Development of respiratory activity and oxidative phosphorylation in flight muscle mitochondria of the blowfly, Lucilia cuprina

J. InsectPhysiol., 1969,Vol. 15,pp. 305to 317.Pergamon Press. Printed in Great Britain

DEVELOPMENT OF RESPIRATORY ACTIVITY AND OXIDATIVE PHOSPHORYLATION IN FLIGHT MUSCLE MITOCHONDRIA OF THE BLOWFLY, LUCILIA CUPRINA A: C. WALKER*

and L. M. BIRT*

Department of Biochemistry,

University of Sheffield

(Received 5 August 1968) Abstract-Adult development in Lucilia nrprina involves the formation of large numbers of thoracic mitochondria containing an extremely active system for the oxidation of u-glycerophosphate, which provides a convenient marker for studying their development. Mitochondrial fractions have been isolated at various stages of adult development of L. cuprina by density gradient centrifugation and the distributions of protein, o-glycerophosphate dehydrogenase, and oxidase in the gradients studied. Over the period of emergence there is an increase in the mean specific gravity of the mitochondrial population indicating a concentration of enzymic and non-ensymic (structural) protein. Initially the mitochondria accumulate mainly dehydrogenaae protein; accumulation, incorporation, and organization of ‘oxidase’ protein is somewhat slower so that dehydrogenase cannot be fully expressed as oxidase. Over the period of emergence ‘oxidase’ protein is accumulated faster than dehydrogenase and the complexity of internal organization increases so that in post-emergent

tissue the mature dense mitochondria can fully express dehydrogenase as oxidase. Thus development involves the asynchronous incorporation of enxyrnic and non-enxymic protein, the latter being laid down most rapidly over the period of emergence. Phosphorylation capacity develops synchronously with the oxidase. The respiratory chain is extremely sensitive to ADP and oligomycin before adult After emergence, although the mitochondria are still coupled, emergence. there is a marked decrease in respiratory control by ADP and a marked increase in control by Ca*+. The results are considered in relation to the development of flight muscle sarcosomes already described by Lennie and Birt and also in relation to the physiological changes which occur after emergence as the insect prepares for flight. INTRODUCTION CONSIDERABLEattention has been directed over recent years to the development of flight muscle mitochondria in various insects ( BROSEMERet al., 1963 ; BUCHER, 1965 ; HEROLD, 1965) ; this paper is concerned with aspects of the development of such mitochondria in the blowfly, Lucilia cuprina, in relation to other information available on the development of this insect (HOWELLS and BIRT, 1964; D’COSTA * Present address: Department of Biochemistry, National University, Canberra, A.C.T., Australia. 20

305

School of General Studies, Australian

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A. C. WALTERANDL. M.

BIRT

and BIRT, 1966; LENNIE and BIRT, 1967; LENNIE et al., 1967; CROMPTONand BIRT, 1967; GREGORYet al., 1968). Maturation of adult LuciZia involves the rapid formation of large numbers of thoracic mitochondria early in adult life (GRECORYet al., 1968). They contain the characteristic redox system of insect flight muscle sarcosomes for oxidizing or-glycerophosphate which provides a convenient marker for studying their development. Previous investigations on changes in the amounts and distribution of sarcosomal and respiratory enzymes during pharate and early adult life (LENNXE and BIRT, 1967) indicated that development involved the asynchronous incorporation of dehydrogenase and non-dehydrogenase (structural) protein. Incorporation of structural protein appears to be most rapid over the period of emergence and its synthesis may be under the control of mitochondrial DNA (LENNIE et al., 1967). This paper presents a study of the redox and phosphorylating capacities of several mitochondrial fractions separated from tissues at various developmental stages by density gradient centrifugation. The results of this study, together with previously published information (LENNIE and BIRT, 1967) present a more complete pattern of development of flight muscle sarcosomes. MATERIALS AND METHODS All fine chemicals were obtained from Sigma with the exception of: phenazine methosulphate, which was obtained from Koch Light Laboratories; bovine serum albumin from Armour Pharmaceutical Co. Ltd. ; and 2-(p-iodophenyl)-3(p-nitrophenyl)&phenyl tetrazolium chloride (INT) from B.D.H. All other materials used were AnalaR reagents. Rearing of insects

