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Comparison of the enzymatic activities of homologous muscles of flying and flightless beetles
Comp. Biochem. Physiol., 1977, Vol. 57B, pp. 111 to II 6. Pergamon Press. Printed in Great Britain
COMPARISON OF THE ENZYMATIC ACTIVITIES OF H O M O L O G O U S MUSCLES OF FLYING A N D FLIGHTLESS BEETLES J. A. MITCHELL,J. J. A. HEFFRON* AND H. R. HEPBURN Departments of Physiology and Physiological Chemistry, University of the Witwatersrand, Johannesburg 2001, South Africa (Received 21 October 1976)
Abstract--1. Activity levels of the enzymes actomyosin ATPase, phosphorylase, succinic dehydrogenase, 3-hydroxyacyl-CoA dehydrogenase, proline dehydrogenase, sarcoplasmic L-glycerol-3-phosphate dehydrogenase, pyruvate kinase and fructose-l, 6-diphosphatase were determined in homogenates of the tergocoxal muscle of the flightless beetle, Anthia thoracica, and of the tergocoxal and dorsal longitudinal muscle of Pachynoda sinuata, a normal flier. 2. Actomyosin ATPase and phosphorylase activities in the muscles of P. sinuata were 2.5- and 18-fold greater than in the muscle of A. thoracica, respectively. The low phosphorylase activity and glycogen content of the latter suggest that glycogenolysisis a minor source of energy for contraction. 3. The activities of succinic, proline, and glycerol-3-phosphate dehydrogenases were 4-15-fold greater in P. sinuata than in A. thoracica, but the 3-hydroxyacyl-CoAdehydrogenase activities were the same, indicating that fatty acid oxidation is the primary source of ATP in A. thoracica muscle. 4. Electron microscopy showed that the tergocoxal muscle of A. thoracica was afibrillar while the muscles of P. sinuata were fibrillar. 5. It is concluded that evolutionary adaptation to the flightless state results in transition of homologous muscles from a fibrillar to an afibrillar muscle type and from a primarily aerobic glycogen-based energy metabolism to one mainly dependent on fatty acid oxidation.
INTRODUCTION Insect flight muscles are the most active muscles known. It is therefore not surprising that they are characterized by a high oxidative metabolic capacity, mitochondrial density and rate of ATP utilization. Since prolonged muscular activity is essential to sustain flight, it might be expected that the pattern of energy metabolism would be at least qualitatively similar to that of vertebrate cardiac or red skeletal muscle. While many similarities do exist, the much greater rate of metabolism of insect flight muscle necessary to support flight requires some unique metabolic specializations (Crabtree & Newsholme, 1975). In the more primitive insects, the leg and flight muscles are of the synchronous type, similar to vertebrate striated muscle. Specialization in the more advanced orders of insects has led to the evolution of a more efficient muscle type not found elsewhere in the animal kingdom, namely, the asynchronous muscle characteristic of the more powerful and rapidly flying insects. However, in some species of Coleoptera, specialization has resulted in secondary loss of the flight mechanism due to atrophy of the flight muscles and wings (Imms, 1964). Some Carabidae show these modifications and although they became flightless, they differ from the primitively wingless Apterygota in that the loss of their hind wings is a secondary adaptation. These morphological changes, together with the physiological and biochemical differences, are of fundamental interest when
considering the insects' mode of locomotion. The changes are probably conservative resulting in more efficient skeletal muscle contraction for the requirements of the different species of insects. Observations of this nature are more meaningful when made on muscles of insects of close taxonomic affinities but which have different modes of locomotion. In this study two species of Coleoptera, the flightless Carabid beetle A. thoracica and the Scarabid beetle P. sinuata, a normal flier, are compared with these views in mind. The aim was to establish the relative importance of glycolytic metabolism, the tricarboxylic acid cycle, lipid and proline oxidation in supplying energy to homologous muscles which subserve different functions in the two Coleopterans. The approach was based on measuring the maximal activities of some key enzymes in each metabolic pathway to provide a means of determining the maximal rate of the pathway in the intact muscles.
* Present address and address for reprints: Department of Biochemistry, University College Dublin, Belfield, Dublin 4, Ireland 111
MATERIALS A N D METHODS
Insect material
Specimens of P. sinuata and A. thoracica were collected in the Johannesburg area and in the Eastern Transvaal, respectively. The dorsal longitudinal and tergocoxal muscles (i.e. the indirect flight muscles) of P. sinuata and the tergocoxal muscle of A. thoracica were used throughout. While the tergocoxal muscles of the two species are homologous, the use of the dorsal longitudinal muscle of P. sinuata makes this comparison of the two species not strictly homologous. Insects were killed by decapitation as soon as possible after capture. The abdomen and legs were removed, and the thorax was split bilaterally before removal of muscle. To slow down any postmortem
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J.A. MITCHELL, J. J. A. HEFFRON AND H. R. HEPBURN
changes, these procedures were carried out, where possible, on ice. Depending on the enzyme to be assayed, muscle from 1 to 5 insects was pooled, weighed quickly and placed in the required volume of ice-cold homogenizing medium. Muscles were minced finely with a scissors and homogenized in glass hand-homogenizers having a total volume of 1 ml. Enzyme assays
Enzymes were assayed in duplicate at 30°C. For those enzymes assayed by direct optical methods, the reaction rates were followed continuously on a Beckman DB-GT double-beam recording spectrophotometer equipped with a constant temperature cell compartment set at 30°C. Initial rates of the reactions were obtained by drawing tangents at the origin of the progress curves. Non-specific enzyme activities in the tissue homogenates were determined by omitting the various substrates from the assay media, and were subtracted from the appropriate gross enzyme activities. Except for actomyosin ATPase, enzyme activities were expressed as/~moles of substrate or product/ rain/g, muscle, wet wt. Actomyosin ATPase (EC 3.6.1.3) was extracted from the muscle of each species in 10 vols of a solution containing 0.6 M KC1, 1 mM dithiothreitol and 25 mM Tris-HC1 pH 7.3 for 24 hr at 2°C according to the method of Heffron & Duggan (1971) for frog muscle. Maximal ATPase activity was determined in a medium containing 5 mM ATPTris, 5 mM CaC12, 0.5 M KC1, 25 mM Tris-HC1 pH 7.3 and 0.8 mM dithiothreitol at 30°C. After adding enzyme the reaction was run for 5, 10, 15 and 20rain and terminated with trichloroacetic acid (TCA) at a final concentration of 5°J;. Precipitated protein was removed by centrifugation and inorganic phosphate (Pi) in the supernatant was determined by the method of Taussky & Shorr (1953). The reaction rate was found to be linear with time for 15 min. ATPase activity was expressed as #moles Pi/mg protein/rain. Protein was assayed by the method of Lowry et al. (1951). Fructose-l, 6-diphosphatase (EC 3.1.3.11) was extracted and assayed according to the method of Newsholme et al. (1972). The homogenate was prepared in 10vols of a medium containing 50mM Tris-HC1 pH 7.5, l mM EDTA, 5 mM MgSO4 and 20 mM fl-mercaptoethanol and centrifuged at 15000 for 30 min at 0°C. The supernatant was used for enzyme assay. The assay medium contained 0 . l m M fructose-l, 6-diphosphate, 1 mM EDTA, 20mM /#mercaptoethanol, 50raM Tris-HCl pH 7.5, 0.20mM nicotinamide adenine dinucleotide phosphate (NADP), 5U phosphoglucose isomerase and 5U glucose-6-phosphate dehydrogenase in a cuvette volume of 2.60 ml. 0.04 ml of enzyme extract was added to the cuvette and the rate of NADP reduction at 340 nm was recorded. 3-Hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) activity of muscle homogenates, prepared in 20 vols of 100 mM phosphate buffer pH 7.3, was assayed by the method of Bass et al. (1969). The homogenate was clarified at 7000 for 30 min at 0°C and the supernatant (0.02 ml) used for enzyme assay. The assay medium contained 0.1 mM acetoacetyl-CoA, 0.25mM reduced nicotinamide adenine dinucleotide (NADH), 5 mM EDTA and 80 mM Tris-HC1 pH 7.0. After adding supernatant to the assay medium, the rate of NADH oxidation at 340 nm was recorded for 3 min. Pyruvate kinase (EC 2.7.1.40) was assayed by a coupled enzymatic method similar to that described by Simon & Robin (1972). The homogenate was prepared in 50 vols of 10 mM EDTA-40mM Tris-HC1 pH 7.4 and clarified at 700 g for 30 min at 0°C. Enzyme activity was determined at 340 nm in a medium containing 83 mM KCI, 10 mM MgSO4, 1.9mM phosphoenolpyruvate, 0.25raM ADP, 4.6U lactate dehydrogenase/ml, 0.30mM NADH and 10 mM Tris-HC1 pH 7.5.
