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THE JOURNAL OF B~OLOCICAL CHEM~~YVol. 250, No. 21, Issue of Novemb er 10, pp. 8361-8375, 1975

Printed m U.S.A.

The Steady State Concentrations of Coenzyme A-SH andCoenzyme A Thioester, Citrate, and Isocitrate duringTricarboxylate Cycle Oxidations in Rabbit Heart Mitochondria

(Received for publ icatio n, May 2, 1975)

RICHARD G. HANSFORD AND ROGER N. JOHNSONFrom the Gerontology Research Center, National Institute of Child Health and Human Development,National Institutes of Health, Baltimore City Hospitals, Baltimore, Maryland 21224

The steady state mitochondrial content of coenzyme A-SH (CoA), acetyl-CoA, succinyl-CoA, and longchain acyl-CoA has been determined during the oxidation of palmitoylcarnitine by rabbit heart mito-chondria. Variation of the substrate concentration during ADP-stimulated (state 3) respiration varies themitochondrial content of long chain acyl-CoA and the rate of 0, uptake, and permits the conclusion thatthe K, of p oxidation for intramitochondrial long chain acyl-CoA is approximately 1 nmol/mg of mito-chondrial protein. At near saturating concentrations of palmitoylcarnitine, plus Lmalate, the addition ofADP ca uses a decrease in acetyl-CoA, an increase in CoA and succinyl-CoA, and no clear change in longchain acyl-CoA content. These ch anges 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 withpyruvate plus L-malate and octanoate plus L-malate as substrates. These results suggest a limitation offlux 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 ofpalmitoylcarnitine plus L-malate and pyruvate plus L-malate, but also during intermediate respiratorystates (state 3%) generated by adding glucose and varying amounts of hexokinase. These intermediatestates are characterized by a high succinyl-CoA content, relative to either state 3 or state 4, and asubo ptim al flux through citrate synthase , estim ated either by pyruvate disapp earance or by 0, uptake.Despite th is, no correlation was found between acety-CoA/succinyl-CoA ratio and flux through citratesynthase with the possible exception of the region between 72 and 100% of the state 3 rate withacetylcarnitine as substrate. Experiments involving the state 4 + 3 + 4 transition with P-oxoglutarateplus L-malate as substrate showed a high succinyl-CoA content in state 4, and inclusion of 2-oxoglutaratein an experiment with pyruvate plus L-malate and varying activ ities of hexokinase gave decreas edacetyl-CoA/succinyl-CoA ratios at low citrate synthase flux. However, increased citrate synthase fluxachieved with increased hexokinase was not associated with a rise in this ratio. Thus it seemed moreprobable that under al l of the conditions studied citrate synthase is regulated by the availability of itsother substrate, oxalacetate. This prompted the estimation of mitochondrial NAD+ and NADH in states3 and 4 as a determinant of oxalacetate concentration, and the estimation of intramitochondrial citrateconcentration, as a determinant of oxalacetate binding to citrate synthase. Mitochondrial citrate contentwas found to rise during state 3 + 4 transitions, but not to decrease during state 4 + 3 transitions. Itscontribution to citrate synthase regulation under these experimental conditions is thus equivocal. Themitochondrial isocitrate content, however, decreased during the state 4 + 3 transition and this isconsistent with an activation of isocitrate dehydrogenase. This was true with either pyruvate plus L-malateor palmitoylcarnitine plus L-malate as substrate. Experiments with uncoupling agents and oligomycinallowed the demonstration that a decreased NAD(P)H/NAD(P) + ratio is more important than adecreased ATP/AD P ratio in the activation of isocitrate dehydrogenase. An evaluation of the possiblecontribution of NAD-isocitrate dehydrogenase (EC 1.1.1.41) and NADP-isocitrate dehydrogenase (EC1.1.1.42) is made. The steady state concentrations of CoA, CoA thioesters, citrate, and isocitrate arecompared with values obtained for perfused heart, with glucose or glucose plus palmitate as substrate.

The oxidation of fatty acids supplies a major portion of the process is to study the steady state concentrations of keyenergy requirements of mamm alian heart (see Neely et al. (1) intermediates and investigate the way in which they changefor a review). One method of studying the control of this when the flux through the pathway is changed (see Rolleston

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8362(2) for a review). This approach has been applied to CoA,acyl-CoA derivatives, and tricarboxylate cycle intermediatesin whole heart (3) but there are at present no data obtainedwith isolated heart mitochondria, oxidizing fa tt y acids. Suchdata may be useful in evaluating results obtained with thewhole heart which will reflect contributions from both mito-chondria and cytosol. This may be especially true for CoA andCoA thioesters, in that these are divided into two intracellularpools by the impermeability of the mitochondrial membrane.This study presents determinations of mitochondrial CoA,acetyl-CoA, succinyl-Coi l, and long chain acyl-CoA, anddocuments the changes that occur as the concentration of thesubstrate, palmitoylcarnitine, is varied and as the mitochon-drial energy state is varied by the increased availabil ity ofADP. Such studies focused attention on the control of citratesynthase (EC 4.1.3.7), and the results of these experiments andothers involving pyruvate and acetylcarnitine as substrateshave been examined to see whether they fi t a model of controlby the ratio acety-CoA/succinyl-CoA advanced by LaNoue etal. (4). In addition, studies have been made of the intramito-chondrial content of citrate and isocitrate and the response tomitochondrial energy state. These experiments allow theidentification of isocitrate dehydrogenase as a control site,which may be important in the context of the control of citratesynthase, because citrate inhibits citrate synthase competi-tively with respect to oxalacetate (5). It is shown that theoxidation-reduction state of nicotinamide nucleotide is moreimportant than the ATP/ADP ratio in determining fluxthrough the isocitrate dehydrogenase step.

EXPERIMENTAL PROCEDUREPreparation of Mitochond ria-Mitochondria were prepared from theheart of one New Zealand white rabbit by a slight modification of the

method of Chance and Hagihara (6). The heart was chopped withscisso rs and then passe d through a tissue press with holes of l-mmdiameter. The heart m uscle was then incubated with 7.5 mg of Nagarse(from Enzyme Developm ent Corp., New York) in a total volum e of 40ml of 0.25 M sucrose/l0 tnM N-tris(hydroxymethyl)methyl-P-amino-ethanesulfonate (TES)/l mM ethyleneglycol bis(&aminoethyl ether).N,N-tetraacetic acid (EGTA) pH 7.2, at 0. Incubation was carriedout in a Dounce homogenizer, with a Teflon pestle for a total of 12 min.Homogenization was carried out by hand, as soon as mechanicallypossible, with a total of five complete passes. The homogenate wasthen centrifuged at 10,000 x g for 6 min and the entire pellet wasresuspended in 60 ml of preparation medium. The suspension wascentrifuged at 350 x g for 10 rain and the supernatant layer w asdecanted. The latter was then centrifuged at 10,000 x g for 6 min toyield mitochondrial pellets. These were resuspended and pooled to givea total volume of 30 ml. The final centrifugation was for 7 min at 7,500x g to permit the removal of an upper fluffy iayer from the pelle t. Themitochondria were resuspended in 2 ml of preparation medium, to givea suspension of approximately 20 mg of protein/ml.

Extraction and Estimation of Isocitrate Dehydrogevmses and Nico-tinamide Nucleotide Transhydrogenase-Two methods were used toremove the latency of NAD -&citrate dehydrogenase. In one proce-dure, 1 ml of mitochondrial suspension containing 15 to 20 mg ofprotein was diluted with 2 ml of 50 mM KP,, pH 7.2, containing 2 m MADP, 1 m u EDTA, and 10 rn~ dithiothreitol. The suspension wassonicated using a Branson sonifier, setting 2, in bursts (3 x 20 s). 40 selapsing between each burst. Cooling during sonication was effected byan ice/water mixture. Suhmitochondrial particles were sedimented at100,000 x g for 45 min and the supernatant fraction was used for theassay of NAD-isocitrate dehydrogenase. The assa !. medium comprised50 tnM KP,, pH 7.2, 10 rn~ MgCI,. 0.5 pg of rotenone/m l. 1 kg ofoligomycin/ml, and NAD+. Isocitrate was added as a mixture of citrateand m-isocitrate containing a 25.fold molar excess of citrate overthreo-n,-isocitrate to start the reaction, and NAD reduction wasfollowed spectrophotometrically at 340 nm.

In the second procedure, mitochondria were added directly to amedium containing 50 trIM KP,, 10 m M M&l,, 1 m M dithiothreitol, 0.5pg of rotenone/ml. 1 py of oligomycin/ml, 0.05t (v/v) Triton X-100,and KAD+. The fina l protein concentration was about 0.2 mg/ml andthe pH was 7.2. The reaction was begun by addition of a citrate--isocit-rate mixture and followed at 340 nm as described above.

