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Studies on the energy release of rice mitochondria under different conditions by means of microcalorimetry
J. Biochem. Biophys. Methods 48 (2001) 1–11 www.elsevier.com / locate / jbbm
Studies on the energy release of rice mitochondria under different conditions by means of microcalorimetry Pei-Jiang Zhou
a,c ,
*, Han-Tao Zhou b , Yi Liu a , Song-Sheng Qu a , Ying-Guo Zhu b ,1 , Zhen-Bin Wu c
a
College of Chemistry and Environmental Sciences, Wuhan University, Wuhan 430072, China b College of Life Sciences, Wuhan University, Wuhan 430072, China c State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China Received 6 June 2000; accepted 10 June 2000
Abstract The thermodynamic and kinetic behaviors of energy release of mitochondria isolated from rice ( Oryza sative L.) were studied by using a LKB 2277 Bioactivity Monitor under different conditions. The thermogenesis curves of energy release of the rice mitochondria (which had been kept at 0–38C for 15 h and 40 day before the determination) were determined respectively at 25 and 308C, and the difference in shape of the thermogenesis curves and thermodynamic and kinetic characteristics were compared. The thermodynamic and kinetic parameters of energy release of the mitochondria in the thermogenesis increasing stage have been calculated, and the experimental thermokinetic equations of the thermogenesis have been established. The results indicated that the lower the temperature, the slower the energy release of the rice mitochondria. Both the thermogenesis and the energy release rate of the rice mitochondria increased after the mitochondria was kept at lower temperature for 40 days. One can use the methods to characterize the ability of the rice mitochondria to release energy under different conditions. 2001 Published by Elsevier Science B.V. Keywords: Rice (Oryza sative L.); Mitochondria; Energy release; Microcalorimetry; Thermokinetics
*Corresponding author. 1 Co-corresponding author. 0165-022X / 01 / $ – see front matter 2001 Published by Elsevier Science B.V. PII: S0165-022X( 00 )00123-8
P.-J. Zhou et al. / J. Biochem. Biophys. Methods 48 (2001) 1 – 11
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1. Introduction Mitochondria are semiautonomous organelles, containing the components necessary for the synthesis of some of their own proteins, and are the power plants of all eukaryotic cells. Mitochondria are the sites of respiration, a process releasing energy from oxidation of organic molecules and synthesis of ATP, the chief chemical energy source for cells. Most eukaryotic cells contain hundreds or thousands of mitochondria, the number of mitochondria per cell being related to the cell’s demand for ATP. Study of mitochondria is not only of theoretical significance, but also of applied value. Many aspects of the relations between mitochondria and the hardiness of plants, cytoplasmic male sterility of plants, disease and aging etc. have been studied in recent years [1,2]. Moreover, few microcalorimetric studies on energy release of mitochondria isolated from plants have been reported before to our knowledge. The mitochondrion, as a cellular center for energy metabolism, serves essential functions in the development of eukaryotic organisms. In higher plants, one of the developmental transitions that appears to be particularly influenced by mitochondrial function is male reproductive (pollen) development. Mutations in the mitochondrial genome most commonly result in the inability of the plant to shed viable pollen. This phenomenon is known as cytoplasmic male sterility (CMS) and is observed in more than 150 plant species [3]. The hybrid seeds can be produced with the help of male sterile lines, and the fine varieties of crops can be supplied by the hybrid seeds to raise the yield of crops. Rice breeding has been very successful during the last two decades and the yields have been raised in many parts of the world. The identification and development of cytoplasmic male sterility, maintainer and restorer lines were a major step in the success of this technology [4]. The studies of the male sterility of rice and hybrid rice began with Long-Ping Yuan in China, who discovered a male sterile individual plant of rice in 1964. After that, the cytoplasmic male sterility, maintainer and its restorer lines of HongLian type rice (Guangchong 41 etc.) were bred by Wuhan University in 1975 in China. At present, the hybrid rice has been widely used in farming in China, and the grain yield has increased greatly for many years [5]. In the paper, the thermogenesis curves of energy release of Guangchong 41 maintainer line rice mitochondria (which had been kept at 0–38C for 15 h and 40 days before the determination) have been determined respectively at 25 and 308C by using a LKB 2277 Bioactivity Monitor, and the thermodynamic and kinetic parameters of energy release of the mitochondria under different conditions have been calculated. The differences of the thermodynamic and kinetic characters of energy release of Guangchong 41 maintainer line rice mitochondria have been compared under different conditions.
2. Materials and methods
2.1. Materials The rice seed (Guangchong 41 maintainer line, in short GB) was provided by
P.-J. Zhou et al. / J. Biochem. Biophys. Methods 48 (2001) 1 – 11
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Research Institute of Genetics, College of Life Sciences, Wuhan University, Wuhan, China.
