoylcarnitine, or octanoate. The importance of succiny-CoA concentration is discussed in a later section. The rationale of this experiment (Fig. 4) was to use a pyruvate concentration nearer to that found in the heart, so as not to obscure possible controls on pyruvate dehydrogenase activity. It is seen nevertheless that the CoA/acetyl-CoA ratio is still much lower, in both states 3 and 4. than the ratios of approximately 11:l and 14:l found by Oram et al. (3) in perfused hearts oxidizing glucose alone, and at low and high work loads, respectively. A further experiment (Table I) used lactate dehydrogenase to generate a low and constant concentration of pyruvate for mitochondrial oxidation. Samples were initially taken during state 4 respiration and the pyruvate concentration was determined to be 14 FM. Even at this substrate concentration, the CoA/acetyl-CoA ratio was still low (0.26). Addition of ATP plus 0.11 mM M&l, gave a doubling of respiratory rate, due to extramitochondrial ATPase, possibly of myofibrillar origin, and a rise in the CoA/acetyl-CoA ratio to 0.35. Further portions of this experiment in which the respiratory rate was further increased by addition of hexokinase are not presented in Table I as the equilibrium pyruvate concentration began to decline. However, even during state 3 respiration and at 6 fiM pyruvate the CoA/acetyl-CoA ratio was 3.4, still considerably less than the values reported during carbohydrate oxidation in the heart. This discrepancy may reflect a high ratio of CoA/acetyl-CoA in the cytosol of the heart, or the presence in the intact organ of a control of pyruvate dehydrogenase not reconstructed in the

One milliliter of mitochondrial suspension (24 mg of protein) was added to 20 ml of 0,.saturated basal medium containing 0.3 mM I.-malate, 1.25 mM NAD+, 0.5 mM ADP, 5 mM glucose, and 12 units/ml of dialyzed lactate dehydrogenase. After 5.5 min of incubation, to cause depletion ofendogenous substrate, the reaction was initiated by the addition of 150 rmol of DL-lactate. Two samples were taken at 7.5 min (state 4). ATP (10 pmol) and M&l, (2 pmol) were added at 8 min to partially activate respiration, and two further samples were taken at

9.5 min. CoA and CoA derivatives, ADP, ATP, and pyruvate were estimated in the samples as described under Experimental Procedure. Pyruvate was 13.7 + 0.4 pM in samples 1 and 2 and 13.0 I 0.5 fiM in samples 3 and 4. 0, uptake was measured in a parallel experiment. Respiratory state
latio iTP/ 1DP Rate of 0 $ con-

sumption

content

CoA

AcetyL
CoA contenr

SuccinylCoA content

minlmg protein

nmollmg

protein

State 4
+ATP +Mg*+

170 72

0.036 0.071

mitochondrial experiments. In this context the control of pyruvate dehydrogenase by phosphorylation-dephosphorylation (21, 22) is thought to underlie the changes in pyruvate dehydrogenase activity which can be shown in rabbit heart mitochondria in the presence of ADP plus uncoupling agents plus Mg2+ (19) or on incubation in the presence of ATP, oligomycin, and fluoride (23). Experiments were not carried out under either of these sets of conditions in the present study, but a partial inactivation of pyruvate dehydrogenase by phosphorylation cannot be ruled out during state 4. Steady State Mitochondrial Contents of CoA and CoA Thioester during Oxidation of Pyruvate Plus Palmitoylcarni-

8366

01 t

MINUTES

MINUTES

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5. Mitochondrial CoA and CoA thioester during the oxidation of both pyruvate and palmitoylcarnitine. The reaction was initiated by the addition of 1.2 ml of mitochondrial suspension (protein _ 22.8 mg/ml) to 20 ml of air-saturated basal medium containing 0.25 mM pyruvate, 10 FM palmitoyl-L-carnitine, and 0.5 mM L-malate. ADP (40
FIG.

pmol) was added at the point indicated. Panel A presents the contents of CoA and CoA thioesters determined by sampling and extraction; panel B, an 0, electrode recording obtained from the same experiment. O---O, CoA; 0-O. acetyl-CoA: l - ~-a, succiny1LCoA; W---m, HClO,-insoluble CoA.

tine and of 2-Oxoglutarate-Fig. 5 presents the results of an experiment in which the substrate was 0.25 mM pyruvate/lO pM palmitoyl-L-carnitine plus 0.5 mM L-malate and in which respiration was stimulated by sufficient ADP to give anaerobiosis without a state 3 - 4 transition. A control experiment established that the palmitoylcarnitine would be depleted at the time of taking sample 6, if palmitoylcarnitine were oxidized to the exclusion of pyruvate. Equally, the concentration of palmitoylcarnitine remaining at a time corresponding to sample 4 would be sufficient to guarantee a maximal rate of palmitoylcarnitine oxidation. It is noted that the content of long chain acyl-CoA fell progressively during state 3 oxidation, this presumably due to the depletion of palmitoylcarnitine. The pattern of CoA, acetyl-CoA, and succinyl-CoA contents resembles more closely that obtained with palmitoylcarnitine plus L-malate (Fig. 1) than that with 0.25 mM pyruvate plus L-malate (Fig. 4). Thus, the CoA/acetyl-Co.4 ratio is very low in state 4 (approximately 0.11, Fig. 5, sample 3) and the CoA/succinyl-CoA ratio is approximately 1 in state 3. The results of this experiment allow comment to be made on the suggestion by Bremer (24) that the mitochondrial oxidation of palmitoylcarnitine inhibits pyruvate oxidation by competition for mitochondrial CoA. The CoA content is indeed decreased in the presence of palmitoylcarnitine (compare Figs. 4 and 5) but is still high relative to the K, of pyruvate dehydrogenase (25, 26). The ratio CoA/acety-CoA is probably more significant for pyruvate dehydrogenase activity (25, 26) and this is marginally decreased by the presence of palmitoylcarnitine during state 3 oxidation (Figs. 4 and 5). However, the CoA/ acetyl-CoA ratio is much lower during state 4 respiration in the presence of both pyruvate and palmitoylcarnitine than it is with pyruvate alone. The respiratory state studied by Bremer (24) is not clear and may well have been between states 3 and 4, in which case a diminished CoA/acetyl-CoA ratio could have had a material effect in diminishing the oxidation of pyruvate.

