Effect of Phosphate and Uncouplers on Substrate Transport And

Plant Physiol. (1977) 59, 139-144

Effect of Phosphate and Uncouplers on Substrate Transport and Oxidation by Isolated Corn Mitochondria1 Received for publication May 25, 1976 and in revised form September 13, 1976

DAVID A. DAY2 AND JOHN B. HANSON Department of Botany, University of Illinois, Urbana, Illinois 61801

ABSTRACT ADP) stimulates the rate by increasing transport (3, 25, 27). However, Pi stimulation of exogenous NADH oxidation by corn A study was made to determine conditions under which malate oxida- mitochondria has also been reported (9). Oxidation of this tion rates in corn (Zea mays L.) mitochondria are limited by transport substrate does not require its penetration of the inner membrane processes. In the absence of added ADP, inorganic phosphate increased (3, 6) and the Pi stimulation was attributed to an accelerated malate oxidation rates by processes inhibited by mersalyl and oligomy- turnover of the coupling mechanism, since it was sensitive to cin, but phosphate did not stimulate uncoupled respiration. However, oligomycin (9). Oligomycin did not inhibit Pi stimulation of the uncoupled oxidation rates were inhibited by butylmalonate and malate oxidation by cauliflower mitochondria (27). mersalyl. When uncoupler was added prior to substrate, subsequent 02 Inhibition of substrate transport by uncouplers has been ob- uptake rates were reduced when malate and succinate, but not exoge- served with animal mitochondria (23), but the low rates of nous NADH, were used. Uncoupler and butylmalonate also inhibited substrate oxidation by plant mitochondria in the presence of swelling in malate solutions and malate accumulation by these mitochon- uncouplers have generally been attributed to a requirement for dria, which were found to have a high endogenous phosphate content. exogenous adenylates (12, 15, 26). Addition of uncoupler after malate or succinate produced an initial rapid The experiments reported here were carried out to determine oxidation which declined as the mitochondris lost solute and contracted. the degree to which malate and succinate oxidation by corn This decline was not affected by addition of ADP or AMP, and was not mitochondria were dependent on Pi-linked transport. The re- observed when exogenous NADH was substrate. Increasing K+ permea- sults confirm that substrate anion transport is energy-linked and bility with valinomycin increased the P-trifluoromethoxy (carboxylcya- dependent on a PMF, since uncouplers inhibit it. The slow nide)phenyl hydrazone inhibition. Kinetic studies showed the slow rate respiration of uncoupled mitochondria appears to be due largely of malate oxidation in the presence of uncoupler to be characterized by a to diffusive entry of substrate. Operation of the dicarboxylate/Pi high Km and a low Vmax, probably reflecting a diffusion-limited process. antiporter is required for maximum transport and oxidation The results indicate that rapid malate and succinate oxidation require rates, but endogenous Pi levels are sufficient to support these the operation of both the phosphate and dicarboxylate transporters, processes in acceptorless respiration. Stimulation of initial mal- which in turn depend on maintenance of a proton motive force across the ate oxidation by exogenous Pi seems to be principally due to Pi inner membrane. In addition, phosphate can stimulate acceptorless mal- interaction with the coupling mechanism. Adenine nucleotides ate oxidation by reaction with the coupling mechanism, and in uncoupled did not affect substrate transport or oxidation per se, with the mitochondria which are depleted of substrate there is a slow rate of exception of the well known ATP activation of succinic dehydro- oxidation which appears to be limited by diffusive entry. genase (20).

