Exp. Geroat. Vol. 12. pp. 1-5. PergamonPress
1977. Printed in Great Britain.
AGE-RELATED CHANGES IN MITOCHONDRIAL FUNCTION IN DROSOPHILA MELANOGASTER A. C. VANN* and G. C. WEBSTER Department of Biological Sciences, Florida Institute of Technology, Melbourne, FL, U.S.A. (Received 15 May 1976)
INTRODUCTION KNOWLEDGE of the age-related changes that occur in cellular structure and function is likely to be essential for an understanding of senescence. However, as Franks (1974) has pointed out, there is still remarkably little information available on these changes. Among the cellular structures, the mitochondria are of particular interest, because the mitochondrial production of ATP is necessary for most cellular activities, and an agerelated decrease in mitochondrial activity could have widespread effects on the continued life of the cell. From the work of several investigators (Weinbach and Garbus, 1956, 1959; Barrows et aL, 1960; Gold et aL, 1968), it seems clear that isolated mitochondria from the liver, brain, kidney, and heart of aged rats exhibit no significant decrease in the efficiency of ATP synthesis. However, there is considerable mitochondrial replacement in these rat organs, even in aged rats (Menzies and Gold, 1971), and Wilson and Franks (1975), in a study of age-related changes in mitochondrial structure in mouse liver, have noted that the abnormal mitochondria found in aging cells are lost during the fractionation of disrupted cells for mitochondrial isolation. Thus, studies of isolated mammalian mitochondria may select for recently-formed mitochondria which have normal phosphorylative efficiency. A different situation appears to exist in aging Diptera. In these organisms, changes in mitochondrial structure, similar to those observed by Wilson and Franks (1975) in mouse liver, have been reported by many investigators (Rockstein and Bhatnagar, 1965; Takahashi et aL, 1970; Sohal and Allison, 1971 ; Saktor and Shimada, 1972; Sohal, 1975). However, in contrast to the relatively rapid replacement of rat mitochondria (Menzies and Gold, 1971), there is little or no turnover of mitochondria of the fruitfly, Drosophila (MaynardSmith et al., 1971), or the blowfly, Calliphora (Tribe and Ashhurst, 1972). Furthermore, increasing age in Calliphora results in a marked decrease in ATP production by isolated mitochondria (Tribe and Ashhurst, 1972). These observations are potentially quite significant, because they indicate that aging mitochondria may lose their ability to produce ATP, unless replaced by the processes of turnover. We were, therefore, interested in learning whether the observations of Tribe and Ashhurst (1972) were an isolated instance, or were representative of the situation when mitochondrial turnover is low or non-existent. Since Maynard-Smith et aL (1970) reported no significant turnover of Drosophila mitochondria, we have studied the effect of age on phosphorylative efficiency of Drosophila mitochondria. Our findings show not only an age-related decrease in ATP synthesis, but also a decrease in mitochondrial ATPase, and the accumulation of a cytoplasmic inhibitor of ATP synthesis. *Present address: Department of Zoology, University of Florida, Gainesville, FL, U.S.A.
2
A . C . VANN AND G. C. WEBSTER
MATERIALS AND METHODS The Oregon R strain of Drosophila melanogaster was grown at 25° in clear plastic containers on a medium (Demerec and Kaufmann, 1967) consisting of (per liter): 500 g banana pulp, 15 g agar, 7 ml of a 10~o solution of methyl-p-hydroxybenzoate in 95 ~o ethanol and 478 ml water. Flies were transferred to fresh medium each week.