Glycerol-3-phosphate dehydrogenase (EC 1.l.l.8) was assayed under conditions similar to those given by Crabtree & Newsholme (19701 for proline dehydrogenase except that the substrate was L-glycerol-3-phosphate. The muscle homogenate was prepared in 100vols of medium containinng 2mM MgCI2, I mM EDTA, 30mM /l-mercaptoethanol and 50mM Tris HCI pH 7.5, and centrifuged at 38,000 g for 20 min at 0'C. The supernatant was assayed for enzyme activity. The assay medium consisted of 20 mM L-glycerol-3-phosphate, 0.25°o 2-(p-iodophenyl)-3-p-(nitrophenyl)-5-phenyl-monotetrazolium chloride (INTt, l mM KCN and 50 mM phosphate buffer pH 7.5. The reaction was terminated with 5°0 TCA (final concentration), the diformazan was extracted into ethyl acetate and determined spectrophotometrically at 490 nm. A standard curve for pure INT diformazan was run with each assay. The reaction rate was linear with time for 6 rain. Proline dehydrogenase (EC 1.5.1.2) was assayed in the whole homogenate prepared as already described for glycerol-3-phosphate dehydrogenase. The assay medium was also the same except that 100raM L-proline was substituted for L-glycerol-3-phosphate (Crabtree & Newsholme, 1970). The amount of diformazan was measured as described above. Succinic dehydrogenase (EC 1.3.99.1) was assayed in the whole homogenate by a modification of the method of Pennington (1961). The homogenate was prepared in 100 vols of 100mM phosphate buffer pH 7.4. The assay medium contained 50mM succinate, 0.05'!i, INT and 50 mM phosphate buffer pH 7.4. The amount of INT diformazan liberated at various times after adding the homogenate was determined as described above. The reaction rate was linear with time for at least 20 min. Phosphorylase (EC 2.4.1.1} was assayed in the direction of glycogen formation as described by Crabtree & Newsholme (1972). The homogenizing medium consisted of 35 mM glycerol-2-phosphate, 20mM NaF, l mM EDTA and 30 mM fl-mercaptoethanol at pH 6.2, and the muscles were homogenized in 25, 50 or 100 vols of medium. The assay medium contained 32mM glycose-l-phosphate. 0.5 mM AMP and 2~o (w/v) glycogen. Pi liberated by the enzyme was determined by the method of Taussky & Shorr (1953). Glycogen
Small pieces of muscle (0.01-0.1 g) were excised and weighed quickly and placed in 3.0 ml 30°~; KOH in "'Pyrex" test tubes. The samples were digested by heating in a boiling water bath for 30min. 0.5 ml saturated Na2SO4 was added to each sample, followed by 4.2 ml of 95'~;, ethanol. After stirring the tubes were again heated to boiling point, then cooled and centrifuged at 1500 0 for 10min. The supernatants were discarded; 2 ml of water and 2.5 ml of 95% ethanol were added to the sediments. The tubes were centrifuged as before, the supernatants were discarded. each sediment was dissolved in 2.5 ml of water and analyzed for glycogen by the anthrone method (Hassid & Abraham, 1957). Electron microscopy
Muscles were fixed in situ in the hemi-thorax by submersion in ice-cold Karnovsky fixative (2.5~!/,, glutaraldehyde, 2.4~o paraformaldehyde and 0.05 M sodium cacodylate pH 7.3) for 1 hr. They were then cut into small pieces (about 1 x 2 mm) and left for a further hour in fresh fixative at room temperature. The specimens were washed for 30 rain in I°/O sucrose-25mM cacodylate pH 7.3 and post-fixed in buffered 1~o OsO4 for 1 hr at room temperature. They were washed again in buffered sucrose and left overnight in the same medium at 4"C. The specimens were then dehydrated in acetone for 30 min, embedded in Araldite, and the tissue blocks were sectioned with glass knives on a Sorvall Porter-Blum MT2 ultramicrotome. The sections.
Enzymes of insect muscle 90-150nm thick, were mounted on copper grids, were stained first in uranyl acetate and then in lead citrate. They were viewed in a Siemens Elmiskop I transmission electron microscope. The microscope was calibrated with latex particles having a mean diameter of 0.312, S.D. + 0.002#m. Chemicals
Chemicals were analytical grade from BDH Chemicals Ltd., Poole, Dorset, U.K. Analytical grade enzymes, NADH, NADP, adenosine mono-, di- and tri-phosphates, glucose-l-phosphate, phosphoenolpyruvate and fructose-l, 6-diphosphate were obtained from Boehringer GmbH, Mannheim, West Germany. INT, INT-diformazan and acetyl-CoA were obtained from Sigma Chemical Co., St. Louis, MO, U.S.A. Anthrone was obtained from Merck, Darmstadt, West Germany. Acetoacetyl-CoA was synthesized from acetyl-CoA and diketen and standardized by the method described by Sugden & Newsholme (1973). Solutions were made up with glass double-distilled water.
113 RESULTS
The ultrastructural features of the tergocoxal muscles of A. thoracica and P. sinuata are shown in Fig. l a and b. The muscle of P. sinuata has short, wide sarcomeres and columns of closely packed, den. ,1' sely staining mltochondrla in longitudinal section. In transverse section, the myofibrils were cylindrical and showed a double hexagonal arrangement of myofilaments with an actin :myosin filament ratio of 3:1. The ultrastructure of the dorsal longitudinal muscle of P. sinuata was similar to that of the tergocoxal muscle (Mitchell, 1976). All these features are characteristic of the fibrillar muscle type (Elder, 1975). On the other hand, the tergocoxal muscle of A. thoracica shows the typical features of afibrillar muscle such as long, narrow sarcomeres, dense and wide Z lines, and small, paired mitochondria on either side of the Z line (Fig.
Fig. I. (a) Longitudinal section of the tergocoxal muscle of P. sinuata, x 10,000; (b) longitudinal section of tergocoxal muscle of A. thoracica, x 10,000; (c) rosettes of glycogen in P. sinuata muscle, x 60,000; (d) glycogen granules in A. thoracica muscle, x 40,000.