2.2. Isolation of rice mitochondria The mitochondria were isolated from the GB rice type above according to Pring’s method [6]. Some changes in the experimental procedure have been made in the isolation process. The seeds of the rice (Oryza sative L) were sown in vermiculite in flats, which were placed in growth chambers at 258C in the absence of light. Seven to 10 days after planting, etiolated mesocotyl and coleoptile tissue was weighed and immediately homogenized for 30 s in a high speed warring blender. The cold homogenization buffer was 0.5 mol l 21 sucrose, 0.005 mol l 21 Na 2 EDTA, 0.1% bovine serum albumin (BSA), and 0.05 mol l 21 Tris–HC1, pH 7.5. The preparation was filtered through eight layers of cheesecloth prior to centrifugation for 13 min at 1000 3 g and 48C. The supernatant was then centrifuged for 20 min at 16 000 3 g and 48C. The pellets were resuspended in reaction buffer (0.3 mol l 21 sucrose, 0.4 mol l 21 MgCl 2 , 0.1% BAS, 0.01 mol l 21 21 Tris–HCl, pH 7.5 and deoxyribonuclease I to 0.05 mg l ) for 60 min at ordinary temperature to digest extro-mitochondrial DNA. The solution was then added to washing buffer (0.6 mol l 21 , 0.02 mol l 21 Na 2 EDTA, 0.1% BSA, 0.01 mol l 21 Tris–HCl, pH 8.0) in the same volume, then centrifuged for 20 min at 16 000 3 g and 48C twice. The pellet (mitochondria) of 0.3 g was resuspended in 0.25 ml of reserved buffer (0.001 mol l 21 Na 2 EDTA, 0.01 mol l 21 Tris–HCl, pH 8.0) and stored in a refrigerator at 0–38C for measurement. All the above were carried out under aseptic conditions.
2.3. Instrument A LKB 2277 Bioactivity Monitor, a new type of heat-flow microcalorimeter was used in this experiment. It is designed to continuously monitor a wide variety of processes and complex systems over the temperature range 20–808C. A schematic representation of the microcalorimetric system is shown in Fig. 1. Each measuring cylinder normally contains a sample and a reference in separate measuring cups (twin system). The heat output from the sample flows from the thermoelectric detector to the large heat sink (in close contact with the water bath). In response the detector produces a voltage which is proportional to the power output from the sample. In order to minimize the systematic error and disturbance effect, a differential or twin detector system is used. This system is very sensitive, the detection limit is 0.1 mW and the baseline stability (over a period of 24 h) is 0.2 mW. The performance of this instrument and the details of its construction have been previously described [7].
2.4. Experimental determination The thermogenesis curves of rice mitochondria were recorded by ampoule method, using two sterile sealed ampoules, one ampoule containing a reference solution such as
4
P.-J. Zhou et al. / J. Biochem. Biophys. Methods 48 (2001) 1 – 11
Fig. 1. Simplified operation diagram.
sterile reserved buffer, and the other containing the sample (suspension of the mitochondria). The sample normally occupied position A in the monitor, and the reference occupied position B. Each ampoule contained 1 ml of sample (which contained about 0.3 g of the mitochondria and was kept at 0–38C for 15 h and 40 days before the determination) or reference and 2 ml of air. The experimental temperature was at 308C and the amplifiers of the monitor were set at 10 mW. All the above were carried out under aseptic conditions.
3. Results and discussion
3.1. Thermogenesis curves and thermodynamics of energy release of rice mitochondria The thermogenesis curves of energy release of the rice mitochondria (which were kept
P.-J. Zhou et al. / J. Biochem. Biophys. Methods 48 (2001) 1 – 11
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Fig. 2. Thermogenesis curve of energy release of rice mitochondria (which had been kept at 0–38C for 15 h before the determination) at 258C.
at 0–38C for 15 h and 40 days before the determination) at 258C and 308C are shown in Figs. 2–4. Mitochondria are semiautonomous organelles, containing large amounts of enzymes, and the enzymes which catalyse oxidation cellular reactions are the specific loci of the oxidation. These enzymes are still active after the isolation of mitochondria and there
Fig. 3. Thermogenesis curve of energy release of rice mitochondria (which had been kept at 0–38C for 15 h before the determination) at 308C.