Finally, the same sort of experiment was repeated with 2-oxoglutarate plus L-malate as substrate (Fig. 6) to explore the acylation pattern of CoA during the operation of the 2-oxoglutarate + malate segment of the tricarboxylate cycle. There is information on succinyl-CoA and CoA content in state 4 respiration (20), but none on the changes evoked by a state 4 - 3 transition. It is seen that the succiny-CoA/CoA ratio is extremely high in state 4, in fair agreement with the results of LaNoue et al. (20) and that it declines upon the generation of state 3 respiration (Fig. 6). It is noted that this is the inverse of the response of the succinyl-CoA/CoA ratio to ADP addition described in the earlier figures. However, this is not unreasonable, as both the high concentration (1 mM) of added 2-oxoglutarate and the relatively low reduction of NAD(P) seen in Fig. 6C (cf. Figs. 8B and 9B) will allow a high activity of P-oxoglutarate dehydrogenase in state 4. The decrease in succinyl-CoA content on adding ADP then represents a greater activation of succinyl-CoA synthetase (EC 6.2.1.4), operating in the direction of succinyl-CoA cleavage, than of 2-oxoglutarate dehydrogenase.
Effect of Pyruvate and of Palmitoylcarnitine upon Acylation of Mitochondrial CoA and Consumption during State 3 Respiration-A Concentration Rate of Oxygen

systematic study was made of the relation between the concentration of added pyruvate, the mitochondrial respiratory rate, and the pattern of CoA acylation (Table II). State 3 respiration was guaranteed throughout the study by the presence of ADP and hexokinase plus glucose. It is seen that over the range of pyruvate concentration 0.024 to 0.16 mM the ratio CoA/acetyl-CoA declines steadily, such that there is an inverse linear relationship between this ratio and the respiratory rate (Table II, plot not shown). Over this range of pyruvate concentration, the respiratory rate doubles, whereas there is very little change in the acetyl-CoA/succinyl-CoA ratio. Data and reasoning to be adduced later suggest that oxygen consumption may be taken

8367
IC)

-I

1
(A)

FLUORESCENCE INCREASE

ADP

60 -

OXYGEN IAPPROX 0.4 ,,g-ATOM/ML)

20 o--o-o o\O Ol 2 / 3 MINUTES \ 4 1 FCCP 8 6 0 1 2 3 MINUTES 4 5 6

O--o ,FO 5

FIG. 6. Mitochondrial CoA and succinyl-CoA during state 4 - 3 -4 transitions with 2-oxoglutarate plus L-malate as substrate. The reaction was initiated by the addition of 1.3 ml of mitochondrial suspension (protein = 30.5 mg/ml) to 20 ml of O,-saturated basal medium containing 1 mM 2-oxoglutarate and 0.5 rnhi L-malate. At the point indicated, 50 pmol of ADP were added. Panel A presents the mitochondrial content of CoA (O-O) and succinyl-CoA (O-O),
TABLE

panel B presents an oxygen electrode recording from the same experiment, and panel C a recording of the fluorescence of mitochondrial NAD(P) obtained in a parallel experiment, using the same incubation conditions, with the exception that ADP was added to 0.5 mM. FCCP is the uncoupling agent carhonyl cyanide p-trifluoromethoxyphenylhydrazone.

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II of mitochondrial CoA during state 3 respiration followed by sampling at 3.9 min, and to 2.4 mM (4.3 min) followed by sampling at 4.6 min. The results shown are the mean values obtained from these duplicate samples. Oxidation was monitored with an OS electrode in the suspension sampled, but the rates of 0, uptake were obtained in a parallel experiment, using an air-saturated medium.
Ratio acetyl-CoA/ succinyl-CoA

Effect

of pyruvate

concentration

upon pattern

of acylation

Reaction was initiated by adding 1 ml of mitochondrial suspension (14.3 mg of protein) to 20 ml of O,-saturated basal medium containing 25 pM pyruvate, 0.5 mM L-malate, 2.5 mM MgCl,, 10 mM glucose, and 9 units/ml of dialyzed hexokinase. ADP was added to 0.5 mM at 2 min and two samples were taken at 2.3 min. Pyruvate was then added to 48 pM (2.5 min) followed by sampling at 3.0 min, to 160 pM (3.25 min) Pyruvate
concentration mhf

Rate of 01
consumption ag-atoms O,!

CoA
content

Acetyl-CoA content

Succinyl-CoA content

Ratio CON acetyl-CoA

mmlmgprotem 0.17 0.24 0.33 0.38

nmollmg O&79 * 0.01 0.65 zt 0.002 0.46 zt 0.01 0.38

protem 0.39 * 0.03 0.44 * 0.01 0.49 * 0.02 0.38 5.2 3.2 1.9
0.91

0.024 0.048 0.16 2.4

0.15 * 0.01 0.20 rk 0.01 0.25 f 0.01 0.42

0.40 0.46 0.49

1.1

as an approximate index of the flux through pyruvate dehydrogenase and citrate synthase. Thus, over the concentration range 0.024 to 0.16 mM pyruvate the flux through these enzymes is directly proportional to the acetyl-CoA/CoA ratio

with acetyl-CoA/succinyl-CoA ratio in any range of substrate concentration tested. When the inverse of the rate of oxygen consumption is plotted uersus the inverse of the content of long chain acyl-CoA a linear plot is obtained (Fig. 7) suggesting a

and unrelated to the acetyl-Colvsuccinyl-CoA ratio, suggesting a limitation of flux by pyruvate dehydrogenase. At high pyruvate concentrations (0.16 to 2.4 mM), however, there is an increase in the acetyl-CoA/succinyl-CoA ratio with increased flux. LaNoue et al. (4) have suggested that citrate synthase activity may be related to the acetyl-CoA/succinyl-CoA ratio, under conditions giving oxidized nicotinamide nucleotide. This may be true in the region 0.16 to 2.4 mM pyruvate in this experiment. An analogous experiment with palmitoylcarnitine as substrate is shown in Table III. Here there is a large fall in the ratio CoA/long chain acyl-CoA with increase in substrate concentration and increased flux. However, there is no correlation of flux

K, for the p oxidation process of 0.95 nmol of long chain acyl-CoA/mg of mitochondrial protein. It is noted that the mitochondrial suspension was depleted of endogenous substrate in this experiment, and in some subsequent studies. It was found that in the absence of this depletion step the mitochondrial suspension contained 0.1 to 0.2 nmol/mg of protein of long chain acyl-CoA, before the addition of substrate. The inference is that these mitochondria contain fatty acids, and judging from the amount of OR consumed during substrate-depletion, this content may be considerable. The nature of the endogenous substrates present in rabbit heart mitochondria, and the effects of their removal, have been thoroughly investigated (27-29).

