Planta
Planta 144, 359-365 (1979)
9 by Springer-Verlag 1979
Effect of Temperature on the Pathways of NADH-Oxidation in Broad-Bean Mitochondria R. Marx and K. Brinkmann Botanisches Institut der Universit/it, Kirschallee 1, D-5300 Bonn 1, Federal Republic of Germany
Abstract. 1. Respiration rates of broad-bean (Vicia faba) mitochondria were studied as a function of tem-
perature. Arrhenius plots of all membrane-bound enzymes, as obtained with saturating substrate concentrations, revealed a break in the lower temperature range. That break was considered to indicate a phase transition of membrane phospholipids, characteristic for chilling-sensitive plants. A second discontinuity at 30 ~ C occurred only with activities linked to energy conservation. - 2. The activation energies for the oxidation of NAD+-linked substrates differ between states 3 and 4. State 3 respiration of NAD+-linked substrates is the result of a superimposition of two branches of electron transport, which can be separated by different sensibilities to rotenone. A characteristic temperature dependency of the respiratory control, as well as a shift of the low temperature break in the Arrhenius plot toward a higher temperature after state 4 to state 3 transition, are calculated to be caused by the superimposition of the two branches. 3. The temperature dependency of the oxidation of extra-mitochondrial N A D H and of succinate differs remarkably from that of the oxidation of matrix-NADH. It has been concluded that the rotenone-resistant oxidation of matrix-NADH and the oxidation of external N A D H are mediated via different pathways with individual regulation sites. Key words: Mitochondria - N A D H oxidation Respiration (rotenone-resistant) - Temperature activ a t i o n - Vicia
Introduction
Temperature activations as calculated from the Arrhenius plots are considered to characterize candidates for rate limitation in a reaction sequence providing Abbreviations: B S A : b o v i n e serum albumin; CCCP=carbonylcyanide- m-chlorophenylhydrazone; TPP = thiaminepyrophosphate
saturating, substrate concentrations (Steam, 1945; Dixon and Webb, 1964). Temperature analyses of the respiratory activity of plant mitochondria have been carried out primarily to investigate chilling injury and mechanisms of membrane-phase transitions (Lyons and Raison, 1970; Raison etal., 1971; Breidenbach etal., 1974; Raison etal., 1977). The aim of this communication is to investigate possible regulation sites of N A D H oxidation in broad-bean mitochondria by means of a temperature analysis. Broad-bean mitochondria oxidize endogeneous N A D H via two pathways (Marx and Brinkmann, 1978). The rotenone-sensitive pathway includes three phosphorylation sites, while the rotenon-resistant, electron transport bypasses site I. On the other hand, and in correspondence to other plant mitochondria, the oxidation of external N A D H is mediated by the N A D H dehydrogenase located on the c-side, and/or by the N A D H cytochrome c-oxidoreductase on the outer membrane (Palmer and Coleman, 1974). Several indications led to the conclusion that the rotenoneresistant pathway is located on the matrix side of the membrane and not mediated via the N A D H dehydrogenase located on the c-side (Marx and Brinkmann, 1978). The overall velocity of the bypass is very low, even in the presence of ADP, suggesting that the rotenone-insensitive electron transport has a regulation site that is different from that of the c-side N A D H dehydrogenase. The objective of our investigation was to compare activation energies of segments of rotenone-sensitive and rotenone-resistant electron transport in order to identify rate-limiting steps. Therefore, the possible role of temperature in the regulation of mitochondrial N A D H oxidation will be discussed.
Materials and Methods Isolation of Mitochondria and Crude Enzymes Mitochondria were prepared from etiolated broad-bean hypocotyls as described previously (Marx and Brinkmann, 1978). For the
0032-0935/79/0144/0359/$01.40
360
R. Marx and K. Brinkmann: Effect of Temperature on NADH-Oxidation
isolation of a crude enzyme extract, the mitochondria were suspended in a 10 mi potassium phosphate buffer, pH 6.9, containing 150 mM sucrose. The suspension was sonicated at 0~176 for 15 s at maximum power (Branson Sonic Power) and the resulting particles were centrifuged off at 100,000 g for 45 rain. The supernatant contained 1-2 mg protein per ml and was assayed as crude malate dehydrogenase and malic enzyme.
Assay Procedures Protein was estimated according to the method of Lowry et al. (1951) with crystalline BSA (fraction V) as the standard. Oxygen uptake was measured polarographically in a stirred vessel using a Clarce-type electrode. The desired temperature was maintained within + 0.2~ C by an Ultra-Kyrosat (Lauda, Germany). The standard reaction medium consisted of 0.4 M sucrose, 10 mM potassium phosphate at pH 6.9, with a final volume of 1.8 ml and 0.5 to 1.0 mg mitochondrial protein per ml. Temperature activation of mitochondrial respiration was measured according to the methods of Lyons et al. (1974). All experiments were carried out with saturating substrate concentrations approximately ten times kin. Thus, possible influences of temperature on the substrate-enzyme affinity which would produce artificial breaks in the Arrhenius plots (Silvius et al., 1978) were excluded from these investigations. Malic enzyme and malate dehydrogenase were assayed by a spectrophotometric measurement of oxidation and a reduction of nicotinamide nucleotides at 366 nm (Bergmeyer, 1974; Macrae, 1971).
Calculations The logarithms of respiratory rates and specific enzyme activities, respectively, were plotted against the reciprocals of absolute temperature. The resulting Arrhenius plots, obtained with 2 to 4 preparations, were normalized to identical values at 20~ C. Small differences of respiratory activities of different preparations revealed no effect on the slope of the Arrhenius line. From these Arrhenius plots, the temperatures at which discontinuities occurred were obtained. For each linear segment the regression line and the 90% region of confidence were obtained9 In addition, individual Arrhenius plots were calculated for each experiment to provide a measure of the variability of the activation energies. With this method, even small differences of activation energies and accurate transition temperatures could be estimated with reliable significance.
Chemicals and Enzymes All chemicals were of analytical-reagent grade; NAD +, NADH, ADP and ATP, and all enzymes used were obtained from Boehringer (Mannheim, Germany). BSA fraction V, antimycin A, and rotenone were obtained from Sigma (Mtinchen, Germany). TPP and CCCP were purchased from Serva (Heidelberg, Germany).
Results
The temperature activations of the oxidation of malate, e-ketoglutarate, citrate, succinate and external N A D H are summarized in Fig. 1. The oxidation rates were studied with the intact mitochondria measuring the O2 uptake in state 3 and state 4. The temperature
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Fig. 1 A-F. Arrhenius plots of the oxidation of (A) malate (45 mM), (B) c~-ketoglutarate (12 mM), (C), citrate (30 mM), (E) external NADH (1 mM), and (F) succinate (12 raM) in state 3 (110 tIM ADP) = 9 and in state 4= o - - o , measured with intact mitochondria incubation medium: 0.4 M sucrose, 10 mM KH2PO4, pH 6.9; (D) Arrhenius plot of malic enzyme and malate dehydrogenase measured with enzyme crude extract (see mat. & meth.) following O.D. at 366 nm
activation of malate dehydrogenase and of malic enzyme is shown in Fig. 1 D, representing non-membrane-bound dehydrogenases. In broad bean mitochondria, a malic enzyme was found to participate in malate oxidation in addition to malate dehydrogenation (unpublished results).
