On Methylating Activity of L-(Methy1-1 4C)-Methionine in Metabolism of Phospholipids by Insect Ceratitis capitata C. JIMENEZ, A.M. MUNICIO, and A. SUAREZ, Department of Biochemistry, Faculty of Sciences, University of Madrid, Spain ABSTRACT

when dimethyl aminoethanol is added to the diet (3,4). On the other hand, by the injection of L-(methylA4C)-methionine to larvae of Vitula edmandsae serratilineela the presence of a transmethylation system capable of methylating phosphatidyl ethanolamine (PE) to form phosphatidyl-N-methyl ethanolamine, phosphatidyl-N,N'-dimethyl ethanolamine, and phosphatidyl choline (PC) was indicated (5). In a series of previous experiments, the levels and specific radioactivities of the main phospholipid classes from Ceratitis capitata at different stages of development were determined when 5 day old larvae were fed on diets containing either 3H-glycerol, 32p-orthophosphate, or 14C-acetate (6). The clear tendency to equalize the specific activities of PC, PE, and phosphatidyl serine suggested the possibility of participation of base conversion mechanisms during the metamorphosis of the insect. The purpose of this contribution is to investigate further the methylating activity of L-(methylA4C)-methionine either when it was fed to larvae of C. capitata or when it was incubated in the presence of larval or pharate adult homogenates of the insect.

The methylating activity of L-(methyl14C)-methionine in different stages of development of the insect Ceratitis capitara was studied in a series of in vitro and in vivo experiments. Larval and pharate adult homogenates of the insect were used in the in vitro conditions, and the utilization of the methyl group of methionine for the synthesis of different classes of phospholipids was evaluated. Incorporation of radioactivity in lipids by pharate adult homogenates was significmltly higher than that by larval homogenates. In both cases, phosphatidyl ethanolamine showed the highest levels of radioactivity incorporation. Free bases from total lipid hydrolysates were resolved and identified by paper chromatography, and the labeling was investigated by radioactivity scanning of paper chromatograms. Significant differences were observed in the activity of both stages of development of the insect. Larval and pharate adult homogenates incorporated mainly the labeled methyl groups into ethanolamine. Monomethyl ethanolamine was the only methyl derivative that appeared in the hydrolysates of lipids synthesized by larval homogenates, whereas mono-, di- and trimethyl ethanolamine clearly were detected in those synthesized by pharate adult homogenates. Administration of L-(methyl14C)-methionine to larvae confirmed the existence of methylation reactions ir~ the metabolic activity of the insect.

MATERIALS A N D METHODS Rearing of Insects

Larval and pharate adult C. capitata (Wiedemann) were used. Diet, temperature, and humidity conditions during culturing were controlled carefully. Culturing of the insect was carried out under the conditions previously described (7). Preparation of Homogenates

INTRODUCTION

It generally is accepted that transmethylation reactions have no signification in the phospholipid metabolism among insects (1). Thus, no evidence for transmethylation products was obtained by the labeling found in fractionated tissue extracts of Tribolium confusum, Phormia regina, and other insectsreared on diets containing labeled methyl donors (2). Nevertheless, larvae of Phormia regina and Musca domestica can synthesize large amounts of phosphatidyl-N,N'-dimethyl ethanolamine 82

Larvae were reared until 2-3 days before the larval-pupal apolysis and were starved 3-4 hr before used. Pharate adults were collected 5 days beyond the larval-pupal apolysis. Both larvae and pharate adult were washed carefully with distilled water before use in the experiments. Larvae and pharate adults were homogenized directly with 3 vol cold homogenizing buffer (0.35 M sucrose-0.05 M tris, pH 7.4) in a Potter-Elvehjem glass homogenizer with a Teflon pestle. Homogenates were handled according to the method described (8). Floating lipids were removed from larval and pharate adult

METHYLATING ACTIVITY IN C. CAPITA TA cpm

83

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7 1;

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6b

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FIG. 1. Radioactivity incorporated into different classes of phospholipids by larval homogenates of

Ceratitis capitata from L-(methyl-14C)-methionine. PE = phosphatidyl ethanolamine, PC = phosphatidyl choline, LPC = lysophosphatidyl choline, and LPE = lysophosphatidyl ethanolamine.

homogenates used as enzyme preparations in the in vitro experiments. In vitro Assay Mixtures