8368 Pattern of Acylation of Mitochondrial CoA in Respiratory States Intermediate between States 3 and 4: Discussion of Effect of Ratio Acetyl-CoAfSuccinyl-CoA on Activity of Citrate Synthase-A prime goal of the present work was to
determine the mitochondrial content of CoA and CoA thioesters under conditions which allowed a meaningful comparison with data from whole heart. This suggested the study of metabolic states intermediate between states 3 and 4 (the state 3% of K&rig et al. (30)) and generated by varying the steady state concentrations of ADP available to the mitochondria. Table IV presents the results of an experiment in which this was done, with palmitoylcarnitine plus L-malate as substrate. Two phases in the response to increased ADP can be clearly discerned. First, a decline in the ATP/ADP ratio from 43 to 25 caused a large oxidation of nicotinamide nucleotide, a 2.5.fold increase in the rate of 0, uptake and a significant rise in succiny-CoA, and a small increase in CoA content. Secondly, a further decrease in the ATP/ADP ratio to 2 gave very little further oxidation of nicotinamide nucleotide, a slight increase in the rate of 0, uptake, and a decrease in succiny-CoA, but a large increase in CoA content. Essentially the same results were obtained in an experiment with pyruvate plus L-malate as substrate (not shown). The progressive decline in acetyl-CoA TABLE
Effect ofpuZmitoylcurnitine concentration upon pattern

content with decreased ATP/ADP ratio reflects citrate synthase activation, probably due to increased nucleotide oxidation and oxalacetate availability, at least in the range of ATP/ADP ratios of 43 to 25. The increase in succinyl-CoA content in the first phase of the experiment suggests increased activity of 2-oxoglutarate dehydrogenase relative to that of succiny-CoA synthetase. This stems from the oxidation of nicotinamide nucleotide, which will activate 2-oxoglutarate

0 l/S

I 1

1 5

nMOLES HC104-INSOLUBLE CoA/MG PROTEIN

FIG. 7. Lineweaver-Burk plot of rate of 0 2 uptake and mitochondrial content of long chain acyl-CoA during state 3 palmitoylcarnitine oxidation at different concentrations of palmitoylcarnitine. The data are taken from the experiment shown in Table III. III
of mitochondriul CoA during state 3 respiration

Downloaded from www.jbc.org by on July 19, 2012

of ~cyZrn5on

One milliliter of mitochondrial suspension (21.4 mg of protein) was added at time zero to 20 ml of 0,.saturated basal medium containing 0.5 mM L-malate, 0.5 mM ADP, 10 mM glucose, 9 units/ml of dialyzed hexokinase, 2.5 mM MgCl,, and 5 mg/ml of bovine serum albumin. After 5.5 min to allow depletion of endogenous substrate, palmitoyl-Lcarnitine was added to 2 PM and two samples were taken (5.8 min). When all of the palmitoylcarnitine had been oxidized, a second Palmitoyl-Lcarnitine concentration Rate consumption
OfO,

addition of palmitoylcarnitine was made, giving 5 FM (6.7 min). Sampling was at 7 min. After substrate exhaustion, palmitoylcarnitine was added to give 10 pM (8.7 min) and samples were taken at 9.1 min. Palmitoylcarnitine was then added to 50 PM (9.6 min) and samples were taken at 10.4 min. The completion of palmitoylcarnitine oxidation after the first two additions was determined from the 0, electrode recording. Succinyl-CoA content HClO,insoluble-CoA content Ratio CoA/ HClO,. insoluble-CoA Ratio acety-CoA/ succinyl-CoA

CoA content

Acetyl-CoA content
nmollmg protein

2 5

0.06 0.10

0.81 * 0.05 0.64 + 0.01

0.37 * 0.03

0.12

0.11
0.073 0.053

* 0.01 * 0.01
* 0.003 + 0.006

10
50

0.16
0.17

0.24 0.23 0.21 0.14

;t 0.01 * 0.01 * 0.002

* 0.01 0.39 * 0.01 0.61 * 0.01

0.20 0.83 * 0.05

4.1
1.6

0.48 0.45

0.61
0.12

0.35
0.37

0.10 * 0.01

* 0.01

TABLE
Pattern ofucylution

IV
by mitochondriu 3 and 4 respiring in state 3 and in states intermediate

ofmitochondriul

CoA

during

oxidation

ofpulmitoylcurnitine between

One and five-tenths milliliters of mitochondrial suspension (protein = 24.6 mg/ml) were added to 18 ml of 0,.saturated basal medium containing 106 PM palmitoyl-Lcarnitine, 0..5 mM L-malate, 10 mM glucose, 0.1 mM M&l,, 0.6 units/ml of dialyzed hexokinase, and 10 mg/ml of bovine serum albumin. After 0.75 min, ATP was added to 0.5 mM. Samples were taken at 2 min. Hexokinase was added to give 2 units/ml at 2.25 min and samples were taken at 3 min. Hexokinase was HeXokinase present Ratio ATPI ADP Rate of0, On: sumpt1on CoA content AcetyllCoA content

then added to 6.3 units/ml at 3.3 min and samples were taken at 4.3 min. Finally, ADP was added to 2 mM at 4.5 min and samples were taken at 5.25 min. The results shown are the mean values obtained from the duplicate samples. Changes due to substrate depletion were avoided in this experiment by the use of a high concentration of palmitoylcarnitine, buffered by serum albumin. HClO,insolubleCoA content Ratio HE%.
msolubie-

SuccinylCoA content

CoA

Ratio acetylGA/ succinylCoA

5 Reduction iYADP,

units/ml
0.6 2.0 6.3 43 40 25 2.0

pg.atoms OJ
minlmg protein 0.048 0.064 0.117 0.171

nmollnq

protein

6.3 (+ADP)

