Carbohydrate metabolism in the flight muscle of the southern armyworm moth, Prodenia eridania

J. Insect Physiol., 1968, Vol. 14, pp. 179 to 198. Pergamon Press. Printed in Great Britain

CARBOHYDRATE METABOLISM IN THE FLIGHT MUSCLE OF THE SOUTHERN ARMYWORM MOTH, PRODENIA ERIDANIA” EDMUND E.

STEVENSON

I. du Pont de Nemours & Co., Central Research Department, Wilmington, Delaware 19898 (Received 1 September 1967)

Abstract-Flight muscle mitochondria isolated from the southern armyworm moth, Prodeniu eriohiu (Cramer), can oxidize pyruvate + malate very rapidly with high P : 0 ratios and good respiratory control by ADP. There is a requirement for Pi, but not for any added cofactor except ADP. or-Glycerol phosphate, acetyl camitine, various citric acid cycle intermediates, and amino acids are oxidized more slowly than pyruvate + malate. A highly active acetyl CoAcamitine transferase is present in these mitochondria, but acetyl CoA synthetase is absent. Homogenates of Prod&a flight muscle can completely and rapidly oxidize glucose, trehalose, glycogen, and phosphorylated hexoses. The enzyme trehalase appears to be localized mainly in particles lighter than mitochondria; its activity can be enhanced some fifteenfold by repeated freezing and thawing and by sonication. The glycogen reserves of newly emerged moths are too small to sustain flight for a significant period of time. The r61e of carbohydrate metabolism in the flight muscle of P. eridaniu, an insect that oxidizes fat for flight energy, is probably to convert ingested carbohydrate to fat for storage. INTRODUCTION

MEMBERSof the order Lepidoptera oxidize only fat during flight (KOZHANTSCHIKOV, 1938; ZEBE, 1954; DOMROESEand GILBERT, 1964) even though carbohydrate in the form of nectar is the food for those adults that can feed. To understand why carbohydrate is not directly utilized it is necessary to know whether the flight muscle of these insects lacks or is deficient in one of the enzymes for glycolysis. DOMROESE and GILBERT (1964) reported that preparations of the flight muscle of Hyalophora cemopiu cannot oxidize carbohydrate at significant rates. MICHEJDA(1964), using a variety of techniques, found that flight muscle mitochondria from H. cecropiu oxidize pyruvate + malate and pyruvate + fumarate rather slowly (so, = 15 100 ~1 O,/mg protein per hr). These reports suggest that the muscle lacks the ability to metabolize carbohydrates. However, I have found that pyruvate + malate can be as rapidly oxidized by the flight muscle mitochondria of the southern armyworm moth, Prodenia eridunia, as by sarcosomes of flies * Contribution No. 1371. 179

180

EDMUND STEVENSON

(PO, r 700 ~1 O,/mg protein per hr ; VAN DEN BERCH and SLATER, 1962; VAN DEN BERGH, 1964; CHILDRESS and SACKTOR, 1966). Various carbohydrates can be completely oxidized by homogenates of the flight muscle of this moth. These findings are described in this paper. MATERIALS

AND METHODS

Animals Armyworm larvae were reared on lima bean foliage and on a modification of the artificial diet of SHOREY (1963). Moths had access to water and were used within a week of emergence. Experimental procedure The procedure for dissecting and homogenizing the flight muscle has been described (STEVENSON, 1966). The h omogenizing media used here are 0.25 M sucrose-5 mM EDTA,* pH 7.0, and 0.154 M KCI-5 mM EDTA, pH 7.0. Mitochondria were isolated as previously described (STEVENSON, 1966). For studies on carbohydrate oxidation the 120 g supernatant fluid, which contains mitochondria, was used. 0% uptake was measured manometrically at 30°C with the mitochondria or supernatant fluid being added from the side-arm after thermal equilibration. Rates of oxidation were computed from the period during which 0, uptake was linear with time. When pyruvate was a substrate, solutions of the sodium salt were prepared just before use since parapyruvate, which inhibits ar-ketoglutarate oxidation (MONTGOMERY and WEBB, 1956), gradually f ormed even in frozen solutions. Pi was determined by the method of SUMNER (1944), and P : 0 ratios were calculated from the differences in Pi between experimental and substrate-free flasks. Assay methods method of Reduced CoA (HSCoA) was assayed by the phosphotransacetylase MICHAL and BERGMEYER(1965), and NADH was assayed enzymatically with malic dehydrogenase. Trehalase activity was measured as the glucose formed from trehalose, the glucose being determined by the glucose oxidase method. Glycogen was isolated from moths within 12 hr after eclosion by extraction with hot 30% KOH and two precipitations with 66% ethanol. Na,S04 was added to ensure quantitative precipitation (VAN HANDEL, 1965). The collected glycogen was determined by the anthrone method (HASSID and ABRAHAM, 1957) with glucose as the standard. Separate experiments established that glycogen recovery was quantitative. Protein was measured by the biuret method (LAYNE, 1957) after precipitation with trichloroacetic acid, with crystalline BSA being used as the * Abbreviations used in this article are: ADP, adenosine diphosphate; ATP, adenosine triphosphate; ATPase, adenosine triphosphatase; BSA, bovine serum albumin; DNP, dinitrophenol; EDTA, ethylenediamine-tetraacetic acid; HSCoA, reduced coenzyme A; NAD, nicotine adenine dinucleotide; NADH, reduced nicotine adenine dinucleotide; NADP, nicotine adenine dinucleotide phosphate; Pi, inorganic phosphate; R.C.I., respiratory control index; R.Q., respiratory quotient; TEA, triethanolamine hydrochloride; Tris, tris(hydroxymethyl)aminomethane.

CARBOHYDRATE

METABOLISM

IN MOTH MUSCLE

181

standard. Because a fine precipitate slowly formed, the insect protein samples were permitted to stand for 12 hr in the biuret reagent and were then centrifuged before the optical densities were determined. ATPase was measured as the Pi released from added ATP in 20 min incubation. Additional tubes were included and incubated for longer times to assure that Pi release was linear with time. The ATPase assay medium was the same as the standard incubation mixture (Fig. 1) except that Pi and substrate were omitted and ATP (2 mM) was included. Source of chemicals Sodium pyruvate, L-malic acid, and TEA were obtained from Calbiochem. Acetyl-DL-carnitine hydrochloride was prepared by the method of FRAENKELand FRIEDMAN(1957) as modified by BREMER(1962) and recrystallized from n-butanol ; it melted at 181.5 to 182*5”C. Crude hexokinase was a product of Nutritional Biochemicals Corporation, and crystalline hexokinase was purchased from Sigma Chemical Co. and from Boehringer-Mannheim. All other chemicals were obtained from Sigma Chemical Co. (NHJ,SO, was removed from crystalline hexokinase either by passage of a diluted solution through Bio-Gel P-30 or by centrifuging the suspension to collect the crystals, dissolving the pellet in 1% glucose-5 mM EDTA, pH 7.4, and then dialysing this solution overnight against several changes of glucose-EDTA. Hexokinase activity was determined by the method of DARROWand COLOWICK (1962). RESULTS Oxidation of pyruvate + malate

