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Respiratory chain metabolism in the Colorado potato beetle—I
J. Ins. Physiol., 1962, Vol. 8, pp. 117 to 126. Pergamon Press Ltd.
Printed in Great Britaitz
RESPIRATORY CHAIN METABOLISM IN THE COLORADO POTATO BEETLE---I RESPI_RATIONAND OXIDATIVE PHOSPHORYLATION IN SARCOSOMES FROM ACTIVE BEETLES D. Laboratory
STEGWEE and
for Entomology,
A. R. VAN KAMMEN-WERTHEIM
Agricultural (Received
University,
26 September
Wageningen,
The
Netherlands
1961)
Abstract-Respiratory chain metabolism was studied with isolated thoracic muscle mitochondria (sarcosomes) of the adult Colorado potato beetle. The composition of the respiratory chain did not d&r estientially f5-om that found in housefly sarcosomes and rat liver mitochondria. Isolated sarcosomes readily oxidized various substrates with concomitant phosphorylation. Respiratory control through the phosphate acceptor level was demonstrated to operate in sarcosomes of the Colorado potato beetle, the housefly, the American cockroach, and the Moroccan locust. Observed differences between insect and mammalian mitochondrial activities are explained by differences in intactness of the mitochondria. INTRODUCTION
DE WILDE and SFGWEE (1958) provided direct evidence of stimulation of succinoxidase activity in the d&pausing Colorado potato beetle, Lq&zo&zr~~ dkcemlineuta Say, by a substance (presumably a hormone) derived from the corpora allata of active beetles. This discovery prompted a more detailed investigation of respiratory chain metabolism in this insect. Mitochondria from the thoracic muscles of insects have proved to be extremely well suited for studying respiration and oxidative phosphorylation. Therefore, these bodies were chosen for the present study. MATERIALS
AND
METHODS
Insect material Active beetles of mixed sexes, between 1 and 2 weeks old, were used in all experiments. The insects were obtained. from laboratory cultures maintained as described previously (DE WILDE and STEGWEE,1958). Houseflies (Musca domestica L.), American cockroaches (Peripluneta amerkana L.), and Moroccan locusts (Low&a migratoria L.) which were occasionally used were also obtained from laboratory stock cultures. Reagents All chemicals used were reagent grade. ADP* and ATP were purchased as the sodium and d&odium salts respectively from Sigma Chemical Co., St. Louis, MO,, * The following abbreviations are used: ADP: adenosine diphosphate; ATP: phate; DNP: 2,4_dinitrophenol; EDTA: ethylene d&nine tetraacetic acid. 8
117
adenosine triphos-
11s
D. STEGWEE AND A. R. VANKAMMEN-WERTHEIM
U.S.A. Hexokinase, type III, and cytochrome c, type II, were also obtained from Sigma. Cytochrome c was further purified according to the method of MARGOLIASH (1954,1957). EDTA, disodium salt,was obtained from E. Merck A.G., Darmstadt, German Federal Republic. Preparation of sarcosomf3 The beetles were immobilized by chilling at 2°C. The heads, elytra, and abdomens were removed, and the isolated thoraces were placed in an ice-cold mortar. After the addition of a small amount of isolation medium (an aqueous solution of 0.32 M sucrose and 0.01 M EDTA, pH 7.4) the thoraces were gently crushed with a pestle. The resulting brei was filtered through four layers of muslin and the residue was washed carefully with small portions of the medium. The combined.filtrate and washings (approximately O-5 ml for each thorax used) were then centrifuged for 4 min at 125 g. The sediment was discarded and the supematant centrifuged for 20 min at 2000 g to sediment the sarcosomes. These were resuspended in 0.32 M sucrose solution, pH 7.4, with the aid of a hand-driven Potter-Elvehjem (Teflon-and-glass) homogenizer. The suspension was again centrifuged at low and high speed as mentioned before. The sedimented sarcosomes were freed of an upper ‘fluffy’ layer and suspended in approximately 1 ml of 0.32 M sucrose solution for each ten thoraces used. All operations were performed in a cold room at 2X. Determination of sarcosomal protein The calorimetric method of LOWRY et aZ. (1951) was used. Preliminary removal of lipids as recommended by CLELAND and SLATER (1953) for mammalian heart muscle sarcosomes proved unnecessary. A standard curve was prepared on the basis of Kjeldahl nitrogen determinations. Estimation of respiratory enqwaes in sarcosomes The relative concentrations of respiratory pigments in the sarcosomes were calculated from difference spectra, obtained with the technique described by HOLTON(1955), using a Beckman DU spectrophotometer with photomultiplier attachment. The calculations were based upon data given by CHANCEand WILLIAMS(1956). Measurement of oxidative phosphorylation Respiration was measured with the conventional Warburg technique. The reaction medium contained 0.012 M phosphate buffer, pH 7.4; 0.008 M KCl; 0.0025 M MgCla; 0.001 M EDTA; O-120/,bovine serum albumin; O*OOlS M ATP; 150 K.M. units of hexokinase; 0.005 M glucose; 0.025 M substrate; l-5-3 mg of sarcosomal protein. Sufficient 0.32 M sucrose solution was added to give a .tinal volume of 2.1 ml. The centre well contained O-1 ml of 20% KOH. The reaction temperature was 25”C, the gas phase air. For the measurement of phosphorylation the reaction was stopped after 15-30 min by adding O-3 ml of 40% trichloroacetic acid. The flasks were immediately chilled in ice and water and 4 ml of ice-cold 5% trichloroacetic acid were added. The flask contents were then centrifuged in
RESPIRATORYCHAIN
METABOLISM
IN THBCOLORADO
119
POTATOBBETLE-I
the cold. Phosphorylation was measured as the disappearance of inorganic phosphate which was determined in the supernatant according to the method of FISKEand SUB~AROW (1925). N o corrections were made for endogenous respiration and phosphorylation. Determination of ATP-ase activity of sarcosomes The sarcosomes (l-3 mg of protein) were incubated for 5 min at 25°C in a medium containing 0*012 M tris (hydroxymethyl) aminomethane buffer, pH 7.4; 0.08 M KCl; 0.001 M EDTA; 0.12% bovine serum albumin; 0.005 M ATP. The volume was made up to 2.1 ml by adding 0.32 M sucrose solution. ATP-ase activity was measured as the release of inorganic phosphate which was determined as described in the preceding section after the reaction was stopped by adding O-3ml of 40% trichloroacetic acid. RESULTS Components of the respiratory chain A suspension of sarcosomes prepared as described above readily oxidized added succinate. The respiratory enzymes involved in this oxidation could be detected spectrophotometrically by measuring the changes in optical density which occurred upon enzymic reduction after the addition of succinate.