0.002 0.012 0.046

NIL i 0.001
1 0.003 + 0.002

0.20 0.14 0.090 0.061

* 0.01 * 0.01
* 0.002 + 0.003

0.12 * 0.01 0.18 i 0.01 0.21 i 0.01 0.14 * 0.01

0.62 0.65 0.72

0.08

i 0.11
+ 0.06

NIL 0.003 0.017

0.058

1.6
0.78 0.44 0.43

53 40 11

0.79 * 0.01

10

8369 dehydrogenase (and isocitrate dehydrogenase, as shown below), which is seen in the range of ATP/ADP ratios from 43 to 25. Even though the intramitochondrial ATP/ADP ratio will be less than 25 when the ratio found by sampling the whole suspension is 25, owing to the effect of mitochondrial membrane potential on ATP*- for ADP3- exchange (31), it is still evidently high enough to limit the activity of succinyl-CoA synthetase (Table IV, lines 3 and 4). The mechanism is presumably the near equilibration of energy charge (32) of guanine and adenine nucleotides catalyzed by nucleoside diphosphate kinase (20). Higher concentrations of ADP serve mainly to activate the succinyl-CoA synthetase reaction (Table IV), in the direction of succiny-CoA cleavage. An experiment in which an abrupt transition is made from state 4 to state 3 (e.g. Fig. 1) does not allow this partial separation of the effects of oxidation-reduction state and ATP/ADP ratio. In a recent detailed study of respiratory states between 3 and 4, Davis and Lumeng (33) have demonstrated a constancy of intramitochondrial phosphate potential over a wide range of extramitochondrial phosphate potential, achieved by adding purified ATPase. In the present study, intramitochondrial adenine nucleotides were not estimated, but the changes in nicotinamide nucleotide oxidation-reduction state, and in the acylation of CoA which were described above, make it reasonable to infer changes in intramitochondrial phosphate potential. LaNoue et al. (4) have proposed a model of tricarboxylate cycle control in which citrate synthase activity is controlled by the ratio acetyl-CoA/succinyl-CoA, in those circumstances where oxalacetate availability is high, but in which flux is still not maximal. Aphysiological conditions involving the use of oligomycin and an uncoupling agent were used to demonstrate this control. It seemed possible to explore the applicability of this mechanism under conditions nearer to physiology by making use of the intermediate states in Table IV. Here nicotinamide nucleotide is highly oxidized and L-malate is present such that oxalacetate availability is high, but the flux is less than maximal. Under these conditions it seemed plausible that flux through citrate synthase might be controlled by the acety-CoA/succinyl-CoA ratio. In an experiment to test this possibility, five separate incubations were made with pyruvate plus L-malate as substrate, and with differing activities of hexokinase present. Because there are no changes of flux in the course of each incubation, contrary to the studies presented so far, this experimental design allows the estimation of the flux through citrate synthase by the disappearance of pyruvate. It is seen (Table V) that flux through citrate synthase increases as the ratio ,4TP/ADP decreases, but that in no portion of the experiment is this increased flux matched by an increase in the ratio acetyl-CoA/succinyl-CoA. In a subsequent experiment (Table VI), acetylcarnitine plus L-malate was used as a substrate, and an estimate of the activity of citrate synthase was made from the rate of 0, uptake. Although not precise, this is a reasonable approximation, because the accumulation of 2-oxoglutarate was found to be extremely small. The accumulation of succinate was not measured and thus it is not known to what extent succinate is oxidized. However, this does not introduce a great uncertainty into the flux calculations, because 0, uptake is divided by 4 in the event of complete oxidation of succinate and by 3 in the absence of any such oxidation. Examination of the stoichiometry of 0, uptake and palmitoyl group disappearance in fact suggests that rat heart mitochondria oxidize palmitovlcarnitine to completion in the presence of L-malate, with no large loss of tricarboxylate cycle intermediates (16). It is seen that the ratio acetyl-CoA/succinyl-CoA falls as the 0, uptake rises until the rate is 72% of that achieved in state 3. This is achieved with an ATP/ADP ratio of 72. The further reduction of this (ATP/ADP) ratio causes a decrease in succinyl-CoA content, for the reasons outlined in the discussion of Fig. 4, and an increase in the ratio acetyl-CoA/succiny-CoA. There is also a slight increase in flux through citrate synthase, measured by 0, uptake. It must be noted, however, that there is also an increased oxidation of nicotinamide nucleotide under these circumstances. Thus, data in Table VI (lines 4 and 5), although consistent with a control of citrate synthase by the ratio acetyl-CoA/succinyl-CoA under conditions of low nicotinamide nucleotide reduction and almost maximal flux, are quite equivocal. It is noteworthy that the large majority of the 2-oxoglutarate produced is oxidized, despite wide variations in the CoA/succinyl-CoA ratio, which might be expected to alter the activity of 2-oxoglutarate dehydrogenase (20). Finally, the experiment of Fig. 6 showed that succinyl-CoA content is extremely high in state 4 2-oxoglutarate oxidation, and raised the possibility that citrate synthase might be controlled by the acetyl-Coivsuccinyl-CoA ratio, when 2-oxoglutarate is present in addition to a source of acetyl units. The experiment shown in

Downloaded from www.jbc.org by on July 19, 2012

TABLE V Relation betweenflux through citrate synthase, oxidation-reduction pyruuate oxidation by mitochondria state ofNAD(P), in state 3, state and mitochondrial 4, and intermediate acetyl-CoAlsuccinyl-CoA states ratio, during

In each of five separate incubations, 0.3 ml of mitochondrial suspension (protein = 18.7 mg/ml) was added to 5 ml of 0,.saturated basal medium containing 0.8 mM pyruvate, 0.5 mM L-malate, 10 rn~
glucose, 0.5 mM ATP, and 0.1 mM MgCl,. After 1 min, the amount of hexokinase shown was added to each incubation, to activate respiration. Duplicate samples were taken after 8 min (line 1) and after 6 min (line 2) and 5 min (lines 3 to 5) of activated respiration. Citrate synthase flux is estimated from the disappearance of pyruvate, Hexokinase present Ratio ATPIADP CoA content Acetyl-CoA content
% total CoA

obtained by comparing the pyruvate found in the samples with the pyruvate found in samples of a control incubation, in which mitochondria were added to .5ml of medium at O, containing 1 FM rotenone, and
in which sampling was conducted immediately. Correction was made for the 1 min of controlled respiration in lines 2 through 5. The whole mitochondrial suspension had been depleted of endogenous substrate (see Experimental Procedure). Ratio acetyl-coA/ succmylLCoA % Reduction otNAD(P)

