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Thoracic muscle protein biosynthesis in Lucilia cuprina
Insect Biochem., 1975, Vol. 5, pp. 43 to 52. Pergamon Press. Printed in Great Britain
T H O R A C I C M U S C L E P R O T E I N B I O S Y N T H E S I S IN LUCILIA C U P R I N A ANNE J. CAMPBELL* and L. M. BIRT t Department of Biochemistry, School of General Studies, Australian National University, Canberra, A.C.T. Australia (Received 24 April 1974)
Abstract--From measurements of the rate of incorporation of injected [14C] leucine into thoracic actomyosin as well as from those of changes in leucine pool turnover, it was found that thoracic actomyosin synthesis was appreciable 2 days before emergence, that it increased rapidly up to emergence and was almost constant during the first day after emergence; by 3 days after emergence it had declined. Most of the synthesis of actomyosin was required for the increase in the protein which occurred over emergence. INTRODUCTION PREVIOUS studies on the quantitative importance of protein synthesis de novo during development of Lucilia in the puparium showed that there were two periods of marked thoracic protein biosynthetic activity, one occurring about 2 days before emergence and the other over emergence (WILLIAMSand BIRT, 1972). It was proposed that the synthetic activity spanning emergence could be ascribed mainly to the formation of proteins for mitochondrial maturation (WILLIAMSand BIRT, 1971a,b; WILLIAMSet al., 1972) and for energizing flight (CAMPBELLand BIRT, 1972; CAMPBELL,et al., 1973) after emergence. As extensive microscopic studies (GREGORY,et al., 1968; BARRITT and BIRT, 1971; PERISTIANIS and GREGORY, 1971) had shown that there is a rapid accumulation of muscle tissue before emergence, it was surmised that the first peak might be largely a consequence of thoracic muscle protein synthesis (WILLIAMSand BIRT,1972). This proposition was also consistent with the sequence of synthetic events described for isolated thoraces of tobacco horn worm, in which it was found that a peak of cytoplasmic protein synthesis preceded one of mitochondrial protein synthesis (CHAN and RICHARDSON, 1969 ; CHAN and REIBLING,1971). The present study was designed to determine whether the synthesis of the thoracic contractile proteins contributed substantially to the first peak of protein synthesis. This was assessed by measuring the rate of incorporation of an injected radioactive amino acid into actomyosin isolated from insects from 2 days before to * Anne J. Campbell, Department of Biochemistry, University of Leicester, University Road, Leicester, LEI 7RH. J" L. M. Birt, Wollongong University College, Wollongong, N.S.W. 2500 Australia. 43
44
ANNE J. CAMPBELL AND L. M. BIRT
3 days after emergence. Actomyosin was selected, as not only does it represent the functional combination of contractile proteins in active muscle, but because its purification is simple and well documented for insects (GILMOUR and CALABY, 1953; MARUYAMA,1954, 1957, 1958; MARUYAMAet al., 1968); purification was followed by measuring Ca~+ATPase activity. MATERIALS AND METHODS
Insect culture Insects were grown at 30°C under continuous illumination as detailed elsewhere
(CAMPBELLand BIRT, 1972; WILLIAMS and BIRT, 1972). Insects were collected within 1 hr of pupariation (day 0) and flies within 1 hr of emergence from the puparium (day 6); flies were fed sugar and water.
Injection of insects Insects were cooled on ice and injected thoracically (CAMPBELLand BIRT,1972), with 25 nCi[14C]leucine (342 mCi per mmole); pharate adults were left on the needle for a further 15 sec. Those from which actomyosin was to be extracted were kept for 30 min at 30°C and then cooled on ice, whereas with those taken for timecourses of incorporation into total protein (acid-insoluble material), incubations were terminated by freezing.
Tissue preparation Whole insects: for time course studies insects were dispersed individually, in 0.5 ml cold 1 0 ~ trichloroacetic acid and acid-insoluble material prepared for counting and applied to discs (Gelman) essentially as outlined by WILLIAMS and BraT (1972). Routinely the total amount of acid-soluble radioactivity remaining after 30 min was determined per insect in order to measure Ka (rate of leucine pool turnover; see WILLIAMS and BIRT, (1972). Actomyosin: unless stated otherwise, all procedures were conducted at 2 to 4°C; all centrifuging was for 15 min and values for g are based on Ray. Insects were thoroughly chilled on ice, thoraces dissected out and homogenized gently in a Dounce glass tissue grinder (type A pestle) in 0.6 M KC1-0.06 M KHCO3, pH 8 using 1 g tissue: 15 ml extractant. The homogenate was stirred in ice for 1 hr, filtered through glass wool on a sintered disc (0 porosity) and centrifuged at 13,300g. The supernatant is designated as homogenate supernatant. Crude actomyosin was precipitated by diluting the homogenate supernatant 10-fold with distilled water; the suspension was stirred for 10 min, sedimented at 20,750 g and the pellet dissolved in the original extractant. The dilution-precipitation procedure was repeated 3 times more and the final actomyosin precipitate was resuspended in 0.6 M KC1 (1-2ml), stirred for 10 min and cleared by centrifuging at 9220g for 10 min. Actomyosin was assayed for Ca~+ATPase activity on the day of preparation but the enzyme was found to be relatively stable for up to 4 days at 2 to 4°C.
THORACIC MUSCLE PROTEIN BIOSYNTHESIS I N LUCILIA CUPRINA
45
Ca-A TPase assay Enzyme activity was measured as the release of inorganic P. The incubation mixture (1-0 ml) contained 20 m M H E P E S - K O H (N-2-hydroxyethylpiperazineN'-2-ethane-sulphonic acid) pH 7.4, 3 m M ATP, 10 m M CaC12, 200 m M KC1; 10 m M sodium azide was also included (MARUYAMAet al., 1968). Reactions at 30°C were initiated with enzyme (10-50/~1) and stopped with 0.33 ml cold 25 trichloroacetic acid. Routinely, time courses of incubation (up to 30 min) at two different enzyme concentrations were carried out, together with zero-time controls. The amount of inorganic P in deproteinized incubation samples (0.2-0.5 ml) was measured microcolorimetrically .(TAUsSKYand SHORR, 1953) at 720 nm. Enzyme activity is expressed as nmoles inorganic P released per minute at 30°C; specific enzyme activity refers to enzyme activity per mg protein. Protein estimations Routinely, protein was precipitated with cold 1 0 ~ trichloroacetic acid, dissolved in 1 M NaOH at 30°C for 5-16 hr and estimated by the method of LOWRY et al. (1951) using Lowry reagent D; bovine serum albumin (fraction V; Sigma) served as a standard. However, for determination of 'total' thoracic protein content (excluding cuticular material), thoraces were homogenized in 1 M NaOH, incubated for 1 hr at room temperature and filtered through glass wool. The protein content of the filtrate was estimated by the biuret reaction (GORNALL,et al., 1949) and correction for absorbance due to particulate matter was applied using the K C N decolorization procedure (SZARKOWSKAand KLINGEr~BERG, 1963). Leucine pool size This was estimated on groups of 10 insects essentially as outlined by CAMPBELL et al., 1973. Analysis of radioactivity Samples were counted with an efficiency of about 80~o in 10 ml scintillation fluid (1200 ml toluene/800 ml ethoxyethanol/12 g butyl PBD (2(4't-butylphenyl)5-(4"-biphenyl)-l,3,4-oxadiazole). It was satisfactory to count a solution of actomyosin (20-100/~1) directly rather than to apply an acid-precipitate of it to discs as was done for time course studies of [14C]leucine incorporation into total insect protein. RESULTS
Ca-A TPase activity In using Ca2+ATPase as a marker for following the purification of actomyosin, it was necessary to establish that the contribution of other cellular ATPases (e.g. from mitochondria and microsomes) was insignificant under the assay conditions. Oligomyein (0.7-2.8tzg/mg protein) which severely inhibits mitochondrial
46
ANNEJ. CAMPBELLANDL. M. BIRT
ATPase at 1/~g per mg mitochondrial protein (SLATERand TERWELLE,1969) did not inhibit Ca2+ATPase of the thoracic homogenate supernatant or of purified actomyosin. Sodium azide (10-20 raM), an inhibitor of mitochondrial ATPase (SACKTOR, 1953) produced no inhibition of either unfractionated or purified preparations. Phospholipase c, a potent inhibitor of microsomal Ca2+ATPase, (MARTONOSI, 1968; MARTONOSI et al., 1968) did not inhibit Ca2+ATPase activity of the homogenate supernatant when incubated under the conditions used by MARTONOSI et al., (1968). Finally, the Ca2+ATPase activity of microsomes from 3 day old flies constituted less than 1 per cent of the total Ca2+ATPase activity of thoracic homogenate supernatants.