Mitochondria isolated from moth flight muscle rapidly oxidize pyruvate with good P : 0 ratios provided L-malate is present (Fig. 1, Table 5). There is an absolute requirement for Pi (Table 1) and 0, uptake ceases when Pi has been exhausted. Endogenous substrate is apparently removed from the mitochondria during isolation, for 0, uptake in the absence of added substrate is insignificant (Table 5). Added NAD (1 mM), NADP (1 mM), cytochrome c (O-01 mM), BSA (O*39o/o), and DL-carnitine (2 or 20 mM) have no effect on 0, uptake. HSCoA (0.35 mM) causes a decrease in Qo, in the absence but not in the presence of BSA (Table 2). 0% uptake is greatly reduced if ADP (Table 3) or MgCl, (Table 1) is omitted. Respiratory Control Indices (R.C.I.; Qo, in presence of ADP divided by Qo, in absence of ADP) were mostly in the range of 12 to 20. Thus, respiratory control by ADP is excellent. Because of the presence of an ATPase, respiratory control could not be demonstrated by the cessation of O2 uptake after a limited amount of ADP had been phosphorylated in the absence of a high-energy trap, as recommended by CHANCE and BALTSCHEFFSKY (1958). This ATPase activity is about 6 pmoles Pi/mg mitochondrial protein per hr and is increased some 50 per cent by 6 x 1O-5 M DNP. Most rapid O2 uptake rates are found if a high-energy phosphate trap is present (Table 1). Hexokinase is most commonly available commercially as a

182

EDMUNDSTEVENSON

INClJBATiCN

TIME,

men

FIG. 1. Oxidation of pyruvate malate and pyruvate + malate by Prodenia flight muscle mitochondria. 0, uptake was measured manometrically at 30°C with shaking (160 oscillations/mm) in an incubation mixture containing TEA, pH 7-O (50 pmoles); KC1 (9.8 poles); MgCla (4.9 pmoles); Pi, pH 7.0 (15 pmoles); glucose (25 pmoles); ADP (1.3 pmoles); dialysed hexokinase (1.28 units; cf. DARROW and COLOWICK,1962); sucrose (B/~moles); EDTA (1.16 pmoles); L-malate (1.3 pmoles); pyruvate (3.25 pmoles); and mitochondria (O-164 mg protein) in a final volume of 0.65 ml. The incubation was started by addition of mitochondria suspension (0.15 ml) from the side-arm after thermal equilibration. CO% was trapped on KOH-soaked paper in the centre well.

crystalline suspension in (NH&SO,. Because (NH&SO, can inhibit pyruvate + malate oxidation (Fig. 2), best results are obtained with dialysed hexokinase. DNP uncouples oxidative phosphorylation but does not enhance the rate of 0, uptake unless a suboptimal concentration of ADP is present (Fig. 3). Fluoroacetate (5 mM) or fluoropyruvate (5 mM) abolishes the oxidation of pyruvate (5 mM) + malate (2 mM). CHILDRESS and SACKTOR (1966) h ave reported that some old samples of Tris strongly inhibited oxidation of pyruvate by blowfly flight muscle mitochondria.

183

CARBOHYDRATE METABOLISMIN MOTHMUSCLE TABLE ~-EFFECT

OF OMISSIONOF INORGANICPHOSPHATE(Pi) ANDHIGH-ENBRGYPHOSPHATE TRAP ON OXIDATIONOF PYFWJ’ATE+ MALATE

Experiment

Qo¶ &l Oa/mg protein per hr)

Omission -

425 32 8 625 116

MgCIa Pi Glucose + hexokinase

Conditions as described in Fig. 1, except that ADP concentration chondria protein: O-228 mg (Expt. l), 0.073 mg (Expt. 2). TABLE ~-EFFECT

Experiment 1

2

Substrate @moles/ml)

Addition (~moles/ml)

Pyruvate (5) + malate (2) Pyruvate (5) + malate (2) Pyruvate (5) + malate (2) Pyruvate (5) + nL-z-Glycerol nL-wGlycero1 nL-a-Glycerol

malate (2) phosphate phosphate phosphate

Qoa

@l O,/mg protein per hr)

HSCoA HSCoA BSA BSA (4

(0.35) (0.35) + (4 mg/ml) mg/ml) -

HSCoA HSCoA BSA BSA (4

(0.35) (0.35) + (4 mg/ml) mg/ml)

378 222 359 384 350 244 359 390

procedure as described in Fig. 1.

TABLE ~-RESPIRATORY CONTROLOF PYRUVATE+

&I O,/mg %ein

Incubation

Mito-

OF SULPHYDRYLCOMPOUNDSON THE OXIDATIONOF VARIOUSSUBSTRATES

nL-or-Glycerol phosphate Incubation

was 0.5 mM.

MALATE

OXIDATION

per hr)

Experiment

+ADP

-ADP

R.C.I.

1 2 3 4 5 6 7

649 745 660 635 660 732 668

101 22 44 31 46 62 38

6.4 33.9 15.0 20.5 14.4 11.8 17.6

procedure described in Fig. 1.

BY ADP

184

EDMUND

STEVENSON

TABLE~-EFFECT OF VARIOUS BUFFERS ON OXIDATION OF PYRUVATE FLIGHT MUSCLE

+ MALATE

Qos

Buffer (r*moles/ml)

BY Prod&

MITOCHONDRIA

P:O

(~1 Ol/mg protein per hr)

Phosphate (23) Phosphate (23) + TEA (77) Phosphate (23) + Trizma (77) Phosphate (23) + Tris (77)

578 533 540

2.0 2.0 1.9

436

2.0

All buffers were at pH 7. Triethanolamine hydrochloride was purchased from Calbiochem. Trizma was a preparation of tris(hydroxymethyl)aminomethane recently obtained from the Sigma Chemical Co. Tris is an old preparation of tris(hydroxymethyl)aminomethane from Commercial Solvents Corp., purchased in February 1957. Oa uptake was measured as described in Fig. 1.

01 0

I

I

I

5 IO 15 AMMONIUM SULFATE

I

1

I

20 25 30 CONCENTRATION, mM

1

35

FIG. 2. Effect of ammonium sulphate on oxidation of pyruvate + malate by Prodenia flight muscle mitochondria. Incubation procedure as described in Fig. 1. Mitochondria protein used, 0.148 mg.

The moth mitochondria are likewise adversely affected (although to a lesser extent) by an old sample of this buffer, but not at all by a new preparation (Table 4).