The Oxidation of NAD +-linked Substrates Within the temperature range of 30-45 ~ C, the temperature activation of state 3 respiration (Fig. 1A-C) exhibits two c o m m o n breaks at 17.5 ~ and 30 ~ C, respectively. A c o m m o n break at 12.5~ occurs in state 4. In the upper-temperature region, a break at 30~ may be c o m m o n to states 3 and 4, at least for malate. Whether or not the break also exists for the oxidation of citrate could not be deduced, as the R C approaches 1 shortly above 30 ~ C. c~-ketoglutarate oxidation is not changed at 30 ~ C. The activation energies within a 90% region of confidence and the transition temperatures have been summarized in Table 1. It is significant that, below 30 ~ C, the activation
R. Marx and K. Brinkmann: Effect of Temperature on NADH-Oxidation
361
Table 1. Summary of transition temperatures, T1, T2 (~ and activation energies, El-E3 (kJ/Mol) of broad bean mitochondrial activities; n =number of preparations, in parenthesis the 90% region of confidence. Substrate concentrations and other assay conditions see Figure 1 Condition
n
T1
T2
E1
E2
E3
Malate Citrate ~-Ketoglutarate
state 3
4 3 2
17.5 17.5 17.5
30.0 30.0 30.0
75.36 ( _+3.97) 73.26 ( + 2.72) 79.54 ( _+3.09)
38.61 ( _+3.14) 36.84 ( _+2.26) 36.00 ( + 1.67)
17.16 (_+3.55) 18.00 (+2.68) 23.28 (_+ 1.68)
Malate Citrate e-Ketoglutarate
4 3 2
12.5 12.5 12.5
30.0
96.29 (+4.77) 90.29 (-+4.39) 98.38 (-+ 8.57)
53.17 (_+ 1.67) 57.35 (-+2.51) 52.33 ( + 3.35)
29.30 (_+4.60)
state 4
2 3
30.0
44.79 (_+2.09) 40.16 ( -+2.92)
29.30 ( -+2.50)
71.17 ( -+2.09) 73.26 ( _+5.86)
44.38 ( -+1.46) 44.38 ( _+1.04)
73.26 ( + 3.35) 78.71 (_+3.30)
43.38 ( _+1.04) 40.19 (_+ 1.88)
Malate dehydrogenase Malic enzyme NADH Succinate
state 3
4 3
10.0 10.0
NADH Succinate
state 4
4 3
10.0 10.0
30.0 30.0
energies o f the oxidation o f the three N A D + - l i n k e d substrates do not differ. However, there is a difference between state 3 and state 4. These results indicate that the rate-limiting step in state 3 is different f r o m that in state 4.
Malate Dehydrogenase and Malic Enzyme The temperature activations, o f the isolated matrix enzymes (Fig. 1 D) differ f r o m that o f the malate oxidoreductase (Fig. 1A). The Arrhenius plots do not exhibit discontinuities at 12 ~ or 17 ~ C. Only the malic enzyme reveals a slight break at 30 ~ C, which m a y be responsible for the discontinuity o f malate oxidation in state 4 (Fig. 1 A). These results indicate that an intact mitochondrial m e m b r a n e is required for the breaks in the lower temperature range.
The Oxidation of Site H Substrates The Arrhenius plots o f the oxidation o f succinate and external N A D H (Fig. 1 E,F) are identical but they differ f r o m that of the N A D § substrates (see also Table 1). The respiration in states 3 and 4 exhibits identical activation energies between 2 ~ and 30 ~ C. This is an i m p o r t a n t difference with respect to the temperature behaviour of site I.
The Branching of NAD +-Linked Pathways As reported previously (Marx and Brinkmann, 1978), b r o a d bean m i t o c h o n d r i a have two pathways for oxi-
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dizing endogeneous N A D H (Fig. 2). Both pathways flow into the c o m m o n pool, u b i q u i n o n e - c y t o c h r o m e b-complex, as shown by the 100% sensitivity to antimycin A in both pathways. It has been demonstrated that in state 3 the rotenone-sensitive, electron transport is superimposed by the rotenone-resistant one. We have considered that the different Arrhenius plots for the respiration in the states 3 and 4 were the result o f the superimposition o f two parallel pathways with different temperature activations. The temperature activation o f the rotenone-resistant respiration should yield information about this question. The comparison o f this temperature activation with that o f exogenous N A D H should give further evidence for a possible relationship between these electron-transporting pathways. The Arrhenius plot of malate oxidation in the presence of rotenone (Fig. 3 and data in Table 2) indi-
362
R. Marx and K. Brinkmann: Effect of Temperature on NADH-Oxidation
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cates that the temperature activation of the rotenoneresistant oxidation of NAD+-linked substrates is identical to that of the state 4 respiration between 0 ~ and 30 ~ C, whether the experiments are carried out in the presence of ADP or CCCP. Thus the Arrhenius plot of state 4 oxidation of NAD+-linked substrates represents the temperature characteristic of the rotenone-resistant electron transport. This temperature activation differs from that of the oxidation of external N A D H (Fig. 3). The activation energies above and below the transition temperature at 10~ C are lower than that of the rotenone-resistant pathway (Table 2), which exhibits a break at 12.5 ~ C. Since temperature activation of the rotenone-sensitive electron transport is always superimposed with the rotenone-resistant one, it cannot be measured experimentally. Assuming that the rotenone-resistant respiration is identical to the activity in state 4, between 0 ~ and 30 ~ C, and assuming that the activities of two parallel reactions with a common product are
additive (Stearn, 1949), one can calculate the temperature characteristic of the rotenono-sensitive pathway by subtracting the activity in state 4 from the overall O2 consumption of NAD+-linked oxidation in state 3. The resulting activities of the rotenone-sensitire part of the oxidation of malate, citrate and e-ketoglutarate were plotted in the Arrhenius diagram in Fig. 4. Since the state 4 respiration reveals the transition temperature as 12.5 ~ C, and state 3's as 17.5 ~ C, the Arrhenius lines do no intersect at the low transition temperature. The activation energies below this break are identical in all NAD+-linked substrates, which indicates a common rate-limiting step. Between 17.5 ~ and 30 ~ C, the rotenone-sensitive oxidation of each substrate exhibits individual temperature activations. Above 30~ ~ C the rotenone-resistant respiration decreases in a non-linear fashion. When this respiration rate is subtracted from the overall Oz consumption, the result is a temperature compensatedlike activity of the rotenone-sensitive oxidation of NAD+-linked substrates.
Effect of Temperature on Oxidative Phosphorylation In state 3, the Arrhenius plots of all tested substrates reveal a discontinuity at 30 ~ C (Fig. 1), while in state 4 and with the exception of malate, the activation energies do not change above the lower transition temperature (Table 1). It should be emphasized that the lowered activation energies above 30~ are not a consequence of thermal denaturation. With preincubation experiments we were able to show that within the time limits of our experiments thermal denaturation started above 42~ ~ C. Since the downward bend at 30 ~ C occurred only in state 3 and not in state 4, we supposed that this break was caused by a process of the phosphorylation. This was confirmed by the temperature characteristic of the uncoupled respiration (Table 3). With the exception of malate, the oxidation of all tested substrates exhibited no discontinuity at 30 ~ C.