J. A. MITCHELL,J. J. A. HEFFRONAND H. R. HEPBURN
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Table 1. Activities of actomyosin ATPase and some glycolytic enzymes in the indirect flight muscles of P. sinuata and tergocoxal muscle of A. thoracica Enzyme
Anthia thoracica
Pachynoda sinuata
Actomyosin ATPase Phosphorylase Pyruvate kinase Fructose-l, 6-diphosphatase
0.105 + 0.023 (6) 1.19 _+ 0.42 (9) 14.3 _+ 1.08 (6) 1,54 Jr 0.18 (6)
0.244 _+ 0.034(6} 21.9 _+ 1.45 (6) 41.1 _+ 2.51 (6) 2.96 _+ 0.40 (6)
Signilicance P P P P
< < < <
0.005 0.0005 0.0005 0.005
ATPase activity is expressed as ,umoles/mg protein/min. Other enzyme activities are expressed as llmoles/g muscle/rain. Values are given as mean _+ S.E.M. with the number of insects in brackets. An unpaired t test was used for statistical analysis of the results. lb). In addition, the myofibrils were lamellar-like and radially oriented in transverse section while the myofilament arrangement showed 11 12 thin filaments in slightly irregular orbit about each thin filament (Mitchell, 1976). Since the dorsal longitudinal and tergocoxal muscles of P. sinuata are both fibrillar muscles and they occur together in the thorax, it was convenient to use both muscles for preparation of homogenates for the enzyme assays. Table 1 summarizes the activities of actomyosin ATPase and some glycolytic enzymes in the indirect flight muscle of P. sinuata and in the homologous tergocoxal muscle of A. thoracica. Actomyosin ATPase activity of P. sinuata muscle was 2.3 times greater than in A. sinuata muscle and the corresponding ratio of phosphorylase activity was 18.4:1. The activity ratios of pyruvate kinase and fructose-l, 6-diphosphatase were 2.9:1 and 1.9:1, respectively. The low phosphorylase activity of the A. thoracica muscle suggested that aerobic glycogenolysis might not be an important source of energy for muscular contraction. This was confirmed by determination of the glycogen content of the muscles of both insects; A. thoracica muscle contained 1.36 _+ 0.39 mg glycogen/g (n = 6) while that of P. sinuata contained 17.6 _+ 1.88 mg/g (6). Further evidence for this was obtained from the nature and density of the glycogen granules in electron micrographs of the muscles. Glycogen is very abundant and exists as large rosettes and groups of single granules in the muscle of P. sinuata (Fig. Ic) but occurs only as single granules distributed in low density in A. thoracica muscle (Fig. ld}. The activities of some important enzymes of oxidative metabolism are shown in Table 2. Sarcoplasmic glycerol-3-phosphate dehydrogenase activity is an in-
dicator of the potential of the muscles to utilize N A D H produced in the sarcoplasm. The activity of this enzyme in P. sinuata was some 15-fold greater than in A. thoracica indicating the high capacity of the muscle of the former to carry out aerobic carbohydrate metabolism, and also substantiating the very low glycolytic capacity of A. thoracica muscle. The maximal activity of 3-hydroxyacyl-CoA dehydrogenase indicates the relative importance of fatty acid oxidation in energy provision for contractile activity. It is shown in Table 2 that of all the enzymes examined in this study only 3-hydroxyacyl-CoA dehydrogenase activity was similar in both insects. The four-fold greater activity of succinic dehydrogenase in P. sinuata compared with A. thoraciea confirms the greater overall aerobic capacity of the fibrillar muscle. Mobilization and oxidation of proline to oxaloacetate is of particular importance in the initial phase of insect flight (Hochacha & Somero, 1973) when the availability of oxaloacetate is the principle factor limiting the rate of metabolite oxidation. The relative activities of proline dehydrogenase provide an estimate of the importance of proline mobilization in providing sufficient oxaloacetate to meet the large demands for ATP during sudden bursts of muscular activity in insects. It is significant then that proline dehydrogenase activity in P. sinuata muscle was 4-fold greater than in A. thoracica (Table 2), similar to the ratio of succinic dehydrogenase activity in the two species. DISCUSSION The results presented here show that the tergocoxal muscle of A. thoracica has the typical ultrastructural features of afibrillar muscle while its homologue in
Table 2. Activities of some oxidative enzymes in the indirect flight muscles of P. sinuata and tergocoxal muscle of A. rhoracica Enzyme Glycerol-3-phosphate dehydrogenase 3-Hydroxyacyl CoA dehydrogenase Succinic dehydrogenase Proline dehydrogenase
Anthia thoracica
Pachynoda sinuata
Significance
0.391 _ 0.090 (6)
6.03 +_ 0.25 (7)
P < 0.0005
7.63 + 1.34 (4)
6.61 _+ 1.45 (6t
N.S.
0.703 +_ 0.048 (9) 2.44 + 0.29 (6)
2.75 + 0.14 (6) 8.94 +_ 0.28 (6)
P < 0.0005 P < 0.0005
Enzyme activities are expressed as #moles/g muscle/min. Values are given as mean +_ S.E.M. with the number of insects in brackets. An unpaired t test was used for statistical analysis. N.S. = not significant.
Enzymes of insect muscle P. sinuata is typically fibrillar. The dorsal longitudinal
muscle of the latter is similar ultrastructurally to its tergocoxal muscle, and for this reason, no attempt was made to separate them after removal from the thorax prior to preparation of the homogenates. The demonstration of an almost 3-fold greater actomyosin ATPase activity in the muscles of P. sinuata compared with that of A. thoracica is consistent with the strong flying ability of the former (cf. Barnett et al., 1975) and with the principle that fibrillar muscle is the fastest-contracting muscle type. Since maximal actomyosin ATPase activity is a good index of the maximal speed of contraction of muscles of widely different species (Barany et al., 1967; Bullard et al., 1973) it may be inferred from these and the present study that the structural and functional features of the contractile apparatus of synchronous muscle are of the conservative type from the evolutionary viewpoint. Unlike the case in vertebrate striated muscle the role of phosphorylase and glycogen stores in insect flight muscle is not to provide ATP under hypoxic or anaerobic conditions. Rather, glycogen is metabolized oxidatively under normal conditions. It is clear from the present results that glycogen metabolism is of little importance for the activity of the tergocoxal muscle of A. thoracica since both the glycogen content and phosphorylase activity are considerably less than in vertebrate red slow-twitch muscle (cf. Crabtree & Newsholme, 1975). In insect flight muscle the activities of the enzymes of the Krebs cycle are usually sufficient to permit complete oxidation of glucose units formed from glycogen at the maximum activity of phosphorylase (cf. Tables 3 8,: 4, Crabtree & Newsholme. 1975). In this study, relative succinic dehydrogenase activities were measured since we omitted the electron transferring agent, phenazine methosulphate, from the assay media. This was necessary because of considerable variation in non-enzymatic reduction of INT by this compound. For this reason the activities of succinic dehydrogenase in both insects cannot be compared directly with the respective phosphorylase activities; however, the results clearly show the greater capacity of the flight muscles of P. sinuata to carry out complete oxidation of glycogen. Insect flight muscles possess a glycerol-3-phosphate cycle which, via the activities of sarcoplasmic NADlinked glycerol-3-phosphate dehydrogenase and the mitochondrial flavoprotein glycerol-3-phosphate dehydrogenase, catalyses oxidation of sarcoplasmic NADH by the mitochondrial electron transport chain (Sacktor, 1970). The present work shows that the flight muscle of P. sinuata had a 15-fold greater activity of sarcoplasmic glycerol-3-phosphate dehydrogenase than the tergocoxal muscle of A. thoracica, an activity ratio similar to that of phosphorylase in the same muscles. This substantiates the earlier conclusion that glycogenolysis is of minor significance in the energy metabolism of the tergocoxal muscle of A. thoracica, and indicates that glycolysis of such sugars as trehalose is also of little importance. Considering the low actomyosin ATPase, phosphorylase and glycerol-3-phosphate dehydrogenase activities, it may be concluded that the tergocoxal muscle of the A. thoracica performs slow but sustained contractions in vivo. This contrasts with the synchronous hind-leg muscles of the locust (Locusta migratoria) which have