Insects were reared as described by LENNIE and BIRT (1967). Insects chosen at a particular stage before emergence were recognized by their characteristic appearance. Two days before emergence the body of the pharate adult is unpigmented but the eyes are coloured; 1 day before emergence both the body and eyes are pigmented. Preparation of mitoch&iaZfractionr

Insects were immobilized on ice, and heads, abdomens, wings, and legs were removed. Pharate adults were freed from the pupal case prior to dissection. Preparation of a mitocho&kZ

suspension by a mo&jication of the method of VAN

BERGH (1962). Thoraces (S/ml isolation medium) were gently pounded for 2 min in an ice-cold glass mortar containing an isolation medium of O-3 M sucrose, 1 mM EDTA, 10 mM Tris-HCl buffer (pH 7*4), and O*Sg/, BSA. The pounded thoraces were stirred gently to tease out any remaining muscle still attached to the body wall and repounded for 30 sec. The suspension was filtered by gentle suction, through four layers of cheese cloth, and the filtrate centrifuged (4 min at 300 g) to remove cell debris and muscle fibres. The supematant fluid was recentrifuged (15 min at 2500 g) and the mitochondrial pellet gently resuspended DEN

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by hand in about 2 ml of isolation medium to give a final protein concentration of approximately 3 mg/ml. All manipulations were carried out between 0 and 3°C. This method was chosen, after testing a number of alternatives, because it proved to be the most effective in providing mitochondrial preparations in which respiration and phosphorylation were coupled. For investigating the effects of Caa+ and EGTA an isolation medium in which EDTA was replaced by 1 mM EGTA was used. Fractimzatiun uf the mitochondkial population. Thoraces (5jmi isolation medium) were homogenized by hand (20 strokes) in a glass homogenizer fitted with a Teflon pestle and containing ice-cold O-3 M sucrose. The homogenate was filtered using gentle suction through a layer of glass wool placed on a sintered funnel (porosity x 1). The mitochondrial suspension was fractionated by means of continuous density gradients of sucrose solution prepared by the method of SALO and KOUNS (1965). Mitochondria from the thoraces of pharate adults were separated on a gradient in which the specific gravity ranged from 1.18 to l-19; preliminary experiments showed that the mean specific gravities of such mitochondria fell within this range. However, mitochondria from insects after emergence were separated on a continuous gradient in which the specific gravity ranged from 1.18 to 1.21. The arrangement of sucrose solutions in both types of gradients is illustrated in Fig. 1. Layers A and 3 are included to provide a clear separation of the mitochondrial fractions from muscle fragments and cell debris, and the original suspending medium. Layer C allows the upward displacement of lipid during centrifugation. The gradients were centrifuged at 0°C for 45 min at 45,000 g. After centrifugation, fractions were removed from the sides of the tubes using a hypodermic needle fitted to a graduated syringe. The mitochondrial fractions were checked for their degree of purity by phase contrast and electron microscopy; and they were almost completely free from contamination by muscle fibres, nuclei, and tracheal fragments. Determination of protein in the density gradient. An upper layer (Layers B and C-Fig. l-and the layer of suspending medium) was first removed. One millilitre aliquots of the gradient were collected by downward displacement with applied gas pressure. Protein was estimated by the Folin-Lowry method as described by LENNIEand BIRT (1967). Determination of respiratory rates Oxygen uptake was measured with a GME oxygraph model KM. The vibrating platinum electrode was covered with a Teflon membrane to prevent poisoning during the incubations. The standard reaction mixture consisted of 15 mM KC1 5 mM MgCl,, 50 mM Tris, 2 mM EDTA, and 35 mM phosphate buffer (pH 7.4); when the effects of Car+ were being tested 2 mM EDTA was replaced by 2 mM EGTA.