Preparation of mitochondria All preparative operations were performed at 0--4°. After immobilization by cooling to 4~, 100-500 flies were homogenized for 30 sec in a Potter-Elvejem glass homogenizer containing 5 ml of preparation medium (see below). The preparation was filtered through gauze, and centrifuged at 150 × g for 5 min. The sediment was discarded. Mitochondria were sedimented from the supernatant fluid by centrifugation at 3000 × g for 8 min (Van den Bergh, 1967). The mitochondrial pellet was rinsed two times in the preparation medium, and suspended in two ml of the same medium. The procedure yielded 1-5 mg mitochondrial protein per ml. Two different media were compared for the preparation of mitochondria. The first contained 0.01 M Tris (hydroxymethyl) aminomethane (pH 7"4), 0.15 M KCI, and 0.001 M sodium ethylenediaminetetraacetate, and was used by Bulos et al. (1972) for the preparation of blowfly mitochondria. The second medium contained 0.30 M sucrose, 0"02 M potassium phosphate (pH 7.2), 0.001 M sodium ethylenediaminetetraacetate and 0.5 ~ cysteine (Dounce et al., 1972), and is similar to sucrose-containing media used for mitochondrial isolation from both animals and plants. Since mitochondria isolated in the sucrose-containing medium exhibited a respiratory control ratio (oxygen consumption in the presence of ADP/oxygen consumption in the absence of ADP) of 1-2 during oxidation of 0.01 M pyruvate-malate, while the respiratory control ratio of mitochondria isolated in the KCl-containing medium was 1.9, the KCl-containing medium was used for all subsequent preparations. The reaction medium for measurement of oxidative phosphorylation consisted of: 0.15 M KCI, 0.01 M tris (hydroxymethyl) aminomethane (pH 7.4), 0.005 M MgCI2, 0.004 M potassium phosphate (pH 7.4), 0.001 M sodium ADP, 0.001 M sodium ethylenediaminetetraacetate, 0.1 M glucose, 1 mg crystalline hexokinase (Sigma, Type III) in 0.1 M glucose, 0'01 M sodium pyruvate, 0.01 M sodium malate and 1-5 mg of mitochondrial protein in a total volume of 5 ml. Oxygen consumption was measured polarographically at 27° using a YSI Model 55 oxygen monitor (Yellow Springs Instruments, Yellow Springs, OH, U.S.A.). Orthophosphate disappearance was measured spectrophotometrically, according to Lindberg and Ernster (1956), after partition of orthophosphate and organic phosphates between benzene-isobutanol and silicotungstic acid. ATPase activity was determined as described by Pullman et al. (1960). ADP : 0 ratios were determined according to Bulos et al. (1972). Protein was determined with the biuret procedure described by Gornall et al. (1949), using 18 % sodium deoxycholate to insure complete solubilization of mitochondrial proteins (Webster, 1969). RESULTS
Effect o f age on oxidative phosphorylation T h e rates o f oxygen c o n s u m p t i o n a n d o r t h o p h o s p h a t e d i s a p p e a r a n c e , catalyzed by Drosophila m i t o c h o n d r i a o f different ages, are presented in Fig. 1. T h a t the m e a s u r e d p h o s p h a t e c o n s u m p t i o n was due to oxidative p h o s p h o r y l a t i o n is indicated b y the observ a t i o n t h a t there was no c h a n g e in o r t h o p h o s p h a t e level in the presence o f 10-5 M 2,4d i n i t r o p h e n o l . T h e rate o f oxygen c o n s u m p t i o n increased steadily with increasing age o f the organisms. T h e rate o f p h o s p h a t e d i s a p p e a r a n c e r o u g h l y p a r a l l e l e d oxygen c o n s u m p t i o n d u r i n g m u c h o f a d u l t life. H o w e v e r , very o l d o r g a n i s m s exhibited a m a r k e d r e d u c t i o n in p h o s p h a t e fixation. This reduction, t o g e t h e r w i t h the c o n t i n u e d increase in the rate o f oxygen c o n s u m p t i o n , resulted in a l m o s t a 50 ~ decrease in the efficiency o f p h o s p h o r y lation, as m e a s u r e d b y the P / O ratio, in the oldest o r g a n i s m s studied. Similar results were o b t a i n e d when p h o s p h o r y l a t i o n efficiency was m e a s u r e d by the A D P : O ratio (Fig. 1).
Effect o f age on mitoehondrial A TPase Since increased age resulted in a decrease in the i n c o r p o r a t i o n o f o r t h o p h o s p h a t e into A T P , b u t no decrease in the rate o f oxygen c o n s u m p t i o n , it seemed possible t h a t increased
AGE-RELATED CHANGES IN M1TOCHONDKIAL FUNCTION IN
-
Drosophila melanogaster
3
.o8
.oe
~o...,,~ / \
.04
x
~ d.- - 0 2
1.2
o
o
~0.6 0.3 !