Succinyl-CoA content

Pvruvate utilization nmollminlmgprotein

units/ml
7 14 18 46 62 38 27 24 2.2 26 24 25 22 25 * + + * * 3 2 1 1 0.4

58 48 43 44 46

i + i i i

3 0.3 1 2 2

14 28 32 35 29

* zt * * *

2 2 2 1 2

4.1 1.7 1.3 1.3 1.6

51 35 23 15 10

11 34 46 53 50

* i i 3 *

1 1 1 0.2 1

a370 TABLE VI state of NAD(P),

in state 3, state

Relation

between

flux

through

citrate acetylcarnitine

synthase,

oxidation-reduction oxidation by mitochondria

and

mitochondrial acetyl-CoAlsuccinyl-CoA 4, arzd intermediate states

ratio,

during

In each of five separate incubations, 0.19 ml of mitochondrial suspension (protein = 20 ma/ml) was added to 5 ml of 0,.saturated basal medium containing 5 mM acetyl-DL-carnitine, 0.5 mM L-malate, 10 mM glucose, 0.5 mM ATP, and 0.1 mM M&l,. lmmediately thereafter, the amount of hexokinase indicated was added. In the Hexokinase present Ratio ATPI ADP Rate OfO, consumption
pg-atoms OJminlmg protein

experiment shown in line 5, ADP was also added to 1 mM, in place of ATP. Duplicate samples were taken from each incubation after 6 min. The rate of 0, uptake corresponding to the conditions of each incubation was determined separately, using an 0, electrode and an air-saturated medium. Succinyl-CoA content Ratio acetyl-GA/ succiny-CoA Ratlo COAI succinylC0A x Reduction iYAoDf(P) Production of z-oxoglutarate
nmollninlmg protein

C0A content

Acety-CoA content

unitslml

70 total

CoA

1.1 2.2 4.3 8.6 + added

128 110 103 74 0.53 ADP

0.034 0.059 0.086 0.117 0.162

5.2 10 21 25 51

zt * * + r

0.4 1 2 2 0.2

75 66 49 23 24

* * * * +

1 1 1 3 0.4

20 23 31 52 26

t * * * zt

1 0.05 1 1 0.2

3.75 2.84 1.60 0.45 0.91

0.26 0.44 0.67 0.49 2.0

73 + 55 47 37 21 15

1.1 1.2 1.2 1.2 1.6

Relation

between

flux

through oxidation

citrate synthase. of pyruuate plus

oxidation-reduction 2.oxoglutarate

TABLE VII state of NAD(P),

by mitochondria in state

and

mitochondrial acetyl-CoA/succinyl-CoA 3, state 4, and intermediate states

ratio,

during

Downloaded from www.jbc.org by on July 19, 2012

In each of 5 incubations, 0.3 ml of mitochondrial suspension (protein = 22.3 mg/ml) was added to 5 ml of 0,.saturated basal medium containing 0.55 mM PyrUvate, 1 mM 2-OXOglUtarate, 0.5 mM L-malate, 10 mM glucose, 0.5 mM ATP, and 0.1 mM M&l,. After 1 min, the amount of hexokinase shown (dialyzed to remove NH,) was added to H&-Kkinase present
units/ml

each incubation, to activate respiration. Duplicate samples were taken after 8 min (incubation shown on line 1) and after 7 min (line 2), 6 min (line 31, and 5 min (lines 4 and 5) of activated respiration. Pyruvate utilization was estimated as described in Table V. The whole mitochondrial suspension had been depleted of endogenous substrate. SuccinyCoA content Ratio acetyl-CON succinyl-CoA Ratio COAI succinylCoA Pyruvate utilization
nmollminlmg protem

ADP

Rate OfO, consumption

Odminlmg protem

CoA content

Acetyl-CoA content

pg-atoms
0.036 0.097 0.217 0.344 0.398 12 8.4 13 12 15 * + * * * 0.2 1.5 1 1 0.3

9%total

CoA

4.6 12 21 46

55 33 22 4.2

34 30 28 29 38

* i + * +

0.2 1 3 3 1

54 62 59 60 47

* i * + *

0.2 0.4 3 2 1

0.64 0.49 0.47 0.48 0.81

0.23 0.14 0.23 0.20 0.31

50 34 25 17 10

6.5 15 28 39 36

i .i i i i

0.5 0.2 0 1 0.4

Table VII used both 2-oxoglutarate and pyruvate as substrate, and the depletion of the latter is taken as an index of flux through citrate synthase. It is seen that the presence of 2-oxoglutarate did indeed generate a succinyl-CoA content greater than that found when pyruvate alone was the substrate (Table V). However, as the ATP/ADP ratio was decreased from more than 55 to 22, the increased flux through citrate synthase was not matched by an increase in the ratio acety-CoA/succinyl-CoA, indeed the latter declined. Thus, the results of this experiment provide no support for the concept of the control of citrate synthase by the ratio acetyl-CoA/succinyl-CoA, even when the stage is set for such a control by the generation of high succinyl-Coil concentrations. Instead, all of the results presented are consistent with control by oxalacetate availability, as measured by the oxidation-reduction state of NAD(P)H, at a constant added malate concentration.
Steady State Concentrations citrate, NAD+, NADH, NADP+, tion of Palmitoylcarnitine Plus
of Mitochondrial

and NADPH L-Ma&e and

Citrate, Zsoduring OxidaPyruvate Plus

L-Ma&e-Fig. 8 presents data from two experiments in which intramitochondrial citrate and isocitrate content were determined by centrifugation and subtraction as described under Experimental Procedure. The respiratory substrate was

palmitoyl-L-carnitine plus L-malate. It is seen that on transition from state 4 to state 3 the intramitochondrial isocitrate content decreased, although there was a slight increase in intramitochondrial citrate. Furthermore, when ADP phosphorylation was completed, the initial change in isocitrate content was reversed and there was a concomitant increase in the intramitochondrial citrate content. The difference in intramitochondrial isocitrate content between state 4 and state 3 was shown to be statistically significant and demonstrates a control of isocitrate dehydrogenase activity during state 4 respiration. The reason for the failure of citrate content to follow exactly the changes in isocitrate content is not clear but may be related to an overall increase in intramitochondrial tricarboxylate cycle intermediates coupled with an inability to maintain strict equilibrium at the aconitase reaction. Such an increase in tricarboxylate cycle intermediates is seen more clearly in the experiment of Fig. 9 where pyruvate and L-malate are the respiratory substrates. Although more than 95% of the 2-oxoglutarate was found to be external to the mitochondria, the rise in total 2-oxoglutarate content during the state 4 ---t 3 transition and its partial reversal in the subsequent state 3 + 4 transition (Fig. 8) is probably indicative of changes in the intramitochondrial concentration of 2-oxoglutarate. This can