.4ctomyosin purification Identification of the protein isolated as actomyosin depended primarily on the specificity of the extraction procedure but in preliminary experiments, the isolated product was monitored for superprecipitation and ultraviolet absorption. Purified actomyosin (0.5 mg protein per ml in 0.1 M KC1, 20 mM Tris-HC1 pH 7.4) and 0-5 mM ATP, formed a fibrous plug at room temperature; this 'superprecipitation' is a characteristic response of actomyosin to ATP (MARUYAMA, 1965). The maximum and minimum absorbances for actomyosin purified from 3 day old flies were 265 and 255 nm respectively, compared to 275 and 255 nm for mature honeybee actomyosin (MARuYAMA, 1957, 1958, 1965); the absorption maximum shifted to 260 and 255 nm in preparations from newly emerged and pharate adults of Lucilia compared to a shift to 260 to 270 nm in pharate adults of honeybee (MARUYAMA,1957). In general, it was found that with purified actomyo-
T A B L E 1 - - P R E P A R A T I O N S OF THORACIC ACTOMYOSIN FROM
Lucilia
OF DIFFERENT AGES
~o Actomyosin Age (days) 4 5 6 7 9
Homogenate supernatant Actomyosin PurificaATPase/rng tion ATPase Protein protein (fold) 76 69 90 69 61
2"2 7"4 23 22 19
889 1429 1843 1307 1720
39 97 4 3 3
ATPase/ thorax 16(12) 110(re) 317(285) 404(27~) 516(315)
/~g Actomyosin/ thorax 18(14) 77(53) 172(155) 309(~13) 300(133)
Actomyosin was purified from 100 thoraces of day 4 insects and from 50 thoraces of insects from days 5-9; pharate adults were of mixed sex; flies were males. Age was from pupariation (day 0); emergence occurred on day 6. ATPase measured as nmoles P released per min. at 30°C. * Total amounts of actomyosin ATPase and protein per thorax were corrected for losses during purification by assuming 100 ~ ATPase recovery from the homogenate supernatant; uncorrected values are shown in parenthesis.
47
THORACIC MUSCLE PROTEIN BIOSYNTHESIS I N LUCIL[A CUPRINA
sin (100/~g per incubation) only about 40 to 50~o of the labile phosphate of ATP was released as P after 20 min; at this protein concentration the rate of enzyme reaction was linear for up to 10 min. The recoveries of actomyosin-Ca~+ATPase and protein were always expressed relative to the homogenate supernatants (Table 1). In insects of different ages, 73 +_1 2 ~ of the homogenate supernatant Ca~+ATPase was found in purified actomyosin. Indirectly, the high recovery ( 9 0 ~ ) o f Ca2+ATPase from the homogenate supernatant from newly emerged flies supports the conclusion derived from the inhibitor studies, that the contribution of other ATPases to the assay of actomyosin Ca2+ATPase was not appreciable. The proportion of protein in the homogenate supernatant finally extracted as actomyosin increased about 10-fold (days 4-6); thereafter little change was noted. The extent of purification declined with increasing insect age to a value (3- or 4-fold), which was similar in newly emerged and older flies. Purification was not monitored through all dilution-precipitation steps as sampling losses would have been too great in view of the small amount of tissue (about 0.5 g) taken initially. In order to account for losses incurred during purification, the amount of enzyme activity and protein in the actomyosin isolated per thorax were corrected on the assumption that there was 100 per cent recovery of Ca2+ATPase activity from each particular homogenate supernatant preparation. The amount of protein in actomyosin per thorax increased 4.1, 2-2, and 1.8 times respectively between days 4 to 5, 5 to 6 and 6 to 7; during these periods the amounts of actomyosin
T A B L E 2 - - S Y N T H E S I S OF ACTOMYOSIN IN
Age (days)
dis/min/#g actomyosin/ hr
4 5 6 7 9
45"4 39"8 32"0 13"6 8"8
Lucilia
dis/rain into Leucine pool actomyosin/ size thorax/hr (nmoles/insect) 817 3065 5504 4202 2640
19 19 23 33 3
OF DIFFERENT AGES
Ka leu.
Kp leu.
Actomyosin synthesis /~g/thorax/hr
21 23 30 48 5"4
0"36 1"40 3"10 3"56 0"22
0"6 2"1 4"7 5"4 0"3
Insects injected with 25nCi[t4C]leucine were incubated for 30 min at 30°C and thoracic actomyosin prepared (Table 1). Age is from pupariation (day 0); emergence occurred on day 6. Pool size valves for pharate adults are those from BraT and CHRISTIAN(1969) for 30 mg insect); those for flies were measured directly. Ka (nmoles leucine per insect per hr) and Kp (nmoles leucine incorporated into actomyosin per thorax per hr) were calculated from equations of Hearon (DINAMARCAand LEWI'~OOK, 1966; see also WILLIAMSand BraT, 1972). Corrected valves for actomyosin protein per thorax were used (Table 1). Actomyosin synthesis calculated as 1.529 x Kp (as outlined in the Text).
48
ANNE J. CAMPBELLAND L. M. BIRT
formed per day were 58, 95, and 137/zg respectively with no further formation of protein between days 7 to 9. The level found in mature flies (day 9) was already reached in 1 day old flies (day 7) (Table 1). In insects of corresponding ages the 'total' thoracic protein (excluding cuticular material) per insect was 1.4 mg (day 4), 2.5 mg (day 5), 1.9 mg (day 6), 2.1 mg (day 7), and 2.7 mg (day 9). The specific enzyme activity measured directly on the purified actomyosin, increased about twice from day 4 to day 5 and was relatively constant thereafter.