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Solubility of oxygen in these media was taken to be O-474 pg atom/ml at 25°C and 0.455 pg atom/ml at 30°C (CHAPPELL, 1964).

S.G.

17ml of grodient

I.180

S.G. I.210

Before

After

emerpenct

emergence

FIG. 1. Diagrammatic representation of the system used for separating mitochondria by means of sucrose density gradient centrifugation.

Layer A-4 ml of sucrose S.G. I.22

3 ml of in 0.3M

homogenate sucrose

Estimation of wglycerophosphate dehydrogenase activity The dehydrogenase-INT reductase assay used was based on the semi-micro method of LEE and LARDY (1965) as modified by LENNIE and BIRT (1967)

RESULTS

Distribution of mitochondrial protein The distribution of mitochondrial protein in fractions of different specific gravities is expressed in the form of a histogram (Fig. 2). There was an increase in the mean specific gravity of the mitochondrial population up to about the end of the first day after emergence of the adult. Almost all of this increase occurred between 1 day before emergence and 1 day after emergence during which time the change in mean specific gravity was from 1.184 to l-194. Before emergence about 85 per cent of the total mitochondrial protein had a specific gravity below 1.190. However, immediately after emergence only 40 per cent of the total mitochondrial protein had a specific gravity below l-190, and the remaining 60 per cent was evenly distributed in the specific gravity range 1.190 to

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1.210. Thereafter, there was no change in the range of specific gravities (l-1801.210) of the mitochondrial population, but within the least dense fraction there was a continuous increase in the mean value of the specific gravity with increasing age.

SO

7

I

I.180

1

i I.190 Specific

I,200

I.210

gravity

FIG. 2. The percentage distribution of mitochondrial protein in fractions of different specific gravity.

The disiribution of wglycemphosphate dehydrogenase activity The specific activities (~1 O,/hr per mg protein) of ol-glycerophosphate dehydrogenase in mitochondrial fractions of different specific gravity are shown in Fig. 3. At all stages of development, increase in specific gravity was accompanied initially by an increase in specific activity, thereafter by a decline. Before emergence

g :: 2 !z ::

,600~Ll

c S.G.

000

I.195

Age,

days

FIG. 3. Distribution of or-glycerophosphate dehydrogenase and oxidase activities in fractions of different specific gravity. or-Glycerophosphate dehydrogenaae activity was determined from the rate of formaza n production which was expressed as ~1 O&r per mg protein; u-glycerophosphate oxidase activity was determined by estimating the rate of oxygen uptake polarographically. O-a-Glycerophosphate dehydrogenase; O-o-glycerophosphate oxidase.

