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Nicotinamide Adenine Dinucleotidel. Received for publication December 31, 1985 and in revised form March 4, 1986. KATHLEEN L. SOOLE*2, IAN B. DRY, ...

Plant Physiol. (1986) 81, 587-592

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The Responses of Isolated Plant Mitochondria to External Nicotinamide Adenine Dinucleotidel Received for publication December 31, 1985 and in revised form March 4, 1986

KATHLEEN L. SOOLE*2, IAN B. DRY, AND JOSEPH T. WISKICH Botany Department, University ofAdelaide, Adelaide 5001 South Australia ABSTRACI The effects of added NAD on substrate oxidation by turnip (Brassica rapa L.) and beetroot (Beta vulgaris L.) mitochondria were investigated. State 3 malate and 2-oxoglutarate oxidation rates with turnip mitochondria were stimulated 25 to 40% by external NAD. Following NADdepletion this stimulation by NAD was increased to 70 to 80%. With purified beetroot mitochondria, state 3 malate and 2-oxoglutarate oxidation rates were only marginally increased (10-15%) by the addition of NAD but after NAD-depletion treatments this stimulation increased to 55%. The effect of added NAD on oxidation rates could be reduced by preloading mitochondria with NAD in the presence of succinate. Oxidation rates were found to be most sensitive to the addition of external NAD when rotenone was present. The uptake of external NAD into beetroot mitochondria appeared to be composed of both an active and a diffusive component. The active component displayed saturation kinetics with an approximate K,. of 0.105 ± 0.046 millimolar. These results provide further evidence, reported previously with potato mitochondria, that NAD can move across the inner membrane of plant mitochondria. They are particularly significant with respect to beetroot mitochondria which in contrast to other plant mitochondria, have not demonstrated any response to added NAD.

internal electron flow via the rotenone-insensitive NADH dehydrogenase (17). NAD uptake is a carrier-mediated process (17) and is the currently accepted explanation of the NAD-stimulation of oxidation rates. Isolated beetroot mitochondria are unable to oxidize external NADH as a respiratory substrate because they lack the NADH dehydrogenase on the outer surface of the inner membrane (5). Beetroot mitochondria also are not stimulated by external NAD when oxidizing NAD-linked substrates (28). This lack of response could be due to matrix levels of NAD being sufficient for maximum turnover of the enzymes involved. Alternatively, it may suggest some link between the presence of the external NADH dehydrogenase and the NAD response. Such a link may be between the external NADH dehydrogenase and increased electron flow or between the external dehydrogenase and the NAD transport mechanism. This study involved a comparison of the characteristics of NAD loss and uptake from beetroot mitochondria with those of a tissue known to have an active external NADH dehydrogenase, i.e. turnip. The experimental approach involved analysis of NAD loss and uptake in isolated mitochondria. Depletion of matrix NAD is a technique previously used in potato mitochondria studies (19) and it was essential that matrix NAD be reduced to rate-limiting levels, to elicit maximum response to exogenous NAD. The results indicate that the lack of response of beetroot mitochondria to added NAD is due to matrix levels of NAD being sufficient, and only after reduction of these levels can a response to added NAD be observed. Further, it was possible to show that responses to NAD were energy dependent and displayed saturation kinetics. Therefore, it can be concluded that no correlation exists between the stimulation of oxidation by added NAD and the presence of the external NADH dehydrogeanse.

