THE JOURNAL OF BIOLOGICAL CIIEMI~TRY Vol. 249, No. 9, Issue of May 10, PP. 2897-2903, 1974

Printed in lJ.SA.

Precursor-Product Relationship between Amphibian Vitellogenin and the Yolk Proteins, Lipovitellin and Phosvitin

(Received for publication, November 19, 1973)

E. WILLEM BERGINK* AND RORIN A. WALLACE

From the University of Tennessee-Oak Ridge Glacluate School of Biomedical Sciences, and the Biology Division, Oak Ridge National Laboratoly,$ Oak Ridge, Tennessee 37803

SUMMARY

The yolk precursor protein vitellogenin was isolated from plasma of estrogen-treated Xenopus laevis males, and the yolk platelet proteins, lipovitellin and phosvitin, were isolated from oocytes of females previously stimulated with human chorionic gonadoptropin. The molecular weights of the poly- peptide chains of vitellogenin and lipovitellin were deter- mined by electrophoresis on sodium dodecyl sulfate poly- acrylamide gels and by gel filtration in 6 M guanidine hydro- chloride. Both methods yielded a molecular weight of ap- proximately 200,000 for delipidated, S-carboxymethylated vitellogenin. Lipovitellin consisted of two polypeptide chains, with molecular weights of 31,000 and 120,000 and a 1: 1 molar ratio, as shown by electrophoresis on sodium dodecyl sulfate polyacrylamide gels. The smaller subunit appeared to be a phosphoprotein with a phosphate content of about 2 %. The heavy subunit contained little or no phos- phate. Treatment with guanidine hydrochloride did not dissociate lipovitellin into the two subunits: after gel filtration of delipidated, S-carboxymethylated lipovitellin in 6 M guanidine hydrochloride only one component was observed, with a molecular weight of about 140,000. Electrophoresis of 32P-labeled phosvitin on sodium dodecyl sulfate poly- acrylamide gels produced two labeled bands with mobilities corresponding to molecular weights of 35,000 and less than 25,000. The total molecular weight of the lipovitellin and phosvitin peptide chains was essentially equal to that of the polypeptide chain of vitellogenin. Vitellogenin incubated with very low concentrations of either trypsin or chymotrypsin dissociated into discrete chains with characteristics very similar to those of the peptide chains present in yolk platelets. The evidence thus supports the view that the vitellogenin precursor is a continuous polypeptide chain which is degraded into specific yolk proteins by proteolytic splitting in regions that are also sensitive to attack by trypsin and chymotrypsin.

* Postdoctoral Investigator supported by subcontract No. 3322 from the Biolorrv Division of Oak Ridee National Laboratorv

“ I

to the University of Tennessee. Present-address, University if Rochester, Medical Center, 260 Crittenden Boulevard, Rochester, N. Y. 14642.

$ Operated by Union Carbide Corporation for the U. S. Atomic Energy Commission.

Yolk formation in oviparous vertebrates appears to involve the uptake of the precursor molecule vitcllogenin from the blood- stream into the developing oocyte. Normally this precursor is produced seasonally in the liver of the female, but its synthesis can be induced in the liver of both male and female animals by administration of estradiol (1). Senopus laevis vitellogenin is a calcium-binding glycolipophosphoprotein (2-4) with a molecular weight of about 450,000 (2, 3). Its amino acid composition is almost identical to that of the total yolk platelet proteins (3, 5), and it cross-reacts with an antibody preparation against the yolk platelet protein lipovitellin (6, 7). Upon incubation with vitel- logcnic oocytes, vitellogenin is selectively taken up and trans- formed into the yolk platelet proteins lipovitellin and phosvitin (8). The nature of the chemical bonds that maintain the vitel- logenin complex intact in the serum and the mechanism responsi- ble for the macromolecular conversion within the oocyte are not yet understood.