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only the rise, which was relatively large (2-Sfold), could be observed. After emergence the initial rise in activity was somewhat less (1-Sfold). The highest specific activities were always found in particles of specific gravity 1.185 to 1.195. With increase in age of the tissue, in the lightest fraction there was a further increase in the specific activity of the dehydrogenase after emergence (twofold); in the 6-day-old adult, the activity in this fraction equalled that of mitochondria in the most dense fraction. In contrast, the specific activities of the two heavier fractions were almost constant at all stages of development. The distribution of a-glycerophosphate oxidase activity The oxidase values expressed in Fig. 3 are the maximum specific activities (~1 O,/mg protein per hr), which could be obtained by adding either ADP or DNP. Before emergence, increase in specific gravity was associated with a very large increase in oxidase activity; 2 days before emergence the increase was fivefold; 1 day before emergence the increase was threefold. Thereafter, there was a striking increase in oxidase activity between the lightest and the intermediate fractions, but no difference in activity between the intermediate and the heaviest fractions. With increase in age the oxidase activity in the lightest fraction increased only slightly up to emergence and decreased thereafter. In the intermediate fraction, however, there was a 40 per cent decline in activity up to emergence after which there was no further significant change. The oxidase activity of the heaviest fraction did not change with increase in age. Thus after emergence the mitochondrial population appears to have a stable oxidase activity within each fraction. Changes in the phosphorylating capacity of the mitochondria Table 1 illustrates changes during development in various aspects of phosphorylation accompanying the oxidation of cu-glycerophosphate and pyruvate + malate by unfractionated mitochondrial preparations. The temperature of incubation had a considerable effect on the retention of coupling which was most stable at temperatures below 30°C. Perhaps this is a reflection on the fact that insects are reared and maintained at 30°C throughout their life history (compare RICHARDSONand TAPPEL, 1962). Respiration was initiated by adding the mitochondrial preparation. When the rate of oxygen uptake was steady, 150 ~1 of ADP (10 mM) was added ; when the ADP stimulation had reached its maximum 5 pg of oligomycin was added; when the decline in oxygen uptake had ceased DNP was added at a sufficient concentration (lo-* M to 1.5 x 10”’ M) to give a maximum increase in rate. Respiratory rates The respiratory rates of cu-glycerophosphate oxidation measured in unfractionated mitochondria increased very rapidly (ninefold) from 1 day before emergence to 1 day after emergence. Thereafter, oxidase activity was stable. This pattern is similar to that for the development of oxidase activity in fractionated mitochondria.

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Pyruvic oxidase activity also increased very rapidly (fivefold) from 1 day before emergence to 1 day after emergence. Thereafter oxidase activity still increased but at a much slower rate. Thus it appears that pyruvic and cu-glycerophosphate oxidases do not develop synchronously (contrast LENNIE and BIRT, 1967). TABLE l-ASPECTS OF THE DWBLOPMENTOF OXIDATIVE PHOSPHORYLAYION

Age (days) -2 -1 0 +l +6

Respiratory rate with ADP @l Os/hr per mg protein)

Respiratory control ratio

(a)

(b)

(a)

(b)

24 63.5 265.5 507 510

9.3 57 145 250 450

cc cc

cc CD cc

3; 1.2

9:

“;b Inhibition by oligomycin

Rate with DNP ( “;b of rate with ADP)

(a)

(b)

(a)

100 83 80 81.3 68.5

100 84 84 88 82

114 85.5 87 75 48.5

Unfractionated mitochondrial preparations were used. Additions were made in the following order: ADP (finally 1 mM) ; oligomycin (5 pg) ; DNP (finally l-5 x 10V4 Mlo-* M). Amount of mitochondrial protein/incubation approximately 2 mg. nL-a-Glycerophosphate, 60 mM; pyruvate, 20 mM f malate, 10 r&I; B.S.A., O-1“/. Temperature of incubation, 30°C. Total incubation vol., 1.6 ml. Respiratory rates determined polarographically. (a) Ls-Glycerophosphate oxidase. (b) Pyruvic oxidase.

Respiratory control Respiratory control of the or-glycerophosphate oxidase system by ADP was absolute before emergence (the ratio, oxygen uptake with ADP/oxygen uptake without ADP : the respiratory control ratio). After emergence the control ratios declined to values similar to those reported elsewhere for adult flies (VAN DEN BERGH, 1962). Increasing the concentration of added ADP in order to overcome the large myokinase activity found (see later) did not increase the lower respiratory control values obtained after emergence. However, with pyruvic oxidase, respiratory control by ADP remained very high (ratio of co) even after emergence. Ol~omycin iffhibition The cY-glycerophosphate oxidase system was markedly inhibited by oligomycin up to 1 day after emergence, but in the 6-day-old tissue the sensitivity of this oxidase system to oligomycin was considerably lower. However, inhibition of the pyruvic oxidase system by oligomycin was still considerable (about 80 per cent) in such material.

