The effects of different isolation media on the respiration and morphology of housefly sarcosomes

J. Insect Physiol., 1966, Vol. 12, pp. 1093 to 1103. Pergamon Press Ltd.

Printed in Great Britain

THE EFFECTS OF DIFFERENT ISOLATION MEDIA ON THE RESPIRATION AND MORPHOLOGY OF HOUSEFLY SARCOSOMES G. C. CARNEY* (Received 11 March 1966) Atomic Energy of Canada Limited, Chalk River Nuclear Laboratories, Biology and Health Phvsics Division, Chalk River, Ontario, Canada Abstract-The morphology and respiratory activity of housefly sarcosomes were compared after isolation in media based on O-154 M KC1 and 0.25 M sucrose respectively. The sucrose medium produced dense, distorted sarcosomes, whereas the KC1 medium resulted in partial swelling and vacuolation. Oxygen uptake was measured polarographically during the first 5 min of incubation in the presence of pyruvate and c+glycerophosphate, respectively. The KCl-isolated sarcosomes showed a higher degree of respiratory control by ADP. This was most marked when the substrate was pyruvate. In contrast, the sarcosomes isolated in sucrose showed a moderate and about equal respiratory response to ADP with both substrates. Both isolation media yielded sarcosomes which oxidized o-glycerophosphate over 30 times as fast as pyruvate. INTRODUCTION

IN THE years which have followed the first demonstration of oxidative phosphorylation in housefly flight muscle sarcosomes, no unanimous agreement has been reached as to the identity of the principal substrate consumed during flight. These sarcosomes are notoriously unstable after isolation, and it is therefore not surprising that different laboratories have obtained widely different results in evaluating the relative importance of alpha-glycerophosphate (CL-GP) and pyruvate as substrates. The difficulties have been compounded by the adoption by different workers of isolation media which differ widely either in their chemical nature (sucrose versus KCl) or with respect to additives. The principal function of the sarcosomes is to supply the muscle fibrils with ATP, the precursor of which is ADP. It is a characteristic of a preparation of viable mitochondria that the respiratory rate is controlled by the amount of available ADP. The addition of ADP to a preparation of functional mitochondria in the presence of a suitable substrate will result in an increase in the respiratory rate as this ADP is oxidatively phosphorylated to ATP. The opinion that c+GP is the principal physiological substrate is strongly expressed in the writings of Sacktor and his co-workers. It is based on experimental evidence that this *Present address: Ohio, U.S.A.

Dept.

of Biology,

University 1093

of Bowling Green,

Bowling Green,

1094

G. C. CARNEY

compound yields respiratory rates which are greatly in excess of those obtained with pyruvate (CHANCEand SACKTOR,1958). The higher respiratory rates obtained by BIRT (1961) with a-GP, as compared with pyruvate in experiments using Lucilia sarcosomes, supports this view. However, both results were obtained using sucrose-based isolation media. The opposing view, that pyruvate plays the major role in supplying the energy necessary for flight, is also based on clear experimental evidence, this time for a higher degree of respiratory control (over tenfold) with pyruvate as against about twofold for c+GP. At the same time, respiratory rates were observed with pyruvate which approached or exceeded those for c+GP (GREGG et al., 1960; VAN DEN BERGH and SLATER, 1962; VAN DEN BERGH, 1964). These experiments were conducted on sarcosomes isolated in saline media based on either sucrose plus phosphate or KCl. The conflicting views therefore appear to depend to a large extent on the saline or non-saline nature of the isolation medium. The morphological integrity of the isolated sarcosomes has received little or no reference in most of the accounts of biochemical studies published in recent years. The early and well-documented findings of WATANABEand WILLIAMS(1953) have shown, however, that these organelles are profoundly altered in form and size by variations in the isolation medium. The isolation media commonly used for metabolic studies have been found, during the present work, to have different effects on the microscopic appearance of the sarcosomes. These visible differences and the demonstration of related differences in their respiratory activity are the subject of the present communication. MATERIALS

