A biochemical characterization of vitellogenins isolated from the marine fish ocean pout (Macrozoarces americanus L.), lumpfish (Cyclopterus lumpus) and Atlantic cod (Gadus morhua)

Comp. Biochem. Physiol. Vol. ll3B, No. 2, pp. 247-253, 1996 Copyright © 1996 Elsevier Science Inc.

ISSN 0305-0491/96/$26.00 SSDI 0305-0491(95)02015-D

ELSEVIER

A Biochemical Characterization of Vitellogenins Isolated from the Marine Fish Ocean Pout (Macrozoarces americanus L.), Lumpfish (Cyclopterus lumpus) and Atlantic Cod (Gadus morhua) guxu Yao* and Laurence W. Crim OCEAN SCIENCE CENTRE AND DEPARTMENTOF BIOLOGY, MEMORIALUNIVERSITYOF" NEWFOUNDLAND,

ST. JOHN'S, NEWFOUNDLAND,CANADAA1C 587

ABSTRACT. Vitellogenins (VTGs) were isolated from three marine teleosts, the ocean pout (Macrozoarces americanus), lumpfish (Cyclopterus lumpus) and Atlantic cod (Gadus morhua), for the analysis of amino-acid composition and other partial biochemical characterizations. All three VTGs contained high levels of essential amino acids, arginine (5.08-6.54%), lysine (8.44-8.74%), isoleucine (5.60-6.82%), leucine (9.61-11.39%) and valine (6.76-6.99%). While histidine (2.27-2.67%) levels were low, the levels of methionine (1.51-3.38%), threonine (4.69-6.09%) and phenylalanine (3.38-4.41%) varied between species. In the category of non-essential amino acids, aspartic acid (8.60-9.68%), serine (6.09-7.68%), glutamic acid (11.45-11.97%) and alanine (6.49-7.79%) were predominant. Similarity was also noted in the VTG content of protein-bound phosphorus (0.62-0.72%) as well as total lipid (17.92-21.34%) among the three species. However, the molecular weights (485-630 KD), total phosphorus (2.15-3.56%) and the lipid-bound phosphorus contents (1.51-2.86%) of the VTGs varied. VTGs from the Atlantic cod and lumpfish did not cross-react with the antibodies made against ocean pout VTG, suggesting that VTG is species-specific. COMeBIOCHEM PHYSIOLl13B, 247-253, 1996. KEY WORDS. Amino acid, liquid chromatography, 17/3-estradiol, lipid, fish, phospholipid, vitellogenin

INTRODUCTION Vitellogenin (VTG) is a major yolk protein precursor in oviparous invertebrates and vertebrates. In oviparous vertebrates including fish, VTG is synthesized in liver rough endoplasmic reticulum and processed in the Golgi apparatus, where phosphorylation, glycosylation and lipidation of VTG occur yielding a glycolipophosphoprotein. The process of hepatic vitellogenesis is hormonally controlled by the ovary, and can be induced by 17/3-estradiol (E2) in adult male or female fish, and even juveniles. After its release into blood, VTG is transported into the ovary, where it is incorporated into the growing oocytes (reviewed by Ng and Idler, 1983; Wallace and Selman, 1985; Mommsen and Walsh, 1988). Because VTG deposits form the principal nutritive reserve of eggs and constitute the major food supply of embryos before external feeding, knowledge of the biochemistry of fish VTG will improve our understanding of fish reproduction and nutri-

tional requirements of larvae. Although VTG has been studied in some teleosts, the majority of which are freshwater species including rainbow trout Oncorhynchus mykiss (7,13,24), goldfish Carassius auratus (10) and carp Cyprinus carpio (34), full characterization of VTG, especially the analysis of amino-acid composition, was rarely reported in marine fish species except the sea trout Salmo trutta (24) and turbot Scophthalmus maximus (29). Knowledge of fish VTG aminoacid composition may assist with the starter diet formulation for fish larvae (17). Because the potential for marine aquaculture is gaining recognition, recent laboratory studies have focused upon the development of alternative cold ocean species for aquaculture (6) including an understanding of their reproductive biology. The objective of this study was to isolate and compare the VTGs, particularly their amino-acid composition, of ocean pout (Macrozoarces americanus ) , lumpfish (C yclopterus lumpus ) and Atlantic cod (Gadus morhua).