For the assay of SADP-isocitrate dehgdrogenase, mitochondria wereadded to the medium containing Triton X-100 used for the SAD-linked enzyme, NADP+, replacing NAD+. All other conditions were asdescrib ed for the NAD-linked enzyme.

For some experiments the mitochondrial suspension was depleted ofendog.enous substrate by adding it to 15 ml of 0,.saturated basalmedium (see below) to which ADP had been added to 0.5 mM , glucoseto 5 mM , M&l, to 2.5 mM , hexokinase to 10 units/ml, and L-malate to0.1 ITIM. After 7 min at 25, the diluted mitochondrial suspension wascooled and centrifuged at 7500 x g for 7 min. The pellet wasresuspended as described previously.

NADPH-NAD + transhydrogenase was assayed in submitochondrialparticles isolated by the method of Hansen and Smith (12) asdescrib ed hy Lee and Ernster (13). The assay procedure (14) used atrapping system of 3 m M pyruvate and an exces s activity of lacta tedehydrogenase (0.7 units/ml final concentration). The reaction wasinitiated by addition of NAD+ to 0.4 mu. The NADPH concentrationwas 0.1 I T I M . Under these conditions the assay was linear with addedparticle protein. Transhydrogenase activity was calculated with re-spect to mitochondrial protein hy estimating the cytochrome a contentof submitochondrial particles and intact mitochondria, using thedifference in absorbance between 605 and 630 nm of air-oxidized,dithionite-reduced cytochrome.

Incubatiorl of Mitochondria and Extraction and Assay of Inter-mediates-All incubations used a basal medium comprising 0.12 MKCl, 20 m M KTES , pH 7.2, and 10 mM KP,, pH 7.2. Other additionsare detailed in the figure legends. All experiments were carried out at25. The sampling, extraction, neutralization, and assay procedures forCoA, acetyl-CoA, and succiny-CoA were exactly as described previ-ously (7). Long chain acy-CoA was determined in the washed pellets ofHClO,-insoluble material by hydrolyzing the thioester a s described by

Reagents and Enzymes-Reagents were of the highest purity availa-ble commercially. The uncoupling agent FCCP was from PierceChemical and oligomycin was from Sigma Chemical Co. All enzymesused were from Boehringer-Mannheim Corp., New York.

RESULTS AND DISCUSSIONThe Steady State Mitochondrial Contents of CoA and CoA

Thioester during Oxidation of Palmitoylcarnitine and Oc-

In this paper, CoA is used to denote coenzyme A-SHThe abbreviation used is: FCCP, carbonyl cyanide p-trifluorome-

thoxyphenylhydrazone.

William son and Corkey (8) and then assaying CoA by the cyclingtechnique (7. 9). Sampling, extracting. neutralization, and assayprocedures for citrate, isocitrate, and 2.oxoglutarate were as describedpreviously (10) with the mitochondrial content of these compoundsbeing determined by the subtraction of a supernatant content,obtained after microcentrifugation. from the total content of theincubation medium containing mitochondria. The extraction andassay of mitochondrial NAD +, NADP+, NADH, and NADPH were asdescribed by William son and Corkey (8). LVhere duplicate experimentswere performed they were matched closely with respect to time ofsampling and, when tricarboxylate cycle intermediates were assayed,with respect to protein concentration. The values presented are meansand Students t test was applied to establish the significan ce ofdifferences between groups of data; where limits of error are given, thestandard error of the mean is shown. with the number of samples inparentheses. In all extraction experiments, 0, uptake was monitoredcontinuously, using a Clark-type 0, electrode. Where rates of 0,uptake are quoted, these were obtained in parallel incubationsinvolving air-saturated medium and a completely closed chamber ofvolume 2 ml. The recordings of intramitochondrial NAD(P)H wereobtained in parallel incubations. using a Farrand fluorimeter, asdescribed previously (11). ATP and ADP were measured in extracts asdescribed by William son and Corkey (8) using either the fluorimeter ora Beckma n-Gilford spectropho tometer, as appropriate. Pyruvate in theextracts was measured spectrophotometrically with lactate dehgdro-genase.

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FIG. 1. Mitochondrial CoA and CoA thioester during state 4 - 3 + 4transitions with palmitoylcarnitine and L-malate a s substrate. At zerot ime, 1.5 ml of mitochondrial suspension (protein 18.4 mg/mll wereadded to 20 ml of a basal medium containing 12 @M palmitoyl-L-carni-tine and 0.5 mM r--malate. The medium was saturated with air. At thepoint indicated, 12 pmol of ADP were added. Samples (2 ml) were

tanoate-Fig. 1 presents the results of an experiment in whichmitochondrial CoA and CoA thioester were estimated duringthe oxidation of palmitoylcarnitine plus 0.5 mM L-malate. Itis seen that during the initial phase of controlled respiration(state 4, according to Chance, and Williams (15)) acetyl-CoAcontent is high, whereas that of CoA is exceedingly low. Onactivation of respiration by ADP (state 3) there is a marked fallin acety-CoA content, with a concomitant increase in CoAcontent. In addition, succiny-CoA content increases slight ly.This latter change is defined with more exactness in a latersection. On re-entry into state 4, the changes reverse, with theabsolute amounts measured being somewhat higher, owing to adecline in long chain acy-CoA content with a decline in theconcentration of the substrate. palmitoylcarnitine. Although inthis experiment the long chain acyl-CoA content appeared torise during the state 4 + 3 transition, the opposite behavior inother experiments (Fig. 5 and not shown) indicates that thereis no clear response to changes in flux . The absence of any largechange suggests that increased carnitine palmitoyltransferaseact ivi ty (EC 2.3.1.a) which would be expected in state 3 owingto the rise in CoA content must be matched by an increase inthe act ivi ty of p oxidation in this state, the latter presumablydue to decreased concentration of NADH and FADH,. Thequantitative contribution of long chain acyl:CoA to the mito-chondrial pool of CoA and thioester is greater than that inperfused heart even at high palmitate concentrations (31,suggesting that the ratio of the concentration of long chainacy-CoA to CoA is higher in the mitochondria than in the

0 1 2 3 4 5 6MINUTES

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

cytosol. The low concentrations of CoA and acetyl-CoA inmitochondria oxidizing palmitoylcarnitine would be diff icul tto measure using conventional stoichiometric assays, but areamenable to the kinetic assay described by Allred and Guy (9).The modification of this assay to include succinyl-CoA synthe-tase (7) permits the measurement of succiny-CoA at equallylow concentrations. The clear decrease in acetyl-CoA contenton activating respiration which is seen in Fig. 1 implicates thetricarboxylate cycle. and specif ically citrate synthase, as beingrate limiting during the controlled (state 4) oxidation ofpalmitoylcarnitine by isolated heart mitochondria. This is inaccord with a conclusion reached by Oram et al. (3) on thecontrol of the oxidation of high concentrations of palmitate byperfused rat heart. There is no real confl ict with the conclusionreached by Pande (16) that the act ivi ty of /3 oxidation limitsthe rate of oxidation of the palmitoyl group by rat heartmitochondria. This is because the earlier study (16), whichcompared the rate of palmitoyl group disappearance in thepresence and absence of a functioning tricarboxylate cycle,exclusively used conditions which maximally activate oxida-tive phosphorylation (ADP) or electron transfer (uncouplingagent). By contrast, the present study is concerned with thosefacto rs which regulate the oxidation of palmitoyl groups andpyruvate when the rate of oxidative phosphorylation is aminimum (state 4, Fig. 1) or is less than maximal (state 3?2,see below). It is noteworthy that Garland et al. (17) foundchanges precisely opposite to those reported here during thestate 4 + 3 transition, in experiments involving the oxidation of

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8364palmitoylcarnitine by rat liver mitochondria. The large in-crease in acety-CoA content, which they reported, suggests anincrease in the act ivi ty of p oxidation relative to citratesynthase, upon the activation of respiration. This emerges as amajor difference between heart and liver mitochondria. Thepresence o f a high content of acetyl-CoA during state 4respiration in the present work (Fig. 1) and the irreversiblenature of the citrate synthase reaction raise the question of thecontrol of this enzyme, and this is discussed in a later section.

