Biochemical analysis of vitellogenin from rainbow trout (Salmo gairdneri) : fatty acid composition of phospholipids Lucie Fremont, A. Riazi To cite this version: Lucie Fremont, A. Riazi. Biochemical analysis of vitellogenin from rainbow trout (Salmo gairdneri) : fatty acid composition of phospholipids. Reproduction Nutrition Développement, 1988, 28 (4A), pp.939-952. hal-00898891 HAL Id: hal-00898891 https://hal.archives-ouvertes.fr/hal-00898891 Submitted on 1 Jan 1988 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Biochemical analysis of vitellogenin from rainbow trout (Salmo gairdneri) : fatty acid composition of phospholipids Lucie FREMONT, A. RIAZI Station de Recherches de Nutrition, /.N.R.A., 78350 Jouy-en-Josas, France. Summary. Vitellogenin was obtained from three year-old vitellogenic trout. Two procedures of isolation were compared : dialysis against distilled water and ultracentrifugation in the density interval 1 .21 -1 .28 g/ml. Similar patterns were observed by gel filtration and electrophoresis for both prepara- tions of vitellogenin, indicating that electric charge and molecular weight were not modified by either procedure. The apparent M, of the native form was 560,000 in gel filtration, whereas that of the monomer was estimated as 170,000 by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Minor proteins were also detected (100,000-88,000-78,000). The main components were protein (79%), and lipids (19 %), Carbohydrates accounted for 0.3 % when protein phosphorus and calcium each represented 0.7 % of total weight. Phospholipids (70 % of total lipids) mainly consisted of phosphatidylcholine in which n-3 docosahexanenoic acid accounted for one-third of total fatty acids. The results show the high levels of essential fatty acids in structural lipids which are known to be involved in embryo development. Introduction. Vitellogenin (VTG), the lipophosphoprotein synthesized during vitellogene- sis by oviparous vertebrates, has been characterized in some fish species including rainbow trout (Hara and Hirai, 1978; Campbell and Idler, 1980; Norberg and Haux, 1985). The complex is generally isolated by precipitation. However VTG can be obtained by ultracentrifugation keeping the complex in a soluble form. Previously (Fremont et al., 1984) we used sequential ultracentrifugation for comparing chemical composition and fatty acid distribution in VTG and plasma lipoproteins (d < 1.21 g/ml) obtained by the same procedure. However, it is quite possible that methods used for isolation change the properties of VTG, specially that related to the insolubility of the complex in aqueous solutions. In the present study we therefore compared some properties of VTG obtained either by ultracentrifugation (VTGU) or by dialysis against bidistilled water (VTG,). In addition, we characterized the phospholipid moiety of VTG which is the main vehicle for the essential fatty acids involved in the embryonic development of trout (Leray et al., 1985). Material and methods. Animals. Three year-old female trout (Salmo gairdneri) from an autumn strain were reared at 9-15 °C in a fish farm under the control of the « Institut National de la Recherche Agronomique, France ». Fish were fed a commercial diet in which protein and lipid accounted for 43-45 % and 8-10 %, respectively, of the dry matter. The essential n-3 polyunsaturated fatty acids (PUFA) were provided at a level of 1 % of the diet (in weight). When serum VTG reached a high level (September-October), blood was taken after one-week starvation by puncture in the dorsal caudal artery. Serum was isolated by a low-speed centrifugation and antiproteolytic agents were added : 0.01 % sodium azide (NaN3) and 0.005 % phenylmethylsulfonylfluoride (PMSF). Samples were stored at 80 °C until analysis. Analytical procedures. - lsolation of vitellogenin. Dialysis. - A volume of serum was introduced into a membrane tubing (Spectrapor) and placed for dialysis in bidistilled water (5 I for 30 ml serum) at 4 °C for 2 days. After 3-4 changes of water, the precipitated VTG was collected by a 30-min centrifugation at 15 000 x g and solubilized in 0.5 M NaCI. It was twice purified by reprecipitation in 10 vol. of distilled water. The final material was designed as VTG!. Ultracentrifugation. - Serum lipoproteins of d < 1.21 g/ml were isolated by flotation in a NaCI-KBr solution according to Havel, Eder and Bragdon (1955) for 40 h at 10 °C. A rotor 50 Ti Beckman was operated at 145 000 x g. After removing lipoproteins, the subnatant fraction was raised to d = 1.28 g/ml. The floating VTG was obtained after a 72-h run at 10 °C and collected as previously described (Fremont et al., 1984). The VTG containing saline solution was exhaustively dialyzed against 0.5 M NaCI to remove KBr and the excess of NaCI. The final material was designed as VTGU. Gelfiltration. - Several trade marks of beads were used : Sepharose CL 6B, Sephacryl S 300 (Pharmacia fine Chemical, Upsala, Sweden), AcA 22 (IBF, France) and Biogel 1.5 M (Biorad, Richmond, USA). The elution solvent was 0.5 M NaCI containing 0.01 %3 NaN and 0.005 % PMSF. In some assays, the influence of ionic strength (N) and Ca2+ were tested. The saline solutions were : 0.5 M NaCl (N = 0.5) ; 0.8 M NaCl, 0.5 M NaCl + 0.1 M MgCl2, 0.5 M NaCl + 0.1 M CaC12 (p = 0.8) ; 1.25 NaCI (!