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Molecular basis of age-dependent changes in the activity of adenine nucleotide translocase
Mechanisms of Ageing and Development, 14 (1980) 137-144
137
MOLECULAR BASIS OF AGE-DEPENDENT CHANGES IN THE ACTIVITY OF ADENINE NUCLEOTIDE TRANSLOCASE*
HANS NOHL t Institut ffir l~harmakologie, Universitdt Miinchen, Veterinitrstrasse 13, 8000 Munich 22 {F.R.G.)
REINHARD KR,~MER lnstitut fiir Physikalische Biochemie, UniversitiitMfinchen, Goethestrasse 33, 8000 Munich 2 (F.R.G.)
(Received December 3, 1979; in revised form April 10, 1980)
SUMMARY (1) Rat-heart mitochondria from 30-month-old animals are 40% less active in translocating adenine nucleotides across the inner membrane than 3-month-old rats. (2) The number of Sites available for binding the specific ligands to the adenine nucleotide carrier remains unchanged during aging. (3) The endogenous pool of the adenine nucleotides exhibits an age-dependent fall by more than 25%, essentially at the expense of ATP. The amount of ATP + ADP representing the exchangeable pool for adenine nucleotide translocation is decreased to the same extent. (4) Negatively charged phospholipids as well as polyunsaturated fatty acids of membrane lipids were found to be reduced with aging. (5) The results are discussed in terms of changes in the phospholipid-protein interactions due to the observed alterations in the physical state of the bulk phase of membrane lipids.
INTRODUCTION It appears now to be established that the cytoplasmic ATP/ADP × Pi ratio operates asthe most important regulatory factor of cellular homeostasis [1]. In order to perform this regulatory function in the cell, the ATP/ADP X Pi ratio must be maintained constant through operation of the control responses. Since oxidative phosphorylation is the principle source of cellular ATP and at the same time provides the major sink for ADP and
*Paper presented at the Sixth European Symposium on Basic Research in Gerontology, Munich, F.R.G., September 4-7, 1979. tTo whom all correspondence should be addressed.
138 inorganic phosphate (Pi), the importance of the exchange between extra- and intramitochondrial ADP, ATP and Pi becomes evident. The communication between the ATP-regenerating and the ATP-consuming systems involves specific carrier proteins facilitating the exchange of ATP/ADP and Pi across the inner mitochondrial membrane. The transport system most extensively investigated is the exchange between extra- and intramitochondrial ADP and ATP. This transport system has been studied from many viewpoints and has yielded an unusually great amount of knowledge. Our interest in studying this most important regulatory system of mitochondrial energy metabolism was based upon findings of our own [2, 3] and of other groups [4, 5] about age-dependent changes in biochemical pathways involved in the homeostasis of mitochondrial energy metabolism. The present investigation describes an age-related decrease in the ADP/ATP exchange rates. Different alterations associated with the latter were discussed to evaluate the molecular basis for the impaired adenine nucleotide translocase activity. In accordance with the other membrane-bound enzymes [3] it appears that the translocase activity is also highly sensitive to changes in the composition of the surrounding membrane lipids.
METHODS Rat-heart mitochondria (RHM) were prepared as described previously [6]. The translocase activity was assayed by back-exchange with prelabelled mitochondria. Labelling was performed by incubating RHM with ~4C-labelled ADP at 0 °C for 40 min. After washing to remove extramitochondrial radioactivity the stock solution was energized by the addition of glutamate (2 mM final conc.) and Pi (1 mM final conc.) at 6 °C for 5 min. The exchange was initiated by the addition of 200 mM unlabelled ADP to the energized RHM. The kinetic experiments were carried out with a rapid mixing, quenching and sampling apparatus as described by Palmieri and Klingenberg [7]. Carboxyatractylate (50 taM final conc.) was mixed with the reactants in order to stop the exchange after the various time intervals indicated in Fig. 1. The samples were instantly centrifuged in an Eppendorf microcentrifuge at 4 °C and the supernatant analysed for ~4C-labelled compounds. Material for the determination of the phosphorylation pattern was taken from the energized stock solution and measured enzymatically [8, 9] as well as by Dowex column chromatography [10]. The number of specific carrier binding sites were determined by use of 3H-labelled carboxyatractylate [11]. For chemical analysis of mitochondrial membrane lipids Folch extracts were made and run on silica-gel DC plates (Merck) for thin-layer chromatography. After a two-dimensional procedure using chloroform-methanol-ammonia (65:35:5) followed by chloroformmethanol--acetone-acetic acid(100%)-H20 (30:10:40:10:5) to separate the phospholipids, phosphorus was determined by the method of Bartlett [12]. Another fraction of the lipid extract was methylated with ZnCI in methanol. The methylated derivatives were separated by thin-layer chromatography using dichloroethane and evaporated to dryness with oxygen-free nitrogen. The methyl esters of the phospholipid fatty acids
139 were extracted by chloroform-methanol (1:1) and analysed in a Packard gas-liquid chromatograph using a flame-ionisation detector. Protein was measured by a modified biuret method using KCN in order to circumvent turbidity caused by contaminating lipids.