MATERIALS AND METHODS Mitochondria were isolated from 3-day-old etiolated corn shoots essentially as described by Hanson (7) except that TES Exchange carriers for the transport of substrate anions exist in buffer (pH 7.5) replaced KH2PO4 and 0.1% BSA was included the inner membrane of animal (2) and plant (22, 27) mitochon- in the grinding medium. A careful comparison was made of this dria. Dicarboxylate anions, such as malate and succinate, enter method of isolation with the density gradient method of Douce et in exchange for Pi via a carrier which can be inhibited by al. (5). No important difference could be detected between the substrate analogues such as 2-n-butylmalonate (22, 24, 27); Pi preparations with respect to malate oxidation rates, respiratory enters in exchange for OH- driven outward by a PMF3 which is control and ADP/O ratios, purity and intactness in electron produced by substrate oxidation (4, 9, 18). Thus, the transport micrographs, and passive swelling in KCI and (NH4)3PO,. Both of dicarboxylate anions into mitochondria is dependent on respi- preparations had very little succinate-Cyt c reductase activity ration-linked proton efflux coupled to Pi/OH- exchange, fol- until treated with detergent. lowed by substrate/Pi exchange; potassium ions also enter to Oxygen consumption and per cent transmission were mea- preserve electrical neutrality (see refs. 2 and 8). sured simultaneously as described previously (7) with full scale When substrate transport to the matrix limits the respiration transmittance set from 20 to 40%. Changes in light transmit- rate of isolated mitochondria, addition of Pi (in the absence of tance can be used to estimate swelling and shrinkage upon movement of osmotically active solutes (14, 21). The standard ' This research was supported by the United States Energy Research reaction medium consisted of 0.25 M sucrose, 10 mM TES and Development Administration Grant E(1 1-1)-790. 5 2 Present address: Department of Biology, University of California, buffer, mM MgCl2, and 0.1% BSA, adjusted to pH 7.2 with Los Angeles, Calif. 90024. NaOH. Mitochondrial protein was approximately 1.5 mg/vessel. 3Abbreviations used: PMF: proton motive force; FCCP: P-trifluoro- Swelling was also monitored by following A changes at 520 nm methoxy(carboxylcyanide) phenyl hydrazone; mers: mersalyl; BM: bu- in a Hitachi model 100-10 spectrophotometer. tymalonate; mal: malate. Mitochondrial protein was estimated by the method of Lowry 139 140 DAY AND HANSON Plant Physiol. Vol. 59, 1977 et al. (16) using BSA (fraction V) as the standard. A The Pi content of the mitochondria was estimated from perchloric acid extracts according to the method of Marsh (17). Mitochondria (0.1 ml) were added to 2 ml of 5.5% cold perchloric acid and allowed to stand for 10 min at 0 C. Precipitated protein was removed by centrifugation and the supernatant ana- lyzed for Pi. Malate dehydrogenase activity was measured spec- trophotometrically by following the oxidation of NADH at 340 B nm, in the presence of oxaloacetate (19). The malate content of mitochondria was measured enzymi- cally according to the method of Hohorst (11). Mitochondria o%Tjdigomycin (about 2 mg) were allowed to accumulate malate for 3 min at 25 C in 3 ml of standard reaction medium containing 0.4 ,umol NADH and 30 AM rotenone. Other additions were made as given in Table IV. The reaction was terminated by centrifugation Pi in a Sorvall RC2 centrifuge at 30,000g for 2 min; the superna- C 20 mers tant was decanted and the pellet rinsed with distilled H20. The 38 l oligomycin mitochondria were resuspended in 1.5 ml of 6% perchloric acid and held in ice for 5 min. Precipitated protein was removed by Mai~ ~ ~~~~m centrifugation and the supernatant neutralized with KOH prior to assaying. Controls were run with 5 AM antimycin A in the incubation medium, which blocks NADH oxidation and thus PiA provides a '"blank" estimate of passive malate absorption. 