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Energy metabolism and ageing in Phormia terrae-novae—II. Non-glycolytic enzymes in the flight muscle catalyzing early and late steps of energy supply, their corresponding substrates and influence on flight performance
Comp. Biochem. Physiol. Vol. 74B, No. 2, pp. 337 to 342, 1983 Printed in Great Britain.
0305-0491/83/020337-06503.00/0 © 1983 Pergamon Press Ltd
ENERGY METABOLISM A N D AGEING IN P H O R M I A TERRAE-NOVAE--II. NON-GLYCOLYTIC ENZYMES IN THE FLIGHT MUSCLE CATALYZING EARLY A N D LATE STEPS OF ENERGY SUPPLY, THEIR C O R R E S P O N D I N G SUBSTRATES A N D INFLUENCE ON FLIGHT PERFORMANCE H. WILPS, K.-G. COLLATZ,L. MEHLER and I. WURFEL Institut fiir Biologie I (Zoologie), Albertstral~e 21a, 7800 Freiburg, Federal Republic of Germany (Received 28 M a y 1982) Abstract--1. The activities of flight muscle enzymes, APK, trehalase, HK, ~-GDH, IDH and MDH and their substrates were determined in different adult life stages of Phormia terrae-novae.
2. The activity profiles of trehalose and APK are different from those of the other enzymes tested, having maxima one week after emergence and well before a drop of flight ability occurs. 3. Actomyosin-ATPase activity was tested in dependence of varying substrate concentrations and age. There seems to be an age related substrate influence on enzyme activity. 4. The concentrations of glycogen, trehalose and glucose correlate with the activity profile of trehalase pointing to a well-regulated energy metabolism in ageing flies.
INTRODUCTION
During recent years especially, the enzymes ~-glycerophosphate-dehydrogenase, actomyosin ATPase (myosin B) and arginine phosphokinase (L-arginine phosphotransferase) were subjects of age dependent studies in dipterans (summarized in Baker, 1976; Chesky, 1978). In general there exist well-programmed flight and enzyme activity changes with age, a direct correlation of both events remains contradictory. In contrast to a number of observations published by Rockstein et al. (Rockstein & Chesky, 1973; Chesky, 1974, 1975a, b; Baker 1975a-c), Beezeley et al. (1974) could not find any relation of activity changes of intramitochondrial c¢-glycerophosphate-dehydrogenase to the flight ability of the flies. The same holds for succinic dehydrogenase and malate dehydrogenase. Investigations of body composition (Collatz & Hoeger, 1980), activities of glycolytic enzymes and actual concentrations of metabolites and nucleotides (Wilps et al., 1983) also failed to indicate a primary cause for correlated changes in flight activity. Consulting the published literature, arginine phosphokinase and actomyosin ATPase appear to be responsible for this phenomenon, but nothing is known about the regulation mechanisms between physical and biochemical ageing events. This work presents age dependent activity determinations of trehalase and hexokinase, which produce metabolites for glycolytic degradation, ~-GDH, I D H Abbreviations: AM-ATPase: actomyosin ATPase. APK: arginine-phosphate-kinase (EC 2.7.3.3). HK: hexose-kinase (EC 2.7.1.1). ct-GDH: glycerol-3-phosphate-kinase (EC 1.1.1.8). MDH: malate dehydrogenase (EC 1.1.1.37). IDH: isocitrate-dehydrogenase (EC 1.1.1.42). PEP: phosphoenole pyruvate.
337
and M D H , involved in substrate support of the respiratory chain and A P K and AM-ATPase, which directly supply the flight muscle with energy. The substrates glycogen, trehalose and citrate were also determined. Glycogen content should show the potential and trehalose concentration the actual mobilizable energy resources. Summing up, the intention of this study was to strengthen the theory of a strictly programmed life course of this species and to look for primary enzyme activity changes which could be responsible for the early loss of flight activity. Sex specific age differences in metabolism and flight performance and their interactions are under investigation. MATERIAL AND METHODS
Rearing conditions were the same as described in the previous paper (Wilps eta[., 1982). Preparation of actomyosin (myosin B) after Maruyama et al. (1968). Thoraxes were gently homogenized in 0.3 M sucrose, I mM EGTA and l mM KHCO3, pH 7.2 and lipids were removed after slow speed centrifugation. After washing in 0.1 M KCI the homogenization was repeated with 0.6 M KC1-0.06 M KHCO 3, pH 8.3 and the actomyosin precipitated by lowering the ionic strength. The preparation was resuspended and precipitated another 3 times for purification. Actomyosin-ATPase activity was estimated in an optical test-system with 3 mM PEP; 2.5 mM NADH2, 4.2U/ml pyruvate-kinase, 9.6 U/ml lactate dehydrogenase, 0.2 8 mM ATP together in 25 mM TRIZMA HCI buffer pH 7.6 containing 50raM KC1, 2.5 mM MgCI/, 2.5 mM EGTA, 2.5 mM CaCI 2. APK was determined with a modified test system from Blethen (1970) with 6 mM MgSO4, 7.5 mM KC1, 3 mM ATP, 0.3 mM NADH, 0.8 mM P-enolepyruvate, 9.6 U/ml lactate-dehydrogenase, 4.2 U/ml PK together in 360mM triethanoleaminebuffer pH 8.6. Trehalose was measured according to Rosinsky et al. (1979), the formed glucose enzymatically assayed.