Table 2. Summary of transition temperatures T1, T2 (~ and activation energies El-E3 (kJ/Mol) of the rotenone-resistant respiration; all experiments were carried out in the presence of 22 gM rotenone; incubation medium and substrate concentrations see Figure 1, concentration of CCCP=0.5 gM; n = n u m b e r of preparations, in parenthesis the 90% region of confidence Condition
n
T1
T2
E1
Malate state 3 Malate+CCCP Citrate+ CCCP c~Ketogl, state 3
2 3 2 1
12.5 12.5 12.5 12.5
30.0 30.0
94.29 96.29 92.24 97.24
E2 ( • 4.58) (+_4.12) (+_6.38) ( +_8.26)
53.59 53.17 53.29 52.33
E3 ( +_2.87) (+_1.87) (+_2.72) ( _+6.75)
25.12 ( • 5.26) 25.12 (_+4.38)
R. Marx and K. Brinkmann: Effect of Temperature on NADH-Oxidation
363
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As a result of the different temperature activations o f the oxidation o f N A D + - l i n k e d substrates in states 3 and 4, it was noted that the temperature influenced the respiratory control (RC). This has been illustrated in Figure 5 with the oxidation o f malate and e-ketoglutarate. With the temperature range between 0 ~ and 35 ~ C, a non-linear dependency of R C occurred. Clear m i n i m u m s o f R C at 12 ~ C were dem o n s t r a t e d which coincided with the transition temperature of state 4 respiration. The following optim u m coincided with the discontinuity o f the Arrhenius lines in state 3. In contrast to these results, the R C o f the oxidation of succinate (not shown) and external N A D H (Fig. 5) exhibited no temperature dependency between 0 ~ and 30 ~ C. The R C decreased only above 30 ~ C. These results indicated that the effect o f temperature on the respiratory control was restricted to the oxidation o f N A D + - l i n k e d substrates, which suggested an i m p o r t a n t role of temperature in regulating N A D H oxidation.
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Discussion
Rate Limiting Steps The oxidation o f N A D § substrates is characterized by a different temperature activation in states 3 and 4. In a previous paper (Marx and Brinkmann, 1978), we have shown that in oxidizing N A D +linked substrates in state 4, the electrons are only transferred via a rotenone-resistant pathway. On the other hand, in state 3, this way is superimposed by the rotenone-sensitive electron transport. Thus, the temperature activation of the rotenone-resistant p a t h w a y is expressed by the activity in s t a t e 4 (Fig. 1 A - C ) , while the real temperature activation o f the rotenone-sensitive p a t h w a y is calculated by substracting the activity of state 4 from that of state 3 (Fig. 4). Within statistical error, state 4 oxidation o f N A D + - l i n k e d substrates exhibits identical energies of activation between 0 ~ and 3 0 ~ and a break at
Table 3. Transition temperatures T1, T2 (~
and activation energies E1-E3 (kJ/Mol) of uncoupled respiration (0.5 gM CCCP); incubation medium and substrate concentrations see Figure 1; n-number of preparations, in parenthesis the 90% region of confidence Substrate
n
T~
T2
E1
E2
E3
Malate Citrate NADH Succinate
3 2 2 3
17.5 17.5 10.0 10.0
30.0
75.16 (_+3.75) 73.26 ( _+3.12) 71.87 (_+3.42) 72.38 ( _+3.65)
38.51 (_+4.11) ~ 37.68 ( + 3.83) 43.96 (_+2.95) 42.12 ( _+3.42)
26.74 (+4.71)
364
R. Marx and K. Brinkmann: Effect of Temperature on NADH-Oxidation
12.5 ~ C. This indicates a common rate-limiting step. This temperature characteristic of the rotenone-resistant electron transport is independent of the energetic state of the respiratory chain (Table 2). Since the substrate dehydration exhibits no break at 12.5 ~ C, as shown for malate in Figure 1 D, we conclude that a process between a rotenone-resistant N A D H dehydrogenase and the ubiquinone-cytochrome b-complex is the rate-limiting step of the rotenone-resistant oxidation of endogenous N A D H . This agrees with results of a previous paper which showed that the overall velocity of the bypass is not enhanced by ADP although it is coupled to phosphorylation sites II and III (Marx and Brinkmann, 1978). The rotenone-sensitive oxidations of N A D § linked substrates exhibit identical activation energies only below the transition field at 12.5~ ~ C, indicating a common rate-limiting step. Different energies of activation above this break, 13, 23 and 36 kj M o l - 1, suggest a limitation by the reactions of dehydration or (less probably) by substrate transport into the mitochondria. In the case of malate, it is striking that above 30~ the activation energies of malic enzyme and of state-4 oxidation are identical (Table 1). The Arrhenius plots of malate oxidation show a discontinuity at 30 ~ C, even in the uncoupled state (Table 3). From these results it is conclusive that above 30 ~ the respiratory activity with malate is controlled by the malic enzyme. Between 0 ~ and 30 ~ C the oxidation of succinate and external N A D H exhibits identical energies of activation in states 3 and 4 (Table 1) and in the artificially uncoupled state (Table 3). F r o m these data we conclude that the oxidation of both substrates is regulated by a common, rate-limiting step of the respiratory chain. The fundamental difference between the temperature characteristic of site It electron transport and the activation energies of the rotenone-resistant pathway with NAD+-linked substrates proves that the rotenone-resistant electron transport does not enter the respiratory chain via the N A D H dehydrogenase on the c-side. The Arrhenius plot of the rotenone-resistant electron transport represents the temperature activation either of a rotenone-insensitive N A D H dehydrogenase on the matrix side, proposed by Palmer and Arron (1975), or that of a transmembrane transhydrogenase, proposed by Day and Wiskich (1947a, b). However, we have not detected any transhydrogenase activity in broad bean mitochondria (Marx and Brinkmann, 1978). Mechanism of Discontinuities in the Arrhenius Plots Within the temperature range of 0~ ~ the Arrhenius plots of mitochondrial activities exhibit one criti-
cal temperature in state 4 and two in state 3. These temperatures are indicated by with a change of the apparent activation energy. Interpretations of such breaks or discontinuities commonly fall into three groups: 1. A discontinuity in the Arrhenius plot of an enzymatic reaction is explained as due to a conformational modification of the enzyme at that temperature (Massey et al., 1966). This primarily causes a change in the substrate (Silvius et al., 1978) or cosubstratebinding-affinity (Palm and Katzendobler, 1972), i.e., the km of the enzyme. 2. In a reaction sequence, the alteration of the activation energy may indicate a switch-over from one to another master reaction (Crozier, 1954; Han, 1972). This mechanism assumes that with increasing temperature, the rate limitation transfers to an enzyme with a lower activation energy. 3. According to Kumamoto et al. (1971) and Raison (1972), breaks in the Arrhenius plots of membrane-bound enzymes are a consequence of phase transitions of the lipid phase in the membrane, causing changes of km as well a s I / m a x . We exclude the first possibility because the experiments were carried out at saturating substrate concentrations. The low temperature breaks (10 ~ and 12.5 ~ C) confirm the results of many authors and indicate a lipid phase transition, characteristic of chillingsensitive plants (Lyons and Raison, 1970; Miller et al., 1974; Pomeroy and Andrews, 1975; for review see Lyons, 1973). The discontinuity at 17.5 ~ C in state3 oxidation of site I substrates is considered to be a shift of the transition temperature from 12.5 ~ to 17.5 ~ C, and is a consequence of the superimposition of two different temperature activations. The Arrhenius plot, shown in Figure 4, exhibits a phase-transition " f i e l d " between 12.5 ~ and 17.5 ~ C. This transition field causes a direct effect of temperature on the respiratory control of site-I oxidation (Fig. 5). The minimum of RC at 12.5~ and the optimum at 17.5~ coincide with the breaks in state 4 and state 3, respectively. The 30 ~ C discontinuities are common to the state3 oxidation of all substrates (Fig. 1), although this break appears to be ofheterogenous origin. The 30 ~ C break of malate oxidation in state 4, or in presence of an uncoupler, corresponds to the discontinuity of the malic enzyme (Fig. 1D). We deduce that the oxidation of the other substrates is limited by a process of energy conservation since CCCP removes that break. F r o m our results we cannot decide whether the rate-limiting step is the adenylate transport, as proposed by Lee and Gear (1974), or a reaction of oxidative phosphorylation. The presented data were obtained with highly saturated substrate concentrations, i.e., unnatural conditions. The question that
R. Marx and K. Brinkmann: Effect of Temperature on NADH-Oxidation
arises is whether the temperature-dependent branching of the NAD+-linked electron transport into two alternative pathways, with two or three coupling sites, plays a regulative role in vivo. It is striking that the RC for the matrix-NAD +-linked oxidation undergoes a characteristic wave-like temperature dependency whereas the RC for the cytosol-NAD +linked oxidation is constant up to 30 ~ C. The temperature characteristic of the RC between 10~ and 20 ~ C could serve for a balance of the adenylate energy charge and the redox state of pyridine nucleotides. Transitionally high substrate concentrations in the mitochondria are expected if the concentration of ATP or of N A D H suddenly rises. In such a situation an increasingly activated pathway, via two instead of three coupling sites, could counterbalance the oxidation of matrix N A D H with less effect on the phosphorylation potential.
