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Time course of 32P-orthophosphate incorporation into different classes of phospholipids by homogenates of the fly, Ceratitis capitata
Insect Biochem., i97x, I, z37-z43. [Scientechnica (Publishers) Ltd.]
z37
TIME COURSE OF 8'P-ORTHOPHOSPHATE INCORPORATION INTO DIFFERENT CLASSES OF PHOSPHOLIPIDS BY HOMOGENATES OF THE FLY, CERATITIS CAPITATA* M. P. CASTILL(3N, R. E. CATAL,MN, A. M. MUNICIO, ANDA. SUAREZ Department of Biochemistry, Faculty of Sciences, University of Madrid, Spain
(Received, I
NOT). i
t97o)
ABSTRACT Ceratitis capitata adult homogenates were incubated with 'zP-orthophosphate for z6 hours and the time course of incorporation into phospholipid phosphorous and the specific radioactivity of the different phospholipid classes were evaluated. De novo biosynthesis of phosphatidylethanolarnine and phosphatidylcholine as well as the acylation of the lyso-derivatives take place during the first few hours (o-6 hours). A transacylation of both diacylglycerophospholipids occurs after 8-x3 hours. A direct metabolic conversion from phosphatidylethanolamine to its plasmalogen, and from phosphatidylcholine to phosphatidylserine, was found. A phosphatide transfer between serine and phosphatidylcholine is suggested. HACK, Gussin, and Lowe (x96z) studied 5 orders of insects showing the presence of the same general types of phospholipids as in the vertebrates. The larvae of the moth, Galleria mellonella, possess a remarkably high concentration of phospholipids in the haemolymph (Wlodawer and Wisniewoska, i965). Regarding the constitution of phospholipids, these authors assert that phosphatidylcholine makes up 60 per cent of the phospholipids from haemolymph while phosphatidylethanolamine constitutes about zo per cent. These data appear not to agree with the previous data from Lenartowicz ( 196 i) and Lenartowicz and Niemierko (x 964) showing the presence of more phosphorylethanolamine than phosphorylcholine in the larval waxmoth haemolymph. Gilbert (t967) explains this discrepancy by suggesting the possibility that phosphorylcholine, which is a precursor of CDP-choline and phosphatidylcholine, is present in higher quantities because of a block in the reactions leading to phosphatidylcholine, while the titre of phosphorylethanolamine remains low because of its rapid conversion to phosphatidylethanolamine. The role of phospholipids in the haemolymph may be involved with lipid transport as it is in mammals. Lenartowicz and Niemierko (I964) suggested that the haemolymph acts as a phospholipid depot, and proposed that the utilization of phosphorylethanolamine during starvation is higher than that of phosphorylcholine. These results were not confirmed by Wlodawer and Wisniewoska (t965) , who found small qualitative changes during starvation. In a developmental study of the phospholipid content of Bombyx mori, Niemierko, Wlodawer, and Wojtczak (i956) showed that the levels of phospholipids, as ratios of the * This work was supported by a grant from the Fondo Nacional para el desarollo de la Investigacion Cientffica.
238
CASTILL6NET AL.
Insect Biochem.
total lipids, reach a maximum at the end of metamorphosis. These investigators postulate a synthesis of phospholipids towards the last days of pharate adult development. Habibulla and Gilbert (x965) support this statement, working on Hyalophora, and suggest that it is mainly due to the development of the mitochondria in the flight muscles. These and other results (Bieber, Hodgson, Cheldelin, Brookes, and Newburgh, 1961; Bridges and Cox, i962; D'Costa and Birt, 1966) indicate few changes in the lipid phosphorous during development. Throughout the life cycle the main components were phosphatidylethanolamine and phosphatidylcholine with minor amounts of lysophosphatides, phosphatidylserine, and polyglycerophosphatides. The major phospholipid fraction has been identified as phosphatidylethanolamine (Bieber and others, I96i; Fast and Brown, I962) or phosphatidylcholine (Sridhara and Bhat, 1962; Chojnacki, i96i ; Chojnacki and Korzybski, 1962, 1963). Fast (1966) suggested that the predominance of phosphatidylethanolamine in the lipids of insects is associated with the occurrence of 5o per cent or more of the fatty acids with chain lengths less than i8 carbon atoms long. However, we found (CastiUdn, Catalan, Municio, and Suarez, 1971) similar amounts of phosphatidylethanolamine and phosphatidylcholine in two species of the same family (Tripetidae) of Diptera, Ceratitis capitata and Dacus oleae, in spite of the large difference between the values for palmitoleic acid exhibited by both insects. Throughout the life cycle of C. capitata we showed (Castilldn and others, x971) the existence of similar patterns of variation in the ratio phospholipid/lyso-derivative; the changes in this ratio were greatest at the larval-pupal transition and after adult emergence. Gilbert (I 967) warns of the importance of evaluating differences between synthesis and turnover and established that only a quantitative evaluation will allow a real differentiation between the two alternatives. Using ssP-orthophosphate Chojnacki and Korzybski (1962, I963) showed a higher rate of incorporation into phosphatidylethanolamine in Celerio euphorbiae adults than into phosphatidylcholine. On the basis of specific activities, they suggested that phosphatidylcholine is incorporated into phosphatidylethanolamine. These authors (Chojnacki and others, i961, x962) also showed the incorporation of 3sP-phosphorylcholine into phosphatidylcholine in C. euphorbiae and Arctia caja. • Crone and Bridges (I963), after injection of 3sP-phosphate into Musca domestica, found a greater specific activity in phosphatidylcholine than in phosphatidylethanolamine. Thomas and Gilbert (1967) studied the rate of incorporation of 3aP-phosphate into the fat body of diapausing and chilled pupa e and adult males of the silkworm Hyalophora cecropia. The results obtained suggested that the incorporation into phospholipids is greatest with adult fat body and least with fat body from diapausing pupae. The majority of the labelled phospholipids released from a pie-labelled fat body consists of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. In this paper quantitation of phospholipid classes and 82P-phosphate incorporation by C. capitata adult homogenates were measured during the course of 26-hour experiments. MATERIALS AND METHODS INSECT CULTURE Culturing of C. capitata (Wied.) was carried out according to the method previously described (Municio, Odriozola, and Pifieiro, x97o). All the experiments were done with newly emerged adults starved for z4 hours and given water ad libitum.