RESULTS
Influence of age on the activity of the adenine nucleotide exchange Preliminary investigations on the ADP/ATP exchange in RHM revealed the existence of a rapid onset phase of translocation accounting for two-thirds of the exchangeable pool within the first 5 sec at 6 °C. From this observation it may be concluded that adenine nucleotide translocation is especially susceptible to the regulatory process in its rapid initial part. To investigate this most interesting phase of the ADP/ATP exchange we used a quench-flow method, in which the kinetics are followed after the mixing process, allowing time resolution down to I00 msec [7]. Figure 1 shows the time course of adenine nucleotide exchange in RHM from 3- and 30-month-old animals. The exchange is based on the total 14C.labelled endogenous adenine nucleotide pool. Five minutes prior to starting the exchange, RHM were incubated with glutamate (2 mM) and potassium phosphate (1 mM). Three seconds after the initiation of the ADP/ATP translocation by the rapid addition and mixing of unlabelled ADP, the amount of adenine nucleotides equilibrated by RHM from young animals was found to be 40% faster than in heart mitochondria from old rats, In the following time period this difference remains unchanged, N~JmberOf CarrierBit
Ph~phorykation Pattern 3 30 month
3 month
1.03 (-'0051
30 month
0,99 (~0031
3
8
~6
v
@
c c
2
ol
Q 0
1
2
3
t.
5
6
Fig. 1. Comparison of the A D P / A T P exchange kinetics in RHM from 3- and 30-month-old animals at 6 °C. Experimental conditions: 1 m g protein X m1-1 was suspended in a 0.25 M sucrose buffer, containing 50 m M Hepes (pH 7.4), 2 m M glutamate and 1 mM Pi. The traces are each representative o f three experiments and were obtained from the arithmetic means. Statistical deviations between different preparations o f one age group were in the same range as those seen between the triplicates. The n u m b e r s o f specific carrier binding sites for adenine nucleotides (ADN) are associated with the corresponding kinetic curves ( m e a n ~: S.E.M.). The bars at the left represent the endogenous a m o u n t o f ATP (T), A D P (D) and AMP (M) in RHM from y o u n g adult and old animals. Other details are given in the text and in the Methods section.
140 indicating identical types of exchange kinetics for both preparations in the second part of translocation.
Age-dependency of the number of adenine nucleotide binding sites on the carrier protein The number of sites available for specific binding of the adenine nucleotides to the carrier may be of relevance for an evaluation of the age-dependent differences in the exchange kinetics. Therefore we have determined the number of specific binding sites for ADP and ATP under the conditions of the above exchange experiments using the 3Hcarboxyatractyloside binding method [11]. The numbers in Fig. 1 associated with the curves indicate the results for the preparations from 30-month-old and 3-month-old rats. A significant change in the availability of ADP/ATP-binding sites at the two different carrier proteins does not exist. Influence of age on the endogenous phosphorylation pattern of RHM Since the adenine nucleotide carrier has been shown to be inactive in translocating AMP [10] the rapid kinetics are linked exclusively to the endogenous amount of ATP plus ADP. Besides the size of this so-called exchangeable pool the energy charge depending on the relative amount of the adenine nucleotides is another factor which may be of importance for the equilibration of the endogenous adenine nucleotides. The analysis of the phosphorylation pattern has been performed with RHM respiring glutamate and inorganic phosphate (same conditions as for the kinetic experiments). The bars on the left in Fig. 1 represent the quantitative and qualitative analysis of adenine nucleotides in RHM from 3- and 30-month-old animals. The total amount of adenine nucleotides in mitochondria from aged animals is significantly decreased, essentially at the expense of ATP. Therefore the exchangeable pool is lowered to an extent of about 20% below that of the control. The energy charge of the RHM can be derived from the following equation: ATP + 1/2ADP ~c =
ATP + ADP + AMP
The calculated value for the aged animals is 0.88, scarcely differing from that of the young controls which was 0.89.