I’hosvitin and lipovitcllin are present in a crystalline state in yolk platelets and can be readily separated (9, 10). Our past at- tempts to isolate phosvitin from the plasma of the frog have been unsuccessful. Rudack and Wallace (11) demonstrated the s>-u- thesis of phosvitin by liver slices prepared from estrogentreated animals, but their results showed a very low level of isotope incor- poration into phosvitin. Chick phosvitin, or a protein with simi- lar properties, has been isolated from the serum of a laying hen (12, 13). Beuving and Gruber (14) reported that it was neces- sary to incubate the serum of estrogen-treated roosters with pan- crcatic lipase in order to obtain phosvitin. In all those studies, however, no special precautions were taken to prevent proteolytic degradation of vitcllogenin.

In order to determine the nature of the bonds that maintain the vitellogenin complex, we assayed the molecular weights of vitel- logenin and the yolk platelet proteins from S. laevis under strongly denaturing and reducing conditions and analyzed the susceptibility of the vitellogenin molecule to proteolytic degrada- tion. We obtained evidence that vitellogenin consists of two polypeptide chains of equal size and that conversion to the yolk proteins phosvitin and lipovitellin occurs by a specific proteolytic process.

MATERIALS AND hlETIIODS

Aknals, Chemicals, a& Isolopes-Care of animals, anestheti- zation, and injection of hormones and isotopes xere performed as previously described (15). Kach animal was bled by heart incision, and 3 to 5 ml of blood were collected in an ice-cooled

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polyethylene centrifuge tube containing 0.5 ml of 0.07 M trisodium citrate-0.05% Dhenvlmethvl sulfonyl fluoride. The plasma was prepared by”c&triiugation. Biochemicals were purchased from Sigma Chemical Co., unless otherwise specified. Carrier-free [32P]orthophosphate in 0.02 N HCl was bought from Schwarz Bio- Research, and L-4,5[3H]leucine (1 mCi per ml, 20 Ci per mmole) was obtained from New England Nuclear.

Biological Labeling of Prolein-Double-labeled vitellogenin wasprepared from 50-g male frogs injected with 1 mg of fl-estradiol, 1 mCi of 32P:, and 1 mCi of [3H]leucine at 1 week, 24 hours, and 10 hours, respectively, before bleeding. Yolk platelet proteins were labeled by injecting 100-g female frogs with 1000 units of human chorionic gonadotropin and 5 mCi of 32Pi 5 days and 24 hours, respectively, before the removal of the ovaries.

Protein ZsoZalio?l-Vitellogenin was isolated from the plasma of estrogen-treated males by chromatography on TISAC-cellulose (O-(triethvlaminoethvl)cellulose) (2. 10). Linovitellin and nhos- vitin were”prepared f;o& platelets obtained f&m ovarian homage- nates (IG). The platelets were dissolved in 0.01 N citrate acid-O.06 M 2-amino-2.methyl-1-propanol-1.0 M NaCl and chromatographed on a TEAE-cellulose column after appropriate dialysis as de- scribed previously (10). The same column was used for the isola- tion of vitellogenin and the yolk platelet proteins: an important step before each run was a column wash with 500 ml of 0.2 M sodium citrate-0.2yo Triton X-100.

Qua&t&on, Delipidation, Enzymatic Digestion, altd Amino Acid ATLaZy& of Proteirls-Protein concentrations were deter- mined by the method of Bramhall et al. (17) with xylene brilliant cyanin G as the dye. Delipidation of proteins was performed by modification of the Bligh and Dyer procedure (10, 18). The protein precipitate obtained by centrifugation was washed se- quentially with 1-butanol, methanol, and ether and air-dried. Labeled vitellogenin (2 mg per ml) was enzymatically digested with diphenyl carbamyl chloride-treated trypsin or a-chymo- trypsin in 0.5 ml of 0.1 M HCl-0.005 M CaCl2, pH 8.0. Incubation with a-chymotrypsin took place in the presence of equal con- centrations of soybean trypsin inhibitor. The digestion mixture was incubated at 37” for 5 min, after which the reaction wits stopped by the addition of 10 mg of phenylmethyl sulfonyl fluoride and 2 ml of 1% sodium dodecyl sulfate in 0.01 M sodium phosphate buffer, pH 6.8, and immediately heated at 100” for 5 min. Amino acid analysis was performed with a model 120 Beck- man automatic amino acid analyzer, using the method of Moore and Stein (19). Approximately-l mg of delipidated, S-carboxy- methvlated lioovitellin was hvdrolvzed in 2 ml of 6 M HCl con- taining 0.1 M kercaptoethanol; for i4 hours at 110” iti vucuo. Gel slices containing the lipovitellin subunits (2 mg of I,, or 1 mg of Lz) were treated in the same way. About 0.5 mg of protein hy- drolysate was applied per column run. No correc*tions were made for the destructibn of-amino acids during hydrolysis.