Actomyosin synthesis At each age studied, the rate of [l*C]leucine incorporation into total acidinsoluble material was linear for at least 30 min, the period selected for labelling of actomyosin. In calculating the rate of actomyosin synthesis (nmoles leucine incorporated into protein per thorax per hour; Kp, Table 2), the rate of leucine turnover in the pool (Ka; Table 2) and leucine pool size were used, as well as the measured incorporation of [14C]leucine into the purified protein. Equations for calculating Ka and Kp are those derived by Hearon in DINAMARCAand LEVENBOOK(1966). The amount of actomyosin synthesized was estimated from Kp as outlined by WILLIAMS and BIRT, 1972); it was assumed that leucine constituted 8.5 ~ of the amino acids of actomyosin. The rate of actomyosin synthesis rose to a maximum value at day 7 and declined considerably by day 9 (Table 2). It is noteworthy that [x4C]leucine incorporation into thoracic actomyosin represented about 4 per cent of that into total acidinsoluble thoracic protein at day 4 and about 25 per cent of that with older insects (days 5-9). It was important to establish that labelling of the actomyosin was due to incorporation into, rather than physical retention by, the protein during isolation. Accordingly, 50 newly emerged flies were injected but not incubated and the actomyosin purified and counted in the usual way. A total of 700 dis/min recovered in the protein purified from controls constituted from 0.5 to 3 . 4 ~ of the total radioactivity incorporated into actomyosin isolated from insects from days 4 to 9. Similar values (0.9-5.9 ~ ) were found in a control experiment using mature flies. DISCUSSION In Lucilia at days 4 and 5, the purified actomyosin protein constituted about 1.3 per cent and 3 per cent of the 'total' alkali-soluble thoracic protein, whereas from days 6 to 9, it constituted on average about 11 per cent. Newly emerged flies contained about half as much thoracic actomyosin as mature adults (day 9) in contrast to the newly emerged honey bee in which levels were close to those of the mature bee (MARUYAMA,1965). In general, in Lucilia of different ages, there were nearly equal amounts of thoracic actomyosin (Table 1) and mitochondrial protein (70, 180, 300, and 400/~g at days 5, 6, 7, and 9; WILLIAMS,1972).
THORACIC MUSCLE PROTEIN BIOSYNTHESIS I N LUCILIA CUPR1NA
49
There is a general similarity between the changes in the amount of actomyosin protein isolated from thoraces of developing Lucilia (Table 1) and the levels of thoracic Ca2+ATPase activity in the same insect (Table 1). The most striking difference is the absence of any increase in the amount of actomyosin protein per thorax after day 7, compared to a further 20 per cent increase in Ca2+ATPase activity from days 7-9. It is possible that the contractile proteins are present at day 7 without full development of their potential ATPase activity or that actomyosin can exist in two forms, active and inactive and that these are not separated by the isolation procedure. From Table 2, it is clear that although there is some synthesis of actomyosin before emergence (days 4-5), it is most rapid in the day afterwards (days 6-7); by day 9 it has declined. Thus actomyosin synthesis will contribute substantially to the bulk incorporation of labelled amino acids at both days 6 and 7 (compare Fig. 4; WILLIAMSand BIaT, 1972) but not to that between days 4 and 5. Therefore some other cellular component remains to be identified as the site of the pronounced incorporation before emergence (days 4-5); it is distinctly possible that this could be the cuticle of the insect. Preliminary measurements of the rate of [14C]leucine incorporation into isolated cuticles, have indicated that the pattern of variation of protein synthesis in whole insect cuticle (as nmoles leucine incorporated into cuticle per insect per hour) was very similar to that obtained for 'bulk' protein (WILLIAMSand BIRT, 1972); it remains to determine what proportion of cuticle synthesis before emergence is due to that in the thorax. Some correlation can be attempted between actomyosin synthesis and the appearance of the protein. In the 2 days spanning emergence (days 5-7) it can be calculated (by assuming average rates of actomyosin synthesis for each of the 24 hr periods subsequent to day 5) that about 90 per cent of the actomyosin formed by the insect was being synthesized de novo. It seems likely that after day 7, the rate of actomyosin synthesis must decline abruptly for no further increase in the amount of protein was measured after this time. A similar rapid decline in cytochrome c synthesis had been found previously in Lucilia flies (WILLIAMSet al., 1972). These calculations of the rate of synthesis of actomyosin from [14C]leucine incorporation assume that the total amino acid pool is that used for synthesis, that there is rapid mixing of the injected amino acid in the pool and that, in the pool, there is relatively little inter-conversion of injected leucine to other metabolites. Previously, the assumption of a single amino acid pool successfully accounted for synthesis of other proteins in Lucilia (WILLIAMSand BIRT, 1972; WILLIAMSet al., 1972; CAMPBELLet al., 1973) and it was also found that the only radioactivity incorporated into specific proteins was that of the injected radioactive amino acid (WILLIAMS et al., 1972; CAMPBELLet al., 1973). The rate of synthesis of actomyosin (/zg/thorax per hour) can be compared with the synthetic rates of other thoracic proteins which have been measured in a similar way (using Ka and Kp). In Lucilia at emergence, actomyosin is synthesized 47 times faster than cytochrome c (from [14C]lysine; WILLIAMSet al., 1972) and 22 times faster than glycerophosphate dehydrogenase (from 14C leucine; CAMPBELL
50
ANNE J. CAMPBELLANDL. M. BIRT
et al., 1973) but only 10 per cent faster than mitochondrial protein (from [14C]