I
I
8
32
,8
AGE (DAYS)
FIG. 1. Oxygen consumption, phosphate incorporation into ATP, P/O ratio, and ADP : O ratio in mitochondria isolated from Drosophila melanogaster of different ages. Reaction system was that described in the Methods Section.
age particularly affected the mitochondrial phosphorylating system. Although the enzymes involved in oxidative phosphorylation are not understood clearly, there is considerable evidence that the mitochondrial FI-ATPase, described originally by Pullman et al. (1960), catalyzes the synthesis of ATP from ADP and phosphate during oxidative phosphorylation, and would thus be critical for the synthesis of ATP. ATPase activity, assayed as described by Pullman et al. (1960) with mitochondria from flies of different ages, is presented in Fig. 2. The ATPase activity increased in adults, and then decreased abruptly in very old Z 1¢1 I--
0 .020
nil.
,s .015 =-.
~
W
.OlD
¢r ~ .005
•
0
n I -1 o
I0
I
I
I
I,
20
30
40
50
AGE
(DAY)
FIG. 2. ATPase activity of mitochondria from Drosophila melanogaster of different ages. ATPase assay was that described by Pullman et ai. (1960).
4
A . C . VANN AND G. C. WEBSTER
TABLE 1. EFFECT OF CYTOPLASM FROM FLIES OF DIFFERENT AGES ON OXIDATIVE PHOSPHORYLATION BY MITOCHONDRIA OF YOUNG FLIES
Age of flies Phosphate incorporated furnishing into ATP Oxygen consumed cytoplasm (hamole/min/mg (la atom/min/mg protein) protein)
P/O
1-9 days 4147 days 45-54 days
0.48 0.32 0-14
0"023 0.017 0.006
0.048 0.053 0.043
Mitochondria were from 1-9 day-old Drosophilamelanogaster.Assay procedure was that described in the Methods section, except 1 ml of the supernatant solution from centrifugation (3000 x g for 8 min) of the cytoplasm from flies of the indicated ages was substituted for 1 ml of assay medium. organisms in the same manner as the P/O ratio. If, as seems likely, F~-ATPase is a key enzyme in ATP synthesis, then the decrease in phosphorylation could be related to the greatly decreased activity of the ATPase.
Inhibition of oxidative phosphorylation by cytoplasmicfactors accumulating during aging Regardless of whether there was a causal relationship between the decrease in ATPase activity and the decrease in phosphorylation, the reason for the decrease was of considerable interest. Since there is little or no replacement of Drosophila mitochondria, the accumulation of defective mitochondria by an error-introducing process (Orgel, 1963) seemed unlikely. Instead, some kind of mitchondrial degradative process seemed more probable. Although a spontaneous internal degradation was possible, we were interested in the possibility of degradation or inhibition of the mitochondrial phosphorylating system by cytoplasmic factors. In order to study this, mitochondria from young (1-9 day-old) flies were assayed for phosphorylation activity in the presence of cytoplasm (centrifuged for 8 min at 3000 x g to remove heavy particles) from flies of increasing age. As can be seen from Table 1, oxidation was relatively unaffected by the presence of cytoplasm. However, phosphate incorporation into ATP was inhibited by cytoplasm from flies of increasing age to the extent that cytoplasm from very old flies almost eliminated the ability of the young mitochondria to form ATP. It appears that increasing age resulted in the accumulation of a factor which inhibited or degraded the phosphorylating ability of the mitochondria. DISCUSSION The results of this study, when viewed along with the findings of other investigators (Weinbach and Garbus, 1959; Gold et al., 1968; Tribe and Ashhurst, 1972) are in accord with the following conclusions regarding the effects of age on mitochondrial activity. In cells, such as the mammalian cells studied thus far, the constant replacement of mitochondria (Menzies and Gold, 1971) results in the isolation, from aged organisms, of mitochondria