8371

0.1

fig-ATOM/ML)

Downloaded from www.jbc.org by on July 19, 2012

3 MlNCiTES

FIG. 8. The mitochondrial content of citrate and isocitrate and the oxidation-reduction state of mitochondrial NAD(P) during state 4 + 3 + 4 transitions with palmitoylcarnitine (Pm. Cn.) plus L-malate as substrate. Mitochondria were added to 19 ml of an air-saturated basal medium containing in addition 10 pM palmitoy-L-carnitine and 0.5 mM L-malate to give a final protein concentration of 1.25 n&ml. Where indicated on the oxygen electrode trace (A), samples were removed and extracted as described under Experimental Procedure; ADP was added to 0.96 rn~ and palmitoylcarnitine was added a second time to avoid substrate depletion, giving an increment of 8.2 k~. The values for sample contents shown in the accompanying table are nanomoles/mg of mitochondrial protein and are means from duplicate experiments. B, fluorimeter trace of intramitochondrial NAD(P)H fluorescence obtained by using 15 FM palmitoylcarnitine but otherwise the conditions of (A) in a 2.ml system. FCCP, added to a concentration of 2.5 be readily appreciated if 2-oxoglutarate leaves the matrix space in exchange for L-malate, as in other mammalian mitochondria (34,35) because the external L-malate concentration is relatively high (20) and not subject to sudden change. The fluorimeter trace (Fig. 8B) derived from intramitochondrial NAD(P)H fluorescence showed a cycle of NAD(P)H oxidation during state 3 as found by others (15, 18, 36). However, when, in experiments of similar design, the nicotinamide nucleotides were extracted from the mitochondria and measured enzymically, it was found (Table VIII) that NADPH, as well as NADH, was oxidized on going from state 4 + 3. This contrasts with the findings of LaNoue et al. (18) and is discussed later in connection with the activity of NADP-isocitrate dehydrogenase. A repetition of these experiments with pyruvate plus Lmalate as substrate (Fig. 9) revealed a more pronounced increase in citrate content throughout the experiment, but the same, statistically significant, decrease in isocitrate content. The pattern of change in 2-oxoglutarate content (Fig. 9) and in oxidation-reduction state of NAD and NADP (Table VIII) was similar to that obtained for palmitoylcarnitine oxidation but the 2-oxoglutarate contents were greater and both nicotinamide nucleotides were somewhat more reduced in state 3 with pyruvate as substrate. This information is consistent with the higher rates of oxygen consumption obtained with pyruvate

FM, was used to establish anaerobiosis. A different mitochondrial preparation was used to obtain this trace and the temperature was 22.

1 Citrate Total Mitochondrial Total Mitochondrial Total 4.91 1.35

Sample
2 3 4

5.98 1.59

6.55
1.51

9.06 2.83 0.59

0.17

Isocitrate Oxoglutarate

0.56
0.16 0.59 significantly

0.43 0.05 2.73

from

0.48 0.04 3.53

2.23

u State 4 (samples 1 and 4) differs 2 and 3), p < 0.005.

state 3 (samples

(mean state 3 rate with pyruvate = 0.346 * 0.032 (n = 12) pgatoms of O,/min/mg of mitochondrial protein; mean state 3 rate with palmitoylcarnitine = 0.164 i 0.011 (n = 12) wgatoms of O,/min/mg of mitochondrial protein). It is noted that 25% or more of the citrate and isocitrate in the total mitochondrial suspension was found to be intramitochondrial (Figs. 8 and 9, state 4). Assuming a matrix space of 1 pl/mg of protein, this means that the intramitochondrial concentration of these ions is 250 times greater than that in the suspending medium. This indicates an extremely low mitochondrial permeability to citrate and isocitrate in rabbit heart and is consistent with other reports of limitingly low tricarboxylate carrier activity in rat heart (37, 38). It is suggested that the decreased mitochondrial isocitrate content seen in state 4 + 3 transitions (Figs. 8 and 9) does not reflect an increased transport of isocitrate out of the mitochondria, as the total suspension content also appears to be slightly decreased. These results (Figs. 8 and 9) demonstrate an activation at the level of isocitrate dehydrogenase during the state 4 + 3 transition. As such they are in agreement with a conclusion reached by LaNoue et al. (18) about control at this locus. However, it should be noted that mitochondrial citrate, but not isocitrate, was measured in the previous work and it seems from Figs. 8 and 9 that it is wrong to assume that aconitase maintains equilibrium under all conditions. Indeed, in the

8372
:B)

FLUORESCENCE INCREASE

/
OXYGEN (APPROX 0.4 pg-ATOM/ML)

I
0 1 2 3 MINUTES 4 5 6 7 8

Downloaded from www.jbc.org by on July 19, 2012

The mitochondrial content of citrate and isocitrate and the oxidation-reduction state of mitochondrial NAD(P) during state 4 + 3 + 4 transitions with pyruvate plus L-malate as substrate. Mitochondria were added to 19 ml of an 0,.saturated basal medium containing in addition 2.5 rnM pyruvate and 0.5 mM L-malate to give a final protein concentration of 0.96 mg/ml. Where indicated on the oxygen electrode trace (A) samples were removed and extracted as described under Experimental Procedure, and ADP was added to a concentration of 3.2 mM. The values for sample contents shown in the accompanying table are nanomolesimg of mitochondrial protein and are means from duplicate experiments. B, fluorimeter trace of intramitochondrial NAD(P)H fluorescence obtained by using 2 ml of an air-saturated basal medium and 0.5 mM ADP, but otherwise the conditions of (A). FCCP, added to a concentration of 2.5 pM, was used to establish anaerobiosis. A different mitochondrial preparation was used to obtain
FIG. 9.

this trace, and the temperature was 22

1 2

Sample
3 4

Citrate

Total Mitochondrial Total Mitochondrial Total

4.85

10.4

1.34
0.66 0.22 3.8

4.91
0.48 0.04 16.7

12.6 5.74 0.53 0.06 23.5

19.2
8.22 1.38 0.49 18.7

Isocitrate Oroglutarate

a State 4 (samples 1 and 4) differs significantly from state 3 (samples 2 and 3), p < 0.01.

