B I O C H I M I C A E T B I O P H Y S I C A A C T A 2 9

BRA 65474

RAPID OXIDATION OF PALMITATE WITH CONCOMITANT PHOSPHORY-

LATION OF ADENOSINE 5 ' -DIPHOSPHATE BY MOTH FLIGHT-MUSCLE

MITOCHONDRIA

EDMUND STEVENSON Central Research Department, Experimental Station, E. I. du Pont de Nemours and Company, Wilmington, Del. (U.S.A.)

(Received January 24th, 1966 )

S U M M A R Y

A procedure is described for isolating mitochondria from the thoracic muscle of the southern armyworm moth, Prodenia eridania. These mitochondria are capable of very rapidly oxidizing palmitate with concomitant phosphorylation of adenosine 5'-diphosphate. There is an absolute requirement for adenosine 5'-diphosphate and inorganic phosphate, but added nicotinamide-adenine dinucleotide, nicotinamide- adenine dinucleotide phosphate and carnitine have no effect. Maximal rates of oxygen uptake are found when a high-energy phosphate trap and some protein are present.

INTRODUCTION

There have been many studies on fat ty acid oxidation by intact mitochondria, but in all cases the rates of 02 consumption have been low (usually less than 7 ° #1 per mg protein per h). Since moths and butterflies1, 2, and locusts s utilize fat for flight, their thoracic muscles should yield mitochondria capable of rapidly oxidizing fat ty acids. However, particles isolated from such sources by other workers 4-* consume 03 at disappointingly low rates--far too low to account for the very rapid utilization of energy by the flying insect.

A procedure is described here for isolating from the thoracic muscle of the southern armyworm moth, Prodenia eridania, mitochondria which rapidly oxidize

r

palmitate with concomitant phosphorylation of ADP.

METHODS

Armyworm larvae were reared on lima bean leaves and on the artificial diet described by SHOREY 8. Moths had access to water, and were used within a week of emergence. Female moths were usually used, but no differences attributable to sex were found.

Biochim. Biophys. Acta, 128 (1966) 29-33

30 E. STEVENSON

After the moths were immobilized by cold the thoraces were isolated, very briefly dipped in 95 % ethanol to wet the scales, quickly rinsed in water, and blotted on absorbent paper. The wet scales were rubbed off and the thoraces were divided sagitally. Thoracic muscle was removed and rinsed in cold 0.25 M sucrose-5 mM EDTA (pH 7.o), and blotted free of excess liquid on filter paper. Muscle from 5-8 moths was accumulated in 2 ml cold sucrose-EDTA in a glass Potter- type homogen- izing tube and homogenized by hand with four up-and-down strokes of the Teflon pestle. Cell debris was removed by centrifugation for 4 min at 12o × g, and the mito- chondria were sedimented at 1475 × g (8 min), washed once in 2 ml fresh sucrose- EDTA and centrifuged as before. The pellet was suspended in an appropriate volume of sucrose-EDTA in a glass homogenizing tube with two strokes of the Teflon pestle.

0 2 consumption was measured with differential manometers. The final incuba- tion mixture contained 9.8 #moles KC1, 4.9 #moles MgC12, 50 #moles tr iethanolamine- HC1 buffer (pH 7.o), 15/,moles potassium phosphate (pH 7.o), 1.3 #mole ADP, 25 #moles glucose, 0.5 unit (ref. 9) (1.8 #g) crystalline hexokinase or o.75 unit (ref. 9) (23o/~g) crude hexokinase, 85 #g bovine serum albumin, 37.6/,moles sucrose, 0.75 #mole EDTA, 0.26/,mole potassium palmitate, 1.3 #mole potassium L-malate and O.lO-O.25 mg mitochondrial protein in a total volume of 0.65 ml. CO 2 was trapped on KOH-soaked filter paper in the center well. After thermal equilibration, incubations were begun by the addition of the mitochondria from the side arm and were carried out at 3 °0 with shaking (16o oscillations/rain). Qo2 values were calculated from the period during which 0 2 uptake is linear with time. No correction has been made for endogenous respiration, which was lO-2O #1 per mg protein per h. Protein was deter- mined by the biuret procedure 1° with crystalline bovine serum albumin as standard.

The various coenzymes and crystalline hexokinase (EC 2.7.1.1) (a (NH4)2SO ~ suspension) were obtained from Sigma Chemical Co. Lalmitic and malic acids were purchased from Calbiochem and bovine serum albumin was obtained from Armour Pharmaceutical Co., Mann Research Laboratories and Pentex Inc. Crude hexokinase was a product of Nutritional Biochemicals Corp.