exhibiting no apparent decrease in phosphorylative efficiency. However, in those adult Dipterans where little or no mitochondrial replacement has been observed, isolated mitochondria show a significant decrease in phosphorylative efficiency. It would be of interest to know whether decrease in mitochondrial turnover, and decrease in efficiency of phosphorylation are general phenomena in aging organisms. In view of the observation of Wilson and Franks (1975) that the "abnormal" mitochondria observed in aged mouse liver cells are not seen in preparations of mitochondria isolated from these cells, it would also be of interest to know whether phosphorylative efficiency (per cell) of intact mam-
AGE-RELATEDCHANGESIN MITOCHONDRIALFUNCTION IN Drosophila melanogaster
5
malian cells decreases with age, and whether there are mammalian tissues composed of cells where mitochondrial replacement is so slow that a decay in phosphorylative efficiency is observed. In Drosophila mitochondria, there appear to be two kinds of decrease in phosphorylative efficiency. First, the mitochondria, when separated from other cellular components, and studied in the usual sucrose-buffer system, exhibit a definite decrease in phosphorylative efficiency. In addition to this, however, there appears to be an accumulation in the cytoplasm of an inhibitor of oxidative phosphorylation. I f both phenomena occur in intact cells, A T P production could be essentially eliminated at an advanced age. The identification of this inhibitor is of considerable interest. It would also be of interest to know whether aging mammalian cells exhibit a similar formation of inhibitory material in the cytoplasm. SUMMARY Aging o f Drosophila melanogaster resulted in a steady increase in the rate of mitochondrial oxidation of pyruvate-malate. At advanced ages, the rate of phosphate incorporation into A T P decreased markedly. This decrease in ATP synthesis was paralleled by a decrease in the activity of mitochondrial ATPase, a probable component of the phosphorylating system. Aging cells steadily accumulated an inhibitor which strongly depressed ATP synthesis when the cytosol of aged cells was incubated with the mitochondria of young cells. REFERENCES BARROWS, C. H., FALZONE, J. A. and SHOCK, N. W. (1960) J. Geront. 15, 130. BULOS,B., SHUKLA,S. arid SAKTOR,B. (1972) Arch. biochem. Biophys. 149, 461. DEMEREC,M. and KAUFMANN,B. P. (1967) Drosophila Guide: Introduction to the Genetics and Cytology of Drosophila melanogaster, p. 10. Carnegie Institution, Washington. FRANKS,L. M. (1974) Gerontologia 20, 51. GOLD, P. H., GEE, M. V. and STREHLER,B. L. (1968) J. Geront. 23, 509. GORNALL,A. C., BARDAWILL,C. J. and DAVID,M. M. (1949) J. biol. Chem. 177, 751. LIZ,a3BERG, O. and ERNSTER,L. (1956) In: Methods of Biochemical Analysis (Edited by D. GLICK),Vol. 3, p. 1. Wiley, New York. MA~Ago-SmTH, J., BOZCUK,A. N. and TFBBUTT,S. (1970) J. Insect Physiol. 16, 601. MEN2aES, g. A. and GOLD,P. H. (1971) d. biol. Chem. 246, 2425. ORGEL, L. (1963) Proe. Nat. Acad. Sei. U.S. 49, 517. PULLMAN,M. E., PENEFSKY,H. S., DATTA,A. and RAcrdsR,E. (1960) J. biol. Chem. 235, 3322. ROCKSTEIN,M. and BHATNAGAR,P. L. (1965) J. Insect Physiol. 11, 481. SAKTOR, B. and SHIMADA,Y. (1972) J. Cell Biol. 52, 465. SOtIAL,R. S. (1975) J. Morph. 145, 337. SOI-IAL,R. S. and ALUSON,V. F. (1971) Exp. Geront. 6, 167. TAKAHASm,A., PmLPOTr,D. E. and MIQtmL,J. (1970) d. Geront. 25, 222. TRINE,M. A. and ASI-II-ItmST,D. E. (1972) J. Cell Sci. 10, 443. VANDENBERGH,S. (1967) In: Methods in Enzymology (Edited by R. W. ESTABROOKand M. E. PULLMAN), Vol. 10, p. 117. Academic Press, New York. WEnSTER,G. C. (1969) J. Cell Biol. 43, 154a. WE~InACH,E. C. and GARnUS,J. (1956) Nature, Lond. 178, 1225. WErNnACH,E. C. and GAP.BUS,J. (1959) J. biol. Chem. 234, 412. WILSON,P. D. and FRANKS,L. M. (1975) Gerontologia 21, 81.