References Bergmeyer, H.U.: Methoden der enzymatischen Analyse. Weinheim: Chemie 1974 Breidenbach, R.W., Wade, N.L., Lyons, J.M.: Effect of chilling temperatures on the activities of glyoxysomal and mitochondrial enzymes from castor bean seedlings. Plant Physiol. 54, 324-327 (1974) Crozier, W.J.: In: The kinetic basis of molecular biology, Johnson, F.H., Eyring, H., Polissar, M.J. (ed.), pp. 197 214. New York: J. Wiley and Sons 1954 Day, D.A., Wiskich, J.T. : The oxidation of malate and exogenous reduced nicotinamide adenine dinucleotide-linked substrates by isolated plant mitochondria. Plant Physiol. 53, 104-109 (i974a) Day, D.A., Wiskich, J.T.: The effect of exogenous nicotinamide adenine dinucleotide on the oxidation of nicotinamide adenine dinucleotide-linked substrates by isolated plant mitochondria. Plant Physiol. 54, 360 363 (1974b) Dixon, K.D., Webb, E.C. : Enzymes, 2nd ed., Chap. IV, London: Longmans Green (1964) Hart, M.H. : Non-linear Arrhenius plots in temperature dependent kinetic studies of enzymes reaction. J. Theor. Biol. 35, 543 568 (1972) Kumamoto, J., Raison, J.K., Lyons, J.M. : Temperature "breaks" in Arrhenius plots: A thermodynamic consequence of a phase change. J. Theor. Biol. 31, 47-51 (1970) Lee, M.P., Gear, A.R.L. : The effect of temperature on mitochondrial membrane-linked reactions. J.Biol. Chem. 249, 7541-7549 (1974)
365
Lowry, O.H., Rosebrough, N.J., Far, A.L., Randall, R.J. : Protein measurement with the folin phenol reagent. J.Biol. Chem. 193, 265 275 (1951) Lyons, J.M., Raison, J.K. : Oxidative activity of mitochondria isolated from plant tissues sensitive and resistant to chilling injury. Plant Physiol. 45, 386-389 (1970) Lyons, J.M. : Chilling injury in plants. Ann. Rev. Plant. Physiol. 24, 445 466 (1973) Lyons, J.M., Raison, J.K., Kumamoto, J. : Polarographic determination of phase changes in mitochondrial membranes in response to temperature. In: Methods in Enzymology Vol. 32, 258-262, Colwick, S.P., Kaplak, N.O., eds. New York: Academic Press 1974 Macrae, A.R.: Isolation and properties of a malic enzyme from cauliflower bud mitochondria. Biochem. J. 122, 495-501 (1971) Marx, R., Brinkmann, K.: Characteristics of rotenone-insensitive oxidation of matrix-NADH by broad bean mitochondria. Planta 142, 83 90 (1978) Massey, V., Curti, B., Ganther, H. : A temperature dependent conformational change in e-amino acid oxidase and its effects on catalysis. J. Biol. Chem. 241, 234%2357 (1966) Miller, R.W., de la Roche, I.A., Pomeroy, M.: Structural and functional responses of wheat mitochondrial membrane to growth at low temperatures. Plant Physiol. 53, 426-433 (1974) Palm, D., Katzendobler, H.: Effect of allosteric ligands on the mechanism and stability of nicotinamide adenine dinucleotide specific isocitrate dehydrogenase from yeast. Biochemistry 11, 1283 1289 (1972) Palmer, J.M., Coleman, J.O.D.: Multiple pathways of NADH oxidation in the mitochondria. Horiz. Biochem. Biophys. 1, 220 260 (1974) Palmer, J.M., Arron, G.P.: The influence of exogenous nicotinamide adenine dinucleotide on the oxidation of malate by Jerusalem artichoke mitochondria. J. Exp. Bot. 27, 418-430 (1976) Pomeroy, M.K., Andrews, C.J.: Effect of temperature on respiration of mitochondria and shoot segments from cold-hardened and nonhardened wheat and rye seedlings. Plant Physiol. 56, 703-706 (1975) Raison, J.K., Lyons, J.M., Mehlhorn, R.J., Keith, A.D. : Temperature induced phase changes in mitochondrial membranes detected by spin iabeling. J. Biol. Chem. 246, 4036-4040 (1971) Raison, J.K. : The influence of temperature-induced phase changes on the kinetics of respiratory and other membrane-associated enzyme systems. Bioenerg. 4, 559-583 (1972) Raison, J.K., Chapman, E.A., White, P.Y.: Wheat mitochondria, oxidative activity and membrane lipid structure as a function of temperature. Plant Physiol. 59, 623-627 (1977) Silvius, J.R., Read, B.D., McElhaney, R.N.: Membrane enzymes: artifacts in Arrhenius plots due to temperature dependence of substrate-binding affinity. Science 199, 902-904 (1978) Steam, A.E. : Kinetics of biological reactions with special reference to enzymic processes. Adv. Enzym. 9, 25 27 (1949)
Received 16 August; accepted 1 October 1978
Planta 144, 359-365 (1979)
9 by Springer-Verlag 1979
Effect of Temperature on the Pathways of NADH-Oxidation in Broad-Bean Mitochondria R. Marx and K. Brinkmann Botanisches Institut der Universit/it, Kirschallee 1, D-5300 Bonn 1, Federal Republic of Germany
Abstract. 1. Respiration rates of broad-bean (Vicia faba) mitochondria were studied as a function of tem-
perature. Arrhenius plots of all membrane-bound enzymes, as obtained with saturating substrate concentrations, revealed a break in the lower temperature range. That break was considered to indicate a phase transition of membrane phospholipids, characteristic for chilling-sensitive plants. A second discontinuity at 30 ~ C occurred only with activities linked to energy conservation. - 2. The activation energies for the oxidation of NAD+-linked substrates differ between states 3 and 4. State 3 respiration of NAD+-linked substrates is the result of a superimposition of two branches of electron transport, which can be separated by different sensibilities to rotenone. A characteristic temperature dependency of the respiratory control, as well as a shift of the low temperature break in the Arrhenius plot toward a higher temperature after state 4 to state 3 transition, are calculated to be caused by the superimposition of the two branches. 3. The temperature dependency of the oxidation of extra-mitochondrial N A D H and of succinate differs remarkably from that of the oxidation of matrix-NADH. It has been concluded that the rotenone-resistant oxidation of matrix-NADH and the oxidation of external N A D H are mediated via different pathways with individual regulation sites. Key words: Mitochondria - N A D H oxidation Respiration (rotenone-resistant) - Temperature activ a t i o n - Vicia
Introduction