Results with various isolation media VAN DEN BERGHand SLATER(1962) reported that housefly flight muscle mitochondria isolated in 0.154 M KCl-1 mM EDTA, pH 7.4, oxidized pyruvate more rapidly than particles isolated in 0.25 M sucrose-l mM EDTA, pH 7.4. However, moth mitochondria isolated in 0.154 M KCl-5 mM EDTA, pH 7.0, oxidized the substrates tested somewhat more slowly than mitochondria isolated in the standard

CARBOHYDRATE

METABOLISM

185

IN MOTH MUSCLE

600

2.4 2.0

1.6

O

5 RINITROPHENOL

IO CONC., M x IO-5

0 F a u 0

15

FIG. 3. Effect of dinitrophenol on Qo, and P : 0 ratio for pyruvate + malate oxidation at different ADP concentrations. Conditions as described in Fig. 1. Qol (ADP = 2 mM); -A-, -0-, Qor (ADP = O-5 mM); --O-, P:O(ADP=2mM);--A--,P:O(ADP=O*SmM). medium.

Many of the particles isolated in the saline medium are more severely damaged than are those isolated in 0.25 M sucrose-5 mM EDTA (Figs. 4 and 5). Mitochondria isolated in 0.25 M sucrose, in 0.25 M sucrose-5 mM TEA, pH 7.0, or in 0.33 M sucrose-5 mM EDTA, pH 7.0, oxidized pyruvate more slowly than did particles isolated in the standard medium. Also, poor 0, uptake resulted if sufficient sucrose was added to the flask to maintain the concentration at 0.25 M during the incubation. Oxidation of other substrates

Oxidation of oxalacetate is fairly rapid (Table 5) and requires the same cofactors as pyruvate + malate oxidation. Other members of the citric acid cycle are oxidized more slowly (Table 5). or-Glycerol phosphate is fairly rapidly oxidized (Table 5), but respiratory control by ADP is poor (R.C.I. = 1.2-1.8). Oxidation of ol-glycerol phosphate is unaffected by varying ADP concentration between O-5 and 2 mM, but is suppressed by HSCoA in the absence of BSA (Table 2). Acetyl CoA-carnitine

transferase

An acetyl CoA-carnitine transferase, catalyzing the reaction acetyl CoA + carnitine + acetyl carnitine + HSCoA, is present in the mitochondria in high activity (Table 6). The enzyme requires

EDMUNDSTEVENSON

186

TABLE ~-OXIDATION OF VARIOUSSUBSTRATESBY Prodenia (~1 OJmg Ezein Substrate (~molesjml) Pyruvate (5) + L-malate (2) Pyruvate (5) + L-proline (10) Oxalacetate (6.5) ol-Glycerol phosphate (38.5) Succinate (25) L-Glutamate (20) + L-malate (20) L-Glutamate (20) c+Ketoglutarate (20) + L-malate (20) a-Ketoglutarate (25) L-Proline (10) Citrate (25) Pyruvate (2.5) L-Malate (25) Fumarate (2) Acetyl-nL-carnitine (10) + L-malate (2) Acetyl phosphate (5) + L-malate (2) nL-j%Hydroxybutyrate (20) nL-/%Hydroxybutyrate (20) + L-malate (2) NADH (22) None

FLIGHT MUSCLEMITOCHONDRIA

per hr)

P:O

Mean

Range

Mean

Range

661 448 424 375 146 180 143 94 45 85 48 14 41 8 330 9 0 33 29 12

398-877 435-468 385-516 311-470 144-147 151-231 115-176 91-96 33-55 84-86 32-66 9-18 o-75 -

2.4 -

2.2-2.6

2.5 1.2 I.3 2.2 2.3 1.3 1.0

2.3-2.6 1~1-1~5 1.0-l .6 2.1-2.3 2.0-2.7 -

307-362 O-36 -

1.9

-

16-46 25-31 8-14

-

-

1.8 -

-

0.8-1.1

-

-

Mitochondria were isolated in sucrose-EDTA. Incubation procedure is described in Fig. 1. ADP concentration was 2 mM when pyruvate I- malate was the substrate couple, and 0.5 mM in all other cases. TABLE 6-ACET~

COA-CARNITINETRANSFERASE IN Prodenia FLIGHT MUSCLEMITOCHOP\‘DRIA

Addition BSA (3.85 mg/ml) L-Cysteine (0.5 ~moles/ml) L-Cysteine (0.5 pmoles/ml) -

Omission HSCoA NaF MgCI, Substrate

Specific activity (pmoles acethydroxamic acid formed/mg protein per hr) 45.2 43.5 44.0 0 46.9 47.5 0

Complete system consisted of TEA buffer, pH 7.0 (50 pmoles); KC1 (9.8 pmoles); MgCl, (49 pmoles); NHaOH, pH 7-O (400 pmoles); NaF (25 pmoles); HSCoA protein (3.5 pmoles); acetyl-DL-Canine (20 /JmOleS); and O-057 mg mitochondrial (sonicated 20 set at 20 kc/s) in a total volume of I.0 ml. Incubation was for 30 min at 30°C with shaking and was stopped by the addition of I.5 ml 4.7% HClO,-3.3% FeCl,0.067 M HCI. Denatured protein was removed by centrifugation and the optical density of supematant fluids measured at 520 rnp, with succinhydroxamic acid as standard (cf. STADTMAN,1957).

FIG. 4. Prodenia flight muscle isolated in 0'154 M KC1-5 m M EDTA, pH 7"0. Pellet of washed mitochondria was fixed in 2°,'0 gluteraldehyde-l°/o OsO4, embedded in EPON 812 and doubly stained with lead citrate-uranyl acetate.

FIG. 5. Prodenia flight muscle mitochondria isolated in 0"25 M sucrose-5 m M E D T A , p H 7"0. Pellet of washed mitochondria was fixed in 2% gluteraldehyde1% OsO, in Millonig's buffer, embedded in E P O N 812 and doubly stained with lead citrate-uranyl acetate.

187

CARBOHYDRATE METABOLISM IN MOTHMUSCLE

neither Mg2+ nor cysteine for maximal activity, but has an absolute requirement for HSCoA. The K, for acetyl-DL-carnitine is 4 x 1O-3 M and is presumably 2 x 10~~ M for the L-isomer. The K, for HSCoA, 3.7 x 10e4 M, is not altered by the presence of cysteine. Maximal transferase activity is found if the mitochondria are sonicated for 30 set at 3.2 A and 20 kc/s (Branson Sonifier, Model S-110). This enzyme has also been found in the flight muscle mitochondria of blowflies (CHILDRESSet al., 1967) and locusts, but not bees (BEENAKKERS and KLINGENBERG, 1964).