1971, I
s~pi INCORPORATION
INTO FLY LIPID$
239
P~.AGI~TS
Carrier-free asP-disodium phosphate was obtained from The Radiochemical Centre, Amersham. Standard phospholiplds and scintillation reagents were purchased from Sigma Chemicals. All other reagents were of the highest analytical grade available and were used without further purification.
In vitro CONDITIONS Whole, living adults were directly homogenized with 3 volumes of cold homogenizing buffer
(o'35 M sucrose-o'o5 M'Iris, pH 7"4) in a Potter-Elvehjem glass homogenizer with a teflon pestle, and filtered through 3 layers of cheese-cloth. The filtrate was centrifuged at 9oo g for lO minutes. These operations were carried out at a temperature of o--4° C. The supernatant, free from the upper lipid layer, was used for the incorporation studies. Total protein determination was performed in this homogenate by the method of Lowry, Rosebrough, Farr, and Randall (I95I). T h e incubation mixture consisted of: o'o I M MgCls; o" 123 M NaCI; 1"98 X 10-2 M ~T~"ICOs ; 5"1 × IO -3 M K H ~ P O ~ ; 7 x io -8 M Tris-HCl. T h e p H was adjusted to 7"4 at zz ° C. with HCI. T o this mixture were added 4o gc. (o.I ml.) carrier-free 82P-disodium phosphate and a volume of homogenate containing io mg. of proteins. T h e totalvolume was 2 ml. Incubation was performed in a shaker at 37 ° C. for differentperiods of time (I-3o hours). Following incubation, the reaction was stopped by the addition of 2"x volumes of cold methanol.
LIPID EXTRACTION Total lipids were extracted by the procedure of Bligh and Dyer (1959) under nitrogen. The combined chloroform extracts were washed with o'75 per cent NaCI solution and concentrated in vacuo. The lipids were taken up in chloroform-methanol (z :t) to i mg. per io M. SEPARATION OF PHOSPHOLIPIDS
Three rag. of the lipid solution were chromatographed on 3oo-~t thin-layer plates of silica gel G. The solvents used were chloroform/methanol/water (65:z5:4) as the first solvent and butanol/ acetic acid/water (6o:2o:zo) in the second dimension. The lipids were visualized by placing the plates in iodine vapour, and following evaporation of the iodine the spots were carefully removed and used for P determination and radioactivity estimation. PHOSPHORUSDETERMINATION Phosphorus was estimated according to the method previously described (Castill6n and others, z97I) based on the procedure of Bartlett (I959). ASSAYOF RADIOACTIVITY For the radioassay of individual phospholipids the spots from the thin-layer plates were transferred to scintillation vials containing io ml. of scintillation solution. The scintillation solution was PPO Io'5 g. ; methyl-POPOP 0.45 g.; naphthalene I5o g. ; dioxane to i5oo ml. ; and water to i8oo ml. Radioactivity was measured in a Nuclear Chicago Model 6755, low temperature, liquid scintillation spectrometer. Identification of the spots to be counted was made according to the methods of locating phospholipids, previously outlined (Castill6n and others, I971). RESULTS AND DISCUSSION
Figs. x- 4 show the evolution of the levels of the most important classes of phospholipids present in the homogenates (prepared from C. capitata adults) as well as the time course of the respective incorporation of s~p-orthophosphate. I n this way the interpretation of the results can be done more accurately, taking into account the important warning of Gilbert (i967).
24o
CASTILL(~NET AL.
Insect Biochera.
Fig. I shows the variations both in the amount of phosphorous and the specific radioactivity of phosphatidylethanoamine and lyso-phosphatidylethanolamine. Regarding the lyso-derivative, it is easy to realize the existence of a remarkable net decrease in its levels during the incubation, with a progressive diminution of the slope of the variation. On the other hand, the specific activity of lyso-phosphatidylethanolamine rises from the beginning, reaching a maximum after io-12 hours of incubation of the homogenate. This maximum is exactly coincident with the minimal values of both the levels and the specific activity of phosphatidylethanolamine. Both the evolution of phosphorous and the specific activity follow a similar pattern, and their initial simultaneous rise suggests a de novo biosynthesis of phosphatidylethanolamine, starting at the beginning of the incubation. The similarity in the magnitude of the specific activities of both ethanolamine glycerophosphatides allows one to suggest a metabolic relationship between them. c.p.m./,,Lg. P
#Z. P 4.0
,,f|
16,000
| I ! I
3.0
12,000
2.0
8000
1.0
4ooo
I
2
~
t
T
4
6
8
IO
I
I
12
14
1--
16
1
I
I
18
20
26
Hours
FIG. i . - - T i m e course of the incorporation of s'P-phosphate into phosphatidylethanolamine (O) and lyso-phosphatidyl ethanolamine (O) by a homogenate from C. capltata adults together with the variation in levels of phosphatidylethanolamine (O) and lysophosphatidylethanolamine ( I ) .