It has been observed that oxidation of NAD-linked substrates by isolated plant mitochondria can be stimulated by added NAD (1-3). A number of possible explanations have been proposed. Two of these rely on the presence of an external NADH dehydrogenase associated with the respiratory electron transport chain of the inner mitochondrial membrane. Coleman and Palmer (1) suggested that the activity of enzymes oxidizing malate, present in the intermembrane space, became evident only in the presence of added NAD, generating external NADH which led to an increased rate of 02 uptake. Subsequently, it was shown that this NAD stimulation could be observed with other NAD-linked TCA cycle substrates and a transmembrane transhydrogenase, located in the inner membrane, which could transfer reducing equivalents from the matrix to external NAD was proposed (2). An important premise which led to these various proposals was that the inner mitochondrial membrane was impermeable to NAD. However, studies by Neuburger and Douce (17, 19), and Tobin et al. (27) have provided convincing evidence that external NAD can enter the mitochondrial matrix. This led to a third explanation of NAD stimulation of substrate oxidation rates, that is, NAD accumulation in the matrix space increased

MATERIALS AND METHODS Beetroots (Beta vulgaris L.) and turnips (Brassica rapa L.) were obtained fresh from local markets. Biochemicals were obtained from Sigma Chemical Company except for ADP, ATP, and FCCP3 which were purchased from Boehringer Mannheim GmH Biochemica (West Germany). Preparation of Mitochondria. Beetroot and turnip mitochondria were prepared as previously described (4, 15). Mitochondria were prepared from aged beetroot tissue discs using the method of Day et al. (5). The beetroot tissue was aged by incubating discs in 0.1 mM CaSO4 for 48 h. Mitochondria were purified on continuous sucrose density gradients, 30 to 60% gradient for beetroot (fresh and aged tissue) and 15 to 60% gradient for

' Financial support from the Australian Research Grants Committee is gratefully acknowledged. 2 Recipient of a Commonwealth Tertiary Allowance.

3Abbreviations: FCCP, carbonyl-cyanide p-trifluoromethoxyphenyl hydrazone; MAL, malate, 2-OG, 2-oxoglutarate; SRM, standard reaction medium; RCR; respiratory control ratio; TPP, thiamine pyrophosphate.

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turnip. Gradient media also contained 1 mM Tes (pH 7.2), 1 mM EDTA, and 0.1% BSA (w/v). The gradients were centrifuged for 90 min at 45,000g on a Beckman model L-2 Ultracentrifuge using a SW 25.2 rotor at 1°C. When mitochondria were being purified from beetroot tissue 1% PVP was included in the purification media. Assay Procedures. 02 uptake was measured polarographically in a sealed vessel using a Rank 02 electrode at 25C. The 02 concentration of the air saturated medium was taken as 240 JM at 25°C. Respiration rates were measured in standard reaction media containing 0.25 M sucrose, 10 mm phosphate buffer, 5 mM MgC92, 10 mM Tes (pH 7.2), and 0.2 to 0.8 mg of protein. Enzyme assays for fractionation of the sucrose gradient for beetroot mitochondria were catalase (14) and fumarase (9). Sucrose density was determined using an ABBE refractometer. Analyses of protein content of mitochondrial suspensions were performed using the method of Lowry et al. (13). Intramitochondrial NAD levels were measured using the method of Klingenberg (11) as adapted by Tobin et al. (27). NAD Depletion and Uptake Studies. Mitochondria were incubated in the SRM containing 0.1% BSA (w/v) and 1 mm EDTA at 4°C while stirring gently. Depending on the substrate being observed, the protein concentration ranged from 0.3 to 1.0 mg/ml. NAD-depleted mitochondria were loaded with NAD by incubation in SRM containing 2 mM NAD and 10 mM pH 7.2 for 60 min at 4°C while gently stirring. The low temperatures were used to help preserve enzyme activities. The mitochondrial suspension was then layered onto a 0.6 M sucrose cushion and centrifuged at l0,OOOg for 20 min. The pellet was resuspended to give a protein concentration of 4 to 7 mg/ml. Integrity Assay. Mitochondria were suspended in 3 ml of SRM (pH 7.2) with 1 mm NAD. External NAD reduction was monitored at 340 nm, in the presence of 10 mm isocitrate with and without 0.04% Triton X-100. Intactness was determined by comparing the rate in the absence of detergent with the rate in the presence of the detergent. Thus, if no rate was detected prior to the addition of the detergent, the mitochondria were 100% intact. RESULTS Purification of the Mitochondria. Depleting mitochondria of NAD required incubating the mitochondria for extended periods of time (up to 20 h). Initial depletion experiments with washed mitochondria resulted in rapid loss of enzyme activity. Mitochondria purified from contaminating protein and organelles, (normally present in a l0,OOOgpellet) maintained their intactness and viability for the time period of the depletion experiments. This is the first report of purification of mitochondria from these tissues and the method resulted in intact mitochondria with minimal contamination from other organelles. Figure 1 represents a fractionated beetroot gradient, indicating the location of different organelles within the gradient. Similar gradients were obtained for preparations from fresh and aged beetroot tissue. NAD Depletion and Uptake Studies with Turnip Mitochondria. Purified mitochondria were depleted ofNAD by incubating them in a dilute suspension to induce diffusive efflux of matrix cofactors. Dilute suspensions lost NAD more rapidly than concentrated suspensions as used by Neuburger and Douce (19) with potato mitochondria. The effects of these depletion treatments were initially characterized with purified turnip mitochondria (Table I) which are known to oxidize external NADH. Aliquots were taken at the times specified and the oxidation rates measured simultaneously, under state 3 conditions in the presence (+NAD) and absence (-NAD) of NAD. The oxidation rates of two NAD-linked substrates (i.e. malate and 2-OG) and the effects