6
P.-J. Zhou et al. / J. Biochem. Biophys. Methods 48 (2001) 1 – 11
Fig. 4. Thermogenesis curve of energy release of rice mitochondria (which had been kept at 0–38C for 40 days before the determination) at 308.
remains large amounts of nutrients in the mitochondria, hence enzymatic reactions can occur and release energy. The heat effects of the process can be monitored using sufficiently sensitive calorimeters and their thermogenesis curves obtained. Analysis of the thermogenesis curves showing the energy release of the rice mitochondria reveals four regions: the lag stage, increasing stage, stationary stage and decline stage. Sometimes the stationary stage is not very obvious, without any clear boundary at the stage. During isolation of the mitochondria, the enzymatic system of the mitochondria is damaged (and is in a resting condition at low temperature) so time is necessary for activity to be recovered and for adaptation to the new conditions. During this period, the lag stage, the thermogenesis curve is a horizontal straight line. These results indicate that the lag stage lasts for about 5 h at 308C and for about 30 h at 258C (Figs. 2 and 3). Once adequate adaptation has taken place, enzymatic activity gradually recovers, and both substance metabolism and energy metabolism starts, and the energy release of rice mitochondria increases, producing a logarithmic curve or sigmoid curve, i.e. the increasing stage. When all enzymes become active, the thermogenesis reaches a stationary stage without much change of the curves. When nutrients of the mitochondria and oxygen in the ampoule are exhausted, the curve enters the decline stage. Feedback inhibition of enzymes by metabolites would also contribute to this decline. The increasing time, maximal heat power and heat effect of energy release of the mitochondria at the increasing stage were determined, and are shown in Table 1. These results indicate that the lower the temperature, the slower the energy release of
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Table 1 Thermodynamic parameters of the thermogenesis of energy release of the rice mitochondria at the thermogenesis-increasing stage Duration of storage at 0–38C
Temperature (8C)
Increasing time (h)
Max power (mW)
Heat effect (J g 21 )
Thermogenesis rate (J h 21 g 21 )
15 h 15 h 40 days
25 30 30
20 9 10
19.5 35.5 42.0
21.40 20.97 21.97
20.07 20.11 20.20
the rice mitochondria over the range of a certain temperature. Both the heat effect and the heat release rate of energy release of the mitochondria (which had been kept at a lower temperature for 40 days before the determination) increased at 308C, and the energy release process was noticeably shortened (Fig. 4).
3.2. Thermokinetics of the thermogenesis of rice mitochondria energy release As previously described, analysis of the thermogenesis curves of energy release of rice mitochondria reveals four stages: lag stage, increasing stage, stationary stage and decline stage. The increasing stage is a comparatively important stage among the four stages, the thermogenesis of the mitochondrial energy release reveals two kinetic increasing forms at the stage. One is a limitless increasing form, the thermogenesis curve assumes the form of a logarithmic curve (Fig. 3). The other is a limited increasing form, the thermogenesis curve assumes the form of a sigmoid curve (Figs. 2 and 4). The limitless increasing rate constants (k) have been calculated from the equation: Pt 5 Po exp (kt) or ln Pt 5 ln Po 1 kt, where Po is the initial thermal power of the thermogenesis
Table 2 Data for Pt and t of the rice mitochondria at the thermogenesis increasing stage Storage at 0–38C for 15 h (258C)
Storage at 0–38C for 15 h (308C)
Storage at 0–38C for 40 days (308C)
t (h)
Pt (mW)
t (h)
Pt (mW)
t (h)
Pt (mW)
2 4 6 8 10 12 14 16 18 20
2.4 2.8 3.2 4.0 5.5 7.5 10.0 12.7 15.7 18.6
1 2 3 4 5 6 7 8 2 2
4.6 5.7 8.1 13.0 16.0 22.5 31.5 35.5 2 2
1 2 3 4 5 6 8 9 2 2
14.5 17.5 21.5 25.0 30.0 33.5 38.0 39.5 40.7 2
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Table 3 Kinetic parameters of energy release of the rice mitochondria at the thermogenesis increasing stage Duration of storage at 0–38C
Temperature (8C)
k (h 21 )
r
15 h 15 h 40 days
25 30 30
0.1226 0.3110 0.3741
0.9952 0.9945 20.9966
at time 5 0, and Pt is the thermal power of the thermogenesis at time 5 t. The data for Pt and t as well as the values of the thermokinetic parameters are shown in Tables 2 and 3, respectively. The limited increasing process of the thermogenesis at the increasing stage presents a sigmoid curve, the curve can best be expressed as the logistic equation (8). In the limited increasing process, the quantity of the thermogenesis increase in the process is timerelated according to: dNt / dt 5 k(1 2 sNt )Nt 5 kNt 2 skN t2 5 kNt 2 b N t2
(1)
where k is the limited increasing rate constant, s is the increase inhibitory factor and Nt represents the quantity of the thermogenesis increase in the limited increasing process at time5t. The deceleration rate constant is given by b 5 sk. Assuming PN is the thermal power of the unit increasing quantity of the thermogenesis of energy release of the mitochondria in the limited increasing process, then Pt 5 PN Nt
(2)
whence dN 1 dP ]t 5 ] ]t dt PN dt
(3)
Compare Eq. (1) with Eq. (3), we have dP Pt 2 sP 2t ]t 5 k ]]] dt PN