Age-related changes in membrane lipids Recent evidence from reconstitution studies of the adenine nucleotide carrier protein suggests that the activity of the tranglocase system is strongly dependent upon a more or less defined lipid environment. For example, the requirement of negatively charged phospholipids has been documented to be essential [13]. In order to test the existence of such an interaction we have analysed heart mitochondria from aged and young adult rats with respect to lipid composition. Table I compares the phospholipid pattern of mitochondrial membranes from old animals with the young controls. The most remarkable result of this analysis is the significant age-dependent decrease of negatively charged phospholipids, such as cardiolipin, phosphatidic acid, phosphatidylserine,
141 TABLE I AGE-DEPENDENT CHANGES IN PHOSPHOLIPID COMPOSITION CHONDRIA
OF RAT-HEART MITO-
Phospholipids were analyzed by two-dimensional thin-layer chromatography. Methodological details are described in the Methods section. The data represent percentage values of total membrane lipids (mean +- S.E.M.).
Phospholipids
Age (months) 3
Cardiolipin Phosphatidic acid Phosphatidylethanolamine Phosphatidylcholine Phosphatidyl serine Sphingomyelin Phosphatidylglycerol
30
13.1 2.8 39.6 38.6 5.4 0.7 0.3
(-+0.5) (-+0.3) (-+0.5) (-+0.5) (-+0.2) (-+0.1) (-+0.1)
8.5 1.8 41.0 43.1 4.3 0.8 -
(-+0.4) (-+0.3) (-+0.5) (-+0.6) (-+0.3) (+-0.2)
T A B L E II INFLUENCE OF AGE ON THE FATTY ACID COMPOSITION OF MEMBRANES FROM RATHEART MITOCHONDRIA The values are expressed as percentages of recording area of the total peak areas of fatty acids in gasliquid chromatography. Other details are given in the Methods section.
Fatty acids
14:0 14:1 15:0 i:16 16:0 16 : 1 17:0 17:1 18:0 18:1 18:2 19:0 18:3 20:0 20:1 21:0 20:4 22:1 22:4 24:0 22:5 22:6 Ratio unsaturated)saturated
Age (months) 3
30
0.15 0.17 0.15 0.29 8.90 0.45 0.41 0.31 17.70 6.10 30.30 0.31 0.14 0.23 0.33 0.34 18.20 0.17 0.57 0.44 2.04 12.60 2.47
0.12 0.08 0.10 0.60 11.67 0.35 0.37 0.32 20.23 6.58 19.05 0.14 0.07 0.21 0.18 0.27 21.57 0.17 0.36 0.28 2.06 15.30 1.94
142 and the complete disappearance of phosphatidylglycerol. Furthermore, a distinct increase of the phosphatidylcholine content could be observed as a function of age. Chemical changes in membrane lipids were further investigated, the fatty acids of the phospholipids being analyzed by means of gas-liquid chromatography. Table 1I depicts the age-dependency of the fatty acid composition in membranes of RHM. Polyunsaturated fatty acids exhibit a distinct decrease in lipids from old rats while the amount of saturated fatty acids was found to be increased. On the other hand, arachidonic acid (20:4) does not follow this trend but increases clearly with aging. The ratio of polyunsaturated to saturated fatty acids taken as an index of chemical changes in fatty acid composition shows a quantitative shift of unsaturated to saturated membrane lipids in the aged animals. This is due mainly to a drastic decrease of linoleic acid (18:2) associated with a distinct increase of palmitic (16:0) and stearic (18:0) acid.
DISCUSSION We have previously reported that enzymes associated with the lipid phase of the mitochondrial membrane exhibit reduced activities as the animals age [2, 3]. Thus a decrease in the ADP/ATP exchange as stated in the present paper was not unexpected. The complexity of this carrier system provides many possibilities which could alter the kinetics of the exchange. One of them is the physicochemical state of the membrane lipids. Evidence for the role of phospholipids in the activity of the adenine nucteotide translocator can be derived from reconstitution experiments with the isolated carrier protein [13, 14]. In accordance with our results, it was reported from this type of experiment that negatively charged phospholipids are essential for full activity of the ADP/ATP exchange. Furthermore, from the studies with the isolated carrier there is evidence that the phosphatidylcholine/phosphatidylethanolamine ratio in the membrane is another regulatory factor [ 14]. Accordingly, an increase in this ratio, as reported in this paper to occur as a function of age, should reduce the activity of the ADP/ATP exchange. Further support for the importance of membrane lipids in regulating the translocase activity comes from investigations with spin-labelled acyl-CoA derivatives [ 15]. From the latter experiment there is evidence that the physical state of the phospholipid environment is effective as a regulatory factor in determining the activity of the ADP/ATP translocator. Age-related changes in the physical state of the lipid membrane must be expected considering the data of the present report with respect to lipid analysis. In another report we have tested this question using electron-spin resonance to follow lipid phase transition [3]. The results of this investigation have indicated that aging is accompanied by a distinct decrease in lipid fluidity of the bulk phase of membrane lipids. Thus, beside the decrease of negatively charged phospholipids and the increase of the phosphatidylcholine/ phosphatidylethanolamine ratio, changes in the freedom of motion of membrane fatty acids may be another factor that regulates the activity of the ADP/ATP exchange in aged animals.