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of rotenone on these rates were determined. The cofactors, CoA and TPP, were added to the reaction medium to ensure that these cofactors were not limiting the rate of oxidation because there is evidence that these cofactors can be lost during the NAD depletion process (20). It can be seen that NAD stimulates both the rotenone-insensitive and the state 3 respiration rates at the beginning of the incubation period. After 16 h incubation the rates of 02 uptake had decreased dramatically but were restored if NAD was provided externally. These results suggest that the decreases in the respiration rates were due to the loss of NAD. During these experiments it was important to demonstrate maintenance of an intact inner membrane, so that any effects of NAD could be assigned to NAD accumulation by flux through intact inner membranes and not to gross bidirectional diffusion through leaky or damaged membranes. Most integrity assays used previously rely on proton diffusion or measure intactness of the outer membrane only using added Cyt c (2, 18). In this study, an integrity assay was used which gave a measure of diffusive pyridine nucleotide flux through the inner membrane. Figure 2 is an example of a trace from which intactness was calculated. The ratio before and after the addition of the detergent gives the proportion of leakiness. In this case, there is no rate prior to the disruption of the membrane indicating that the inner membrane was impermeable to nonspecific flux of pyridine nucleotides. RCRs have also been used as a measure of membrane quality (24) and the results of Table I show that inner membrane intactness as measured by both these methods remained relatively constant throughout the incubation period. Thus, the observed decreases in oxidation rates (Table I) must have been due to the loss of NAD from the matrix, such that the substrate dehydrogense turnover was limited by the concentration of matrix NAD. If NAD was added back to the reaction media, oxidative rates were restored to their original values showing that maximal enzyme activity was maintained. Analyses of intramitochondrial NAD levels (Table III) confirmed that NAD was lost during the incubation period. These results obtained with turnip are comparable to those observed with potato mitochondria where depletion resulted in very slow rates of oxidation in the absence of external NAD (19). NAD-depleted turnip mitochondria could be reloaded with NAD by incubation with NAD and succinate. With these 'loaded' mitochondria, the rate of 02 uptake in the absence of NAD exceeded the rate observed with freshly isolated mitochondria and the addition of NAD had minimal effect (Table I). NAD analyses (Table III) confirmed that NAD had been taken up and retained in the matrix and was now at a level which was no longer rate-limiting for substrate dependent 02 uptake. These results provide a vital indicator of the ability to take up NAD and retain it in the matrix. It is interesting to note that the rotenone-insensitive rates of respiration of the NAD-loaded mitochondria still showed a significant stimulation in the presence of external NAD, demonstrating that rotenone-insensitive respiration is more sensitive to matrix NAD levels (Table 1). This is in agreement with the observation that the rotenone-insensitive dehydrogenase has a lower affinity for NADH than the rotenone-sensitive dehydrogenase and thus requires a higher level of NADH in the matrix to operate at maximal rates (16). NAD Depletion and Uptake Studies with Beetroot Mitochondria. The results of the depletion studies with purified beetroot mitochondria are presented in Table II. State 3 respiration rates in freshly isolated mitochondria were minimally stimulated by external NAD as previously reported (28), but some stimulation of the rotenone-insensitive rate was observed. Following incubation for 16 h, the state 3 respiration rate dropped significantly (i.e. 30-60%) while rotenone-insensitive respiration decreased between 60 and 80%. The rates of both substrates showed similar