Molecular Weioht Measurelne?~(s~Pol\,acrvlamide nel elec trophoresis was clrried out on 3.3 and 5Cj,“acr~?amide geis in 0.1% sodium dodecyl sulfate (20). The ratio of acrylamide to meth- ylene acrylamide (both purchased from I’Jastman Chemical Co.) was 37:l in both cases. For the electrophoresis of labeled pro- teins, ethylenediacrylate (Monomer-Polymer Lab, Bordon Chem- ical Co.) was used as cross-linker in a ratio of 0.1 ml of diacrglate to 2.22 g of acrylamide. No difference in mobility was observed when this cross-linking agent, which can be hydrolyzed under alkaline conditions, was used. Protein samples were denatured in a solution containing 1% sodium dodecyl sulfate-loj, mercapto- ethanol at 100” rather than 37” in order to prevent proteolysis due to impurities in the sample (21). 1)ennturing conditions, preparation of the gels, and protein staining with Coomassie brilliant blue were performed as described by Weber et al. (22).

(;el chromatography was carried out according to the pro- cedures of Mann and Fish (23). A 47( agarose column (Bio-Rad. 90 X 1.5 cm) was used with a flow rate of 2.5 ml per hour at 15.cm head. The column was eauilibrated with 6 hf GuIICl’ (Schwarz- Mann, ultrapure). Protefn was dissolved in 6 M GuIiCl-0.1 M

mercaptoethanol at a concentration of 10 mg per ml and was carboxymethylated with a slight excess of iodoncetic acid at pII 8.6 according to the procedure of Crestfield (24). Distribution coefficients of the proteins (kd) were determined by measuring the elution positions of 1 to 3 mg of protein (V,), blue dextran (V,),

1 The abbreviation used is: GuHCl, guanidine hydrochloride

and fluorescein-labeled glycine (Vi) at 230 nm. Both V, and Vi markers were present during every run. The calibration pro- teins used were ovalbumin, serum albumin, catalase, phospho- rylase a, p-galactosidase, and Escherichia coli RNA polymerase.

Gel Scanning and Radioactivity Analysis-Gels were scanned at 570 nm with a Gilford spectrophotometer equipped with a gel transport mechanism. =P in gel slices or in fractions from the GuHCl-agarose column was determined by placing the gel slice or a sample of the column fraction on a filter paper disc. After drying, the discs were counted in a Beckman Lowbeta II planchet counter. Radioactivity in 311, aP-gel slices was determined by hydrolyzing them in 1 ml of concentrated ammonia added to the scintillation vials. Excess ammonia was evaporated at 50”, and 6 of ml scintillation fluid (10% naphthalene, 0.5% 2,5-diphenyl- oxazole) were added. The vials were counted for 31-I and 32P in a Beckman LS-100 scintillation counter.

RESULTS

Isolation of Intact Vitellogenin-Vitellogellill from the serum of

estrogen-treated males can be recovered quantitatively from the other serum proteins by chromatography on TEAE-cellulose (25).