(4)
integrating Eq. (4) yields Pt (1 2 s) ln ]]]] 5 kt sPN PN 1 2 ] Pt
S
D
(5)
or PN Pt 5 ]]]]]] s 1 (1 2 s) exp (2kt)
(6)
P.-J. Zhou et al. / J. Biochem. Biophys. Methods 48 (2001) 1 – 11
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Eq. (5) can be rewritten as
S
D S D
PN /s 12s ln ]] 2 1 5 ln ]] 2 kt Pt s
(7)
or
S
D S D
a 12s ln ] 2 1 5 ln ]] 2 kt Pt s
(8)
where a 5 PN /s. Using the data Pt and t of the limited increasing stage obtained from the thermogenesis curve of energy release of the mitochondria, suitable values of a have been chosen in the calculations to give the best linearity of Eq. (8). Hence the limited increasing rate constant (k), the increase inhibitory factor (s), the deceleration rate constant ( b ) and the thermal power of the unit increasing quantity of the thermogenesis of the mitochondrial energy release in the limited increasing process (PN ) can be obtained. The data for Pt and t as well as the thermokinetic parameters of the limited increase of the thermogenesis of the rice mitochondria energy release at 308C are shown in Tables 2 and 3, respectively. According to Tables 1–3 the corresponding experimental thermokinetic equations can be established. At the limitless increasing phase of the thermogenesis of rice mitochondria energy release (which was kept at 0–38C for 15 h before the determination): Pt 5 1.684 exp (0.1226t) Pt 5 3.334 exp (0.311t)
37 h < t < 57 h at 258C t < 7 h at 308C
At limited increasing phase of the thermogenesis of rice mitochondria energy release (which kept at 0–38C for 40 days before the determination): Pt 5 10.48 /(0.2283 1 0.7717) exp (20.3741t) t < 9 h at 308C These results indicated that the kinetic behavior in the increasing stage of energy release of the rice mitochondria at 258C is similar to that of energy release of the mitochondria at 308C, their thermogenesis kinetic increasing forms limitlessly increase but the increasing rate at 258C is lower than that of at 308C. Because the mitochondria had been kept at a low temperature for a long time, the membranes of the mitochondria were damaged, and the enzyme activities of the mitochondria are down, and oxidation and phosphorylation is uncoupled in the mitochondria. The energy of the mitochondria is released as heat after uncoupling. So the thermogenesis kinetic increasing form of the mitochondria was limited. In the limited increasing stage, the thermogenesis increased fast, and released more heat, its stationary stage was short, and the whole energy release process was noticeably shortened. Evidently the damaging mechanism of plants at low
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P.-J. Zhou et al. / J. Biochem. Biophys. Methods 48 (2001) 1 – 11
temperature has much to do with the energy release of mitochondria [8], the problem is worth investigating further.
4. Simplified description of the method and its applications In recent years, microcalorimetry has been increasingly used in studies of various biological phenomena including studies at the cellular and subcellular level. The very broad application range for non-specific methods like calorimetry can be attractive both in thermodynamic measurements and in analytical work. As practically all processes are accompanied by heat effect, calorimetry is particularly well suited to the discovery of unexpected or unknown processes in samples of any aggregation state. Further, in contrast to spectroscopic methods, calorimetry does not require optically clear objects. In particular when heat conduction calorimeters are used, the experiments can be conducted over long periods or time — weeks or longer. These properties can make isothermal microcalorimeters ideal as monitors for slow and complex processes like the metabolism of plant mitochondria. In the paper, the thermogenesis curves of energy release of the mitochondria isolated from rice have been determined by using a LKB 2277 Bioactivity Monitor under different conditions. These curves contain much information. Through analysis of the curves, the thermodynamic and kinetic characteristics of the mitochondrial thermogenesis under different conditions have been studied, and the thermodynamic and kinetic parameters of the energy release process have been obtained, and some new experimental thermokinetic equations of the thermogenesis increasing stage in the energy release process of the rice mitochondria have been established. The characteristics of energy release of the rice mitochondria under different conditions can be identified with the microcalorimetric method. One can use the method and these results to characterize the ability of rice and other plants to release mitochondrial energy under different conditions.
Acknowledgements We gratefully acknowledge the financial support of the National Natural Science Foundation of China and State Key Laboratory of Freshwater Ecology and Biotechnology for this project.
References [1] Brennicke A, Kuck U. In: Plant mitochondria, New York: VCH Publishers Inc, 1993, pp. 251–401. [2] Wallace DC. Mitochondrial genetics: a paradigm for ageing and degenerative disease. Science 1992;256:628–32. [3] He S, Abad AR, Gelvin SB, Mackenzie SA. A cytoplasmic male sterility-associated mitochondrial protein causes pollen disruption in transgenic tobacco. Pro Natl Acad Sci USA 1996;93:11763–8.