NAD AND PLANT MITOCHO)NDRIA

0 r43~~~~~~~

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0

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TOP

-.

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TWE NO.

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FIG. 1. Fractionation of beetroot sucrose gradient. Location of markers in a gradient 35 to 60% sucrose for beetroot tissue mitochondrial preparation, fractionated into 1.2 ml volumes. (0), Catm 120 alase activity (nmol H202 decomposed/min ,d sample-'); (0), % transmission at 254 nm; (A), % sucrose (w/w); (O), fumarase activity (nmol fumarate/min- 120 Ml sample-').

t

Table I. NAD Depletion and Uptake of Turnip Mitochondria was measured as described in "Materials and Methods." Conditions for malate oxidation were 10 mM malate, 10 mm glutamate, 1 mM pyruvate, 200 gM CoA, 200 gM TPP, and 1.3 mM ADP. For 2-00 oxidation conditions were 10 mM 2-OG, 5 mM malonate, 1 mM malate, 200 Mm CoA, 200 jLM TPP, and 1.3 mM ADP. Both substrate oxidations were measured in reaction media at pH 7.2; 2.5 gM rotenone and 1 mM NAD were included where indicated. RCR were determined from the -rotenone, +NAD treatments. Loaded refers to rates after NAD uptake as described in "Materials and Methods." Mitochondrial integrity was maintained at 100%. State 3 Respiration

02 uptake

RCR

- Rotenone

I Onmole.

NADH I min

+ Rotenone

+NAD -NAD +NAD nmol 02 minm mg'I protein

-NAD Malate oxidation Depleted Oh 16h Loaded

2-OG oxidation Depleted Oh 16h Loaded

0

II

5.5 4.8 4.8

4.3 3.9 3.8

146 47 202

94 24 145

189 160 206

174 174

168

48 7

110

13 7 45

116 109 128

80 73

ISOCITRATE

Triton X- 100

FIG. 2. External NAD reduction in freshly isolated beetroot mitochondria. An example of a trace from which inner membrane integrity was assessed. Reaction conditions were 10 mM isocitrate and 1 mM NAD in standard reaction media with subsequent addition of 0.4% Triton X100. Rates are expressed as nmol NADH produced/min mg-' protein.

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trends and integrity assays indicated maintenance of inner membrane quality. Thus, the results clearly demonstrate that NAD uptake occurs in beetroot mitochondria in a similar fashion to that observed with turnip mitochondria, because after being loaded with NAD the rates of oxidation (in the absence of NAD) were similar to those measured with freshly isolated mitochondria (Table II). In freshly isolated beetroot mitochondria it was possible to see a significant stimulation by NAD of rotenone-insensitive respi-

ration. This observation has not previously been reported as earlier studies were performed using unpurified preparations of beetroot mitochondria (28). It may be that during the purification procedure some NAD was lost from the mitochondria eliciting an NAD response. Intramitochondrial NAD analyses (Table III) confirm the 02 uptake data suggesting that NAD was lost during incubation and accumulated the uptake experiment, back to the level of freshly isolated mitochondria. While an NAD response to beetroot mitochondria could therefore be demonstrated, a distinct difference still existed between