Temperature activations as calculated from the Arrhenius plots are considered to characterize candidates for rate limitation in a reaction sequence providing Abbreviations: B S A : b o v i n e serum albumin; CCCP=carbonylcyanide- m-chlorophenylhydrazone; TPP = thiaminepyrophosphate
saturating, substrate concentrations (Steam, 1945; Dixon and Webb, 1964). Temperature analyses of the respiratory activity of plant mitochondria have been carried out primarily to investigate chilling injury and mechanisms of membrane-phase transitions (Lyons and Raison, 1970; Raison etal., 1971; Breidenbach etal., 1974; Raison etal., 1977). The aim of this communication is to investigate possible regulation sites of N A D H oxidation in broad-bean mitochondria by means of a temperature analysis. Broad-bean mitochondria oxidize endogeneous N A D H via two pathways (Marx and Brinkmann, 1978). The rotenone-sensitive pathway includes three phosphorylation sites, while the rotenon-resistant, electron transport bypasses site I. On the other hand, and in correspondence to other plant mitochondria, the oxidation of external N A D H is mediated by the N A D H dehydrogenase located on the c-side, and/or by the N A D H cytochrome c-oxidoreductase on the outer membrane (Palmer and Coleman, 1974). Several indications led to the conclusion that the rotenoneresistant pathway is located on the matrix side of the membrane and not mediated via the N A D H dehydrogenase located on the c-side (Marx and Brinkmann, 1978). The overall velocity of the bypass is very low, even in the presence of ADP, suggesting that the rotenone-insensitive electron transport has a regulation site that is different from that of the c-side N A D H dehydrogenase. The objective of our investigation was to compare activation energies of segments of rotenone-sensitive and rotenone-resistant electron transport in order to identify rate-limiting steps. Therefore, the possible role of temperature in the regulation of mitochondrial N A D H oxidation will be discussed.
Materials and Methods Isolation of Mitochondria and Crude Enzymes Mitochondria were prepared from etiolated broad-bean hypocotyls as described previously (Marx and Brinkmann, 1978). For the
0032-0935/79/0144/0359/$01.40
360
R. Marx and K. Brinkmann: Effect of Temperature on NADH-Oxidation
isolation of a crude enzyme extract, the mitochondria were suspended in a 10 mi potassium phosphate buffer, pH 6.9, containing 150 mM sucrose. The suspension was sonicated at 0~176 for 15 s at maximum power (Branson Sonic Power) and the resulting particles were centrifuged off at 100,000 g for 45 rain. The supernatant contained 1-2 mg protein per ml and was assayed as crude malate dehydrogenase and malic enzyme.
Assay Procedures Protein was estimated according to the method of Lowry et al. (1951) with crystalline BSA (fraction V) as the standard. Oxygen uptake was measured polarographically in a stirred vessel using a Clarce-type electrode. The desired temperature was maintained within + 0.2~ C by an Ultra-Kyrosat (Lauda, Germany). The standard reaction medium consisted of 0.4 M sucrose, 10 mM potassium phosphate at pH 6.9, with a final volume of 1.8 ml and 0.5 to 1.0 mg mitochondrial protein per ml. Temperature activation of mitochondrial respiration was measured according to the methods of Lyons et al. (1974). All experiments were carried out with saturating substrate concentrations approximately ten times kin. Thus, possible influences of temperature on the substrate-enzyme affinity which would produce artificial breaks in the Arrhenius plots (Silvius et al., 1978) were excluded from these investigations. Malic enzyme and malate dehydrogenase were assayed by a spectrophotometric measurement of oxidation and a reduction of nicotinamide nucleotides at 366 nm (Bergmeyer, 1974; Macrae, 1971).
Calculations The logarithms of respiratory rates and specific enzyme activities, respectively, were plotted against the reciprocals of absolute temperature. The resulting Arrhenius plots, obtained with 2 to 4 preparations, were normalized to identical values at 20~ C. Small differences of respiratory activities of different preparations revealed no effect on the slope of the Arrhenius line. From these Arrhenius plots, the temperatures at which discontinuities occurred were obtained. For each linear segment the regression line and the 90% region of confidence were obtained9 In addition, individual Arrhenius plots were calculated for each experiment to provide a measure of the variability of the activation energies. With this method, even small differences of activation energies and accurate transition temperatures could be estimated with reliable significance.
Chemicals and Enzymes All chemicals were of analytical-reagent grade; NAD +, NADH, ADP and ATP, and all enzymes used were obtained from Boehringer (Mannheim, Germany). BSA fraction V, antimycin A, and rotenone were obtained from Sigma (Mtinchen, Germany). TPP and CCCP were purchased from Serva (Heidelberg, Germany).
Results
The temperature activations of the oxidation of malate, e-ketoglutarate, citrate, succinate and external N A D H are summarized in Fig. 1. The oxidation rates were studied with the intact mitochondria measuring the O2 uptake in state 3 and state 4. The temperature
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Fig. 1 A-F. Arrhenius plots of the oxidation of (A) malate (45 mM), (B) c~-ketoglutarate (12 mM), (C), citrate (30 mM), (E) external NADH (1 mM), and (F) succinate (12 raM) in state 3 (110 tIM ADP) = 9 and in state 4= o - - o , measured with intact mitochondria incubation medium: 0.4 M sucrose, 10 mM KH2PO4, pH 6.9; (D) Arrhenius plot of malic enzyme and malate dehydrogenase measured with enzyme crude extract (see mat. & meth.) following O.D. at 366 nm
activation of malate dehydrogenase and of malic enzyme is shown in Fig. 1 D, representing non-membrane-bound dehydrogenases. In broad bean mitochondria, a malic enzyme was found to participate in malate oxidation in addition to malate dehydrogenation (unpublished results).