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[4] Mignouna H, Virmani SS, Briquet M. Mitochondria DNA modifications associated with cytoplasmic male sterility in rice. Theor Appl Genet 1987;74:666–9. [5] Zhou P-J, Hu Y-C, Wang C-X, Song Z-H, Wang T-Z, Qu S-S et al. Determination of the thermogenesis curves and studies of the thermodynamics and thermokinetics of seed germination. J Biochem Biophys Methods 1999;38:171–80. [6] Pring DR, Levings III CS. Heterogeneity of maize cytoplasmic genomes among male-sterile cytoplasms. Genetics 1978;89:121–36. [7] Suurkuusk J, Wado¨ I. A multichannel microcalorimetry system. Chem Sci 1982;20:155–63. [8] Graham D, Patterson BD. Responses of plants to low, non-freezing temperatures: proteins, metabolism, and acclimation. Annu Rev Plant Physiol 1982;33:347–72.
Studies on the energy release of rice mitochondria under different conditions by means of microcalorimetry Pei-Jiang Zhou
a,c ,
*, Han-Tao Zhou b , Yi Liu a , Song-Sheng Qu a , Ying-Guo Zhu b ,1 , Zhen-Bin Wu c
a
College of Chemistry and Environmental Sciences, Wuhan University, Wuhan 430072, China b College of Life Sciences, Wuhan University, Wuhan 430072, China c State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China Received 6 June 2000; accepted 10 June 2000
Abstract The thermodynamic and kinetic behaviors of energy release of mitochondria isolated from rice ( Oryza sative L.) were studied by using a LKB 2277 Bioactivity Monitor under different conditions. The thermogenesis curves of energy release of the rice mitochondria (which had been kept at 0–38C for 15 h and 40 day before the determination) were determined respectively at 25 and 308C, and the difference in shape of the thermogenesis curves and thermodynamic and kinetic characteristics were compared. The thermodynamic and kinetic parameters of energy release of the mitochondria in the thermogenesis increasing stage have been calculated, and the experimental thermokinetic equations of the thermogenesis have been established. The results indicated that the lower the temperature, the slower the energy release of the rice mitochondria. Both the thermogenesis and the energy release rate of the rice mitochondria increased after the mitochondria was kept at lower temperature for 40 days. One can use the methods to characterize the ability of the rice mitochondria to release energy under different conditions. 2001 Published by Elsevier Science B.V. Keywords: Rice (Oryza sative L.); Mitochondria; Energy release; Microcalorimetry; Thermokinetics
*Corresponding author. 1 Co-corresponding author. 0165-022X / 01 / $ – see front matter 2001 Published by Elsevier Science B.V. PII: S0165-022X( 00 )00123-8
P.-J. Zhou et al. / J. Biochem. Biophys. Methods 48 (2001) 1 – 11
2
1. Introduction Mitochondria are semiautonomous organelles, containing the components necessary for the synthesis of some of their own proteins, and are the power plants of all eukaryotic cells. Mitochondria are the sites of respiration, a process releasing energy from oxidation of organic molecules and synthesis of ATP, the chief chemical energy source for cells. Most eukaryotic cells contain hundreds or thousands of mitochondria, the number of mitochondria per cell being related to the cell’s demand for ATP. Study of mitochondria is not only of theoretical significance, but also of applied value. Many aspects of the relations between mitochondria and the hardiness of plants, cytoplasmic male sterility of plants, disease and aging etc. have been studied in recent years [1,2]. Moreover, few microcalorimetric studies on energy release of mitochondria isolated from plants have been reported before to our knowledge. The mitochondrion, as a cellular center for energy metabolism, serves essential functions in the development of eukaryotic organisms. In higher plants, one of the developmental transitions that appears to be particularly influenced by mitochondrial function is male reproductive (pollen) development. Mutations in the mitochondrial genome most commonly result in the inability of the plant to shed viable pollen. This phenomenon is known as cytoplasmic male sterility (CMS) and is observed in more than 150 plant species [3]. The hybrid seeds can be produced with the help of male sterile lines, and the fine varieties of crops can be supplied by the hybrid seeds to raise the yield of crops. Rice breeding has been very successful during the last two decades and the yields have been raised in many parts of the world. The identification and development of cytoplasmic male sterility, maintainer and restorer lines were a major step in the success of this technology [4]. The studies of the male sterility of rice and hybrid rice began with Long-Ping Yuan in China, who discovered a male sterile individual plant of rice in 1964. After that, the cytoplasmic male sterility, maintainer and its restorer lines of HongLian type rice (Guangchong 41 etc.) were bred by Wuhan University in 1975 in China. At present, the hybrid rice has been widely used in farming in China, and the grain yield has increased greatly for many years [5]. In the paper, the thermogenesis curves of energy release of Guangchong 41 maintainer line rice mitochondria (which had been kept at 0–38C for 15 h and 40 days before the determination) have been determined respectively at 25 and 308C by using a LKB 2277 Bioactivity Monitor, and the thermodynamic and kinetic parameters of energy release of the mitochondria under different conditions have been calculated. The differences of the thermodynamic and kinetic characters of energy release of Guangchong 41 maintainer line rice mitochondria have been compared under different conditions.