It has been shown that no decrease in acety-CoA contentoccurs on increasing the pressure development of rat heartsoxidizing octanoate (3), contrary to the decrease observed withpalmitate. For this reason an experiment was carried outsimilar in.design to that shown in Fig. 1, but using 0.25 mMoctanoate plus 0.5 mM L-malate as substrate (Fig. 2). This wasfound to be an optimal concentration of octanoate in the ab-sence of serum albumin, giving a State 3 rate of oxygen con-sumption of 0.275 lg.atoms of 0 Jmin/mg of protein. It is seenthat the decrease in acetyl-CoA content with the activation ofrespiration was marked, and indeed greater than that obtainedwith palmitoylcarnitine (Fig. 1). The discrepancy between thisresult and that obtained with perfused hearts (3) is unex-plained, but may reflect species difference.

Steady State Mitochondrial Contents of CoA and CoAThioester during Oxidation of Pyruuate, Presen t a t VaryingConcentrations-There is some ambiguity in the literatureabout the mitochondrial content of CoA and acety-CoA duringcontrolled (state 4) and active (state 3) pyruvate plus L-malateoxidation. Thus, acetyl-CoA content was found to be the samein states 3 and 4 (18) and this was confirmed for mostincubation times in a later study (4). However, the latter workdid demonstrate an elevated CoA content in state 3 andanother study involving rabbit heart mitochondria demon-strated a fall in acetyl-CoA content on uncoupling, but did notpresent data for state 3 (19). Because of this ambiguity, anexperiment was performed of the design shown in Fig. 1, butwith 2.5 mM pyruvate plus 0.5 mM L-malate as substrate (Fig.

(Ai r ADPqo I-I

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3). It is seen that the state 4 + 3 transition is associated withchanges similar to those described for Fig. 1, and that theacetyl-CoA content clearly falls . The CoA/acety-CoA ratio issomewhat higher in state 4 than that shown in Fig. 1,suggesting that pyruvate dehydrogenase is less active in state 4than is p oxidation as a mechanism of generating acety l groups.Repetition of the experiment at a lower pyruvate concentration(0.25 mM) gave the results shown in Fig. 4. It is noted that thepattern of changes seen in response to the activation ofrespiration is very similar to that seen at 2.5 mM pyruvate, butthat the CoA/acetyl-CoA ratios are higher in both state 3 andstate 4, consistent with a decreased pyruvate dehydrogenaseact ivi ty at the lower substrate concentration. There is a clearrise in succinyl-CoA content on activating respiration, as is

06 ADP STATE 3 -4I

05 t

OLMIN U TES

FIG. 2. Mitochondrial CoA and CoA thioester during state 1- 3 - 4transitions with octanoate plus L-malate as substrate. The reaction wasinitiated 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 m Moctanoate and 0.5 m M L-malate. At the point indicated, 40 pmol ofADP were added. The medium was saturated with 0,. O--O. CoA;O---O, acetyl-CoA ; l - - -0, succiny-C oA.

I XYGEN~(APPROX 0.1pg-ATOM/ML)

I / / 1 I0 1 2 3 4 5.MINUTES

FIG. 3. Mitochondrial CoA and CoA thioester during state 1+ 3 - 4 with air. ADP (13 pmol) was added at the point indicated. Panel Atransitions with a high concentration of pyruvate plus r.-malate as presents the mitochondrial CoA and thioester content, panel R, ansubstrate. One milliliter of mitochondrial suspension (13 mg of oxygen electrode trace from the same experiment. O---O, CoA;protein) was added at zero time to 20 ml of a basal m edium containing 0-O acetyl-CoA ; l - - -0, succin yl-CoA .2.5 rnrvr pyruvate and 0.5 rnM L-malate . The mediu m was saturated

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(A)O./0.6- \

AD P1

zLi 05- I)L

IOOI I I , I I1 2 3 4 5 6

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OXYGEN (APPROX0.1 MS-ATOM/ML)

1 L,4

16 : I

0 1 2 3 4 5 6MINUTES

FIG. 4. Mitochondrial CoA and CoA thioester during state 4 + 3 - 4 transitions with a low concentration of pyruvate plus r.-malate assubstrate. The reaction was initiated by the addition of 1.4 ml ofmitochondrial suspension (protein 24.4 mg/ml) to 20 ml of a basal mediumcontaining 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:l - -0. succinyl-CoA.found in mitochondria from blowfly flight muscle (7). Such achange is noted as a response to increased work load in theperfused rat heart (1) but has not been shown with isolatedheart mitochondria. Thus, LaNoue et al. (4) show rathersimilar succinyl-CoA contents in states 3 and 4 with acetylcar-nitine plus malate as substrate and show a slight fall insuccinyl-CoA content on addition of uncoupling agent (20).There are no values in the literature for succiny-CoA contentof heart mitochondria oxidizing either pyruvate, palmitoylcar-nitine, or octanoate. The importance of succiny-CoA concen-tration is discussed in a later section. The rationale of thisexperiment (Fig. 4) was to use a pyruvate concentration nearerto that found in the heart, so as not to obscure possible controlson pyruvate dehydrogenase act ivi ty. It is seen neverthelessthat the CoA/acetyl-CoA ratio is still much lower, in bothstates 3 and 4. than the ratios of approximately 11:l and 14:lfound by Oram et al. (3) in perfused hearts oxidizing glucosealone, and at low and high work loads, respectively. A furtherexperiment (Table I) used lactate dehydrogenase to generate alow and constant concentration of pyruvate for mitochondrialoxidation. Samples were initially taken during state 4 respira-tion and the pyruvate concentration was determined to be 14FM. Even at this substrate concentration, the CoA/acetyl-CoAratio was stil l low (0.26). Addition of ATP plus 0.11 mM M&l,gave a doubling of respiratory rate, due to extramitochondrialATPase, possibly of myofibril lar origin, and a rise in theCoA/acetyl-CoA ratio to 0.35. Further portions of this experi-ment in which the respiratory rate was further increased byaddition of hexokinase are not presented in Table I as theequilibrium pyruvate concentration began to decline. How-ever, even during state 3 respiration and at 6 fiM pyruvate theCoA/acetyl-CoA ratio was 3.4, still considerably less than thevalues reported during carbohydrate oxidation in the heart.This discrepancy may reflect a high ratio of CoA/acetyl-CoA inthe cytosol of the heart, or the presence in the intact organ of acontrol o f pyruvate dehydrogenase not reconstructed in the

TABLE IPattern of acylation of mitochondrial CoA during oxidation of

pyruuate, generated by lactate dehydrogenase reactionOne millilite r of mitochondrial suspension (24 mg of protein) was

added to 20 ml of 0,.saturated basal medium containing 0.3 mMI.-malate, 1.25 mM NAD+, 0.5 mM ADP, 5 mM glucose, and 12 units/mlof dialyzed lactate dehydrogenase. After 5.5 min of incubation, tocause depletion ofendogenous substrate, the reaction was initiated bythe addition of 150 rmol of DL-actate. Two samp les were taken at 7.5min (state 4). ATP (10 pmol) and M&l, (2 pmol) were added at 8 minto partially activate respiration, and two further samples were taken at9.5 min. CoA and CoA derivatives, ADP, ATP, and pyruvate wereestimated in the samples as described under Experimental Proce-dure. Pyruvate was 13.7 + 0.4 pM in samples 1 and 2 and 13.0 I 0.5 fiMin samples 3 and 4. 0, uptake was measured in a parallel experiment.

Respiratorystate

State 4+ATP

+Mg*+

latio Rate ofiTP/ AcetyL0 $ con- CoA Succinyl-1DP sumption content CoA CoAcontenr content

minlmgprotein170 0.036

72 0.071

nmollmg protein

mitochondrial experiments. In this context the control ofpyruvate dehydrogenase by phosphorylation-dephosphoryla-tion (21, 22) is thought to underlie the changes in pyruvatedehydrogenase act ivi ty which can be shown in rabbit heartmitochondria in the presence of ADP plus uncoupling agentsplus Mg2+ (19) or on incubation in the presence of ATP,oligomycin, and fluoride (23). Experiments were not carriedout under either of these sets of conditions in the present study,but a partial inactivation of pyruva te dehydrogenase byphosphorylation cannot be ruled out during state 4.