2-n-Butylmalonate was a generous gift from J. T. Wiskich 2%T 0.1IfsAmole 02 (Botany Dept., Adelaide University, Adelaide, South Aus- Imin. tralia); other chemicals were obtained from Sigma Chemical Co. FIG. 1. Effect of phosphate, mersalyl, and oligomycin on malate (St. Louis, Mo.). oxidation. Oxygen uptake and swelling were monitored as described under "Materials and Methods." Mitochondria (2.1 mg protein) were added to 4.5 ml of standard reaction medium and allowed to equilibrate RESULTS for 2 min. Other additions as indicated were: (A): 5 mM malate, 5 mM mm Pi, and 0.8,Mmol ADP; (B): 5 mm malate, 5 mm Pi, and 6,ug oligomy- Effect of Pi and Transport Inhibitors. The addition of 5 (C): malate, in the absence of Pi and ADP, cin; 5 mm 5 mm Pi, 25,UM mersalyl, and 6 ug oligomycin. malate to corn mitochondria, Rates are expressed as nmol 02 min-' mg protein-'. Glutamate (5 mM) initiated a slow rate of respiration associated with a small degree was included in the reaction medium. of swelling (Fig. 1); subsequent addition of Pi stimulated the rate of 02 consumption and resulted in more extensive swelling of the A mitochondria (Fig. LA). Similar results have been observed with cauliflower mitochondria (27). The stimulated 02 uptake was partially inhibited by mersalyl (Fig. IC) and more severely by 38 ADP oligomycin (Fig. 1, B and C). Identical effects have been observed with mitochondria oxidizing exogenous NADH, and sug- 127 gest that Pi stimulation of acceptorless respiration involves both Pi Pi transport and its interaction with the coupling mechanism, as molote discussed previously (9). The latter effect may involve interac- tions with endogenous, bound nucleotides. Addition of oligomycin also produced an increase in swelling (Fig. 1, B and C); this 2%T 0.11,umole 02 could have been due to the prevention of proton 'leakage" back B ( - ) butylmolonate if through the ATPase (18), thus establishing a greater PMF to min drive solute uptake. 20 20 Butylmalonate, a competitive inhibitor of dicarboxylate/Pi exchange, did not inhibit initial malate oxidation or the Pi 38 stimulation, although BM did partially inhibit swelling with and without Pi (Fig. 2). In contrast, state 3 respiration with malate P67 was inhibited by equimolar BM up to 50% (Fig. 2). If malate molate transport is via a malate/Pi antiport, there must be sufficient .~~~~~~~~~~~~~~~ADP endogenous Pi and antiport activity to sustain the low level of acceptorless respiration without added Pi and in the presence of FIG. 2. Effect of butylmalonate and phosphate on malate oxidation. BM. Transport does not seem to limit initial malate oxidation Assay conditions were the same as those in Figure 1, except that 1.5 mg rates in corn mitochondria. However, during ATP synthesis mitochondrial protein was used and 5 mm 2-n-butylmalonate was in- when the energy demand is high, BM can limit respiration by cluded in the reaction medium in B. 02 uptake is expressed as limiting transport. nmol - min-'* mg protein-'. Jung and Hanson (13) reported the endogenous Pi content of corn mitochondria to be 19 to 21 nmol/mg protein. We found Pi rapid rates of oxidation independent of the presence of Pi (Table content to fall in the range of 25 to 35 nmol/mg protein. Assum- I, A and B). However, if FCCP were added initially, uncoupled ing a matrix volume of about 2 Al/mg protein (14), a minimum oxidation rates were much inhibited (Table IC). Also, if BM or concentration of 10 mm endogenous Pi should be available for mersalyl were added initially, maximum uncoupled rates were malate/Pi exchange.