AND METHODS

Sarcosomes were isolated from the thoraces of previously chilled houseflies 3 to 12 days old. The thoraces were gently crushed for 2 min in 3 ml of isolation medium in a glass mortar resting in chipped ice. Twenty ml of fresh isolation medium were then added to the mortar and the brei was transferred to a previously moistened filter pad consisting of 8 layers of double-mesh cheesecloth. The residue was carefully washed with a further 40 ml of isolation medium. Gentle suction was used to assist the filtration. The filtrate was centrifuged for 4 min at 200 xg. The supematant was then removed in such a way as to leave behind the last 2 ml which covered the pellet of debris. The sarcosomes were deposited from the supernatant by centrifugation at 1800 xg for 15 min. All centrifugations were carried out at 4°C. The pellet of sarcosomes was washed (surface only) with fresh isolation medium and resuspended in the same medium to a final volume of 1.0 ml. Protein was estimated by the copper-Folin method (LOWRY et al., 1951), using crystalline bovine serum albumin as a standard. The isolation media used were 0.25 M sucrose plus 0.001 M EDTA at pH 7.4 and 0.154 M KC1 plus 0.001 M EDTA at pH 7.4. The incubation mixture, which was similar to that used by VAN DENBER~XX and SLATER(1962), contained 15 mM KCl, 5 mM Mg Cl,, 50 mM Tris, 2 mM EDTA, and 35 mM potassium phosphate buffer. The substrates, which were obtained from Sigma, were 60 mM a-GP

RESPIRATION

AND MORPHOLOGY

OF HOUSEFLY

SARCOSOMES

1095

or 20 mM pyruvate as indicated. The pH was 7.4. Oxygen uptake was measured by a vibrating platinum electrode with recorder (‘Oxygraph’, Gilson Medical Electronics). The volume in the reaction vessel during equilibration was 2-5 ml. To this was added 0.02 to O-1 ml portions of sarcosome suspension. A solution of ADP (Sigma) which had previously been adjusted to pH 7.4 was added in 0.02 ml portions (4 pmoles) where indicated. The temperature of incubation was 28°C. The respiratory rates were calculated from the slope of the polarograph tracings during the first 5 min of the reaction. The observations of optical density were made by placing a portion of the incubation medium in a cuvette resting in a metal water jacket (Thomas ‘Roto-Cell’) in the Bauch and Lomb ‘Spectronic 20’ spectrophotometer. The circulating water was at the same temperature as the water which passed around the cell of the polarograph. By this means the temperature of the incubation medium when determining optical density was kept as close as possible to the temperature used for the respiration studies. The morphology of the sarcosomes was studied using a 100 x Wild phase contrast objective, N.A.1.3. Total magnification was 1250 x . RESULTS

Morphology

AND

DISCUSSION

of sarcosomes isolated in dzzeerent media

A close examination of the isolated sarcosomes used in the present work was prompted by the findings of WATANABEand WILLIAMS (1953) with regard to the morphological differences observed in sarcosomes placed in different media. There was in fact a distinct difference in appearance between sarcosomes isolated in 0.25 M sucrose with 1 mM EDTA and those isolated in O-154 M KC1 with 1 mM EDTA. The vast majority of sarcosomes assumed the appearance shown in Fig. 1A and 1B respectively. This was the case both in the presence and in the absence of EDTA. Furthermore, sarcosomes suspended in the sucrosesaline isolation medium used by GREGG et al. (1959, 1960) were virtually identical in appearance to those isolated in the KU-based medium used by VAN DEN BERGH (1964). In both the organelles were swollen and vacuolated (Fig. 2B and C). This was in sharp contrast to the dense, distorted appearance of sarcosomes suspended in 0.25 M sucrose with 1 mM EDTA (Figs. 1A and 2A). The mannitol medium used by SACKTORand DICK (1962) was also tested in this laboratory and was found to have substantially the same effects on sarcosome morphology as the sucroseEDTA medium. An observation made during the present work which could be confused with the conclusions of Watanabe and Williams concerns the significance of the C-shaped profiles of some of the sarcosomes illustrated in Figs. 1B and 2B and C. The above authors described a similar structural change (their Fig. 3s) which they ascribe to an infolding of the outer membrane. It is important to note that the C-shapes observed in the present work are not the result of an infolding. The outer membrane was observed to extend across the open part of the ‘C’ as a curved line continuous with the circular outline of the remainder of the sarcosome.

G. C. CARNEY

1096

The particular type of deformation which occurs in the saline-isolated sarcosomes described in the present work is therefore the result of vacuolation and is not a transitional shrinkage stage towards the dense rod-shaped forms typically observed in 0.25 M sucrose.

1 A.

SALACOSOMES

SUSPENDED

0*25 M SUCROSE , I mM

6.

SARCOSOMES SUSPENDED 0.154 M

KCL,

ImM

IN

EDTA.

IN

EDTA.

FIG. 1. Detailed appearance of sarcosomes as observed under phase-contrast.