Steady State Mitochondrial Contents of CoA and CoAThioester during Oxidation of Pyruvate Plus Palmitoylcarni-

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01t

MIN U TESFIG . 5. Mitochondrial CoA and CoA thioester during the oxidation

of both pyruvate and palmitoylcarnitine. The reaction was initiated bythe addition of 1.2 ml of mitochondrial suspension (protein _ 22.8mg/ml) to 20 ml of air-saturated basal medium containing 0.25 m Mpyruvate, 10 FM palmit oyl-L-carnitin e, and 0.5 m M L-malate. ADP (40tine and of 2-Oxoglutarate-Fig. 5 presents the results of anexperiment in which the substrate was 0.25 m M pyruvate/lO pMpalmitoyl-L-carnitine plus 0.5 m M L-malate and in whichrespiration was stimulated by suff icient ADP to give anaerobi-osis without a state 3 - 4 transition. A control experimentestablished that the palmitoylcarnitine would be depleted atthe time of taking sample 6, if palmitoylcarnitine were oxidizedto the exclusion of pyruvate. Equally, the concentration ofpalmitoylcarnitine remaining at a time corresponding to sam-ple 4 would be suff icient to guarantee a maximal rate ofpalmitoylcarnitine oxidation. It is noted that the content oflong 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 contentsresembles more closely that obtained with palmitoylcarnitineplus L-malate (Fig. 1) than that with 0.25 m M pyruvate plusL-malate (Fig. 4). Thus, the CoA/acetyl-Co.4 ratio is very lowin state 4 (approximately 0.11, Fig. 5, sample 3) and theCoA/succinyl-CoA ratio is approximately 1 in state 3. Theresults of this experiment allow comment to be made on thesuggestion by Bremer (24) that the mitochondrial oxidation ofpalmitoylcarnitine inhibits pyruvate oxidation by competitionfor mitochondrial CoA. The CoA content is indeed decreasedin 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 signifi-cant for pyruvate dehydrogenase act ivi ty (25, 26) and this ismarginally decreased by the presence of palmitoylcarnitineduring state 3 oxidation (Figs. 4 and 5). However, the CoA/acetyl-CoA ratio is much lower during state 4 respiration in thepresence of both pyruvate and palmitoylcarnitine than it iswith 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 havehad a material eff ect in diminishing the oxidation of pyruvate.

MIN U TESpmol) was added at the point indicated. Panel A presents the contentsof 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, succin y1LC oA; W---m,HClO,-insoluble CoA.

Finally, the same sort of experiment was repeated with2-oxoglutarate plus L-malate as substrate (Fig. 6) to explorethe acylation pattern of CoA during the operation of the2-oxoglutarate + malate segment of the tricarboxylate cyc le.There is information on succinyl-CoA and CoA content in state4 respiration (20), but none on the changes evoked by a state 4- 3 transition. It is seen that the succiny-CoA/CoA ratio isextremely high in state 4, in fair agreement with the results o fLaNoue et al. (20) and that i t declines upon the generation ofstate 3 respiration (Fig. 6). It is noted that this is the inverse ofthe response of the succinyl-CoA/CoA ratio to ADP additiondescribed in the earlier figures. However, this is not unreasona-ble, as both the high concentration (1 m M) of added 2-oxogluta-rate and the relatively low reduction of NAD(P) seen in Fig. 6C(cf. Figs. 8B and 9B) will allow a high a ctivit y of P-oxoglutaratedehydrogenase in state 4. The decrease in succinyl-CoAcontent on adding ADP then represents a greater activation ofsuccinyl-CoA synthetase (EC 6.2.1.4), operating in the direc-tion of succinyl-CoA cleavage, than of 2-oxoglutarate dehydro-genase.

Effect of Pyruvate and of Palmitoylcarnitine Concentrationupon Acylation of Mitochondrial CoA and Rate of OxygenConsumption during State 3 Respiration-A systematic studywas made of the relation between the concentrat ion of addedpyruvate, the mitochondrial respiratory rate, and the patternof CoA acylation (Table II). State 3 respiration was guaranteedthroughout the study by the presence of ADP and hexokinaseplus glucose. It is seen that over the range of pyruvateconcentration 0.024 to 0.16 m M the ratio CoA/acetyl-CoAdeclines steadily, such that there is an inverse linear relation-ship between this ratio and the respiratory rate (Table II, plotnot shown). Over this range of pyruvate concentration, therespiratory rate doubles, whereas there is very little change inthe acetyl-CoA/succinyl-CoA ratio. Data and reasoning to beadduced later suggest that oxygen consumption may be taken

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(A) I ADP I

60 -

20 -o--o-o

o\O / \ O--oOl ,F O2 3 4 5 6

MINUTES

OXYGEN IAPPROX0.4 ,,g-ATOM/ML)

80 1 2 3 4 5 6

MINUTES

IC)

1LUORESCENCE

8367-I

INCREASE

1FCCP

FIG. 6. Mitochondrial CoA and succinyl-CoA during state 4 - 3 -4transitions with 2-oxoglutarate plus L-malate as substrate. The reac-

panel B presents an oxygen electrode recording from the sameexperiment, and panel C a recording of the fluorescence of mitochon-

tion 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 drial NAD(P) obtained in a parallel experiment, using the sameincubation conditions, with the exception that ADP was added to 0.5containing 1 m M 2-oxoglutarate and 0.5 rnhi L-malate. At the pointindicated, 50 pmol of ADP were added. Panel A presents the m M . FCCP is the uncoupling agent carhonyl cyanide p-trifluoromethox-yphenylhydrazone.mitoc hond rial content of CoA (O-O) and succin yl-CoA (O-O),

TABL E IIEffect ofpyruvate concentration upon pattern of acylation of mitochondrial CoA during state 3 respiration

Reaction was initiated by adding 1 ml of mitochondrial suspension followed by sampling at 3.9 min, and to 2.4 m M (4.3 min) followed by(14.3 mg of protein) to 20 ml of O,-saturated basal medium containing sampling at 4.6 min. The results shown are the mean values obtained25 pM pyruvate, 0.5 m M L-malate, 2.5 m M MgCl,, 10 m M glucose, and 9 from these duplicate samples. Oxidation was monitored with an OSunits/ml of dialyzed hexokinase. ADP was added to 0.5 mM at 2 min electrode in the suspension sampled, but the rates of 0, uptake wereand two samples were taken at 2.3 min. Pyruvate was then added to 48 obtained in a parallel experiment, using an air-saturated medium.pM (2.5 min) followed by sampling at 3.0 min, to 160 pM (3.25 min)

Pyruvate Rate of 01 CoAconcentration Acetyl-CoA Succinyl-CoA Ratio CONconsumption content content content acetyl-CoARatioacetyl-CoA/succinyl-CoA

mhf0.0240.0480.162.4

ag-atoms O,!mmlmgprotem0.170.240.330.38

O&79 * 0.010.65 zt 0.0020.46 zt 0.01

0.38

nmollmg protem0.15 * 0.010.20 rk 0.010.25 f 0.01

0.42

0.39 * 0.03 5.2 0.400.44 * 0.01 3.2 0.460.49 * 0.02 1.9 0.49

0.38 0.91 1.1

as an approximate index of the flux through pyruvate dehydro- with acetyl-Co A/succin yl-CoA ratio in any range of substrategenase and citrate synthase. Thus, over the concentration concentration tested. When the inverse of the rate of oxygenrange 0.024 to 0.16 mM pyruvate the flux through these consumption is plotted uersus the inverse of the content of longenzymes is directly proportional to the acetyl-CoA/CoA ratio chain acyl-CoA a linear plot is obtained (Fig. 7) suggesting aand unrelated to the acetyl-Colvsuccinyl -CoA ratio, suggest- K, for the p oxidation process of 0.95 nmol of long chaining a limitation of flux by pyruvate dehydrogenase. At high acyl-CoA/mg of mitochondrial protein. It is noted that thepyruva te concentrations (0.16 to 2.4 m M ) , however, there is an mitochondrial suspension was depleted of endogenous sub-increase in the acetyl-CoA/succinyl-CoA ratio with increased strate in this experiment, and in some subsequent studies. Itflux . LaNoue et al. (4) have suggested that citrate synthase was found that in the absence of this depletion step theact ivi ty may be related to the acetyl-CoA/succinyl-CoA ratio, mitochondrial suspension contained 0.1 to 0.2 nmol/mg ofunder conditions giving oxidized nicotinamide nucleotide. This protein of long chain acyl-CoA, before the addition of sub-may be true in the region 0.16 to 2.4 m M pyruvate in this strate. The inference is that these mitochondria contain fa tt yexperiment. acids, and judging from the amount of OR consumed during

An analogous experiment with palmitoylcarnitine as sub- substrate-depletion, this content may be considerable. Thestrate is shown in Table II I. Here there is a large fal l in the ratio nature of the endogenous substrates present in rabbit heartCoA/long chain acyl-CoA with increase in substrate concentra- mitochondria, and the effects of their removal, have beention and increased flux. However, there is no correlation of flux thoroughly investigated (27-29).