80r- r * - : : - "~ 70J- / Po,mit~t. (0.4,

60]- / MoIot. (2)

5oF /

3o t- /

O0 2 0 4 0 6 0 BO tO = ~ - ' ' ' i " t 0 1 4 0 INCUBATION TIME.

minutes

Fig. I

RESULTS

Palmitate is rapidly oxidized by moth mitochondria if malate is present (Fig. I and Table nI). Rapid 02 uptake begins only after an initial lag and ceases when Pi has been depleted. Optimal palmitate concentration is about 0.5 raM, and optimal p H

Biochim. Biophvs. Acta, 128 (1966) 29-33

PALMITATE OXIDATION BY MOTH SARCOSOME8 3 I

T A B L E I

E F F E C T O F VARIOUS PROTEINS ON PALMITATE OXIDATION BY THORACIC-MUSCLE MITOCHONDRIA

E x p t . I : condi t ions as descr ibed in t e x t ; 0.2 7 mg p ro te in was used. E x p t . 2: condi t ions as des- cr ibed in t ex t , excep t t h a t A D P concen t r a t ion was 3 . I / , m o l e s / m l , a n d bovine se rum a lbumin , c rys t a l l i ne hexok inase and glucose were o m i t t e d unless o therwise ind ica t ed (because crude hexo- k inase con ta ins inver tase , sucrose in t he m i t o c h o n d r i a l suspens ion is hydro lyzed , so glucose need no t be added). Mi tochondr ia l p ro te in was o.18 mg.

Expt. Omission d ddition (l~g) Qo~ No. (Izl Offing

protein per h)

I - - - - 431 c rys t a l l i ne hexok inase + glucose - - 252 glucose - - 239 bovine se rum a l b u m i n - - 194

2 - - - - 261 - - crude hexok inase (80) 429 - - bovine s e rum a l b u m i n (80) 318 -- cy toch rome c (80) 352

is about 7.0. Omission of either PI or ADP reduces O 3 uptake by over 95%. Addition of 2 mM DL-carnitine, I mM NAD +, I mM NADP +, or o.oi mM cytochrome c has no effect on the Qo2.

I f bovine serum albumin, glucose, or glucose plus crystalline hexokinase are omitted the rate of oxidation is decreased (Table I). Since bovine serum albumin from three vendors gave similar results, the effect is not peculiar to material from one supplier. When crude hexokinase (containing much extraneous protein) is used the bovine serum albumin can be omitted with little effect on Oz uptake (Expt. 2, Table I). Bovine serum albumin can be replaced also by cytochrome c (Table I).

T A B L E I I

PALMITATE OXIDATION AND OXIDATIVE PHOSPHORYLATION

Condi t ions as descr ibed in t ex t , excep t as fol lows: in Exp t s . I and 2 crude hexok inase was used, w i t h a glucose concen t r a t i on of 77/~moles/ml, and an A D P concen t r a t ion of 1. 5 # m o l e / m l (Expt . i) and 0. 5 /~mole /ml (Expt . 2). I n E x p t . 3, crude hexok inase was used, w i th no added glucose; AD P concent ra t ion , I. 5 #mole /ml . The mi tochondr i a l p ro te in used was : E x p t . I, o.21 mg ; E x p t . 2, o . io mg; E x p t . 3, o.17 mg.

Expt. P/O ratio Qo2 (tzl Ozlmg No. protein per h)

I 2. 3 326 2 1.7 475 3 2.2 505

P/O ratios are about 2.o (Table II). These mitochondria probably lack an ace- tate thiokinase, for acetate is not oxidized (Table III). In the presence of 5 mM fluoro- pyruvate, oxidation of palmitate proceeds slowly and soon ceases altogether.

DISCUSSION

There have been few successful studies on long-chain fat ty acid oxidation by

Biochim. Biophys. Acta, 128 (1966) 29-33

32 E. STEVENSON

T A B L E I I I

OXIDATION OF VARIOUS ACIDS BY PRODENIA THORACIC-MUSCLE MITOCHONDRIA

C o n d i t i o n s as d e s c r i b e d in t e x t ; o t h e r w i s e c r y s t a l l i n e h e x o k i n a s e was used.