Chromatography yields a single vitellogenin component, which

rechromatographs reproducibly and shows a homogenous peak distribution. When such material was denatured in sodium

dodecyl sulfate-mercaptoethanol and subjected to electrophoresis on sodium dodecyl sulfate polyacrylamide gels, a main band along

with several fainter bands was observed (Fig. 1A). We ob-

served a different pattern, however, when precautions were taken to prevent degradation by contaminating proteolytic enzymes

during the isolation procedure. Thus, when the blood was im-

mediately collected into a citrate solution containing phenyl- methyl sulfonyl fluoride, and sterile glassware and buffer solu-

tions were used, the final vitellogenin preparation appeared more homogeneous after electrophoresis (Fig. 1B). The main band

also had a distinctly higher molecular weight than the main band

in our initial preparations, and on the basis of repeated esperience with even more homogeneous preparations, apparently represents

the native, undegraded polypeptide present in vitellogenin.

These results indicate that an undenatured vitellogenin prepara- tion, apparently homogeneous by chromatographic criteria, can

in fact be degraded. Xolecular Il’eight Delerminations with Sodium Dodecyl Sulfate

Polyacrylamide Gel Electrophoresis-The sizes of the constituent vitellogenin polypeptides were determined by comparing their mobility on sodium dodecyl sulfate polyacrylamide gels with marker proteins. Fig. 2 provides a plot of the logarithm of molecular weights against clcctrophoretic mobilities. The mo- lecular weight of undegraded vitellogenin polypeptide was be- tween 195,000 and 210,000, based on six determinations with 3.3 and 5% gels. Prior delipidation of vitellogenin as described under “Materials and Methods” had no effect on the mobility. Lipovitellin isolated from yolk platelets showed two major bands

after electrophoresis (Fig. 1C). The heavy subunit (L]) had a molecular weight between 110,000 and 120,000; the light subunit (L2) had a molecular weight of 31,000. The same results were obtained with delipidated lipovitellin.

Phosphate Content of Polypeptide Chains Present in Yolk Plate- lets-Yolk platelets were isolated 24 hours after the injection of 9’i from a female previously stimulated with human chorionic gonadotropin. The estcnt of labeling was a measure of the phos- phate content since the specific activity of phosphate bound to lipovitellin and phosvitin is the same after this period of labeling

(26). Lipovitellin, phosvitin, and the total yolk platelet proteins

were denatured in 1 y. sodium dodecyl sulfate-phosphate buffer containing 0.1 y. mercaptoethanol, and the denatured proteins

were run on separate gels (Fig. 3). The larger subunit of lipo-

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MIGRATION DISTANCE

FIG. 1. Spectrophotometric scan of polyacrylamide gels (5% acrylamide, 0.1% sodium dodecyl sulfate). The electro- phoretically separated proteins were stained with Coomassie brilliant blue. A, vitellogenin degraded during the isolation pro- cedure; B, vitellogenin; C, lipovitellin; D, vitellogenin incubated with trypsin for 5 min at 37” in an enzyme to substrate ratio of 1:5060 (w/w); E, vitellogenin incubated with chymotrypsin for 5 min at 37” in an enzyme to substrate ratio of 1:1060.

vitellin contained little or no phosphate (Fig. 3, A and C), while the smaller subunit appeared to be a phosphoprotein with a phos- phate content 20 to 250/, that of phosvitin (Fig. 3C). Phosvitin, isolated by TEAE-cellulose chromatography (lo), formed two labeled bands on the gel (Fig. 3B). One had a mobility corre- sponding to a molecular weight of 35,000; the other band con- tained about 35% the activity of the larger component and mi- grated with the marker dye, indicating a molecular weight of less than 25,000. This latter component was not Pi or phospholipid, since (a) those substances migrate faster, and (5) the phosvitin- derived component was also observed after electrophoresis of the delipidated yolk platelet proteins (Fig. 3C). The relative amounts of label present in the two phosvitin-derived components were the same for isolated phosvitin (Fig. 3B) and for the yolk platelets (Fig. 3C). We assume that the second component con- sists of a small phosphopeptide(s), although we do not have direct evidence for its peptide nature. It did not stain with Coomassie

0 0.4 086 0.8 180 RELATIVE MOBILITY

FIG. 2. Semilog plot of molecular weight of marker proteins, lipovitellin, and vitellogenin versus the relative mobility of the polypeptides to bromophenol blue on a polyacrylamide gel (3.3% acrylamide, 0.1% sodium dodecyl sulfate). BSA, bovine serum albumin.