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Table II. NAD Depletion and Uptake of Beetroot Mitochondria Conditions were as described for Table I. Mitochondrial integrity was maintained at 100%. State 3 Respiration

RCR

- Rotenone

+ Rotenone

-NAD +NAD -NAD +NAD nmol 02 min-' mg' protein

Malate oxidation Depleted 0h 16 h Loaded

4.4 3.0 3.0

41 26 34

59 57 45

16 6 15

26 27 23

2-OG oxidation Depleted 0h 16 h Loaded

2.9 2.8 2.3

27 16 25

32 31 29

9 6 13

16 17 14

Table III. Intramitochondrial NAD Analyses NAD levels were measured as described in "Materials and Methods." These analyses correspond to treatments represented in Tables I and II. Values represent means of replicated measurements. Treatment NAD Content h nmol/mg protein Turnip 0 1.75 16 0.38 NAD-loaded 5.70 Beetroot 0 3.27 16 2.42 4.99 NAD-loaded

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(NON-LIN for microcomputers [7]) and only the points at low concentrations of NAD, where the diffusive component was assumed to be minimal, were used. This gave an estimate of Km of 0.105 ± 0.046 mm. Computer analyses were used to calculate this parameter from the raw data and not from the transformed data as in Lineweaver-Burk plots as it is claimed that greater accuracy can be obtained from computer analyses (25). Energy Dependence of NAD Uptake. The addition of FCCP to mitochondria prior to the addition of NAD markedly affected the stimulation by NAD and the effect was also dependent on the concentration of NAD present (Fig. 4, A and B). The NAD response was measured during rotenone-insensitive respiration as this allowed for FCCP to be added to this system at a time when it had been turning over maximally, hence the addition of the uncoupler at this stage minimized any influence it may have had on substrate transport. FCCP markedly decreased the degree of stimulation of 02 uptake by low concentrations of NAD (Fig. 4A) but appeared to have little effect at high NAD concentrations (Fig. 4B). However, FCCP did increase the time taken to achieve maximal rates of 02 uptake at high NAD concentrations. This result is due to the diffusive component of NAD uptake which can lead to a significant increase of internal NAD concentrations. Diffusion may still occur at low NAD concentrations, so some stimulation can be observed, but obviously at these low concentrations, the active component ( i.e. that inhibited by FCCP) is essential to generate high internal concentrations of NAD. The energy-dependence at low NAD concentrations of NAD uptake, agrees with a recent report (21). DISCUSSION Explanations of the response of isolated plant mitochondria to NAD have been controversial and beetroot mitochondria which neither respond to NAD, nor oxidize exogenous NADH have been featured in these controversies (28). Even though NAD uptake is now generally accepted as an explanation of the stimulation of respiration by added NAD, the lack of response of beetroot mitochondria remained anomalous. The behavior of beetroot mitochondria to added NAD suggested a connection between the presence of an external NADH dehydrogenase associated with the respiratory chain and the capacity to transport NAD. The results of this study clearly demonstrate that the lack of response of freshly isolated beetroot mitochondria to external NAD is not because they lack the external NADH dehydrogenase but because their matrix levels of NAD are sufficient to saturate the system, and that the addition of further NAD has no effect. Clearly, once matrix NAD levels of beetroot mitochondria were depleted experimentally, an NAD response could be observed. The response was not due to membrane damage during depletion, and so demonstrates that NAD can move across the inner membrane of beetroot mitochondria. Furthermore, the results provide evidence of an NAD carrier in the beetroot inner membrane that facilitates this transport process.