The assay mixture for incorporating L( m e t h y l -1 4C)-methionine contained/1 ml: adenosine 5'-triphosphate, 2.4 rag; a-glycerophosphate, 2.6 mg; MgC12, 2.0 rag; NaHCO3, 1.6 rag; NaC1, 7.2 rag; KH2PO4, 0.70 mg; tris-base, 0.8 mg; sodium penicillin, 0.4 mg; streptomycin sulphate, 0.4 rag; phosphatidylN,N'-dimethyl ethanolamine, 4 mg; and sodium desoxycholate, 8 mg. The mixture was sonicated for 15 sec. To 0.5 ml mixture was added 0.5 ml respective homogenate containing 10-12 mg proteins and 0.1 ml (12.5 /~Ci) of labeled methionine (specific activity, 56 mCi/mmol; The Radiochemical Centre, Amersham, England). Incubations were carried out in a shaker at 37 C for different times. In vivo Assays

Five day old larvae (5 g) were starved 5 hrs and fed afterwards for 3 hr on 0.5 g diet containing 250/ICi L-(methy1-14 C)-methionine. After this time, 1 g pool of larvae were washed carefully and submitted to the extraction of lipids. The rest of the pool of larvae were left to pupate under the ordinary conditions of diet and collected as 5 day old pharate adults. Insects at this stage of development also were submitted to the general procedure of extraction of lipids. Extraction of Lipids

At the end of the incubation, the reaction was stopped by the addition of chloroform, and

~

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90

60

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FIG. 2. Radioactivity incorporated into different classes of phospholipids by pharate adult homogenates

of Ceratitis capitata from L-(methyl-14C)-methionine. PE = phosphatidyl ethanolaminc, LPC = lysophosphatidyl choline, PC = phosphatidyl choline, PC = phosphatidyl choline, and LPE = lysophosphatidyl ethanolamine. total lipids were obtained according to the method of Bligh and Dyer (9). Larvae and pharate adults from the in vivo experiments also were extracted according to the procedure of Bligh and Dyer (9). Phosphorous was estimated according to the method previously described (10) based upon the procedure of Bartlett (1 1). Fractionation of Classes of Lipids

The total lipids (3 g) were separated on thin layer plates prepared by coating a slurry of 65 g Silica Gel G (E. Merck, Darmstadt, Germany) in 240 ml distilled water to a thickness of 0.3 ram. The solvents used were chloroform-methanolwater (65:25:4, v/v/v) as the first solvent and butanol-acetic acid-water (60:20:20, v/v/v) in the second direction. Lipids were visualized by placing the plates in iodine vapor, and, following evaporation of the iodine, the spots were removed carefully and used for either P deterruination or radioactivity estimation. Hydrolysis of kipids

Total lipids were hydrolyzed with N HC1 (0.1 ml/mg lipids) at 100 C for 16 hr. After hydrolysis, the mixture was extracted with 3 vol heptane (3 times), and the aqueous solution was analyzed by paper chromatography. Whatman no. 1 paper, impregnated in N KC1 solution and dried, was used for paper chromatography. The upper phase of the mixture phenol-n-butanol-80% formic acid-KC1 saturated water (50:50:3:10, w/v/v/v) was used as developing system. Location of radioactive LIPIDS, VOL. 10, NO. 2

C. JIMENEZ, A.M. MUNIC10 AND A. SUAREZ

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30

60

120

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FIG. 5. Changes of the quantitative composition (~g P/I 0 mg proteins) of different phospholipid classes FIG. 3. Specific activities (sp act) (logarithmic during incubation of L-(methyl-14:C)-methioninewith scale) of phosphatidyl ethanolamine (PE) and phos- pharate adult homgenates of Ceratitis capitata. PE = phatidyl choline (PC) vs time of incubation with larval phosphatidyl ethanolamine, PC = phosphatidyl choand pharate adult homogenates of Ceratitis capitata. line, LPE = lysophosphatidyl ethanolamine, and LPC = lysophosphatidyl choline. *c ;,~ 3b