The sharp decrease of lyso-phosphatidylethanoamine and the counterbalance of its level with that of phosphatidylethanolamine might be interpreted as an initial acylation reaction. Therefore, during the initial stages of the in vitro experiments of a~P-orthophosphate incorporation by C. capitata adult homogenates, the following would occur : precursors --, phosphatidylethanolamine o-6 hours
lyso-phosphatidylethanolamine -~ phosphatidylethanolamine. The falling levels of lyso-phosphatidylethanolamine after 8 hours of incubation, and the clear and considerable increase of its specific activity coinciding with the diminution in amount of phosphatidylethanolamine, suggests the following metabolic change :-8-13 hours
phosphatidylethanolamine
-~ lyso-phosphatidylethanolamine.
i97z, I
81Pi INCORPORATIONINTO FLY LIPIDS
24I
It seems that the levels of the de novo synthesized diacylglycerophosphorylethanolamines exert a regulation on the activity of the enzymes implicated in the transacylation system. In this connexion a certain rhythmical variation of the ratio, phosphatidylethanolamine]lyso-derivative, has been previously described (Castill6n and others, I97z). Fig. 2 shows the variation of the amounts of phosphorus and of the specific radioactivity of phosphatidylcholine and its lyso-derivative. The levels of specific radioactivity reached by choline derivatives are about one-third of that of ethanolamine. From these data it is clear that there is a great difference in the metabolic activity of both types of phosphoglycerides. The pattern of changes in lyso-phosphatidylcholine levels is similar to that exhibited by the lyso-derivative of phosphatidylethanolamine. Therefore, a continuous decrease from the initial value occurs. The specific activity rises gradually to reach its maximal value at i6 hours. pg. P
,
c,p.m !t~g. P
4000 1.5
3000 I.O
2000
O'S IO00
/7 2
/" 4
6
8
I0
12
14
""'- .......
-,
16
26
18
20
Hours
FiG. 2.--Tirne course of the incorporation of 82p-phosphate into phosphatidyleholine (O) and lyso-phosphatidylcholine (F1) by a homogenate from C. capitata adults together with the variation in levels of phosphatidylcholine (O) and lyso-phosphatidylcholine (m).
Concerning phosphatidylcholine variations, both amount and specific activity follow a scheme analogous to that of phosphatidylethanolamine. The simultaneous rise of both amount and specific activity also suggests a de novo initial biosynthesis of phosphatidylcholine. The maximal levels of lyso-phosphatidylcholine were achieved about z hours after the highest value for phosphatidylcholine was reached. These changes allow us to suggest an initial transformation :-o-6 hours
lyso-phosphatidylcholinc -* phosphatidylcholine to be subsequently reversed during the interval 6-13 hours. Fig. 3 shows the variation of ethanolamine-plasmalogens. The quantitative values for this phospholipid follow a pattern that clearly resembles the evolution of activity of this compound. The specific activity shows a maximum between x2 and x8 hours of incubation. T h e values for the specific activity reached by this phospholipid are of the same
242
Insect Biochem.
ET AL.
CASTILLON
magnitude as those of the phosphatidylethanolamine. This fact might account for a direct formation of the alkyl linkage from the acyl group of phosphatidylethanolamine. Fig. 4 shows the corresponding data for phosphatidylserine. The quantitative values of this phospholipid appear to be only slightly increased after io-z 5 hours, coinciding /~g. p
~
c.p.m,]~Lg, P 16,0oo
1.5 14,000 -
12,000
t'O
I 0,000
SO00 6OO0 O.S "~ 4000
I
-
am
I
2 4 6 8 t0 12 14 16 18 20 26 Hours FIC. 3 . - - T i m e course of t h e i n c o r p o r a t i o n o f 8~P-phosphate into e t h a n o l a m i n e - p l a s m a logens ( O ) a n d t h e v a r i a t i o n in t h e i r level ( O ) . vg. p
c.p.m,/#g. P
5000
0~6
4OOO
0.S 3OOO 0.4
0'3
~
2000
0"2 "
-
1000
0.1
..... I , 2
I
t
t
!
4
6
8
I0
....
I
I
I
!
1
12
14
16
18
20
,,,I
26
Hours
FzG. 4 . - - T i m e course of t h e i n c o r p o r a t i o n of 82P-phosphate into p h o s p h a t i d y l s e r i n e ( O ) a n d t h e v a r i a t i o n in its level ( O ) .
with a major change in the specific radioactivity. Taking into account these variations and the similarity of specific radioactivity of both phosphatidylcholine and phosphatidylserine, the suggestion can be made as to the existence of a phosphatide transfer between serine and choline. This type of direct base exchange between ethanolamine and serine has been discussed by Crone (1967) in Musca domestica and by Beaudoin, Villeneuve, and Lemonde (1968) during the metamorphosis of Tribolium confusum.
