The Effect of Exogenous Nicotinamide Adenine Dinucleotide on the ...

2 downloads 0 Views 586KB Size Report
Plant Physiol. (1974) 54, 360-363. The Effect of Exogenous Nicotinamide Adenine Dinucleotide on the Oxidation of Nicotinamide Adenine Dinucleotide-linked.
Plant Physiol. (1974) 54, 360-363

The Effect of Exogenous Nicotinamide Adenine Dinucleotide the Oxidation of Nicotinamide Adenine Dinucleotide-linked Substrates by Isolated Plant Mitochondrial

on

Received for publication November 7, 1973 and in revised form April 16, 1974

DAVID A. DAY AND JOSEPH T. WISKICH Department of Botany, University of Adelaide, Adelaide, 5001, Australia ABSTRACT

The oxidation of malate, citrate, and a-ketoglutarate by cauliflower (Brassica oleacea L.) bud mitochondria was inhibited by rotenone. This inhibition was relieved upon addition of NAD+ to the medium, and ADP/O values were lowered to less than 2 when both rotenone and NAD+ were present. Dinitrophenol did not affect the relief of rotenone inhibition by exogenous NAD+.

The oxidation of exogenous NADH via the respiratory chain of plant mitochondria is insensitive to rotenone and yields ADP/O values between 1 and 2 (5, 7-9, 13, 17, 19, 20), indicating that part of the chain, including the first phosphorylation site, is bypassed. On the other hand, the oxidation of NADH generated within the mitochondrion, for example by the oxidation of malate, is coupled to three sites of phosphorylation and is strongly inhibited by rotenone (6, 7, 9, 16, 20, 21). Recently it has been shown (6, 7) that in the presence of added NAD+, malate oxidation can also bypass the rotenonesensitive site and the first site of phosphorylation, and in this respect resembles exogenous NADH oxidation. Coleman and Palmer (6) suggested that malic enzyme, which requires NAD+ as a cofactor, was situated in the intermembrane space of Jerusalem artichoke mitochondria and thus was responsible for the above effect of added NAD+. Contrary to this, we suggested that a transmembrane transfer of reducing equivalents was involved, since even in the presence of NAD+ and rotenone, malate oxidation was stimulated by inorganic phosphate and inhibited by n-butyl malonate (an inhibitor of the malate/ phosphate exchange carrier on the inner membrane [ref. 4 and Wiskich, unpublished results]). The present study demonstrates similar effects of NAD+ on the oxidation of citrate and a-ketoglutarate by isolated cauliflower bud mitochondria.

Preparation of Mitochondria. Mitochondria were isolated from cauliflower buds as described previously (7), except that the medium contained 0.5% BSA and the tissue was disrupted using a Moulinex liquidizer. An additional wash in 0.3 M sucrose was also included in the procedure. Oxygen Consumption. Oxygen uptake was measured polarographically in a sealed Perspex vessel with a circulating water bath, using a Clarke electrode (Yellow Springs Instrument Co., Cleveland, Ohio) connected to a 1-mv recorder (Electronik, Honeywell Controls, Ltd., Great Britain). A standard reaction medium of 0.25 M sucrose containing 0.01 M phosphate buffer (K salts, pH 7.2), 5 mim MgCl,, 0.5 mM EDTA, and 10 mm tris-HCI buffer (pH 7.2) was used. Each assay contained 1.5 to 2.5 mg of mitochondrial protein. The total volume of the assay mixture was 3.2 to 3.4 ml and was maintained at 25 C. ADP/O and Respiratory Control Ratios. These were determined from the oxygen electrode traces obtained upon addition of ADP, according to the method of Chance and Williams

(3).

Protein Determinations. Protein content was estimated using the procedure of Lowry et al. (11). Cytochrome c Reduction. A Beckman spectrophotometer (Model DB), connected to a Beckman linear-log 12.5-cm stripchart recorder, was used for the measurement of Cyt c reductases. Activity was determined by following absorbance at 550 nm at room temperature, using cuvettes with a 1-cm light path. The reaction mixture consisted of a 0.1-ml mitochondrial suspension, 0.05 mM Cyt c, and 10 mm KCN in 3 ml of the standard reaction medium described above. The reaction was initiated by the addition of 0.5 mm NADH, 15 mm malate, or 15 mm succinate to the cuvette. A molar extinction coefficient (reduced minus oxidized) of 19.8 x 10' cm-' was used (14).