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8368Pattern of Acylation of Mitochondrial CoA in Respiratory

States Intermediate between States 3 and 4: Discussion ofEffect of Ratio Acetyl-CoAfSuccinyl-CoA on Activ ity of Cit-rate Synthase-A prime goal of the present work was todetermine the mitochondrial content of CoA and CoA thioes-ters under conditions which allowed a meaningful comparisonwith data from whole heart. This suggested the study ofmetabolic states intermediate between states 3 and 4 (the state3% of K&rig et al. (30)) and generated by varying the steadystate concentrations of ADP available to the mitochondria.Table IV presents the results of an experiment in which thiswas done, with palmitoylcarnitine plus L-malate as substrate.Two pha ses in the response to increased ADP can be clearlydiscerned. First, a decline in the ATP/AD P ratio from 43 to 25caused a large oxidation of nicotinamide nucleotide, a 2.5.foldincrease in the rate of 0, uptake and a significan t rise insucciny-CoA, and a smal l increase in CoA content. Secondly,a further decrease in the ATP/AD P ratio to 2 gave very littlefurther oxidation of nicotinamide nucleotide, a slight increasein the rate of 0, uptake, and a decrease in succiny-CoA, but alarge increase in CoA content. Essentially the same resultswere obtained in an experiment with pyruvate plus L-malate assubstrate (not shown). The progressive decline in acetyl-CoA

content with decreased ATP/AD P ratio reflects citrate syn-thase activation, probably due to increased nucleotide oxida-tion and oxalacetate availability, at least in the range ofATP/AD P ratios of 43 to 25. The increase in succinyl-CoAcontent in the first phase of the experiment suggests increasedactivity of 2-oxoglutarate dehydrogenase relative to that ofsucciny-CoA synthetase. This stems from the oxidation ofnicotinamide nucleotide, which will activate 2-oxoglutarate

0 I 10 1 2 3 4 5l/S nMOLES HC104-INSOLUBLE

CoA/MG PROTEINFIG. 7. Lineweaver-Burk plot of rate of 0 2 uptake and mitoc hondria l

content of long chain acyl-CoA during state 3 palmitoylcarnitineoxidation at different concentrations of palmitoylcarnitine. The dataare taken from the experiment shown in Table III.

TABLE II IEffect ofpuZmitoylcurnitine concentration upon pattern of ~cyZrn5on of mitochondriul CoA during state 3 respiration

One milliliter of mitochondrial suspension (21.4 mg of protein) was addition of palmitoylcarnitine was made, g iving 5 FM (6.7 min).added at time zero to 20 ml of 0,.saturated basal medium containing Sampling was at 7 min. After substrate exhaustion, palmitoylcarnitine0.5 mM L-malate, 0.5 mM ADP, 10 mM glucose, 9 units/ml of dialyzed was added to give 10 pM (8.7 min) and samples were taken at 9.1 min.hexokinase, 2.5 mM MgCl,, and 5 mg/ml of bovine serum albumin. Palmitoylcarnitine was then added to 50 PM (9.6 min) and samplesAfter 5.5 min to allow depletion of endogenous substrate, palmitoyl-L- were taken at 10.4 min. The completion of palmitoylcarnitine oxida-carnitine was added to 2 PM and two samples were taken (5.8 min). tion after the first two additions was determined from the 0, electrodeWhen all of the palmitoylcarnitine had been oxidized, a second recording.

Palmitoyl-L- Ratecarnitine OfO, CoAconcentration consumption contentAcetyl-CoA Succinyl-CoAcontent content

HClO,-insoluble-CoAcontentRatio CoA/ RatioHClO,. acety-CoA/insoluble-CoA succinyl-CoA

nmollmg protein2 0.06 0.81 * 0.05 0.12 * 0.01 0.24 ;t 0.01 0.20 * 0.01 4.1 0.485 0.10 0.64 + 0.01 0.11 * 0.01 0.23 * 0.01 0.39 * 0.01 1.6 0.45

10 0.16 0.37 * 0.03 0.073 * 0.003 0.21 * 0.002 0.61 * 0.01 0.61 0.3550 0.17 0.10 * 0.01 0.053 + 0.006 0.14 * 0.01 0.83 * 0.05 0.12 0.37

TABLE IVPattern ofucylution ofmitochondriul CoA during oxidation ofpulmitoylcu rnitine by mitochondriu respiring in state 3 and in state s intermed iate

between 3 and 4One and five-tenths milliliters of mitochondrial suspension (protein then added to 6.3 units/ml at 3.3 min and samples were taken at 4.3

= 24.6 mg/ml) were added to 18 ml of 0,.saturated basal medium min. Finally, ADP w as added to 2 mM at 4.5 min and samples werecontaining 106 PM palmitoyl-Lcarnitine, 0..5 mM L-malate, 10 mM taken at 5.25 min. The results shown are the mean values obtainedglucose, 0.1 mM M&l,, 0.6 units/ml of dialyzed hexokinase, and 10 from the duplicate samples. Changes due to substrate depletion weremg/ml of bovine serum albumin. After 0.75 min, ATP was added to 0.5 avoided in this experiment by the use of a high concentration ofmM. Samples were taken at 2 min. Hexokinase was added to give 2 palmitoylcarnitine, buffered by serum albumin.units/ml at 2.25 min and samples were taken at 3 min. Hexokinase was

HeXo-kinasepresentRatio RateATPI of0,ADP On:sumpt1on

CoAcontent AcetyllCoAcontentSuccinyl-CoAcontent

HClO,-insoluble-CoAcontentRatio

HE%.msolubie-CoA

Ratioacetyl- 5ReductionGA/succinyl-CoA iYADP,pg.atomsunits/ml OJminlmg nmollnq proteinprotein

0.6 43 0.048 NIL 0.20 * 0.01 0.12 * 0.01 0.62 i 0.08 NIL 1.6 532.0 40 0.064 0.002 i 0.001 0.14 * 0.01 0.18 i 0.01 0.65 i 0.11 0.003 0.78 406.3 25 0.117 0.012 1 0.003 0.090 * 0.002 0.21 i 0.01 0.72 + 0.06 0.017 0.44 116.3 (+ADP) 2.0 0.171 0.046 + 0.002 0.061 + 0.003 0.14 * 0.01 0.79 * 0.01 0.058 0.43 10

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dehydrogenase (and isocitrate dehydrogenase, as shown be-low), which is seen in the range o f ATP/ADP ratios from 43 to25. Even though the intramitochondrial ATP/ADP ratio will beless than 25 when the ratio found by sampling the wholesuspension is 25, owing to the ef fec t of mitochondrial mem-brane potential on ATP*- for ADP3- exchange (31), it is stillevidently high enough to limit the act ivi ty of succinyl-CoAsynthetase (Table IV, lines 3 and 4). The mechanism ispresumably the near equilibration of energy charge (32) ofguanine and adenine nucleotides catalyzed by nucleosidediphosphate kinase (20). Higher concentrations of ADP servemainly to activate the succinyl-CoA synthetase reaction(Table IV), in the direction of succiny-CoA cleavage. Anexperiment in which an abrupt transition is made from state 4to state 3 (e.g. Fig. 1) does not allow this partial separation ofthe effects of oxidation-reduction state and ATP/ADP ratio.

In a recent detailed study of respiratory states between 3 and4, Davis and Lumeng (33) have demonstrated a constancy ofintramitochondrial phosphate potential over a wide range ofextramitochondrial phosphate potential, achieved by addingpurified ATPase. In the present s tudy, intramitochondrialadenine nucleotides were not estimated, but the changes innicotinamide nucleotide oxidation-reduction state, and in theacylation of CoA which were described above, make it reasona-ble to infer changes in intramitochondrial phosphate potential.

LaNoue et al. (4) have proposed a model of tricarboxylatecycle control in which citrate synthase activity is controlled bythe ratio acetyl-CoA/succinyl-CoA, in those circumstanceswhere oxalacetate availabili ty is high, but in which f lux is stillnot maximal. Aphysiological conditions involving the use ofoligomycin and an uncoupling agent were used to demonstratethis control. It seemed possible to explore the applicability ofthis mechanism under conditions nearer to physiology bymaking use of the intermediate states in Table IV. Herenicotinamide nucleotide is highly oxidized and L-malate ispresent such that oxalacetate availabili ty is high, but the fluxis less than maximal. Under these conditions it seemedplausible that flux through citrate synthase might be con-trolled by the acety-CoA/succinyl-CoA ratio. In an experimentto test this possibility, fi ve separate incubations were madewith pyruvate plus L-malate as substrate, and with differingactivi ties of hexokinase present. Because there are no changesof flux in the course of each incubation, contrary to the studies