Substrate (ttmoles/ml) Qo2 Number of (#l OJmg protein per h) experiments

2Vlean Range

P a l m i t a t e ( 0 . 4 ) + m a l a t e (2) 371 338-575 6 A c e t a t e ( I O ) + m a l a t e (2) 34 24 -43 2 M a l a t e (2) 28 14-41 2 P a l m i t a t e (0.4) o o 2 P a l m i t a t e ( 0 . 4 ) + m a l a t e (2)* 458 350 -748 6

* I n t h e s e e x p e r i m e n t s c r u d e h e x o k i n a s e w a s used.

insect preparations. BODE AND KLINGENBERG 6 found that locust flight-muscle mito- chondria oxidize free palmitate slowly (Q02 = 22) but palmityl carnitine somewhat more rapidly (Q02 = 123). DOMROESE AND GILBERT ~ showed that mitochondria from the moth Hyalophora cecropia oxidize [14Clpalmitate, but the authors measured only 14C02 release. Other workers could not demonstrate long-chain fatty acid oxidation by particles from locust muscle4, 5 or silkworm gut 12. Thus the rates of O~ uptake reported here are by far the highest yet published for the oxidation of long-chain fatty acids by mitochondria from any source.

Mitochondria from locust muscle and mammalian tissues oxidize the carnitine esters of fatty acids 6,13 and the free acids plus carnitine 6,13-15 more rapidly than the free acids alone. The present data suggest that moth mitochondria do not oxidize palmitate via the carnitine ester, for washing sufficient to remove endogenous sub- strate does not produce a carnitine requirement. It seems unlikely that carnitine could act as an acyl carrier (which would have to cross mitochondrial membranes readily) and yet be so tightly bound within the particles that it could resist this washing.

Bovine serum albumin probably reversibly binds palmitate, and gradually releases it to maintain a very low concentration of the free acid. This binding may protect the mitochondria from what would otherwise be too high a level of free fatty acid, and can explain the enhanced 02 uptake in the presence of added bovine serum albumin. The effect is not specific for this protein, for added cytochrome c, which exerts no effect in the presence of bovine serum albumin, can replace it. In addition, when crude hexokinase containing considerable extraneous protein is used, bovine serum albumin need not be added.

Adenine nueleotides play a dual role in this system: ATP is necessary for fatty acid activation and ADP is needed as a phosphate acceptor. An adenylate kinase probably converts added ADP to ATP for the activation of the first few molecules of palmitate and after that, ATP generated by fatty acid oxidation should suffice to activate the remaining palmitate. ADP can be regenerated from ATP by the high- energy phosphate trap or by an adenosine triphosphatase. Since 02 uptake occurs in the absence of the high-energy phosphate trap but not in the absence of P i , ATPase activity must be significant. Preliminary experiments indicate it to be about 7 #moles P i released per mg protein per h.

igiochim. Biophys. Acta, 128 (1966) 29 -33

PALMITATE OXIDATION BY MOTH SARCOSOMES

ACKNOWLEDGEMENT

The author acknowledges the technical assistance of Mrs. C. BEVERIDGE.

33

R E F E R E N C E S

i I. KOZHANTSHIKOV, Bull. Entomol. Res., 29 (1938) lO3. 2 E. ZEBE, Z. Vergleich. Physiol., 36 (1954) 29 TM

3 A. KROGH AND T. WEIS-FOGH, J. Exptl. Biol., 28 (1951) 344. 4 K. R. REES, Biochem. J., 58 (1954) 196. 5 H. MEYER, ]3. PREISS AND S. ]3AUER, Biochem. J., 76 (196o) 27. 6 C. BODE AND M. KLINGENBERG, Biochem. Z., 341 (1965) 271. 7 K. A. DOMROESE AND L. I. GILBERT, J. Exptl. Biol., 41 (1964) 573- 8 H. SHOREV, J. Econ. Entomol., 56 (1963) 536. 9 R. A. DARROW AND S. P. COLO'*VICK, in S. P. COLOWICK AND N . O . KAPLAN, Methods in

Enzymology, Vol. 5, Academic Press, New York, 1962, p. 226. i o E. LAVNE, in S. P. COLOWlCK AND N. O. KAPLAN, Methods in Enz~:mology, Vol. 3, Academic

Press, New York, 1955, p. 45 o. i I S. SRIDHARA, J. Insect Physiol., I I (1965) 33. I2 S. J. FRIEDBERG AND 1~.. BRESSLER, Biochim. Biophys. Acta, 98 (1965) 335. 13 I. ]3. FRITZ AND I~. T. N. YUE, J. Lipid Res., 4 (1963) 279. 14 I. B. FRITZ AND N. R. MARQUIS, Proc. Natl. Acad. Sci. U.S., 54 (1965) 1226.

Biochim. Biophys. Acta, 128 (1966) 29-33