- not obtained (TableI, D and E). No such inhibition of uncou- High respiration rates can also be secured by uncoupling. If pled rates was obtained with NADH, a substrate which is not the mitochondria were allowed to accumulate malate or succi- transported (data not shown). Neither was state 3 respiration nate before the addition of uncoupler, FCCP initially produced with NADH affected by BM (TableII). There was no effect of Plant Physiol. Vol. 59, 1977 MITOCHONDRIAL TRANS PORT AND OXIDATION 141 BM on malic dehydrogenase (Table Il). Collectively, the evi- III) and active swelling (Fig. 4). In both cases, energy was dence indicates that malate and succinate oxidation rates can be supplied by NADH oxidation with 20 ,UM rotenone to block limited by substrate transport under conditions of high energy malate oxidation; this allows study of malate transport uncon- demand, as with state 3 respiration or uncoupling. Under these founded by variations in malate supply as the source of energy. conditions, collapse of the PMF by uncoupling, inhibition of Pi Active malate accumulation of about 10 nmol/mg protein transport by mersalyl, and inhibition of the dicarboxylate/Pi occurred in the absence of exogenous Pi (Table III), an amount exchange by BM will lower substrate uptake and respiration. well within the 25 to 35 nmol of endogenous Pi available for The oxidation rate of nontransported NADH is not subject to exchange (some malate may have leaked out during reisolation). these limitations. However, exogenous Pi doubled the malate accumulation, indi- Uncoupling and Transport. Kinetic data on malate oxidation under state 3 and uncoupled conditions are shown in Figure 3. 0.1 / Uncoupling was with FCCP added before malate, thus collapsing any PMF prior to malate availability for transport. The apparent Km for malate rose from 0.25 mm in state 3 to 3.6 mm with uncoupling; the respective Vmax were 125 and 41.5 nmol 02/ min. If it is assumed that FCCP completely collapses the PMF, the malate oxidized in uncoupled mitochondria must be entering by diffusion, and the change in kinetics reflects the introduction 0.06 of malate as entry the rate-limiting process. (Malate transport I/v I may also limit state 3 oxidation, but this is difficult to determine.) It is not known if the passive penetration of malate is due 0.04 to an inherent property of the inner membrane, some change associated with mitochondria isolation, or an increased permea- bility introduced by uncoupling. More direct determination of the effect of uncoupling on 0.02 malate transport was made by analysis for malate content (Table

Table I. The effect of FCCP, mersalyl and butylmalonate on malate and succinate oxidation. 0 2 3 4 5 Oxygen consumption was measured as described in Materials and Methods. Final concentrations were: 5 mM malate, 5 mM succinate, 5 mM 7/ [malate] mM- KH2POA, 2 FCCP, 5 mM butylmalonate and wM 25 PM mersalyl. Glutamate FIG. 3. Effect of on (5 MlJ was also present when malate was substrate, and ATP (0.5 imole) FCCP the kinetics of malate oxidation. Mito- and oligomycin (1 ug/ml) when succinate was used. chondria (1.5 mg protein) were added to 4.5 ml standard reaction medium containing 5 mm glutamate, 5 mm Pi, and either 3 ADP Initial Rate Of 02 Amol Consumption (o *) or 2 uM FCCP (0 0). Malate, at varying concentrations, Order of Addition Malate Succinate was added to start the reaction, and 02 consumption was measured as described under "Materials and Methods." nmoles/min*mg protein A. Substrate 22 43 Table III. Malate accumulation by corn mitochondria. P. 34 65 FCP 140 135 Malate was estimated as described in Materials and Methods. The malate content of the antimycin A 'blank' was 18.5 (±6.9) nmoles- B. Substrate 22 44 mg protein-1 and this was subtracted from the other values to give those shown. FCCP 140 115 Averages from 5 determinations ± standard deviation. C. FCCP Assay Conditions Malate Content Substrate 46 88 2-n-butylmalonate 44 70 nmoles-mg prot1 D. 2-n-butylmalonate 2 mM malate 9.84 Substrate 18 38 (±1.29) FCCP 46 72 2 mM malate + 3 mM Pi 19.64 (±1.89) E. Mersalyl 2 mM malate, 3 mM + 2 uM FCCP Substrate 22 -- P1 1.2 (±0.8) FCCP 88 2 mM malate, 3 mM Pi + 3 mM butylmalonate 4.2 (±1.7)