1977. Printed in Great Britain.
AGE-RELATED CHANGES IN MITOCHONDRIAL FUNCTION IN DROSOPHILA MELANOGASTER A. C. VANN* and G. C. WEBSTER Department of Biological Sciences, Florida Institute of Technology, Melbourne, FL, U.S.A. (Received 15 May 1976)
INTRODUCTION KNOWLEDGE of the age-related changes that occur in cellular structure and function is likely to be essential for an understanding of senescence. However, as Franks (1974) has pointed out, there is still remarkably little information available on these changes. Among the cellular structures, the mitochondria are of particular interest, because the mitochondrial production of ATP is necessary for most cellular activities, and an agerelated decrease in mitochondrial activity could have widespread effects on the continued life of the cell. From the work of several investigators (Weinbach and Garbus, 1956, 1959; Barrows et aL, 1960; Gold et aL, 1968), it seems clear that isolated mitochondria from the liver, brain, kidney, and heart of aged rats exhibit no significant decrease in the efficiency of ATP synthesis. However, there is considerable mitochondrial replacement in these rat organs, even in aged rats (Menzies and Gold, 1971), and Wilson and Franks (1975), in a study of age-related changes in mitochondrial structure in mouse liver, have noted that the abnormal mitochondria found in aging cells are lost during the fractionation of disrupted cells for mitochondrial isolation. Thus, studies of isolated mammalian mitochondria may select for recently-formed mitochondria which have normal phosphorylative efficiency. A different situation appears to exist in aging Diptera. In these organisms, changes in mitochondrial structure, similar to those observed by Wilson and Franks (1975) in mouse liver, have been reported by many investigators (Rockstein and Bhatnagar, 1965; Takahashi et aL, 1970; Sohal and Allison, 1971 ; Saktor and Shimada, 1972; Sohal, 1975). However, in contrast to the relatively rapid replacement of rat mitochondria (Menzies and Gold, 1971), there is little or no turnover of mitochondria of the fruitfly, Drosophila (MaynardSmith et al., 1971), or the blowfly, Calliphora (Tribe and Ashhurst, 1972). Furthermore, increasing age in Calliphora results in a marked decrease in ATP production by isolated mitochondria (Tribe and Ashhurst, 1972). These observations are potentially quite significant, because they indicate that aging mitochondria may lose their ability to produce ATP, unless replaced by the processes of turnover. We were, therefore, interested in learning whether the observations of Tribe and Ashhurst (1972) were an isolated instance, or were representative of the situation when mitochondrial turnover is low or non-existent. Since Maynard-Smith et aL (1970) reported no significant turnover of Drosophila mitochondria, we have studied the effect of age on phosphorylative efficiency of Drosophila mitochondria. Our findings show not only an age-related decrease in ATP synthesis, but also a decrease in mitochondrial ATPase, and the accumulation of a cytoplasmic inhibitor of ATP synthesis. *Present address: Department of Zoology, University of Florida, Gainesville, FL, U.S.A.
2
A . C . VANN AND G. C. WEBSTER
MATERIALS AND METHODS The Oregon R strain of Drosophila melanogaster was grown at 25° in clear plastic containers on a medium (Demerec and Kaufmann, 1967) consisting of (per liter): 500 g banana pulp, 15 g agar, 7 ml of a 10~o solution of methyl-p-hydroxybenzoate in 95 ~o ethanol and 478 ml water. Flies were transferred to fresh medium each week.