338
a[.
H. WILPS e[
The HK, :~-GDH. MDH and IDH activity determination was according to Bergmeyer (1974). Assay procedure for glycogen, trehalose were as described previously (Wilps et al.. 1982). Glycogen was enzymatically split with amyloglucosidase, trehalose with acid hydrolysis in 3 N TCA for 30 rain. The formed glucose was enzymatically determined•
RESULTS
The activity course of flight muscle enzymes are summarized in Fig. 1. The activities show a rapid rise during the first days after emergence reaching a maxi-
mum at about the 12th day, after which they decrease. The activity profiles of A P K and trehalase are in strong contrast to those of HK, ~-GDH, IDH and M D H . Only the former reach their maximum at day 6. Then activity decreases, first sharply followed by a plateau between the 8th and 12th day. Up to the 25th day of life the activity decreases steadily. Figure 2 presents the influence of age and substrate concentration on the AM-ATPase activity, Fig. 3 the amount of extractable AM per g thorax wet weight. As described for P F K (Wilps e t al., 1982) not only the AM-ATPase itself, but also the inhibiting concentration of the substrate, are subjected to the ageing
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/
~
.
TREHALASE
L/\ o
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g
i
lb
12
li.
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Fig. 1. Age dependent activity changes in P h o r m i a flight muscle enzymes (:~-GDH, HK, APK, trehalose. MDH, IDH), Values in U/rag protein.
del~
Energy metabolism and ageing of Phormia terrae-novae--II.
24 2 1 ~
339
AN-ATPose
~
~ z 4 ~-p
6 8
Fig. 2. Influence of age and substrate concentration on the flight muscle actomyosin ATPase from Phormia.
process. Between day 8-12 the specific activity is almost unaffected by ATP concentrations from 2 to 6/iM. At day 17 the maximum of activity is reached with 4 ~M ATP. As age advances the substrate saturation concentration shifts to lower ATP values resulting in a clearly inhibited effect of 4/iM ATP at day 24. Using a linear transformation of sigmoid enzyme kinetics we determined K m and Vm,x at different age stages (Fig. 4). The Vmax of ATP cleavage rises from day 1 up to the 17th day by a factor of 10 and declines again. The lowest apparent K m values were found at the 6th-8th day followed by a rise with advancing age. Immediately after emergence only 0.5/~M ATP are necessary in vitro to reach Vmax. At day 8 with the lowest apparent K m the corresponding saturation concentration is 6/~M, the same needed to reach Vm,~of P F K (Part I). The amount of extractable actomyosin decreases slightly up to the 4th day followed by an increase in the next 4 days. From the 10th-17th day no change are visible. The AM decreases steadily with proceed-
ing age to a level, at day 24, 1/2 of the amount of day 8 (Fig. 3). Figure 5 shows the concentration of trehalose, glycogene and citrate. Between the 3rd and 6th day glycogen reserves decrease strongly corresponding with the increase of trehalose. Trehalose decreases by about 40~o to day 8. Up to the 14th day (for glycogen) and 18th day (for trehalose) these substrates remain at the 60Yo level of the original concentration. Followed by a minimum they regain their former value. Citrate concentration displays a maximum on the 9th day and again between the 12th and 23rd day.
DISCUSSION
All investigated enzymes and substrates vary in an ordered and regular manner. Related to the flight per-
3O AM
Vmox
2O
-10
,oi 2
l.
6
8
10
12
1~
16
18
20
22
24 26 doys
Fig. 3. Amount of extractable actomysin (mg/g wet wt.) from flight muscles of Phormia of different ages.