The Oxidation of NAD +-linked Substrates Within the temperature range of 30-45 ~ C, the temperature activation of state 3 respiration (Fig. 1A-C) exhibits two c o m m o n breaks at 17.5 ~ and 30 ~ C, respectively. A c o m m o n break at 12.5~ occurs in state 4. In the upper-temperature region, a break at 30~ may be c o m m o n to states 3 and 4, at least for malate. Whether or not the break also exists for the oxidation of citrate could not be deduced, as the R C approaches 1 shortly above 30 ~ C. c~-ketoglutarate oxidation is not changed at 30 ~ C. The activation energies within a 90% region of confidence and the transition temperatures have been summarized in Table 1. It is significant that, below 30 ~ C, the activation
R. Marx and K. Brinkmann: Effect of Temperature on NADH-Oxidation
361
Table 1. Summary of transition temperatures, T1, T2 (~ and activation energies, El-E3 (kJ/Mol) of broad bean mitochondrial activities; n =number of preparations, in parenthesis the 90% region of confidence. Substrate concentrations and other assay conditions see Figure 1 Condition
n
T1
T2
E1
E2
E3
Malate Citrate ~-Ketoglutarate
state 3
4 3 2
17.5 17.5 17.5
30.0 30.0 30.0
75.36 ( _+3.97) 73.26 ( + 2.72) 79.54 ( _+3.09)
38.61 ( _+3.14) 36.84 ( _+2.26) 36.00 ( + 1.67)
17.16 (_+3.55) 18.00 (+2.68) 23.28 (_+ 1.68)
Malate Citrate e-Ketoglutarate
4 3 2
12.5 12.5 12.5
30.0
96.29 (+4.77) 90.29 (-+4.39) 98.38 (-+ 8.57)
53.17 (_+ 1.67) 57.35 (-+2.51) 52.33 ( + 3.35)
29.30 (_+4.60)
state 4
2 3
30.0
44.79 (_+2.09) 40.16 ( -+2.92)
29.30 ( -+2.50)
71.17 ( -+2.09) 73.26 ( _+5.86)
44.38 ( -+1.46) 44.38 ( _+1.04)
73.26 ( + 3.35) 78.71 (_+3.30)
43.38 ( _+1.04) 40.19 (_+ 1.88)
Malate dehydrogenase Malic enzyme NADH Succinate
state 3
4 3
10.0 10.0
NADH Succinate
state 4
4 3
10.0 10.0
30.0 30.0
energies o f the oxidation o f the three N A D + - l i n k e d substrates do not differ. However, there is a difference between state 3 and state 4. These results indicate that the rate-limiting step in state 3 is different f r o m that in state 4.
Malate Dehydrogenase and Malic Enzyme The temperature activations, o f the isolated matrix enzymes (Fig. 1 D) differ f r o m that o f the malate oxidoreductase (Fig. 1A). The Arrhenius plots do not exhibit discontinuities at 12 ~ or 17 ~ C. Only the malic enzyme reveals a slight break at 30 ~ C, which m a y be responsible for the discontinuity o f malate oxidation in state 4 (Fig. 1 A). These results indicate that an intact mitochondrial m e m b r a n e is required for the breaks in the lower temperature range.
The Oxidation of Site H Substrates The Arrhenius plots o f the oxidation o f succinate and external N A D H (Fig. 1 E,F) are identical but they differ f r o m that of the N A D § substrates (see also Table 1). The respiration in states 3 and 4 exhibits identical activation energies between 2 ~ and 30 ~ C. This is an i m p o r t a n t difference with respect to the temperature behaviour of site I.
The Branching of NAD +-Linked Pathways As reported previously (Marx and Brinkmann, 1978), b r o a d bean m i t o c h o n d r i a have two pathways for oxi-
[matrix ---_NADH
Rotenone
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25.12 (_+1.39) 19.67(_+2.51)
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Antimvcin A
[ external NADH I Fig. 2. Scheme of the branching of NAD+-linked pathways in broad bean mitochondria (Marx and Brinkmann, 1978)
dizing endogeneous N A D H (Fig. 2). Both pathways flow into the c o m m o n pool, u b i q u i n o n e - c y t o c h r o m e b-complex, as shown by the 100% sensitivity to antimycin A in both pathways. It has been demonstrated that in state 3 the rotenone-sensitive, electron transport is superimposed by the rotenone-resistant one. We have considered that the different Arrhenius plots for the respiration in the states 3 and 4 were the result o f the superimposition o f two parallel pathways with different temperature activations. The temperature activation o f the rotenone-resistant respiration should yield information about this question. The comparison o f this temperature activation with that o f exogenous N A D H should give further evidence for a possible relationship between these electron-transporting pathways. The Arrhenius plot of malate oxidation in the presence of rotenone (Fig. 3 and data in Table 2) indi-
362
R. Marx and K. Brinkmann: Effect of Temperature on NADH-Oxidation
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cates that the temperature activation of the rotenoneresistant oxidation of NAD+-linked substrates is identical to that of the state 4 respiration between 0 ~ and 30 ~ C, whether the experiments are carried out in the presence of ADP or CCCP. Thus the Arrhenius plot of state 4 oxidation of NAD+-linked substrates represents the temperature characteristic of the rotenone-resistant electron transport. This temperature activation differs from that of the oxidation of external N A D H (Fig. 3). The activation energies above and below the transition temperature at 10~ C are lower than that of the rotenone-resistant pathway (Table 2), which exhibits a break at 12.5 ~ C. Since temperature activation of the rotenone-sensitive electron transport is always superimposed with the rotenone-resistant one, it cannot be measured experimentally. Assuming that the rotenone-resistant respiration is identical to the activity in state 4, between 0 ~ and 30 ~ C, and assuming that the activities of two parallel reactions with a common product are
additive (Stearn, 1949), one can calculate the temperature characteristic of the rotenono-sensitive pathway by subtracting the activity in state 4 from the overall O2 consumption of NAD+-linked oxidation in state 3. The resulting activities of the rotenone-sensitire part of the oxidation of malate, citrate and e-ketoglutarate were plotted in the Arrhenius diagram in Fig. 4. Since the state 4 respiration reveals the transition temperature as 12.5 ~ C, and state 3's as 17.5 ~ C, the Arrhenius lines do no intersect at the low transition temperature. The activation energies below this break are identical in all NAD+-linked substrates, which indicates a common rate-limiting step. Between 17.5 ~ and 30 ~ C, the rotenone-sensitive oxidation of each substrate exhibits individual temperature activations. Above 30~ ~ C the rotenone-resistant respiration decreases in a non-linear fashion. When this respiration rate is subtracted from the overall Oz consumption, the result is a temperature compensatedlike activity of the rotenone-sensitive oxidation of NAD+-linked substrates.
Effect of Temperature on Oxidative Phosphorylation In state 3, the Arrhenius plots of all tested substrates reveal a discontinuity at 30 ~ C (Fig. 1), while in state 4 and with the exception of malate, the activation energies do not change above the lower transition temperature (Table 1). It should be emphasized that the lowered activation energies above 30~ are not a consequence of thermal denaturation. With preincubation experiments we were able to show that within the time limits of our experiments thermal denaturation started above 42~ ~ C. Since the downward bend at 30 ~ C occurred only in state 3 and not in state 4, we supposed that this break was caused by a process of the phosphorylation. This was confirmed by the temperature characteristic of the uncoupled respiration (Table 3). With the exception of malate, the oxidation of all tested substrates exhibited no discontinuity at 30 ~ C.