The comparatively large diameter of housefly sarcosomes (2 to 3 p) enables internal details to be seen using the light microscope which would be almost invisible in, for example, beef heart sarcosomes which only approach 1 ,u in diameter. In housefly sarcosomes it is possible to detect not only the presence or absence of vacuoles, but to distinguish a definite pattern in the form of vacuolation. During observation under the microscope, the sarcosomes frequently move and roll

FIG 2. .A--5arcosomes suspended in 0.25 M sucrose plus 1 mM EDT.\, pH 7.4; R- Sarcoscjmes suspended in 0.154 lb1 KC1 plus 1 mA1 EDT& pH 7.4; C ~Sari cosomes suspended in medium containing 0.25 YI sucrose, 0.1 hl potassium 3 rnlII M&I,, and 3 mRI each of citrate pho lsphate, 5 m\I Tris, 3 mM EDTA, succinate and pyruvate, pH 7-4. White line represents 5 ,u.

RESPIRATIONAND MORPHOLOGYOFHOUSEFLYSARCOSOMES so that a three-dimensional

1097

picture can be constructed.. The various patterns of light and dark areas shown in Fig. IB are largely interchangeable and represent a similar structural deformation which is viewed from different angles in the different sarcosomes. The different morphology of the sarcosomes isolated in the two ways has a parallel in the findings of workers who have studied mammalian heart sarcosomes. For example, the effects of various isolation media on the morphology of beef heart and rabbit heart sarcosomes have been described in the electron micrographic studies of ZIEGLER et al. (1958) and DESHPANDE et al. (1961) respectively. Many of the structural deformations observed in the present work can be recognized in the electron micrographs of these authors, although the mammalian sarcosomes apparently require a higher concentration of sucrose to bring about shrinkage. There is little doubt that the shaded areas in Fig. 1B represent the electron opaque areas (cristae) in the electron micrographs mentioned above. The resemblance ,is particularly clear in one of the electron micrographs of ZIEGLER et al. (their Fig. 3L). This pattern of vacuolation is probably an intermediate stage in the swelling process which, if carried out in distilled water, leads to a large optically empty sphere with its contents compacted into a narrow crescent (WATANABE and WILLIAMS,

1953; SMITH, 1963).

0’35

I I

I 2

I 3

I 4

I 5

MINUTES

FIG. 3. Swelling rates of sarcosomes during incubation as indicated by rate of & Isolated in 0.25 M sucrose, 1 mM change of optical density at 600 rnp. ED'I'A; substrate, ol-GP. A, Isolated in 0.25 M sucrose, 1 mM EDTA; substrate, pyruvate. 0, Isolated in 0.154 M KCl, 1 mM EDTA; substrate, c+GP. @, Isolated in O-154 M KCI, 1 mM EDTA; substrate, pyruvate. Figures next to graphs show the average sarcosome protein concentration of the suspensions in mg/ml. Each point is the mean of at least four experiments. No ADP present. 68

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Swelling behaviour in the incubation mixture Optical density studies carried out in the present work indicated that the sarcosomes swelled after being mixed with the incubation medium. Sarcosomes which had been isolated in the sucros+EDTA medium showed a faster rate of swelling than their counterparts isolated in KCl-EDTA (Fig. 3). This would be expected on the basis of the observations made through the microscope which showed that the sucrose-isolated sarcosomes were extremely dense. Conversely, the sarcosomes isolated in KCl-EDTA were partly swollen and vacuolated. Samples taken after 5 to 10 min of incubation and observed microscopically showed that the majority of the sarcosomes were greatly swollen and vacuolated irrespective of the substrate used or their original means of isolation. Respiration of sarcosomes isolated in two &$&rent media A series of experiments was carried out in which sarcosomes from a singIe batch of flies were isolated using two different media, 0.25 M sucrose plus 1 mM EDTA, and O-154 M KC1 plus 1 mM EDTA respectively. The oxygen uptake of the sarcosomes was measured first in the absence and then in the presence of ADP,

_

240pM

OXYGEN

,

ZERO

60

set

,

OXYGEN-

FIG. 4. Superimposed polarograph tracings showing the effect of ADP on the respiratory rate of housefly sarcosomes. Isolation medium, 0.154 M KC1 plus 1 mM EDTA. Incubation medium as in text. Substrate, c+GP. Final sarcosome protein concentration, 0.16 mg/ml. Final ADP concentration, 16 mM._ Cell volume, 2.5 ml. Temperature, 28°C. Figures next to tracings are qOa values. Vertical arrows indicate times of addition of ADP and sarcosomes (S) respectively. All tracings were obtained from the same batch of sarcosomes isolated from 6-day-old flies.