Preparation of mitochondria All preparative operations were performed at 0--4°. After immobilization by cooling to 4~, 100-500 flies were homogenized for 30 sec in a Potter-Elvejem glass homogenizer containing 5 ml of preparation medium (see below). The preparation was filtered through gauze, and centrifuged at 150 × g for 5 min. The sediment was discarded. Mitochondria were sedimented from the supernatant fluid by centrifugation at 3000 × g for 8 min (Van den Bergh, 1967). The mitochondrial pellet was rinsed two times in the preparation medium, and suspended in two ml of the same medium. The procedure yielded 1-5 mg mitochondrial protein per ml. Two different media were compared for the preparation of mitochondria. The first contained 0.01 M Tris (hydroxymethyl) aminomethane (pH 7"4), 0.15 M KCI, and 0.001 M sodium ethylenediaminetetraacetate, and was used by Bulos et al. (1972) for the preparation of blowfly mitochondria. The second medium contained 0.30 M sucrose, 0"02 M potassium phosphate (pH 7.2), 0.001 M sodium ethylenediaminetetraacetate and 0.5 ~ cysteine (Dounce et al., 1972), and is similar to sucrose-containing media used for mitochondrial isolation from both animals and plants. Since mitochondria isolated in the sucrose-containing medium exhibited a respiratory control ratio (oxygen consumption in the presence of ADP/oxygen consumption in the absence of ADP) of 1-2 during oxidation of 0.01 M pyruvate-malate, while the respiratory control ratio of mitochondria isolated in the KCl-containing medium was 1.9, the KCl-containing medium was used for all subsequent preparations. The reaction medium for measurement of oxidative phosphorylation consisted of: 0.15 M KCI, 0.01 M tris (hydroxymethyl) aminomethane (pH 7.4), 0.005 M MgCI2, 0.004 M potassium phosphate (pH 7.4), 0.001 M sodium ADP, 0.001 M sodium ethylenediaminetetraacetate, 0.1 M glucose, 1 mg crystalline hexokinase (Sigma, Type III) in 0.1 M glucose, 0'01 M sodium pyruvate, 0.01 M sodium malate and 1-5 mg of mitochondrial protein in a total volume of 5 ml. Oxygen consumption was measured polarographically at 27° using a YSI Model 55 oxygen monitor (Yellow Springs Instruments, Yellow Springs, OH, U.S.A.). Orthophosphate disappearance was measured spectrophotometrically, according to Lindberg and Ernster (1956), after partition of orthophosphate and organic phosphates between benzene-isobutanol and silicotungstic acid. ATPase activity was determined as described by Pullman et al. (1960). ADP : 0 ratios were determined according to Bulos et al. (1972). Protein was determined with the biuret procedure described by Gornall et al. (1949), using 18 % sodium deoxycholate to insure complete solubilization of mitochondrial proteins (Webster, 1969). RESULTS
Effect o f age on oxidative phosphorylation T h e rates o f oxygen c o n s u m p t i o n a n d o r t h o p h o s p h a t e d i s a p p e a r a n c e , catalyzed by Drosophila m i t o c h o n d r i a o f different ages, are presented in Fig. 1. T h a t the m e a s u r e d p h o s p h a t e c o n s u m p t i o n was due to oxidative p h o s p h o r y l a t i o n is indicated b y the observ a t i o n t h a t there was no c h a n g e in o r t h o p h o s p h a t e level in the presence o f 10-5 M 2,4d i n i t r o p h e n o l . T h e rate o f oxygen c o n s u m p t i o n increased steadily with increasing age o f the organisms. T h e rate o f p h o s p h a t e d i s a p p e a r a n c e r o u g h l y p a r a l l e l e d oxygen c o n s u m p t i o n d u r i n g m u c h o f a d u l t life. H o w e v e r , very o l d o r g a n i s m s exhibited a m a r k e d r e d u c t i o n in p h o s p h a t e fixation. This reduction, t o g e t h e r w i t h the c o n t i n u e d increase in the rate o f oxygen c o n s u m p t i o n , resulted in a l m o s t a 50 ~ decrease in the efficiency o f p h o s p h o r y lation, as m e a s u r e d b y the P / O ratio, in the oldest o r g a n i s m s studied. Similar results were o b t a i n e d when p h o s p h o r y l a t i o n efficiency was m e a s u r e d by the A D P : O ratio (Fig. 1).
Effect o f age on mitoehondrial A TPase Since increased age resulted in a decrease in the i n c o r p o r a t i o n o f o r t h o p h o s p h a t e into A T P , b u t no decrease in the rate o f oxygen c o n s u m p t i o n , it seemed possible t h a t increased
AGE-RELATED CHANGES IN M1TOCHONDKIAL FUNCTION IN
-
Drosophila melanogaster
3
.o8
.oe
~o...,,~ / \
.04
x
~ d.- - 0 2
1.2
o
o
~0.6 0.3 !
