The larval fat body of Sarcophaga bullata (Diptera) as a system for studying phospholipid biosynthesis

r. Insect Physiol.,

1966,

6’01. 12, pp, 619 to 624.

Pergamon

Press Ltd.

Printed

in Great

Britain

THE LARVAL FAT BODY OF SARCOPHAGA BULLATA (DIPTERA) AS A SYSTEM FOR STUDYING PHOSPHOLIPID BIOSYNTHESIS* H. D. CRONE?, Science

Research

R. W. NEWBURGH,

Institute,

Oregon

and

State University,

(Received 22 January

C. MEZEI

Corvallis,

Oregon

1966)

Abstract-The

larval fat body of Sarcophaga bullata was shown to be an effective system for the study of phosphatidylcholine biosynthesis. 32P-phosphorylcholine was incorporated to the extent of 4.7 per cent in 1 hr at 30°C. This substrate was a precursor of phosphatidylcholine.

INTRODUCTION

RECENT studies have done much to elucidate the chemical nature of insect phospholipids (FAST, 1964), and work has been done on the metabolism of these compounds in vivo by feeding or injecting isotopic tracers (BRIDGES et al., 1962; TAYLOR and HODGSON, 1965). The detailed study of insect phospholipid metabolism in vitro is represented at the present time by the work of CHOJNACKI and KORZYBSKI (1963), who have studied the incorporation of the 32P-phosphorylcholine fragment from cytidine diphosphorylcholine into phosphatidylcholine in homogenates of adult locust and silkworm tissues. This paper is an investigation of the suitability of a whole fat body preparation from Sarcophaga bullata larvae as a means of studying phospholipid. metabolism of Diptera in vitro. The results show that phosphorylcholine is a precursor of phosphatidylcholine in this organism. METHODS

AND

RESULTS

32P-phosphorylcholine was prepared by the method of WEISBURGER and SCHNEIDER (1961), using 150 pmoles of choline chloride and an equimolar amount of 32P-phosphoric acid. Eighteen per cent of the material was recovered from the Dowex-1 formate column as pure 32P-phosphorylcholine. Paper chromatography showed less than 1-O per cent of the radioactivity to be inorganic phosphate. The initial specific radioactivity of the preparation was 0.27 PC per pg atom of phosphorus. The 14C-1,2-choline HBr was purchased from Tracerlab. Adenosine * This work was supported by a grant-in-aid from the U.S. Army Chemical Center to R. W. N. f H. D. C. was on leave of absence from the Agricultural Research Council of the United Kingdom. Present address: Biochemistry Department, Pest Infestation Laboratory, London Road., Slough, Bucks. 38

619

620

H.D.