Correspondence to: L. W. Crim, Ocean Science Centre and Department of

Biology, Memorial University of Newfoundland, St. John's, Newfoundland, Canada A1C 5S7. Tel (709) 737-3708; Fax (709) 737-3220. "Present address: Department of Zoology, University of Guelph, Guelph, Ontario, Canada NtG 2W1. Received: 30 November 1994; revised 25 May 1995; accepted 6 August

1995.

MATERIALS A N D M E T H O D S

Experimental Fish Immature male and female ocean pout M. americanus (BW 399.2 +- 157 g) and lumpfish C. lumpus (158.9 -+ 12 g) were

248

held in separate indoor tanks (cap. 400 L), supplied with aerated flow-through seawater (3-4°C) under a simulated natural photoperiod. Experiments for the two species were conducted in the winter (December-January) of 1990. The experiment with post-spawned (spent) adult male Atlantic cod G. morhua (801.9 + 225 g) was conducted in May-June 1992 with fish held in outdoor tanks (cap. 5000 L) supplied with ambient seawater (6-7°C). All fish were fed chopped capelin to satiation twice a week during the experiments.

Hormonal Induction of VTG Synthesis Synthesis of VTG in fish was induced by injection of the steroid hormone 17/3-estradiol (E2). The hormone was dissolved in ethanol and diluted 1 : 1 (v/v) in saline (0.9% NaC1) to reach a hormone concentration of 10 mg/ml, and injected intraperitoneally (ip) into fish at a dose of 10 mg/kg BW (34). Control fish (N = 3 - 4 for each species) received only an injection of blank ethanol: saline (1 : 1) solution (without hormone). All fish received three injections and the injection scheme was once per week for the ocean pout but once per 2 weeks for the lumpfish and cod. In the study of ocean pout, a small volume of blood (0.5 ml) was taken from both control and E2-treated fish weekly during the treatment period. The fish were anaesthetized in 2-phenoxyethanol (100-300 ppm) and blood was withdrawn from the caudal vein into chilled, aprotinin-coated Eppendorf tubes before returning fish to their holding tanks after blood sampling. Blood was centrifuged at 15,000 × g for 3 rain and plasma was collected and analysed by native polyacrylamide gel electrophoresis (native PAGE) (31). Proteins on the gel were stained with Coomassie Brilliant Blue G-250 (BIORAD) and acetylated Sudan Black B (BDH), respectively, to identify the E2-induced, new lipoprotein or VTG in the plasma (18,31). One week after the final hormone treatment, a large volume of blood was withdrawn from the caudal vein of each fish (control and E2-treated fish) into chilled tubes containing 20 TIU aprotinin ml-1 blood (31,34) and processed as described above. The plasma was collected and stored at - 20°C before use.

Purification of VTG Plasma from both control and E2-treated fish were submitted to three purification steps including gel filtration, ultrafiltration and ion-exchange chromatography. Prior to chromatography, the plasma was diluted 1:1 in VTG buffer (50 mM Tris, 0.5 M NaC1, 10 mM EDTA, 100 TIU/L aprotinin, pH 8.0; (31) at 4°C, centrifuged at 15,000 × g for 3 min and the supernatant retained for use. Gel filtration using two columns of superfine Sephacryl $300 (90 × 1.5 cm and 70 × 1.5 cm) connected together to increase resolving efficiency was conducted. After equilibrating the gel with large volumes

Z. Yao and L. W. Crim

of VTG buffer and adding 2 ml of diluted plasma ( 1 ml plasma equivalent), the columns were eluted with buffer at 12 ml/hr and 1.2-ml fractions were collected for monitoring of the protein content at 280 nm. By comparing the elution profiles of plasma collected from E2-treated or control males and females, the E2-induced or female-specific plasma VTG peaks were identified and the purity of separated proteins analysed by native PAGE. Next, the VTG fractions were diluted 10fold (v/v) in Tris buffer (50 mM Tris, 100 TIU aprotinin/L, pH 8.0) and equilibrated in an Amicon ultrafiltration apparatus (membrane XM 100) at 20 psi before the VTG solution was further chromatographed on a DEAE-Sephacel column (30 × 1.5 cm) at an elution rate of 50 ml/hr by NaC1 stepgradients (0.00-0.30 M in Tris buffer (24,34). Final purity of VTG was examined again by native PAGE. All purification procedures were conducted in a cold room (4°C) to reduce the proteolysis of VTG.