ieucine; WILLIAMS,1972). In the 2 days spanning emergence, about 7 5 - 8 0 ~ of the total thoracic protein synthesized (0-54 mg; WILLIAMS and BIRT, 1972) can be attributed to the appearance of both mitochondrial (0.18-0.2 mg: LENNIE et al., 1967; WILLIAMS, 1972) and actomyosin (0.23 rag) protein. The rapid synthesis of both these protein complexes over emergence can be correlated with the development of the capacity for flight soon after eclosion. A comparison between both the formation and synthesis of actomyosin and the ultrastructural development of the thoracic muscle (GRECORY et al., 1968; BARRITT and BIRT, 1971; PERISTIANIS and GREGORY, 1971) can also be attempted. The relevant microscopical observations are (a) by day 5 the indirect flight muscle has reached almost all of its final volume (BARRITTand BIRT, 1971) and (b) the sarcomere length probably does not change significantly from emergence to maturity and each sarcomere is approximately cylindrical (GREGORYet al., 1968). From these observations, and by setting the value for the increase in the number of myofilaments per fibril during the first day after emergence as 1, it can be calculated that the number of myofilaments increase 1.6 times after the first day post-emergence; if the same assumptions are used for the 3 days preceding emergence, the relative increase is 1.3 times (though there is a much greater likelihood of error in the latter calculation as there is no precise information on sarcomere size and number in the pharate adults). These considerations suggest that there is substantial formation of muscle filaments after the first day post-emergence, in sharp contrast to conclusions reached by studying the synthesis (Table 2) and appearance (Table 1) of the thoracic actomyosin. This disparity could be interpreted as indicating that the developing muscle accumulates actomyosin for some time before the final assembly of the myofilaments or that actomyosin is more difficult to extract from the older insects. Considerable speculation exists as to the method and sequence of assembly of the proteins in the contractile apparatus; a number of reports suggest that assembly proceeds asynchronously both at the biochemical (OGAWA, 1962; HITCHCOCK, 1970) and ultrastructural level (HAY, 1963 ; ALLEN and PEPE, 1965 ; PRZYBYLSKIand BLUMBERG,1966; FISCHMAN,1967). The results with Lucilia suggest that the formation of actomyosin (which has reached about 75 per cent of the adult level by day 7 estimated by either ATPase or actomyosin protein levels) precedes myofilament formation (by day 7 about 60 per cent of the adult levels are present). It can be envisaged that the sequential development of particular properties of the contractile apparatus proceeds by (a) synthesis de novo of individual proteins of the contractile apparatus (b) development of enzyme activity (c) myofilament formation. The rate of synthesis of actin and myosin may accelerate from days 4-6, as does the rate of assembly into functional units (ATPase) so that the specific enzyme activity of actomyosin increases (Table 1). At the period of maximal synthesis (days 6-7), the formation of actin and myosin exceeds the rate of assembly into functional units, i.e. specific activity declines by day 7 (Table 1). Assembly but not synthesis
THORACIC MUSCLE PROTEIN BIOSYNTHESIS IN LUCILL4 CUPRINA
51
continues appreciably f r o m days 7 to 9, so that again the specific activity increases (Table 1). As myofilaments are laid down in the greatest n u m b e r s after the first day post-emergence, in contrast to the development of enzyme activity, this implies that there is a delay of incorporation of enzymatically active complexes into the complete filament array of the functional muscle. A capacity for self-assembly of filaments f r o m their c o m p o n e n t molecule,s has been shown in vitro (e.g. HUXLEY, 1963). It is clear that a correlation of ultrastructural development with biochemical analyses in developing muscle in Lucilia offers a fruitful field for further enquiry.
Acknowledgements--We thank Mr. A. VAN GEBWEN (C.S.I.R.O. Entomology, Canberra) for the supply of insects and Mr. M. DESMET, Biochemistry Department, S.G.S. for performing the amino acid analyses. A.J.C. was a recipient of a Ph.D. Scholarship from the Australian National University. The work was supported by funds from the Australian Research Grants Committee (D76/16645).
REFERENCES ALLEN E. R. and PEPE F. A. (1965) Ultrastructure of developing muscle cells in the chick embryo. A m . J . Anat. 116, 115-148. BARRITT L. C. and BIRT L. M. (1971) Development of Lucilia cuprina: correlation of biochemical and morphological events..7. Insect Physiol. 17, 1169-1183. CAMPBELL A. J. and BIRT L. M. (1972) Studies on the appearance of soluble d-glycerophosphate dehydrogenase activity during the development of the sheep blowfly, Lucilia. Insect Biochem. 2, 279-296. CAMPBELL A. J., SHAW D. C., and BIRT L. M . (1973) Biosynthesis of soluble a-glycerophosphate dehydrogenase during the adult development of the sheep blowfly, Lucilia. Insect Biochem. 4, 135. CHAN S. K. and KEIBLINGA. (1971) Biochemical studies in the developing thoracic muscles of the tobacco hornworm. I I I Amino acid incorporation in the cytoplasmic ribosomal fraction. Biochem. biophys. Acta 228, 252-258. CHAN S. K. and Rlct-mm)SON L. (1969) Biochemical studies on the developing thoracic muscles of the tobacco hornworm. J. biol. Chem. 244, 1039-1042. FISCHMAN D. A. (1967) An electron microscope study of myofibril formation in embryonic chick skeletal muscle. J. Cell Biol. 32, 557-575. GILMOURD. and CALABYJ. H. (1953) Physical and enzymic properties of actomyosins from the femoral and thoracic muscles of an insect. Enzymologia 16, 23-33. GORNALLA. G., BPd~DAWmLC. S., and DAVIDM. M. (1949) Determination of serum proteins by means of the biuret reaction, ft. biol. Chem. 177, 751-766. GREOORYD. W., LENNIE R. W., and BIRT L. M. (1968) An electron-microscopic study of flight muscle, development in the blowfly, Lucilia cuprina, jY. R. microsc. Soc. 88, 151-175. HAY E. D. (1963) The fine structure of differentiating muscle in the salamander tail. Z. Zellforsch. microsk. Anat. 59, 6-34. HITCHCOCKS. E. (1970) The appearance of a functional contractile apparatus in developing muscle. Devl. Biol. 23, 399-423. HUXLEYH. E. (1963) Electron microscope studies on the structure of natural and synthetic protein filaments from striated muscle..~, molec. Biol. 7, 281-308. LV2qNIER. W., GREGORYD. W., and BIRT L. M. (1967) Changes in the nucleic acid content and structure of thoracic mitochondria during development of the blowfly, Lucilia cuprina. .7. Insect Physiol. 13, 1745-1756.