I
I
8
32
,8
AGE (DAYS)
FIG. 1. Oxygen consumption, phosphate incorporation into ATP, P/O ratio, and ADP : O ratio in mitochondria isolated from Drosophila melanogaster of different ages. Reaction system was that described in the Methods Section.
age particularly affected the mitochondrial phosphorylating system. Although the enzymes involved in oxidative phosphorylation are not understood clearly, there is considerable evidence that the mitochondrial FI-ATPase, described originally by Pullman et al. (1960), catalyzes the synthesis of ATP from ADP and phosphate during oxidative phosphorylation, and would thus be critical for the synthesis of ATP. ATPase activity, assayed as described by Pullman et al. (1960) with mitochondria from flies of different ages, is presented in Fig. 2. The ATPase activity increased in adults, and then decreased abruptly in very old Z 1¢1 I--
0 .020
nil.
,s .015 =-.
~
W
.OlD
¢r ~ .005
•
0
n I -1 o
I0
I
I
I
I,
20
30
40
50
AGE
(DAY)
FIG. 2. ATPase activity of mitochondria from Drosophila melanogaster of different ages. ATPase assay was that described by Pullman et ai. (1960).
4
A . C . VANN AND G. C. WEBSTER
TABLE 1. EFFECT OF CYTOPLASM FROM FLIES OF DIFFERENT AGES ON OXIDATIVE PHOSPHORYLATION BY MITOCHONDRIA OF YOUNG FLIES
Age of flies Phosphate incorporated furnishing into ATP Oxygen consumed cytoplasm (hamole/min/mg (la atom/min/mg protein) protein)
P/O
1-9 days 4147 days 45-54 days
0.48 0.32 0-14
0"023 0.017 0.006
0.048 0.053 0.043
Mitochondria were from 1-9 day-old Drosophilamelanogaster.Assay procedure was that described in the Methods section, except 1 ml of the supernatant solution from centrifugation (3000 x g for 8 min) of the cytoplasm from flies of the indicated ages was substituted for 1 ml of assay medium. organisms in the same manner as the P/O ratio. If, as seems likely, F~-ATPase is a key enzyme in ATP synthesis, then the decrease in phosphorylation could be related to the greatly decreased activity of the ATPase.
Inhibition of oxidative phosphorylation by cytoplasmicfactors accumulating during aging Regardless of whether there was a causal relationship between the decrease in ATPase activity and the decrease in phosphorylation, the reason for the decrease was of considerable interest. Since there is little or no replacement of Drosophila mitochondria, the accumulation of defective mitochondria by an error-introducing process (Orgel, 1963) seemed unlikely. Instead, some kind of mitchondrial degradative process seemed more probable. Although a spontaneous internal degradation was possible, we were interested in the possibility of degradation or inhibition of the mitochondrial phosphorylating system by cytoplasmic factors. In order to study this, mitochondria from young (1-9 day-old) flies were assayed for phosphorylation activity in the presence of cytoplasm (centrifuged for 8 min at 3000 x g to remove heavy particles) from flies of increasing age. As can be seen from Table 1, oxidation was relatively unaffected by the presence of cytoplasm. However, phosphate incorporation into ATP was inhibited by cytoplasm from flies of increasing age to the extent that cytoplasm from very old flies almost eliminated the ability of the young mitochondria to form ATP. It appears that increasing age resulted in the accumulation of a factor which inhibited or degraded the phosphorylating ability of the mitochondria. DISCUSSION The results of this study, when viewed along with the findings of other investigators (Weinbach and Garbus, 1959; Gold et al., 1968; Tribe and Ashhurst, 1972) are in accord with the following conclusions regarding the effects of age on mitochondrial activity. In cells, such as the mammalian cells studied thus far, the constant replacement of mitochondria (Menzies and Gold, 1971) results in the isolation, from aged organisms, of mitochondria