RESULTS Malate and citrate were readily oxidized by cauliflower bud mitochondria, but a-ketoglutarate oxidation required the addition of ADP to the reaction mixture (Fig. 1). State 3 rates of oxygen uptake and respiratory control ratios associated with MATERIALS AND MEETHODS citrate and a-ketoglutarate oxidation were less than those obwhen malate was substrate (Fig. 1). ADP/O ratios obFresh cauliflower (Brassica oleacea L.) was purchased lo- served served with malate and citrate were close to three (2.7-2.9) cally. Bovine serum albumen was obtained from the Commona-ketoglutarate oxidation gave ADP/O values of 3.3 to wealth Serum Laboratories (Melbourne, Aust.), enzymes were but 3.6 (Fig. 1). An induction period was necessary for maximal from Boehringer and Soehne (Mannheim, Germany), and other rates of a-ketoglutarate oxidation (Fig. 1C). Similar results biochemicals were from Sigma Chemical Co. (St. Louis). have been obtained by others (2, 19, 22). The oxidation of all three substrates was inhibited by rotenone, but this inhibition ' Support was provided by a Commonwealth Postgraduate Award was relieved to varying degrees by the addition of 1 mm NAD+ (Table I). Malonate was included in the reaction medium when to D. A. D. and by the Australian Research Grants Committee. 360

Plant Physiol. Vol.

54,

1974

NAD-LINKED SUBSTRATE OXIDATION

Mw

361

Mw

ADP

ADP

lo nMOLES 02 ADP

2 MIN. I

FiG. 1. Malate, citrate, and a-ketoglutarate oxidation by cauliflower bud mitochondria. Mitochondria were added to 3 ml of standard assay medium as described under "Materials and Methods." Additions as indicated were: A: 1.26 mg of mitochondrial protein, 20 mm malate, and 0.15 mM ADP; B: 2 mg mitochondrial protein, 20 mm citrate, and 0.15 mm ADP; C: 2.52 mg of mitochondrial protein, 10 mmia-ketoglutarate and 0.15 mm ADP. In addition, 20 mri glutamate was added to the reaction medium in A, and 10 mm malonate in B and C. Oxygen uptake is expressed as nmoles 02/min-mg protein. KG: as-ketoglutarate; Mw: washed mitochondria.

Table I. Effect of Exogenous NAD+ on Malate, Citrate, and a-Ketoglutarate Oxidation Conditions of assay are described in Figure 1.

C C ITRATE

Pi

/~~~~~~~

0

State 3 Respiration

Substrate

Experiment Control

+12 Am rotenone

+12 pm rotenone and 1 mm NAD+

nmoles 02/min-mg protein

L-Malate

63 78 29 31 24 29

1 2 1 2 1 2

Citrate

ra-Ketoglutarate

9 12 7 10 6 9

48 50

Respiration

Experiment

Respiratory Control Ratio State 3 State 4

ROTENONE

27 33 18 18

Table II. Effect of NAD+ and Rotenone on Citrate Oxidation Conditions of assay are described in Figure 1. The substrate was 20 mm citrate. Other Additions

105

ADP/O

FIG. 2. NADH and citrate oxidation by cauliflower bud mitochondria. Mitochondria were added to 3 ml standard reaction medium as described under "Materials and Methods." Additions, as indicated were: A: 1.5 mg of mitochondrial protein, 1 mm NADH, 0.46 mM ADP, and 16 ,uM of rotenone; B: 1.45 mg of mitochondrial protein, 20 mm citrate, 10 mm phosphate, and 0.23 mm ADP. Malonate (8 mM) was included in the reaction medium. Rates are expressed as nmoles 02/min mg protein. Mw: washed mitochondria. -

nmoles of 02/

min-mg protein

None 1 mM NAD+ and 12 Mm rotenone

1 2 1 2

53 59 40 45

26 28 30 33

2.0 2.1 1.3 1.36

2.7

2.7 1.6 1.8

citrate and a-ketoglutarate were used, to prevent the oxidation of succinate and to aid the entry of these substrates into the matrix via the tricarboxylate/dicarboxylate exchange carrier of the inner membrane (ref. 4 and Wiskich, unpublished results).

Table II shows that ADP/O ratios and respiratory control ratios associated with citrate oxidation were lowered when NAD+ and rotenone were added to the medium. The same effect has been observed with malate oxidation (6, 7). These results suggest that transfer of reducing equivalents across the inner membrane can occur with all NAD-linked substrates under certain conditions. Alternatively, the inner membrane of these mitochondria could be permeable to pyridine nucleotides. However this does not seem likely, since the oxidation of exogenous NADH was completely insensitive to high rotenone concentrations (Fig. 2).

362

DAY AND WISKICH

Table III. Effect of DNP ont the Recovery of Mtla/te alid Citrate Oxidationt by NAD+ Conditions of assay are described in Figure 1. Final concentrations were: malate and citrate, 20 mm. Respiration

Additions to Reaction \ essel

Citrate

Malate Expt 1

Expt 2

Expt 1

Expt 2

ninnles Qof 02'n in *Img protein

1. 2. 3. 4.