8

6

4

2

40

N p 20

E u n :: 100

A LIPOVITELLIN

,

PHOSVITIN

2 c’ ” YOLK PLATELET PROTEINS

,-

) - I A -z/ . 0 10 20 30 40 50 60 70

MIGRATION DISTANCE (mm1

FIG. 3. Electrophoresis of “P-labeled proteins on a polyac- rylamide gel (5y0 acrylamide, 0.1% sodium dodecyl sulfate). A, 3:P-labeled, delipidated lipovitellin; B, 32P-labeled phosvitin; C, 32P-labeled yolk platelet proteins. The isolated, intact platelets were dissolved in 1% sodium dodecyl sulfate-O.1 M mercapto- ethanol-O.1 M phosphate buffer, pI-1 6.8.

brilliant blue (similar to the larger phosvitin component), it pre- cipitated incompletely in 10% trichloroacetic acid, and it was lost from Visking Nojax casings (8/32) during dialysis of the de- natured phosvitin solution.

Amino Acid Analysis of Lipovitellin and Lipovitellin Subunits- We applied sodium dodecyl sulfate-denatured, S-carboxymethyl- ated lipovitellin to several gels and located the subunit compo- nents after electrophoresis by scanning the unstained gel at 280 nm. The appropriate regions were excised and processed as described under “Materials and Methods.” Amino acid analyses of lipovitellin and the subunits L, and Lz are given in Table I. Ammonia formed during hydrolysis of polyacrylamide dis- turbed the analysis of the basic amino acids. The amino acid

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TABLE I Amino acid composition of lipovitellin and the constituent

subunits L1 and Lz

Amino acid” R

Aspartic acid Threonine Serine Glutamic acid. Proline Glycine Alanine Valine. Methionine Isoleucine Leucine Tyrosine. Phenylalanine j$-Cystine. S-Carboxymcthylcystine. Lysine. . Histidine Arginine

-

-

Lipovitellin

edshaa and

Follet (3)

1.00 0.64 0.92 1.58 0.57 0.61 1.16 0.58 0.23 0.47 0.97 0.34 0.48 0.10

0.83 0.33 0.64

Lb Zresent study

1.00 1.00 1.00 0.69 0.63 0.66 0.94 0.85 1.25 1.49 1.49 1.44 0.62 0.64 0.96 0.62 0.64 0.98 1.25 1.25 1.20 0.66 0.67 0.76 0.25 0.11 0.11 0.52 0.59 0.45 1.01 1.04 0.63 0.35 0.25 0.42 0.49 0.42 0.42

0.11 0.77 0.31 0.62

0.12 -c

-

0.00 - -

= Amino acid residues are expressed as moles of amino acid per mole of aspartic acid.

* Present study. c -, obscured by gel-derived ammonia.

800.

600 B In

/

l ‘VITELLOGENIN

. LIPOVITELLIN

/

‘B GALACTOSIDASE

/

. PHOSPHORYLASE o

,* BSA

i

.’ CATALASE

400 / l OVALEUMIN

/

0 L?, I I I I 0 OS5 006 0.7 08 1.0

erf -’ I-k,)

FIG. 4. Calibration plot for the Bio-Rad 4% agarose column. M is the polypeptide molecular weight and erf-1 (l-k+) is the in- verse function complement of the partition coefficient. BSA, bovine serum albumin.

1

compositions of the two lipovitellin subunits were somewhat different. The small subunit contained more serine, proline, glycine, and tyrosine and less isoleucine and leucine than the large subunit. All of the cysteine appeared to be carboxymethylated and was present in the large subunit.