Table II. The effect of butylmalonate on malate, succinate and NADH oxidation. Assay conditions are described in Materials and Methods, except KH PO4 (5mM) and ADP (1.5 ,mole) were also present. Final substrate concentrations were: 5 mM malate, 5 mM succinate and 1 mM NADH. Oxygen uptake is expressed as nmoles-min-l-mg-1 and malate dehydrogenase activity as moles NADH min~1 mg-1. For the latter assay, the mitochondria were disrupted with 0.05% sodium deoxycholate. Assay Control Rate +5 mM butylmalonate

malate, State 3 02 uptake 145 88 succinate, State 3 02 uptake 121 65 NADH, State 3 02 uptake 282 285 MDH activity 14.18 14.71 142 DAY AND HANSON Plant Physiol. Vol. 59, 1977

A B chain (Fig. 5, A and C). The final uncoupled rate with malate as _ NADH malate substrate was approximately the same whether FCCP was added pi before or after malate (cf. Fig. 5, A and C), and was not relieved by ADP or AMP (Fig. 5A). The gradual inhibition upon FCCP malate addition was also observed when FCCP was added after the 101 mitochondria had undergone a state 3/state 4 cycle (Fig. 5B), but not when FCCP was added during exogenous NADH oxidation (Fig. 5E). State 3 rates with malate showed no long term + FCCP inhibition (Fig. SD). Inclusion of AMP in the reaction medium did not prevent the decrease in respiration (Table IV). mers When succinate was the substrate, ATP was needed to elicit maximum oxidation rates upon addition of FCCP (Fig. 6). + butylmalonal When ATP plus oligomycin was present, a decline in 02 uptake was Swel ling observed, similar to that found with malate as substrate. In the presence of oligomycin, ADP could not substitute for ATP (data not shown). The effect of ATP probably lies in its activation of succinate dehydrogenase (20). A Control Control 40 C NADH 109 FCCP FC+CP\ ADP %<, mulate ADPor AMP

48 NADH I0.01 OD520 mal 208 maol 2 min. C u 43

ADP\tOt FCCP FIG. 4. Swelling of corn mitochondria in malate solutions. Mitochon- E t 1 dria (about mg protein) were added to 3 ml standard reaction medium mal and absorbance monitored as described under "Materials and Methods." 2 Additions as indicated were: (A): 0.2 ,mol NADH, 1 mM Pi, 10 mM FCCI; malate; (B): 0.2 ,umol NADH, 10 mm malate, 1 mm Pi; (C): 0.2 ,umol NADH, 10 mm malate, 2 ,LM FCCP. Where indicated in A and B, 2 AM 95 2%T|O.l Iumole 02 FCCP, 5 ZiM antimycin A, 10 mm 2-n-butylmalonate or 25 Mm mersalyl 2min were included in the reaction medium. Rotenone (20 AM) was routinely added to the medium during swelling measurements. cating that the matrix pool must be resupplied by Pi transport to FIG. 5. Effect of FCCP and adenine nucleotides on malate and achieve maximum malate uptake. FCCP was 94% effective in NADH oxidations. Assay conditions were the same as those in Figure 1 inhibiting active malate accumulation, lending credence to the except that (a) 1.3 mg mitochondrial protein was used; (b) 5 mm Pi was above speculation that diffusion must be supplying most of the included in the reaction medium in all cases, and (c) 2 Mm FCCP was included in the medium in C. Other as malate oxidized by uncoupled mitochondria (Fig. 3). Butylma- additions indicated were, 2 Mm FCCP, 0.5 umol AMP, 1 mm NADH, 5 mM malate, and either 1 ,umol