Substrate ADP, 0.3 mm Rotenone, 12,um NAD+, I mM

36 68 32 62

29 104 40 79

23 30 15 29

18 45 24 45

1. 2. 3. 4.

Substrate DNP, 30,llM Rotenone, 12 NAD+, 1 mm

38 68 20 46

29 104 36 72

28 35 18 35

21 46 22 52

AM

Table IV. Cytoclhiome c Reductase Activity in Caluliflower Bud Mitoclioldiria Rate of C)yt. c Reduction

Substrate

nIoles/, in i1n ng protein

3.4 23.5

Malate Malate + NAD+, 0.5 mm

9.7 114

Succinate Succinatel NADH, 0.5 mM NADH, 0.5 mm + antimycin A, 5 ,uM

68.2 51.4

1 Mitochondria were incubated in distilled water for 5 min before assaying.

The fact that citrate and malate oxidation were dependent on added phosphate (Fig. 2 and reference 7) also suggests that the inner membrane was intact. Table IV shows the rates of Cyt c reduction by cauliflower bud mitochondria. Both malate and succinate-Cyt c reductase activities were very low unless NAD+ was added (with malate) or the mitochondria were disrupted. In addition, the NADH-Cyt c reductase was largely insensitive to antimycin A. These results suggest that the outer membrane was also intact. DNP uncoupled malate and citrate oxidation both in the presence and absence of exogenous NAD+ and rotenone, but did not prevent the relief of rotenone inhibition by NAD+ (Table II1), suggesting that this process is not dependent on a supply of energy.

was attributable to intermembrane enzyme activity (such as malic enzyme as suggested by Coleman and Palmer [6]), then enzymes capable of oxidizing citrate and a-ketoglutarate must also be present in this outer compartment. However, there is no evidence to support this. Glyoxysomes, which are capable of oxidizing malate and reducing NAD (1 and 12), cannot be implicated, since NAD+ relieved rotenone inhibition of a-ketoglutarate and citrate oxidation (Table I), and glyoxysomes do not possess enzymes which can oxidize these substrates (1). A transfer of reducing equivalents across the inner membrane is probably involved; transport of NAD+ and NADH across this membrane is unlikely, since results with animal mitochondria have shown it to be impermeable to pyridine nucleotides (10, 18). It is unlikely that the inner membranes of the mitochondria used in this study were damaged, thereby allowing passage of exogenous NAD+ into the matrix because of the following: (a) The oxidation of exogenous NADH is not inhibited by rotenone at concentrations which strongly inhibit malate oxidation (Fig. 2 and ref. 7). (b) Malate and citrate enter via the tricarboxylate and dicarboxylate transporters on the inner membrane, and are therefore dependent on inorganic phosphate (Fig. 2; ref. 7). Malate oxidation was also strongly inhibited by n-butylmalonate (7), an inhibitor of the dicarboxylate transporter (4). (c) Respiratory control and ADP/O ratios were high for all substrates, indicative of tight coupling and intact membranes. (d) If the mitochondria were swollen and damaged, then one would expect their outer membranes to be broken, yet succinate-Cyt c reductase activity was very low (Table IV). Lack of this activity has been used by Douce et al. (8) to demonstrate intactness of the outer membrane of mung bean mitochondria. Point (a), above, indicates that NADH does not penetrate into the matrix. Points (b) and (c) show that the inner membrane is intact with respect to organic acid entry and maintenance of a high-energy state. Any damage to the inner membrane would increase the passive diffusion of organic acids and decrease respiratory control and ADP/O ratios. Point (d) demonstrates that very little damage due to swelling has occurred, since swelling results in breakage of the less flexible outer membrane. In fact, this forms the rationale behind some methods used to isolate the outer mitochondrial membrane (8). Hence, any pyridine nucleotide passage across the membrane would have to be via a transporter, for which there is no precedence. Whichever is transported, either reducing equivalents or nucleotide, the transfer appears to be unidirectional ([H] or NADH out; NAD+ in), since exogenous NADH oxidation is insensitive to rotenone. We propose the existence of a transhydrogenase across the inner membrane of plant mitochondria, which is capable of transferring reducing equivalents from within the matrix to exogenous NAD+ in the intermembrane space, upon oxidation of NAD-linked substrate. The NADH thus formed in the outer compartment is oxidized via the external NADH-dehydrogenases (8).