Nolar Ratio-The stoichiometry of the polypeptide chains present in lipovitellin was determined by scanning the gels stained with Coomassie brilliant blue at 570 nm (Fig. 10. In addition, the ratio of the amount of protein present in each subunit was

directly determined (17) after gel parts containing the subunits were excised and dialyzed against 0.1 M 2-amino-2-methyl-l- propanol in order to solubilize the gel. The ratio of the relative amounts of protein obtained with the two methods was about 1:4, in agreement with a molar ratio of 1: 1. We conclude from these observations that the lipovitellin molecules consist of only two polypeptide subunits, with molecular weights of 31,000 and 120,000.

GuHCl Gel Filtration on 4% Agarose-Results with delipidated vitellogenin, and the protein standards are plotted in Fig. 4 as recommended by Ackers (27). The elution positions of lipo- vitellin and vitellogenin were determined by measuring absorb- ance at 230 nm and that of 32P-labeled protein by measuring the radioactivity present in eluted fractions. The molecular weights obtained with vitellogenin fell in the range 185,000 to 195,000. Lipovitellin provided only one peak, with a molecular weight in the range 140,000 to 150,000 (three determinations). We tested azP-labeled delipidated lipovitellin and observed the same molecu- lar weight; no label was detectable in lower molecular weight fractions. Incomplete dissociation of lipovitellin in 6 M GuHCl was not due to incomplete reduction of cysteine, since complete dissociation occurred when the 140,000 molecular weight compo- nent was further denatured in sodium dodecyl sulfate in the ab- sence of reducing agents.

Incubation of Vitellogenin with Trypsin and Chymotrypsin- [3H, 32P]Vitellogenin was incubated with trypsin or chymotrypsin to provide information about the susceptibility of the precursor protein to proteolytic attack. Vitellogenin, incubated in the ab- sence of enzymes, migrated essentially as a single component (Fig. 5A). The second peak of 32P-labeled material consisted of phospholipids, since the peak was absent upon electrophoresis of delipidated vitellogenin (Fig. 5B). Dissociation into specific components occurred after 5 min of incubation at 37” with very low concentrations of trypsin (Fig. 5, C and D) or chymotrypsin (Fig. 5, E and F). Trypsin (enzyme to substrate ratio = 1: 104) degraded about 98% of the precursor molecule (Fig. 5C). The ‘H label (associated with leucine) was predominantly present in a nonphosphopeptide. The electrophoretic mobility of this com- ponent was indistinguishable from that of the heavy (L1) subunit of lipovitellin (Fig. 1D). The a2P label appeared in peptide chains with molecular weights of about 30,000 to 40,000. Incubation with a higher concentration of trypsin (enzyme to substrate ratio = 1:103) showed a second, high molecular weight nonphospho-

peptide (Fig. 50), apparently a degradation product of the LY like peptide. About 90% of the 32P label was present in peptides with low molectilar weights and low leucine contents.

In the presence of the same low concentrations of chymotrypsin, a similar susceptibility to proteolysis was evidenced. Chymo- trypsin (enzyme trypsin inhibitor to substrate ratio = 1: 103 pro- moted the formation of several peptides (Fig. 5E). The poly- peptide in Slice 25 (Fig. 5F) showed the same characteristics as the Ll-like polypeptide produced by trypsin action: a low phos- phate content, a mobility similar to the mobility of L1 (Fig. lE), and conversion to a second nonphosphopept,ide on incubation with a higher enzyme concentration (chymotrypsin-trypsin inhi- bitor to substrate ratio = 1: 103). The peptide in Slice 28 (Fig. 5E) has a higher phosphate to leucine ratio than vitellogenin. It is an intermediate product, very susceptible to further proteolytic attack since it is entirely absent upon incubation with a higher chymotrypsin concentration (Fig. 5F). Incubation with di- phenyl carbamyl chloride-treated trypsin as well as chymotrypsin (in the presence of soybean trypsin inhibitor) thus converted vitellogenin into nonphosphopeptides with high molecular

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100 A B

8o L

60-

40-

20.