Amino.Acid Analysis of VTG Purified VTGs were dialysed 1:1000 (v/v) against distilled water for 48 hr at 4°C and freeze-dried. The samples were oxidized with performic acid for determination of cysteine and methionine as cysteic acid and methionine sulphone, respectively, and then hydrolysed at ll0°C in 6.0 N HC1 for 24 hr. The individual amino acids were separated, identified and quantified following the methods of Blackburn (4) in an autoamino-acid analyser. Content of each amino acid in VTG was expressed as a percentage of the total amino acids.

Other Characterizations of VTG Molecular weights (MW), lipid and phosphorus contents of the isolated VTGs were also determined. The MW of different VTGs was estimated by gel filtration comparing the elution volume of VTG to that of the standard calibration proteins of known MW, including thyroglobulin (669 KD), catalase (232 KD), bovine serum albumin (67 KD) and chymotrypsinogen A (25 KD) (Pharmacia Chemical Co., Quebec, Canada). Blue Dextran 2000 was used as a leading dye for this MW estimation. For lipid determination, a 50/*1 aliquot of VTG solution (2 mg/ml) or Tris-buffer control were twice extracted (1:80, v/v) with a solution of methanol:chloroform:saline (2:1 : 1), modified from Norberg and Haux (24). After centrifugation at 3000 × g for 10 min, the chloroform phase, which contained the lipids, was retained and dried under N 2 at low heat (35°-40°C). Total lipid content of VTG was determined from the chloroform extracts following the methods of Marsh and Weinstein (21). Total and lipid-bound phosphorus contents of VTG were determined from non-extracted VTG solution and the chloroform extracts, respectively, with the method of Bartlett (2). Protein-bound phosphorus content of VTG was calculated by subtracting the lipid-bound phosphorus from the total phosphorus content of VTG.

Vitellogenins from Marine Teleosts

249

Cross,Reactivity of the Three VTGs to Ocean Pout

following repeated E2 treatment (Fig. 1A, gel columns c and d). This E2-induced new protein also reacted with Sudan Black B (Fig. 1B). In contrast, this protein was not present in fish plasma before E2 treatment or in control fish after receiving blank-hormone injections (Fig. 1A, gel columns a and e). Similar results were observed in studies of E2 induction of VTG production in the lumpfish and Atlantic cod (data not shown). Four distinct protein peaks were resolved by Sephacryl $300 gel filtration of the plasma of E2-treated immature ocean pout and untreated adult female ocean pout (Figs. 2A,B). However, in control immature fish and untreated adult male ocean pout plasma, the second peak (PK II) was not present. The PK II plasma proteins from both E2-treated immature fish and untreated maturing females had similar elution volumes (Figs. 2A,B), indicating similar MWs, and both were stained with Sudan Black B and bound to Affi-Gel Con-A (data not shown). Electrophoresis by native PAGE indicated that PK II fractions contained proteins of similar MW to the E2-induced proteins in plasma (Fig. 1A, columns f and g). However, in addition to the major protein band, the presence of several light bands in PK II fractions were also revealed by native PAGE (Fig. 1A, columns f and g; Fig. 3, columns a and b). Further purification of PK II proteins by DEAE-Sephacel ion-exchange chromatography yielded a sharp elution peak at an ionic strength of 0.20-0.21 M NaC1 (Fig. 2C), which contained the highly purified VTG according to native PAGE (Fig. 3). Following the same procedures, pure VTGs were also isolated from the plasma of E2-treated immature lumpfish and adult male Atlantic cod.