CRONE,

R. W. NEWBURCH,AND C. MEZEI

triphosphate (ATP), cytidine triphosphate (CTP), cytidine diphosphate (CDP), and Coenzyme A (CoA) were purchased from Sigma Chemical Co. The alumina used for column chromatography was basic grade I alumina 100-200 mesh from M. Woelm, Eschwege, Germany. Other reagents and solvents used were of analytical grade. Phosphorus was determined after a sulphuric acid-hydrogen peroxide digestion by the heating method of BARTLETT (1959). Radioactivity of lipid samples was determined by taking to dryness in a stream of air, adding 10 ml of a solution of 6 g PPO and 0.1 g POPOP in 586 ml toluene and 414 ml methanol, and counting in a liquid scintillation spectrometer. The radioactivity of water-soluble compounds was determined by plating out aliquots of solution into aluminium planchets lined with filter paper discs and counting under a Geiger-Mi_iller tube. An approximate correlation between the two methods was established by counting radioactive lipid under both conditions. Eggs or young larvae were collected from Sarcophaga bulkta by leaving a piece of raw liver in the cage for about 1 hr. The larvae were reared at 30°C in beakers containing raw liver and damp sawdust, the ratio of larvae to nutrient being approximately constant. After 3 days the larvae were transferred to clean damp sawdust for 12 hr and then used for the experiments. To remove the fat body material, the larvae were washed externally, pinned down in cold 0.75 y0 w/v NaCl solution, and opened along the dorsal midline. The fat bodies were gently teased away from the attachments to the gut and body wall, and transferred on a pair of fine forceps to a 5 ml conical flask in ice. Care was taken to damage the fat bodies as little as possible. The material from 5 larvae was collected for each incubation; this wet tissue had an approximate volume of O-2 ml. The fat bodies were homogenized in a glass homogenizer for the cell-free preparations. For extraction, the incubation flasks were removed from the waterbath and cooled in ice. The contents of each flask were then passed onto a cooled glass funnel 2 cm wide, mounted on a filter flask, and plugged with a small piece of glass wool. The aqueous medium was removed by mild suction, and the incubation flask was rinsed twice with O-5 ml cooled fresh medium which was then passed through the filter. The glass plug with the tissue on it was transferred to a centrifuge tube and 2 ml 2 : 1 v/v chloroform-methanol added. The tube contents were then ground with a fine glass rod until the glass fibre was powdered and the tissue thoroughly disintegrated. The tube was centrifuged, the supernatant transferred to a stoppered tube, and the solid was extracted twice more with 1.5 ml of the solvent mixture. The cell-free preparations were extracted directly, omitting the filtration and grinding. The crude extracts were then washed with 0.2 vol of 0.75% NaCl solution. The lower layer was dried down under a stream of nitrogen. In and most, the whole of the lipid was taken up in 1 : 1 v/v chloroform-methanol applied to an alumina column. In some, the lipid was taken up in a definite volume of this solvent and suitable aliquots removed for phosphorus estimation, radioactivity determination, and column separation.

PHOSPHOLIPID

SYNTHESIS

IN

THE

FAT

BODY

621

The alu.mina columns were formed by suspending 1 g of the absorbent in 1 : 1 v/v chloroform-methanol and pouring the slurry into a glass column of 6 mm internal diameter. The column was washed with about 20 ml of the solvent mixture. Then the sample was applied in a minimal amount of solvent, and one fraction of 10 ml of th.e 1 : 1 chloroform-methanol solvent was collected. A further 10 ml fraction contained no significant radioactivity or phosphorus. The first fraction was mixed and suitable aliquots removed for radioactivity and lipid phosphorus determinations. It was (demonstrated by the following procedure that the only phosphoruscontaining component of this fraction was diacyl phosphatidylcholine. When chromatographed on a column of 3 g silicic acid and eluted with chloroform, 19 : 1 v/v chloroform-methanol, 1 : 1 chloroform-methanol, and methanol in fractions of 50 ml, 104 per cent of the radioactivity was recovered in the 1 : 1 C-M and methanol fractions. The other fractions were not radioactive. When an aliquot of the combined 1 : 1 C-M and methanol fractions was run on a basic thinlayer chromatogram by the method of SKIPSKI et al. (1963), one phosphoruscontaining spot only was observed, associated with the only detectable radioactivity, and corresponding in position to authentic samples of lecithin. Direct thin-layer chromatography of the alumina eluate was not possible due to the excessive proportion of neutral lipid present. Alkaline hydrolysis of the silicic acid eluate by the method of DAWSON(1960) resulted in 98 per cent by phosphorus determination or 99.7 per cent by radioactivity determination becoming water-soluble, showing a virtual absence of choline plasmalogen and sphingomyelin in accordance with the observations of CRONE and BRIDGES (1963) on hhca domestica. The lipid extracts from two incubations were compared with respect to radioactivity and phosphorus content with the fractions obtained after alumina chromatography. The phosphatidylcholine contained 27 per cent of the total lipid phosphorus, and 98 per cent of the radioactivity in the lipid extract. It was concluded that phosphatidylcholine was the only lipid significantly labelled by the 32P-phosphorylcholine. The chemical nature of the radioactivity remaining water-soluble after the incubations was examined in two cases in which the incubation medium was unbuffered saline. After lipid extraction the tissue was extracted twice with 1 ml of 10% w/v trichloracetic acid solution. These extracts were combined with the aqueous wa.shings of the lipid extract and the incubation medium. The mixture was thoroughly shaken, allowed to stand 3 hr at 5”C, and then centrifuged. The supematant was extracted twice with equal volumes of ether, and then aliquots were chromatographed on paper in water-saturated phenol. One peak of radioactivity was observed near the solvent front. There was no trace of radioactive inorganic phosphate. It was concluded that there was no phosphatase activity in the preparation. Table 1 gives the percentage incorporation of label into phosphatidylcholine under various conditions of incubation. The mean value for the buffered medium (4.7 per cent of the 0.64 pmoles phosphorylcholine added) represents 30 rnp moles.