? E u
0.06
0.03
0.04
0.02
eo2
0.01 0
0 E
-0.01
i -0.02 a ." 3r -0.04 .z$
-0.02
5 -0.06 'D
-0.03
?j .'L-0.08 z 0 -0.10
-0.04
360
360
500 420 440
460 480 500
520 540
560
580
600 620
Wavelength, mp FIG. 1. Spectrum representingthe differences in absorption between aerobic and anaerobic
sarcosomes of the Colorado potato beetle. The sarcosomes were suspended in O-32 M -sucrose, pH 7.4, to a concentration of 3.8 mg of protein per ml. Anaerobic conditions were obtained by adding 0.01 ml of a saturated sodium succinate solution to 2.5 ml of the suspension in a 1 cm cuvette. Anaerobiosis was considered complete when no further increaseof optical density at 550 mp was observed.
Fig. 1 shows the difference spectrum, reduced vs.. oxidized, of a suspension of sarcosomes. The absorption bands of the different respiratory pigments show very
120
D. STEGWEEANDA. R. VANI(AMMEN-WERTHRIM
clearly the a-bands of cytochrome (a + u3), cytochrome 6, and cytochrome c at 603, 563, and 551 rnp respectively, the #&band of cytochrome c at 520 rnp, the trough due to the disappearance of absorption when flavoprotein becomes reduced at 465 mp, and the y-band of cytochrome a at 444 rnp. The sequence of the optical density changes and the concentrations of the various pigments relative to cytochrome a are given in Table 1. TABLE ~-SEQUENCE OF OPTICAL DENSITY CHANGES RESPIRATORYCHAINCOMPONENTS
AND OFSARCOSOMESOFTHE
RELATIVB CONCENTRATION OF THE COLORADO POTATO BEETLR
Component* Spectral band Optical density relative to cytochrome at
Concentration relative to cytochrome at
Flavoprotein” (465 nw)
2.0 2.9
* CytJ,,
etc., designate the reduced forms of cytochrome a3, etc. t The figures represent average values from three experiments.
Oxidatise phosphorylation Freshly prepared sarcosomes oxidized not only succinate but also cy-ketoglutarate, glutamate, and or-glycerophosphate. They were inactive towards fl-hydroxybutyrate. In the presence of ADP, Mg +-+-ions, and serum albumin the oxidation of the various substrates was accompanied by an appreciable phosphorylation. DNP at lOAM uncoupled this phosphorylation to a large degree, though not completely. In the presence of DNP a slight decrease of oxygen consumption was observed. The values for the respiration rates and phosphorylating efficiencies in the presence of different substrates are summarized in Table 2. No values are given for the rates of endogenous respiration. These were variable but sometimes considerable, approaching the respiration rate with succinate as a substrate. By way of comparison, a few data obtained with sarcosomes of Musca dome&a, Periplaneta americana, and Locusta migratoria have been included. In contrast to these, Leptinotarsa, sarcosomes did not oxidize a-glycerophosphate more rapidly than succinate. The P : 0 ratios found are in agreement with those reported in the literature. It should be noted that cytochrome c was absent from the reaction medium. Addition of cytochrome c increased the respiration rate of Leptinotarsa sarcosomes, however without a corresponding increase of the rate of phosphorylation. The result was a considerable lowering of the P : 0 ratio. This is shown in Table 3.
Respiratory control The presence of the hexokinase/glucose ‘trapping’ system for ATP provided for a continuous supply of ADP in the reaction medium. Without ADP the amount of inorganic phosphate esterified was very small. However, the respiration rate was
RESPIRATORY CHAIN METABOLISM
121
IN THE COLORADO POTATO BEE’IZE-I
not lowered significantly by the omission of ADP. Actually, the amount of ADP per flask could be varied between 0 and 30 PM without affecting the respiration rate (this result was obtained by adding ADP as such and omitting hexokinase TABLE
2-&~SPIRATION RATES AND PHOSPHORYLATING EFFICIENCIES OF s~~Coso~~s OF THE COLORADO POTATO BEETLE AND SOME OTHER INSECTS
Substrate
Insect
P : O$
Succinate cw-Glycerophosphate cw-Ketoglutarate* Glutamate
Z&i 32 (2)
Musca
Succinate cw-Glycerophosphate
46 (1) 194 (1)
Peri&?aneta
Succinate wGlycerophosphate
30 (1) loo (1)
Locusta
Succinate c+Glycerophosphate
Leptinotarsa
38 (13)t
* In the presence of 0.01 M malonate. t Figures in brackets represent the number of experiments. $ fitoms inorgdc phosphate taken up per patom oxygen consumed. TABLE 3--THE
Concentration of cyto;c,;)me c
+ Substrate
O-025 M
EFFECT OF
CYTOCHROME c .UPON OXIDATIVE PHOSPHORYLATION SARCOSOMFS OF THE COLORADO POTATO BEETLE
Phosphate uptake (t.Wmg proteinlhr)
IN
P:O
succinate. The figures represent the average values from two experiments.
and glucose). These findings are in agreement with the reports by SACKTOR and COCHRAN (1958) and CHANGE and SACKTOR (1958) on the housefly and by COCHRKN and WG (1960) on the American cockroach. They would indicate that in Leptimtarsa sarcosomes there is also no respiratory control through the phosphorylating system. The demonstration, however, of the existence of such a respiratory control mechanism in isolated sarcosomes of Locusta migratoria by BGXER et al.
D. STEGWB~ANDA. R. VANKAMMEN-WERTHEIM
122
(1959) and KLINGENBERG and B&HER (1959) made us suspicious as to the validity of this conclusion. Upon closer examination it appeared that when Leptinotursa sarcosomes were pre-incubated with the substrate for about 15 min at 25”C, the subsequent addition of ADP caused a distinct increase of the respiration rate. This led us to re-examine the effect of ADP upon respiration of housefly and American cockroach sarcosomes. Locusta migratoria was also included in our investigations so as to enable a comparison with the work of KLINGENBERG and B~CHER (1959). Table 4 summarizes the results of these experiments. In all cases TABLE ~-RESPIRATORY CO~OL IN SAR~OSOMES OF THE COLORADOPOTATOBEETLE ANDSOMEOTHERINSECTS*
Respir;~;l Insect
1
Substrate
Ql Leptinotarsa
Succinate
u-Glycerophosphate or-Ketoglutaratet
46 (5) 54 (2) 45 (1)
Ih.