Oxidative decarboxylation of pyruvate Pyruvate is oxidized only in the presence of an acetyl acceptor. When the citric acid cycle is operating, the acceptor is oxalacetate, and added carnitine has no effect on the rate of oxidation. However, if the citric acid cycle is blocked (by omission of malate or by the addition of fluoroacetate), carnitine will accept acetyl groups and permit O2 uptake (Table 7). Because of the presence of acetyl CoA-carnitine transferase, acetyl carnitine can be oxidized if malate is present, and acetyl CoA can be rather slowly oxidized if both malate and carnitine are present (Table 8). The 0, uptake rate is about TABLE ~-OXIDATION OF PYRUVATEIN PRESENCEOF CAFWITINEBUT IN ABSENCEOF FUNCTIONING CITRICACIDCYCLE Addition (~moles/ml)

DL-Carnitine (20) DL-Carnitine (20) Fluoroacetate (5) Fluoroacetate (5) -IDL-carnitine (20)

Omission

QOZ

(~1 O.Jmg protein per hr)

Malate Malate -

863 0 817 106 44 161

Incubation conditions as described in Fig. 1, except that pyruvate concentration was 10 mM. Mitochondria protein used, 0.102 mg. TABLE S-OXIDATION OF ACETYLCOENZYMEA BY Prodenia FLIGHTMUSCLEMITOCHONDRIA Substrate (pmoles/ml)

Addition (pmoles/ml)

Acetyl CoA (5) +malate (2) Acetyl CoA (5) + malate (2) DL-Carnitine (2) Acetyl-DL-camitine (10) +malate (2) -

!&p, @l Oa/mg protein per hr) 38 ~126 378

Incubation procedure as described in Fig. 1, except that BSA (3.85 mg/ml) was present. Mitochondria protein used, O-233 mg.

188

EDMUND STEVENSON

half-maximal at an acetyl-DL-carnitine concentration of 5 x lOA M, which agrees well with the K, of the transferase for this substrate (vide ~upra). HSCoA (O-35 mM) and mercaptoethanol (0.5 mM) lower the rate of oxidation of acetyl carnitine + malate to that of malate alone, and added carnitine (2 mM racemic mixture) does not reverse this effect. Attempts to demonstrate presence of acetyl CoA synthetase

Acetyl CoA synthetase activity could not be demonstrated in either mitochondria or whole homogenates of flight muscle. In attempts to measure this enzyme, a basic assay mixture was used consisting of 50 mM TEA buffer, pH 7.0; 4.9 mM MgCl,; 20 mM acetate; 400 mM NHsOH, pH 7.0; 3.5 mM HSCoA; 10 mM ATP; sonicated homogenate or mitochondria (0.06-1.3 mg protein). Control tubes lacked acetate or ATP. At the end of the incubation (1 hr at 30°C) the acethydroxamic acid that formed was assayed by the method of KORNBERG and PRICER (1953). The limit of uncertainty of the method was N 10 mpmoles/mg protein per hr. Variations in the incubation mixture involved omitting MgCls; adding 10 mM NaF; adding 10 mM NaF + 10 mM cysteine; adding cysteine and omitting HSCoA; and varying the HSCoA concentration from 1.4 to 7.0 mM. TABLE g--OXIDATION EDTA

HOMOGENATE

Experiment 1

OF CARBOHYDRATESBY OF

Prodeniu

FLIGHT

Substrate (~moles/ml) Glucose-6-phosphate (5) + fumarate (2) Fructose-1,6-diphosphate + fumarate (2) Fumarate (2)

(5)

2

Glucose (10) + fumarate (2) Glycogen (2%) + fumarate (2) Fumarate (2)

3

Glucose-6-phosphate + fumarate (2) Glucose-6-phosphate + fumarate (2) Fumarate (2) Fumarate (2)

(5)

120 g

MUSCLE

SUPERNATANT

AND

ITS

FRACTION

INHIBITION

BY

OF

SUCROSE-

FLUOROPYRWATR

Qot

Inhibitor (pmoles/ml)

(~1 Oz/mg total protein per hr)

-

180

-

173 50

-

174 154 54

-

162

(5) Fluoropyruvate (5) Fluoropyruvate (5)

14 69 5

Incubation mixture contained 50 pmoles TEA buffer, pH 7.0; 9.8 pmoles KCl; 4.9 pmoles MgCI,; 15 pmoles potassium phosphate, pH 7.0; 1 pmole ADP; 1.0 pmole ATP, 0.65 pmole NAD, 57.6 pmoles sucrose, 0.75 pmole EDTA, and substrates as indicated in a final volume of 0.65 ml. The protein in the 120 g supernatant fraction was 0.41 mg (Expt. l), 0.31 mg (Expt. 2) and 0.10 mg (Expt. 3).

CARBOHYDRATE METABOLISM

IN MOTH MUSCLE

189

Oxidation of carbohydrates

The 120g supernatant fluid of flight muscle homogenates can oxidize phosphorylated hexoses, glucose, and glycogen (Table 9). Prevention of glucose6-phosphate oxidation by the addition of fluoropyruvate demonstrates that these carbohydrates can be completely oxidized to CO, + H,O via the citric acid cycle. Trehalose is oxidized only slowly by homogenates prepared in sucrose-EDTA because the sucrose inhibits trehalase. However, trehalose can be oxidized by 120g supernatant fluid prepared in KCl-EDTA (Table 10). TABLE

lo-OXIDATION

OF CARBOHMRATES

BY 120 g SUPERNATANT FLUID OF

MUSCLE HOMOGENIZED

IN

Substrate (~1 O,/mg

Ij.kmoles/ml)

Prodmziu

FLIGHT

KCl-EDTA

Qoa

protein

per hr) -

25

Malate

(2)

Malate

(2) + pyruvate

Malate

(2) + glucose

Malate

(2) + trehalose

78

(5)

101

(10)

85

(10)

The

incubation

mixture

9, except that sucrose was absent and Homogenate protein was 0.33 mg.

is as described

KC1 concentration was 50.7 mM.

in Table

Trehalase Prodenia flight muscle contains a trehalase with a pH optimum at 6.5 and a I&, for trehalose of 3.6 x 10~~ M. Presence or absence of phosphate has no effect on activity, indicating that trehalose is cleaved hydrolytically and not phosphorolytically. If flight muscle is gently homogenized in KCl-EDTA, the specific activity in the whole homogenate is 0.20 to 0.35 pmoles trehalose/mg protein per hr. When the homogenate is fractionated by differential centrifugation, most of the activity is found in the fraction sedimenting between 5000 g and 30,000 g (Table 11). TABLE

1 ~-LOCALIZATION

OF TREHALASE BY DIFFERENTIAL

CENTRIFUGATION

OF

Prodeniu

FLIGHT MUSCLE HOMOGENATE Fraction

120 g pellet 120-5000 g pellet SOOO-30,000 g pellet 30,000 g supematant fluid

Total

activity

0.92 0.45 1.24 0.87

Specific

activity

0.09 0.21 6.40 0.39

Flight muscle was gently homogenized in 0.154 M KCl-5 mM EDTA, pH 7.0, and separated into indicated fractions. Each fraction was sonicated 25 set to disrupt particles. Incubation mixtures contained 48 ,umoles phosphate buffer, pH 7.0, and 12 pmoles trehalase in a final volume of 0.6 ml. After 30 min with shaking at 3O”C, the tubes were heated to stop the reaction and glucose determined on an aliquot of the deproteinized incubation mixture by the glucose oxidase method. Activity is expressed as pmoles trehalose hydrolysed/mg protein per hr.