143 The number of specific binding sites of the carrier protein is not altered with aging, despite the development of drastic changes in the membrane lipids. This indicates that the availability of binding sites for the specific ligands is not subject to regulation by surrounding lipids, and cannot account for changes in the capacity to translocate adenine nucleotides. It has been shown earlier that kinetic analysis of the ADP/ATP exchange in the millisecond range does not reflect exchange kinetics as expected if the exchangeable pool were homogeneously reactive [16]. The nature of the biphasicity of the adenine nucleotide exchange is difficult to analyse and goes beyond the limits of the present work. However, considering the great amount of knowledge concerning this transport system it can be assumed that the exchange within the time range measured in this paper partially follows first-order kinetics. Thus the size of the exchangeable pool would be of importance for the evaluation of the first-order reaction constant but without effect on the translocation rates. The age-related decrease of the endogenous ADP + ATP pool is therefore not critical with respect to the translocation rates but must be taken into account in evaluating the regulation of the rate constants. The data of the present report are consistent with the following concept. Age-dependent changes in membrane lipids from RHM which we showed earlier to result as a consequence of radical-induced peroxidation [17] provide an important factor which influences the association of the lipidcarrier-protein components. In accordance with studies from reconstitution experiments, the activity of the ADP/ATP exchange might depend (a) on the amount of negatively charged phospholipids such as cardiolipin, (b) on the ratio of phosphatidylcholine to phosphatidylethanolamine, and (c) on maintaining a high lipid fluidity. Other factors that can influence the activity of the adenine nucleotide exchange, such as the energy charge, or the number of specific binding sites, do not alter, and thus cannot be responsible for the age-related changes in the activity of the ADP/ATP exchange.
ACKNOWLEDGMENTS The authors are greatly indebted to Professors Klingenberg and Hegner for their permanent interest in this study and for the facilities provided. We thank Petra Hauptmann for skilful technical assistance and Dr. Sybille Soboll who performed the enzymatic analysis of the phosphorylation pattern. This work was supported by grants from the Deutsche Forschungsgemeinschaft.
REFERENCES 1 M. Erecifiska, M. Stubbs, Y. Miyata, C. M. Ditre and D. F. Wilson, Distribution of metabolites between mitochondria and cytosol of cultured fibroblastoid rat heart cells. Biochirn. Biophys. Acta, 462 (1977) 20-35. 2 H. Nohl, V. Breuninger and D. Hegner, Influence of mitochondrial radical formation on energylinked respiration. Eur. J. Biochem., 90 (1978) 385-390.