Succinate c+Glycerophosphate
46 (1) 194 (1)
Pmiplaneta
Succinate ar-Glycefophosphate
LOCUSta
control
QB
Succinate a+Glycerophosphate Pyruvate + malate$
* Reaction inedium without ATP and hexokinasefglucose. ADP was added from the sidearm to a final concentration of 0902 M. t cz-Ketoglutarate OG?S M + malonate O-01 M. $ Pyruvate O-025 M + malate 0.02 M. $ Average values. The number of experiments is given in brackets. 7 Qlythe ratio of respiration rates after and before addition of ADP. Qs=the ratio of respiration rates after and before addition of ADP and after expenditure of the added ADP.
a distinct respiratory control was observed. Sarcosomes of the Colorado potato beetle showed no decrease of respiration rate after expenditure of the added ADP (Qz = 0). This is probably due to high ATP-ase activity, whereby ADP is regenerated. Measurements of ATP-ase in Leptinotuwa sarcosomes, prepared in the usual way, showed that under the present conditions there was indeed a considerable Mg++- activated ATP-ase activity (Table 5). The addition of DNP at lO+M showed that there is about as much ‘latent’ ATP-ase. In the sarcosomes which had received a somewhat milder treatment (only one cycle of centrifuging, which means only once resuspended in the
RESPIRATORY
CHAIN METABOLISM
IN THE COLORADO POTATO
BEETLE-1
123
homogenizer) the total ATP-ase activity was equal, but much less was ‘apparent’ and much more ‘latent’, i.e. only showing up in the presence of DNP. In view of the reported effect of cytochrome c upon respiration and especially of the lowering of the P : 0 ratio it is worthwhile to note that cytochrome c at 6 x 10GM had no effect whatsoever upon ATP-ase activity. TABLE 5-ATP-ASE
Additions*
None p:Z-+ Vg+++DNP
ACTIVITY OF SARCOSOMES OF THE COLORADO POTATOBEETLE
Inorganicphosphatereleased control(pM/mg protein/hr)t
over
PreparationA
PreparationB
2.34
4.84
2.34 3.21 13.20
5:E 13.20
* MgCls and CaCl, were addedso as to give a final concentration of 2.5 x lo-sM. The final concentration of DNP was lO-“M. t Preparation A: suspension after one cycle of low and high speed centrifuging (sarcosoxnes resuspended once). Preparation B: suspension after two cycles of centrifuging (sarcosomes resuspended twice). DISCUSSION On the whole, the results presented in the preceding sections need little comment. The composition of the respiratory chain in sarcosomes of the adult Colorado beetle is quite comparable to that found in housefly sarcosomes by CHANCE and SACKTOR (1958) and not essentially different from that found, e.g., in rat liver mitochondria by CHANCE and WILLIAMS (1956). The data on oxidative phosphorylation show, in agreement with the Endings of SACKTOR and COCHRAN (1958), COCHRAN and KING (1960), and NEWBURGH et al. (1960) that low P : 0 ratios are not characteristicfor oxidative phosphorylation in insect mitochondria. The lowering of the P : 0 ratio by added cytochrome c contrasts with the report by MINNAERT and VAN KAMMEN-WERTHEIM (1960) on rat liver mitochondria, where no effect of cytochrome c upon the P : 0 could be found : oxygen consumption and phosphorylation were equally stimulated. In the case of L.eptimtarsa sarcosomes the phosphorylation rate was only slightly increased by cytochrome at 10q5M, whereas the respiration rate was almost doubled. The resulting lowering of the P : 0 ratio might be termed ‘uncoupling’, but this seems somewhat confusing especially since we were able to demonstrate that cytochrome c had no effect upon ATP-ase activity. For the present we are inclined to think of stimulation of non-phosphorylating respiration, such as occurs in damaged sarcosomes, by added cytochrome c. Thus this stimulation would be indicative of the still relatively poor condition of our sarcosome preparations.
124
D. STEGWEE ANDA. R. VANKAMMEN-WERTHEIM
True uncoupling was brought about by DNP. The concomitant lowering of the respiration rate agrees with findings by SACKTOR and COCHRAN (1958) for the housefly. CLARKEand BALDWIN(1960) found with sarcosomes from -Locusta mi&-at&a also a depression of respiration by DNP. Sarcosomes from Calliphora erythrocephala, however, showed stimulation of respiration by DNP (SLATERand LEWIS,1954). It seems probable that the effect of DNP upon respiration depends on what is the rate-limitingprocess in oxidative phosphorylation (LEHNINGER et al., 1959). Sarcosomes from different sources might well be different in this respect, thus showing opposite responses to DNP. The relatively slow rate of oxidation of cY-glycerophosphate,or, for that matter, the rapid rate of oxidation of several other tri-carboxylic acid cycle intermediates by Leptinotarsa sarcosomes deserves some comment. CHANCE and SACKTOR (1958) with housefly sarcosomes, and COCHRAN and KING (1960) with American cockroach sarcosomes, found respiration rateswith a:-glycerophosphatewhich exceeded those with succinate 4- to lo-fold. The latterauthorsconcluded that this constitutes a characteristicdifference between insect sarcosomes and mammalianmitochondria. On the other hand, B~~CHJZR et al. (1959) believe that the relatively rapid rate of or-glycerophosphate oxidation is typical for damaged mitochondria. In their sarcosome preparationsof Locusta ma’gratoriathe ratio of the oxidation of or-glycerophosphate and pyruvate + malate was only 2. In the only experiment we did with Locusta this ratio was 2.4. In housefly and American cockroach sarcosomes, however, we found ratios which were quite comparable to those mentioned above, whereas with the Colorado potato beetle the respiration rates with ar-glycerophosphate and succinate were invariably about equal. Therefore, we conclude that the rapid rate of oxidation of or-glycerophosphateis typical, not for insect sarcosomes in general, but for sarcosomes of certain insect species, such as Musca domestica In other species, such as Locusta migratoria, the and Periplaneta adana. relative rate of ar-glycerophosphateoxidation may be slower, whereas in species like Leptinotarsa decemkxzta this does not at all exceed the oxidation rate of other substrates. It seems premature to speculate about the significance of the relatively high rate of endogenous respirationfound with Leptinotarsa sarcosomes. According to B@GHER et al. (1959) Locusta sarcosomes also show a considerable endogenous respiration. Investigations of the nature of this phenomenon are planned. The failure to detect respiratory control through the phosphate acceptor level has led COCHRAN and KING (1960) to assume that phosphorylating respiratory control in isolated insect sarcosomes is ‘loosely coupled’. SACKTOR (1959), for the same reason, even proposed an entirely different mechanism for respiratory control in insect mitochondria. However, as was stated before, KLINGENBERG and B~CHER(1959) clearly demonstrated the occurrence of phosphorylating respiratory control in isolated sarcosomes of Locusta migratoria. VAN DENBERGHand SLATER (1960) obtained respiratory control with isolated housefly sarcosomes under special conditions. Recently SACKTOR and PACER (1961) were able to show that ADP stimulates ar-glycerophosphateoxidation in teased flight muscle.