EDMUNDSTEVENSON

190

TABLE 12-INCREASE IN TREHAL~SEACTIVITY OF TBBLL~LASB-RICH FRACTIONBY SONICATION ANDBY DECXYCHOLATB TREATMENT

Experiment

Treatment

Trehalase specific activity &moles trehalose/mg protein per hr)

-

1

Sonication, Sonication, Sonication,

4.2 9.6 16.4 17.1 3.3 25.2

1 min 3 min 5 min

-

Deoxycholate

Flight muscle was homogenized in 0.154 M KCI-5 mm EDTA, pH 7.0, and the material sedimenting between 1475 g and 37,000 g used. In Expt. 1 the particles were suspended in KCI-EDTA and sonicated in a Branson Sonifier Model S-110 at 3 A (15 set bursts with cooling between bursts). In Expt. 2, the particles were suspended in KCI-EDTA and an aliquot exposed to a final concentration of 0.5% sodium deoxycholate, pH 7.3. In both experiments, trehalase activity was determined as described in Table 11. TABLE

13--INCREASE

IN FLIGHT MUSCLE HOMOGENATETBBHALASEACTIVITY BY FBBBZING AND THAWING

No. of Freeze-thaw cycles

Specific activity &moles trehalose hydrolysed/mg protein per hr)

0 2 5 8 11 14 17

0.24 0.99 2.11 2.80 2.98 3.25 3.09

Flight muscle was rinsed in cold distilled water, blotted, then homogenized in cold distilled water with an all-glass Dual1 homogenizer. Aliquots of the homogenate were frozen in dry ice and thawed in a 30°C bath. Incubation procedure and subsequent glucose determination are described in Table Il. Each tube contained 0.131 mg homogenate protein. Mitochondria are in the pellet sedimenting between 120 and 1475 g, and the 1475 g supernatant fluid is devoid of the ability to oxidize pyruvate + malate. The activity of the trehalase-rich fraction is enhanced by sonication and deoxycholate treatment (Table 12). After sonication of these particles enzymatic activity is found in the 105,OOOg supernatant fluid and in the fraction sedimenting between 37,000 and 105,OOOg, with only a small amount remaining in the 37,000g pellet. When whole homogenates of flight muscle from males were subjected to repeated freezing and thawing in distilled water, the specific activities increased

CARBOHYDRATE METABOLISM IN MOTHMUSCLE

191

about fifteenfold (Table 13). Sonication (Branson Sonifier, Model S-110, 20 kc/s, 8 A) had the same effect. If the homogenates were frozen and thawed in 120 mM phosphate buffer, pH 7.0, or sonicated in KCl-EDTA, the activity increase was less. Glycogen content of moths

The glycogen contents of newly emerged male (abdomens and thoraces) and female (thoraces) moths are listed in Table 14. Determination of the glycogen in female abdomens was not attempted because the developing eggs would be likely to contain much glycogen not available for flight. There was very little anthronepositive material in the thoraces that was not precipitable by ethanol, indicating that the amount of circulating carbohydrate (probably trehalose, ZEBE, 1958) is small compared to the amount of glycogen. TABLE 14-GLYCOGEN CONTENTOF Prodenia THORACES ANDABDOMENS Mean weights Sex Male Female

No. of moths mg/moth 6 6

108 174

Glycogen

mg/thorax

mg/abdomen

pglthorax

pg/abdomen

pg/moth

32 40

72 -

111 165

634 --

745 -

Moths that had emerged the previous night were used. Extractions and determination procedures are described under Materials and Methods. Above values are as glycogen, not hexose. DISCUSSION

Flight muscle mitochondria from Prodeniu eridunia are large particles, morphologically similar to those from fly flight muscle. When isolated in 0.25 M sucrose5 mM EDTA, pH 7.0, these moth mitochondria are slightly damaged. The cristae are swollen in some areas, and on some particles there is a ‘bleb’ formation where apparently the outer membrane has separated (Fig. 5). However, the inner membrane and matrix are intact, for the electron-dense material has a sharp boundary. The mitochondria isolated in 0.154 M KCl-5 mM EDTA, pH 7.0, are more heterogenous (Fig. 4), some of the particles being seriously damaged and others closely resembling those seen in in situ sections of insect flight muscle. VAN DEN BERGH and SLATER (1962) h ave found housefly flight muscle mitochondria isolated in O-154 M KCl-1 mM EDTA, pH 7.4, to have rates of oxidation of pyruvate + malate and P : 0 ratios similar to those reported here, whereas the fly mitochondria they isolated in 0.25 M sucrose-l mM EDTA, pH 7.4, oxidize pyruvate + malate more slowly. Moth flight muscle mitochondria isolated in sucrose-EDTA can oxidize palmitate + malate (STEVENSON, unpublished results) and pyruvate + malate more rapidly than those isolated in the saline medium. The sucrose-EDTA

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particles also have retained their metabolic properties intact as indicated by very rapid oxidation of pyruvate + malate with good P : 0 ratios and excellent respiratory control, all without the need for added co-factors except ADP, and by the failure to oxidize exogenous NADH. In fact, these moth mitochondria can oxidize pyruvate -I- malate as rapidly as can fly sarcosomes (VAN DEN BERGHand SLATER,1962; VAN DEN BERGH, 1964; CHILDRESSand SACKTOR,1966). MICHEJDA (1964), using a variety of procedures, was unable to demonstrate rapid oxidation of pyruvate + malate by flight muscle of the silkmoth, Hyalophora cecropia. Although the P : 0 ratios reported here fall short of those obtained with carefully isolated mitochondria from mammalian tissues, they are similar to the values found by other investigators working with insect flight muscle mitochondria (GREGG et al., 1960; VAN DEN BERGHand SLATER,1962; COCHRAN,1963; VAN DEN BERGH, 1964; CHILDRESSand SACKTOR,1966). However, not all these workers have demonstrated rapid oxidation of pyruvate -I- malate. The R.C.I. vary greatly (Table 2) ; the reason may be that it is difficult to measure accurately and simultaneously two values that differ greatly. The data on Table 2 show that the (- ADP) va 1ues are subject to greater percentage variation than are the (+ ADP) values ; this is to be expected since the incubation mixtures lacking phosphate acceptor took up volumes of 0, (5-10 ~1) too small to be measured manometrically with precision. However, these indices are so large that it is obvious that respiratory control in moth flight muscle can be achieved through ADP availability, Pyruvate oxidation is not enhanced by 3 x 10m5M dinitrophenol unless the ADP level is suboptimal. 0, uptake when the substrate is palmitate + pyruvate + malate is only slightly faster than when the substrate is pyruvate + malate or palmitate + malate (STEVENSON,unpublished results). Thus, it appears that the citric acid cycle with its electron transport chain is operating at capacity when oxidizing either substrate couple. The relatively low rates of oxidation of most of the added citric cycle intermediates may be due to impermeability of the mitochondrial membrane to these substrates. VAN DEN BERGHand SLATER(1962) have shown housefly mitochondria to possess such a barrier. It seems likely that oxidation of oxalacelate and of pyruvate + malate are in reality the same process. This is possible if most of the added oxalacelate is decarboxylated to pyruvate, which is then oxidized in the usual way. Other workers have suggested that proline may be a primary substrate for flight in the tsetse fly (BURSELL, 1963) and in locusts (BROSEMERand VEERABHADRAPPA, 1965). SACKTORand WORMSER-SHAVIT(1966) have hypothesized that proline plays a key role in supplying citric acid ‘sparker’ in the first minutes of flight in Phormia regina. The present data show that in moth mitochondria proline can substitute for malate as a ‘sparker’ (as can fumarate, glutamate, and a-ketoglutarate), but they do not permit the conclusion that proline is a primary substrate or that it occupies a special place in flight metabolism. These moth flight muscle mitochondria do not oxidize cu-glycerol phosphate as