Table 2. Summary of transition temperatures T1, T2 (~ and activation energies El-E3 (kJ/Mol) of the rotenone-resistant respiration; all experiments were carried out in the presence of 22 gM rotenone; incubation medium and substrate concentrations see Figure 1, concentration of CCCP=0.5 gM; n = n u m b e r of preparations, in parenthesis the 90% region of confidence Condition
n
T1
T2
E1
Malate state 3 Malate+CCCP Citrate+ CCCP c~Ketogl, state 3
2 3 2 1
12.5 12.5 12.5 12.5
30.0 30.0
94.29 96.29 92.24 97.24
E2 ( • 4.58) (+_4.12) (+_6.38) ( +_8.26)
53.59 53.17 53.29 52.33
E3 ( +_2.87) (+_1.87) (+_2.72) ( _+6.75)
25.12 ( • 5.26) 25.12 (_+4.38)
R. Marx and K. Brinkmann: Effect of Temperature on NADH-Oxidation
363
RC
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As a result of the different temperature activations o f the oxidation o f N A D + - l i n k e d substrates in states 3 and 4, it was noted that the temperature influenced the respiratory control (RC). This has been illustrated in Figure 5 with the oxidation o f malate and e-ketoglutarate. With the temperature range between 0 ~ and 35 ~ C, a non-linear dependency of R C occurred. Clear m i n i m u m s o f R C at 12 ~ C were dem o n s t r a t e d which coincided with the transition temperature of state 4 respiration. The following optim u m coincided with the discontinuity o f the Arrhenius lines in state 3. In contrast to these results, the R C o f the oxidation of succinate (not shown) and external N A D H (Fig. 5) exhibited no temperature dependency between 0 ~ and 30 ~ C. The R C decreased only above 30 ~ C. These results indicated that the effect o f temperature on the respiratory control was restricted to the oxidation o f N A D + - l i n k e d substrates, which suggested an i m p o r t a n t role of temperature in regulating N A D H oxidation.
~" A~A~.,~.. A
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Discussion
Rate Limiting Steps The oxidation o f N A D § substrates is characterized by a different temperature activation in states 3 and 4. In a previous paper (Marx and Brinkmann, 1978), we have shown that in oxidizing N A D +linked substrates in state 4, the electrons are only transferred via a rotenone-resistant pathway. On the other hand, in state 3, this way is superimposed by the rotenone-sensitive electron transport. Thus, the temperature activation of the rotenone-resistant p a t h w a y is expressed by the activity in s t a t e 4 (Fig. 1 A - C ) , while the real temperature activation o f the rotenone-sensitive p a t h w a y is calculated by substracting the activity of state 4 from that of state 3 (Fig. 4). Within statistical error, state 4 oxidation o f N A D + - l i n k e d substrates exhibits identical energies of activation between 0 ~ and 3 0 ~ and a break at
Table 3. Transition temperatures T1, T2 (~
and activation energies E1-E3 (kJ/Mol) of uncoupled respiration (0.5 gM CCCP); incubation medium and substrate concentrations see Figure 1; n-number of preparations, in parenthesis the 90% region of confidence Substrate
n
T~
T2
E1
E2
E3
Malate Citrate NADH Succinate
3 2 2 3
17.5 17.5 10.0 10.0
30.0
75.16 (_+3.75) 73.26 ( _+3.12) 71.87 (_+3.42) 72.38 ( _+3.65)
38.51 (_+4.11) ~ 37.68 ( + 3.83) 43.96 (_+2.95) 42.12 ( _+3.42)
26.74 (+4.71)
364
R. Marx and K. Brinkmann: Effect of Temperature on NADH-Oxidation
12.5 ~ C. This indicates a common rate-limiting step. This temperature characteristic of the rotenone-resistant electron transport is independent of the energetic state of the respiratory chain (Table 2). Since the substrate dehydration exhibits no break at 12.5 ~ C, as shown for malate in Figure 1 D, we conclude that a process between a rotenone-resistant N A D H dehydrogenase and the ubiquinone-cytochrome b-complex is the rate-limiting step of the rotenone-resistant oxidation of endogenous N A D H . This agrees with results of a previous paper which showed that the overall velocity of the bypass is not enhanced by ADP although it is coupled to phosphorylation sites II and III (Marx and Brinkmann, 1978). The rotenone-sensitive oxidations of N A D § linked substrates exhibit identical activation energies only below the transition field at 12.5~ ~ C, indicating a common rate-limiting step. Different energies of activation above this break, 13, 23 and 36 kj M o l - 1, suggest a limitation by the reactions of dehydration or (less probably) by substrate transport into the mitochondria. In the case of malate, it is striking that above 30~ the activation energies of malic enzyme and of state-4 oxidation are identical (Table 1). The Arrhenius plots of malate oxidation show a discontinuity at 30 ~ C, even in the uncoupled state (Table 3). From these results it is conclusive that above 30 ~ the respiratory activity with malate is controlled by the malic enzyme. Between 0 ~ and 30 ~ C the oxidation of succinate and external N A D H exhibits identical energies of activation in states 3 and 4 (Table 1) and in the artificially uncoupled state (Table 3). F r o m these data we conclude that the oxidation of both substrates is regulated by a common, rate-limiting step of the respiratory chain. The fundamental difference between the temperature characteristic of site It electron transport and the activation energies of the rotenone-resistant pathway with NAD+-linked substrates proves that the rotenone-resistant electron transport does not enter the respiratory chain via the N A D H dehydrogenase on the c-side. The Arrhenius plot of the rotenone-resistant electron transport represents the temperature activation either of a rotenone-insensitive N A D H dehydrogenase on the matrix side, proposed by Palmer and Arron (1975), or that of a transmembrane transhydrogenase, proposed by Day and Wiskich (1947a, b). However, we have not detected any transhydrogenase activity in broad bean mitochondria (Marx and Brinkmann, 1978). Mechanism of Discontinuities in the Arrhenius Plots Within the temperature range of 0~ ~ the Arrhenius plots of mitochondrial activities exhibit one criti-