2. Materials and methods
2.1. Materials The rice seed (Guangchong 41 maintainer line, in short GB) was provided by
P.-J. Zhou et al. / J. Biochem. Biophys. Methods 48 (2001) 1 – 11
3
Research Institute of Genetics, College of Life Sciences, Wuhan University, Wuhan, China.
2.2. Isolation of rice mitochondria The mitochondria were isolated from the GB rice type above according to Pring’s method [6]. Some changes in the experimental procedure have been made in the isolation process. The seeds of the rice (Oryza sative L) were sown in vermiculite in flats, which were placed in growth chambers at 258C in the absence of light. Seven to 10 days after planting, etiolated mesocotyl and coleoptile tissue was weighed and immediately homogenized for 30 s in a high speed warring blender. The cold homogenization buffer was 0.5 mol l 21 sucrose, 0.005 mol l 21 Na 2 EDTA, 0.1% bovine serum albumin (BSA), and 0.05 mol l 21 Tris–HC1, pH 7.5. The preparation was filtered through eight layers of cheesecloth prior to centrifugation for 13 min at 1000 3 g and 48C. The supernatant was then centrifuged for 20 min at 16 000 3 g and 48C. The pellets were resuspended in reaction buffer (0.3 mol l 21 sucrose, 0.4 mol l 21 MgCl 2 , 0.1% BAS, 0.01 mol l 21 21 Tris–HCl, pH 7.5 and deoxyribonuclease I to 0.05 mg l ) for 60 min at ordinary temperature to digest extro-mitochondrial DNA. The solution was then added to washing buffer (0.6 mol l 21 , 0.02 mol l 21 Na 2 EDTA, 0.1% BSA, 0.01 mol l 21 Tris–HCl, pH 8.0) in the same volume, then centrifuged for 20 min at 16 000 3 g and 48C twice. The pellet (mitochondria) of 0.3 g was resuspended in 0.25 ml of reserved buffer (0.001 mol l 21 Na 2 EDTA, 0.01 mol l 21 Tris–HCl, pH 8.0) and stored in a refrigerator at 0–38C for measurement. All the above were carried out under aseptic conditions.
2.3. Instrument A LKB 2277 Bioactivity Monitor, a new type of heat-flow microcalorimeter was used in this experiment. It is designed to continuously monitor a wide variety of processes and complex systems over the temperature range 20–808C. A schematic representation of the microcalorimetric system is shown in Fig. 1. Each measuring cylinder normally contains a sample and a reference in separate measuring cups (twin system). The heat output from the sample flows from the thermoelectric detector to the large heat sink (in close contact with the water bath). In response the detector produces a voltage which is proportional to the power output from the sample. In order to minimize the systematic error and disturbance effect, a differential or twin detector system is used. This system is very sensitive, the detection limit is 0.1 mW and the baseline stability (over a period of 24 h) is 0.2 mW. The performance of this instrument and the details of its construction have been previously described [7].
2.4. Experimental determination The thermogenesis curves of rice mitochondria were recorded by ampoule method, using two sterile sealed ampoules, one ampoule containing a reference solution such as
4
P.-J. Zhou et al. / J. Biochem. Biophys. Methods 48 (2001) 1 – 11
Fig. 1. Simplified operation diagram.
sterile reserved buffer, and the other containing the sample (suspension of the mitochondria). The sample normally occupied position A in the monitor, and the reference occupied position B. Each ampoule contained 1 ml of sample (which contained about 0.3 g of the mitochondria and was kept at 0–38C for 15 h and 40 days before the determination) or reference and 2 ml of air. The experimental temperature was at 308C and the amplifiers of the monitor were set at 10 mW. All the above were carried out under aseptic conditions.
3. Results and discussion
3.1. Thermogenesis curves and thermodynamics of energy release of rice mitochondria The thermogenesis curves of energy release of the rice mitochondria (which were kept
P.-J. Zhou et al. / J. Biochem. Biophys. Methods 48 (2001) 1 – 11
5
Fig. 2. Thermogenesis curve of energy release of rice mitochondria (which had been kept at 0–38C for 15 h before the determination) at 258C.
at 0–38C for 15 h and 40 days before the determination) at 258C and 308C are shown in Figs. 2–4. Mitochondria are semiautonomous organelles, containing large amounts of enzymes, and the enzymes which catalyse oxidation cellular reactions are the specific loci of the oxidation. These enzymes are still active after the isolation of mitochondria and there
Fig. 3. Thermogenesis curve of energy release of rice mitochondria (which had been kept at 0–38C for 15 h before the determination) at 308C.