VTG Antibodies The cross-reactivity of the three VTGs to the antibodies raised against ocean pout VTG was tested. In brief, 1.0 mg purified ocean pout VTG was emulsified in Freund's complete adjuvant and injected subcutaneously into New Zealand White rabbits (BW 2-3 kg). One month after the primary injection, the rabbits were twice boosted at 1- to 1.5-month intervals with 1.0-mg doses of VTG emulsified in Freund's incomplete adjuvant. Five months after the initial injection, a large volume of blood (50 ml) was withdrawn from the ear vein of immunized rabbits into large culture tubes and placed at 4.0°C overnight for collection of antiserum (31). A radioimmunoassay (RIA) was developed in Polypropylene culture tubes (12 × 75 mm) using the ocean pout VTG antiserum and iodine 125labelled VTG as tracer following the method of So et al., (31). Plasmas obtained from control and E2-treated immature ocean pout, untreated adult male and female ocean pout, adult male and female Atlantic cod and lumpfish were diluted in RIA buffer (80 mM barbitol, 0.5% bovine serum albumin, 0.01% thimerosal, pH 8.6) and their cross-reactivity in the ocean pout VTG RIA tested. RESULTS

A study of E2 induction of VTG synthesis in the ocean pout by native-PAGE electrophoretic analysis of the plasma revealed that within 1 week of E2 treatment, a new protein of large MW appeared in the plasma (Fig. 1A, gel column b) and the quantity of this protein continued to increase dramatically

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A FIG. 1. Electrophoretic results of 5 ~tl plasma from control and E2-treated immature male ocean pout on native PAGE. Proteins on the gel were stained with (panel A) Coomassie Brilliant Blue and (panel B) Sudan Black B. In panel A, c o l u m n a: immature male fish before E2 treatment; columns b, c and d: fish after 1, 2 and 3 E2 treatments, respectively; c o l u m n e: control fish after 3 blank treatments; columns f and g: PK II fractions from the E2-treated fish plasma by Sephacryl $300 filtration. In panel B, columns a and b: control fish; c and d: E2 treated fish. The label of "VTG." in the figure indicates the position on gel where the n e w protein (VTG) appeared (see columns b, c, d, f and g in panel A, and columns c and d in panel B).

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Analysis of the amino-acid composition of all three VTGs revealed the presence of relatively high levels of the essential amino acids including arginine (5.08-6.54%), lysine (8.44-8.74%), isoleucine (5.60-6.82%), leucine (9.6111.39%) and valine (6.76-6.99%), but lower levels of histidine (2.27-2.67%). The levels of methionine ( 1.51-3.38%), phenylalanine (3.38-4.41%) and threonine (4.69-6.09%) varied between these three fish species. Among the nonessential amino acids, the levels of aspartic acid (8.609.68%), serine (6.09-7.68%), glutamic acid (11.45-11.97%) and alanine (6.49-7.79%) were consistently high (Table 1). For the VTG of all three marine species, the Sephacryl $300 chromatography estimates of the MW of VTG (Fig. 4) ranged from 485 to 630 KD (Table 2). Likewise, VTG total phosphorus and lipid-bound phosphorus contents varied

greatly, ranging from 2.15-3.56% and 1.51-2.86%, respectively (Table 2). By contrast, a similarity between VTGs was found for total lipid (17.9-21.4%) and protein-bound phosphorus (0.62-0.72%). Injection of New Zealand White rabbits with ocean pout VTG effectively induced the production of VTG antibodies, which were used to develop a VTG RIA (Fig. 5) with a reproducible range of 1.3-675 ng/ml (interassay and intraassay variations, 1.76%, N = 3; 1.64%, N = 6, respectively). TABLE 1. Amino acid composition of VTGs from the ocean pout (M. amer/canus), lumpfish (C. lumpus) and Atlantic cod (G. morhua) Amino acid (%)* Aspartic acid Threonine Serine Proline Glutamic acid Glycine Alanine Cystine Cysteic acid Valine Methionine Cystathionine Isoleucine Leucine Tyrosine Phenylalanine Ethanolamine Ornithine Lysine Histidine Arginine

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Lumpfish

Cod

8.60 4.69 6.88 4.13 11.97 2.80 7.57 I. 32 0.47 6.83 3.38 0.10 5.60 9.61 3.58 4.41 0.14 0.07 8.74 2.59 6.54

9.68 6.09 7.68 3.91 11.86 2.65 6.49 0.90 0.14 6.99 1.51 0.11 6.82 11.39 3.09 3.38 0.16 0.07 8.44 2.27 6.38

8.79 5.14 6.09 4.13 11.45 3.02 7.79 1.69 -6.76 2.48 0.13 6.58 10.99 4.21 3.97 0.20 0.12 8.71 2.67 5.08

"All values are expressed as percentages of total amino acids.