RESPIRATORY CHAINMETABOLISM IN THE COLORADO POTATOBEETLE-I
125
The present results show that it is possible to obtain sarcosomes with respiratory control not only from the Colorado potato beetle, but also from the housefly and the American cockroach. The values for the respiratory control ratios were low as compared to those found for mammalian mitochondria (CHANCE, 1959). This is probably merely a reflection of the comparatively poor condition of the isolated sarcosomes. As was discussed before, the stimulation of respiration by cytochrome c also suggested that the sarcosomes were damaged. Another indication is found in the high ATP-ase activity of Leptinotarsa sarcosomes, which appeared to be clearly dependent on the way the sarcosomes were treated, less severe treatment resulting in a lower ATP-ase activity. On the other hand, the high P : 0 ratios found and the fact that our preparations did show respiratory control indicate that the sarcosomes were probably less severely damaged than those used by other workers. The main conclusion from the present work is that there seem to be no real qualitative differences between the activities of Leptimtarsa sarcosomes and of mammalian mitochondria. Such differences as are found can be ascribed to a marked difference in stability. From the results obtained with sarcosomes from other insect species it would appear that this is a more general phenomenon, insect sarcosomes probably being much more labile to the relatively harsh treatments applied during isolation. AcJmowledgemwts-The authors express their appreciation to Miss EUEN C. KIMMBL for skilful technical assistance. A grant from the National Council far Agricultural Research (T.N.O.) is gratefully acknowledged. Thanks are due to the members of the staff and personnel of the Laboratory for Virology, Wageningen, for their kind hospitality. REFERENCES B~%XXERT., KLINGENBBRGM., and ZEBE E. (1959) Discussion of Dr. Sacktor’s paper. Proc. 4th itrt. Congr. Bioch. 12, 153-160 (1958). CHANCEB. (1959) Quantitative aspects of the control of oxygen utilixation. Ciba Foundation Symposium on the Regulation ofCell Metabolism, pp. 91-129. Churchill, London. CHANCE B. and SACKTORB. (1958) Respiratory metabolism of insect flight muscle-II. Kinetics of respiratory enzymes in flight muscle sarcosomes. Arch. B&hem. Biophys. 76, 509-531. CHANCE B. and WILLIAMS G. R. (1956) The respiratory chain and oxidative phosphorylation. Advanc. Enzymol. 17, 65-134. CLARKE K. U. and BALDWINR. W. (1960) The effect of insect hormones and of 2: 4-dinitrophenol on the mitochondria of Locusta migratoria L. J. I&. Physiol. 5, 37-46. C=AND K. W. and SLATER E. C. (1953) The effect of tonicity of the medium on the respiratory and phosphorylative activity of heart-muscle sarcosomes. Biochem. J. 53, 557-567. COCHRAN D. G. and KING K. W. (1960) Oxidative phosphorylation in sarcosomes from thoracic muscles of the American cockroach. Biochim. biophys. Acta 37, 562-563. FISKE C. H. and SUBBAROWY. (1925) The calorimetric determination of phosphorus. J. biol. Chem. 66, 375-400. HOLTON F. A. (1955) Spectrophotometric studies of the cytochrome system of heart muscle. Biochem. J. 61,46-61. KLINGENBERG M. and B~SCHERT. (1959) Flugmuskelmitochondrien aus Locusto migrate mit Atmungskontrolle. Biochem. 2. 331, 312-333.
D. %EGWEEANJJ
126
A.R.vAN
KAMM~N-WERTHEIM
LEHNINGERA. L., WAXINS C. L., and REMMERTL. F. (1959) Control points in phosphorylating respiration and the action of a mitochondrial respiration releasing factor. Ciba Foundation Symposium on the Regulation of Cell Metabolism, pp. 130-149. Churchill, London. LOWRY 0. H., ROSBBROUGH N. J., FARR A. L., and RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 1@3,265-275. MARG~LIASHE. (1954) The use of ion-exchangers in the preparation and purification of cytochromes. Biochem. J. 56, 529-535. mG0LIAs.H E. (1957) Cytochrome c (addendum) and reduced cytochrome c. Biochem.
Preparations 5, 33-39. MINNAERTK. and VANKUXMEN-W~RTHEIMA. R. (1960) Measurement of the P : 0 ratio associated with the oxidation of ascorbate by rat-liver mitochondria. B&him.. biophys.
Acta 44; 593-595. NEWBURGHR. W., POTTERL. N., and CHELDELINV. H. (1960) Oxidative phosphorylation in Phormk regina Ianrae. J. Ins. Physiol. 4, 348-349. SACKTOR B. (1959) A biochemical basis of flight muscle activity. Proc. 4th int. Congr. B&hem. 12, 138-152 (1958). SACICTOR B. and Cocmt~~ D. G. (1958) The respiratory metabolism of insectflightmuscle-I. Manometric
studies of oxidation and concomitant
phosphorylation
with sarcosomes.
Arch. Biodzem. Biophys. 74, 266-267. SACKTORB. and PACKER L. (1961) The stimulation of a-glycerophosphate oxidation by adenosine diphosphate in teased flight muscle. Biochim. biophys. Acta 49,402-403. SLA~R E. C. and Lxw~ S. E. (1954) Stimulation of respiration by 2 : 4-dinitrophenol.
B&hem. J. 58, 337-358. VAN DENBZRGHS. and SLATERE. C. (1960) The respiratory activity and respiratory control of sarcosomes isolated from the thoracic muscle of the housefly. &o&m. biophys.