~97~, x
s~Pi INCORPORATION INTO FLY LIPIDS
Z43
REFERENCES
BARTLETT,G. R. (x959), 'Phosphorous assay in column chromatography', .,¢. biol. Chem., 234~ 466-468. BEAUDOIN,A. R., VXLLENEUW,J. L., and LPMOND~,A. (I968), 'Variations des phospholipides au cours de la m~tamorphose de Tribolium confusum',~. Insect Physiol., I4b 83x'-84o. BtEBER, L. L., HODGSON, E., CI~LDELIN, V. H., BROOKm, V. J., and NEWBURGH, R. W. (I96x), ' Phospholipid patterns in the blowfly, Phormia regina',.~, biol. Chem., 236, z59o-z595. BLtGH, E. G., and DYER, W. J. (x959), ' A rapid method of total lipid extraction and purification', Can. a~. Biochem. Physiol., $9, 9xI-9x7. BRtDGSS, R. G., and Cox, J. T. (I962), ' Changes in lipids of the housefly during metamorphosis and development', Pest Infest. Res., 6, 53-54. CASTXLL6N, M. P., CATALAN, R. E., MUNICXO,A. M., and SuAm~Z,A. (I97I), 'Biochemistry of the development of the insects Dacus oleae and Ceratitis capitata. Evolution of phospholipids', Comp. Biochem. Physiol., 38B, xo9-It7. CHOJNACKI, T. (I96z), 'Biosyntheze fosfolipidow u awadow. II. Badania nad wlaczeniem 8aPeortofosforanu u motyla Celerio euphorbiae', Acta biochim, pol., 8, t67-x75. CHOJNACKI,T., and KOnZYBSKI,T. (x96z), 'Biosynthesis of phospholipids in insects. I I L Incorporation of a2P-orthophosphate into phospholipids of Arctia caia', Acta biochim, pol., 9, 95-t 1o. CHOJNACKI,T., and KORZYSSKt,T. (1963) , ' On the specificity of cytidine coenzyme in the incorporation of phosphorylcholine into phospholipids by tissue homogenates of various animal species', Acta biochim, pol., IO, 455-46I. CRONE, H. D. (x967), ' The relationship between phosphatide serine and ethanolamine in larvae of the housefly Musca domestica', jY. Insect Physiol., IS, 8 I--9o. CRONE, H. D., and BRIDGm, R. G. (x963), ' T h e phospholipids of the housefly, Musca domestica', Biochem.y., 89, ti--zi. D'CosTA, M. A., and BraT, L. M. (x966), 'Changes in the lipid content during the metamorphosis of the blowfly, Lucilia', J. Insect Physiol., I2, 1377-I 394. FAST, P. G. (x966), 'A comparative study of the phospholipids and fatty acids of some insects', Lipid.s, I, 2o9-2x 5. FasT, P. G., and BROWN,A. W. A. (I962), ' Lipids from DDT-resistant and susceptible larvae of Aedes aegypti', Ann. ent. Soc. Am., 55~ 663-672. GILB~T, L. I. (x967), ' Lipid metabolism and function in insects', Adv. Insect Physiol., 4, 69-z xx. HABmULLA, M., and Gmn~RT, L. I. (I965), cited in GILBERT(X967). HACK, M. H., GUSSIN, A. E., and LOWE, M. E. (z96z), 'Comparative lipid biochemistry. I. Phosphatides of invertebrates', Comp. Biochem. Physiol., $, z x7-zz I. L~ARTOWICZ, E. (I96x), ' T h e effect of low temperature upon phosphorous metabolism in Galleria mellonella larvae', Acta biochim, poL, 8, 15-z4. LI~Pa~TOWlCZ, E., and NIEMI~O, S. (I964), 'Phosphorylethanolamine and phosphorylcholine in the haemolymph of larvae of Galleia mellonella during starvation',aT. Insect Physiol., to, 89--96. LowRY, O. H., ROSEBROUCH,N. J., FAtaL A. L., and RANDALL,R. J. (195I), 'Protein measurement with the Folin phenol reagent', ft. biol. Chem., I95~ z65-z75. M~.r~Icxo, A. M., ODRtOZOt.A,J. M., and Pxgrsmo, A. (x97o), ' Biochemistry of the development of the insect Ceratitis capitata. In vitro biosynthesis of fatty acids from x~C-acetate during metamorphosis', Comp. Biochem. Physiol., 37~ 387-395. NmMm~a~o, S., WLODAW~R,P., and WOJTCZAK,A. F. (~956),' Lipid and phosphorous metabolism during growth of the silkworm (Bombyx mori)', Acta BioL exp., Vats., x% z55-z76. SRtDH~, S., and BHAT, J. V. (x96z), 'Phospholipids of the silkworm, Bombyx mori', Curr. Sci., 31~ 240. THOMAS, K. K., and GXLB~RT,L. I. (~967), 'In vitro studies on the release and transport of phospholipids', j~. Insect Physiol., ~ , 963--980. WLOD~W~, P., and W~sNmwosg~, A. (~965), 'Lipids in the haemolymph of waxmoth larvae during starvation ', a~. Insect Physiol., l I , II-2o.