0 20 40 60 80 0 20 40 60 80

50

40

30

20

10

15 CT

i2 IO x

E 2

5 - 2

I I

10

5

0

MIGRATION DISTANCE (mm)

FIG. 5. Electrophoresis of [3H,3ZP]vitellogenin on polyacryl- D, vitellogenin incubated with trypsin at an enzyme to substrate amide gels (5y0 acrylamide, 0.1% sodium dodecyl sulfate). ratio of 1:lOOO; E, vitellogenin incubated in the presence of chy- - - -9 3H radioactivity; --, 32P radioactivity. A, vitellogenin motrypsin at an enzyme to substrate ratio of l:lO,OOO. F, vi- incubated with trypsin inhibitor at an inhibitor to substrate ratio tellogenin incubated in the presence of chymotrypsin at an en- of 1:lOOO (w/w); II, delipidated vitellogenin; C, vitellogenin in- zyme to substrate ratio of 1:lOOO. All incubations were per- cubated with trypsin at an enzyme to substrate ratio of l:lO,OOO; formed at 37” for 5 min.

weights and phosphopeptides with low molecular weights. Soy- bean trypsin inhibitor alone had no effect.

DISCUSSION

Vitellogenin, the precursor molecule of yolk platelet proteins, apparently consists of two large polypeptide chains with molecu- lar weights of 200,000. The number of chains in the native mol- ecule can be calculated from the molecular weight of serum vitellogenin, 440,000 to 460,000 after correction for a lipid con- tent of 12% (2, 3).

The molecular weight of the vitellogenin polypeptides was ob- tained by two different methods: electrophoresis on sodium dodecyl sulfate polyacrylamide gels and gel chromatography in the presence of GuHCl. Deviation from normal mobility be- havior during electrophoresis in the presence of sodium dodecyl sulfate occurs if abnormal amounts of sodium dodecyl sulfate are bound to the protein due to the presence of covalently bound carbohydrates or the infiuence of the polypeptide primary struc- ture. Significant deviations due to carbohydrates occur only with proteins containing high amounts of carbohydrates (28), and the carbohybrate content of vitellogenin is low (0.7%) in this respect (3). Moreover, the absence of variation in the molecular weight values obtained with gels of different porosities is an indi- cation of the noninvolvement of carbohydrate groups (28). De-

viation because of primary structure effects is also very unlikely. The presence of phosphate groups apparently has no effect on electrophoretic behavior in sodium dodecyl sulfate since the elec- trophoretic mobility of phosvitin is in good agreement with the molecular weight estimated by other methods (9). Delipidation prior to denaturation of vitellogenin in sodium dodecyl sulfate solution was not necessary since the ratio of phosphate to leucine was not changed by such treatment (Fig. 5, A and B). This ob- servation is consistent with a similar conclusion derived from a delipidation study of low density lipoprotein from human serum in 3% sodium dodecyl sulfate (29).

Our molecular weight determination in GuHCl was based on the assumption that vitellogenin with S-carboxymethylated cysteines is totally dissociated in 6 M GuHCl. This assumption is not valid for every protein, as was shown to be the case for lipovitellin. The two lipovitellin subunits remained associated, due entirely to noncovalent binding between the subunits, which could be separated in sodium dodecyl sulfate. Moreover, the light chain did not contain cysteine, and all such residues of the heavy chain were S-carboxymethylated. We conclude that the lipovitellin polypeptides have a pronounced hydrophobic nature which inhibits the formation of randomly coiled linear homo- polymers.