Acta 40,176-177. WILDE J. DE and STEGWEED. (1958) Two major effects of the corpus allatum in the adult Colorado beetle, Arch. n&d. Zool. 13 (Suppl.), 277-289.
Printed in Great Britaitz
RESPIRATORY CHAIN METABOLISM IN THE COLORADO POTATO BEETLE---I RESPI_RATIONAND OXIDATIVE PHOSPHORYLATION IN SARCOSOMES FROM ACTIVE BEETLES D. Laboratory
STEGWEE and
for Entomology,
A. R. VAN KAMMEN-WERTHEIM
Agricultural (Received
University,
26 September
Wageningen,
The
Netherlands
1961)
Abstract-Respiratory chain metabolism was studied with isolated thoracic muscle mitochondria (sarcosomes) of the adult Colorado potato beetle. The composition of the respiratory chain did not d&r estientially f5-om that found in housefly sarcosomes and rat liver mitochondria. Isolated sarcosomes readily oxidized various substrates with concomitant phosphorylation. Respiratory control through the phosphate acceptor level was demonstrated to operate in sarcosomes of the Colorado potato beetle, the housefly, the American cockroach, and the Moroccan locust. Observed differences between insect and mammalian mitochondrial activities are explained by differences in intactness of the mitochondria. INTRODUCTION
DE WILDE and SFGWEE (1958) provided direct evidence of stimulation of succinoxidase activity in the d&pausing Colorado potato beetle, Lq&zo&zr~~ dkcemlineuta Say, by a substance (presumably a hormone) derived from the corpora allata of active beetles. This discovery prompted a more detailed investigation of respiratory chain metabolism in this insect. Mitochondria from the thoracic muscles of insects have proved to be extremely well suited for studying respiration and oxidative phosphorylation. Therefore, these bodies were chosen for the present study. MATERIALS
AND
METHODS
Insect material Active beetles of mixed sexes, between 1 and 2 weeks old, were used in all experiments. The insects were obtained. from laboratory cultures maintained as described previously (DE WILDE and STEGWEE,1958). Houseflies (Musca domestica L.), American cockroaches (Peripluneta amerkana L.), and Moroccan locusts (Low&a migratoria L.) which were occasionally used were also obtained from laboratory stock cultures. Reagents All chemicals used were reagent grade. ADP* and ATP were purchased as the sodium and d&odium salts respectively from Sigma Chemical Co., St. Louis, MO,, * The following abbreviations are used: ADP: adenosine diphosphate; ATP: phate; DNP: 2,4_dinitrophenol; EDTA: ethylene d&nine tetraacetic acid. 8
117
adenosine triphos-
11s
D. STEGWEE AND A. R. VANKAMMEN-WERTHEIM
U.S.A. Hexokinase, type III, and cytochrome c, type II, were also obtained from Sigma. Cytochrome c was further purified according to the method of MARGOLIASH (1954,1957). EDTA, disodium salt,was obtained from E. Merck A.G., Darmstadt, German Federal Republic. Preparation of sarcosomf3 The beetles were immobilized by chilling at 2°C. The heads, elytra, and abdomens were removed, and the isolated thoraces were placed in an ice-cold mortar. After the addition of a small amount of isolation medium (an aqueous solution of 0.32 M sucrose and 0.01 M EDTA, pH 7.4) the thoraces were gently crushed with a pestle. The resulting brei was filtered through four layers of muslin and the residue was washed carefully with small portions of the medium. The combined.filtrate and washings (approximately O-5 ml for each thorax used) were then centrifuged for 4 min at 125 g. The sediment was discarded and the supematant centrifuged for 20 min at 2000 g to sediment the sarcosomes. These were resuspended in 0.32 M sucrose solution, pH 7.4, with the aid of a hand-driven Potter-Elvehjem (Teflon-and-glass) homogenizer. The suspension was again centrifuged at low and high speed as mentioned before. The sedimented sarcosomes were freed of an upper ‘fluffy’ layer and suspended in approximately 1 ml of 0.32 M sucrose solution for each ten thoraces used. All operations were performed in a cold room at 2X. Determination of sarcosomal protein The calorimetric method of LOWRY et aZ. (1951) was used. Preliminary removal of lipids as recommended by CLELAND and SLATER (1953) for mammalian heart muscle sarcosomes proved unnecessary. A standard curve was prepared on the basis of Kjeldahl nitrogen determinations. Estimation of respiratory enqwaes in sarcosomes The relative concentrations of respiratory pigments in the sarcosomes were calculated from difference spectra, obtained with the technique described by HOLTON(1955), using a Beckman DU spectrophotometer with photomultiplier attachment. The calculations were based upon data given by CHANCEand WILLIAMS(1956). Measurement of oxidative phosphorylation Respiration was measured with the conventional Warburg technique. The reaction medium contained 0.012 M phosphate buffer, pH 7.4; 0.008 M KCl; 0.0025 M MgCla; 0.001 M EDTA; O-120/,bovine serum albumin; O*OOlS M ATP; 150 K.M. units of hexokinase; 0.005 M glucose; 0.025 M substrate; l-5-3 mg of sarcosomal protein. Sufficient 0.32 M sucrose solution was added to give a .tinal volume of 2.1 ml. The centre well contained O-1 ml of 20% KOH. The reaction temperature was 25”C, the gas phase air. For the measurement of phosphorylation the reaction was stopped after 15-30 min by adding O-3 ml of 40% trichloroacetic acid. The flasks were immediately chilled in ice and water and 4 ml of ice-cold 5% trichloroacetic acid were added. The flask contents were then centrifuged in
RESPIRATORYCHAIN
METABOLISM
IN THBCOLORADO
119
POTATOBBETLE-I
the cold. Phosphorylation was measured as the disappearance of inorganic phosphate which was determined in the supernatant according to the method of FISKEand SUB~AROW (1925). N o corrections were made for endogenous respiration and phosphorylation. Determination of ATP-ase activity of sarcosomes The sarcosomes (l-3 mg of protein) were incubated for 5 min at 25°C in a medium containing 0*012 M tris (hydroxymethyl) aminomethane buffer, pH 7.4; 0.08 M KCl; 0.001 M EDTA; 0.12% bovine serum albumin; 0.005 M ATP. The volume was made up to 2.1 ml by adding 0.32 M sucrose solution. ATP-ase activity was measured as the release of inorganic phosphate which was determined as described in the preceding section after the reaction was stopped by adding O-3ml of 40% trichloroacetic acid. RESULTS Components of the respiratory chain A suspension of sarcosomes prepared as described above readily oxidized added succinate. The respiratory enzymes involved in this oxidation could be detected spectrophotometrically by measuring the changes in optical density which occurred upon enzymic reduction after the addition of succinate.