cal temperature in state 4 and two in state 3. These temperatures are indicated by with a change of the apparent activation energy. Interpretations of such breaks or discontinuities commonly fall into three groups: 1. A discontinuity in the Arrhenius plot of an enzymatic reaction is explained as due to a conformational modification of the enzyme at that temperature (Massey et al., 1966). This primarily causes a change in the substrate (Silvius et al., 1978) or cosubstratebinding-affinity (Palm and Katzendobler, 1972), i.e., the km of the enzyme. 2. In a reaction sequence, the alteration of the activation energy may indicate a switch-over from one to another master reaction (Crozier, 1954; Han, 1972). This mechanism assumes that with increasing temperature, the rate limitation transfers to an enzyme with a lower activation energy. 3. According to Kumamoto et al. (1971) and Raison (1972), breaks in the Arrhenius plots of membrane-bound enzymes are a consequence of phase transitions of the lipid phase in the membrane, causing changes of km as well a s I / m a x . We exclude the first possibility because the experiments were carried out at saturating substrate concentrations. The low temperature breaks (10 ~ and 12.5 ~ C) confirm the results of many authors and indicate a lipid phase transition, characteristic of chillingsensitive plants (Lyons and Raison, 1970; Miller et al., 1974; Pomeroy and Andrews, 1975; for review see Lyons, 1973). The discontinuity at 17.5 ~ C in state3 oxidation of site I substrates is considered to be a shift of the transition temperature from 12.5 ~ to 17.5 ~ C, and is a consequence of the superimposition of two different temperature activations. The Arrhenius plot, shown in Figure 4, exhibits a phase-transition " f i e l d " between 12.5 ~ and 17.5 ~ C. This transition field causes a direct effect of temperature on the respiratory control of site-I oxidation (Fig. 5). The minimum of RC at 12.5~ and the optimum at 17.5~ coincide with the breaks in state 4 and state 3, respectively. The 30 ~ C discontinuities are common to the state3 oxidation of all substrates (Fig. 1), although this break appears to be ofheterogenous origin. The 30 ~ C break of malate oxidation in state 4, or in presence of an uncoupler, corresponds to the discontinuity of the malic enzyme (Fig. 1D). We deduce that the oxidation of the other substrates is limited by a process of energy conservation since CCCP removes that break. F r o m our results we cannot decide whether the rate-limiting step is the adenylate transport, as proposed by Lee and Gear (1974), or a reaction of oxidative phosphorylation. The presented data were obtained with highly saturated substrate concentrations, i.e., unnatural conditions. The question that
R. Marx and K. Brinkmann: Effect of Temperature on NADH-Oxidation
arises is whether the temperature-dependent branching of the NAD+-linked electron transport into two alternative pathways, with two or three coupling sites, plays a regulative role in vivo. It is striking that the RC for the matrix-NAD +-linked oxidation undergoes a characteristic wave-like temperature dependency whereas the RC for the cytosol-NAD +linked oxidation is constant up to 30 ~ C. The temperature characteristic of the RC between 10~ and 20 ~ C could serve for a balance of the adenylate energy charge and the redox state of pyridine nucleotides. Transitionally high substrate concentrations in the mitochondria are expected if the concentration of ATP or of N A D H suddenly rises. In such a situation an increasingly activated pathway, via two instead of three coupling sites, could counterbalance the oxidation of matrix N A D H with less effect on the phosphorylation potential.
References Bergmeyer, H.U.: Methoden der enzymatischen Analyse. Weinheim: Chemie 1974 Breidenbach, R.W., Wade, N.L., Lyons, J.M.: Effect of chilling temperatures on the activities of glyoxysomal and mitochondrial enzymes from castor bean seedlings. Plant Physiol. 54, 324-327 (1974) Crozier, W.J.: In: The kinetic basis of molecular biology, Johnson, F.H., Eyring, H., Polissar, M.J. (ed.), pp. 197 214. New York: J. Wiley and Sons 1954 Day, D.A., Wiskich, J.T. : The oxidation of malate and exogenous reduced nicotinamide adenine dinucleotide-linked substrates by isolated plant mitochondria. Plant Physiol. 53, 104-109 (i974a) Day, D.A., Wiskich, J.T.: The effect of exogenous nicotinamide adenine dinucleotide on the oxidation of nicotinamide adenine dinucleotide-linked substrates by isolated plant mitochondria. Plant Physiol. 54, 360 363 (1974b) Dixon, K.D., Webb, E.C. : Enzymes, 2nd ed., Chap. IV, London: Longmans Green (1964) Hart, M.H. : Non-linear Arrhenius plots in temperature dependent kinetic studies of enzymes reaction. J. Theor. Biol. 35, 543 568 (1972) Kumamoto, J., Raison, J.K., Lyons, J.M. : Temperature "breaks" in Arrhenius plots: A thermodynamic consequence of a phase change. J. Theor. Biol. 31, 47-51 (1970) Lee, M.P., Gear, A.R.L. : The effect of temperature on mitochondrial membrane-linked reactions. J.Biol. Chem. 249, 7541-7549 (1974)
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Lowry, O.H., Rosebrough, N.J., Far, A.L., Randall, R.J. : Protein measurement with the folin phenol reagent. J.Biol. Chem. 193, 265 275 (1951) Lyons, J.M., Raison, J.K. : Oxidative activity of mitochondria isolated from plant tissues sensitive and resistant to chilling injury. Plant Physiol. 45, 386-389 (1970) Lyons, J.M. : Chilling injury in plants. Ann. Rev. Plant. Physiol. 24, 445 466 (1973) Lyons, J.M., Raison, J.K., Kumamoto, J. : Polarographic determination of phase changes in mitochondrial membranes in response to temperature. In: Methods in Enzymology Vol. 32, 258-262, Colwick, S.P., Kaplak, N.O., eds. New York: Academic Press 1974 Macrae, A.R.: Isolation and properties of a malic enzyme from cauliflower bud mitochondria. Biochem. J. 122, 495-501 (1971) Marx, R., Brinkmann, K.: Characteristics of rotenone-insensitive oxidation of matrix-NADH by broad bean mitochondria. Planta 142, 83 90 (1978) Massey, V., Curti, B., Ganther, H. : A temperature dependent conformational change in e-amino acid oxidase and its effects on catalysis. J. Biol. Chem. 241, 234%2357 (1966) Miller, R.W., de la Roche, I.A., Pomeroy, M.: Structural and functional responses of wheat mitochondrial membrane to growth at low temperatures. Plant Physiol. 53, 426-433 (1974) Palm, D., Katzendobler, H.: Effect of allosteric ligands on the mechanism and stability of nicotinamide adenine dinucleotide specific isocitrate dehydrogenase from yeast. Biochemistry 11, 1283 1289 (1972) Palmer, J.M., Coleman, J.O.D.: Multiple pathways of NADH oxidation in the mitochondria. Horiz. Biochem. Biophys. 1, 220 260 (1974) Palmer, J.M., Arron, G.P.: The influence of exogenous nicotinamide adenine dinucleotide on the oxidation of malate by Jerusalem artichoke mitochondria. J. Exp. Bot. 27, 418-430 (1976) Pomeroy, M.K., Andrews, C.J.: Effect of temperature on respiration of mitochondria and shoot segments from cold-hardened and nonhardened wheat and rye seedlings. Plant Physiol. 56, 703-706 (1975) Raison, J.K., Lyons, J.M., Mehlhorn, R.J., Keith, A.D. : Temperature induced phase changes in mitochondrial membranes detected by spin iabeling. J. Biol. Chem. 246, 4036-4040 (1971) Raison, J.K. : The influence of temperature-induced phase changes on the kinetics of respiratory and other membrane-associated enzyme systems. Bioenerg. 4, 559-583 (1972) Raison, J.K., Chapman, E.A., White, P.Y.: Wheat mitochondria, oxidative activity and membrane lipid structure as a function of temperature. Plant Physiol. 59, 623-627 (1977) Silvius, J.R., Read, B.D., McElhaney, R.N.: Membrane enzymes: artifacts in Arrhenius plots due to temperature dependence of substrate-binding affinity. Science 199, 902-904 (1978) Steam, A.E. : Kinetics of biological reactions with special reference to enzymic processes. Adv. Enzym. 9, 25 27 (1949)
Received 16 August; accepted 1 October 1978