present work, mitochondrial citrate increases from state 4 to 3. Nevertheless, the data on isocitrate content (Figs. 8 and 9) lead to the conclusion reached in the earlier work (18). LaNoue et al. showed that the total citrate formed during the oxidation of pyruvate plus L-malate by heart mitochondria was less in the presence of oligomycin and ADP than in state 4, which would indicate a response of isocitrate dehydrogenase to intramitochondrial adenine nucleotide energy charge. They also showed, however, that flux through this enzyme was much greater in the presence of oligomycin plus the uncoupling agent FCCP than in state 4 respiration, indicating a sensitivity of isocitrate dehydrogenase to the oxidation-reduction state of nicotinamide nucleotide. These respiratory states differ in that both show a high adenine nucleotide energy charge, but the uncoupled state shows much less nicotinamide nucleotide reduction. An attempt to determine the relative importance of nicotinamide nucleotide oxidation-reduction state and adenine nucleotide energy charge in the control of isocitrate dehydrogenase in the mitochondrion is shown in Figs. 10 and 11. It is seen (Fig. 10) that addition of oligomycin plus ADP to mitochondria oxidizing pyruvate plus L-malate in state 4 results in no change in intramitochondrial isocitrate content, whereas the subsequent addition of the uncoupling agent FCCP gives rise to a statistically significant decrease in isocitrate content. When the sequence in which ADP and FCCP are added is reversed

(Fig. 11) it is seen that FCCP gives a decrease in intramitochondrial isocitrate content and the subsequent addition of ADP gives rise to no further change. Inspection of the fluorimeter traces of Figs. 10 and 11, showing the high NAD(P)H/ NAD(P)+ ratio in the presence of oligomycin plus ADP, and low ratio in the presence of FCCP, then allows the statement that under conditions of both high and low energy charge the overriding influence on isocitrate dehydrogenase activity is the oxidation-reduction state of nicotinamide nucleotide. The demonstration of control at the isocitrate dehydrogenase reaction in experiments involving isolated mitochondria (Figs. 8 and 9) is in agreement with the finding that increasing the work load of the isolated, perfused heart decreases the heart isocitrate content, when the substrate is either glucose or glucose plus palmitate (1). The question of the significance of control at this locus arises. An important point appears to be the inhibition of citrate synthase by citrate, which is competitive with oxalacetate (5). All of the thioester studies reported here tend to minimize the control of citrate synthase by acetyl-CoA availability, and therefore implicate control by the availability of oxalacetate. This control would be tightened by the accumulation of citrate in state 4, as suggested by LaNoue et al. (4). In this context, the control of isocitrate oxidation becomes important. Pathway of Isocitrate Oxidation-There has been debate

8373
TABLE VIII Mitochondrial content ofnicotimmide nucleotides during state 4 - 3 + 4 transitions with pyruuate plus L-malate and with palmitoylcarnitine plus L-malate as substrate

Mitochondria were added to 20 ml of an air-saturated basal medium containing in addition either 2.5 mM pyruvate and 0.5 mM L-malate or 50 pM palmitoyl-L-carnitine, 5 mg of bovine serum albumin/ml, and 0.5 mM L-malate to give a final protein concentration which varied between 0.7 and 1.4 mg/ml. After 2.5 min of state 4 respiration samples (2 x 2 ml) were withdrawn simultaneously (sample l), one of which was expelled into 1.3 ml of 16% (v/v) HClO,, the other being discharged into 1 ml of 1 M KOH in ethanol. ADP (0.9 IIIM) was added 0.5 min later and two more double samples (samples 2 and 3) were removed for analysis during the phase of state 3 respiration. When state 4 respiration had been resumed for at least 1 min, a fourth double sample (sample 4) was removed and treated as before. The values in the table are from duplicate experiments using different mitochondrial preparations. Total NAD = 5.78 + 0.47 (16) nmol/mg; total NADP = 1.55 + 0.19 (16) nmol/mg.
Reduction Substrate Sample 1 (70) Samples 2and3 S.3mple
4*

(A)

OXYGEN (APPROX 0.4 pg-ATOM/ML)

4 MINUTES

10,

OLIGO I

ADP 1 FCCP

Pyruvate plus L-malate Palmitoylcarnitine plus L-malate State 4 (samples 2 and 3). *p <O.OOl. p < 0.05.

NAD NADP NAD NADP

59.5 * 0.5 (2) 98.5 + 1.5 (2) 58 * 2 (2) 97 * 0 (2)

15.3 + 2.5 (4) 92.3 i 2.0 (4) 5.5 * 0.3 (4) 77.8 sz 1.1 (4) from

42 + 3 (2)b 98 i 2 (2) 40
l

Downloaded from www.jbc.org by on July 19, 2012

7 (2)b

96 + l(2)

FLUORESCENCE INCREASE

1 and 4) differs

significantly

state 3 (samples
2 ml.

(reviewed by Nicholls and Garland (39)) as to whether the NAD- or NADP-linked isocitrate dehydrogenase is involved in isocitrate oxidation. The finding of high activities and regulatory properties of the former, when extracted and assayed under optimal conditions (40, 41), and its presence in a constant proportion group with other tricarboxylate cycle enzymes (41) suggested that the NAD-linked enzyme is involved. Equally, the highly reduced state of mitochondrial NADP and its lack of response to alterations in tricarboxylate cycle flux (18), and the low activity of transhydrogenase in coupled mitochondria (39) argued against participation of the NADP-linked enzyme. However, the finding in the present work that NADP becomes slightly less reduced (Table VIII) on transition from state 4 to 3 with either pyruvate plus L-malate or palmitoylcarnitine plus L-malate as substrate, again raised the possibility that NADP-isocitrate dehydrogenase (EC 1.1.1.42) might contribute significantly to the total flux from isocitrate to 2-oxoglutarate. The activities of both NADP- and NAD-linked enzymes were measured in mitochondrial extracts in an attempt to place limits on their relative contributions. NAD-isocitrate dehydrogenase (EC 1.1.1.41) was measured in mitochondria solubilized either with Triton X-100 or by sonication, and in the presence of rotenone to prevent reoxidation of NADH by the respiratory chain. The V,,, was determined to be 72 f 4 nmol/min/mg of mitochondrial protein, at 25 (mean of four determinations using different mitochondrial preparations). The K, for NAD+ was found to be 0.3 mM and the K, for NADH to be 20 PM. The K, for threo-n,-isocitrate was found to be 50 PM in the absence of ADP and 10 FM in the presence of 2 mM ADP. From these data it