RESPIRATION AND MORPHOLOGYOF HOUSEFLYSARCOSOMES

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with a-GP and pyruvate as substrates. The respiratory control ratio as defined here is the ratio of the respiratory rates in the presence and in the absence of added ADP. A similar ratio may also be obtained by adding ADP after the experiment has been started and comparing the respiratory rates before and after the addition. The latter method was not adopted in the present work because the value of the ratio so obtained was altered to a variable extent depending on the time of addition of ADP. The decrease in the response to ADP with only a small change in this time period is illustrated in Fig. 4. Reproducible respiratory control ratios could, however, be obtained by comparing respiratory rates in the absence of ADP with those in which ADP was added just before the sarcosomes. The values for q0, and the respiratory control ratios are shown in Table 1. TABLE I-EFFECT

OF ISOLATIONMEDIUM ON RESPIRATORY RATE AND RESPIKATORY CONTROL IN HOUSEFLYSARCOSOMES Sucrose-EDTA

Experiment No.

Ply age (davs) _

isolation

No

ADP

2 3 4 5 6 7

0.7 0.2 0.2 0.1 0.2 0.2 0.05

Mean

1 2 3 4 5 6 7 Mean

3 4 6 9 9 11 12

Protein

b-s/ml) ADP

wGlycerophosphate

.I

isolation 4%

RCR

Protein

(mgb-4

3 4 6 9 9 11 12

KCl-EDTA

RCR

No ADP

ADP

522 235 385

970 604

as substrate

291 253 579 347 589 418 961

528 480 873 420 870 469 1642

1.8 1.8 1.5 1.2 1.5 1.1 1.7

491

755

1.5

0.7 1.0

Pyruvate as substrate 10 12 1.2 8 12 I.5

1-o 0.5 1.0 I.0 0.4

8 11 14 10 17

14 25 25 18 22

1.8 2.3 1.8 1.8 1.3

10

18

1.7

0.05 0.3 0.2 0.3 0.2 0.2 0.05

0.4 I.1 0.8 0.5 0.8 0.8 0.4

330 251 1110

660 862 312 2482

I.9 2.6 3.6 I.9 2.6 I.2 2.2

455

1041

2.3

4 4 7 4 5 5 5

26 19 48 16 52 36 47

6.5 4.8 6.9 4.0

5

35

349

1396

10.0

7.2 9.4 7.0

The respiratory rates of housefly sarcosomes isolated in 0.25 M sucrose plus 1 mM EDTA and 0.154 M KC1 plus 1 mM EDTA respectively. Substrate concentrations: c+GP, 60 mM; pyruvate, 20 mM. Isolated and incubated at pH 7.4. Incubation medium as in text. RCR-respiratory control ratio, the ratio of the respiratory rates in the presence and in the absence of ADP.

1100

G. C. CARNEY

The first impression to be obtained from these data is that ol-GP is oxidized at a much faster rate than pyruvate, irrespective of the isolation medium employed. However, when one compares the effects of the two isolation media on the oxidation of a given substrate, certain differences become apparent. The oxidation of ol-GP was slightly faster in the case of sarcosomes isolated in the KC1 medium, provided ADP was present in the incubation mixture. This difference between the two batches of sarcosomes was much more obvious when the substrate was pyruvate. In this case, the sarcosomes isolated in the KC1 medium oxidized pyruvate at a slower rate in the absence of ADP. In the presence of ADP, however, the respiratory rate was increased on the average by a factor of 7 bringing it to twice the level of the sucrose-isolated sarcosomes, even when the latter were supplied with ADP, It would appear, therefore, that the higher respiratory rates observed with KClisolated sarcosomes are the result of a greater response to ADP and not of a general stimulation of respiration by the saline medium. These results are based on measurements of oxygen uptake during the first 5 min of incubation. Caution must be exercised therefore in drawing close comparisons between these data and those obtained by other workers using longer periods of incubation. Much higher respiratory rates with pyruvate have been reported by GREGGet al. (1960) and VAN DEN BERGHand SLATER(1962) using manometric methods. On several occasions during the present work it was observed that the rate. of oxidation of pyruvate speeded up considerably after 5 or 6 min of incubation. In a manometric determination this phenomenon would lead to higher q0, values after the customary period of temperature equilibragion. The ability of insect mitochondria to oxidize pyruvate has been described by SACKTOR( 1964) as a variable property which may be lost after brief exposure to temperatures above 0°C. The sarcosomes used in the present work were always able to oxidize pyruvate, but brief exposure to room temperature brought about large variations in respiratory rates and respiratory control. The ability of the KC1 isolation medium to increase the response to ADP may be the result of an alteration in the permeability of the sarcosome membranes. A permeability effect was suggested by SACKTOR (1964) while citing some unpublished work of VAN DEN BERGH which indicated that the use of a KC1 isolation medium increased the requirement of the sarcosomes for cytochrome c. This evidence led Sacktor to conclude that the KC1 medium, while resulting in a loss of cytochrome c, might also facilitate the entry of pyruvate and NADH. The present work indicates that KC1 might also facilitate the entry of ADP. The distinct morphological difference between sucrose-isolated and salineisolated sarcosomes described in the present work suggests that an important physical~difference may also exist in the sarcosome membranes or the arrangement of the cristae. Evidence that induced changes in mitochondrial size may alter their permeability and metabolic properties has been presented in the case of rat liver mitochondria (BARTLEY,1961; LEHNINGER,1961) and crab mitochondria (MUNDAY and THOMPSON, 1962).