6
P.-J. Zhou et al. / J. Biochem. Biophys. Methods 48 (2001) 1 – 11
Fig. 4. Thermogenesis curve of energy release of rice mitochondria (which had been kept at 0–38C for 40 days before the determination) at 308.
remains large amounts of nutrients in the mitochondria, hence enzymatic reactions can occur and release energy. The heat effects of the process can be monitored using sufficiently sensitive calorimeters and their thermogenesis curves obtained. Analysis of the thermogenesis curves showing the energy release of the rice mitochondria reveals four regions: the lag stage, increasing stage, stationary stage and decline stage. Sometimes the stationary stage is not very obvious, without any clear boundary at the stage. During isolation of the mitochondria, the enzymatic system of the mitochondria is damaged (and is in a resting condition at low temperature) so time is necessary for activity to be recovered and for adaptation to the new conditions. During this period, the lag stage, the thermogenesis curve is a horizontal straight line. These results indicate that the lag stage lasts for about 5 h at 308C and for about 30 h at 258C (Figs. 2 and 3). Once adequate adaptation has taken place, enzymatic activity gradually recovers, and both substance metabolism and energy metabolism starts, and the energy release of rice mitochondria increases, producing a logarithmic curve or sigmoid curve, i.e. the increasing stage. When all enzymes become active, the thermogenesis reaches a stationary stage without much change of the curves. When nutrients of the mitochondria and oxygen in the ampoule are exhausted, the curve enters the decline stage. Feedback inhibition of enzymes by metabolites would also contribute to this decline. The increasing time, maximal heat power and heat effect of energy release of the mitochondria at the increasing stage were determined, and are shown in Table 1. These results indicate that the lower the temperature, the slower the energy release of
P.-J. Zhou et al. / J. Biochem. Biophys. Methods 48 (2001) 1 – 11
7
Table 1 Thermodynamic parameters of the thermogenesis of energy release of the rice mitochondria at the thermogenesis-increasing stage Duration of storage at 0–38C
Temperature (8C)
Increasing time (h)
Max power (mW)
Heat effect (J g 21 )
Thermogenesis rate (J h 21 g 21 )
15 h 15 h 40 days
25 30 30
20 9 10
19.5 35.5 42.0
21.40 20.97 21.97
20.07 20.11 20.20
the rice mitochondria over the range of a certain temperature. Both the heat effect and the heat release rate of energy release of the mitochondria (which had been kept at a lower temperature for 40 days before the determination) increased at 308C, and the energy release process was noticeably shortened (Fig. 4).
3.2. Thermokinetics of the thermogenesis of rice mitochondria energy release As previously described, analysis of the thermogenesis curves of energy release of rice mitochondria reveals four stages: lag stage, increasing stage, stationary stage and decline stage. The increasing stage is a comparatively important stage among the four stages, the thermogenesis of the mitochondrial energy release reveals two kinetic increasing forms at the stage. One is a limitless increasing form, the thermogenesis curve assumes the form of a logarithmic curve (Fig. 3). The other is a limited increasing form, the thermogenesis curve assumes the form of a sigmoid curve (Figs. 2 and 4). The limitless increasing rate constants (k) have been calculated from the equation: Pt 5 Po exp (kt) or ln Pt 5 ln Po 1 kt, where Po is the initial thermal power of the thermogenesis
Table 2 Data for Pt and t of the rice mitochondria at the thermogenesis increasing stage Storage at 0–38C for 15 h (258C)
Storage at 0–38C for 15 h (308C)
Storage at 0–38C for 40 days (308C)
t (h)
Pt (mW)
t (h)
Pt (mW)
t (h)
Pt (mW)
2 4 6 8 10 12 14 16 18 20
2.4 2.8 3.2 4.0 5.5 7.5 10.0 12.7 15.7 18.6
1 2 3 4 5 6 7 8 2 2
4.6 5.7 8.1 13.0 16.0 22.5 31.5 35.5 2 2
1 2 3 4 5 6 8 9 2 2
14.5 17.5 21.5 25.0 30.0 33.5 38.0 39.5 40.7 2
P.-J. Zhou et al. / J. Biochem. Biophys. Methods 48 (2001) 1 – 11
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Table 3 Kinetic parameters of energy release of the rice mitochondria at the thermogenesis increasing stage Duration of storage at 0–38C
Temperature (8C)
k (h 21 )
r
15 h 15 h 40 days
25 30 30
0.1226 0.3110 0.3741
0.9952 0.9945 20.9966
at time 5 0, and Pt is the thermal power of the thermogenesis at time 5 t. The data for Pt and t as well as the values of the thermokinetic parameters are shown in Tables 2 and 3, respectively. The limited increasing process of the thermogenesis at the increasing stage presents a sigmoid curve, the curve can best be expressed as the logistic equation (8). In the limited increasing process, the quantity of the thermogenesis increase in the process is timerelated according to: dNt / dt 5 k(1 2 sNt )Nt 5 kNt 2 skN t2 5 kNt 2 b N t2
(1)
where k is the limited increasing rate constant, s is the increase inhibitory factor and Nt represents the quantity of the thermogenesis increase in the limited increasing process at time5t. The deceleration rate constant is given by b 5 sk. Assuming PN is the thermal power of the unit increasing quantity of the thermogenesis of energy release of the mitochondria in the limited increasing process, then Pt 5 PN Nt