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rapidly as pyruvate + malate. This contrasts with the results other workers have obtained with fly flight muscle mitochondria (VAN DEN BERGHand SLATER, 1962; VAN DEN BERGH, 1964; CHILDRESS and SACKTOR, 1966). Even so, if the role of a-glycerol phosphate is to carry into the mitochondrion reducing equivalents derived extramitochondrially from glycolysis, the activity of ol-glycerol phosphate oxidase in Prodenia flight muscle mitochondria (about 33 pmoles a-glycerol phosphate/mg per hr) is sufficient to permit glycolysis to operate faster than the rate at which pyruvate can be oxidized by the mitochondria (about 12 pmoles pyruvate/mg protein per hr). Thus, mitochondrial a-glycerol phosphate oxidase activity would not limit the glycolytic rate in moth flight muscle. The moth mitochondria are like sarcosomes isolated from other insect flight muscle in that respiratory control of ol-glycerol phosphate oxidation by ADP is poor (GREGG et al., 1960; VAN DEN BERGH and SLATER, 1962; STEGWEE and VAN KAMMEN-WERTHEIM, 1962; COCHRAN,1963). The ol-glycerol phosphate shuttle is but one of the pathways that have been suggested for the mitochondrial oxidation of extramitochondrially generated reducing equivalents. Similar cycles involving malate-oxalacetate and ,!I-hydroxybutyrate-acetoacetate have also been hypothesized. Since neither malate (25 mM) nor /3_hydroxybutyrate (20 mM racemic mixture) is rapidly oxidized, it appears that neither of these two cycles is functioning in moth flight muscle. The moth mitochondria can oxidize acetyl carnitine + malate because the ester can enter the mitochondria where the acetyl group is transferred to endogenous HSCoA and then oxidized via the citric acid cycle. However, acetyl CoA + malate is oxidized only as rapidly as malate alone, probably because the acetyl CoA cannot enter the mitochondria. Acetyl CoA + malate + carnitine can be oxidized, most likely because the acetyl groups can be transferred from coenzyme A to carnitine and the acetyl carnitine can then cross the mitochondria membrane. This suggests that the acetyl CoA-carnitine transferase must be available to substrates outside the particle. BEENAKKERSand HENDERSON(1967) have postulated that there are two acetyl CoA-carnitine transferase in locust mitochondria, one located in the space between the outer and inner membrane and the other embedded in the inner membrane. The data obtained ,with moth mitochondria are not inconsistent with the model those workers suggest, but another possibility is that the enzyme they believe to be in the space between the two membranes is actually embedded in the outer membrane. That added carnitine is necessary for the oxidation of added acetyl CoA demonstrates that the particles have been washed free of endogenous carnitine, as previously suggested (STEVENSON, 1966). Oxidation of acetyl CoA + malate + carnitine proceeds more slowly than oxidation of acetyl carnitine + malate, but this may not be due to limiting acetyl CoA-carnitine transferase activity since, as Table 6 shows, this enzyme is quite active. Instead, it seems likely that oxidation may be inhibited by the HSCoA released as acetyl groups are transferred from acetyl CoA to carnitine. This inhibition is not caused specifically by HSCoA, but is probably due to -SH groups, for mercaptoethanol has the same effect. The 13

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site of this inhibition is not immediately apparent. Acetyl CoA-carnitine transferase itself does not seem to be inhibited by -SH groups since added cysteine has no effect on the activity of this enzyme. ONTKO and JACKSON(1964) have reported that added HSCoA inhibits palmitate oxidation by rat liver mitochondria, and they suggest that the effect may be due to HSCoA driving the palmityl CoAcarnitine transferase reaction in the direction of palmityl carnitine formation. An analogous explanation cannot account for the ‘HSCoA effect’ on acetyl carnitine oxidation in moth mitochondria because added carnitine does not reverse the HSCoA-induced inhibition. ONTKO and JACKSON(1964) found that oxidation of pyruvate + malate was unaffected by added HSCoA, but their incubation medium contained BSA. When this protein is present, pyruvate + malate oxidation by moth mitochondria is also unaffected by HSCoA. Oxidation of pyruvate by mitochondria from rat liver (BREMER,1966) and fly muscle (CHILDRESSet al., 1967) is enhanced by added carnitine which can withdraw as acetyl carnitine some of the acetyl groups formed from pyruvate. Apparently little acetyl carnitine is formed from pyruvate by isolated moth mitochondria if the citric acid cycle is operating, for added carnitine does not enhance 0s uptake. But if the citric acid is not operating, a large excess of carnitine will permit 0s uptake by removing acetyl groups and freeing HSCoA to permit oxidative decarboxylation of more pyruvate. It has already been reported that added carnitine does not enhance the oxidation of palmitate by moth mitochondria, indicating that carnitine is not involved in transferring long-chain acyl groups across the mitochondrial membrane (STEVENSON,1966). Because of all these results it appears that in moth muscle the role of carnitine is to transport acetyl groups derived through pyruvate from carbohydrate to the extramitochondrial site of fatty acid synthesis. Other workers have also designated carnitine as a carrier of acetyl groups out of mitochondria in mammalian tissues (BREMER,1962; NORUM and BREMER,1963; BRESSLERand KATZ, 1965). CHILDRESSet al. (1967) have suggested that carnitine permits oxidation of pyruvate in the first few minutes of flight in the blowfly, when they believe the citric acid cycle is not operating because of a deficiency in sparking intermediates (cf. SACKTORand WORMSERSHAVIT, 1966). There is at present no evidence to indicate that the citric acid cycle is non-functional in moths at the beginning of flight. I reported earlier (STEVENSON,1966) that Prodenia flight muscle mitochondria cannot oxidize acetate and suggested that acetyl CoA synthetase might be lacking or else’ present in very low activity. This suggestion is now supported by the finding that the enzyme is not detectable by direct assay. Because fluoroacetate can block the citric acid cycle, acetyl CoA synthetase cannot be completely absent but its activity must be very low. ZEBE and MCSHAN (1959) reported that Prodeniu flight muscle can incorporate acetate into long-chain fatty acids. This would require an acetyl CoA synthetase, but the rates of incorporation they found were low-in the general vicinity of experimental uncertainty of the synthetase assay I have used (N 10 mpmoles/mg homogenate protein per hr). BEENAKKERSand HENDERSON(1967) h ave reported that flight muscle mitochondria from Locusta