2
4
6
8
10
12
14
16
18
20
22
2~ 26 doys
Fig. 4. Age dependent changes in Vm.xand K m of the flight muscle actomyosin ATPase from Phormia.
340
H. WlLl,s et al.
GLYCOGEN
M
TREHALOSE
I
/"
6
8
10
12
14
16
18
20
22
24
26
dOyS
28
2
/,
6
8
10
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14
16
18
20
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28
/ ~"~'~C,T.ATE
I,~
11 10 O9 ~8
/,
7\/ 6
8
10
12
1/,
16
18
20
22
24
26 28 days
Fig. 5. Age dependent concentration changes of glycogen, trehalose and citrate in ltight muscles of Phormia. Values in itM/g wet wt.
formance (Collatz et al., 1981) it is clearly visible that the maximum of APK and trehalose activity is reached before, that of AM-ATPase qOer, the maximum of flight ability• The highest activities of the other enzymes fall to#ether with maximum of flight ability. Comparable enzyme activity profiles were reported for APK, AM-ATPase and :~-GDH from Musca and Phormia regina (Baker, 1976; Chesky, 1978; Rockstein et al., 1981). We were able to confirm the existence of an ageing program on metabolic level as it was shown for all key enzymes of carbohydrate metabolism (Wilps et al., 1982) and was formerly assumed for the above mentioned enzymes (Rockstein & Miquel, 1973}. Completely different results in some instances however were found comparing the reported timing of age dependent and flight correlated activity changes of single enzymes to each other and with our own observations on Phormia terrae-not'ae. The activity changes of z~-GDH of Musca domestica alone were described in 3 different manners. After Chesky (1978) its decrease of activity occurs clearly after the drop of flight performance, after Baker (1975) this happens before the flight maximum (in both cases the flight durance was measured), Beezeley et al. (1974t, in contrast, found no correspondence between enzyme activity changes and flight performance. Actomyosin A TPase and Ar#inine phosphokinase
As already stated the activity changes of enzymes in carbohydrate catabolism and Krebs-cycle are not responsible for the programmed loss of flight ability.
The same holds for the actomyosin ATPase with its maximum of activity at day 17. This enzyme however is remarkable in another way. The kinetic data of the latter enzyme with the parallel increase of apparent K m and b{,,,,x up to the 20th day support the view that - similar to that reported for P F K (Part i} there exists a possibility to compensate the progressive loss of substrate affinity during the middle time of life by rising the Vmax. This "compensating effect" shifts to "senescent conditions" following the 20th day after which the apparent K m and V,,~x values diverge more and more, marking a progressive impairment of the catalytic property of the ATPase. Further it should be noted that lowest apparent K m and the maximum of extractable actomyosm fall together. Both these parameters point to a high flight capacity, although the maximum of flight ability itself is reached 2 days later, due to the rapid increase of the enzyme activities of the carbohydrate catabolism. The decrease in the amount of actomyosin in old flies is not accompanied by a structural muscle dystrophy as electron microscopic observations show (Collatz & Wilps, 19821. The isolated natural actomyosin of the housefly (Chesky, 1975) exhibits maximal ATPase activity with only 1.6 mM ATP. 6 mM ATP proved to be fully inhibitory in all adult stages of Musca between 1 and 12 days. However, the significance of such a comparison is weakened due to the different preparations possible for this complex protein system. Chesky (1978) proposed the AM-ATPase to be inti-
341
Energy metabolism and ageing of Pltorrnicr twrcre-novue-II.