K ~ Word Index: Phospholipids in insects, Ceratitis capitata, 3zP-phosphate incorporation by insects, biosynthesis of phosphatidylcholine and phosphatidylethanolamine.
z37
TIME COURSE OF 8'P-ORTHOPHOSPHATE INCORPORATION INTO DIFFERENT CLASSES OF PHOSPHOLIPIDS BY HOMOGENATES OF THE FLY, CERATITIS CAPITATA* M. P. CASTILL(3N, R. E. CATAL,MN, A. M. MUNICIO, ANDA. SUAREZ Department of Biochemistry, Faculty of Sciences, University of Madrid, Spain
(Received, I
NOT). i
t97o)
ABSTRACT Ceratitis capitata adult homogenates were incubated with 'zP-orthophosphate for z6 hours and the time course of incorporation into phospholipid phosphorous and the specific radioactivity of the different phospholipid classes were evaluated. De novo biosynthesis of phosphatidylethanolarnine and phosphatidylcholine as well as the acylation of the lyso-derivatives take place during the first few hours (o-6 hours). A transacylation of both diacylglycerophospholipids occurs after 8-x3 hours. A direct metabolic conversion from phosphatidylethanolamine to its plasmalogen, and from phosphatidylcholine to phosphatidylserine, was found. A phosphatide transfer between serine and phosphatidylcholine is suggested. HACK, Gussin, and Lowe (x96z) studied 5 orders of insects showing the presence of the same general types of phospholipids as in the vertebrates. The larvae of the moth, Galleria mellonella, possess a remarkably high concentration of phospholipids in the haemolymph (Wlodawer and Wisniewoska, i965). Regarding the constitution of phospholipids, these authors assert that phosphatidylcholine makes up 60 per cent of the phospholipids from haemolymph while phosphatidylethanolamine constitutes about zo per cent. These data appear not to agree with the previous data from Lenartowicz ( 196 i) and Lenartowicz and Niemierko (x 964) showing the presence of more phosphorylethanolamine than phosphorylcholine in the larval waxmoth haemolymph. Gilbert (t967) explains this discrepancy by suggesting the possibility that phosphorylcholine, which is a precursor of CDP-choline and phosphatidylcholine, is present in higher quantities because of a block in the reactions leading to phosphatidylcholine, while the titre of phosphorylethanolamine remains low because of its rapid conversion to phosphatidylethanolamine. The role of phospholipids in the haemolymph may be involved with lipid transport as it is in mammals. Lenartowicz and Niemierko (I964) suggested that the haemolymph acts as a phospholipid depot, and proposed that the utilization of phosphorylethanolamine during starvation is higher than that of phosphorylcholine. These results were not confirmed by Wlodawer and Wisniewoska (t965) , who found small qualitative changes during starvation. In a developmental study of the phospholipid content of Bombyx mori, Niemierko, Wlodawer, and Wojtczak (i956) showed that the levels of phospholipids, as ratios of the * This work was supported by a grant from the Fondo Nacional para el desarollo de la Investigacion Cientffica.
238
CASTILL6NET AL.
Insect Biochem.
total lipids, reach a maximum at the end of metamorphosis. These investigators postulate a synthesis of phospholipids towards the last days of pharate adult development. Habibulla and Gilbert (x965) support this statement, working on Hyalophora, and suggest that it is mainly due to the development of the mitochondria in the flight muscles. These and other results (Bieber, Hodgson, Cheldelin, Brookes, and Newburgh, 1961; Bridges and Cox, i962; D'Costa and Birt, 1966) indicate few changes in the lipid phosphorous during development. Throughout the life cycle the main components were phosphatidylethanolamine and phosphatidylcholine with minor amounts of lysophosphatides, phosphatidylserine, and polyglycerophosphatides. The major phospholipid fraction has been identified as phosphatidylethanolamine (Bieber and others, I96i; Fast and Brown, I962) or phosphatidylcholine (Sridhara and Bhat, 1962; Chojnacki, i96i ; Chojnacki and Korzybski, 1962, 1963). Fast (1966) suggested that the predominance of phosphatidylethanolamine in the lipids of insects is associated with the occurrence of 5o per cent or more of the fatty acids with chain lengths less than i8 carbon atoms long. However, we found (CastiUdn, Catalan, Municio, and Suarez, 1971) similar amounts of phosphatidylethanolamine and phosphatidylcholine in two species of the same family (Tripetidae) of Diptera, Ceratitis capitata and Dacus oleae, in spite of the large difference between the values for palmitoleic acid exhibited by both insects. Throughout the life cycle of C. capitata we showed (Castilldn and others, x971) the existence of similar patterns of variation in the ratio phospholipid/lyso-derivative; the changes in this ratio were greatest at the larval-pupal transition and after adult emergence. Gilbert (I 967) warns of the importance of evaluating differences between synthesis and turnover and established that only a quantitative evaluation will allow a real differentiation between the two alternatives. Using ssP-orthophosphate Chojnacki and Korzybski (1962, I963) showed a higher rate of incorporation into phosphatidylethanolamine in Celerio euphorbiae adults than into phosphatidylcholine. On the basis of specific activities, they suggested that phosphatidylcholine is incorporated into phosphatidylethanolamine. These authors (Chojnacki and others, i961, x962) also showed the incorporation of 3sP-phosphorylcholine into phosphatidylcholine in C. euphorbiae and Arctia caja. • Crone and Bridges (I963), after injection of 3sP-phosphate into Musca domestica, found a greater specific activity in phosphatidylcholine than in phosphatidylethanolamine. Thomas and Gilbert (1967) studied the rate of incorporation of 3aP-phosphate into the fat body of diapausing and chilled pupa e and adult males of the silkworm Hyalophora cecropia. The results obtained suggested that the incorporation into phospholipids is greatest with adult fat body and least with fat body from diapausing pupae. The majority of the labelled phospholipids released from a pie-labelled fat body consists of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. In this paper quantitation of phospholipid classes and 82P-phosphate incorporation by C. capitata adult homogenates were measured during the course of 26-hour experiments. MATERIALS AND METHODS INSECT CULTURE Culturing of C. capitata (Wied.) was carried out according to the method previously described (Municio, Odriozola, and Pifieiro, x97o). All the experiments were done with newly emerged adults starved for z4 hours and given water ad libitum.