144 3 H. Nohl, Influence of age on thermotropic kinetics of enzymes involved in mitochondrial energymetabolism. Z. Gerontol., 12 (1979) 9 - 1 8 . 4 J. C. Chen, J. B. Warshaw and D. R. Sanadi, Regulation of mitochondrial respiration in senescence. J. Cell. Comp. Physiol., 80 (1972) 141-148. 5 B. A. Bulos, S. P. Shukla and B. Sacktor, Bioenergetics of mitochondria from flight muscles of aging blowflies: partial reaction of oxidation and phosphorylation. Arch. Biochem. Biophys., 166 (1975) 6 3 9 - 6 4 4 . 6 L. Szarkowska and M'. Klingenberg, On the role of ubiquinone in mitochondria. Biochem. Z., 338 (1963) 674-697. 7 F. Palmieri and M. Klingenberg, Direct methods for measuring metabolite transport and distribution in mitochondria. Methods Enzymol., 56 (1979) 279-301, 8 W. Lamprecht and I. Trautschold, in H. U. Bergmeyer (ed.), Methoden der Enzymatischen Analyse, Verlag Chemie, Weinheim, 1970, pp. 2024-2033. 9 D. Jaworek, W. Gruber and H. U. Bergmeyer, in H. U. Bergmeyer (ed.), Methoden der Enzymatischen Analyse. Verlag Chemic, Weinheim, 1970, pp. 2051-2055. 10 E. Pfaff and M. Klingenberg, Adenine nucleotide translocation of mitochondria. Eur. J. Bioehem., 6 (1968) 6 6 - 7 9 . 11 M. Klingenberg, K. Grebe and B. Scherer, The binding of atractylate and carboxyatractylate to mitochondria. Eur. J. Biochem., 52 (1975) 351-363. 12 G. R. Bartlett, Phosphorus assay in column chromatography. J. Biol. Chem., 234 (1959) 4 6 6 - 4 7 1 . 13 R. Kr/imer and M. Klingenberg, Reconstitution of adenine nucleotide transport with purified ADP/ATP-carrier protein. FEBS Lett., 82 (1977) 363-367. 14 R. Kr~imer, unpublished results (1979). 15 P. F. Devaux, A. Bienvenue, G. Lauquin, A. D. Brisson, P. M. Vignais and P. V. Vignais, Interaction between spin-labelled acyl-coenzyme A and the mitochondrial adenosine diphosphate carrier. Biochemistry, 14 (1975) 1272-1280. 16 H. Nohl and M. Klingenberg, Kinetics of ADP, ATP transport in mitochondria as studied by the quench-flow method. Biochim. Biophys. Acta, 503 (1978) 155-169. 17 H. Nohl and D. Hegner, Do mitochondria produce oxygen radicals in vivo? Eur. J. Biochem., 82 (1978) 563-567.
137
MOLECULAR BASIS OF AGE-DEPENDENT CHANGES IN THE ACTIVITY OF ADENINE NUCLEOTIDE TRANSLOCASE*
HANS NOHL t Institut ffir l~harmakologie, Universitdt Miinchen, Veterinitrstrasse 13, 8000 Munich 22 {F.R.G.)
REINHARD KR,~MER lnstitut fiir Physikalische Biochemie, UniversitiitMfinchen, Goethestrasse 33, 8000 Munich 2 (F.R.G.)
(Received December 3, 1979; in revised form April 10, 1980)
SUMMARY (1) Rat-heart mitochondria from 30-month-old animals are 40% less active in translocating adenine nucleotides across the inner membrane than 3-month-old rats. (2) The number of Sites available for binding the specific ligands to the adenine nucleotide carrier remains unchanged during aging. (3) The endogenous pool of the adenine nucleotides exhibits an age-dependent fall by more than 25%, essentially at the expense of ATP. The amount of ATP + ADP representing the exchangeable pool for adenine nucleotide translocation is decreased to the same extent. (4) Negatively charged phospholipids as well as polyunsaturated fatty acids of membrane lipids were found to be reduced with aging. (5) The results are discussed in terms of changes in the phospholipid-protein interactions due to the observed alterations in the physical state of the bulk phase of membrane lipids.
INTRODUCTION It appears now to be established that the cytoplasmic ATP/ADP × Pi ratio operates asthe most important regulatory factor of cellular homeostasis [1]. In order to perform this regulatory function in the cell, the ATP/ADP X Pi ratio must be maintained constant through operation of the control responses. Since oxidative phosphorylation is the principle source of cellular ATP and at the same time provides the major sink for ADP and
*Paper presented at the Sixth European Symposium on Basic Research in Gerontology, Munich, F.R.G., September 4-7, 1979. tTo whom all correspondence should be addressed.
138 inorganic phosphate (Pi), the importance of the exchange between extra- and intramitochondrial ADP, ATP and Pi becomes evident. The communication between the ATP-regenerating and the ATP-consuming systems involves specific carrier proteins facilitating the exchange of ATP/ADP and Pi across the inner mitochondrial membrane. The transport system most extensively investigated is the exchange between extra- and intramitochondrial ADP and ATP. This transport system has been studied from many viewpoints and has yielded an unusually great amount of knowledge. Our interest in studying this most important regulatory system of mitochondrial energy metabolism was based upon findings of our own [2, 3] and of other groups [4, 5] about age-dependent changes in biochemical pathways involved in the homeostasis of mitochondrial energy metabolism. The present investigation describes an age-related decrease in the ADP/ATP exchange rates. Different alterations associated with the latter were discussed to evaluate the molecular basis for the impaired adenine nucleotide translocase activity. In accordance with the other membrane-bound enzymes [3] it appears that the translocase activity is also highly sensitive to changes in the composition of the surrounding membrane lipids.