DNP Sendvity The release of the oligomycin inhibition of cr-glycerophosphate oxidation by DNP was also most marked up to 1 day after emergence; thereafter there was a decrease in sensitivity to DNP.

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P/O ratios An attempt was made to measure P/O ratios during the oxidation of or-glycerophosphate for which the maximal P/O is 2. Oxygen uptake was measured polarographically and initially phosphate esterified was estimated as glucosedphosphate formed with a glucose-hexokinase ‘trap’. This method, however, proved unsuitable as the control system (no substrate, not incubated) contained a large amount of high-energy phosphate (almost ten times the amount of endogenous ATP in the insect; CROMPTONand BIRT, 1967). The concentration of high-energy phosphate continued to increase during 10 min incubation of the control system, even though there was no detectable endogenous respiration. These results could be explained by the activity of the myokinase present in these mitochondria. Subsequently a second method (CHANCEand WILLIAMS, 1956) was employed to estimate P/O ratios. A known amount of ADP was added to the system and oxygen uptake over the stimulated period was estimated. Difficulty was experienced with estimating the point at which the added ADP was exhausted, as the return to the controlled rate was gradual, probably because of the high myokinase activity. Before emergence, the estimation was even more difficult as the initiation of respiration was completely dependent on ADP and the rate of respiration never returned completely to zero. However, the values obtained with insects from 2 days before emergence to 6-day-old adults were in the range 1.4 to 1.8. Thus, judged by a variety of criteria, both ol-glycerophosphate and pyruvate oxidations appeared to be coupled to phosphorylation from 2 days before emergence to the 6-day-old adult. Eflects of EGTA and Ca2+ on or-glycemphosphate oxidase and dehydrogenase Work already published has shown that respiratory control of or-glycerophosphate oxidase by ADP was relatively low in adult LuciZia (BIRT, 1961), although pyruvate respiration was still highly coupled. Furthermore, in relatively intact Calliphora mitochondria, the rate of oxidation of 2 mM DL-a-glycerophosphate was severely inhibited in presence of EGTA; calcium ions were required for the restoration of oxidation of this substrate (HANSFORDand CI+PPELL, 1965). It appears that or-glycerophosphate oxidation, unlike pyruvate oxidation, is controlled not only by ADP but also by Caa*. L-cu-Glycerophosphate oxidase. The dependence of or-glycerophosphate respiration on Ca2+ in the mitochondria of developing adults of Lucilia was investigated in media in which the or-glycerophosphate concentration was lowered to 3 mM (HANSFORDand CHAPPELL, 1967). This system was compared with one which contained 35 mM L-ar-glycerophosphate and a second which did not contain EGTA; the respiratory rate was then measured in the presenceof 3 mM a-glycerophosphate and an incubation medium which included 2 mM EGTA. The investigation was made on unfractionated preparations. Pyruvate respiration was completely independent of Caa+ at all stages of development. Conversely respiration with or-glycerophosphate was almost completely dependent on Gas+ at all stages of development; moreover, under the

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conditions described added ADP was ineffective in stimulating respiration unless Ca2+ was also present. During development there was a gradual increase in the stimulatory effect of Ca2+ in the absence of ADP. In addition, in the immature fly, there was a considerable dependence of respiration on ADP; in the 8-day-old insect respiration was completely independent of ADP. With these preparations it was also apparent that respiratory-linked phosphorylation was less stable in the absence of Ca2+. L-cY-Glycerophosphate dehydrogenase. An unfractionated preparation of mitochondria was also used for this series of experiments. The concentration of DLQglycerophosphate was 3 mM in the assay system. EGTA (10 mM) inhibited the dehydrogenase by 20 per cent. Increasing the concentration of EGTA to 100 mM subsequently caused a further decrease in activity of approximately 32 per cent. Additions of Ca2+ ion (in the range 5 mM to 20 mM) did not relieve the inhibition Thus it appears that a-glycerophosphate dehydrogenase itself is inhibited considerably by EGTA. However, the extent of inhibition of the dehydrogenase was less than that of the oxidase under comparable conditions; this suggests that EGTA may affect both the dehydrogenase and the transport of a-glycerophosphate into the mitochondrion. It should be noted, however, that the r6le of calcium as an allosteric effector (HANSFORDand CHAPPELL, 1967) can only be demonstrated when a-glycerophosphate dehydrogenase is attached to the membrane. As the mitochondria in the INT-reductase system will be disrupted extensively, since the tonicity of the assay system is very low, it is probable that a full effect of Caa+ stimulation cannot be demonstrated. DISCUSSION