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LOWRY O. H., ROSEBROUGHN. J., FARR A. L., and RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent. ~. biol. Chem. 193, 265-275. MARTONOSI A. (1968) Sarcoplasmic reticulum IV. Solubilization of microsomal adenosine triphosphatase, y. biol. Chem. 243, 71-81. MARTONOSIA., DONLEYJ., and HALPIN R. A. (1968) Sarcoplasmic reticulum III. The role of phospholipids in the adenosine triphosphatase activity and Ca 2+ transport. ~. biol. Chem. 243, 61-70. I~¢IARUYAMAK. (1954) Studies on adenosine triphosphatases of various insect muscles. ~. Fac. Sci. Univ. Tokyo 7, 231-270. ~JIARUYAMAK. (1957) A further study of insect actomyosin. Sci. Pap. Coll. gen. Educ. Univ. Tokyo F, 213-241. 1VIARUYAMAK. (1958) Interaction of insect actomyosin with adenosine triphosphate, o7. Cell. comp. Physiol. 51, 173-187. ~/IARUYAMAK. (1965) The biochemistry of the contractile elements of insect muscle. In The Physiology of Insecta (Ed. by ROCKSTEINM.). 2, 451-482. Academic Press, New York. MARUYAMA K., PmNGLE J. W. S., and 'IMEaEAR R. T. (1968) The calcium sensitivity of ATPase activity of myofibrils and actomyosins from insect flight and leg muscles. Proc. R. Soc. (B) 169, 229-240. OGAWA Y. (1962) Synthesis of skeletal muscle proteins in early embryos and regenerating tissue of chick and Triturus. Exp. Cell Res. 26, 269-274. PERISTIANISG. C. and GREaORY D. W. (1971) Early stages of flight muscle development in the blowfly Lucilia: a light and electron microscopic study. J. Insect Physiol. 17, 10051022. PRZYBYLSKIR. J. and BLUMBERaJ. M. (1966) Ultrastructural aspects of myogenesis in the chick. Lab. Invest. 15, 836-863. SACKTORB. (1953) Investigations on the mitochondria of the housefly, Mustica domestical 1. Adenosinetriphosphatases. 3r. gen. Physiol. 36, 371-387. SLATER E. and TER WELLE H. F. (1969) Applications of oligomycin and related inhibitors. In Inhibitors: Tools in Cell Research (Ed. by BUCHER Th. and SIES H.) pp. 258-278. Springer-Verlag, New York. SZARKOWSKAL. and KLINaENBERGM. (1963) On the role of ubiquinone in mitochondria. Biochem. Z. 338, 674-697. TAVSSKYH. H. and SHOI~ E. (1953) A microcolorimetric method for the determination of inorganic phosphorus. J. biol. Chem. 202, 675-685. WILLIAMS K. L. (1972) Protein synthesis during the metamorphosis of Lucilia cuprina, Ph.D. Thesis, Australian National University, Canberra. WILLIAMS, K. L. and BIRT L. M. (1971a) Incorporation in vitro of 1~C leucine into mitochondrial protein of Lueilia cuprina 1. Basic requirements. Eur. ~. Biochem. 22, 87-95. WILLIAMS K. L. and BIRT L. M. (1971b) Incorporation in vitro of 14C leucine into the mitochondrial protein of Lueilia cuprina 2. Energy requirements. Eur .~. Biochem. 22, 96-103. WILLIAMS K. L. and BIRT L. NI. (1972) A study of the quantitative significance of protein synthesis during the metamorphosis of the sheep blowfly, Lucilia. Insect Biochem. 2, 305-320. WILLIAMS K. L., SMITH E., SHAW D. C., and Brat L. M. (1972) Studies of the levels and synthesis of cytochrome c during adult development of the blowfly, Lucilia cuprina. .7. biol. Chem. 247, 6024-6030.
T H O R A C I C M U S C L E P R O T E I N B I O S Y N T H E S I S IN LUCILIA C U P R I N A ANNE J. CAMPBELL* and L. M. BIRT t Department of Biochemistry, School of General Studies, Australian National University, Canberra, A.C.T. Australia (Received 24 April 1974)
Abstract--From measurements of the rate of incorporation of injected [14C] leucine into thoracic actomyosin as well as from those of changes in leucine pool turnover, it was found that thoracic actomyosin synthesis was appreciable 2 days before emergence, that it increased rapidly up to emergence and was almost constant during the first day after emergence; by 3 days after emergence it had declined. Most of the synthesis of actomyosin was required for the increase in the protein which occurred over emergence. INTRODUCTION PREVIOUS studies on the quantitative importance of protein synthesis de novo during development of Lucilia in the puparium showed that there were two periods of marked thoracic protein biosynthetic activity, one occurring about 2 days before emergence and the other over emergence (WILLIAMSand BIRT, 1972). It was proposed that the synthetic activity spanning emergence could be ascribed mainly to the formation of proteins for mitochondrial maturation (WILLIAMSand BIRT, 1971a,b; WILLIAMSet al., 1972) and for energizing flight (CAMPBELLand BIRT, 1972; CAMPBELL,et al., 1973) after emergence. As extensive microscopic studies (GREGORY,et al., 1968; BARRITT and BIRT, 1971; PERISTIANIS and GREGORY, 1971) had shown that there is a rapid accumulation of muscle tissue before emergence, it was surmised that the first peak might be largely a consequence of thoracic muscle protein synthesis (WILLIAMSand BIRT,1972). This proposition was also consistent with the sequence of synthetic events described for isolated thoraces of tobacco horn worm, in which it was found that a peak of cytoplasmic protein synthesis preceded one of mitochondrial protein synthesis (CHAN and RICHARDSON, 1969 ; CHAN and REIBLING,1971). The present study was designed to determine whether the synthesis of the thoracic contractile proteins contributed substantially to the first peak of protein synthesis. This was assessed by measuring the rate of incorporation of an injected radioactive amino acid into actomyosin isolated from insects from 2 days before to * Anne J. Campbell, Department of Biochemistry, University of Leicester, University Road, Leicester, LEI 7RH. J" L. M. Birt, Wollongong University College, Wollongong, N.S.W. 2500 Australia. 43
44
ANNE J. CAMPBELL AND L. M. BIRT
3 days after emergence. Actomyosin was selected, as not only does it represent the functional combination of contractile proteins in active muscle, but because its purification is simple and well documented for insects (GILMOUR and CALABY, 1953; MARUYAMA,1954, 1957, 1958; MARUYAMAet al., 1968); purification was followed by measuring Ca~+ATPase activity. MATERIALS AND METHODS
Insect culture Insects were grown at 30°C under continuous illumination as detailed elsewhere
(CAMPBELLand BIRT, 1972; WILLIAMS and BIRT, 1972). Insects were collected within 1 hr of pupariation (day 0) and flies within 1 hr of emergence from the puparium (day 6); flies were fed sugar and water.
Injection of insects Insects were cooled on ice and injected thoracically (CAMPBELLand BIRT,1972), with 25 nCi[14C]leucine (342 mCi per mmole); pharate adults were left on the needle for a further 15 sec. Those from which actomyosin was to be extracted were kept for 30 min at 30°C and then cooled on ice, whereas with those taken for timecourses of incorporation into total protein (acid-insoluble material), incubations were terminated by freezing.
Tissue preparation Whole insects: for time course studies insects were dispersed individually, in 0.5 ml cold 1 0 ~ trichloroacetic acid and acid-insoluble material prepared for counting and applied to discs (Gelman) essentially as outlined by WILLIAMS and BraT (1972). Routinely the total amount of acid-soluble radioactivity remaining after 30 min was determined per insect in order to measure Ka (rate of leucine pool turnover; see WILLIAMS and BIRT, (1972). Actomyosin: unless stated otherwise, all procedures were conducted at 2 to 4°C; all centrifuging was for 15 min and values for g are based on Ray. Insects were thoroughly chilled on ice, thoraces dissected out and homogenized gently in a Dounce glass tissue grinder (type A pestle) in 0.6 M KC1-0.06 M KHCO3, pH 8 using 1 g tissue: 15 ml extractant. The homogenate was stirred in ice for 1 hr, filtered through glass wool on a sintered disc (0 porosity) and centrifuged at 13,300g. The supernatant is designated as homogenate supernatant. Crude actomyosin was precipitated by diluting the homogenate supernatant 10-fold with distilled water; the suspension was stirred for 10 min, sedimented at 20,750 g and the pellet dissolved in the original extractant. The dilution-precipitation procedure was repeated 3 times more and the final actomyosin precipitate was resuspended in 0.6 M KC1 (1-2ml), stirred for 10 min and cleared by centrifuging at 9220g for 10 min. Actomyosin was assayed for Ca~+ATPase activity on the day of preparation but the enzyme was found to be relatively stable for up to 4 days at 2 to 4°C.