METHODS Rat-heart mitochondria (RHM) were prepared as described previously [6]. The translocase activity was assayed by back-exchange with prelabelled mitochondria. Labelling was performed by incubating RHM with ~4C-labelled ADP at 0 °C for 40 min. After washing to remove extramitochondrial radioactivity the stock solution was energized by the addition of glutamate (2 mM final conc.) and Pi (1 mM final conc.) at 6 °C for 5 min. The exchange was initiated by the addition of 200 mM unlabelled ADP to the energized RHM. The kinetic experiments were carried out with a rapid mixing, quenching and sampling apparatus as described by Palmieri and Klingenberg [7]. Carboxyatractylate (50 taM final conc.) was mixed with the reactants in order to stop the exchange after the various time intervals indicated in Fig. 1. The samples were instantly centrifuged in an Eppendorf microcentrifuge at 4 °C and the supernatant analysed for ~4C-labelled compounds. Material for the determination of the phosphorylation pattern was taken from the energized stock solution and measured enzymatically [8, 9] as well as by Dowex column chromatography [10]. The number of specific carrier binding sites were determined by use of 3H-labelled carboxyatractylate [11]. For chemical analysis of mitochondrial membrane lipids Folch extracts were made and run on silica-gel DC plates (Merck) for thin-layer chromatography. After a two-dimensional procedure using chloroform-methanol-ammonia (65:35:5) followed by chloroformmethanol--acetone-acetic acid(100%)-H20 (30:10:40:10:5) to separate the phospholipids, phosphorus was determined by the method of Bartlett [12]. Another fraction of the lipid extract was methylated with ZnCI in methanol. The methylated derivatives were separated by thin-layer chromatography using dichloroethane and evaporated to dryness with oxygen-free nitrogen. The methyl esters of the phospholipid fatty acids
139 were extracted by chloroform-methanol (1:1) and analysed in a Packard gas-liquid chromatograph using a flame-ionisation detector. Protein was measured by a modified biuret method using KCN in order to circumvent turbidity caused by contaminating lipids.
RESULTS
Influence of age on the activity of the adenine nucleotide exchange Preliminary investigations on the ADP/ATP exchange in RHM revealed the existence of a rapid onset phase of translocation accounting for two-thirds of the exchangeable pool within the first 5 sec at 6 °C. From this observation it may be concluded that adenine nucleotide translocation is especially susceptible to the regulatory process in its rapid initial part. To investigate this most interesting phase of the ADP/ATP exchange we used a quench-flow method, in which the kinetics are followed after the mixing process, allowing time resolution down to I00 msec [7]. Figure 1 shows the time course of adenine nucleotide exchange in RHM from 3- and 30-month-old animals. The exchange is based on the total 14C.labelled endogenous adenine nucleotide pool. Five minutes prior to starting the exchange, RHM were incubated with glutamate (2 mM) and potassium phosphate (1 mM). Three seconds after the initiation of the ADP/ATP translocation by the rapid addition and mixing of unlabelled ADP, the amount of adenine nucleotides equilibrated by RHM from young animals was found to be 40% faster than in heart mitochondria from old rats, In the following time period this difference remains unchanged, N~JmberOf CarrierBit
Ph~phorykation Pattern 3 30 month
3 month
1.03 (-'0051
30 month
0,99 (~0031
3
8
~6
v
@
c c
2
ol
Q 0
1
2
3
t.
5
6
Fig. 1. Comparison of the A D P / A T P exchange kinetics in RHM from 3- and 30-month-old animals at 6 °C. Experimental conditions: 1 m g protein X m1-1 was suspended in a 0.25 M sucrose buffer, containing 50 m M Hepes (pH 7.4), 2 m M glutamate and 1 mM Pi. The traces are each representative o f three experiments and were obtained from the arithmetic means. Statistical deviations between different preparations o f one age group were in the same range as those seen between the triplicates. The n u m b e r s o f specific carrier binding sites for adenine nucleotides (ADN) are associated with the corresponding kinetic curves ( m e a n ~: S.E.M.). The bars at the left represent the endogenous a m o u n t o f ATP (T), A D P (D) and AMP (M) in RHM from y o u n g adult and old animals. Other details are given in the text and in the Methods section.
140 indicating identical types of exchange kinetics for both preparations in the second part of translocation.