RESPIRATION ANDMORPHOLOGY OF HOUSEFLY SARCOSOMES

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Other factors which could affect the degree of respiratory control include the age of the insects and the concentration of mitochondria in the reaction vessel. Lower respiratory control ratios have been reported in mitochondria from older flies (BIRT, 1961). As was the case in the experiments of VAN DEN BERGH and SLATER (1962), the present results showed no age effect. Reducing the concentration of mitochondria has been found to increase the degree of respiratory control in housefly sarcosomes (BIRT, 1961) and cockroach sarcosomes (COCHRAN, 1963). The converse situation has been described in rabbit heart mitochondria (TARJAN and VON KORFF, 1965) where increased respiratory control was observed at higher concentrations of mitochondria. The present results show no consistent effect of sarcosome concentration on respiratory control. The same situation has been reported in the case of honeybee sarcosomes (BALBONI, 1965). There are some grounds for expecting the respiratory control ratios in the case of pyruvate to exceed those with ol-GP. It has been found, for example, that the oxidation of NAD-linked substrates like pyruvate usually shows a more pronounced response to ADP (HATEFI et al., 1961; TARJAN and VON KORFF, 1965). The oxidation of a-GP is, however, apparently linked to a flavoprotein rather than NAD (CHANCEand SACKTOR, 1958; KLINGENBERG and BUCHER, 1961). What remains unexplained is the fact that when the sucrose isolation medium is used, both substrates showed virtually the same degree of respiratory control. This result does not correspond with the findings of VAN DEN BERGH and SLATER (1962), who obtained high respiratory control ratios with sucrose-isolated sarcosomes oxidizing pyruvate. These authors measured respiratory control by adding ADP after a period of incubation. It should be noted, however, that BALBONI (1965), in measuring respiratory control by the same general procedure as the .above authors and using a sucrose-based isolation medium, found higher respiratory control’ ratios and respiratory rates with a-GP than with pyruvate in honeybee sarcosomes. CONCLUSIONS The present results show that the two types of isolation media commonly used for the extraction of housefly sarcosomes result in metabolic and morphological differences in the organelles. This finding has significance in connexion with the controversy over the relative importance of c+GP and pyruvate as physiological substrates (SACKTOR, 1964; CHEFURKA, 1965). The experiments on respiration showed that if KC1 had been the only isolation medium employed, pyruvate would have been judged as being an important substrate as a result of the high degree of respiratory control obtained. On the other hand, an entirely different impression would be gained from the corresponding data using the sucroseisolated sarcosomes. Here pyruvate was oxidized at one-fortieth the rate of ol-GP, while both substrates showed about the same degree of respiratory control. From the standpoint of preserving morphological integrity both the isolation media were found to be unsatisfactory. On the other hand, the optimal medium proposed by WATANABEand WILLIAMS (1953) may not necessarily confer maximum