rnately correlated with the flight ability, probably being the primary cause of its loss in Dipterans. He also compared his measurements of this enzyme in Musca (Rockstein & Chesky, 1973; Chesky, 1974, 1975) with results from Baker (197Sa. b) on Musca arginine phosphokinase and found a close connection between the activity profiles of both enzymes. After these results the maximum APK activity is separated from that of AM-ATPase by only a few hours. Related to the wing-beat frequency the APK-peak was reached hefire regarding the flight duration, however @er the maximum of flight performance. After the results of Rockstein tjf al. (1981) the flight reduction is followed by a decrease of APK-activity. On the other hand, measurements of APK activity changes in Drosopkiia mrianoyltstrr and the shorter living mutant D. vestigial (Baker, 1975b) result in the observation of declining activities at exactly the same day of adult life. This indicates an independence from flight performance and even from life span. Whatever the activity changes of AM-ATPase and APK causes. the peaks are separated in P~art~i~? set-rae-nowe thoraxes by 11 days. Therefore any postulated interrelationship and common influence on the flight activity cannot hold for our blowfly. Only the APK and the trehalase exhibit activity peaks clearly one week before the best flight ability (flight duration and distance). The amount of arginine phosphate however comprises only lCrlS% of the creatine phosphate of the vertebrates (Saktor, 1970) and only 302, of the steady state concentrations of ATP under optimal flight conditions. Therefore we propose that there is an indirect influence of the cleaving enzyme on the muscle energy metabolism via the loss of arginine phosphate. A regulation of cellular metabolism by intracellular phosphate can occur through the hydrolysis of phosphoreactine (Erecinska et cd., 1977). This happens especially in vertebrate tissues such as muscles in which a nearly constant level of ATP is required and leads to a potent stimulation of the respiration. Because Pi is an ailosteric activator of the PFK this key enzyme would be affected by diminished effecters concentrations. Trrhulasr
and suhstrures
The activity changes of trehalase remains to be mentioned and cannot be discussed without consideration of the corresponding changes in trehalose and glycogen. The first week of adult life and achievement of high flight performance are controlled by rising trehalase activities and a transformation from glycogen into trehalose at the same time. Trehalase activity as well as glycogen and trehalose concentrations are already reduced by 40% when Phormia reach their maximum Aight performance. At this time the needed fuel for the high flight performance is obviously provided only by the ingested sugar. The maxima of glycolytic enzyme activities and especially that of the HK (Wilps et al., 1982) support this. An early drop of trehalose concentration was also reported from the flight muscle of the housefly (Rockstein & Srivastava, 1967) and seems to precede the reduction of flight activity. With this result one can assume that the highest flight perfbrmance of Musca was reached even at relatively low trehalose concentration. The trehalase activity of the tobacco horn-
worm on the other hand did not vary to a great extent with advancing age (Dahlman, 1972). In conclusion, these results strongly support the hypothesis that the regulation of flight performance as in a metabolic program occurs via the “early energy yielding system”. It requires additional age dependent studies of the influence of neurosecretory and brain glands on the trehalose and glycogen mobilizing and synthesizing enzymes. This part of the ageing program is under investigation. Acktlowledyrtt~ents--We
wish to thank
Mrs. Marion
Koch and Miss Gisela Reible for technical assistance and Mrs. Judith Whittaker-Mehler for critical reading of this text. This work was supported by the “Deutsche Forschungsgemeinschaft”. REFERENCES G. T. (1975a) Identical age-related patterns of enzyme activity changes in Phormia rrginu and I)rosop~iiu nl~lu~~~asrer. Exp. Grront. 10, 23 l-237. BAKERG. T. (1975b) Age-related activity changes in arginine phosphokinase in the housefly, Musco domrstiru. J. Geront. 30, 163-169. BAKER G. T. (1975~) Age dependent arginine phosphokinase activity changes in male vestigial and wild-type Drosophila melanogaster. Gerontologia 21, 203-210. BAKERG. T. (1976) Insect flight muscle: maturation and senescence, Gerot~rol~~iu22, 334-36 1. BERGMEYER H. U. (19743(ed.) Methoden der enzymatischen Analyse. Bd. I, II. Verlag Chemie, Weinheim. BEEZELEY A. E., MCCARTHYJ. L. & SOHALR. S. (1974) Changes in alpha-glycerophosphate, succinic and malic dehydrogenases in flight muscles of the housefly, Muscu domestics, with age. Exp. Gerottt. 9, 7 1-74. BLETHENS. (1970) Argininkinase. In forbids iti E~t;ymoiogp. XVII Academic Press, New York. CHESKYJ. A. (1974) The intrinsic nature of the loss of actomyosin ATPase activity during senescence in the male housefly Mum domestica L. Mech. uge. dru. 3, 241-244. CHESKYJ. A. (1975a) Actomyosin ATP-sensitivity in the aging male housefly. Muscn domestica. k-p. Geront. 10, 165-169. CHESKYJ. A. (1975b) Properties of natural actomyosin from the male housefly, Musca domrsticu. insrct. BioBAKER
them. 5, 509-518.
CHESKYJ. A. (1978) Comparative aspects of declining muscle function. Aging 6, 179-210. COLLATZK.-G. & HOEGERU. (1980) Age-related changes in the body composition of mated and unmated blowflies P~i~rmiu Murray-novae.Exp. Gcront. 15, 433-441, COLL~TZ K.-G., WILPS H. (1982) Altern bei Insekten. Naturwiss. Rdsch. In press. COLLATZK.-G.. STAMMLER G., WILPS H. & MEHLER L. (1981) Programmed loss of flight ability in the early adult life of the blowfly Phormia trrrae-nocae as a possible mechanism of intraspecific niche building with respect to the duration of life. Camp. Bjo~~ef?z. Physioi. 68.4.571-577.