beetroot and turnip mitochondria. It can be seen that the rates Of 02 uptake of turnip mitochondria decreased by 50 to 100% during the incubation while the respiration rate of beetroot mitochondria declined by only 50% (Table II). This difference was also seen in the NAD analyses where turnip lost virtually all of their NAD but beetroot only exhibited a slight drop in level over the same incubation conditions (Table III). The question therefore remained as to whether this was in any way a reflection of the presence or absence or an external NADH dehydrogenase. Thus, depletion and uptake experiments were performed with mitochondria from aged beetroot tissue which develop an active external NADH dehydrogenase (23). The results revealed a similar response to that observed with fresh beetroot tissue (data not shown) and consequently the difference observed between beetroot and turnip mitochondria most probably reflects a tissue difference and is not a function of the presence or absence of the external NADH dehydrogenase. The demonstration of an NAD carrier in beetroot mitochonKinetics of NAD Uptake. The kinetics of the NAD transport process have been described by Neuburger and Douce (17) who dria provides further evidence to abandon the transmembrane measured ['4C]NAD uptake into intact potato mitochondria. In transhydrogenase hypothesis (6) and indicates that NAD uptake this study, the kinetics of uptake were measured by the response is the simplest explanation for the observed phenomenon. The of the rotenone-insensitive rate of 02 uptake to external NAD. response observed with beetroot is not unique as mitochondria The use of the rotenone-insensitive rate gave a greater sensitivity from mung bean hypocotyls show high levels of matrix NAD to the NAD response. The time taken for 02 concentration to comparable to those measured for beetroot mitochondria; they fall by a specified increment after the addition of each concen- also show little response to exogenous NAD, but, in contrast to tration of NAD was determined. This was performed with par- beetroot mitochondria, have the capacity to oxidize exogenous tially depleted beetroot mitochondria with 2-OG as the substrate NADH (27). Demonstration of a response in beetroot mitochonrepresenting a matrix-located enzyme. The plot of the reciprocal dria has eliminated any connection between the ability to reof time against the NAD concentration is shown in Figure 3. spond to NAD and the ability to oxidize external NADH. The plot indicates both a saturatable and a diffusive component Investigation of the response revealed that NAD accumulation of NAD uptake. Calculation of the Km was complex due to these was energy dependent and displayed saturation kinetics with an two components. Therefore, a computer analysis was used approximate K, of 0. 105 ± 0.046 mm. The possible limitations

NAD AND PLANT MITOCHONDRIA

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FIG. 3. Michaelis-Menten plot for NAD stimulation in NAD-depleted beetroot mitochondria. 2OG was the substrate of oxidation and reaction conditions were as described for Table I. 2.5 gM rotenone was present. l/T represents the reciprocal of the time taken for the 02 concentration to decrease by 26 nmol after the addition of NAD.

v-

E

I-

0.1

0.0 0

200

400 600 NAD CONC (upM )

FIG. 4. Effect of uncoupler on NAD stimulation in NAD-depleted beetroot mitochondria. Malate oxidation conditions were as described in Table I. Subsequent additions were as follows: 1.3 mm ADP, 2.5 AM rotenone, 1 AM FCCP and for (A) 40 ,uM NAD and (B) 1 mM NAD. Oxidation rates are expressed as nmol 02/min-mg-' protein.

of the method used to determine the kinetic parameter are that the method of measurement of NAD uptake involves more than just the actual accumulation of NAD. 02 uptake in the presence of rotenone is the measured parameter and so it may be that the Km being measured is that of the NAD carrier, the substrate dehydrogenase, or the rotenone-insensitive NADH dehydrogenase. The Km values reported for the latter two enzymes are lower than the Km measured for the carrier (2-OG dehydrogenase has a reported Km of 4.5 ,uM for NAD [26] and the rotenoneinsensitive dehydrogenase a Km of 80 A,M [16]). The estimated Km for the carrier is slightly lower than the only other published value of 0.3 ± 0.04 mm for potato mitochondria (17). The difference may represent the different techniques used to estimate this parameter. The technique used in this study is more sensitive