DISCUSSION The slower state 3 oxygen-uptake rates, which resulted in lower respiratory control ratios observed with citrate and aketoglutarate, probably reflect lower levels of dehydrogenase activity (15) compared with the malate system. The ADP activation of a-ketoglutarate oxidation, and the high ADP/0 values, are due to the associated substrate-level phosphorylation (2, 22). The fact that exogenous NAD+ relieved the rotenone inhibition of all three substrates suggests that a transmembrane transfer of reducing equivalents is involved. If the NAD+ effect

Plant Physiol. Vol. 54, 1974

LITERATURE CITED 1. 2. 3. 4.

5.

BREIDENnBACH, R.

W., A. KAHN, AN-D H. BEEVERS. 1968. Characterization of givoxysomes from castor bean endosperm. Plant Physiol. 43: 705-713. CHANCE, B. AND M\. BALTCHEFFSKY. 1958. Spectroscopic effects of a(lenosine diphosphate upon the respiratory pigments of rat-heart-mulsele sarcosomes. Biochem. J. 68: 283-295. CHANCE, B. AND G. R. WILLIAMS. 1956. The respiratory chaini an(l oxidative phosphorylation. Advan. Enzymol. 17: 55-134. CHAPPELL, J. B. 1968. Systems used in the transport of substances into illitochondria. Brit. Med. Bull. 24: 150-157. COLEMAN, J. 0. D. AN-D J. AI. PALMER. 1971. The role of Ca++ in the oxi(lation of exocenous NADH by plant mitochondria. FEBS Lett. 17: 203-208.

Plant Physiol. Vol. 54, 1974

NAD-LINKED SUBSTRATE OXIDATION

6. COLEMAN, J. 0. D. AND J. M. PALMER. 1972. The oxidation of malate by isolated plant mitochondria. Eur. J. Biochem. 26: 499-509. 7. DAY, D. A. AND J. T. WISKiCH. 1974. The oxidation of malate and exogenous NADH by isolated plant mitochondria. Plant Physiol. 53: 104-109. 8. DOUCE, R., E. A. MIANELLA, AND W. D. BONN-ER, Jr. 1973. The external NADH dehydrogenases of intact plant mitochondria. Biochim. Biophys. Acta 292: 105-116. 9. IKUJMIA, H. AND W. D. BONNER, Jr. 1967. Properties of higher plant mitochondria. I. Isolation and some properties of tightly coupled mitochondria from dark grown mung bean hypocotyls. Plant Physiol. 42: 67-75. 10. LEHNINGER, A. L. 1951. Phosphorylation coupled to oxidation of dihydrodiphosphopyridine nucleotide. J. Biol. Chem. 190: 345-359. 11. LOWRY, 0. H., N. J. RoSEBROUGH, A. L. FARR, AND R. J. RANDALL. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. 12. MIARCUS, A. AND J. VELASCO. 1960. Enzymes of the glyoxylate cycle in germinating peanuts and castor bean. J. Biol. Chem. 235: 563-567. 13. MIILLER, R. J. AND D. E. KOEPPE. 1971. The effect of Ca++ and inhibitors on corn mitochondrial respiration. Plant Physiol. 47: 832-835. 14. 'Morton, R. K. 1958. The cytochromes. Rev. Pure Appl. Chem. 8: 161-220.

363

15. NICHOLLS, D. G. AND P. B. GARLAND. 1969. The control of isocitrate oxidation by rat liver mitochondria. Biochem. J. 114: 215-225. 16. PALMER, J. M. AND J. 0. D. COLEMAN. 1972. Pathways of malate oxidation in isolated plant mitochondria. Biochem. J. 127: 42. 17. PALMER, J. M. AND H. C. PASSAM. 1970. The oxidation of exogenous NADH by plant mitochondria. Biochem. J. 122: 16. 18. ROBINsoN-, B. H. AND M. L. HALPERIN. 1970. Transport of reduced nicotinamide-adenine dinucleotide into mitochondria of rat white adipose tissue. Biochem. J. 116: 229-233. 19. WAKIYAMIA, S. AND Y. OGURA. 1970. Oxidative phosphorylation and the electron transport system of castor bean mitochondria. Plant Cell Physiol. 11: 835-848. 20. WILSON, R. H. AND J. B. HANSON. 1969. Effect of respiratory inhibitors on NADH, succinate and malate oxidation in corn mitochondria. Plant Physiol. 44: 1334-1341. 21. WISKICH, J. T. AND W. D. BONNER. 1963. Preparation and properties of sweet potato mitochondria. Plant Physiol. 38: 594-604. 22. WISKICH, J. T., R. E. YOUNG, AND J. B. BIALE. 1964. Metabolic processes in cytoplasmic particles of the Avocado fruit. VI. Controlled oxidations and coupled phosphorylations. Plant Physiol. 39: 312-322.