THORACIC MUSCLE PROTEIN BIOSYNTHESIS I N LUCILIA CUPRINA
45
Ca-A TPase assay Enzyme activity was measured as the release of inorganic P. The incubation mixture (1-0 ml) contained 20 m M H E P E S - K O H (N-2-hydroxyethylpiperazineN'-2-ethane-sulphonic acid) pH 7.4, 3 m M ATP, 10 m M CaC12, 200 m M KC1; 10 m M sodium azide was also included (MARUYAMAet al., 1968). Reactions at 30°C were initiated with enzyme (10-50/~1) and stopped with 0.33 ml cold 25 trichloroacetic acid. Routinely, time courses of incubation (up to 30 min) at two different enzyme concentrations were carried out, together with zero-time controls. The amount of inorganic P in deproteinized incubation samples (0.2-0.5 ml) was measured microcolorimetrically .(TAUsSKYand SHORR, 1953) at 720 nm. Enzyme activity is expressed as nmoles inorganic P released per minute at 30°C; specific enzyme activity refers to enzyme activity per mg protein. Protein estimations Routinely, protein was precipitated with cold 1 0 ~ trichloroacetic acid, dissolved in 1 M NaOH at 30°C for 5-16 hr and estimated by the method of LOWRY et al. (1951) using Lowry reagent D; bovine serum albumin (fraction V; Sigma) served as a standard. However, for determination of 'total' thoracic protein content (excluding cuticular material), thoraces were homogenized in 1 M NaOH, incubated for 1 hr at room temperature and filtered through glass wool. The protein content of the filtrate was estimated by the biuret reaction (GORNALL,et al., 1949) and correction for absorbance due to particulate matter was applied using the K C N decolorization procedure (SZARKOWSKAand KLINGEr~BERG, 1963). Leucine pool size This was estimated on groups of 10 insects essentially as outlined by CAMPBELL et al., 1973. Analysis of radioactivity Samples were counted with an efficiency of about 80~o in 10 ml scintillation fluid (1200 ml toluene/800 ml ethoxyethanol/12 g butyl PBD (2(4't-butylphenyl)5-(4"-biphenyl)-l,3,4-oxadiazole). It was satisfactory to count a solution of actomyosin (20-100/~1) directly rather than to apply an acid-precipitate of it to discs as was done for time course studies of [14C]leucine incorporation into total insect protein. RESULTS
Ca-A TPase activity In using Ca2+ATPase as a marker for following the purification of actomyosin, it was necessary to establish that the contribution of other cellular ATPases (e.g. from mitochondria and microsomes) was insignificant under the assay conditions. Oligomyein (0.7-2.8tzg/mg protein) which severely inhibits mitochondrial
46
ANNEJ. CAMPBELLANDL. M. BIRT
ATPase at 1/~g per mg mitochondrial protein (SLATERand TERWELLE,1969) did not inhibit Ca2+ATPase of the thoracic homogenate supernatant or of purified actomyosin. Sodium azide (10-20 raM), an inhibitor of mitochondrial ATPase (SACKTOR, 1953) produced no inhibition of either unfractionated or purified preparations. Phospholipase c, a potent inhibitor of microsomal Ca2+ATPase, (MARTONOSI, 1968; MARTONOSI et al., 1968) did not inhibit Ca2+ATPase activity of the homogenate supernatant when incubated under the conditions used by MARTONOSI et al., (1968). Finally, the Ca2+ATPase activity of microsomes from 3 day old flies constituted less than 1 per cent of the total Ca2+ATPase activity of thoracic homogenate supernatants.
.4ctomyosin purification Identification of the protein isolated as actomyosin depended primarily on the specificity of the extraction procedure but in preliminary experiments, the isolated product was monitored for superprecipitation and ultraviolet absorption. Purified actomyosin (0.5 mg protein per ml in 0.1 M KC1, 20 mM Tris-HC1 pH 7.4) and 0-5 mM ATP, formed a fibrous plug at room temperature; this 'superprecipitation' is a characteristic response of actomyosin to ATP (MARUYAMA, 1965). The maximum and minimum absorbances for actomyosin purified from 3 day old flies were 265 and 255 nm respectively, compared to 275 and 255 nm for mature honeybee actomyosin (MARuYAMA, 1957, 1958, 1965); the absorption maximum shifted to 260 and 255 nm in preparations from newly emerged and pharate adults of Lucilia compared to a shift to 260 to 270 nm in pharate adults of honeybee (MARUYAMA,1957). In general, it was found that with purified actomyo-
T A B L E 1 - - P R E P A R A T I O N S OF THORACIC ACTOMYOSIN FROM
Lucilia
OF DIFFERENT AGES
~o Actomyosin Age (days) 4 5 6 7 9
Homogenate supernatant Actomyosin PurificaATPase/rng tion ATPase Protein protein (fold) 76 69 90 69 61
2"2 7"4 23 22 19
889 1429 1843 1307 1720
39 97 4 3 3
ATPase/ thorax 16(12) 110(re) 317(285) 404(27~) 516(315)
/~g Actomyosin/ thorax 18(14) 77(53) 172(155) 309(~13) 300(133)
Actomyosin was purified from 100 thoraces of day 4 insects and from 50 thoraces of insects from days 5-9; pharate adults were of mixed sex; flies were males. Age was from pupariation (day 0); emergence occurred on day 6. ATPase measured as nmoles P released per min. at 30°C. * Total amounts of actomyosin ATPase and protein per thorax were corrected for losses during purification by assuming 100 ~ ATPase recovery from the homogenate supernatant; uncorrected values are shown in parenthesis.
47
THORACIC MUSCLE PROTEIN BIOSYNTHESIS I N LUCIL[A CUPRINA
sin (100/~g per incubation) only about 40 to 50~o of the labile phosphate of ATP was released as P after 20 min; at this protein concentration the rate of enzyme reaction was linear for up to 10 min. The recoveries of actomyosin-Ca~+ATPase and protein were always expressed relative to the homogenate supernatants (Table 1). In insects of different ages, 73 +_1 2 ~ of the homogenate supernatant Ca~+ATPase was found in purified actomyosin. Indirectly, the high recovery ( 9 0 ~ ) o f Ca2+ATPase from the homogenate supernatant from newly emerged flies supports the conclusion derived from the inhibitor studies, that the contribution of other ATPases to the assay of actomyosin Ca2+ATPase was not appreciable. The proportion of protein in the homogenate supernatant finally extracted as actomyosin increased about 10-fold (days 4-6); thereafter little change was noted. The extent of purification declined with increasing insect age to a value (3- or 4-fold), which was similar in newly emerged and older flies. Purification was not monitored through all dilution-precipitation steps as sampling losses would have been too great in view of the small amount of tissue (about 0.5 g) taken initially. In order to account for losses incurred during purification, the amount of enzyme activity and protein in the actomyosin isolated per thorax were corrected on the assumption that there was 100 per cent recovery of Ca2+ATPase activity from each particular homogenate supernatant preparation. The amount of protein in actomyosin per thorax increased 4.1, 2-2, and 1.8 times respectively between days 4 to 5, 5 to 6 and 6 to 7; during these periods the amounts of actomyosin
T A B L E 2 - - S Y N T H E S I S OF ACTOMYOSIN IN
Age (days)
dis/min/#g actomyosin/ hr
4 5 6 7 9
45"4 39"8 32"0 13"6 8"8
Lucilia
dis/rain into Leucine pool actomyosin/ size thorax/hr (nmoles/insect) 817 3065 5504 4202 2640
19 19 23 33 3
OF DIFFERENT AGES
Ka leu.