lonate gave 79% inhibition. (in A and B) or 5 ,umol (in D) ADP. 02 uptake is expressed as The swelling studies (Fig. 4) give a more dynamic picture. nmol * min-' - mg protein-'. Very little malate swelling occurs if respiration is blocked with or with Malate antimycin uncoupled FCCP (Fig. 4A). transport Table IV. The effect of uncouplers and adenine nucleotides on malate with only endogenous phosphate present was partially inhibited oxidation. by both BM and mersalyl (Fig. 4B), suggesting the participation Oxygen concumption was measured as described in Materials of endogenous phosphate in malate uptake. Addition of Pi and Methods. Final concentrations were: 5 mM malate, 5 mM KH2PO 2 vM FCCP, 5 mM arsenate (As), 1 iimole ADP, 1 imole AMP; 5 mM glutamate caused additional swelling, which was strongly inhibited by BM was included in the reaction medium. Numbers in parentheses represent and completely so by mersalyl (Fig. 4B). Uncoupling caused loss two separate experiments. of malate and shrinkage (Fig. 4C). Results similar to these were Additions to medium Initial Rate Final Rate Inhibition obtained when succinate replaced malate (data not shown). Although FCCP, when added after substrate, stimulated 02 nmoles 02 * min-1 * mg-i % uptake (Table I; Fig. SA), the initial uncoupled rate decreased A. malate, Pi, FCCP (1) 165 77 53 with time, the final rate being 40 to 50% of that initially ob- (2) 157 71 55 served (Fig. 5A). Concurrently, the mitochondria contracted, B. malate, As, ADP (1) 138 88 37 indicating a loss of solute from the matrix. This contraction was (2) 139 90 36 observed whether Pi was present or not (Fig. 4C). Addition of C. malate, AMP, Pi, FCCP(1) 154 77 50 exogenous NADH fully relieved the uncoupler-inhibited respi- (2) 201 86 57 ration, demonstrating no interference with the electron transport Plant Physiol. Vol. 59, 1977 MITOCHONDRIAL TRANSPORT AND OXIDATION 143 A Phosphate may stimulate malate (or succinate) oxidation in three ways. (a) By exchanging for hydroxyl ions, Pi will dissipate a portion of the PMF (providing K+ also enters) and hence stimulate 02 uptake and swelling. This stimulation is slight and is inhibited by mersalyl (Fig. 1C). (b) Once inside the matrix, Pi interacts with the coupling mechanism in some fashion and stimulates electron transport (9). This stimulation is sensitive to oligomycin (Fig. 1, B and C). (c) Pi in the matrix may exchange for external malate in dicarboxylate transport and thus increase substrate supply (Table III; Fig. 4). When the demand for substrate is high, respiration can be inhibited by n-butylmalonate (Fig. 2; Table II). If malate transport is limiting, then Pi stimulation is insensitive to oligomycin, and uncouplers have little effect on 02 uptake in the absence of ADP (27). When malate trans-

2%TJO.1 IMmole 02 port is not limiting (e.g. when endogenous Pi is high and energy B B t48 demand is low), Pi stimulation is sensitive to oligomycin and 4 2 min added Pi is not required for uncoupler release of electron transport. ~cc Uncouplers, such as FCCP, may inhibit substrate transport by 132 collapsing proton gradients. The effect of uncouplers on substrate oxidation will vary depending on the order of addition. Succ When FCCP is added before substrate, transport is inhibited due to the absence of a proton motive force, and thus 02 uptake is limited by the diffusive entry of substrate. When FCCP is added after substrate accumulation has occurred, there is a burst of uncoupled respiration, but the transporting systems and uncou- 65 pler compete for the available PMF and the level of substrate in the matrix declines (Fig. SA). Hence, substrate transport gradu- FIG. 6. Effect of ATP and FCCP on succinate oxidation. Assay ally becomes limiting to oxidation. When K+ movement is also conditions were as in Figure 1, except that 1.7 mg mitochondrial protein was used and 5 mm succinate, 2 AmM FCCP, and 0.5 umol ATP were added as indicated to the standard medium. In B, 0.5 umol ATP, 15 umM A B rotenone, and 6 lAg oligomycin were included in the medium. 