8369presented so far , this experimental design allows the estima-tion o f the flux through citrate synthase by the disappearanceof pyruvate. It is seen (Table V) that fl ux through citratesynthase increases as the ratio ,4TP/ADP decreases, but that inno portion of the experiment is this increased flux matched byan increase in the ratio acetyl-CoA/succinyl-CoA. In a subse-quent experiment (Table VI), acetylcarnitine plus L-malatewas used as a substrate, and an estimate of the activ ity ofcitrate 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 beextremely small. The accumulation of succinate was notmeasured and thus it is not known to what extent succinate isoxidized. However, this does not introduce a great uncertaintyinto the flux calculations, because 0, uptake is divided by 4 inthe event of complete oxidation of succinate and by 3 in theabsence of any such oxidation. Examination of the stoichiome-try of 0, uptake and palmitoyl group disappearance in fac tsuggests that rat heart mitochondria oxidize palmitovlcarni-tine to completion in the presence o f L-malate, with no largeloss of tricarboxylate cycle intermediates (16). It is seen thatthe ratio acetyl-CoA/succinyl-CoA falls as the 0, uptake risesuntil the rate is 72% of that achieved in state 3. This isachieved with an ATP/ADP ratio of 72. The further reductionof this (ATP/ADP) ratio causes a decrease in succinyl-CoAcontent, for the reasons outlined in the discussion of Fig. 4, andan increase in the ratio acetyl-CoA/succiny-CoA. There is alsoa slight increase in flux through citrate synthase, measured by0, uptake. It must be noted, however, that there is also anincreased oxidation of nicotinamide nucleotide under thesecircumstances. Thus, data in Table VI (lines 4 and 5), althoughconsistent with a control of citrate synthase by the ratioacetyl-CoA/succinyl-CoA under conditions of low nicotinamidenucleotide reduction and almost maximal flux, are quiteequivocal. It is noteworthy that the large majority of the2-oxoglutarate produced is oxidized, despite wide variations inthe CoA/succinyl-CoA ratio, which might be expected to alterthe act ivi ty of 2-oxoglutarate dehydrogenase (20). Finally, theexperiment of Fig. 6 showed that succinyl-CoA content isextremely high in state 4 2-oxoglutarate oxidation, and raisedthe possibility that citrate synthase might be controlled by theacetyl-Coivsucciny l-CoA ratio, when 2-oxoglutarate is presentin addition to a source of acety l un its. The experiment shown in

TABLE VRelation betweenflux through citrate synthase, oxidation-reduction state ofNAD(P), and mitochondrial acetyl-CoAlsuccinyl-CoA ratio, during

pyruuate oxidation by mitochondria in state 3, state 4, and intermediate statesIn each of five separate incubations, 0.3 ml of mitochondrial obtained by comparing the pyruvate found in the samples with thesuspension (protein = 18.7 mg/ml) was added to 5 ml of 0,.saturated pyruvate found in samples of a control incubation, in which mitochon-

basal medium containing 0.8 m M pyruvate, 0.5 mM L-malate, 10 rn~ dria were added to .5ml o f medium at O, containing 1 FM rotenone, andglucose, 0.5 mM ATP, and 0.1 mM MgCl,. After 1 min, the amount of in which sampling was conducted immediately. Correction was madehexokinase shown w as added to each incubation, to activate respira- for the 1 min of controlled respiration in lines 2 through 5. The wholetion. Duplicate samples were taken after 8 min (line 1) and after 6 min mitochondrial suspension had been depleted of endogenous substrate(line 2) and 5 min (lines 3 to 5) of activated respiratio n. Citrate (see Experimenta l Procedure).synthase flux is estimated from the disappearance of pyruvate,

Hexokinasepresentunits/ml

7141846

Ratio CoAATPIADP content

62 26 * 338 24 + 227 25 + 124 22 * 1

2.2 25 * 0.4

Acetyl-CoAcontent% total CoA

58 i 348 + 0.343 i 144 i 246 i 2

Succinyl-CoAcontent

14 * 228 zt 232 * 235 * 129 * 2

Ratioacetyl-coA/succmylLCoA

4.11.71.31.31.6

% PvruvateReductionotNAD(P) utilizationnmollminlmgprotein

51 11 * 135 34 i 123 46 i 115 53 3 0.210 50 * 1

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a370TABLE VI

Relation between flux through citrate synthase , oxidation-reduction state of NAD(P), and mitochondrial acetyl-CoAlsuccinyl-CoA ratio, duringacetylcarnitine oxidation by mitochondria in state 3, state 4, arzd interme diate states

In each of five separate incubations, 0.19 ml of mitochondrial experiment shown in line 5, ADP was also added to 1 mM, in place ofsuspension (protein = 20 ma/ml) was added to 5 ml of 0,.saturated ATP. Duplicate samples were taken from each incubation after 6 min.basal medium containing 5 mM acetyl-DL-carnitine, 0.5 mM L-malate, The rate of 0, uptake corresponding to the conditions of each10 mM glucose, 0.5 mM ATP , and 0.1 mM M&l,. lmmediately incubation was determined separately, using an 0, electrode and anthereafter, the amount of hexokinase indicated was added. In the air-saturated medium.

HexokinasepresentRatio Rate Ratio Ratlo xATPI OfO, C0A Acety-CoA Succinyl-CoA acetyl-GA/ COAI Reduction ProductionADP consumption content content content of z-oxo-succiny-CoA succinyl-C0A iYAoDf(P) glutarate

unitslml pg-atomsOJminlmgprotein 70 total CoAnmollninlmgprotein

1.12.24.38.6 +

added ADP

128 0.034 5.2 zt 0.4 75 * 1 20 t 1 3.75 0.26 73 + 55 1.1110 0.059 10 * 1 66 * 1 23 * 0.05 2.84 0.44 47 1.2103 0.086 21 * 2 49 * 1 31 * 1 1.60 0.67 37 1.274 0.117 25 + 2 23 * 3 52 * 1 0.45 0.49 21 1.20.53 0.162 51 r 0.2 24 + 0.4 26 zt 0.2 0.91 2.0 15 1.6

TABLE VIIRelation between flux through citrate synthase . oxidation-reduction state of NAD(P), and mitochondrial acetyl-CoA/succinyl-CoA ratio, during

oxidation of pyruuate plus 2.oxoglutarate by mitochond ria in state 3, state 4, and intermediate statesIn each of 5 incubations, 0.3 ml of mitochondrial suspension (protein each incubation, to activate respiration. Duplicate samples were taken

= 22.3 mg/ml) was added to 5 ml of 0,.saturated basal medium after 8 min (incubation shown on line 1) and after 7 min (line 2), 6 mincontaining 0.55 mM PyrUvate, 1 mM 2-OXOglUtarate, 0.5 mM L-malate, (line 31, and 5 min (lines 4 and 5) of activated respiration. Pyruvate10 mM glucose, 0.5 mM ATP , and 0.1 mM M&l,. After 1 min, the utilization was estimated as described in Table V. The whole mito-amount of hexokinase shown (dialyzed to remove NH,) was added to chondrial suspension had been depleted of endogenous substrate.

H&-K-kinasepresent ADPRateOfO,consumption

CoA Acetyl-CoAcontent contentSucciny- RatioCoA acetyl-CONcontent succinyl-CoA

RatioCOAIsuccinyl-CoAPyruvateutilization

units/ml

4.6122146

553322

4.2

pg-atomsOdminlmgprotem0.0360.0970.2170.3440.398

9% total CoA12 * 0.2 34 * 0.2

8.4 + 1.5 30 i 113 * 1 28 + 312 * 1 29 * 315 * 0.3 38 + 1

Table VII used both 2-oxoglutarate and pyruvate as substrate,and the depletion of the latter is taken as an index of fluxthrough citrate synthase. It is seen that the presence of2-oxoglutarate did indeed generate a succinyl-CoA contentgreater than that found when pyruvate alone was the substrate(Table V). However, as the ATP/ADP ratio was decreased frommore than 55 to 22, the increased flux through citrate synthasewas not matched by an increase in the ratio acety-CoA/succi-nyl-CoA, indeed the latter declined. Thus, the results of thisexperiment provide no support for the concept o f the control ofcitrate synthase by the ratio acetyl-CoA/succinyl-CoA, evenwhen the stage is set for such a control by the generation ofhigh succinyl-Coil concentrations. Instead, all of the resultspresented are consistent with control by oxalacetate availabil-ity , as measured by the oxidation-reduction state of NAD(P)H,at a constant added malate concentration.