Kp leu.
Actomyosin synthesis /~g/thorax/hr
21 23 30 48 5"4
0"36 1"40 3"10 3"56 0"22
0"6 2"1 4"7 5"4 0"3
Insects injected with 25nCi[t4C]leucine were incubated for 30 min at 30°C and thoracic actomyosin prepared (Table 1). Age is from pupariation (day 0); emergence occurred on day 6. Pool size valves for pharate adults are those from BraT and CHRISTIAN(1969) for 30 mg insect); those for flies were measured directly. Ka (nmoles leucine per insect per hr) and Kp (nmoles leucine incorporated into actomyosin per thorax per hr) were calculated from equations of Hearon (DINAMARCAand LEWI'~OOK, 1966; see also WILLIAMSand BraT, 1972). Corrected valves for actomyosin protein per thorax were used (Table 1). Actomyosin synthesis calculated as 1.529 x Kp (as outlined in the Text).
48
ANNE J. CAMPBELLAND L. M. BIRT
formed per day were 58, 95, and 137/zg respectively with no further formation of protein between days 7 to 9. The level found in mature flies (day 9) was already reached in 1 day old flies (day 7) (Table 1). In insects of corresponding ages the 'total' thoracic protein (excluding cuticular material) per insect was 1.4 mg (day 4), 2.5 mg (day 5), 1.9 mg (day 6), 2.1 mg (day 7), and 2.7 mg (day 9). The specific enzyme activity measured directly on the purified actomyosin, increased about twice from day 4 to day 5 and was relatively constant thereafter.
Actomyosin synthesis At each age studied, the rate of [l*C]leucine incorporation into total acidinsoluble material was linear for at least 30 min, the period selected for labelling of actomyosin. In calculating the rate of actomyosin synthesis (nmoles leucine incorporated into protein per thorax per hour; Kp, Table 2), the rate of leucine turnover in the pool (Ka; Table 2) and leucine pool size were used, as well as the measured incorporation of [14C]leucine into the purified protein. Equations for calculating Ka and Kp are those derived by Hearon in DINAMARCAand LEVENBOOK(1966). The amount of actomyosin synthesized was estimated from Kp as outlined by WILLIAMS and BIRT, 1972); it was assumed that leucine constituted 8.5 ~ of the amino acids of actomyosin. The rate of actomyosin synthesis rose to a maximum value at day 7 and declined considerably by day 9 (Table 2). It is noteworthy that [x4C]leucine incorporation into thoracic actomyosin represented about 4 per cent of that into total acidinsoluble thoracic protein at day 4 and about 25 per cent of that with older insects (days 5-9). It was important to establish that labelling of the actomyosin was due to incorporation into, rather than physical retention by, the protein during isolation. Accordingly, 50 newly emerged flies were injected but not incubated and the actomyosin purified and counted in the usual way. A total of 700 dis/min recovered in the protein purified from controls constituted from 0.5 to 3 . 4 ~ of the total radioactivity incorporated into actomyosin isolated from insects from days 4 to 9. Similar values (0.9-5.9 ~ ) were found in a control experiment using mature flies. DISCUSSION In Lucilia at days 4 and 5, the purified actomyosin protein constituted about 1.3 per cent and 3 per cent of the 'total' alkali-soluble thoracic protein, whereas from days 6 to 9, it constituted on average about 11 per cent. Newly emerged flies contained about half as much thoracic actomyosin as mature adults (day 9) in contrast to the newly emerged honey bee in which levels were close to those of the mature bee (MARUYAMA,1965). In general, in Lucilia of different ages, there were nearly equal amounts of thoracic actomyosin (Table 1) and mitochondrial protein (70, 180, 300, and 400/~g at days 5, 6, 7, and 9; WILLIAMS,1972).
THORACIC MUSCLE PROTEIN BIOSYNTHESIS I N LUCILIA CUPR1NA
49
There is a general similarity between the changes in the amount of actomyosin protein isolated from thoraces of developing Lucilia (Table 1) and the levels of thoracic Ca2+ATPase activity in the same insect (Table 1). The most striking difference is the absence of any increase in the amount of actomyosin protein per thorax after day 7, compared to a further 20 per cent increase in Ca2+ATPase activity from days 7-9. It is possible that the contractile proteins are present at day 7 without full development of their potential ATPase activity or that actomyosin can exist in two forms, active and inactive and that these are not separated by the isolation procedure. From Table 2, it is clear that although there is some synthesis of actomyosin before emergence (days 4-5), it is most rapid in the day afterwards (days 6-7); by day 9 it has declined. Thus actomyosin synthesis will contribute substantially to the bulk incorporation of labelled amino acids at both days 6 and 7 (compare Fig. 4; WILLIAMSand BIaT, 1972) but not to that between days 4 and 5. Therefore some other cellular component remains to be identified as the site of the pronounced incorporation before emergence (days 4-5); it is distinctly possible that this could be the cuticle of the insect. Preliminary measurements of the rate of [14C]leucine incorporation into isolated cuticles, have indicated that the pattern of variation of protein synthesis in whole insect cuticle (as nmoles leucine incorporated into cuticle per insect per hour) was very similar to that obtained for 'bulk' protein (WILLIAMSand BIRT, 1972); it remains to determine what proportion of cuticle synthesis before emergence is due to that in the thorax. Some correlation can be attempted between actomyosin synthesis and the appearance of the protein. In the 2 days spanning emergence (days 5-7) it can be calculated (by assuming average rates of actomyosin synthesis for each of the 24 hr periods subsequent to day 5) that about 90 per cent of the actomyosin formed by the insect was being synthesized de novo. It seems likely that after day 7, the rate of actomyosin synthesis must decline abruptly for no further increase in the amount of protein was measured after this time. A similar rapid decline in cytochrome c synthesis had been found previously in Lucilia flies (WILLIAMSet al., 1972). These calculations of the rate of synthesis of actomyosin from [14C]leucine incorporation assume that the total amino acid pool is that used for synthesis, that there is rapid mixing of the injected amino acid in the pool and that, in the pool, there is relatively little inter-conversion of injected leucine to other metabolites. Previously, the assumption of a single amino acid pool successfully accounted for synthesis of other proteins in Lucilia (WILLIAMSand BIRT, 1972; WILLIAMSet al., 1972; CAMPBELLet al., 1973) and it was also found that the only radioactivity incorporated into specific proteins was that of the injected radioactive amino acid (WILLIAMS et al., 1972; CAMPBELLet al., 1973). The rate of synthesis of actomyosin (/zg/thorax per hour) can be compared with the synthetic rates of other thoracic proteins which have been measured in a similar way (using Ka and Kp). In Lucilia at emergence, actomyosin is synthesized 47 times faster than cytochrome c (from [14C]lysine; WILLIAMSet al., 1972) and 22 times faster than glycerophosphate dehydrogenase (from 14C leucine; CAMPBELL