02 con- - FCCP sumption is expressed as nmol -min- - mg protein-'. Arsenate uncoupling, which proceeds by normal transport and phosphorylation pathways (1), gave less final inhibition of malate oxidation than did FCCP (Table IV). However, initial uncoupled rates were not as great as with FCCP (Table IV), and the shrinkage was not as greal (data not shown), suggesting that PMF was not so thoroughly collapsed. The data show that the gradual decline in respiration is independent of the mechanism of uncoupling. Valinomycin, by facilitating operation of the K+/H+ antiporter vol of the inner membrane, acts as an uncoupler for corn mitochon- dnia (10). It was ineffective with mitochondria which had lost their solutes (K+, malate and Pi) due to uncoupling, and actually suppressed malate oxidation further (Fig. 7A). When added C prior to uncoupler, valinomycin caused a rapid influx of solute and attainment of high steady-state swelling; 02 uptake was also stimulated (Fig. 7C). Valinomycin stimulated swelling and respiration in a similar manner when exogenous NADH was substrate, but the respiratory increment was greater (Fig. 7B). Evidently, most of the increased swelling in Figure 7B was due to Pi influx. Addition of FCCP at this point resulted in a marked 2%TjO.I IMmole solute loss and a decline in respiratory rate when malate was substrate (Fig. 7C), but respiration increased when exogenous 2min. NADH was substrate (Fig. 7B). The inhibition of malate oxidation was probably caused by an efflux of malate from the matrix and an inhibition of uptake by destruction of the transmembrane PMF. Transmembrane equilibration of K+ and H+ when both FCCP and valinomycin were present would have caused a more rapid and greater decline in membrane gradients than when either substance was used alone.

DISCUSSION FIG. 7. Effect of valinomycin and FCCP on malate and NADH oxidation. Assay conditions were the same as those in Figure 1 except From the viewpoint of the chemiosmotic hypothesis as applied that 1.4 mg protein was used and 0.3 iLg valinomycin, 2 Mm FCCP, and to transport (see introductory section), the data presented here 0.3 ,umol NADH were added as shown. 02 uptake is expressed as suggest the following. nmol * min-' * mg protein-'. 144 DAY AND HANSON Plant Physiol. Vol. 59, 1977 facilitated, the collapse of the PMF is more pronounced and mitochondria. Plant Physiol. 56: 13-18. inhibition of 02 uptake is greater (Fig. 7A). 1 1. HOHORST. H-J. 1965. In: H. U. Bergmeyer, ed., Methods of Enzymatic Analysis. Academic Press, New York. pp. 328-334. Although no requirement for adenylates in uncoupled respira- 12. IKUMA, H. AND W. D. BONNER, JR. 1967. Properties of higher plant mitochondria. 1. tion was found in this study, it may be that isolated corn mito- Isolation and some properties of tightly coupled mitochondria from dark grown mung bean chondria have a higher content of endogenous adenine nucleo- hypocotyls. Plant Physiol. 42: 67-75. 13. JUNG, D. W. AND J. B. HANSON. 1975. Activation of 2,4-DNP-stimulated ATPase activity tides than those isolated from other plant tissues (13), and hence in cauliflower and corn mitochondria. Arch. Biochem. Biophys. 168: 358-368. the effects observed by Laties (15) and Sotthibandhu and Palmer 14. KIRK, B. 1. AND J. B. HANSON. 1973. The stoichiometry of respiration-driven potassium (26) would not have been obvious here. Our investigation transport in corn mitochondria. Plant Physiol. 