Steady State Concentrations of Mitochondrial Citrate, Zso-citrate, NAD+, NADH, NADP+, and NADPH during Oxida-tion of Palmitoylcarnitine Plus L-Ma&e and Pyruvate PlusL-Ma&e-Fig. 8 presents data from two experiments in whichintramitochondrial citrate and isocitrate content were deter-mined by centrifugation and subtraction as described underExperimental Procedure. The respiratory substrate was

nmollminlmgprotem54 * 0.2 0.64 0.23 50 6.5 i 0.562 i 0.4 0.49 0.14 34 15 .i 0.259 * 3 0.47 0.23 25 28 i 060 + 2 0.48 0.20 17 39 i 147 * 1 0.81 0.31 10 36 i 0.4

palmitoyl-L-carnitine plus L-malate. It is seen that on transi-tion from state 4 to state 3 the intramitochondrial isocitratecontent decreased, although there was a slight increase inintramitochondrial citra te. Furthermore, when ADP phospho-rylation was completed, the initial change in isocitrate contentwas reversed and there was a concomitant increase in theintramitochondrial citrate content. The difference in in-tramitochondrial isocitrate content between state 4 and state 3was shown to be statis tically significant and demonstrates acontrol of isocitrate dehydrogenase act ivi ty during state 4respiration. The reason for the failure of citrate content tofollow exact ly the changes in isocitrate content is not clear butmay be related to an overall increase in intramitochondrialtricarboxylate cycle intermediates coupled with an inability tomaintain strict equilibrium at the aconitase reaction. Such anincrease in tricarboxylate cycle intermediates is seen moreclearly in the experiment of Fig. 9 where pyruvate and L-malateare the respiratory substrates. Although more than 95% of the2-oxoglutarate was found to be external to the mitochondria,the rise in total 2-oxoglutarate content during the state 4 ---t 3transition and its partial reversal in the subsequent state 3 + 4transition (Fig. 8) is probably indicative of changes in theintramitochondrial concentration of 2-oxoglutarate. This can

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8371

0.1 f ig-ATOM/ML)

/ I I0 1 2 3 4 5 6MlNCiTES

FIG. 8. The mitochondrial content of citrate and isocitrate and theoxidation-reduction state of mitochondrial NAD(P) during state 4 + 3+ 4 transitions with palmitoylcarnitine (Pm. Cn.) plus L-malate assubstrate. Mitochondria were added to 19 ml of an air-saturated basalmedium containing in addition 10 pM palmitoy-L-carnitine and 0.5mM L-malate to give a final protein concentration of 1.25 n&ml. Whereindicated on the oxygen electrode trace (A), samples were removed andextracted as described under Experimental Procedure; ADP wasadded to 0.96 rn~ and palmitoylcarnitine was added a second time toavoid substrate depletion, giving an increment of 8.2 k~. The values forsample contents shown in the accompanying table are nanomoles/mgof mitochondrial protein and are means from duplicate experiments.B, fluorimeter trace of intramitochondrial NAD(P)H fluorescenceobtained by using 15 FM palmitoylcarnitine but otherwise the condi-t ions of (A) in a 2.ml system. FCCP, added to a concentration of 2.5be readily apprecia ted if 2-oxoglutarate leaves the matrixspace in exchange for L-malate, as in other mam malianmitochondria (34,35) because the external L-malate concentra-tion is relatively high (20) and not subject to sudden change.The fluorimeter trace (Fig. 8B) derived from intramitochon-drial NAD(P)H fluorescence showed a cycle of NAD(P)Hoxidation during state 3 as found by others (15, 18, 36).However, when, in experiments of similar design, the nicotina-mide nucleotides were extracted from the mitochondria andmeasured enzymically, it was found (Table VIII) that NADPH,as well as NADH, was oxidized on going from state 4 + 3. Thiscontrasts with the findings of LaNoue et al. (18) and isdiscusse d later in connection with the activity of NADP-iso-citrate dehydrogenase.

A repetition of these experiments with pyruvate plus L-malate as substrate (Fig. 9) revealed a more pronouncedincrease in citrate content throughout the experiment, but thesame, statistically significant, decrease in isocitrate content.The pattern of change in 2-oxoglutarate content (Fig. 9) and inoxidation-reduction state of NAD and NADP (Table VIII) wassimilar to that obtained for palmitoylcarnitine oxidation butthe 2-oxoglutarate conte nts were greater an d both nicoti na-mide nucleotides were somewhat more reduced in state 3 withpyruvate as substrate. This information is consistent with thehigher rates of oxygen con sumption obtained with pyruvate

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

Sample1 2 3 4Citrate Total 4.91 5.98 6.55 9.06

Mitochondrial 1.35 1.59 1.51 2.83Isocitrate Total 0.56 0.43 0.48 0.59

Mitochondrial 0.16 0.05 0.04 0.17Oxoglutarate Total 0.59 2.73 3.53 2.23

u State 4 (samples 1 and 4) differs significantly from state 3 (samples2 and 3), p < 0.005.(mean state 3 rate wit h pyruvate = 0.346 * 0.032 (n = 12) pg-atoms of O,/min/mg of mitochondrial protein; mean state 3rate with palmitoylcarnitine = 0.164 i 0.011 (n = 12) wg-atoms of O,/min/mg of mitochondrial protein). It is noted that25% or more of the citrate and isocitrate in the total mito-chondrial suspension was found to be intramitochondrial (Figs.8 and 9, state 4). Assum ing a matrix space of 1 pl/mg ofprotein, this means that the intramitochondrial concentrationof these ions is 250 times greater than that in the suspendingmedium. This indicates an extremely low mitochondrialpermeability to citrate and isocitrate in rabbit heart and isconsistent with other reports of limitingly low tricarboxylatecarrier activity in rat heart (37, 38). It is suggested that thedecreased mitochondrial isocitrate content seen in state 4 + 3transitions (Figs. 8 and 9) does not reflect an increased trans-port of isocitrate out of the mitochondria, as the totalsuspension content also appears to be slightly decreased.

These results (Figs. 8 and 9) demonstrate an activation atthe level of isocitrate dehydrogenase during the state 4 + 3transition. As such they are in agreement with a conclusio nreached by LaNoue et al. (18) about control at this locus.However, it should be noted that mitochondrial citrate, but notisocitrate, was measured in the previous work and it seemsfrom Figs. 8 and 9 that it is wrong to assume that aconitasemaintains equilibrium under all conditions. Indeed, in the

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/OXYGEN (APPROX 0.4 pg-ATOM/ML)

I81 2 3 4 5 6 7MIN U TES

FIG. 9. The mitochondrial content o f citrate and isocitrate and theoxidation-reduction state of mitochondrial NAD(P) during state 4 + 3+ 4 transitions with pyruva te plus L-malate as substrate. Mitochondriawere added to 19 ml of an 0,.saturated basal medium containing inaddition 2.5 rnM pyruva te and 0.5 m M L-malate to give a final proteinconcentration of 0.96 mg/ml. Where indicated on the oxygen electrodetrace (A) samples were removed and extracted as described underExperimental Procedure, and ADP was added to a concentrat ion of3.2 m M . The values for sample contents shown in the accompanyingtable are nanomolesimg of mitochondrial protein and are means fromduplicate experiments. B, fluorimeter trace of intramitochondrialNAD(P)H fluorescence obtained by using 2 ml of an air-saturatedbasal medium and 0.5 m M ADP, but otherwise the conditions of (A).FCCP, added to a concentration of 2.5 pM, was used to establishanaerobiosis. A dif ferent mitochondrial preparation was used to obtainpresent work, mitochondrial citrate increases from state 4 to 3.Nevertheless, the data on isocitrate content (Figs. 8 and 9) leadto the conclusion reached in the earlier work (18). LaNoue etal. showed that the total citrate formed during the oxidation ofpyruvate plus L-malate by heart mitochondria was less in thepresence of oligomycin and ADP than in state 4, which wouldindicate a response of isocitrate dehydrogenase to intramito-chondrial adenine nucleotide energy charge. They also showed,however, that flux through this enzyme was much greater inthe presence of oligomycin plus the uncoupling agent FCCPthan in state 4 respiration, indicating a sens itiv ity of isocitratedehydrogenase to the oxidation-reduction state of nicotina-mide nucleotide. These respiratory states diff er in that bothshow a high adenine nucleotide energy charge, but the uncou-pled state shows much less nicotinamide nucleotide reduction.An attempt to determine the relative importance of nicotina-mide nucleotide oxidation-reduction state and adenine nucleo-tide energy charge in the control of isocitrate dehydrogenase inthe mitochondrion is shown in Figs. 10 and 11. It is seen (Fig.10) that addition of oligomycin plus ADP to mitochondriaoxidizing pyruvate plus L-malate in state 4 results in no changein intramitochondrial isocitrate content, whereas the subse-quent addition of the uncoupling agent FCCP gives rise to astatis tically significant decrease in isocitrate content. Whenthe sequence in which ADP and FCCP are added is reversed

:B )

FLUORESCENCEINCREASE

this trace, and the temperature was 22

CitrateSample1 2 3 4

Total 4.85 10.4 12.6 19.2Mitochondrial 1.34 4.91 5.74 8.22Isocitrate Total 0.66 0.48 0.53 1.38Mitochondrial 0.22 0.04 0.06 0.49Oroglutarate Total 3.8 16.7 23.5 18.7

a State 4 (samples 1 and 4) diff ers signi ficantly from state 3 (samples2 and 3), < 0.01.