Age-dependency of the number of adenine nucleotide binding sites on the carrier protein The number of sites available for specific binding of the adenine nucleotides to the carrier may be of relevance for an evaluation of the age-dependent differences in the exchange kinetics. Therefore we have determined the number of specific binding sites for ADP and ATP under the conditions of the above exchange experiments using the 3Hcarboxyatractyloside binding method [11]. The numbers in Fig. 1 associated with the curves indicate the results for the preparations from 30-month-old and 3-month-old rats. A significant change in the availability of ADP/ATP-binding sites at the two different carrier proteins does not exist. Influence of age on the endogenous phosphorylation pattern of RHM Since the adenine nucleotide carrier has been shown to be inactive in translocating AMP [10] the rapid kinetics are linked exclusively to the endogenous amount of ATP plus ADP. Besides the size of this so-called exchangeable pool the energy charge depending on the relative amount of the adenine nucleotides is another factor which may be of importance for the equilibration of the endogenous adenine nucleotides. The analysis of the phosphorylation pattern has been performed with RHM respiring glutamate and inorganic phosphate (same conditions as for the kinetic experiments). The bars on the left in Fig. 1 represent the quantitative and qualitative analysis of adenine nucleotides in RHM from 3- and 30-month-old animals. The total amount of adenine nucleotides in mitochondria from aged animals is significantly decreased, essentially at the expense of ATP. Therefore the exchangeable pool is lowered to an extent of about 20% below that of the control. The energy charge of the RHM can be derived from the following equation: ATP + 1/2ADP ~c =
ATP + ADP + AMP
The calculated value for the aged animals is 0.88, scarcely differing from that of the young controls which was 0.89.
Age-related changes in membrane lipids Recent evidence from reconstitution studies of the adenine nucleotide carrier protein suggests that the activity of the tranglocase system is strongly dependent upon a more or less defined lipid environment. For example, the requirement of negatively charged phospholipids has been documented to be essential [13]. In order to test the existence of such an interaction we have analysed heart mitochondria from aged and young adult rats with respect to lipid composition. Table I compares the phospholipid pattern of mitochondrial membranes from old animals with the young controls. The most remarkable result of this analysis is the significant age-dependent decrease of negatively charged phospholipids, such as cardiolipin, phosphatidic acid, phosphatidylserine,
141 TABLE I AGE-DEPENDENT CHANGES IN PHOSPHOLIPID COMPOSITION CHONDRIA
OF RAT-HEART MITO-
Phospholipids were analyzed by two-dimensional thin-layer chromatography. Methodological details are described in the Methods section. The data represent percentage values of total membrane lipids (mean +- S.E.M.).
Phospholipids
Age (months) 3
Cardiolipin Phosphatidic acid Phosphatidylethanolamine Phosphatidylcholine Phosphatidyl serine Sphingomyelin Phosphatidylglycerol
30
13.1 2.8 39.6 38.6 5.4 0.7 0.3
(-+0.5) (-+0.3) (-+0.5) (-+0.5) (-+0.2) (-+0.1) (-+0.1)
8.5 1.8 41.0 43.1 4.3 0.8 -
(-+0.4) (-+0.3) (-+0.5) (-+0.6) (-+0.3) (+-0.2)
T A B L E II INFLUENCE OF AGE ON THE FATTY ACID COMPOSITION OF MEMBRANES FROM RATHEART MITOCHONDRIA The values are expressed as percentages of recording area of the total peak areas of fatty acids in gasliquid chromatography. Other details are given in the Methods section.
Fatty acids
14:0 14:1 15:0 i:16 16:0 16 : 1 17:0 17:1 18:0 18:1 18:2 19:0 18:3 20:0 20:1 21:0 20:4 22:1 22:4 24:0 22:5 22:6 Ratio unsaturated)saturated
Age (months) 3
30
0.15 0.17 0.15 0.29 8.90 0.45 0.41 0.31 17.70 6.10 30.30 0.31 0.14 0.23 0.33 0.34 18.20 0.17 0.57 0.44 2.04 12.60 2.47
0.12 0.08 0.10 0.60 11.67 0.35 0.37 0.32 20.23 6.58 19.05 0.14 0.07 0.21 0.18 0.27 21.57 0.17 0.36 0.28 2.06 15.30 1.94
142 and the complete disappearance of phosphatidylglycerol. Furthermore, a distinct increase of the phosphatidylcholine content could be observed as a function of age. Chemical changes in membrane lipids were further investigated, the fatty acids of the phospholipids being analyzed by means of gas-liquid chromatography. Table 1I depicts the age-dependency of the fatty acid composition in membranes of RHM. Polyunsaturated fatty acids exhibit a distinct decrease in lipids from old rats while the amount of saturated fatty acids was found to be increased. On the other hand, arachidonic acid (20:4) does not follow this trend but increases clearly with aging. The ratio of polyunsaturated to saturated fatty acids taken as an index of chemical changes in fatty acid composition shows a quantitative shift of unsaturated to saturated membrane lipids in the aged animals. This is due mainly to a drastic decrease of linoleic acid (18:2) associated with a distinct increase of palmitic (16:0) and stearic (18:0) acid.