60

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RESULTS AND DISCUSSION

In vitro and in vivo series of experiments were carried out to test the behavior of L-(methyl-14C)-methionine as methyl donor during different phases of development of the insect C. capitata. The in vitro experiments consist in a time L-PE ~ study of incorporation of radioactivity into the ______-----~ L'PC main phospholipid classes, PE, PC, and their lysoderivatives (lysophosphatidyl ethanolamine [LPE] and lysophosphatiyl choline [LPC]) by 15 30 6O 120 IS0 mm either larval or pharate adult homogenates of FIG. 4. Changes of the quantitative composition the insect. 0zg P[10 mg proteins) of different phospholipid classes Figures I and 2 show the distribution of the during incubation of L-(methyl-14-C)-methioninewith larval homogenates of Ceratitis capitata. PC = phos- incorporated radioactivity by larval and pharate phatidyl choline, PE = phosphatidyl ethanolamine, adult homogenates, respectively. Incorporation LPE = lysophosphatidyl ethanolamine, and LPC = took place rapidly, and PE accounted in both lysophosphatidyl choline. cases for the highest levels; however, incorporation was noticeably higher in the pharate adult spots on papers was carried out by autoradioghomogenates than in those of the larval stage. raphy using Valca, H-27, films. PC exhibited low levels of radioactivity, and the PE/PC content ratio was higher in the pharate Measurements of Radioactivity For the radioassay of individual classes of adult than in the larval homogenates. The lipids, the spots from the thin layer plates were incorporation of radioactNity in the lysoderivatransferred to scintillation vials containing 10 tires was the same as that of PC in both ml scintillation solution (4 g diphenyloxazole homogenates. These findings clearly show that larval and [PPO], 0.1 g diphenytoxazole-benzene pharate adult homogenates of the insect exhibit [POPOP], toluene to 1 liter). Paper chromatograms were cut into 3 mm a different incorporation capacity of the radiostrips, and the radioactivity present in each one activity from L-(methy1-14C)-methionine into was determined by liquid scintillation counting. the different phospholipid classes. This fact Data given in the figures represent the mean stresses again the previous findings on the values of three individual determinations different metabolic activity of the two stages of (p<0.01). Radioactivity was measured in a development of the insect (8,12,13). The labeled carbon atom of the methyl Nuclear Chicago model 67 66 liquid-scintillation spectrometer. Identification was carried out group of the methionine was incorporated into from Rf data of the literature and using pure PE, mainly by pharate adult homogenates, in agreement with results of Moulton, et al., (14) standard samples. LIPIDS, VOL. 10, NO. 2

METHYLATING ACTIVITY IN C. CAPITA TA

CPM

RATE

ADULTS

FIG. 6. Scanning of radioactivity on paper chromatograms after separating the bases from lipid hydrolysates. (E = ethanolamine, M = monomethyl ethanolamine, D --- dimethyl ethanolamine, T = choline, V = valine, and B = 9-methyl choline). In vitro incorporation of radioactivity from L-(methyl14C)-methionine by larval and pharate adult homogenares of Ceratitis capitata. using M. d o m e s t i c a larvae. Thus, the results obtained with C. capitata are consistent with the idea that one carbon reactions that occur in vertebrate metabolism also are performed in insects. On the other hand, the low levels of radioactivity which appeared in PC allow one to conclude that the in vitro methylation pathway for PC synthesis, although clear, was not of much quantatative significance. In Figure 3, the specific activities of PE and PC vs time are given. These results are consistent with the higher incorporation of the methionine methyl group into PE than into PC and confirm both the incorporation of the methyl group into the (1 C) pool and the ability of homogenates to perform transmethylation reactions from methionine using ethanolamine or PE as acceptors. Figures 4 and 5 show the results of the quantitation of the phospholipids during the time course experiments using larval and pharate adult homogenates, respectively. Since the contents of both PE and PC undergo only a slight decrease during the experiments with larval homogenates, levels of labeled PC (Fig. 3) could be a measure of the synthesis of the phospholipid through the methylation pathway. Patterns of variation of the contents of PE and LPE during the time course experiments using pharate adult homogenates (Fig. 5) showed a sharp decrease during the first 60 rain of incubation. PC and LPC showed a low variation during the experiment. The concentrations of phospholipids in the enzyme preparations used in these in vitro experiments differ from the composition of the insect (10) be-

85

ADULTS

FIG. 7. Scanning of radioactivity on paper chromatograms after separating the bases from lipid hydrolysates. (S = serine. Others as in Figure 6). In vivo incorporation of radioactivity from dietary L-(methyl-l-4C)-methionine in larvae and pharate adults of Ceratitis capitata. cause of the elimination of a layer of the less polar floating lipids during the preparation of the homogenates. From Figures 3 and 5, it is clear that, in spite of the net diminution of the contents of PE (Fig. 5) during the incubation with pharate adult homogenates, the specific activity of the phospholipid was higher than that exhibited in the experiments using larval homogenates (Fig. 3). Thus, it can be concluded that the synthesis of PE from methionine methyl groups can not counterbalance the net degradation of the phospholipid content and that transmethylation reactions occur more efficiently in the pharate adult than in the larval stage of development of the insect under the in vitro conditions. These findings could explain, at least partially, the tendency to equalize the specific activities of PE and PC during development of the insect when larvae were fed on several labeled precursors (6). To learn more about the methylation reactions in the in vitro experiments, aliquots of both total lipid extracts were hydrolyzed, and the bases were separated and identified by paper chromatography. Figure 6 shows the radioactivity patterns of labeled bases from the phospholipids synthesized in the in vitro experiments using either larval or pharate adult homogenates of the insect. In agreement with the findings on the distribution of radioactivity in phospholipids, ethanolamine exhibited the highest levels of incorporation in both stages of development of the insect; however, the levels achieved in the presence of pharate adult homogenates were much higher than those LIPIDS, VOL. 10, NO. 2