1971, I
s~pi INCORPORATION
INTO FLY LIPID$
239
P~.AGI~TS
Carrier-free asP-disodium phosphate was obtained from The Radiochemical Centre, Amersham. Standard phospholiplds and scintillation reagents were purchased from Sigma Chemicals. All other reagents were of the highest analytical grade available and were used without further purification.
In vitro CONDITIONS Whole, living adults were directly homogenized with 3 volumes of cold homogenizing buffer
(o'35 M sucrose-o'o5 M'Iris, pH 7"4) in a Potter-Elvehjem glass homogenizer with a teflon pestle, and filtered through 3 layers of cheese-cloth. The filtrate was centrifuged at 9oo g for lO minutes. These operations were carried out at a temperature of o--4° C. The supernatant, free from the upper lipid layer, was used for the incorporation studies. Total protein determination was performed in this homogenate by the method of Lowry, Rosebrough, Farr, and Randall (I95I). T h e incubation mixture consisted of: o'o I M MgCls; o" 123 M NaCI; 1"98 X 10-2 M ~T~"ICOs ; 5"1 × IO -3 M K H ~ P O ~ ; 7 x io -8 M Tris-HCl. T h e p H was adjusted to 7"4 at zz ° C. with HCI. T o this mixture were added 4o gc. (o.I ml.) carrier-free 82P-disodium phosphate and a volume of homogenate containing io mg. of proteins. T h e totalvolume was 2 ml. Incubation was performed in a shaker at 37 ° C. for differentperiods of time (I-3o hours). Following incubation, the reaction was stopped by the addition of 2"x volumes of cold methanol.
LIPID EXTRACTION Total lipids were extracted by the procedure of Bligh and Dyer (1959) under nitrogen. The combined chloroform extracts were washed with o'75 per cent NaCI solution and concentrated in vacuo. The lipids were taken up in chloroform-methanol (z :t) to i mg. per io M. SEPARATION OF PHOSPHOLIPIDS
Three rag. of the lipid solution were chromatographed on 3oo-~t thin-layer plates of silica gel G. The solvents used were chloroform/methanol/water (65:z5:4) as the first solvent and butanol/ acetic acid/water (6o:2o:zo) in the second dimension. The lipids were visualized by placing the plates in iodine vapour, and following evaporation of the iodine the spots were carefully removed and used for P determination and radioactivity estimation. PHOSPHORUSDETERMINATION Phosphorus was estimated according to the method previously described (Castill6n and others, z97I) based on the procedure of Bartlett (I959). ASSAYOF RADIOACTIVITY For the radioassay of individual phospholipids the spots from the thin-layer plates were transferred to scintillation vials containing io ml. of scintillation solution. The scintillation solution was PPO Io'5 g. ; methyl-POPOP 0.45 g.; naphthalene I5o g. ; dioxane to i5oo ml. ; and water to i8oo ml. Radioactivity was measured in a Nuclear Chicago Model 6755, low temperature, liquid scintillation spectrometer. Identification of the spots to be counted was made according to the methods of locating phospholipids, previously outlined (Castill6n and others, I971). RESULTS AND DISCUSSION
Figs. x- 4 show the evolution of the levels of the most important classes of phospholipids present in the homogenates (prepared from C. capitata adults) as well as the time course of the respective incorporation of s~p-orthophosphate. I n this way the interpretation of the results can be done more accurately, taking into account the important warning of Gilbert (i967).
24o
CASTILL(~NET AL.
Insect Biochera.
Fig. I shows the variations both in the amount of phosphorous and the specific radioactivity of phosphatidylethanoamine and lyso-phosphatidylethanolamine. Regarding the lyso-derivative, it is easy to realize the existence of a remarkable net decrease in its levels during the incubation, with a progressive diminution of the slope of the variation. On the other hand, the specific activity of lyso-phosphatidylethanolamine rises from the beginning, reaching a maximum after io-12 hours of incubation of the homogenate. This maximum is exactly coincident with the minimal values of both the levels and the specific activity of phosphatidylethanolamine. Both the evolution of phosphorous and the specific activity follow a similar pattern, and their initial simultaneous rise suggests a de novo biosynthesis of phosphatidylethanolamine, starting at the beginning of the incubation. The similarity in the magnitude of the specific activities of both ethanolamine glycerophosphatides allows one to suggest a metabolic relationship between them. c.p.m./,,Lg. P
#Z. P 4.0
,,f|
16,000
| I ! I
3.0
12,000
2.0
8000
1.0
4ooo
I
2
~
t
T
4
6
8
IO
I
I
12
14
1--
16
1
I
I
18
20
26
Hours
FIG. i . - - T i m e course of the incorporation of s'P-phosphate into phosphatidylethanolamine (O) and lyso-phosphatidyl ethanolamine (O) by a homogenate from C. capltata adults together with the variation in levels of phosphatidylethanolamine (O) and lysophosphatidylethanolamine ( I ) .