DISCUSSION We have previously reported that enzymes associated with the lipid phase of the mitochondrial membrane exhibit reduced activities as the animals age [2, 3]. Thus a decrease in the ADP/ATP exchange as stated in the present paper was not unexpected. The complexity of this carrier system provides many possibilities which could alter the kinetics of the exchange. One of them is the physicochemical state of the membrane lipids. Evidence for the role of phospholipids in the activity of the adenine nucteotide translocator can be derived from reconstitution experiments with the isolated carrier protein [13, 14]. In accordance with our results, it was reported from this type of experiment that negatively charged phospholipids are essential for full activity of the ADP/ATP exchange. Furthermore, from the studies with the isolated carrier there is evidence that the phosphatidylcholine/phosphatidylethanolamine ratio in the membrane is another regulatory factor [ 14]. Accordingly, an increase in this ratio, as reported in this paper to occur as a function of age, should reduce the activity of the ADP/ATP exchange. Further support for the importance of membrane lipids in regulating the translocase activity comes from investigations with spin-labelled acyl-CoA derivatives [ 15]. From the latter experiment there is evidence that the physical state of the phospholipid environment is effective as a regulatory factor in determining the activity of the ADP/ATP translocator. Age-related changes in the physical state of the lipid membrane must be expected considering the data of the present report with respect to lipid analysis. In another report we have tested this question using electron-spin resonance to follow lipid phase transition [3]. The results of this investigation have indicated that aging is accompanied by a distinct decrease in lipid fluidity of the bulk phase of membrane lipids. Thus, beside the decrease of negatively charged phospholipids and the increase of the phosphatidylcholine/ phosphatidylethanolamine ratio, changes in the freedom of motion of membrane fatty acids may be another factor that regulates the activity of the ADP/ATP exchange in aged animals.
143 The number of specific binding sites of the carrier protein is not altered with aging, despite the development of drastic changes in the membrane lipids. This indicates that the availability of binding sites for the specific ligands is not subject to regulation by surrounding lipids, and cannot account for changes in the capacity to translocate adenine nucleotides. It has been shown earlier that kinetic analysis of the ADP/ATP exchange in the millisecond range does not reflect exchange kinetics as expected if the exchangeable pool were homogeneously reactive [16]. The nature of the biphasicity of the adenine nucleotide exchange is difficult to analyse and goes beyond the limits of the present work. However, considering the great amount of knowledge concerning this transport system it can be assumed that the exchange within the time range measured in this paper partially follows first-order kinetics. Thus the size of the exchangeable pool would be of importance for the evaluation of the first-order reaction constant but without effect on the translocation rates. The age-related decrease of the endogenous ADP + ATP pool is therefore not critical with respect to the translocation rates but must be taken into account in evaluating the regulation of the rate constants. The data of the present report are consistent with the following concept. Age-dependent changes in membrane lipids from RHM which we showed earlier to result as a consequence of radical-induced peroxidation [17] provide an important factor which influences the association of the lipidcarrier-protein components. In accordance with studies from reconstitution experiments, the activity of the ADP/ATP exchange might depend (a) on the amount of negatively charged phospholipids such as cardiolipin, (b) on the ratio of phosphatidylcholine to phosphatidylethanolamine, and (c) on maintaining a high lipid fluidity. Other factors that can influence the activity of the adenine nucleotide exchange, such as the energy charge, or the number of specific binding sites, do not alter, and thus cannot be responsible for the age-related changes in the activity of the ADP/ATP exchange.
ACKNOWLEDGMENTS The authors are greatly indebted to Professors Klingenberg and Hegner for their permanent interest in this study and for the facilities provided. We thank Petra Hauptmann for skilful technical assistance and Dr. Sybille Soboll who performed the enzymatic analysis of the phosphorylation pattern. This work was supported by grants from the Deutsche Forschungsgemeinschaft.
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