DAHLMAND. L. (1972) Age-dependent trehalase activity in adult tobacco hornworm tissues. Insect. Biochem. 2, 143-149. ERECINSKAM., STUBBS M., MIYATAY., DITRE C. M. & WILSOND. F. (1977) Regulation of cellular metabolism by intracellular phosphate. Biochim. Bi~~~~s. Actu 462, X-35.
MARUYAMA K., PRINGLEJ. W. S. & TREGEARR. T. (1968) The calcium sensitivity of ATPase activity of myofibrils and actomyosins from insect Right and leg muscles. Proc. R. Sot. B 169, 229-240.
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ROCKSTEIN M. & MIQUEL J. (1973) Aging in insects. In The Physiology ol' Insects, 2rid edn, Vol. 1, Ch. 6. Academic Press, New York. ROCKSTEIN M., LOPEZ J., M1QUEL J. & BOZCUK A. N. (19811 Age related activity changes in actomyosin ATPase and arginine phosphokinase in male Drosophila melanogaster Meig. Gerontology 27, 301 305. ROCKSTEIN M. & SRIVASTAVAP. N. (1967) Trehalose in the flight muscle of the housefly, Musca domestica L. in relation to age. Experientia 23, 1 4. ROCKSTEIN M. & CHESKY I. (1973) Age-related changes in natural actomyosin of the male housefly, Musea domestica L. J. Geront. 28, 455 459.
ROSINSKY G., WRZESZCZ A. & OBUCHOWICZ L. (19791 Dig ferences in trehalase activity in the intestine of fed and starved larvae of Tenebrio molitor. Insect. BioclTem. 9, 485-488. SACRTOR B. (1970) Regulation of intermediary metabolism. with special reference to the control mechanisms in insect flight muscle, Ado. Insect. Physiol, 7, 267 347. WILPS M., COLLATZ K.-G. & MEHLIR L. 11983t Energ) metabolism and aging of Phormia terrae-m~cae 1, Programmed changes of enzyme activities and substr~tc concentrations in carbohydrate metabolism. ('omp. Biochem. Physiol. 74B. 337 342.
0305-0491/83/020337-06503.00/0 © 1983 Pergamon Press Ltd
ENERGY METABOLISM A N D AGEING IN P H O R M I A TERRAE-NOVAE--II. NON-GLYCOLYTIC ENZYMES IN THE FLIGHT MUSCLE CATALYZING EARLY A N D LATE STEPS OF ENERGY SUPPLY, THEIR C O R R E S P O N D I N G SUBSTRATES A N D INFLUENCE ON FLIGHT PERFORMANCE H. WILPS, K.-G. COLLATZ,L. MEHLER and I. WURFEL Institut fiir Biologie I (Zoologie), Albertstral~e 21a, 7800 Freiburg, Federal Republic of Germany (Received 28 M a y 1982) Abstract--1. The activities of flight muscle enzymes, APK, trehalase, HK, ~-GDH, IDH and MDH and their substrates were determined in different adult life stages of Phormia terrae-novae.
2. The activity profiles of trehalose and APK are different from those of the other enzymes tested, having maxima one week after emergence and well before a drop of flight ability occurs. 3. Actomyosin-ATPase activity was tested in dependence of varying substrate concentrations and age. There seems to be an age related substrate influence on enzyme activity. 4. The concentrations of glycogen, trehalose and glucose correlate with the activity profile of trehalase pointing to a well-regulated energy metabolism in ageing flies.