The sharp decrease of lyso-phosphatidylethanoamine and the counterbalance of its level with that of phosphatidylethanolamine might be interpreted as an initial acylation reaction. Therefore, during the initial stages of the in vitro experiments of a~P-orthophosphate incorporation by C. capitata adult homogenates, the following would occur : precursors --, phosphatidylethanolamine o-6 hours
lyso-phosphatidylethanolamine -~ phosphatidylethanolamine. The falling levels of lyso-phosphatidylethanolamine after 8 hours of incubation, and the clear and considerable increase of its specific activity coinciding with the diminution in amount of phosphatidylethanolamine, suggests the following metabolic change :-8-13 hours
phosphatidylethanolamine
-~ lyso-phosphatidylethanolamine.
i97z, I
81Pi INCORPORATIONINTO FLY LIPIDS
24I
It seems that the levels of the de novo synthesized diacylglycerophosphorylethanolamines exert a regulation on the activity of the enzymes implicated in the transacylation system. In this connexion a certain rhythmical variation of the ratio, phosphatidylethanolamine]lyso-derivative, has been previously described (Castill6n and others, I97z). Fig. 2 shows the variation of the amounts of phosphorus and of the specific radioactivity of phosphatidylcholine and its lyso-derivative. The levels of specific radioactivity reached by choline derivatives are about one-third of that of ethanolamine. From these data it is clear that there is a great difference in the metabolic activity of both types of phosphoglycerides. The pattern of changes in lyso-phosphatidylcholine levels is similar to that exhibited by the lyso-derivative of phosphatidylethanolamine. Therefore, a continuous decrease from the initial value occurs. The specific activity rises gradually to reach its maximal value at i6 hours. pg. P
,
c,p.m !t~g. P
4000 1.5
3000 I.O
2000
O'S IO00
/7 2
/" 4
6
8
I0
12
14
""'- .......
-,
16
26
18
20
Hours
FiG. 2.--Tirne course of the incorporation of 82p-phosphate into phosphatidyleholine (O) and lyso-phosphatidylcholine (F1) by a homogenate from C. capitata adults together with the variation in levels of phosphatidylcholine (O) and lyso-phosphatidylcholine (m).
Concerning phosphatidylcholine variations, both amount and specific activity follow a scheme analogous to that of phosphatidylethanolamine. The simultaneous rise of both amount and specific activity also suggests a de novo initial biosynthesis of phosphatidylcholine. The maximal levels of lyso-phosphatidylcholine were achieved about z hours after the highest value for phosphatidylcholine was reached. These changes allow us to suggest an initial transformation :-o-6 hours
lyso-phosphatidylcholinc -* phosphatidylcholine to be subsequently reversed during the interval 6-13 hours. Fig. 3 shows the variation of ethanolamine-plasmalogens. The quantitative values for this phospholipid follow a pattern that clearly resembles the evolution of activity of this compound. The specific activity shows a maximum between x2 and x8 hours of incubation. T h e values for the specific activity reached by this phospholipid are of the same
242
Insect Biochem.
ET AL.
CASTILLON
magnitude as those of the phosphatidylethanolamine. This fact might account for a direct formation of the alkyl linkage from the acyl group of phosphatidylethanolamine. Fig. 4 shows the corresponding data for phosphatidylserine. The quantitative values of this phospholipid appear to be only slightly increased after io-z 5 hours, coinciding /~g. p
~
c.p.m,]~Lg, P 16,0oo
1.5 14,000 -
12,000
t'O
I 0,000
SO00 6OO0 O.S "~ 4000
I
-
am
I
2 4 6 8 t0 12 14 16 18 20 26 Hours FIC. 3 . - - T i m e course of t h e i n c o r p o r a t i o n o f 8~P-phosphate into e t h a n o l a m i n e - p l a s m a logens ( O ) a n d t h e v a r i a t i o n in t h e i r level ( O ) . vg. p
c.p.m,/#g. P
5000
0~6
4OOO
0.S 3OOO 0.4
0'3
~
2000
0"2 "
-
1000
0.1
..... I , 2
I
t
t
!
4
6
8
I0
....
I
I
I
!
1
12
14
16
18
20
,,,I
26
Hours
FzG. 4 . - - T i m e course of t h e i n c o r p o r a t i o n of 82P-phosphate into p h o s p h a t i d y l s e r i n e ( O ) a n d t h e v a r i a t i o n in its level ( O ) .
with a major change in the specific radioactivity. Taking into account these variations and the similarity of specific radioactivity of both phosphatidylcholine and phosphatidylserine, the suggestion can be made as to the existence of a phosphatide transfer between serine and choline. This type of direct base exchange between ethanolamine and serine has been discussed by Crone (1967) in Musca domestica and by Beaudoin, Villeneuve, and Lemonde (1968) during the metamorphosis of Tribolium confusum.