H. D.

622 TABLE

CRONE, R. W.

~-INCORPORATION

NEWBURGH,

AND C. MEZEI

OF 32P-~~~~~~~~~~~~~~~~

PHOSPHATIDYLCHOLINE

RADIOACTIVITY

INTO

THE

OF FAT BODIES UNDER VARIOUS CONDITIONS Percentage Additions

Medium 0.75 y0 NaCl

incorporation

None

4.0 (5) f 0.9

None

4.7 (4) f 1.4

50 mM

Tris,

pH

7.3

50 mM

Tris,

pH

7.3

1 mM

50 mM

Tris,

pH

7.3

3 mM

CTP

5.5 (3) + 1.8

50 mM Tris, pH 7.3

CTP 1 mM ATP

5.5 (1) 4.7 (2) f 0.8

Medium

Starvation period *

Percentage incorporation

50 mM Tris, pH 7.3 50 mM Tris, pH 7.3

12hr 6 hr

3.7 (4) f 0.3 6.2 (4) + 1.1

The figures give the mean incorporation as a percentage of the initial radioactivity in the incubations, with the number of determinations in parentheses and the standard deviation. The incubation mixtures were composed of the fat body tissue, 1 ml of 0.75% NaCl or 1 ml of 50 mM Tris HCl, 3 mM MgCI,, pH 7.3, and 50 ~1 of the 3ZP-phosphorylcholine solution (0.64pmoles). The flasks were stoppered and shaken in a waterbath at 30°C for 1 hr. Four incubations were performed at a time, one of each batch being a control with no additions when the effect of such additions was being studied. * Time spent on clean sawdust before dissection. The mean phosphatidylcholine minations)

content

was 600 rnp moles,

of 5 fat bodies

thus the amount

after incubation

of incorporation

(30 deter-

is equivalent

to

5 per cent of the phosphatidylcholine in each preparation. The degree of incorporation using whole tissue does not vary markedly with the nature of the medium. TABLE

2--INCORPORATION

OF 14C-1 ,2-CHOLINE

INTO

PHOSPHATIDYL

CHOLINE

OF

CELL-FREE PREPARATIONS OF FAT BODIES Specific Additions

None. Enzyme inactivated by heating None 2.5 pmoles ATP 2.5 pmoles ATP, 1.0 pmole CoA 2.5 pmoles ATP, 5.0 pmoles CDP, 1 pmole CoA ATP, CDP, CoA, non-radioactive choline (1 pmole)

activity

(cpm/pg

P)

1.0 13.1 23.0 17.9 28.3 14.1

The standard incubation mixture consisted of 40 pmoles glycylglycine buffer, pH 8.5 ; 12 pmoles MgCI,; 15 pmoles cysteine; 1 pmole W-1,2-choline HBr, 2.5 x 10e5 cpm. Total volume = 0.5 ml.