LXstribution of protein The co’mplex development of the mitochondrial population involves an increase in mitochondrial specific gravity as the insect matures. This increase is discontinuous; almost all of it occurs over the period of emergence. Thus it is in this period that a marked concentration of protein in the mitochondria can be anticipated. This concentration is probably reflected in the increase in the number of cristae/mitochondrion (14-321~ mitochondrial length) which occurs at this stage (LENNIE et al., 1%7). As this increase is in turn preceded by a fourfold rise in the amount of mitochondrial DNA (LENNIE d al., 1%7), it is possible that the mitochondria at this stage of development are synthesizing at least some of their own protein (mainly structural protein) and that this synthesis accounts for the rise in their specific gravity. The incorporation of protein is paralleled by an accumulation of phospholipid (D’COSTA and BIRT, 1966) ; such a synchrony may be expected in the elaboration of the structural elements of the mitochondria and would demand a fine integration of the control of protein and phospholipid incorporation into the membranes.

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The pattern of deetelopment of a-glycerophosphate dehya?ogenase and oxidase

The development of dehydrogenase, oxidase, and ATP synthetase activities in the period studied involves sequential changes, which can be correlated with the model suggested by LENNIEand BIRT (1967). The features of this model have been described by a diagrammatic representation of the appearance of respiratory and non-respiratory protein in a single particle (LENNIE and BIRT, 1967) and may be summarized as follows : Initially (Stage A) the mitochondrion is assumed to acquire predominantly enzyme protein, which continues to accumulate throughout development. However, over the period of emergence the particle accumulates predominantly nonenzymic (structural) protein (Stage B) at the expense of the protein reserves of the pupa. The two principal features of the scheme are: (1) the asynchronous incorporation of structural and enzymic protein, and (2) the rapid incorporation of structural protein over the period of emergence. L-a-Glycerophosphate dchydrogenase activity. In the light and intermediate fractions there was an increase in a-glycerophosphate dehydrogenase activity before emergence; in the intermediate and the dense fractions there was a decrease in activity after emergence. Thus the accumulation of enzyme protein is more rapid than that of structural protein before emergence and less rapid after emergence, i.e. the data provides further clear evidence of asynchrony. The difference between the dehydrogenaae activities of the light and the intermediate fractions after emergence was much less marked; a-glycerophosphate dehydrogenase was still accumulating in excess of structural protein in the light fraction. Dehydrogenase activity was lower in the dense fraction than in the intermediate fraction, probably because structural protein is being incorporated faster than enzyme protein in this fraction. In contrast the ratio of dehydrogenase protein to non-dehydrogenase protein remains constant in the intermediate and heavy fractions from 2 days before emergence to the 7-day-old adult. L-a-Glycerophosphate oxidase activity. Two days before emergence, in the light fraction, oxidase activity is only half the dehydrogenase activity. In the intermediate fraction at this stage of development oxidase activity is equal to dehydrogenase activity and both values are much higher than that of the lighter fraction. This is probably not because of an increase in ‘oxidase protein’ (either enzymic, for example cytochrome, or structural) but is more likely to result from an increase in internal organization of the membranes. This is indicated by the observations that (i) dehydrogenase activity has also increased considerably in the middle fraction, i.e. it has not been ‘diluted’ by oxidase protein; (ii) the total increase in specific gravity between the light and the intermediate fractions is relatively small; thus protein already present in the mitochondria of the light fraction becomes more highly organized in the slightly more dense intermediate fraction. One day before emergence there is an increase in oxidase activity between the light and the intermediate fractions. However, in the intermediate fraction the dehydrogenase can no longer be fully expressed as oxidase. Thus with increasing