51: 357-362. clearly indicates that high rates of malate and succinate oxidation 15. LATIES, G. G. 1973. The potentiating effect of ADP in the uncoupling of oxidative phosphorylation in potato mitochondria. Biochemistry 12: 3350-3355. require the operation of dicarboxylate and Pi transporting sys- 16. LOWRY, 0. H.. N. J. ROSEBROUGH, A. L. FARR, AND R. J. RANDALL. 1951. Protein tems, and that such transport is dependent on the generation and measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. maintenance of a PMF across the inner membrane. 17. MARSH, B. B. 1959. The estimation of inorganic phosphate in the presence of adenosine triphosphate. Biochim. Biophys. Acta 32: 357-361. LITERATURE CITED 18. MITCHELL, P. 1966. Chemiosmotic Coupling in Oxidative and Photosynthetic Phosphoryla- tion. Glynn Research, Ltd., Bodmin, England. 1. BERTAGNOLLI, B. L. AND J. B. HANSON. 1975. Functioning of the adenine nucleotide 19. OCHOA, S. 1955. Malic dehydrogenase from pig heart. Methods Enzymol. 1: 735. transporter in the arsenate uncoupling of corn mitochondria. Plant Physiol. 52: 431-435. 20. OESTREICHER, G.. P. HOGUE, AND T. P. SINGER. 1973. Regulation of succinate dehydro- 2. CHAPPELL, J. B. 1968. Systems used for the transport of substances into mitochondria. Br. genase in higher plants. II. Activation by substrates, reduced CoQ. nucleotides and Med. Bull. 24: 150-157. anions. Plant Physiol. 52: 622-626. 3. DAY, D. A. AND J. T. WISKICH. 1974. The oxidation of malate and exogenous NADH by 21. OVERMAN, A. R., G. H. LORIMER, AND R. J. MILLER. 1970. Diffusion and osmotic transfer isolated plant mitochondria. Plant Physiol. 53: 104-109. in corn mitochondria. Plant Physiol. 45: 126-132. 4. DE SANTIS, A., G. BORRACCINO, 0. ARRIGONI, AND F. PALMIERI. 1975. The mechanism of 22. PHILLIPS. M. L. AND G. R. WILLIAMS. 1973. Anion transporters in plant mitochondria. phosphate permeation in purified bean mitochondria. Plant Cell Physiol. 16: 911-923. Plant Physiol. 51: 225-228. 5. DoUCE, R., E. L. CHRISTENSEN, AND W. D. BONNER, JR. 1972. Preparation of intact plant 23. PREZIOSO. G.. F. PLAMIERI, AND E. QUAGLIARIELLO. 1972. Kinetic study of the effect of mitochondria. Biochim. Biophys. Acta 275: 148-160. uncouplers on substrate transport by rat liver mitochondria. Bioenergetics 3: 277-287. 6. DOUCE, R., C. A. MANELLA, AND W. D. BONNER, JR. 1973. The external NADH 24. ROBINSON, B. H. AND J. B. CHAPPELL. 1967. The inhibition of malate. tricarboxylate and dehydrogenases of intact plant mitochondria. Biochim. Biophys. Acta 292: 105-116. oxoglutarate entry into mitochondria by 2-n-butylmalonate. Biochem. Biophys. Res. 7. HANSON, J. B. 1972. Ion transport induced by polycations and its relationship to loose Commun. 28: 249-255. coupling of corn mitochondria. Plant Physiol. 50: 347-354. 25. SAUER, L. A. AND R. PARK. 1973. A stimulation by phosphate of malate transport and 8. HANSON, J. B. AND D. E. KOEPPE. 1975. In: D. A. Baker and J. L. Hall, eds., Ion oxidation in rat adrenal mitochondria. Biochemistry 12: 643-649. Transport in Plant Cells and Tissues. North Holland Publishing Co., Amsterdam. pp. 79- 26. SOT-rHIBANDHU, R. AND J. M. PALMER. 1975. The activation of non-phosphorylating 99. electron transport by adenine nucleotides in Jerusalem-artichoke (Helianthus tuberosus) 9. HANSON, J. B.. B. L. BERTAGNOLLI, AND W. D. SHEPHERD. 1972. Phosphate-induced mitochondria. Biochem. J. 152: 637-645. stimulation of acceptorless respiration in corn mitochondria. Plant Physiol. 49: 707-714. 27. WISKICH, J. T. 1975. Phosphate-dependent substrate transport into mitochondria. Oxida- 10. HENSLEY, J. R. AND J. B. HANSON. 1975. The action of valinomycin in uncoupling corn tive studies. Plant Physiol. 56: 121-125.