86

c. JIMENEZ, A.M. MUNICIO AND A. SUAREZ

using larval homogenates. Labeling of m e t h y l e t h a n o l a m i n e s was very scarce with larval homogenates, m o n o m e t h y l derivative being the only one that e x h i b i t e d a significant incorporation. Using labeled m e t h i o n i n e , it is clear f r o m Figure 6 t h a t an increased p r o p o r t i o n of labeled m o n o m e t h y l and d i m e t h y l ethanolamine, choline and /3-methyl-choline was d e t e c t e d following the incubation with pharate adult h o m o g e nates. Evidence of labeled/3-methyl-choline was gained by means of in vivo e x p e r i m e n t s (15) in which larvae were fed on (methyl-14C)-choline and L-methionine simultaneously; paper chrom a t o g r a p h y of total lipid hydrolysates led to a radioactive spot coincidental w i t h the position of a pure standard. These results support further the more active m e t h y l a t i o n capacity of the pharate adult homogenates. Figure 7 shows the patterns of labeling of bases f r o m lipids synthesized in vivo when larvae were fed on L-(methy1-14C)-methionine; bases also were analyzed f r o m lipids of pharate adults c o m i n g from further d e v e l o p m e n t of an aliquote of the pool of labeled larvae. Both patterns are very similar f r o m a qualitative point of view, e t h a n o l a m i n e and serine being the most labeled bases. Larvae exhibited slightly higher levels of radioactivity in choline than those present in the pharate adult stage. These results prove the presence of the three m e t h y l a t e d ethanolamines in b o t h stages of d e v e l o p m e n t of the insect, larvae and pharate adults. These experiments indicated the existence in C. capitata of a transfer of m e t h y l groups f r o m m e t h i o n i n e to PE in a similar way to the described process in the vertebrate tissues (16). Evidence also is given of the presence of m e t h y l a t e d i n t e r m e d i a t e s to the synthesis of PC. Nevertheless, in which proportion the operating mechanism for the synthesis of the labeled PC is either the direct m e t h y l a tion of PE or the i n c o r p o r a t i o n of m e t h y l a t e d

LIPIDS, VOL. 10, NO. 2

ethanolamines by the cytidine 5'-triphosphate pathway remains an open question. Paper c h r o m a t o g r a p h y o f the phospholipid hydrolysates shows clearly the presence of a radioactive ninhydrine-positive spot that was identified as valine. This fact agrees again with the participation of the m e t h i o n i n e m e t h y l groups in the (1 C) metabolic patterns of the insect. Since free amino acids are n o t carried over the lipid extracts, the presence of valine in the a q u e o u s solution after the hydrolysis of phospholipids indicates the previous existence of phosphatidyl valine. REFERENCES 1. Dadd, R.H., Ann. Rev. Entomol. 18:381 (1973). 2. Beaudoin, A.R., and A. Lemonde, J. Insect Physiol. 16: 511 (1970). 3. Bieber, L.L., and R.W. Newburgh, J. Lipid Res. 4:397 (1963). 4. Bridges, R.G., J. Ricketts, and J.T. Cox, J. Insect Physiol. 11:225 (1965). 5. Blankenship, J.W., and G.J. Miller, Ibid. 17:2061 (1971). 6. Castill6n, M.P., R.E. Catalan, A.M. Municio, and A. Suarez, Insect Biochem. 4:395 (I 974). 7. Municio, A.M., J.M. Odriozola, and A. Pifieiro,

Comp. Biochem. Physiol. 37:387 (1970). 8. Municio, A.M., J.M. Odriozola, A. Pifieiro, and A.

Ribera, Biochim. Biophys. Acta 248:212 (1971). 9. Bligh, E.G., and W.J. Dyer, Can. J. Biochem. Physiol. 39:911 (1959). 10. Castill6n, M.P., R.E. Catalan, A.M. Municio, and A. Suarez, Comp. Biochem. Physiol. 38B:109 (1971). 11. Bartlett, G.R., J. Biol. Chem. 234:466 (1959). 12. Municio, A.M., J.M. Odriozola, A. Pifieiro, and A. Ribera, Biochim. Biophys. Acta 280:248 (1972). 13. Municio, A.M., J.M. Odriozola, A. Pifieiro, and A. Ribera, Insect Biochem. 3:19 (1973). 14. ,Moulton, B., F. Rottman, S.S. Kumar, and L.L Bieber, Biochim. Biophys. Acta 21 O: 182 (1970). 15. Jimenez, C., Doctoral Thesis, University of Madrid, Madrid, Spain, 1974. 16. Katyal, S.L., and B. Lombardi, Lipids 9:81 (1974). [ Received June 7, 1974]