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since it measures turnover and amplifies the effect of NAD uptake. Others (17) have measured ['4C]NAD uptake, allowing for any ['4C]NAD present in the sucrose impermeable space but not for any ['4C]NAD nonspecifically bound to the membrane. This study also demonstrated the presence of an energy-independent, diffusive component of uptake. Day and Wiskich (3) observed that the addition of uncoupler did not inhibit NAD stimulation of oxygen uptake in cauliflower mitochondria. They added I mM NAD and reported that its effect was energy independent. They did not record a delay in response but just that the response still occurred. Clearly, they were observing the diffusive component of NAD transport. In this respect, NAD uptake is reminiscent of the pyruvate carrier of the inner mitochondrial membrane. At 5 mM, pyruvate transport across the membrane is part diffusive and part carrier-mediated but above 10 mm pyruvate it appears diffusive, i.e. respiration is not inhibited by the transport inhibitor, hydroxycinnamate (12). Hampp (8) has recently estimated cytosolic NAD concentration to be between 0.4 and 0.8 mm, therefore the NAD concentrations used in this study are quite close to concentrations the mitochondria may be experiencing in vivo. At these concentrations the contribution from the diffusive component appears significant, therefore it is important to consider this characteristic of NAD uptake. It is also important to consider the diffusive component of uptake in kinetic studies as it may be influencing Km determination and therefore result in an inaccurate estimation of this parameter. Clearly, the energy dependence and the saturation kinetics of the NAD response in beetroot mitochondria provides further evidence for uptake being a carrier-mediated process in these mitochondria. In summary, evidence presented in this study, together with previous studies (6, 19, 27) demonstrate conclusively that the stimulation of respiration by added NAD is due to NAD uptake into the matrix stimulating electron flow through the internal NADH oxidation pathways. It can be concluded that no correlation exists between NAD uptake and exogenous NADH oxidation, as one activity has been shown to occur in the total absence of the other. Reports by Hunter et al. (10) and Purvis and Greenspan (22) reveal that NAD can be lost from and taken up into the matrix of rat liver mitochondria. This reincorporation of NAD was found to be energy dependent as with beetroot mitochondria and was only observed in depleted rat liver mitochondria. This led to the proposal of an NAD translocase located in the inner membrane (22). It now appears that the results obtained from

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plant mitochondria on NAD uptake confirm what researchers were observing in the early 1960s with mammalian mitochondna. It may be indicating that carrier-mediated NAD uptake is a ubiquitous property of mitochondria. LITERATURE CITED 1. COLEMAN JOD, JM PALMER 1972 The oxidation of malate by isolated plant mitochondria. Eur J Biochem 26: 499-509 2. DAY DA, JT WISKICH 1974 The oxidation of malate and exogenous reduced nicotinamide adenine dinucleotide by isolated plant mitochondria. Plant Physiol 53: 104-109 3. DAY DA, JT WISKICH 1974 The effect of exogenous nicotinamide adenine dinucleotide on the oxidation of NAD-linked substrates by isolated plant mitochondria. Plant Physiol 54: 360-363 4. DAY DA, JT WISKICH 1975 Isolation and properties of the outer membrane of plant mitochondria. Arch Biochem Biophys 171: 117-123 5. DAY DA, JR RAYNER, JT WISKICH 1976 Characteristics of external NADH oxidation by beetroot mitochondria. Plant Physiol 58: 38-42 6. DAY DA, M NEUBURGER, R DOUCE, JT WISKICH 1983 Exogenous NAD effects on plant mitochondria. A reinvestigation of the transhydrogenase hypothesis. Plant Physiol 73: 1024-1027 7. DUGGLEBY RG 1981 A non-linear regression program for small computers. Anal Biochem 110: 9-18 8. HAMPP R 1985 Compartmentation of metabolite pools in protoplasts, chloroplasts, mitochondrial cytosol/vacuole. In PE Pilet, ed, The Physiological Properties of Plant Protoplasts. Springer-Verlag, Berlin, pp 87-98 9. HILL RA, RA BRADSHAW 1969 An assay for fumarase. Methods Enzymol 13: 9 1-99 10. HUNTER FE, R MALISON, WF BRIDGERS, B SCHUTZ, A ATCHINSON 1959 Reincorporation of diphosphopyridine nucleotide into mitochondrial enzyme systems. J Biol Chem 234: 693-699 11. KLINGENBERG M 1967 An assay for NAD. In HU Bergmeyer, ed, Methods of Enzymatic Analysis, Vol 4. Academic Press, New York, pp 2045-2059 12. LANOUE KF, AL SCHOOLWERTH 1979 Metabolic transport in mitochondria. Annu Rev Biochem 48: 871-922 13. LOWRY OH, NJ ROSEBROUGH, AL FARR, RJ RANDALL 1951 Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275 14. LUCK A 1965 Catalase. In HU Bergmeyer, ed, Methods of Enzymatic Analysis.