CARBOHMRATB METABOLISM IN MOTHMUSCLE migratoriu oxidize

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acetate + HSCoA slowly (Qol = 10 ~1 O,/mg mitochondrial protein per hr), but oxidize acetyl carnitine considerably more rapidly (Qoa = 100). It appears from this that locust flight muscle mitochondria also lack significant acetyl CoA synthetase activity. Lepidoptera use exclusively fat as a source of flight energy (KOZHANTSCHIKOV, 1938; ZEBE, 1954), and it has also been reported that flight muscle preparations from the cemopia moth cannot utilize carbohydrate at significant rates (DOMROESE and GILBERT, 1964). However, I have found that the glycolytic sequence in Prodenia flight muscle is intact, for glucose, glycogen, and trehalose are all oxidized. Qo, values found when carbohydrate is being oxidized by flight muscle homogenates are less than when pyruvate + malate is being oxidized by mitochondria because the Qo, values are computed on the basis of the different amounts of protein in mitochondria suspensions and in homogenates, and because the homogenate incubation mixture does not include the high-energy phosphate trap contained in the mitochondria incubation mixture. Since in the experiments with homogenates the Qo, values for glucose + malate are higher than those for pyruvate + malate, Prodenia flight muscle must be able to metabolize carbohydrate rapidly. Inhibition of trehalase by sucrose has been reported by GUSSIN and WYATT (1965) and also observed in this work. Because of this inhibition, a saline-based medium was used to fractionate the homogenate for studies on this enzyme. GUSSIN and WYATT (1965) reported that flight muscle of cemopia contains a trehalase with a K, for trehalose of 3.6 x 1O-3 M and a pH optimum of 6.5. They found the enzyme to be mainly in the fraction sedimenting between 37,000 and 105,OOOg (which they called the sarcotubular fraction) and its activity to be enhanced by treatments that disrupt membranes. The Prodeniu flight muscle trehalase described here is similar to the cecropia enzyme except that it is isolated in the 5000-30,OOOg pellet. This difference may be due to a less complete disruption of the subcellular particles by the relatively gentle homogenization procedure used in the present experiments. Nevertheless, it is also possible that the Prodeniu enzyme is within the lysosomes, in which case it would be of no physiological significance during flight. The activity of the Prodenia enzyme in the whole homogenate can be increased some fifteenfold by fifteen freeze-thaw cycles, whereas the cemopia enzyme activity could be increased maximally only about three- to sixfold by five freeze-thaw cycles. In both cases the presence of solutes decreases the extent of enhancement of activity by freeze-thaw treatment. It is difficult to judge the in situ activity of trehalase. However, it is pertinent to consider the results of ZEBE (1954), who found that the greatly elevated R.Q. values in resting moths fed large amounts of carbohydrate (R.Q. = 1.6-2.7) are sharply diminished if the insects are forced to fly (R.Q. = O-69-0*94), but are nevertheless higher than in flying moths that have not recently ingested carbohydrate (R.Q. = 0.58-0.74). W ere muscle trehalase highly active in situ, the moths would be expected to use recently ingested carbohydrate for flight and the R.Q. values would certainly be above 1.0. Instead, the values found appear to be the sum of the high R.Q. and relatively low metabolic rate of lipogenesis

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from carbohydrate and of the low R.Q. and very high metabolic rate of flight supported by fat combustion. Obviously something is preventing carbohydrate utilization for flight energy, and since the activity of muscle trehalase in gently treated homogenates is low, it is reasonable to assume that this enzyme is the limiting factor. The glycogen reserves of newly emerged Prodenia are meagre, a fact previously reported by BABERS (1941). Hyalophora cecropia also has but a scanty supply of glycogen (DOMROESE and GILBERT, 1964) which cannot be replenished since this moth is unable to feed. If a flying moth consumes 0, at the rate of 40 $/mg live wt. per hr (ZEBE, 1954), the glycogen in the thorax of a male Prodenia would be sufficient to sustain flight for about 75 set, and the abdominal glycogen, if it could be mobilized, would permit flight for 7 min more. In this laboratory the moths have been flown (tethered flight) for several hours. There is evidence that trehalose, but not glucose, is present in the blood of Prodeniu adults (ZEBE, 1958), so extramuscular carbohydrate most likely is transported to the muscles as this disaccharide. I have found that ethanol-soluble, anthrone-positive material is present in only minor quantities in Prodenia, indicating that the total amount of trehalose in the blood is small. In summary, the physiological evidence presented by ZEBE (1954) indicates that when carbohydrate is available (immediately after feeding) it is not used to support flight, and the quantitative data on the total carbohydrate reserves of the moth before feeding or after digestion is completed demonstrate that this fuel could support flight for only a short time even if it were metabolically available. Although Prodenia can replenish its glycogen stores by ingesting carbohydrate (BABERS, 1941), I have found that moths permitted access only to water can still fly a week after eclosion. Obviously the small amount of glycogen in a newly emerged moth could not last for such a long period of time. A question arises then about the significance of the ability of moth flight muscle to metabolize carbohydrate. The importance of this may not be to provide energy for flight, but rather (1) to generate citric acid cycle ‘sparker’ (either via a pyruvate carboxylation or via a malic enzyme) to permit oxidation of acetyl CoA derived from long-chain fatty acids; (2) to provide glycerol for triglyceride biosynthesis; and (3) to make available acetyl groups for the formation of fat from carbohydrate ingested as nectar. ZEBE (1954) has shown that moths fed carbohydrate quickly convert it to fat (R.Q. = l-5-2*7), and that this conversion is especially rapid in the Noctuidae, of which Prodenia is a member. The present work shows that flight muscle homogenates can oxidize carbohydrate to CO, + H,O, a process in which acetyl CoA is an intermediate. Since ZEBE and MCSHAN (1959) have shown that Prodenia flight muscle homogenates can incorporate [Wj-acetate into long-chain fatty acids, it seems likely that the muscle is also able to convert carbohydrate to lipid, even though those authors could not demonstrate this. Their failure may be due to the incompatible requirements of the oxidative conversion of glucose to acetyl CoA and the reductive conversion of acetyl CoA to fatty acids.