FIG. 10. The effect of the addition of ADP plus oligomycin and of the subsequent addition of uncoupling agent, upon the mitochondrial content of citrate and isocitrate. Mitochondria were added to 21 ml of an 0,.saturated basal medium containing in addition 2.5 mM pyruvate, 0.5 mM L-malate, and 0.5 mM ATP to give a final protein concentration of 1.0 mg/ml. Where indicated on the oxygen electrode trace (A), samples were removed and extracted as described under Experimental Procedure; additions to the following concentrations were 2.8 c(g of oligomycin/ml (oligo), 4 mM ADP, and 1.2 PM FCCP. The

values for sample contents shown in the accompanying table are nanomoles/mg of mitochondrial protein and are means from duplicate experiments. B, fluorimeter trace of intramitochondrial NAD(P)H
fluorescence obtained bv using 2 ml of an air-saturated basal medium but otherwise the conditions of (A). A different mitochondrial preparation was used to obtain this trace and the temperature was 22.
Sample 1
2 3 4 5

Citrate

Total Mitochondrial Total Mitochondrial

8.87 4.07 1.02 0.48

10.3 3.92 1.08 0.39

9.82 2.90 1.13 0.44 + ADP

11.0 2.60 0.86 0.15

11.3 2.06 0.84 0.15

Isocitrate

< 0.005 for FCCP

(4 and 5) uersus oligo

(2 and 3).

8374
ATP

FLUORESCENCE INCREASE

2 IllIll.
ADP

MINUTES

FIG. 11. The effect of the addition of oligomycin plus uncoupling agent, and of the subsequent addition of ADP, upon the mitochondrial content of citrate and isocitrate. Mitochondria were added to 21 ml of an air-saturated basal medium containing in addition 2.5 mM pyruvate, 0.5 mM L-malate, and 0.5 mM ATP to give a final protein concentration of 0.8 mg/ml. Where indicated on the oxygen electrode trace (A), samples were removed and extracted as described under Experimental Procedure; additions to the following concentrations were 2.4 pg of oligomycin/ml (oligo), 1.5 pM FCCP, and 3.7 mM ADP. The values for sample contents shown in the accompanying table are nanomoles/mg of mitochondrial protein and are means from duplicate experiments. B, fluorimeter trace of intramitochondrial NAD(P)H fluorescence obtained by using the conditions of (A) in a 2-ml system. A

different mitochondrial preparation the temperature was 22.

1

was used to obtain

Sample

this trace

and

2 9.45 2.15 0.66 0.03

3 10.2 1.86 0.73 0.07

Downloaded from www.jbc.org by on July 19, 2012

Citrate

Total Mitochondrial Total Mitochondrial + FCCP

7.49 2.86 0.88 0.30

10.9 2.29 0.68 0.04 4 (1).

11.3 2.46 0.75 0.110

Isocitrate p

< 0.005 for oligo

(2 and 3) uersus state

o-------~

20
NADP

40

60

80
(%)

100

REDUCTION

FIG. 12. The dependence of the activity of NADP-isocitrate dehydrogenase upon the degree of reduction of NADP+. Mitochondria (0.17 mg of protein) were added to 2.5 ml of a medium comprising 50 mM KP,, 10 mM MgCl,, 1 mM dithiothreitol, 0.5 rg of rotenone/ml, 0.025% (v/v) Triton X-100, and varied amounts of NADP+ and NADPH to give a combined concentration of 130 pM. The final pH was 7.2 and the temperature was 25. A mixture of 2.5 mM citrate and 0.1 mM three-D,-isocitrate was added to start the reaction and initial rates of NADP+ reduction were measured spectrophotometrically at 340 nm.

would appear that the activity recovered is not sufficient to account for flux through this reaction of at least 70 nmol/ min/mg of protein obtained during state 3 pyruvate oxidation. dehydrogenase was found to be The V,,, of NADP-isocitrate 450 nmol/min/mg of mitochondrial protein, th K, for threon,-isocitrate to be 5 PM and the K, for NADP 2 pM. When enzymic activity was studied as a function of the percentage reduction of NADP, it was found (Fig. 12) that the activity is approximately 40% of V,,, at 92% NADP reduction, and 65% of vmx at 78% reduction. These are the oxidation-reduction conditions found to apply during the state 3 oxidation of

pyruvate and palmitoylcarnitine, respectively (Table VIII). Thus, NADP-isocitrate dehydrogenase may make an appreciable contribution to the oxidation of isocitrate, with the limitation that any such contribution is contingent upon nicotinamide nucleotide transhydrogenase activity (EC 1.6.1.1). Measurements of transhydrogenase in submitochondrial particles derived by sonication gave rates of NADPH oxidation of 36 nmol/min/mg of mitochondrial protein in the presence of oligomycin and FCCP, and 25 nmol/min/mg of mitochondrial protein in particles energized by succinate oxidation in the presence of oligomycin (251~). These figures are not unlike those determined in intact rat liver mitochondria (39) although the heart particle enzyme did not show the extreme inhibition during coupled respiration demonstrated in intact mitochondria (39). It seems probable that this was an artifact of the particle preparation and that the transhydrogenase is extensively inhibited under physiological conditions. This is made the more likely by further experiments which showed that palmitoyl-CoA was a potent inhibitor of transhydrogenase activity, as reported for the bovine heart enzyme (42), 50% inhibition being achieved with less than 1 j.tM palmitoyl-CoA. Because palmitoyl-CoA competes with NADP(H) (42), the relative concentrations of these species within the mitochondrial matrix become important; it is unlikely that NADPH is present at more than 20 times the concentration used in the assay (i.e. 0.1 mM) but the corresponding palmitoyl-CoA concentration could be as much as 1000 times that used in the assay, depending on how much of it is free in solution. Therefore, it becomes a real possibility that the transhydrogenase activity in uiuo is limitingly low and that flux through the NADP-isocitrate dehydrogenase is limited correspondingly. Given a high activity of the NADP-isocitrate

8375 dehydrogenase coupled with a low, transhydrogenase-limited flux through this enzyme, it may be concluded that during the metabolic transitions performed in this work the NADP-isocitrate dehydrogenase reaction was essentially at equilibrium. This would then account for the decreased NADPH content during state 3 as a consequence of the decreased isocitrate content (Figs. 8 and 9). If this supposition is correct, then the NAD-isocitrate dehydrogenase reaction is sufficiently removed from equilibrium in state 4 to exert its postulated regulatory role. Acknowledgments-We would like to recognize technical help of Miss Ono Lescure and fruitful with Dr. Bertram Sacktor throughout the work.
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