INTRODUCTION
During recent years especially, the enzymes ~-glycerophosphate-dehydrogenase, actomyosin ATPase (myosin B) and arginine phosphokinase (L-arginine phosphotransferase) were subjects of age dependent studies in dipterans (summarized in Baker, 1976; Chesky, 1978). In general there exist well-programmed flight and enzyme activity changes with age, a direct correlation of both events remains contradictory. In contrast to a number of observations published by Rockstein et al. (Rockstein & Chesky, 1973; Chesky, 1974, 1975a, b; Baker 1975a-c), Beezeley et al. (1974) could not find any relation of activity changes of intramitochondrial c¢-glycerophosphate-dehydrogenase to the flight ability of the flies. The same holds for succinic dehydrogenase and malate dehydrogenase. Investigations of body composition (Collatz & Hoeger, 1980), activities of glycolytic enzymes and actual concentrations of metabolites and nucleotides (Wilps et al., 1983) also failed to indicate a primary cause for correlated changes in flight activity. Consulting the published literature, arginine phosphokinase and actomyosin ATPase appear to be responsible for this phenomenon, but nothing is known about the regulation mechanisms between physical and biochemical ageing events. This work presents age dependent activity determinations of trehalase and hexokinase, which produce metabolites for glycolytic degradation, ~-GDH, I D H Abbreviations: AM-ATPase: actomyosin ATPase. APK: arginine-phosphate-kinase (EC 2.7.3.3). HK: hexose-kinase (EC 2.7.1.1). ct-GDH: glycerol-3-phosphate-kinase (EC 1.1.1.8). MDH: malate dehydrogenase (EC 1.1.1.37). IDH: isocitrate-dehydrogenase (EC 1.1.1.42). PEP: phosphoenole pyruvate.
337
and M D H , involved in substrate support of the respiratory chain and A P K and AM-ATPase, which directly supply the flight muscle with energy. The substrates glycogen, trehalose and citrate were also determined. Glycogen content should show the potential and trehalose concentration the actual mobilizable energy resources. Summing up, the intention of this study was to strengthen the theory of a strictly programmed life course of this species and to look for primary enzyme activity changes which could be responsible for the early loss of flight activity. Sex specific age differences in metabolism and flight performance and their interactions are under investigation. MATERIAL AND METHODS
Rearing conditions were the same as described in the previous paper (Wilps eta[., 1982). Preparation of actomyosin (myosin B) after Maruyama et al. (1968). Thoraxes were gently homogenized in 0.3 M sucrose, I mM EGTA and l mM KHCO3, pH 7.2 and lipids were removed after slow speed centrifugation. After washing in 0.1 M KCI the homogenization was repeated with 0.6 M KC1-0.06 M KHCO 3, pH 8.3 and the actomyosin precipitated by lowering the ionic strength. The preparation was resuspended and precipitated another 3 times for purification. Actomyosin-ATPase activity was estimated in an optical test-system with 3 mM PEP; 2.5 mM NADH2, 4.2U/ml pyruvate-kinase, 9.6 U/ml lactate dehydrogenase, 0.2 8 mM ATP together in 25 mM TRIZMA HCI buffer pH 7.6 containing 50raM KC1, 2.5 mM MgCI/, 2.5 mM EGTA, 2.5 mM CaCI 2. APK was determined with a modified test system from Blethen (1970) with 6 mM MgSO4, 7.5 mM KC1, 3 mM ATP, 0.3 mM NADH, 0.8 mM P-enolepyruvate, 9.6 U/ml lactate-dehydrogenase, 4.2 U/ml PK together in 360mM triethanoleaminebuffer pH 8.6. Trehalose was measured according to Rosinsky et al. (1979), the formed glucose enzymatically assayed.
338
a[.
H. WILPS e[
The HK, :~-GDH. MDH and IDH activity determination was according to Bergmeyer (1974). Assay procedure for glycogen, trehalose were as described previously (Wilps et al.. 1982). Glycogen was enzymatically split with amyloglucosidase, trehalose with acid hydrolysis in 3 N TCA for 30 rain. The formed glucose was enzymatically determined•
RESULTS
The activity course of flight muscle enzymes are summarized in Fig. 1. The activities show a rapid rise during the first days after emergence reaching a maxi-
mum at about the 12th day, after which they decrease. The activity profiles of A P K and trehalase are in strong contrast to those of HK, ~-GDH, IDH and M D H . Only the former reach their maximum at day 6. Then activity decreases, first sharply followed by a plateau between the 8th and 12th day. Up to the 25th day of life the activity decreases steadily. Figure 2 presents the influence of age and substrate concentration on the AM-ATPase activity, Fig. 3 the amount of extractable AM per g thorax wet weight. As described for P F K (Wilps e t al., 1982) not only the AM-ATPase itself, but also the inhibiting concentration of the substrate, are subjected to the ageing
15" "6
K_
3
Of. 4
6
8
;.
10 12 14 16 18 20 22 24 26 28 days
.~ i/
100"
4
6
e
IO
12
1~
/ ~
•
*e
io
2'2
iL
2'6
&
4o ~
i~
I'6 i8 ~o 2'2 A doy~
\,.,.
'~e
APK
/
~
.