50
ANNE J. CAMPBELLANDL. M. BIRT
et al., 1973) but only 10 per cent faster than mitochondrial protein (from [14C]
ieucine; WILLIAMS,1972). In the 2 days spanning emergence, about 7 5 - 8 0 ~ of the total thoracic protein synthesized (0-54 mg; WILLIAMS and BIRT, 1972) can be attributed to the appearance of both mitochondrial (0.18-0.2 mg: LENNIE et al., 1967; WILLIAMS, 1972) and actomyosin (0.23 rag) protein. The rapid synthesis of both these protein complexes over emergence can be correlated with the development of the capacity for flight soon after eclosion. A comparison between both the formation and synthesis of actomyosin and the ultrastructural development of the thoracic muscle (GRECORY et al., 1968; BARRITT and BIRT, 1971; PERISTIANIS and GREGORY, 1971) can also be attempted. The relevant microscopical observations are (a) by day 5 the indirect flight muscle has reached almost all of its final volume (BARRITTand BIRT, 1971) and (b) the sarcomere length probably does not change significantly from emergence to maturity and each sarcomere is approximately cylindrical (GREGORYet al., 1968). From these observations, and by setting the value for the increase in the number of myofilaments per fibril during the first day after emergence as 1, it can be calculated that the number of myofilaments increase 1.6 times after the first day post-emergence; if the same assumptions are used for the 3 days preceding emergence, the relative increase is 1.3 times (though there is a much greater likelihood of error in the latter calculation as there is no precise information on sarcomere size and number in the pharate adults). These considerations suggest that there is substantial formation of muscle filaments after the first day post-emergence, in sharp contrast to conclusions reached by studying the synthesis (Table 2) and appearance (Table 1) of the thoracic actomyosin. This disparity could be interpreted as indicating that the developing muscle accumulates actomyosin for some time before the final assembly of the myofilaments or that actomyosin is more difficult to extract from the older insects. Considerable speculation exists as to the method and sequence of assembly of the proteins in the contractile apparatus; a number of reports suggest that assembly proceeds asynchronously both at the biochemical (OGAWA, 1962; HITCHCOCK, 1970) and ultrastructural level (HAY, 1963 ; ALLEN and PEPE, 1965 ; PRZYBYLSKIand BLUMBERG,1966; FISCHMAN,1967). The results with Lucilia suggest that the formation of actomyosin (which has reached about 75 per cent of the adult level by day 7 estimated by either ATPase or actomyosin protein levels) precedes myofilament formation (by day 7 about 60 per cent of the adult levels are present). It can be envisaged that the sequential development of particular properties of the contractile apparatus proceeds by (a) synthesis de novo of individual proteins of the contractile apparatus (b) development of enzyme activity (c) myofilament formation. The rate of synthesis of actin and myosin may accelerate from days 4-6, as does the rate of assembly into functional units (ATPase) so that the specific enzyme activity of actomyosin increases (Table 1). At the period of maximal synthesis (days 6-7), the formation of actin and myosin exceeds the rate of assembly into functional units, i.e. specific activity declines by day 7 (Table 1). Assembly but not synthesis
THORACIC MUSCLE PROTEIN BIOSYNTHESIS IN LUCILL4 CUPRINA
51
continues appreciably f r o m days 7 to 9, so that again the specific activity increases (Table 1). As myofilaments are laid down in the greatest n u m b e r s after the first day post-emergence, in contrast to the development of enzyme activity, this implies that there is a delay of incorporation of enzymatically active complexes into the complete filament array of the functional muscle. A capacity for self-assembly of filaments f r o m their c o m p o n e n t molecule,s has been shown in vitro (e.g. HUXLEY, 1963). It is clear that a correlation of ultrastructural development with biochemical analyses in developing muscle in Lucilia offers a fruitful field for further enquiry.
Acknowledgements--We thank Mr. A. VAN GEBWEN (C.S.I.R.O. Entomology, Canberra) for the supply of insects and Mr. M. DESMET, Biochemistry Department, S.G.S. for performing the amino acid analyses. A.J.C. was a recipient of a Ph.D. Scholarship from the Australian National University. The work was supported by funds from the Australian Research Grants Committee (D76/16645).
REFERENCES ALLEN E. R. and PEPE F. A. (1965) Ultrastructure of developing muscle cells in the chick embryo. A m . J . Anat. 116, 115-148. BARRITT L. C. and BIRT L. M. (1971) Development of Lucilia cuprina: correlation of biochemical and morphological events..7. Insect Physiol. 17, 1169-1183. CAMPBELL A. J. and BIRT L. M. (1972) Studies on the appearance of soluble d-glycerophosphate dehydrogenase activity during the development of the sheep blowfly, Lucilia. Insect Biochem. 2, 279-296. CAMPBELL A. J., SHAW D. C., and BIRT L. M . (1973) Biosynthesis of soluble a-glycerophosphate dehydrogenase during the adult development of the sheep blowfly, Lucilia. Insect Biochem. 4, 135. CHAN S. K. and KEIBLINGA. (1971) Biochemical studies in the developing thoracic muscles of the tobacco hornworm. I I I Amino acid incorporation in the cytoplasmic ribosomal fraction. Biochem. biophys. Acta 228, 252-258. CHAN S. K. and Rlct-mm)SON L. (1969) Biochemical studies on the developing thoracic muscles of the tobacco hornworm. J. biol. Chem. 244, 1039-1042. FISCHMAN D. A. (1967) An electron microscope study of myofibril formation in embryonic chick skeletal muscle. J. Cell Biol. 32, 557-575. GILMOURD. and CALABYJ. H. (1953) Physical and enzymic properties of actomyosins from the femoral and thoracic muscles of an insect. Enzymologia 16, 23-33. GORNALLA. G., BPd~DAWmLC. S., and DAVIDM. M. (1949) Determination of serum proteins by means of the biuret reaction, ft. biol. Chem. 177, 751-766. GREOORYD. W., LENNIE R. W., and BIRT L. M. (1968) An electron-microscopic study of flight muscle, development in the blowfly, Lucilia cuprina, jY. R. microsc. Soc. 88, 151-175. HAY E. D. (1963) The fine structure of differentiating muscle in the salamander tail. Z. Zellforsch. microsk. Anat. 59, 6-34. HITCHCOCKS. E. (1970) The appearance of a functional contractile apparatus in developing muscle. Devl. Biol. 23, 399-423. HUXLEYH. E. (1963) Electron microscope studies on the structure of natural and synthetic protein filaments from striated muscle..~, molec. Biol. 7, 281-308. LV2qNIER. W., GREGORYD. W., and BIRT L. M. (1967) Changes in the nucleic acid content and structure of thoracic mitochondria during development of the blowfly, Lucilia cuprina. .7. Insect Physiol. 13, 1745-1756.
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