115
a high phosphorylase activity that enables them to perform extremely rapid and vigorous contractions similar to vertebrate white muscles (cf Crabtree & Newsholme, 1975). The finding of similar 3-hydroxyacyl-CoA dehydrogenase activities in both species suggests that fatty acid oxidation is the predominant energy-generating pathway in A. thoracica muscle, while the same pathway in P. sinuata may play a similar but supplementary role for sustained periods of flight. Proline dehydrogenase is the first enzyme involved in the oxidation of proline and its maximum activity is a useful estimate of the importance of proline as a fuel for muscular activity in insects (Crabtree & Newsholme, 1970). On this basis, the flight muscles of P. sinuata show a three-fold greater capacity for proline oxidation than the tergocoxal muscle of A. thoracica. Both types of muscle can utilize proline as an energy source for contractile activity. Since the flight muscles of P. sinuata are capable of rapid action in vivo, it is likely that the proline oxidation pathway is also important for provision of adequate amounts of oxalo-acetate for maximal operation of the Krebs cycle in the early phases of flight (cf. Hochacha & Somero, 1973). The muscles of both species have high pyruvate kinase activities. In the case of P. sinuata, the two-fold greater activity of pyruvate kinase compared with phosphorylase indicates that the maximum glycolytic rate is determined by the latter. It is difficult to explain the significance of the very high pyruvate kinase/phosphorylase ratio in A. thoracica though it may be postulated that phosphorylase, being the first enzyme of the glycogenolytic pathway, is subject to greater evolutionary adaptive pressures than enzymes further along the pathway. In insect flight muscles, the fructose-6-phosphate cycle is catalyzed by the enzymes phosphofructokinase and fructose-i, 6-diphosphatase. It has been suggested that its main role is to amplify the sensitivity of the glycolytic flux to AMP, although in the flight muscles of the bumble-bee, the cycle functions as an ATPase for heat production (Crabtree & Newsholme, 1975). In the present study, the relatively low activities of fructose-i, 6-diphosphatase in both muscle types, indicating the maximal capacity for cycling of fructose-6phosphate, suggest that the role of the cycle is to provide amplification for glycolytic control rather than heat. The overall changes in enzymatic constitution of the tergocoxal muscle of A. thoracica occurring during the transition to the flightless state result in development of a synchronous muscle type having a virtually completely aerobic metabolism. Fatty acid oxidation is the primary source of energy while proline oxidation may act as a supplementary source. Further, it appears that fatty acid oxidation is a very conservative metabolic pathway in insect muscles generally, while the glycolytic pathway is responsive to the energy requirements of the various modes of functional specialization occurring in the insect kingdom.
Acknowledgements--We thank the South African Council for Scientific and Industrial Research for financial support, and Dr. Wolfgang Prinz for synthesizing acetoacetylCoA.
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J.A. MITCHELL,J. 3. A. HEFFRONAND H. R. HEPBURN REFERENCES
BARANYM., CONOVERT. E., SCHLISELFIELDL. H., GAETJENS E. & GOFFARTM. (1967) Relation of properties of myosin to those of intact muscles of the cat and sloth. Eur. J. Biochem. 2, 156-164. BARNETTP. S., HEFFRONJ. J. A. & HEPBURN H. R. (1975) Some thermal characteristics of insect flight: Enzyme optima versus intra-thoracic temperature. S. Afr. J. Sci. 71, 373-374. BASS A., BRDICZKA D., EYER P., HOFER S. t~¢ PETTED. (1969) Metabolic differentiation of distinct muscle types at the level of enzymatic organization. Eur. J. Biochem. 10, 198-206. BULLARDB., DARROWSKAR. & WINKELMANL. (1973) The contractile and regulatory proteins of insect flight muscle. Biochem. J. 135, 277 286. CRABTREE B. & NEWStfOLMEE. A. (1970) The activities of proline dehydrogenase, glutamate dehydrogenase, aspextate-oxoglutarate aminotransferase and alanineoxoglutarate aminotransferase in some insect flight muscles. Biochem. J. 117, 1019-1021. CRAR~EE B. & NEWSHOLMEE. A. (1972) The activities of phosphorylase, hexokinase, phosphofructokinase, lactate dehydrogenase and glycerol-3-phosphate dehydrogenase in muscles from vertebrates and invertebrates. Biochem. J. 126, 49-58. CRARTREE B. & NEWSHOLME E. A. (1975) Comparative aspects of fuel utilization and metabolism by muscle. In Insect Muscle (Edited by USHEgWOOD P. N. R.) pp. 405-500, Academic Press, London. ELDER H. Y. (1975) Muscle structure. In Insect Muscle (Edited by USHERWOODP. N. R.) pp. t 74. Academic Press, London. HASSID W. Z. & ABRAHAMS. (1957) Chemical procedures for the analysis of polysaccharides. In Methods of Enzymology (Edited by COLOWIO~ S. P. & KAPLAN N. O.) Vol. 3, 34-37. Academic Press, New York.
HEFFRON J. J. A. & DUGGAN P. F. (1971) Adenosinetriphosphatase activity and superprecipitation of actomyosin from the frog, Rana temporaria. Int. J. Biochem. 2, 324-336. HOCHACHA P. W. (~ SOMEROG. ~1973) Strategies oJ' Biochemical Adaptation. pp. 72-73, W. B. Saunders Co., Philadelphia. IMMS A. D. (1964) A General Textbook ¢ff" Entomolo(4y, Parts I & III, Methuen & Co. Ltd, London, LOWRY O. H., ROSEBROUGHN. J,, FARR A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265--275. MITCHELL J. A. (1976) Muscle ultrastructure and enzymology of flying and flightless beetles. M. Sc. thesis. University of the Witwatersrand, Johannesburg. NEWSHOLMEE. A., CRABTREEB., HIGGINS S. J., THORNTON S. D. & STARTC. (1972) The activities of fructose diphosphatase in flight muscles from the bumble-bee and the role of this enzyme in heat generation. Biochem. J. 128, 89- 97. PENNINGTON R. J. (19611 Biochemistry of dystrophic muscle. Mitochondrial succinate-tetrazolium reductase and adenosine triphosphatase. B'iochem. J. 80, 649-654. SACKTORB. (1970) Regulation of intermediary metabolism, with special reference to the control mechanisms in insect flight muscle. Adv. Insect Physiol. 7, 267 347. SIMON L. M. & ROBIN E. D. (1972) Relative anaerobic glycolytic capacity and pyruvate kinase activity of rabbit tissues. Int. J. Biochem. 3, 329-332. SUGDEN P. H. & NEWSr~OLME E. A. (1973) Activities of hexokinase, phosphofructokinase, 3-oxo-acid coenzyme A-transferase and acetoacetyl-coenzyrne A thiolase in nervous tissue from vertebrates and invertebrates. Biochem. J. 134, 97-101. TAUSSKY H. H. & SHORR E. (t953) A microcolorimetric method for the determination of inorganic phosphorus. J. biol. Chem. 202, 675-.-685.