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specific gravity there is still a preferential accumulation of dehydrogenase protein and the incorporation of structural protein is still relatively slow; the internal membrane organization, on which full expression of oxidase depends, is still relatively deficient. At emergence, oxidase activity in the light fraction is equal to dehydrogenase activity; this is a similar situation to that found in the middle fraction 2 days before emergence and suggests that mitochondria in the light fraction now resemble those in the middle fraction more closely (cf. Fig. 3). In the middle fraction dehydrogenase is still accumulating faster than oxidase protein and there is probably still a lack of internal organization. In the dense fraction dehydrogenase activity has fallen somewhat, but oxidase activity is maintained at a value equal to that in the middle fraction ; thus oxidase protein is being accumulated more rapidly than dehydrogenase protein and the internal organization approximates its final state of complexity. One day after emergence the light fraction is still in the stage of preferential dehydrogenase accumulation and the internal organization is deficient (i.e. similar to the situation found in the middle fraction before emergence). This accumulation of dehydrogenase continues in the middle fraction. In the dense fraction oxidase protein is now ‘diluting’ the dehydrogenase activity, but internal organization is now sufficient to allow dehydrogenase to be fully expressed as oxidase. Even at an early stage in the development of the adult, respiration is linked to phosphorylation ; there is no evidence for an uncoupled oxidase in younger tissues. Thus the development of the structures coupling oxidation and phosphorylation must be synchronous with that of the oxidase assembly, so that as soon as the oxidase complex is functional, there is present a system capable of generating ATP. Summmy of the ak~elopment of j?ight muscle mitoc~. Before emergence, mitochondria are relatively deficient in oxidase protein and internal organization. Over emergence there is an increase in the incorporation and organization of oxidase protein. After emergence sufficient internal organization of the mitochondrial membranes has occurred to permit the oxidase system to express most of the dehydrogenase. These conclusions are supported by the evidence obtained by WALKERand BIRT (1968) from electron microscope examinations of comparable mitrochondrial preparations. SigniJTcance in changes in the control of phosphorylating capacity The decline after adult emergence in the ADP control of or-glycerophosphate oxidase may be explained by assuming that some compound can substitute for .4DP in an energy-conserving reaction; thus electron flow continues in the absence of ADP, i.e. the cY-glycerophosphate oxidase system is loosely coupled. As there is no comparable change in the ADP control of pyruvic oxidase it appears that this substitution must occur at a site or sites in the phosphorylation assembly other than the NAD-linked site and before the common path of electron transfer from flavoprotein and NAD is reached. The situation found in L. cuptina is unlike that of the bee (BALBONI,1967) where

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respiratory control ratios never exceed 1.9 at any stage of development with any substrate: however, the physiological behaviour of the bee is very different from L. cuprina, as this insect does not begin to fly until 12-20 days after emergence of the adult.