251

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Introduction of diluted plasmas from E2-treated immature fish or untreated adult female ocean pout into assay tubes yielded parallel competitive displacement curves with the VTG standard (Fig. 5A). However, no parallel displacement was observed when diluted plasmas were added from control immature or untreated adult male ocean pout demonstrating absence of VTG in these fish. Species specificity of VTG was demonstrated by the lack of RIA displacement when diluted plasmas were added from adult male and female Atlantic cod and lumpfish (Fig. 5B). DISCUSSION

The present study has demonstrated that VTG production can be induced by E2 treatment of these three different marine species including immature male and female ocean pout and TABLE 2. A comparison of the MW and phosphorus composition of VTGs from the ocean pout (M. americanus), lumpfish ( C. lumpus) and Atlantic cod ( G. morhua) Items (%)

Total lipid Total phosphorus lipid-bound P protein-bound P MW (KD)

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Lumpftsh

Cod

17.92 3.56 2.86 0.68 577.8

21.18 2.45 1.73 0.72 630.5

21.34 2.15 1.51 0.62 485.2

P: phosphorus; M W : m o l e c u l a r w e i g h t .

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102

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VTG concentration (ng/ml)

FIG. 5. Tests for the cross-reaction between ocean pout VTG antibodies and plasmas obtained from (A) control and E2treated immature ocean pout, untreated adult male and female ocean pout, and (B) the plasmas of adult male and female lumpfish and Atlantic cod. im: immature; OP: ocean pout; LP: lumpfish.

lumpfish and adult male Atlantic cod consistent with previous studies of either marine (30) or freshwater fish species (5,7, 11,20,24,25). Evidence favouring the conclusion that this protein isolated from the plasma of the cod, lumpfish and ocean pout is VTG includes: (1) that this protein is E2-induced and has a MW similar to the adult female-specific protein; (2) that this protein is stained by Sudan Black B indicating its lipoprotein nature (18,31); (3) its binding affinity to Con-A Affi-Gel suggesting the presence of a carbohydrate component (16) and (4) the positive Bartlett (2) reaction indicating the presence of a phosphorus component in this protein. In other words, this protein has the typical biochemical glycolipophosphoprotein characteristics of VTG (22,23). In addition, antibodies raised against this isolated protein cross-reacted with an ovarian homogenate of the prespawning ocean pout gonad (data not shown) suggesting that this is an egg yolk protein precursor immunologically related to ovarian egg yolk protein. Finally, VTG antibodies strongly cross-reacted with the plasma of mature females suggesting that E2-induced VTG from immature males and females is identical to the natural VTG obtained from adult females. Fish VTGs are susceptible to proteolysis and may suffer degradation during handling and storage procedures such as freezing and thawing, which can be prevented by addition of

252

the trypsin enzyme inhibitor, aprotinin (30,34). In the present study, proteolysis of VTG was reduced, as indicated by the single VTG protein band on native PAGE, by immediate addition of aprotinin upon withdrawal of the blood from fish and addition of this trypsin inhibitor to all VTG purification buffers during isolation and handling procedures that were conducted at 4°C. It is a prerequisite that the protein isolation procedures produce a high degree of purity before analysis of the aminoacid composition and lipid and phosphorus contents of VTG. In the present study, the purification procedures adopted included firstly gel filtration using Sephacryl $300, a superfine gel of high resolving capacity (31), followed by XM 100 membrane ultrafiltration, which both desalts and removes small protein contaminants from the VTG solution, and finally ionexchange on DEAE-Sephacel, which yielded a highly purified VTG for further characterization. Except for threonine, cystine, cysteic acid and methionine, the amino-acid contents of ocean pout, lumpfish and cod VTG are strikingly similar and this amino-acid content appears to be characteristic for teleost VTG as previously reported for the turbot (Scophthalmus maximus) (29), the rainbow trout (7,15), the goldfish (10) and the carp (34). To a certain degree, these are also the major amino-acid constituents of VTG found in the amphibian, Xenopus laevis (10) indicating that VTGs of different origins have a similar protein core. This seems to support the hypothesis that VTG genes of nonmammalian vertebrates are highly conserved (19, 28,33). Conservation of the VTG structure based upon the N-terminal amino-acid sequences was recently demonstrated for several species of teleosts (12). Besides comparable amino-acid contents, VTG from these three marine species also shares a similarity in the content of total lipid and protein-bound phosphorus, which is consistent with the values previously reported for other teleosts (7,10,24). However, the lipid-bound phosphorus content was more variable and the negligible degree of cross-reactivity between ocean pout VTG antibodies and the VTG from lumpfish and Atlantic cod suggests that these three VTGs are structurally variable, immunologically distinct and are therefore species-specific. On the other hand, since vitellogenesis is a major event in fish reproduction and VTG serves as an energy source for embryo development and the growth of young larvae, a comparative study of fish VTG may improve our understanding of the relationships between fish nutritional requirements and successful reproduction. For example, the consistency in VTG lipid content between species, indicates a similarity in lipidation while differences in VTG phospholipid content (lipid-bound phosphorus) suggests a variable extent of lipid phosphorylation in VTG after VTG translation. In the rainbow trout, over 70% of total VTG lipid is present in the form of phospholipid (24) and plays a key role in the surface structure of VTG (14). Since ovarian incorporation of VTG is a receptor-mediated process (9,32) and depends on the interaction between the VTG complex and receptors in the oocyte membrane, differences in VTG phospholipid con-