(2)
whence dN 1 dP ]t 5 ] ]t dt PN dt
(3)
Compare Eq. (1) with Eq. (3), we have dP Pt 2 sP 2t ]t 5 k ]]] dt PN
(4)
integrating Eq. (4) yields Pt (1 2 s) ln ]]]] 5 kt sPN PN 1 2 ] Pt
S
D
(5)
or PN Pt 5 ]]]]]] s 1 (1 2 s) exp (2kt)
(6)
P.-J. Zhou et al. / J. Biochem. Biophys. Methods 48 (2001) 1 – 11
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Eq. (5) can be rewritten as
S
D S D
PN /s 12s ln ]] 2 1 5 ln ]] 2 kt Pt s
(7)
or
S
D S D
a 12s ln ] 2 1 5 ln ]] 2 kt Pt s
(8)
where a 5 PN /s. Using the data Pt and t of the limited increasing stage obtained from the thermogenesis curve of energy release of the mitochondria, suitable values of a have been chosen in the calculations to give the best linearity of Eq. (8). Hence the limited increasing rate constant (k), the increase inhibitory factor (s), the deceleration rate constant ( b ) and the thermal power of the unit increasing quantity of the thermogenesis of the mitochondrial energy release in the limited increasing process (PN ) can be obtained. The data for Pt and t as well as the thermokinetic parameters of the limited increase of the thermogenesis of the rice mitochondria energy release at 308C are shown in Tables 2 and 3, respectively. According to Tables 1–3 the corresponding experimental thermokinetic equations can be established. At the limitless increasing phase of the thermogenesis of rice mitochondria energy release (which was kept at 0–38C for 15 h before the determination): Pt 5 1.684 exp (0.1226t) Pt 5 3.334 exp (0.311t)
37 h < t < 57 h at 258C t < 7 h at 308C
At limited increasing phase of the thermogenesis of rice mitochondria energy release (which kept at 0–38C for 40 days before the determination): Pt 5 10.48 /(0.2283 1 0.7717) exp (20.3741t) t < 9 h at 308C These results indicated that the kinetic behavior in the increasing stage of energy release of the rice mitochondria at 258C is similar to that of energy release of the mitochondria at 308C, their thermogenesis kinetic increasing forms limitlessly increase but the increasing rate at 258C is lower than that of at 308C. Because the mitochondria had been kept at a low temperature for a long time, the membranes of the mitochondria were damaged, and the enzyme activities of the mitochondria are down, and oxidation and phosphorylation is uncoupled in the mitochondria. The energy of the mitochondria is released as heat after uncoupling. So the thermogenesis kinetic increasing form of the mitochondria was limited. In the limited increasing stage, the thermogenesis increased fast, and released more heat, its stationary stage was short, and the whole energy release process was noticeably shortened. Evidently the damaging mechanism of plants at low
10
P.-J. Zhou et al. / J. Biochem. Biophys. Methods 48 (2001) 1 – 11
temperature has much to do with the energy release of mitochondria [8], the problem is worth investigating further.
4. Simplified description of the method and its applications In recent years, microcalorimetry has been increasingly used in studies of various biological phenomena including studies at the cellular and subcellular level. The very broad application range for non-specific methods like calorimetry can be attractive both in thermodynamic measurements and in analytical work. As practically all processes are accompanied by heat effect, calorimetry is particularly well suited to the discovery of unexpected or unknown processes in samples of any aggregation state. Further, in contrast to spectroscopic methods, calorimetry does not require optically clear objects. In particular when heat conduction calorimeters are used, the experiments can be conducted over long periods or time — weeks or longer. These properties can make isothermal microcalorimeters ideal as monitors for slow and complex processes like the metabolism of plant mitochondria. In the paper, the thermogenesis curves of energy release of the mitochondria isolated from rice have been determined by using a LKB 2277 Bioactivity Monitor under different conditions. These curves contain much information. Through analysis of the curves, the thermodynamic and kinetic characteristics of the mitochondrial thermogenesis under different conditions have been studied, and the thermodynamic and kinetic parameters of the energy release process have been obtained, and some new experimental thermokinetic equations of the thermogenesis increasing stage in the energy release process of the rice mitochondria have been established. The characteristics of energy release of the rice mitochondria under different conditions can be identified with the microcalorimetric method. One can use the method and these results to characterize the ability of rice and other plants to release mitochondrial energy under different conditions.
Acknowledgements We gratefully acknowledge the financial support of the National Natural Science Foundation of China and State Key Laboratory of Freshwater Ecology and Biotechnology for this project.
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