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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 per- chloric acid extracts according to the method of Marsh (17). Mitochondria (0.1 ml) were added to 2 ml of 5.5% cold per- chloric 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 ob- served 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 oligomy- cin 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.
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