? E u
0.06
0.03
0.04
0.02
eo2
0.01 0
0 E
-0.01
i -0.02 a ." 3r -0.04 .z$
-0.02
5 -0.06 'D
-0.03
?j .'L-0.08 z 0 -0.10
-0.04
360
360
500 420 440
460 480 500
520 540
560
580
600 620
Wavelength, mp FIG. 1. Spectrum representingthe differences in absorption between aerobic and anaerobic
sarcosomes of the Colorado potato beetle. The sarcosomes were suspended in O-32 M -sucrose, pH 7.4, to a concentration of 3.8 mg of protein per ml. Anaerobic conditions were obtained by adding 0.01 ml of a saturated sodium succinate solution to 2.5 ml of the suspension in a 1 cm cuvette. Anaerobiosis was considered complete when no further increaseof optical density at 550 mp was observed.
Fig. 1 shows the difference spectrum, reduced vs.. oxidized, of a suspension of sarcosomes. The absorption bands of the different respiratory pigments show very
120
D. STEGWEEANDA. R. VANI(AMMEN-WERTHRIM
clearly the a-bands of cytochrome (a + u3), cytochrome 6, and cytochrome c at 603, 563, and 551 rnp respectively, the #&band of cytochrome c at 520 rnp, the trough due to the disappearance of absorption when flavoprotein becomes reduced at 465 mp, and the y-band of cytochrome a at 444 rnp. The sequence of the optical density changes and the concentrations of the various pigments relative to cytochrome a are given in Table 1. TABLE ~-SEQUENCE OF OPTICAL DENSITY CHANGES RESPIRATORYCHAINCOMPONENTS
AND OFSARCOSOMESOFTHE
RELATIVB CONCENTRATION OF THE COLORADO POTATO BEETLR
Component* Spectral band Optical density relative to cytochrome at
Concentration relative to cytochrome at
Flavoprotein” (465 nw)
2.0 2.9
* CytJ,,
etc., designate the reduced forms of cytochrome a3, etc. t The figures represent average values from three experiments.
Oxidatise phosphorylation Freshly prepared sarcosomes oxidized not only succinate but also cy-ketoglutarate, glutamate, and or-glycerophosphate. They were inactive towards fl-hydroxybutyrate. In the presence of ADP, Mg +-+-ions, and serum albumin the oxidation of the various substrates was accompanied by an appreciable phosphorylation. DNP at lOAM uncoupled this phosphorylation to a large degree, though not completely. In the presence of DNP a slight decrease of oxygen consumption was observed. The values for the respiration rates and phosphorylating efficiencies in the presence of different substrates are summarized in Table 2. No values are given for the rates of endogenous respiration. These were variable but sometimes considerable, approaching the respiration rate with succinate as a substrate. By way of comparison, a few data obtained with sarcosomes of Musca dome&a, Periplaneta americana, and Locusta migratoria have been included. In contrast to these, Leptinotarsa, sarcosomes did not oxidize a-glycerophosphate more rapidly than succinate. The P : 0 ratios found are in agreement with those reported in the literature. It should be noted that cytochrome c was absent from the reaction medium. Addition of cytochrome c increased the respiration rate of Leptinotarsa sarcosomes, however without a corresponding increase of the rate of phosphorylation. The result was a considerable lowering of the P : 0 ratio. This is shown in Table 3.
Respiratory control The presence of the hexokinase/glucose ‘trapping’ system for ATP provided for a continuous supply of ADP in the reaction medium. Without ADP the amount of inorganic phosphate esterified was very small. However, the respiration rate was
RESPIRATORY CHAIN METABOLISM
121
IN THE COLORADO POTATO BEE’IZE-I
not lowered significantly by the omission of ADP. Actually, the amount of ADP per flask could be varied between 0 and 30 PM without affecting the respiration rate (this result was obtained by adding ADP as such and omitting hexokinase TABLE
2-&~SPIRATION RATES AND PHOSPHORYLATING EFFICIENCIES OF s~~Coso~~s OF THE COLORADO POTATO BEETLE AND SOME OTHER INSECTS
Substrate
Insect
P : O$
Succinate cw-Glycerophosphate cw-Ketoglutarate* Glutamate
Z&i 32 (2)
Musca
Succinate cw-Glycerophosphate
46 (1) 194 (1)
Peri&?aneta
Succinate wGlycerophosphate
30 (1) loo (1)
Locusta
Succinate c+Glycerophosphate
Leptinotarsa
38 (13)t
* In the presence of 0.01 M malonate. t Figures in brackets represent the number of experiments. $ fitoms inorgdc phosphate taken up per patom oxygen consumed. TABLE 3--THE
Concentration of cyto;c,;)me c
+ Substrate
O-025 M
EFFECT OF
CYTOCHROME c .UPON OXIDATIVE PHOSPHORYLATION SARCOSOMFS OF THE COLORADO POTATO BEETLE
Phosphate uptake (t.Wmg proteinlhr)
IN
P:O
succinate. The figures represent the average values from two experiments.
and glucose). These findings are in agreement with the reports by SACKTOR and COCHRAN (1958) and CHANGE and SACKTOR (1958) on the housefly and by COCHRKN and WG (1960) on the American cockroach. They would indicate that in Leptimtarsa sarcosomes there is also no respiratory control through the phosphorylating system. The demonstration, however, of the existence of such a respiratory control mechanism in isolated sarcosomes of Locusta migratoria by BGXER et al.
D. STEGWB~ANDA. R. VANKAMMEN-WERTHEIM
122
(1959) and KLINGENBERG and B&HER (1959) made us suspicious as to the validity of this conclusion. Upon closer examination it appeared that when Leptinotursa sarcosomes were pre-incubated with the substrate for about 15 min at 25”C, the subsequent addition of ADP caused a distinct increase of the respiration rate. This led us to re-examine the effect of ADP upon respiration of housefly and American cockroach sarcosomes. Locusta migratoria was also included in our investigations so as to enable a comparison with the work of KLINGENBERG and B~CHER (1959). Table 4 summarizes the results of these experiments. In all cases TABLE ~-RESPIRATORY CO~OL IN SAR~OSOMES OF THE COLORADOPOTATOBEETLE ANDSOMEOTHERINSECTS*
Respir;~;l Insect
1
Substrate
Ql Leptinotarsa
Succinate
u-Glycerophosphate or-Ketoglutaratet
46 (5) 54 (2) 45 (1)
Ih.