COMPARISON OF THE ENZYMATIC ACTIVITIES OF H O M O L O G O U S MUSCLES OF FLYING A N D FLIGHTLESS BEETLES J. A. MITCHELL,J. J. A. HEFFRON* AND H. R. HEPBURN Departments of Physiology and Physiological Chemistry, University of the Witwatersrand, Johannesburg 2001, South Africa (Received 21 October 1976)
Abstract--1. Activity levels of the enzymes actomyosin ATPase, phosphorylase, succinic dehydrogenase, 3-hydroxyacyl-CoA dehydrogenase, proline dehydrogenase, sarcoplasmic L-glycerol-3-phosphate dehydrogenase, pyruvate kinase and fructose-l, 6-diphosphatase were determined in homogenates of the tergocoxal muscle of the flightless beetle, Anthia thoracica, and of the tergocoxal and dorsal longitudinal muscle of Pachynoda sinuata, a normal flier. 2. Actomyosin ATPase and phosphorylase activities in the muscles of P. sinuata were 2.5- and 18-fold greater than in the muscle of A. thoracica, respectively. The low phosphorylase activity and glycogen content of the latter suggest that glycogenolysisis a minor source of energy for contraction. 3. The activities of succinic, proline, and glycerol-3-phosphate dehydrogenases were 4-15-fold greater in P. sinuata than in A. thoracica, but the 3-hydroxyacyl-CoAdehydrogenase activities were the same, indicating that fatty acid oxidation is the primary source of ATP in A. thoracica muscle. 4. Electron microscopy showed that the tergocoxal muscle of A. thoracica was afibrillar while the muscles of P. sinuata were fibrillar. 5. It is concluded that evolutionary adaptation to the flightless state results in transition of homologous muscles from a fibrillar to an afibrillar muscle type and from a primarily aerobic glycogen-based energy metabolism to one mainly dependent on fatty acid oxidation.
INTRODUCTION Insect flight muscles are the most active muscles known. It is therefore not surprising that they are characterized by a high oxidative metabolic capacity, mitochondrial density and rate of ATP utilization. Since prolonged muscular activity is essential to sustain flight, it might be expected that the pattern of energy metabolism would be at least qualitatively similar to that of vertebrate cardiac or red skeletal muscle. While many similarities do exist, the much greater rate of metabolism of insect flight muscle necessary to support flight requires some unique metabolic specializations (Crabtree & Newsholme, 1975). In the more primitive insects, the leg and flight muscles are of the synchronous type, similar to vertebrate striated muscle. Specialization in the more advanced orders of insects has led to the evolution of a more efficient muscle type not found elsewhere in the animal kingdom, namely, the asynchronous muscle characteristic of the more powerful and rapidly flying insects. However, in some species of Coleoptera, specialization has resulted in secondary loss of the flight mechanism due to atrophy of the flight muscles and wings (Imms, 1964). Some Carabidae show these modifications and although they became flightless, they differ from the primitively wingless Apterygota in that the loss of their hind wings is a secondary adaptation. These morphological changes, together with the physiological and biochemical differences, are of fundamental interest when
considering the insects' mode of locomotion. The changes are probably conservative resulting in more efficient skeletal muscle contraction for the requirements of the different species of insects. Observations of this nature are more meaningful when made on muscles of insects of close taxonomic affinities but which have different modes of locomotion. In this study two species of Coleoptera, the flightless Carabid beetle A. thoracica and the Scarabid beetle P. sinuata, a normal flier, are compared with these views in mind. The aim was to establish the relative importance of glycolytic metabolism, the tricarboxylic acid cycle, lipid and proline oxidation in supplying energy to homologous muscles which subserve different functions in the two Coleopterans. The approach was based on measuring the maximal activities of some key enzymes in each metabolic pathway to provide a means of determining the maximal rate of the pathway in the intact muscles.
* Present address and address for reprints: Department of Biochemistry, University College Dublin, Belfield, Dublin 4, Ireland 111
MATERIALS A N D METHODS
Insect material
Specimens of P. sinuata and A. thoracica were collected in the Johannesburg area and in the Eastern Transvaal, respectively. The dorsal longitudinal and tergocoxal muscles (i.e. the indirect flight muscles) of P. sinuata and the tergocoxal muscle of A. thoracica were used throughout. While the tergocoxal muscles of the two species are homologous, the use of the dorsal longitudinal muscle of P. sinuata makes this comparison of the two species not strictly homologous. Insects were killed by decapitation as soon as possible after capture. The abdomen and legs were removed, and the thorax was split bilaterally before removal of muscle. To slow down any postmortem
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J.A. MITCHELL, J. J. A. HEFFRON AND H. R. HEPBURN
changes, these procedures were carried out, where possible, on ice. Depending on the enzyme to be assayed, muscle from 1 to 5 insects was pooled, weighed quickly and placed in the required volume of ice-cold homogenizing medium. Muscles were minced finely with a scissors and homogenized in glass hand-homogenizers having a total volume of 1 ml. Enzyme assays
Enzymes were assayed in duplicate at 30°C. For those enzymes assayed by direct optical methods, the reaction rates were followed continuously on a Beckman DB-GT double-beam recording spectrophotometer equipped with a constant temperature cell compartment set at 30°C. Initial rates of the reactions were obtained by drawing tangents at the origin of the progress curves. Non-specific enzyme activities in the tissue homogenates were determined by omitting the various substrates from the assay media, and were subtracted from the appropriate gross enzyme activities. Except for actomyosin ATPase, enzyme activities were expressed as/~moles of substrate or product/ rain/g, muscle, wet wt. Actomyosin ATPase (EC 3.6.1.3) was extracted from the muscle of each species in 10 vols of a solution containing 0.6 M KC1, 1 mM dithiothreitol and 25 mM Tris-HC1 pH 7.3 for 24 hr at 2°C according to the method of Heffron & Duggan (1971) for frog muscle. Maximal ATPase activity was determined in a medium containing 5 mM ATPTris, 5 mM CaC12, 0.5 M KC1, 25 mM Tris-HC1 pH 7.3 and 0.8 mM dithiothreitol at 30°C. After adding enzyme the reaction was run for 5, 10, 15 and 20rain and terminated with trichloroacetic acid (TCA) at a final concentration of 5°J;. Precipitated protein was removed by centrifugation and inorganic phosphate (Pi) in the supernatant was determined by the method of Taussky & Shorr (1953). The reaction rate was found to be linear with time for 15 min. ATPase activity was expressed as #moles Pi/mg protein/rain. Protein was assayed by the method of Lowry et al. (1951). Fructose-l, 6-diphosphatase (EC 3.1.3.11) was extracted and assayed according to the method of Newsholme et al. (1972). The homogenate was prepared in 10vols of a medium containing 50mM Tris-HC1 pH 7.5, l mM EDTA, 5 mM MgSO4 and 20 mM fl-mercaptoethanol and centrifuged at 15000 for 30 min at 0°C. The supernatant was used for enzyme assay. The assay medium contained 0 . l m M fructose-l, 6-diphosphate, 1 mM EDTA, 20mM /#mercaptoethanol, 50raM Tris-HCl pH 7.5, 0.20mM nicotinamide adenine dinucleotide phosphate (NADP), 5U phosphoglucose isomerase and 5U glucose-6-phosphate dehydrogenase in a cuvette volume of 2.60 ml. 0.04 ml of enzyme extract was added to the cuvette and the rate of NADP reduction at 340 nm was recorded. 3-Hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) activity of muscle homogenates, prepared in 20 vols of 100 mM phosphate buffer pH 7.3, was assayed by the method of Bass et al. (1969). The homogenate was clarified at 7000 for 30 min at 0°C and the supernatant (0.02 ml) used for enzyme assay. The assay medium contained 0.1 mM acetoacetyl-CoA, 0.25mM reduced nicotinamide adenine dinucleotide (NADH), 5 mM EDTA and 80 mM Tris-HC1 pH 7.0. After adding supernatant to the assay medium, the rate of NADH oxidation at 340 nm was recorded for 3 min. Pyruvate kinase (EC 2.7.1.40) was assayed by a coupled enzymatic method similar to that described by Simon & Robin (1972). The homogenate was prepared in 50 vols of 10 mM EDTA-40mM Tris-HC1 pH 7.4 and clarified at 700 g for 30 min at 0°C. Enzyme activity was determined at 340 nm in a medium containing 83 mM KCI, 10 mM MgSO4, 1.9mM phosphoenolpyruvate, 0.25raM ADP, 4.6U lactate dehydrogenase/ml, 0.30mM NADH and 10 mM Tris-HC1 pH 7.5.