(Fig. 11) it is seen that FCCP gives a decrease in intramito-chondrial isocitrate content and the subsequent addition ofADP gives rise to no further change. Inspection of the fluorime-ter traces of Figs. 10 and 11, showing the high NAD(P)H/NAD(P)+ ratio in the presence of oligomycin plus ADP, andlow ratio in the presence of FCCP, then allows the statementthat under conditions of both high and low energy charge theoverriding influence on isocitrate dehydrogenase act ivi ty is theoxidation-reduction state of nicotinamide nucleotide.

The demonstration of control at the isocitrate dehydrogenasereaction in experiments involving isolated mitochondria (Figs.8 and 9) is in agreement with the finding that increasing thework load of the isolated, perfused heart decreases the heartisocitrate content, when the substrate is either glucose orglucose plus palmitate (1). The question of the significance ofcontrol at this locus arises. An important point appears to bethe inhibition of citrate synthase by citrate, which is competi-tive with oxalacetate (5). All of the thioester studies reportedhere tend to minimize the control of citrate synthase byacetyl-CoA availabil ity, and therefore implicate control by theavailability of oxalacetate. This control would be tightened bythe accumulation of citrate in state 4, as suggested by LaNoueet al. (4). In this context, the control of isocitrate oxidationbecomes important.

Pathway of Isocitrate Oxidation-There has been debate

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TABLE VIIIMitochondrial content ofnicotimmide nucleotides during state 4 - 3

+ 4 transitions with pyruuate plus L-malate and withpalmitoylcarnitine plus L-malate as substrate

Mitochondria were added to 20 ml of an air-saturated basal mediumcontaining in addition either 2.5 mM pyruvate and 0.5 mM L-malate or50 pM palmitoyl-L-carnitine, 5 mg of bovine serum albumin/m l, and 0.5mM L-malate to give a final protein concentration which variedbetween 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 whichwas expelled into 1.3 ml of 16% (v/v) HClO,, the other being dischargedinto 1 ml of 1 M KOH in ethanol. ADP (0.9 IIIM) was added 0.5 min laterand two more double samples (samples 2 and 3) were removed foranalysis during the phase of state 3 respiration. When state 4respiration had been resumed for at least 1 min, a fourth double sample(sample 4) was removed and treated as before. The values in thetable are from duplicate experiments using different mitochondrialpreparations. Total NAD = 5.78 + 0.47 (16) nmol/mg; total NADP =1.55 + 0.19 (16) nmol/mg.

Reduction (70)Substrate Sample Samples S.3mple1 2and3 4*

Pyruvate NAD 59.5 * 0.5 (2) 15.3 + 2.5 (4) 42 + 3 (2)bplus L-malate NADP 98.5 + 1.5 (2) 92.3 i 2.0 (4) 98 i 2 (2)

Palmitoylcar- NAD 58 * 2 (2) 5.5 * 0.3 (4) 40 l 7 (2)bnitine plusL-malate NADP 97 * 0 (2) 77.8 sz 1.1 (4) 96 + l(2)State 4 (samples 1 and 4) differs significantly from state 3 (samples

2 and 3).*p

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8374

MINUTES

FIG. 11. The effect of the addition of oligomycin plus uncouplingagent, and of the subsequent addition of ADP, upon the mitochondrialcontent of citrate and isocitrate. Mitochondria were added to 21 ml ofan air-saturated basal medium containing in addition 2.5 mM pyru-vate, 0.5 mM L-malate, and 0.5 mM ATP to give a final proteinconcentration of 0.8 mg/ml. Where indicated on the oxygen electrodetrace (A), sam ples were removed and extracted as describ ed underExperimental Procedure; additions to the following concentrationswere 2.4 pg of oligomycin/ml (oligo), 1.5 pM FCCP, and 3.7 mM ADP.The values for sample co ntents shown in the accompanying table arenanomoles/mg of mitochondrial protein and are means from duplicateexperiments. B, fluorimeter trace of intramitochondrial NAD(P)Hfluorescence obtained by using the conditions of (A) in a 2-ml system. A

o-------~ 20 40 60 80 100NADP REDUCT ION (%)

FIG. 12. The dependence of the activity of NADP-isocitrate dehydro-genase upon the degree of reduction of NADP+. Mitochondria (0.17 mgof protein) were added to 2.5 ml of a medium comprising 50 mM KP,, 10mM MgCl,, 1 mM dithio threito l, 0.5 rg of rotenone/m l, 0.025% (v/v)Triton X-100, and varied amounts of NADP+ and NADPH to give acombined concentration of 130 pM. The final pH was 7.2 and thetemperature was 25. A mixture of 2.5 m M citrate and 0.1 m Mthree-D,-isocitrate was added to start the reaction and initial rates ofNADP+ reduction were measured spectrophotometrically at 340 nm.would appear that the act ivi ty recovered is not suffi cient toaccount for flux through this reaction of at least 70 nmol/min/mg of protein obtained during state 3 pyruvate oxidation.The V,,, of NADP-isocitrate dehydrogenase was found to be450 nmol/min/mg of mitochondrial protein, th K, for threo-n,-isocitrate to be 5 PM and the K, for NADP 2 pM. Whenenzymic act ivi ty was studied as a function of the percentagereduction of NADP, it was found (Fig. 12) that the act ivi ty isapproximately 40% of V,,, at 92% NADP reduction, and 65%of vmx at 78% reduction. These are the oxidation-reductionconditions found to apply during the state 3 oxidation of

A TP

FLUORESCENCEINCREASE

2 IllIll.

ADP

different mitochondrial preparation was used to obtain t his trace andthe temperature was 22.

Sample1 2 3 4 5Citrate Total 7.49 9.45 10.2 10.9 11.3Mitochondrial 2.86 2.15 1.86 2.29 2.46

Isocitrate Total 0.88 0.66 0.73 0.68 0.75Mitochondrial 0.30 0.03 0.07 0.04 0.110p < 0.005 for olig o + FCCP (2 and 3) uersus state 4 (1).

pyruva te and palmitoylcarnitine, respect ively (Table VIII).Thus, NADP-isocitrate dehydrogenase may make an apprecia-ble contribution to the oxidation of isocitrate, with thelimitation that any such contribution is contingent uponnicotinamide nucleotide transhydrogenase act ivi ty (EC1.6.1.1). Measurements of transhydrogenase in submitochon-drial particles derived by sonication gave rates of NADPHoxidation of 36 nmol/min/mg of mitochondrial protein in thepresence of oligomycin and FCCP, and 25 nmol/min/mg ofmitochondrial protein in particles energized by succinateoxidation in the presence of oligomycin (251~). These figures arenot unlike those determined in intact rat liver mitochondria(39) although the heart particle enzyme did not show theextreme inhibition during coupled respiration demonstrated inintact mitochondria (39). It seems probable that this was anartifact of the particle preparation and that the transhydrogen-ase is extens ively inhibited under physiological conditions.This is made the more likely by further experiments whichshowed that palmitoyl-CoA was a potent inhibitor of transhy-drogenase act ivi ty, as reported for the bovine heart enzyme(42), 50% inhibition being achieved with less than 1 j.tMpalmitoyl-CoA. Because palmitoyl-CoA competes withNADP(H) (42), the relative concentrations of these specieswithin the mitochondrial matrix become important; it isunlikely that NADPH is present at more than 20 times theconcentration used in the assay (i.e. 0.1 mM) but the corre-sponding palmitoyl-CoA concentration could be as much as1000 times that used in the assay, depending on how much o f itis free in solution. Therefore, it becomes a real possibility thatthe transhydrogenase act ivi ty in uiuo is limitingly low and thatflux through the NADP-isocitrate dehydrogenase is limitedcorrespondingly. Given a high act ivi ty of the NADP-isocitrate

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8375dehydrogenase coupled with a low, transhydrogenase-limitedflux through this enzyme, it may be concluded that during themetabolic transitions performed in this work the NADP-isocit-rate dehydrogenase reaction was essentially at equilibrium.This would then account for the decreased NADPH contentduring state 3 as a consequence of the decreased isocitratecontent (Figs. 8 and 9). If this supposition is correct, then theNAD-isocitrate dehydrogenase reaction is suff iciently removedfrom equilibrium in state 4 to exert its postulated regulatoryrole.

Acknowledgments-We would like to recognize the skilledtechnical help of Miss Ono Lescure and frui tful discussionswith Dr. Bertram Sacktor throughout the work.

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