Succinate c+Glycerophosphate
46 (1) 194 (1)
Pmiplaneta
Succinate ar-Glycefophosphate
LOCUSta
control
QB
Succinate a+Glycerophosphate Pyruvate + malate$
* Reaction inedium without ATP and hexokinasefglucose. ADP was added from the sidearm to a final concentration of 0902 M. t cz-Ketoglutarate OG?S M + malonate O-01 M. $ Pyruvate O-025 M + malate 0.02 M. $ Average values. The number of experiments is given in brackets. 7 Qlythe ratio of respiration rates after and before addition of ADP. Qs=the ratio of respiration rates after and before addition of ADP and after expenditure of the added ADP.
a distinct respiratory control was observed. Sarcosomes of the Colorado potato beetle showed no decrease of respiration rate after expenditure of the added ADP (Qz = 0). This is probably due to high ATP-ase activity, whereby ADP is regenerated. Measurements of ATP-ase in Leptinotuwa sarcosomes, prepared in the usual way, showed that under the present conditions there was indeed a considerable Mg++- activated ATP-ase activity (Table 5). The addition of DNP at lO+M showed that there is about as much ‘latent’ ATP-ase. In the sarcosomes which had received a somewhat milder treatment (only one cycle of centrifuging, which means only once resuspended in the
RESPIRATORY
CHAIN METABOLISM
IN THE COLORADO POTATO
BEETLE-1
123
homogenizer) the total ATP-ase activity was equal, but much less was ‘apparent’ and much more ‘latent’, i.e. only showing up in the presence of DNP. In view of the reported effect of cytochrome c upon respiration and especially of the lowering of the P : 0 ratio it is worthwhile to note that cytochrome c at 6 x 10GM had no effect whatsoever upon ATP-ase activity. TABLE 5-ATP-ASE
Additions*
None p:Z-+ Vg+++DNP
ACTIVITY OF SARCOSOMES OF THE COLORADO POTATOBEETLE
Inorganicphosphatereleased control(pM/mg protein/hr)t
over
PreparationA
PreparationB
2.34
4.84
2.34 3.21 13.20
5:E 13.20
* MgCls and CaCl, were addedso as to give a final concentration of 2.5 x lo-sM. The final concentration of DNP was lO-“M. t Preparation A: suspension after one cycle of low and high speed centrifuging (sarcosoxnes resuspended once). Preparation B: suspension after two cycles of centrifuging (sarcosomes resuspended twice). DISCUSSION On the whole, the results presented in the preceding sections need little comment. The composition of the respiratory chain in sarcosomes of the adult Colorado beetle is quite comparable to that found in housefly sarcosomes by CHANCE and SACKTOR (1958) and not essentially different from that found, e.g., in rat liver mitochondria by CHANCE and WILLIAMS (1956). The data on oxidative phosphorylation show, in agreement with the Endings of SACKTOR and COCHRAN (1958), COCHRAN and KING (1960), and NEWBURGH et al. (1960) that low P : 0 ratios are not characteristicfor oxidative phosphorylation in insect mitochondria. The lowering of the P : 0 ratio by added cytochrome c contrasts with the report by MINNAERT and VAN KAMMEN-WERTHEIM (1960) on rat liver mitochondria, where no effect of cytochrome c upon the P : 0 could be found : oxygen consumption and phosphorylation were equally stimulated. In the case of L.eptimtarsa sarcosomes the phosphorylation rate was only slightly increased by cytochrome at 10q5M, whereas the respiration rate was almost doubled. The resulting lowering of the P : 0 ratio might be termed ‘uncoupling’, but this seems somewhat confusing especially since we were able to demonstrate that cytochrome c had no effect upon ATP-ase activity. For the present we are inclined to think of stimulation of non-phosphorylating respiration, such as occurs in damaged sarcosomes, by added cytochrome c. Thus this stimulation would be indicative of the still relatively poor condition of our sarcosome preparations.
124
D. STEGWEE ANDA. R. VANKAMMEN-WERTHEIM
True uncoupling was brought about by DNP. The concomitant lowering of the respiration rate agrees with findings by SACKTOR and COCHRAN (1958) for the housefly. CLARKEand BALDWIN(1960) found with sarcosomes from -Locusta mi&-at&a also a depression of respiration by DNP. Sarcosomes from Calliphora erythrocephala, however, showed stimulation of respiration by DNP (SLATERand LEWIS,1954). It seems probable that the effect of DNP upon respiration depends on what is the rate-limitingprocess in oxidative phosphorylation (LEHNINGER et al., 1959). Sarcosomes from different sources might well be different in this respect, thus showing opposite responses to DNP. The relatively slow rate of oxidation of cY-glycerophosphate,or, for that matter, the rapid rate of oxidation of several other tri-carboxylic acid cycle intermediates by Leptinotarsa sarcosomes deserves some comment. CHANCE and SACKTOR (1958) with housefly sarcosomes, and COCHRAN and KING (1960) with American cockroach sarcosomes, found respiration rateswith a:-glycerophosphatewhich exceeded those with succinate 4- to lo-fold. The latterauthorsconcluded that this constitutes a characteristicdifference between insect sarcosomes and mammalianmitochondria. On the other hand, B~~CHJZR et al. (1959) believe that the relatively rapid rate of or-glycerophosphate oxidation is typical for damaged mitochondria. In their sarcosome preparationsof Locusta ma’gratoriathe ratio of the oxidation of or-glycerophosphate and pyruvate + malate was only 2. In the only experiment we did with Locusta this ratio was 2.4. In housefly and American cockroach sarcosomes, however, we found ratios which were quite comparable to those mentioned above, whereas with the Colorado potato beetle the respiration rates with ar-glycerophosphate and succinate were invariably about equal. Therefore, we conclude that the rapid rate of oxidation of or-glycerophosphateis typical, not for insect sarcosomes in general, but for sarcosomes of certain insect species, such as Musca domestica In other species, such as Locusta migratoria, the and Periplaneta adana. relative rate of ar-glycerophosphateoxidation may be slower, whereas in species like Leptinotarsa decemkxzta this does not at all exceed the oxidation rate of other substrates. It seems premature to speculate about the significance of the relatively high rate of endogenous respirationfound with Leptinotarsa sarcosomes. According to B@GHER et al. (1959) Locusta sarcosomes also show a considerable endogenous respiration. Investigations of the nature of this phenomenon are planned. The failure to detect respiratory control through the phosphate acceptor level has led COCHRAN and KING (1960) to assume that phosphorylating respiratory control in isolated insect sarcosomes is ‘loosely coupled’. SACKTOR (1959), for the same reason, even proposed an entirely different mechanism for respiratory control in insect mitochondria. However, as was stated before, KLINGENBERG and B~CHER(1959) clearly demonstrated the occurrence of phosphorylating respiratory control in isolated sarcosomes of Locusta migratoria. VAN DENBERGHand SLATER (1960) obtained respiratory control with isolated housefly sarcosomes under special conditions. Recently SACKTOR and PACER (1961) were able to show that ADP stimulates ar-glycerophosphateoxidation in teased flight muscle.