~97~, x
s~Pi INCORPORATION INTO FLY LIPIDS
Z43
REFERENCES
BARTLETT,G. R. (x959), 'Phosphorous assay in column chromatography', .,¢. biol. Chem., 234~ 466-468. BEAUDOIN,A. R., VXLLENEUW,J. L., and LPMOND~,A. (I968), 'Variations des phospholipides au cours de la m~tamorphose de Tribolium confusum',~. Insect Physiol., I4b 83x'-84o. BtEBER, L. L., HODGSON, E., CI~LDELIN, V. H., BROOKm, V. J., and NEWBURGH, R. W. (I96x), ' Phospholipid patterns in the blowfly, Phormia regina',.~, biol. Chem., 236, z59o-z595. BLtGH, E. G., and DYER, W. J. (x959), ' A rapid method of total lipid extraction and purification', Can. a~. Biochem. Physiol., $9, 9xI-9x7. BRtDGSS, R. G., and Cox, J. T. (I962), ' Changes in lipids of the housefly during metamorphosis and development', Pest Infest. Res., 6, 53-54. CASTXLL6N, M. P., CATALAN, R. E., MUNICXO,A. M., and SuAm~Z,A. (I97I), 'Biochemistry of the development of the insects Dacus oleae and Ceratitis capitata. Evolution of phospholipids', Comp. Biochem. Physiol., 38B, xo9-It7. CHOJNACKI, T. (I96z), 'Biosyntheze fosfolipidow u awadow. II. Badania nad wlaczeniem 8aPeortofosforanu u motyla Celerio euphorbiae', Acta biochim, pol., 8, t67-x75. CHOJNACKI,T., and KOnZYBSKI,T. (x96z), 'Biosynthesis of phospholipids in insects. I I L Incorporation of a2P-orthophosphate into phospholipids of Arctia caia', Acta biochim, pol., 9, 95-t 1o. CHOJNACKI,T., and KORZYSSKt,T. (1963) , ' On the specificity of cytidine coenzyme in the incorporation of phosphorylcholine into phospholipids by tissue homogenates of various animal species', Acta biochim, pol., IO, 455-46I. CRONE, H. D. (x967), ' The relationship between phosphatide serine and ethanolamine in larvae of the housefly Musca domestica', jY. Insect Physiol., IS, 8 I--9o. CRONE, H. D., and BRIDGm, R. G. (x963), ' T h e phospholipids of the housefly, Musca domestica', Biochem.y., 89, ti--zi. D'CosTA, M. A., and BraT, L. M. (x966), 'Changes in the lipid content during the metamorphosis of the blowfly, Lucilia', J. Insect Physiol., I2, 1377-I 394. FAST, P. G. (x966), 'A comparative study of the phospholipids and fatty acids of some insects', Lipid.s, I, 2o9-2x 5. FasT, P. G., and BROWN,A. W. A. (I962), ' Lipids from DDT-resistant and susceptible larvae of Aedes aegypti', Ann. ent. Soc. Am., 55~ 663-672. GILB~T, L. I. (x967), ' Lipid metabolism and function in insects', Adv. Insect Physiol., 4, 69-z xx. HABmULLA, M., and Gmn~RT, L. I. (I965), cited in GILBERT(X967). HACK, M. H., GUSSIN, A. E., and LOWE, M. E. (z96z), 'Comparative lipid biochemistry. I. Phosphatides of invertebrates', Comp. Biochem. Physiol., $, z x7-zz I. L~ARTOWICZ, E. (I96x), ' T h e effect of low temperature upon phosphorous metabolism in Galleria mellonella larvae', Acta biochim, poL, 8, 15-z4. LI~Pa~TOWlCZ, E., and NIEMI~O, S. (I964), 'Phosphorylethanolamine and phosphorylcholine in the haemolymph of larvae of Galleia mellonella during starvation',aT. Insect Physiol., to, 89--96. LowRY, O. H., ROSEBROUCH,N. J., FAtaL A. L., and RANDALL,R. J. (195I), 'Protein measurement with the Folin phenol reagent', ft. biol. Chem., I95~ z65-z75. M~.r~Icxo, A. M., ODRtOZOt.A,J. M., and Pxgrsmo, A. (x97o), ' Biochemistry of the development of the insect Ceratitis capitata. In vitro biosynthesis of fatty acids from x~C-acetate during metamorphosis', Comp. Biochem. Physiol., 37~ 387-395. NmMm~a~o, S., WLODAW~R,P., and WOJTCZAK,A. F. (~956),' Lipid and phosphorous metabolism during growth of the silkworm (Bombyx mori)', Acta BioL exp., Vats., x% z55-z76. SRtDH~, S., and BHAT, J. V. (x96z), 'Phospholipids of the silkworm, Bombyx mori', Curr. Sci., 31~ 240. THOMAS, K. K., and GXLB~RT,L. I. (~967), 'In vitro studies on the release and transport of phospholipids', j~. Insect Physiol., ~ , 963--980. WLOD~W~, P., and W~sNmwosg~, A. (~965), 'Lipids in the haemolymph of waxmoth larvae during starvation ', a~. Insect Physiol., l I , II-2o.
K ~ Word Index: Phospholipids in insects, Ceratitis capitata, 3zP-phosphate incorporation by insects, biosynthesis of phosphatidylcholine and phosphatidylethanolamine.