TREHALASE
L/\ o
L
g
i
lb
12
li.
i6 ie /0 i2
i~
N
i8
Fig. 1. Age dependent activity changes in P h o r m i a flight muscle enzymes (:~-GDH, HK, APK, trehalose. MDH, IDH), Values in U/rag protein.
del~
Energy metabolism and ageing of Phormia terrae-novae--II.
24 2 1 ~
339
AN-ATPose
~
~ z 4 ~-p
6 8
Fig. 2. Influence of age and substrate concentration on the flight muscle actomyosin ATPase from Phormia.
process. Between day 8-12 the specific activity is almost unaffected by ATP concentrations from 2 to 6/iM. At day 17 the maximum of activity is reached with 4 ~M ATP. As age advances the substrate saturation concentration shifts to lower ATP values resulting in a clearly inhibited effect of 4/iM ATP at day 24. Using a linear transformation of sigmoid enzyme kinetics we determined K m and Vm,x at different age stages (Fig. 4). The Vmax of ATP cleavage rises from day 1 up to the 17th day by a factor of 10 and declines again. The lowest apparent K m values were found at the 6th-8th day followed by a rise with advancing age. Immediately after emergence only 0.5/~M ATP are necessary in vitro to reach Vmax. At day 8 with the lowest apparent K m the corresponding saturation concentration is 6/~M, the same needed to reach Vm,~of P F K (Part I). The amount of extractable actomyosin decreases slightly up to the 4th day followed by an increase in the next 4 days. From the 10th-17th day no change are visible. The AM decreases steadily with proceed-
ing age to a level, at day 24, 1/2 of the amount of day 8 (Fig. 3). Figure 5 shows the concentration of trehalose, glycogene and citrate. Between the 3rd and 6th day glycogen reserves decrease strongly corresponding with the increase of trehalose. Trehalose decreases by about 40~o to day 8. Up to the 14th day (for glycogen) and 18th day (for trehalose) these substrates remain at the 60Yo level of the original concentration. Followed by a minimum they regain their former value. Citrate concentration displays a maximum on the 9th day and again between the 12th and 23rd day.
DISCUSSION
All investigated enzymes and substrates vary in an ordered and regular manner. Related to the flight per-
3O AM
Vmox
2O
-10
,oi 2
l.
6
8
10
12
1~
16
18
20
22
24 26 doys
Fig. 3. Amount of extractable actomysin (mg/g wet wt.) from flight muscles of Phormia of different ages.
2
4
6
8
10
12
14
16
18
20
22
2~ 26 doys
Fig. 4. Age dependent changes in Vm.xand K m of the flight muscle actomyosin ATPase from Phormia.
340
H. WlLl,s et al.
GLYCOGEN
M
TREHALOSE
I
/"
6
8
10
12
14
16
18
20
22
24
26
dOyS
28
2
/,
6
8
10
12
14
16
18
20
22
2L
26
28
/ ~"~'~C,T.ATE
I,~
11 10 O9 ~8
/,
7\/ 6
8
10
12
1/,
16
18
20
22
24
26 28 days
Fig. 5. Age dependent concentration changes of glycogen, trehalose and citrate in ltight muscles of Phormia. Values in itM/g wet wt.
formance (Collatz et al., 1981) it is clearly visible that the maximum of APK and trehalose activity is reached before, that of AM-ATPase qOer, the maximum of flight ability• The highest activities of the other enzymes fall to#ether with maximum of flight ability. Comparable enzyme activity profiles were reported for APK, AM-ATPase and :~-GDH from Musca and Phormia regina (Baker, 1976; Chesky, 1978; Rockstein et al., 1981). We were able to confirm the existence of an ageing program on metabolic level as it was shown for all key enzymes of carbohydrate metabolism (Wilps et al., 1982) and was formerly assumed for the above mentioned enzymes (Rockstein & Miquel, 1973}. Completely different results in some instances however were found comparing the reported timing of age dependent and flight correlated activity changes of single enzymes to each other and with our own observations on Phormia terrae-not'ae. The activity changes of z~-GDH of Musca domestica alone were described in 3 different manners. After Chesky (1978) its decrease of activity occurs clearly after the drop of flight performance, after Baker (1975) this happens before the flight maximum (in both cases the flight durance was measured), Beezeley et al. (1974t, in contrast, found no correspondence between enzyme activity changes and flight performance. Actomyosin A TPase and Ar#inine phosphokinase
As already stated the activity changes of enzymes in carbohydrate catabolism and Krebs-cycle are not responsible for the programmed loss of flight ability.