exhibiting no apparent decrease in phosphorylative efficiency. However, in those adult Dipterans where little or no mitochondrial replacement has been observed, isolated mitochondria show a significant decrease in phosphorylative efficiency. It would be of interest to know whether decrease in mitochondrial turnover, and decrease in efficiency of phosphorylation are general phenomena in aging organisms. In view of the observation of Wilson and Franks (1975) that the "abnormal" mitochondria observed in aged mouse liver cells are not seen in preparations of mitochondria isolated from these cells, it would also be of interest to know whether phosphorylative efficiency (per cell) of intact mam-
AGE-RELATEDCHANGESIN MITOCHONDRIALFUNCTION IN Drosophila melanogaster
5
malian cells decreases with age, and whether there are mammalian tissues composed of cells where mitochondrial replacement is so slow that a decay in phosphorylative efficiency is observed. In Drosophila mitochondria, there appear to be two kinds of decrease in phosphorylative efficiency. First, the mitochondria, when separated from other cellular components, and studied in the usual sucrose-buffer system, exhibit a definite decrease in phosphorylative efficiency. In addition to this, however, there appears to be an accumulation in the cytoplasm of an inhibitor of oxidative phosphorylation. I f both phenomena occur in intact cells, A T P production could be essentially eliminated at an advanced age. The identification of this inhibitor is of considerable interest. It would also be of interest to know whether aging mammalian cells exhibit a similar formation of inhibitory material in the cytoplasm. SUMMARY Aging o f Drosophila melanogaster resulted in a steady increase in the rate of mitochondrial oxidation of pyruvate-malate. At advanced ages, the rate of phosphate incorporation into A T P decreased markedly. This decrease in ATP synthesis was paralleled by a decrease in the activity of mitochondrial ATPase, a probable component of the phosphorylating system. Aging cells steadily accumulated an inhibitor which strongly depressed ATP synthesis when the cytosol of aged cells was incubated with the mitochondria of young cells. REFERENCES BARROWS, C. H., FALZONE, J. A. and SHOCK, N. W. (1960) J. Geront. 15, 130. BULOS,B., SHUKLA,S. arid SAKTOR,B. (1972) Arch. biochem. Biophys. 149, 461. DEMEREC,M. and KAUFMANN,B. P. (1967) Drosophila Guide: Introduction to the Genetics and Cytology of Drosophila melanogaster, p. 10. Carnegie Institution, Washington. FRANKS,L. M. (1974) Gerontologia 20, 51. GOLD, P. H., GEE, M. V. and STREHLER,B. L. (1968) J. Geront. 23, 509. GORNALL,A. C., BARDAWILL,C. J. and DAVID,M. M. (1949) J. biol. Chem. 177, 751. LIZ,a3BERG, O. and ERNSTER,L. (1956) In: Methods of Biochemical Analysis (Edited by D. GLICK),Vol. 3, p. 1. Wiley, New York. MA~Ago-SmTH, J., BOZCUK,A. N. and TFBBUTT,S. (1970) J. Insect Physiol. 16, 601. MEN2aES, g. A. and GOLD,P. H. (1971) d. biol. Chem. 246, 2425. ORGEL, L. (1963) Proe. Nat. Acad. Sei. U.S. 49, 517. PULLMAN,M. E., PENEFSKY,H. S., DATTA,A. and RAcrdsR,E. (1960) J. biol. Chem. 235, 3322. ROCKSTEIN,M. and BHATNAGAR,P. L. (1965) J. Insect Physiol. 11, 481. SAKTOR, B. and SHIMADA,Y. (1972) J. Cell Biol. 52, 465. SOtIAL,R. S. (1975) J. Morph. 145, 337. SOI-IAL,R. S. and ALUSON,V. F. (1971) Exp. Geront. 6, 167. TAKAHASm,A., PmLPOTr,D. E. and MIQtmL,J. (1970) d. Geront. 25, 222. TRINE,M. A. and ASI-II-ItmST,D. E. (1972) J. Cell Sci. 10, 443. VANDENBERGH,S. (1967) In: Methods in Enzymology (Edited by R. W. ESTABROOKand M. E. PULLMAN), Vol. 10, p. 117. Academic Press, New York. WEnSTER,G. C. (1969) J. Cell Biol. 43, 154a. WE~InACH,E. C. and GARnUS,J. (1956) Nature, Lond. 178, 1225. WErNnACH,E. C. and GAP.BUS,J. (1959) J. biol. Chem. 234, 412. WILSON,P. D. and FRANKS,L. M. (1975) Gerontologia 21, 81.