Glycerol-3-phosphate dehydrogenase (EC 1.l.l.8) was assayed under conditions similar to those given by Crabtree & Newsholme (19701 for proline dehydrogenase except that the substrate was L-glycerol-3-phosphate. The muscle homogenate was prepared in 100vols of medium containinng 2mM MgCI2, I mM EDTA, 30mM /l-mercaptoethanol and 50mM Tris HCI pH 7.5, and centrifuged at 38,000 g for 20 min at 0'C. The supernatant was assayed for enzyme activity. The assay medium consisted of 20 mM L-glycerol-3-phosphate, 0.25°o 2-(p-iodophenyl)-3-p-(nitrophenyl)-5-phenyl-monotetrazolium chloride (INTt, l mM KCN and 50 mM phosphate buffer pH 7.5. The reaction was terminated with 5°0 TCA (final concentration), the diformazan was extracted into ethyl acetate and determined spectrophotometrically at 490 nm. A standard curve for pure INT diformazan was run with each assay. The reaction rate was linear with time for 6 rain. Proline dehydrogenase (EC 1.5.1.2) was assayed in the whole homogenate prepared as already described for glycerol-3-phosphate dehydrogenase. The assay medium was also the same except that 100raM L-proline was substituted for L-glycerol-3-phosphate (Crabtree & Newsholme, 1970). The amount of diformazan was measured as described above. Succinic dehydrogenase (EC 1.3.99.1) was assayed in the whole homogenate by a modification of the method of Pennington (1961). The homogenate was prepared in 100 vols of 100mM phosphate buffer pH 7.4. The assay medium contained 50mM succinate, 0.05'!i, INT and 50 mM phosphate buffer pH 7.4. The amount of INT diformazan liberated at various times after adding the homogenate was determined as described above. The reaction rate was linear with time for at least 20 min. Phosphorylase (EC 2.4.1.1} was assayed in the direction of glycogen formation as described by Crabtree & Newsholme (1972). The homogenizing medium consisted of 35 mM glycerol-2-phosphate, 20mM NaF, l mM EDTA and 30 mM fl-mercaptoethanol at pH 6.2, and the muscles were homogenized in 25, 50 or 100 vols of medium. The assay medium contained 32mM glycose-l-phosphate. 0.5 mM AMP and 2~o (w/v) glycogen. Pi liberated by the enzyme was determined by the method of Taussky & Shorr (1953). Glycogen
Small pieces of muscle (0.01-0.1 g) were excised and weighed quickly and placed in 3.0 ml 30°~; KOH in "'Pyrex" test tubes. The samples were digested by heating in a boiling water bath for 30min. 0.5 ml saturated Na2SO4 was added to each sample, followed by 4.2 ml of 95'~;, ethanol. After stirring the tubes were again heated to boiling point, then cooled and centrifuged at 1500 0 for 10min. The supernatants were discarded; 2 ml of water and 2.5 ml of 95% ethanol were added to the sediments. The tubes were centrifuged as before, the supernatants were discarded. each sediment was dissolved in 2.5 ml of water and analyzed for glycogen by the anthrone method (Hassid & Abraham, 1957). Electron microscopy
Muscles were fixed in situ in the hemi-thorax by submersion in ice-cold Karnovsky fixative (2.5~!/,, glutaraldehyde, 2.4~o paraformaldehyde and 0.05 M sodium cacodylate pH 7.3) for 1 hr. They were then cut into small pieces (about 1 x 2 mm) and left for a further hour in fresh fixative at room temperature. The specimens were washed for 30 rain in I°/O sucrose-25mM cacodylate pH 7.3 and post-fixed in buffered 1~o OsO4 for 1 hr at room temperature. They were washed again in buffered sucrose and left overnight in the same medium at 4"C. The specimens were then dehydrated in acetone for 30 min, embedded in Araldite, and the tissue blocks were sectioned with glass knives on a Sorvall Porter-Blum MT2 ultramicrotome. The sections.
Enzymes of insect muscle 90-150nm thick, were mounted on copper grids, were stained first in uranyl acetate and then in lead citrate. They were viewed in a Siemens Elmiskop I transmission electron microscope. The microscope was calibrated with latex particles having a mean diameter of 0.312, S.D. + 0.002#m. Chemicals
Chemicals were analytical grade from BDH Chemicals Ltd., Poole, Dorset, U.K. Analytical grade enzymes, NADH, NADP, adenosine mono-, di- and tri-phosphates, glucose-l-phosphate, phosphoenolpyruvate and fructose-l, 6-diphosphate were obtained from Boehringer GmbH, Mannheim, West Germany. INT, INT-diformazan and acetyl-CoA were obtained from Sigma Chemical Co., St. Louis, MO, U.S.A. Anthrone was obtained from Merck, Darmstadt, West Germany. Acetoacetyl-CoA was synthesized from acetyl-CoA and diketen and standardized by the method described by Sugden & Newsholme (1973). Solutions were made up with glass double-distilled water.
113 RESULTS
The ultrastructural features of the tergocoxal muscles of A. thoracica and P. sinuata are shown in Fig. l a and b. The muscle of P. sinuata has short, wide sarcomeres and columns of closely packed, den. ,1' sely staining mltochondrla in longitudinal section. In transverse section, the myofibrils were cylindrical and showed a double hexagonal arrangement of myofilaments with an actin :myosin filament ratio of 3:1. The ultrastructure of the dorsal longitudinal muscle of P. sinuata was similar to that of the tergocoxal muscle (Mitchell, 1976). All these features are characteristic of the fibrillar muscle type (Elder, 1975). On the other hand, the tergocoxal muscle of A. thoracica shows the typical features of afibrillar muscle such as long, narrow sarcomeres, dense and wide Z lines, and small, paired mitochondria on either side of the Z line (Fig.
Fig. I. (a) Longitudinal section of the tergocoxal muscle of P. sinuata, x 10,000; (b) longitudinal section of tergocoxal muscle of A. thoracica, x 10,000; (c) rosettes of glycogen in P. sinuata muscle, x 60,000; (d) glycogen granules in A. thoracica muscle, x 40,000.