The same holds for the actomyosin ATPase with its maximum of activity at day 17. This enzyme however is remarkable in another way. The kinetic data of the latter enzyme with the parallel increase of apparent K m and b{,,,,x up to the 20th day support the view that - similar to that reported for P F K (Part i} there exists a possibility to compensate the progressive loss of substrate affinity during the middle time of life by rising the Vmax. This "compensating effect" shifts to "senescent conditions" following the 20th day after which the apparent K m and V,,~x values diverge more and more, marking a progressive impairment of the catalytic property of the ATPase. Further it should be noted that lowest apparent K m and the maximum of extractable actomyosm fall together. Both these parameters point to a high flight capacity, although the maximum of flight ability itself is reached 2 days later, due to the rapid increase of the enzyme activities of the carbohydrate catabolism. The decrease in the amount of actomyosin in old flies is not accompanied by a structural muscle dystrophy as electron microscopic observations show (Collatz & Wilps, 19821. The isolated natural actomyosin of the housefly (Chesky, 1975) exhibits maximal ATPase activity with only 1.6 mM ATP. 6 mM ATP proved to be fully inhibitory in all adult stages of Musca between 1 and 12 days. However, the significance of such a comparison is weakened due to the different preparations possible for this complex protein system. Chesky (1978) proposed the AM-ATPase to be inti-
341
Energy metabolism and ageing of Pltorrnicr twrcre-novue-II.
rnately correlated with the flight ability, probably being the primary cause of its loss in Dipterans. He also compared his measurements of this enzyme in Musca (Rockstein & Chesky, 1973; Chesky, 1974, 1975) with results from Baker (197Sa. b) on Musca arginine phosphokinase and found a close connection between the activity profiles of both enzymes. After these results the maximum APK activity is separated from that of AM-ATPase by only a few hours. Related to the wing-beat frequency the APK-peak was reached hefire regarding the flight duration, however @er the maximum of flight performance. After the results of Rockstein tjf al. (1981) the flight reduction is followed by a decrease of APK-activity. On the other hand, measurements of APK activity changes in Drosopkiia mrianoyltstrr and the shorter living mutant D. vestigial (Baker, 1975b) result in the observation of declining activities at exactly the same day of adult life. This indicates an independence from flight performance and even from life span. Whatever the activity changes of AM-ATPase and APK causes. the peaks are separated in P~art~i~? set-rae-nowe thoraxes by 11 days. Therefore any postulated interrelationship and common influence on the flight activity cannot hold for our blowfly. Only the APK and the trehalase exhibit activity peaks clearly one week before the best flight ability (flight duration and distance). The amount of arginine phosphate however comprises only lCrlS% of the creatine phosphate of the vertebrates (Saktor, 1970) and only 302, of the steady state concentrations of ATP under optimal flight conditions. Therefore we propose that there is an indirect influence of the cleaving enzyme on the muscle energy metabolism via the loss of arginine phosphate. A regulation of cellular metabolism by intracellular phosphate can occur through the hydrolysis of phosphoreactine (Erecinska et cd., 1977). This happens especially in vertebrate tissues such as muscles in which a nearly constant level of ATP is required and leads to a potent stimulation of the respiration. Because Pi is an ailosteric activator of the PFK this key enzyme would be affected by diminished effecters concentrations. Trrhulasr
and suhstrures
The activity changes of trehalase remains to be mentioned and cannot be discussed without consideration of the corresponding changes in trehalose and glycogen. The first week of adult life and achievement of high flight performance are controlled by rising trehalase activities and a transformation from glycogen into trehalose at the same time. Trehalase activity as well as glycogen and trehalose concentrations are already reduced by 40% when Phormia reach their maximum Aight performance. At this time the needed fuel for the high flight performance is obviously provided only by the ingested sugar. The maxima of glycolytic enzyme activities and especially that of the HK (Wilps et al., 1982) support this. An early drop of trehalose concentration was also reported from the flight muscle of the housefly (Rockstein & Srivastava, 1967) and seems to precede the reduction of flight activity. With this result one can assume that the highest flight perfbrmance of Musca was reached even at relatively low trehalose concentration. The trehalase activity of the tobacco horn-
worm on the other hand did not vary to a great extent with advancing age (Dahlman, 1972). In conclusion, these results strongly support the hypothesis that the regulation of flight performance as in a metabolic program occurs via the “early energy yielding system”. It requires additional age dependent studies of the influence of neurosecretory and brain glands on the trehalose and glycogen mobilizing and synthesizing enzymes. This part of the ageing program is under investigation. Acktlowledyrtt~ents--We
wish to thank
Mrs. Marion
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