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Academic Press, New York, p 886 15. MILLARD DL, JT WISKICH, RN ROBERTSON 1965 Ion uptake and phosphorylation in mitochondria. Plant Physiol 40: 1129-1135 16. MOLLER IM, JM PALMER 1982 Direct evidence for the presence of a rotenoneresistant NADH dehydrogenase on the inner surface of the inner membrane of plant mitochondria. Physiol Plant 53: 413-420 17. NEUBURGER M, R DOUCE 1978 Transport of NAD through the inner membrane of plant mitochondria. In G Ducet, C Lance, eds, Plant Mitochondria. Elsevier, Amsterdam, pp 109-116 18. NEUBURGER M, E-P JOURNET, R BLIGNY, J-P CARDE, R DOUCE 1982 Purification of plant mitochondria by isopycnic centrifugation in density gradients of percoll. Arch Biochem Biophys 217: 312-323 19. NEUBURGER M, R DOUCE 1983 Slow passive diffusion of NAD between intact isolated plant mitochondria and suspending medium. Biochem J 216: 443450 20. NEUBURGER M, DA DAY, R DOUCE 1984 Transport of coenzyme A in plant mitochondria. Arch Biochem Biophys 229: 253-258 21. NEUBURGER M, DA DAY, R DOUCE 1985 Transport of NAD+ in percollpurified potato tuber mitochondria. Inhibition of NAD+ influx and efflux by N4-azido-2-nitrophenyl4-aminobutyryl-3'-NAD+. Plant Physiol 78: 405-410 22. PURVIS JL, MD GREENSPAN 1965 Energy-linked incorporation of diphosphopyridine nucleotide into rat liver mitochondria. Biochim Biophys Acta 99: 191-194 23. RAYNER JR, JT WISKICH 1983 Development of NADH oxidation by red beet mitochondria on slicing and aging of the tissue. Aust J Plant Physiol 10: 5563 24. ROMANI RJ, SE TUSKES, S OZELKOK 1974 Survival of plant mitochondria in vitro. Form and function after 4 days at 25'C. Arch Biochem Biophys 164: 743-751 25. SAGNELLA GA 1985 Model fitting, parameter estimation, linear and nonlinear regression. TIBS 10: 100-103 26. SANADI DR 1969 a-Ketoglutarate dehydrogenase. Methods Enzymol 13: 5255 27. TOBIN AB, DJERDJOUR B, E-P JOURNET, M NEUBURGER, R DOUCE 1980 Effect of NAD+ on malate oxidation in intact plant mitochondria. Plant Physiol 66: 225-229 28. WISKICH JT, JR RAYNER 1978 Interactions between malate and NADH oxidation in plant mitochondria. In G Ducet, C Lance, eds, Plant Mitochondria. Elsevier, Amsterdam, pp 101-107