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G. C. CARNEY

phosphorylating capacity on the sarcosomes (LEWIS and SLATER, 1954). While the present data support the view that CX-GP is the principal physiological substrate for housefly sarcosomes, much improvement seems necessary in selecting media for both isolation and incubation which minimize morphological changes. Acknowbdgements-I wish to thank Dr. D. K. MYERS for many helpful suggestions, Miss C. M. RAE for her technical assistance, and Mrs. J. M. STALKERof the Pesticide Testing Laboratory, Ottawa, for supplying the insects. REFERENCES BALBONI E. R. (1965) Influence of preparatory procedure on the oxidative activity of honeybee flight muscle sarcosomes. r. Insect Physiol. 11, 1559-1572. BARTLEYW. (1961) Solute movements during volume changes in rat-liver mitochondria. Biochem. J. 80, 46-57. BIRT L. M. (1961) Flight-muscle mitochondria of Lucila cup&a and Musca domestica. Estimation of the pyridine nucleotide content and of the response of respiration to adenosine diphosphate. Biochem. J. 80, 623-631. CHANCEB. and SACKTORB. (1958) Respiratory metabolism of insect flight muscle--II. Kinetics of respiratory enzymes in flight muscle sarcosomes. Archs biochem. Biophys. 76, 509-531. CHEFURKA W. (1965) Carbohydrate metabolism in insects. A. Rev. Ent. 10, 345-382. COCHRAND. G. (1963) Respiratory control in cockroach-muscle mitochondria. Biochim. biophys. Acta 78, 393-403. DESHPANIXP. D., HICKMAND. D., and VON KORFFR. W. (1961) Morphology of isolated rabbit heart muscle mitochondria and the oxidation of extra-mitochondrial reduced diphosphopyridine nucleotide. r. biophys. biochem. Cytol. 11, 77-93. GREGGC. T., HEISLERC. R., and REMMERTL. F. (1959) Pyruvate and ar-glycerophosphate oxidation in insect tissues. Biochim. biophys. Acta 31, 593-595. GREGG C. T., HEISLERC. R., and REMMERTL. F. (1960) Oxidative phosphorylation and respiratory control in housefly mitochondria. Biochim. biophys. Acta 45, 561-570. HATEFI Y., JURTSHUKP., and HAAVIK A. G. (1961) Studies on the electron transport system-XXXII. Respiratory control in beef heart mitochondria. Archs biochem. Biophys. 94, 148-155. KLINGENBERGM. and BUCHERT. (1961) Glycerin-l-P und Flugmuskel-Mitochondrien. Biochem. Z. 334, 1-17. LEHNINGERA. L. (1961) Ionic environment and the contraction of isolated rat liver mitochondria by adenosine triphosphate. Biochim. biophys. Acta 48, 324-331. LEWIS S. E. and SLATERE. C. (1954) Oxidative phosphorylation in insect sarcosomes. Bioch.enz. J. 58, 207-217. LOWRY 0. H., ROSEBROUGH N. J., FARRA. L., and RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. MUNDAY K. A. and THOMPSONB. D. (1962) The effect of osmotic pressure on the activity of Car&us maenas mitochondria. Comp. Biochem. Physiol. 6, 277-287. SACKTORB. (1954) Investigations in the mitochondria of the housefly Musca domestica L.III. Requirements for oxidative phosphorylation. J, gen. Phys-iol. 37, 343-359. SACKTORB. (1964) Energetics and respiratory metabolism of muscular contraction. In The Physiology of Insecta 2 (Ed. by ROCKSTEINM.), pp. 483-580. Academic Press, New York. SACKTORB. and DICK A. (1962) Pathways of hydrogen transport in the oxidation of extramitochondrial reduced diphosphopyridine nucleotide in flight muscle. J. biol. Chem. 237, 3259-3263.

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SMITH D. S. (1963) The structure of flight muscle sarcosomes in the blowfly, Cdiphoru erythrocephala (Diptera). J. Cell Biol. 19, 115-138. T~JAN E. and VON KORFF R. W. (1965) Personal communication. VAN DEN BERGH S. G. (1964) Pyruvate oxidation and the permeability of housefly sarcosomes. Biochem. J. 93, 128-136. VAN DEN BERGHS. G. and SLATES E. C. (1962) Th e respiratory activity and permeability of housefly sarcosomes. Biochem. J. 82, 362-371. WATANABEM. I. and WILLIAMSC. M. (1953) Mitochondria in the flight muscles of insectsII. Effects of the medium on the size, form and organization of isolated sarcosomes. J. gen. Physiol. 36, 71-90. ZIEGLER D. M., LINNANEA. W., GREEN D. E., Doss C. M. S., and RIS H. (1958) Studies on the electron transport system-XI. Correlation of the morphology and enzymic properties of mitochondrial and submitochondrial particles. Biochim. biophys. Acta 28, 524-538.