J. A. MITCHELL,J. J. A. HEFFRONAND H. R. HEPBURN
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Table 1. Activities of actomyosin ATPase and some glycolytic enzymes in the indirect flight muscles of P. sinuata and tergocoxal muscle of A. thoracica Enzyme
Anthia thoracica
Pachynoda sinuata
Actomyosin ATPase Phosphorylase Pyruvate kinase Fructose-l, 6-diphosphatase
0.105 + 0.023 (6) 1.19 _+ 0.42 (9) 14.3 _+ 1.08 (6) 1,54 Jr 0.18 (6)
0.244 _+ 0.034(6} 21.9 _+ 1.45 (6) 41.1 _+ 2.51 (6) 2.96 _+ 0.40 (6)
Signilicance P P P P
< < < <
0.005 0.0005 0.0005 0.005
ATPase activity is expressed as ,umoles/mg protein/min. Other enzyme activities are expressed as llmoles/g muscle/rain. Values are given as mean _+ S.E.M. with the number of insects in brackets. An unpaired t test was used for statistical analysis of the results. lb). In addition, the myofibrils were lamellar-like and radially oriented in transverse section while the myofilament arrangement showed 11 12 thin filaments in slightly irregular orbit about each thin filament (Mitchell, 1976). Since the dorsal longitudinal and tergocoxal muscles of P. sinuata are both fibrillar muscles and they occur together in the thorax, it was convenient to use both muscles for preparation of homogenates for the enzyme assays. Table 1 summarizes the activities of actomyosin ATPase and some glycolytic enzymes in the indirect flight muscle of P. sinuata and in the homologous tergocoxal muscle of A. thoracica. Actomyosin ATPase activity of P. sinuata muscle was 2.3 times greater than in A. sinuata muscle and the corresponding ratio of phosphorylase activity was 18.4:1. The activity ratios of pyruvate kinase and fructose-l, 6-diphosphatase were 2.9:1 and 1.9:1, respectively. The low phosphorylase activity of the A. thoracica muscle suggested that aerobic glycogenolysis might not be an important source of energy for muscular contraction. This was confirmed by determination of the glycogen content of the muscles of both insects; A. thoracica muscle contained 1.36 _+ 0.39 mg glycogen/g (n = 6) while that of P. sinuata contained 17.6 _+ 1.88 mg/g (6). Further evidence for this was obtained from the nature and density of the glycogen granules in electron micrographs of the muscles. Glycogen is very abundant and exists as large rosettes and groups of single granules in the muscle of P. sinuata (Fig. Ic) but occurs only as single granules distributed in low density in A. thoracica muscle (Fig. ld}. The activities of some important enzymes of oxidative metabolism are shown in Table 2. Sarcoplasmic glycerol-3-phosphate dehydrogenase activity is an in-
dicator of the potential of the muscles to utilize N A D H produced in the sarcoplasm. The activity of this enzyme in P. sinuata was some 15-fold greater than in A. thoracica indicating the high capacity of the muscle of the former to carry out aerobic carbohydrate metabolism, and also substantiating the very low glycolytic capacity of A. thoracica muscle. The maximal activity of 3-hydroxyacyl-CoA dehydrogenase indicates the relative importance of fatty acid oxidation in energy provision for contractile activity. It is shown in Table 2 that of all the enzymes examined in this study only 3-hydroxyacyl-CoA dehydrogenase activity was similar in both insects. The four-fold greater activity of succinic dehydrogenase in P. sinuata compared with A. thoraciea confirms the greater overall aerobic capacity of the fibrillar muscle. Mobilization and oxidation of proline to oxaloacetate is of particular importance in the initial phase of insect flight (Hochacha & Somero, 1973) when the availability of oxaloacetate is the principle factor limiting the rate of metabolite oxidation. The relative activities of proline dehydrogenase provide an estimate of the importance of proline mobilization in providing sufficient oxaloacetate to meet the large demands for ATP during sudden bursts of muscular activity in insects. It is significant then that proline dehydrogenase activity in P. sinuata muscle was 4-fold greater than in A. thoracica (Table 2), similar to the ratio of succinic dehydrogenase activity in the two species. DISCUSSION The results presented here show that the tergocoxal muscle of A. thoracica has the typical ultrastructural features of afibrillar muscle while its homologue in
Table 2. Activities of some oxidative enzymes in the indirect flight muscles of P. sinuata and tergocoxal muscle of A. rhoracica Enzyme Glycerol-3-phosphate dehydrogenase 3-Hydroxyacyl CoA dehydrogenase Succinic dehydrogenase Proline dehydrogenase
Anthia thoracica
Pachynoda sinuata
Significance
0.391 _ 0.090 (6)
6.03 +_ 0.25 (7)
P < 0.0005
7.63 + 1.34 (4)
6.61 _+ 1.45 (6t
N.S.
0.703 +_ 0.048 (9) 2.44 + 0.29 (6)
2.75 + 0.14 (6) 8.94 +_ 0.28 (6)
P < 0.0005 P < 0.0005
Enzyme activities are expressed as #moles/g muscle/min. Values are given as mean +_ S.E.M. with the number of insects in brackets. An unpaired t test was used for statistical analysis. N.S. = not significant.
Enzymes of insect muscle P. sinuata is typically fibrillar. The dorsal longitudinal
muscle of the latter is similar ultrastructurally to its tergocoxal muscle, and for this reason, no attempt was made to separate them after removal from the thorax prior to preparation of the homogenates. The demonstration of an almost 3-fold greater actomyosin ATPase activity in the muscles of P. sinuata compared with that of A. thoracica is consistent with the strong flying ability of the former (cf. Barnett et al., 1975) and with the principle that fibrillar muscle is the fastest-contracting muscle type. Since maximal actomyosin ATPase activity is a good index of the maximal speed of contraction of muscles of widely different species (Barany et al., 1967; Bullard et al., 1973) it may be inferred from these and the present study that the structural and functional features of the contractile apparatus of synchronous muscle are of the conservative type from the evolutionary viewpoint. Unlike the case in vertebrate striated muscle the role of phosphorylase and glycogen stores in insect flight muscle is not to provide ATP under hypoxic or anaerobic conditions. Rather, glycogen is metabolized oxidatively under normal conditions. It is clear from the present results that glycogen metabolism is of little importance for the activity of the tergocoxal muscle of A. thoracica since both the glycogen content and phosphorylase activity are considerably less than in vertebrate red slow-twitch muscle (cf. Crabtree & Newsholme, 1975). In insect flight muscle the activities of the enzymes of the Krebs cycle are usually sufficient to permit complete oxidation of glucose units formed from glycogen at the maximum activity of phosphorylase (cf. Tables 3 8,: 4, Crabtree & Newsholme. 1975). In this study, relative succinic dehydrogenase activities were measured since we omitted the electron transferring agent, phenazine methosulphate, from the assay media. This was necessary because of considerable variation in non-enzymatic reduction of INT by this compound. For this reason the activities of succinic dehydrogenase in both insects cannot be compared directly with the respective phosphorylase activities; however, the results clearly show the greater capacity of the flight muscles of P. sinuata to carry out complete oxidation of glycogen. Insect flight muscles possess a glycerol-3-phosphate cycle which, via the activities of sarcoplasmic NADlinked glycerol-3-phosphate dehydrogenase and the mitochondrial flavoprotein glycerol-3-phosphate dehydrogenase, catalyses oxidation of sarcoplasmic NADH by the mitochondrial electron transport chain (Sacktor, 1970). The present work shows that the flight muscle of P. sinuata had a 15-fold greater activity of sarcoplasmic glycerol-3-phosphate dehydrogenase than the tergocoxal muscle of A. thoracica, an activity ratio similar to that of phosphorylase in the same muscles. This substantiates the earlier conclusion that glycogenolysis is of minor significance in the energy metabolism of the tergocoxal muscle of A. thoracica, and indicates that glycolysis of such sugars as trehalose is also of little importance. Considering the low actomyosin ATPase, phosphorylase and glycerol-3-phosphate dehydrogenase activities, it may be concluded that the tergocoxal muscle of the A. thoracica performs slow but sustained contractions in vivo. This contrasts with the synchronous hind-leg muscles of the locust (Locusta migratoria) which have