Z. Yao and L. W. Crim

tent might therefore influence the surface structure of VTG thus affecting ovarian incorporation of VTG. Phospholipids of fish VTG also serve as vehicles for transporting various substances including the polyunsaturated essential fatty acids (13,14), thyroxine (T4) and triiodothyronine (T3) (1) and certain ions such as Zn + + (26), Ca + + and Mg + + (3,8) into oocytes. The majority of phospholipids deposited in eggs, mostly via VTG, are essential for both biomembrane synthesis and energy production by the embryo as was demonstrated in the Atlantic halibut (Hippoglossus hippoglossus), Atlantic cod, plaice (Pleuronectes platessa) and turbot (27). The differences in VTG phospholipid content from the various species might also reflect their variable reproductive strategies, which, however, remain to be defined. It has been suggested that the protein-bound phosphorus content of VTG represents the level of amino-acid phosphorylation, especially that of serine-forming phosphoserine (7,10,14). The consistent protein-bound phosphorus content of VTG apparently relates to the relatively consistent levels of serine found in most VTGs. To date, the amino-acid (nutritional) requirements of fish are usually investigated by determining the carcass amino-acid composition or by studying the effects of deleting individual amino acids from the diet. For example, lysine is essential for growth of adult rainbow trout and salmon and its deficiency induces fin rot and is the source of mortality. Fish exposed to methionine deficiencies develop bilateral lens cataracts and suffer reduced growth (reviewed by Ketola, 17). Deficiencies of some essential amino acids can be partly spared by other non-essential amino acids, e.g. cystine partly spares methionine in the rainbow trout, while tyrosine spares phenylalanine (17). Our knowledge of the amino-acid requirement and how individual dietary amino acids affect growth and health of marine species is very limited. However, our analysis of the VTG amino-acid composition, based upon the three marine species studied here, shows that lysine is present at relatively high levels (8.44%-8.74%) while the levels of cystine (0.90-1.69%), methionine (1.51-3.38%), tyrosine (3.094.21%) and phenylalanine (3.38-4.41%) are low and differ among the three species. This suggests the presence of dissimilarities in the amino-acid requirement of different marine fish larvae and perhaps this information should be taken into account when formulating a starter diet for young marine fish of aquaculture importance. This research was supported by Canadian Centre for Fisheries innovation (CCFI) and Natural Sciences and Engineering Research Council (NSERC) Research Grant #A9729 to L. W. Crim, OSC Contribution No. 248. References

I. Babin, P. J. Binding of thyroxin and 3,5,3'-triiodothyronine to trout plasma lipoproteins. Am. J. Physiol. 262:712-726;1992. 2. Bartlett, G. R. Phosphorus assay in column chromatography. J. Biol. Chem. 234:466-468;1959. 3. Bjomsson, B. T.; Haux, C. Distribution of calcium, magnesium

Vitellogenins from Marine Teleosts

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