=1.25); 0.5 M MgC’2, 0.5 M CaC’2 (1-i = 1.5). Before each fractionation, the column was equilibrated with the elution solvent for at least 2 days. The sample of VTG (20-50 mg protein) was applied to the column (1.5 x 90 cm or 1.6 x 100 cm) and eluted with the tested buffer at a flow rate of 10 (or 12) ml h-’ ; fractions of 2.5 ml were collected after monitoring absorbance at 280 nm. The column was calibrated for molecular weights (M,) with standard proteins : thyroglobulin (669,000), ferritin (440,000), and catalase (232,000). The markers Escherichia coli and tryptophane were used to determine the void volume (Vo) and the total volume (Vt) respectively. The apparent Mr of VTG which was eluted at V. was estimated from the linear relation KAV = f log Mr with KAv = Ve - Vo/Vt - VO- Electrophoretic methods. - Agarose gel electrophoresis was carried out according to Noble (1968) as previously described (Fremont et al., 1981). Proteins were visualized by Amidoblack 10 B. Polyacrylamide gel electrophoresis (PAGE) in non-denaturing conditions was performed by the method of Davis (1964) on 5 % gels using the vertical slab apparatus of Biorad (Protean 32 cell). The molecular weights of subunits were estimated by SDS-PAGE in linear gradient of 3-15 % polyacrylamide according to Laemmli (1970) using the following markers : myosine (200,000), galactosi- dase (116,250), phosphorylase b (92,500), bovine serum albumin (66,200), ovalbumin (45,000). In some assays, samples were incubated at 100 °C for 3 min in the presence of a reducing agent (one-fifth of the volume of 400 mM dithiothreitol). The sample of protein (ca 50 pg per well) was deposited in glycerol. Bromophenol blue was used as a tracing dye. The running time was 4-5 h at 50 mA per slab in electrode buffer TRIS-glycine, pH 8.3. Afterwards, migration gels were stained for proteins with Coomassie Brillant blue R 250. - Chemical analysis. Lipids were extracted with chloroform-methanol (2 : 1 ) according to Folch et a/. (1957) Triglycerides and cholesterol were quantified using enzymatic proce- dures (Biochemica test combination, Boehringer, Mannheim, France). Lipid phosphorus was estimated by a modification of the method of Bartlett (1959). All reactions were performed in the same tube using a heating block to mineralize the lipid extract with 10 NH2S04. The color was developed at 100° after adding the Fiske and Subbarow reagent and the optical density read at 830 nm. The phosphorus content was estimated from a standard curve obtained with H4 KP0 and phospholipids calculated by multiplying the phosphorus value by 25 considering lecithin as the major phospholipid. Phosphoproteins were assayed as acid-insoluble-nonlipid phosphorus according to Bergink et al. (1973). After washing the precipitate by solvents, protein phosphorus was estimated in the same way as lipid phosphorus. For qualitative identification, lipid classes were separated by thin-layer chromatography on Silicagel H (Merck). The developing solvent system was hexane - diethyl ether - formic acid (80-20-2 v/v/v) for total lipids and nonpolar lipids ; the solvent system was methanol/hexane/acetic acid/boric acid (40-20-30-10-1.8 v/v/v/v/w) for phospholipids (Gilfillan et al., 1983). For quantitative analysis, a silica cartridge (25 mm x 10 mm) (SEP-Pack waters, Framingham, USA) was used to separate non-phosphorus lipids from phospholipids with 30 ml of chloroform followed by 5 ml of chloroform/methanol (Juaneda and Rocquelin, 1985). Phospholipid molecular classes were separated by high-performance liquid chromatography (HPLC) using a Beckman appara- tus. The column (10 x 250 mm) was packed with silica (5 Nm ultrasphere-Si) ; the chromatographic system was programmed for elution using two solvent systems (Geurts Van Kessel et al., 1977). System A: hexane/2 propanol/water (6-8-0.75 by vol.) and system B : hexane/2 propanol/water (6-8-1 .4 by vol.). A linear gradient elution ranging from 0 to 100 % of solvent B was used for 10 min.