115
a high phosphorylase activity that enables them to perform extremely rapid and vigorous contractions similar to vertebrate white muscles (cf Crabtree & Newsholme, 1975). The finding of similar 3-hydroxyacyl-CoA dehydrogenase activities in both species suggests that fatty acid oxidation is the predominant energy-generating pathway in A. thoracica muscle, while the same pathway in P. sinuata may play a similar but supplementary role for sustained periods of flight. Proline dehydrogenase is the first enzyme involved in the oxidation of proline and its maximum activity is a useful estimate of the importance of proline as a fuel for muscular activity in insects (Crabtree & Newsholme, 1970). On this basis, the flight muscles of P. sinuata show a three-fold greater capacity for proline oxidation than the tergocoxal muscle of A. thoracica. Both types of muscle can utilize proline as an energy source for contractile activity. Since the flight muscles of P. sinuata are capable of rapid action in vivo, it is likely that the proline oxidation pathway is also important for provision of adequate amounts of oxalo-acetate for maximal operation of the Krebs cycle in the early phases of flight (cf. Hochacha & Somero, 1973). The muscles of both species have high pyruvate kinase activities. In the case of P. sinuata, the two-fold greater activity of pyruvate kinase compared with phosphorylase indicates that the maximum glycolytic rate is determined by the latter. It is difficult to explain the significance of the very high pyruvate kinase/phosphorylase ratio in A. thoracica though it may be postulated that phosphorylase, being the first enzyme of the glycogenolytic pathway, is subject to greater evolutionary adaptive pressures than enzymes further along the pathway. In insect flight muscles, the fructose-6-phosphate cycle is catalyzed by the enzymes phosphofructokinase and fructose-i, 6-diphosphatase. It has been suggested that its main role is to amplify the sensitivity of the glycolytic flux to AMP, although in the flight muscles of the bumble-bee, the cycle functions as an ATPase for heat production (Crabtree & Newsholme, 1975). In the present study, the relatively low activities of fructose-i, 6-diphosphatase in both muscle types, indicating the maximal capacity for cycling of fructose-6phosphate, suggest that the role of the cycle is to provide amplification for glycolytic control rather than heat. The overall changes in enzymatic constitution of the tergocoxal muscle of A. thoracica occurring during the transition to the flightless state result in development of a synchronous muscle type having a virtually completely aerobic metabolism. Fatty acid oxidation is the primary source of energy while proline oxidation may act as a supplementary source. Further, it appears that fatty acid oxidation is a very conservative metabolic pathway in insect muscles generally, while the glycolytic pathway is responsive to the energy requirements of the various modes of functional specialization occurring in the insect kingdom.
Acknowledgements--We thank the South African Council for Scientific and Industrial Research for financial support, and Dr. Wolfgang Prinz for synthesizing acetoacetylCoA.
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J.A. MITCHELL,J. 3. A. HEFFRONAND H. R. HEPBURN REFERENCES
BARANYM., CONOVERT. E., SCHLISELFIELDL. H., GAETJENS E. & GOFFARTM. (1967) Relation of properties of myosin to those of intact muscles of the cat and sloth. Eur. J. Biochem. 2, 156-164. BARNETTP. S., HEFFRONJ. J. A. & HEPBURN H. R. (1975) Some thermal characteristics of insect flight: Enzyme optima versus intra-thoracic temperature. S. Afr. J. Sci. 71, 373-374. BASS A., BRDICZKA D., EYER P., HOFER S. t~¢ PETTED. (1969) Metabolic differentiation of distinct muscle types at the level of enzymatic organization. Eur. J. Biochem. 10, 198-206. BULLARDB., DARROWSKAR. & WINKELMANL. (1973) The contractile and regulatory proteins of insect flight muscle. Biochem. J. 135, 277 286. CRABTREE B. & NEWStfOLMEE. A. (1970) The activities of proline dehydrogenase, glutamate dehydrogenase, aspextate-oxoglutarate aminotransferase and alanineoxoglutarate aminotransferase in some insect flight muscles. Biochem. J. 117, 1019-1021. CRAR~EE B. & NEWSHOLMEE. A. (1972) The activities of phosphorylase, hexokinase, phosphofructokinase, lactate dehydrogenase and glycerol-3-phosphate dehydrogenase in muscles from vertebrates and invertebrates. Biochem. J. 126, 49-58. CRARTREE B. & NEWSHOLME E. A. (1975) Comparative aspects of fuel utilization and metabolism by muscle. In Insect Muscle (Edited by USHEgWOOD P. N. R.) pp. 405-500, Academic Press, London. ELDER H. Y. (1975) Muscle structure. In Insect Muscle (Edited by USHERWOODP. N. R.) pp. t 74. Academic Press, London. HASSID W. Z. & ABRAHAMS. (1957) Chemical procedures for the analysis of polysaccharides. In Methods of Enzymology (Edited by COLOWIO~ S. P. & KAPLAN N. O.) Vol. 3, 34-37. Academic Press, New York.
HEFFRON J. J. A. & DUGGAN P. F. (1971) Adenosinetriphosphatase activity and superprecipitation of actomyosin from the frog, Rana temporaria. Int. J. Biochem. 2, 324-336. HOCHACHA P. W. (~ SOMEROG. ~1973) Strategies oJ' Biochemical Adaptation. pp. 72-73, W. B. Saunders Co., Philadelphia. IMMS A. D. (1964) A General Textbook ¢ff" Entomolo(4y, Parts I & III, Methuen & Co. Ltd, London, LOWRY O. H., ROSEBROUGHN. J,, FARR A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265--275. MITCHELL J. A. (1976) Muscle ultrastructure and enzymology of flying and flightless beetles. M. Sc. thesis. University of the Witwatersrand, Johannesburg. NEWSHOLMEE. A., CRABTREEB., HIGGINS S. J., THORNTON S. D. & STARTC. (1972) The activities of fructose diphosphatase in flight muscles from the bumble-bee and the role of this enzyme in heat generation. Biochem. J. 128, 89- 97. PENNINGTON R. J. (19611 Biochemistry of dystrophic muscle. Mitochondrial succinate-tetrazolium reductase and adenosine triphosphatase. B'iochem. J. 80, 649-654. SACKTORB. (1970) Regulation of intermediary metabolism, with special reference to the control mechanisms in insect flight muscle. Adv. Insect Physiol. 7, 267 347. SIMON L. M. & ROBIN E. D. (1972) Relative anaerobic glycolytic capacity and pyruvate kinase activity of rabbit tissues. Int. J. Biochem. 3, 329-332. SUGDEN P. H. & NEWSr~OLME E. A. (1973) Activities of hexokinase, phosphofructokinase, 3-oxo-acid coenzyme A-transferase and acetoacetyl-coenzyrne A thiolase in nervous tissue from vertebrates and invertebrates. Biochem. J. 134, 97-101. TAUSSKY H. H. & SHORR E. (t953) A microcolorimetric method for the determination of inorganic phosphorus. J. biol. Chem. 202, 675-.-685.