PHOSPHOLIPID

SYNTHESIS IN THE FAT BODY

623

An enhancement of phosphatidylcholine synthesis in the presence of ATP and CDP was found using cell-free systems (Table 2). The phospholipid composition of the fat bodies was examined by chromatography on an alumina column and by thin-layer chromatography. Phosphatidylcholine (31 per cent of the total lipid phosphorus), phosphatidylethanolamine (56 per cent), and polyglyceroIphosphatide (3 per cent) were found. These results agree with those of ALLEN and NEWBURGH(1965), except that polyglycerolphosphatide was found, and that a lower proportion of the lysophosphatides than previously reported was present. DISCUSSION The amount of incorporation of phosphorylcholine in an unsupplemented medium in 1 hr was 4.7 per cent of the substrate supplied, or 5 per cent of the product concentration. This may be compared with the results of CHOJNACKIand KORZYBSKI(1963) for tissue homogenates. For homogenates of larval Bombyx mori fat bodies these authors quote an incorporation for cytidinemonophosphate-[32P]phosphorylcholine of 17.5 per cent or a ‘relative activity x 1000’ of 10.8 (10.8 new molecules I.abelled per 1000 phospholipid molecules in the incubate). These authors did not fractionate the lipid extract to determine the phosphatidylcholine specific activity alone, but using a probable value of 60 per cent for phosphatidylcholine content (SR.IDHARA and BHAT, 1962), a relative activity x 1000 of 18 with respect to the one compound is obtained. This compares to a value of 50 for the present work. The incorporation as a percentage of the substrate supplied is dependent on the relative concentration of the substrate, which was higher in the present work than in that of CHOJNACKIand KORZYBSKI. These authors state, however, that increasing the precursor concentration did not greatly increase labelling. The performance of the present preparation is therefore very good, especially as it is being compared with a one-stage reaction. The amount of incorporation by the whole tissue is not greatly influenced by changes in the external medium, the only marked effect being due to shortening the period of starvation. The cell-free preparations show stimulation by ATP and by cytidine: coenzyme. Since t:he isotopic label is transferred directly from 32P-phosphorylcholine to phosphatidylcholine without appearing in observable amounts in other lipids or water-soluble compounds, it is concluded that the route of synthesis in Diptera is the same as that through phosphorylcholine for other insect orders and mammals. REFERENCES ALLEN R. R. and NEWBURGH R. W. (1965) Phospholipid composition of fat-bodies of Surcophczga buZZata. J. Insect Physiol. 11, 1601-1603. BARTLETT G. R. (1959) Phosphorus assay in column chromatography. 3’. biol. Chem. 234, 466-468. BRIDGES R. G., CRONE H. D., and BEARD J. R. (1962) A study of the phospholipids of dieldrin-,resistant and susceptible houseflies, with particular reference to those of the thoracic ganglion. In Radioisotopes and Radiation in Entomology, I.A.E.A., Vienna,

145-153.

624

H. D. CRONE, R. W. NEWBURGH,

ANDC. MEZEI

CHOJNACKIT. and KORZYBSKI T. (1963) On the specificity of &dine coenzyme in the incorporation of phosphorylcholine into phospholipids by tissue homogenates of various animal species. Acta b-iochim. Polon. 10, 455-461. CRONEH. D. and BRIDGESR. G. (1963) The phospholipids of the housefly, Mzrsca domestica. Biochem. J, 89, 11-21. DAWSON R. M. C. (1960) A hydrolytic procedure for the identification and estimation of individual phospholipids in biological samples. Biochem. J. 75, 45-53. FAST P. G. (1964) Insect lipids: A review. &L&Z. ent. Sot. Can. 37. SKIPSKI V. P., PETERSONR. F., SANDERSJ., and BARCLAYM. (1963) Thin-layer chromatography of phospholipids using silica gel without calcium sulphate binder. J. Lipid Res. 4, 227-228. SRIDHARAS. and BHAT J. V. (1962) Phospholipids of the silkworm Bombyx mori L. Curr. Sci. 31, 240. TAYLOR J. F. and HODGSONE. (1965) The origin of phospholipid ethanolamine in the blowfly, Phormia regina (Meig.). r. Insect Physiol. 11, 281-285. WEISBURGERJ. H. and SCHNEIDERW. C. (1961) An improved preparation of phosphorylethanolamine and phosphorylcholine. J. org. Chem. 26, 1658-1660.