RESPIRATORY CHAINMETABOLISM IN THE COLORADO POTATOBEETLE-I
125
The present results show that it is possible to obtain sarcosomes with respiratory control not only from the Colorado potato beetle, but also from the housefly and the American cockroach. The values for the respiratory control ratios were low as compared to those found for mammalian mitochondria (CHANCE, 1959). This is probably merely a reflection of the comparatively poor condition of the isolated sarcosomes. As was discussed before, the stimulation of respiration by cytochrome c also suggested that the sarcosomes were damaged. Another indication is found in the high ATP-ase activity of Leptinotarsa sarcosomes, which appeared to be clearly dependent on the way the sarcosomes were treated, less severe treatment resulting in a lower ATP-ase activity. On the other hand, the high P : 0 ratios found and the fact that our preparations did show respiratory control indicate that the sarcosomes were probably less severely damaged than those used by other workers. The main conclusion from the present work is that there seem to be no real qualitative differences between the activities of Leptimtarsa sarcosomes and of mammalian mitochondria. Such differences as are found can be ascribed to a marked difference in stability. From the results obtained with sarcosomes from other insect species it would appear that this is a more general phenomenon, insect sarcosomes probably being much more labile to the relatively harsh treatments applied during isolation. AcJmowledgemwts-The authors express their appreciation to Miss EUEN C. KIMMBL for skilful technical assistance. A grant from the National Council far Agricultural Research (T.N.O.) is gratefully acknowledged. Thanks are due to the members of the staff and personnel of the Laboratory for Virology, Wageningen, for their kind hospitality. REFERENCES B~%XXERT., KLINGENBBRGM., and ZEBE E. (1959) Discussion of Dr. Sacktor’s paper. Proc. 4th itrt. Congr. Bioch. 12, 153-160 (1958). CHANCEB. (1959) Quantitative aspects of the control of oxygen utilixation. Ciba Foundation Symposium on the Regulation ofCell Metabolism, pp. 91-129. Churchill, London. CHANCE B. and SACKTORB. (1958) Respiratory metabolism of insect flight muscle-II. Kinetics of respiratory enzymes in flight muscle sarcosomes. Arch. B&hem. Biophys. 76, 509-531. CHANCE B. and WILLIAMS G. R. (1956) The respiratory chain and oxidative phosphorylation. Advanc. Enzymol. 17, 65-134. CLARKE K. U. and BALDWINR. W. (1960) The effect of insect hormones and of 2: 4-dinitrophenol on the mitochondria of Locusta migratoria L. J. I&. Physiol. 5, 37-46. C=AND K. W. and SLATER E. C. (1953) The effect of tonicity of the medium on the respiratory and phosphorylative activity of heart-muscle sarcosomes. Biochem. J. 53, 557-567. COCHRAN D. G. and KING K. W. (1960) Oxidative phosphorylation in sarcosomes from thoracic muscles of the American cockroach. Biochim. biophys. Acta 37, 562-563. FISKE C. H. and SUBBAROWY. (1925) The calorimetric determination of phosphorus. J. biol. Chem. 66, 375-400. HOLTON F. A. (1955) Spectrophotometric studies of the cytochrome system of heart muscle. Biochem. J. 61,46-61. KLINGENBERG M. and B~SCHERT. (1959) Flugmuskelmitochondrien aus Locusto migrate mit Atmungskontrolle. Biochem. 2. 331, 312-333.
D. %EGWEEANJJ
126
A.R.vAN
KAMM~N-WERTHEIM
LEHNINGERA. L., WAXINS C. L., and REMMERTL. F. (1959) Control points in phosphorylating respiration and the action of a mitochondrial respiration releasing factor. Ciba Foundation Symposium on the Regulation of Cell Metabolism, pp. 130-149. Churchill, London. LOWRY 0. H., ROSBBROUGH N. J., FARR A. L., and RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 1@3,265-275. MARG~LIASHE. (1954) The use of ion-exchangers in the preparation and purification of cytochromes. Biochem. J. 56, 529-535. mG0LIAs.H E. (1957) Cytochrome c (addendum) and reduced cytochrome c. Biochem.
Preparations 5, 33-39. MINNAERTK. and VANKUXMEN-W~RTHEIMA. R. (1960) Measurement of the P : 0 ratio associated with the oxidation of ascorbate by rat-liver mitochondria. B&him.. biophys.
Acta 44; 593-595. NEWBURGHR. W., POTTERL. N., and CHELDELINV. H. (1960) Oxidative phosphorylation in Phormk regina Ianrae. J. Ins. Physiol. 4, 348-349. SACKTOR B. (1959) A biochemical basis of flight muscle activity. Proc. 4th int. Congr. B&hem. 12, 138-152 (1958). SACICTOR B. and Cocmt~~ D. G. (1958) The respiratory metabolism of insectflightmuscle-I. Manometric
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Arch. Biodzem. Biophys. 74, 266-267. SACKTORB. and PACKER L. (1961) The stimulation of a-glycerophosphate oxidation by adenosine diphosphate in teased flight muscle. Biochim. biophys. Acta 49,402-403. SLA~R E. C. and Lxw~ S. E. (1954) Stimulation of respiration by 2 : 4-dinitrophenol.
B&hem. J. 58, 337-358. VAN DENBZRGHS. and SLATERE. C. (1960) The respiratory activity and respiratory control of sarcosomes isolated from the thoracic muscle of the housefly. &o&m. biophys.
Acta 40,176-177. WILDE J. DE and STEGWEED. (1958) Two major effects of the corpus allatum in the adult Colorado beetle, Arch. n&d. Zool. 13 (Suppl.), 277-289.