Effect

of Ca2+

on ar-glycerophosphate oxiduse

The change in the pattern of respiratory control by ADP and Ca2+ may be a reflection of the onset of the extraordinary high activity of the insect flight muscle after emergence. In Lucilia this muscle contracts some twenty times faster than vertebrate muscle (VAN DEN BERGH, 1962). In the emerged adult, which begins to fly a few hours after eclosion, it is possible that the process requiring ATP (muscular contraction) and the process generating ATP are controlled by the release of the same component, calcium. In contrast, the respiratory activity of the pharate adult, in which the muscles are not engaged in locomotion, is more directly linked to energy transformations producing ADP. Acknowledgements-We wish to thank the Medical Research Council and the Scientific Research Council for financial assistance and Professor W. BARTLEY for encouragement and support. REFERENCES B.~LBONI E. R. (1967) The respiratory metabolism of insect flight muscle during adult maturation. 3. Insect Physiol. 13, 1849-1856. BIRT L. M. (1961) Flight muscle mitochondria of Lucilia cuprina and Musca domestica. Estimation of the pyridine nucleotide content and of the response of respiration to adenosine diphosphate. Biochem. 3. 80, 623-63 1. BIRT L. hl. (1966) The distribution of nicotinamide nucleotides during the life cycle of the blowfly Lucilia cupina Wied. Biochem.3. 101, 429-434. BROSEMERR. W., VOGEL W., and BUCHERT. (1963) Morphologische und enzvmatische Muster bei der Entwicldung indirekter Flugmuskeh von Locusta migratoria. Biochem. 2. 338, 854-910. BUCHERT. (1965) Formation of the specific structural and enzymic pattern of the insect fhght muscle. Symp. biochem. Sot. 25, 15-28. CHANCEJ. B. and WILLIAMSG. R. (1956) Respiratory enzymes in oxidative phosphorylation -VI. The effects of adenosine diphosphate on axide-treated mitochondria. 3. biol. them. 221,477-489. CHAPPELL J. B. (1964) The oxidation of citrate iso-citrate and cis-aconitate by isolated mitochondria. Biochem. 3. 90, 225-237. CROMPTONM. and BIRT L. M. (1967) Changes in the amounts of carbohydrates, phosphagen and related compounds during the metamorphosis of the blowfly Lucilia cupina. 3. Insect Physiol. 13, 1575-1592. D’COSTA M. A. and BIRT L. M. (1966) Changes in the lipid content during the metamorphosis of the blowfly Lucilia. .7. Insect Physiol. 12, 1377-1394. GREGORYD. W., LENNIE R. W., and BIRT L. M. (1968) An electronmicroscopic study of flight muscle development on the blowfly Lucilia cup&a. 3. R. micr. Sot. 88, 151-17s. HANSFORDR. G. and CHAPPELLJ. B. (1967) The effect of Ca*+ on the oxidation of glycerol phosphate by blowfly flight muscle mitochondria. B.B.R.C. 27, 6, 686-692. HEROLD R. C. (1965) Development and ultra structural changes of sarcosomes during honey bee flight muscle development. Dev. Biol. 12, 269-286.

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HOWELLS A. J. and BIRT L. M. (1964) Amino acid dependent pyrophosphate exchange during the life cycle of the blowfly Lucilia cuprina. Comp. Biochem. Physiol. 11, 61-83. LEE Y. and LARDY H. A. (1965) Influence of thyroid hormones on L-a-glycerophosphate dehydrogenase and other dehydrogenases in various organs of the rat. J. biol. Chem. 240, 1427-1436. LENNIE R. W. and BIRT L. M. (1967) Aspects of the de\ elopment of flight muscle sarcosomes in the sheep blowfly Lucilia cuprina in relation to changes in the distribution of protein and some respiratory enzymes during metamorphosis. Biochem. J. 102, 338-350. LENNIE R. 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. J. Insect Physiol. 13, 1745-1756. RICHARDSON T. and TAPPEL A. L. (1962) Swelling of fish mitochondria. r. Cell Biol. 13, 43-33. SALO T. and KOUNSD. M. (1965) An improved gradient making device for density gradient centrifugation. Analyt. Biochem. 13, 74-79. VAN DEN BERCH S. G. (1962) Respiration and energy production in the flight muscle of the housefly Musca domestica L. Doctoral Thesis, University of Amsterdam. WALKERA. C. and BIRT L. M. (1%8) An electron microscope study of thoracic mitochondrial membranes in developing adults of the blowfly Lucilia cuprina. J. Insect Physiol. In press.