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Although the muscle possesses the ability to oxidize hexose and pyruvate rapidly, this capacity is most likely not fully used since muscle trehalase may limit the rate at which glucose is made available. A low level of trehalase activity would limit carbohydrate utilization for the very intense activity of flight, but would not affect the more leisurely processes of lipogenesis. Achnozoledgements-I am grateful to Mr. RICHARDHEBWT for preparing the electron micrographs and assisting in their interpretation and to Mrs. JANET GORMAN and Mrs. SHARONGERREDfor valuable technical assistance. REFERENCES BABERSF. H. (1941) Glycogen in Prodenia eriduniu, with special reference to ingestion of glucose. r. agric. Res. 62, 509-531. BEENAI~KERS A. M. T. and HENDERSONP. T. (1967) The localization and function of carnitine acetyltransferase in the flight-muscles of the locust. EuropeanJ. Biochem. 1, 187-192. BEENAKKERS A. M. T. and KLINGENBERG M. (1964) Carnitine-coenzyme A transacetylase in mitochondria from various organs. Biochim. biophys. Acta 84, 205--207. BREMERJ. (1962) Carnitine in intermediary metabolism. Reversible acetylation of camitine by mitochondria. J. biol. Chem. 237, 2228-2231. BREMERJ. (1965) The effect of acylcamitines on the metabolism of pyruvate in rat-heart mitochondria. Biochim. biophys. Acta 104, 581-590. BREMERJ. (1966) Comparison of acylcamitines and pyruvate as substrates for rat-liver mitochondria. Biochim. biophys. Actu 116, l-l 1. BRESSLER R. and KATZ R. I. (1965) The effect of camitine on the rate of incorporation of precursors into fatty acids. J. biol. Chem. 240, 622-627. BROSEMER R. W. and VEERABHALIRAPPA P. S. (1965) Pathway of proline oxidation in insect flight muscle. Biochim. biophys. Acta 110, 102-112. BURSELLE. (1963) Aspects of the metabolism of amino acids in the tsetse fly, Gloss&a (Diptera). J. Insect Physiol. 9, 439-452. CHANCEB. and BALTSCHEFF~KY H. (1958) Spectroscopic effects of adenosine diphosphate upon the respiratory pigments of rat-heart muscle sarcosomes. Bi0chem.J. 68, 283-295. 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. CHILDRFZSS C. C. and SACKTORB. (1966) Pyruvate oxidation and the permeability of mitochondria from blow ily flight muscle. Science, N. Y. 154, 268-270. CHILDRESS C. C., SACKTORB., and TRAYNORD. R. (1967) Function of camitine in the fatty acid oxidase-deficient insect flight muscle. r. biol. Chem. 242, 754-760. COCHRAND. G. (1963) Respiratory control in cockroach-muscle mitochondria. Biochim. biophys. Acta 78, 393403. DARROWR. A. and COLOWICKS. P. (1962) Methods in Enzymology (Ed. by COLOWICKS. P. and KAPLAN N. 0.) 5, 226. Academic Press, New York. DOMROESEK. A. and GILBERT L. I. (1964) The role of lipid in adult development and flight-muscle metabolism in Hyalophoru cecropia. 3. exp. Biol. 41, 573-590. FRAENKELG. and FRIEDMANW. (1957) Vitamins and Hormones (Ed. by HARRIS R. S., MARRIANG. F., and THIMANN K. V.) 15, 73. Academic Press, New York. GREGG C. T., HEISLERC. R. and REMMERTL. R. (1960) Oxidative phosphorylation and respiratory control in housefly mitochondria. Biochim. biophys. Acta 45, 561-570. GUSSINA. E. S. and WYATT G. R. (1965) Membrane-bound trehalase from cecropia silkmoth muscle. Archs biochem. Biophys. 112, 626-634.

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HA~SIDW. 2. and ABRAHAMS. (1957) Methods in Enzymology (Ed. by COLOWICKS. P. and KAPLANN. 0.) 3, 43. Academic Press, New York. KORNBERG A. and PRICERW. E. JR. (1953) Enzymatic synthesis of the coenzyme A derivatives of long chain fatty acids. J. biol. Chem. 204, 329-343. KOZHANTSCHIKOV I. (1938) Carbohydrate and fat metabolism in adult Lepidoptera. Bull. ent. Res. 29, 103-114. LA~NE E. (1957) Methods in Enzymology (Ed. by COLOWICK S. P. and KAPLANN. 0.) 3,447. Academic Press, New York. MICHAL G. and BERGMEYER H. (1965) Methods in Enzymatic Analysis (Ed. by BERGMEYER H.), p. 517. Academic Press, New York. MICHEJDAJ. (1964) Physiology and structure of flight-muscle sarcosomes in silkworm, Hyalophora cecropia. Bull. Sot. Amis Sci. Lett. Poznan 4, 61-102. MONTGOMERYC. M. and WEBB J. L. (1956) Metabolic studies on heart mitochondria-II. The inhibitory action of parapyruvate on the tricarboxylic acid cycle. r. biol. Chem. 221, 359-368. NORUM K. R. and BREMERJ. (1963) Acyl coenzyme A as an intermediate in the mitochondrial acylation of camitine by ol-keto acids. Biochim. biophys. Acta 78, 77-84. ONTKO J. A. and JACKSOND. (1964) Factors affecting the rate of oxidation of fatty acids in animal tissues. Effect of substrate concentration, pH, and coenzyme A in rat liver preparations. J. biol. Chem. 239, 3674-3682. SACKTORB. and WORMSER-SHAVIT E. (1966) Regulation of metabolism in working muscle in &pro-I. Concentrations of some glycolytic, tricarboxylic acid cycle, and amino acid intermediates in insect flight muscle during flight. r. biol. Chem. 241, 624-631. SHOREYH. H. (1963) A simple artificial rearing medium for the cabbage looper. J. econ. Ent. 56, 536-537. STADTMANE. R. (1957) Methods in Enzymology (Ed. by COLOWICKS. P. and KAPLANN. 0.) 3, 230-231. Academic Press, New York. STEGWEED. and VAN KAMMEN-WERTHEIM A. R. (1962) Respiratory chain metabolism in the Colorado potato beetle-I. Respiration and oxidative phosphorylation in sarcosomes from active beetles. J. Insect Physiol. 8, 117-126. STEVENSON E. (1966) Rapid oxidation of palmitate with concomitant phosphorylation of adenosine S’diphosphate by moth flight-muscle rnitochondria. Biochim. biophys. Acta 128, 29-33. SUMNERJ. B. (1944) A method for the calorimetric determination of phosphorus. Science, N. Y. loo, 413-414. VAN DENBERGHS. G. (1964) Pyruvate oxidation and permeability of housefly sarcosomes. Biochem. J. 93, 128-136. VAN DENBERGHS. G. and SLATERE. C. (1962) The respiratory activity and permeability of housefly sarcosomes. Biochem. J. 82, 362-371. VAN HANDELE. (1965) Estimation of glycogen in small amounts of tissue. Analyt. Biochem. 11, 256-265. ZEBEE. (1954) Uber den Stoffwechsel der Lepidopteren. 2. wergkich. Physiol. 36, 290-317. ZEBEE. (1958) Untersuchungen zum Fettstoffwechsel der Insekten. Zool. Anz. 11,13 l-l 37. ZEBEE. and MCSHAN W. H. (1959) Incorporation of [r4C] acetate into long chain fatty acids by the fat body of Prodeniu eridania (Lep.). Biochim. biophys. Acta 31, 513-518.