The dissociation of X. Zaevis lipovitellin in sodium dodecyl

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sulfate solutions into a light chain with a molecular weight of 31,000 (LI) and a heavy chain with a molecular weight of 110,000 to 120,000 (Lz) is comparable to the dissociation of fl-lipovitellin from chicken egg into two fractions with molecular weights of 30,000 and 110,000 to 140,000 (30). It was recently shown that the heavy lipovitellin component from chicken egg undcrgocs further dissociation into subunits with weights of 45,000 after prolonged dialysis of succinyl-S-carbosymethylated fragments (31). This result differs from our observations and may be the consequence of a real difference between the two types of lipo- vitellin. However, we must caution that such a prolonged trcat- mcnt involves the risk of cleavage of polgpeptide chains, as has been shown to be the case for hacmocyanin (32, 33) and low deli- sity lipoprotein (34, 35). The heavy subunit of x’. laevis lipo- vitellin remained intact under a variety of denaturing conditions, such as boiling in 3% sodium dodccyl sulfate containing 1% mer- captoethanol or denaturing in GuIICl followed by alkylation, dialysis against 0.1 y0 sodium dodecyl sulfate, and elcctrophorcsis in sodium dodecyl sulfate polgacrylamide gels. Again, this result was not due to an incomplete reduction of the heavy lipo- vitcllin subunit since amino acid analysis of alkglated lipovitellin indicated that all the cysteine residues were carbosymethylated.

The molar ratio of L, and L, subunits was determined initially by scanning of the gels stained with Coomassie brilliant blue. However, this method relies on the assumption that different polypeptide chains have a similar intensity of stain per mass of protein, which is not generally true since, as an immediate csam- plc, phosvitin did not stain with Coomassic brilliant blue under the conditions employed. The USC of gels with a hydrolyzable cross-linker (ethylenediacrylate) in combination with a method for the determination of low concentrations of extracted protein (171 provided us with an alternate method. The ratio of color yield with Coomassie brilliant blue and the ratio of protein pres- ent in L1 and Lz were in both cases 4 : 1, and a molar ratio of 1: 1 was thus obtained for the two subunits of lipovitellin. Native lipovitellin is a dimer in neutral salt solutions and has a molecular weight of about 400,000 and a lipid content of 16 to 22% (9, 10, 36). Therefore, native lipovitellin monomer apparently consists of a single heavy chain (L,) and a single light chain (L,). The lighter subunit is a phosphoprotein and correspondingly has a higher serine content than the heavier subunit,. The relative amount of serine indicated for the smaller subunit (Table I) may be even greater since we did not correct for the destruction of labile phosphoserine in the 24.hour hydrolysis period (37). The total amount of phosphate bound to the light lipovitellin subunit is about 20 to 25% of the amount of phosphate bound to phos- vitin. We could calculate the same ratio using our molecular weight estimation and the protein-phosphate content of lipo- vitellin and phosvitin (0.4 y0 and 8 to 10 %, respectively; see Refs. 3 and 10).

In contrast to the yolk granules from chicken egg, which con- tain two types of lipovitellin, the yolk platelets from amphibian oocytes contained only one type of lipovitellin, as shown by chro- matography on TEAE-cellulose (8, 10) and gel electrophoresis in sodium dodecyl sulfate polyacrylamide gels (Fig. 1C; Ref. 38). [32P]I’hosvitin isolated by TEAE-cellulose chromatography showed two labeled components. A component with a molecular weight of 35,000 contained 67 y0 of the radioactivity and revealed a sharp homogenous peak in sodium dodecyl sulfate polyacryl- amide gels and in 10% polyacrylamide gels with Tris-HCl buffer, pH 8.5 (not shown). The nature of the other labeled component, which migrates with the dye front in sodium dodecyl sulfate polyacrylamide gels, is unknown. It may be a naturally occur-

ring tlcgradation product of phosvitin or another phosphopeptide obtained from a diffcrcnt region of the vitcllogenin precursor.

The total molecular weight of the lipovitellin subunits and phosvitin, assuming molecular weights of 31,000, 120,000, and 35,000, is 180,000 to 190,000. This value is close to the molecu- lar weight of the vitellogcnin polypeptidc chain. We thus pro- pose that vitellogenin is split into three small polypeptitle chains after it is taken up by the oocyte. Our incubation experiments with vitellogcnin and protcolytic enzymes support this proposi- tion since we observed polgpcptitlcs very similar to those which can be isolated from the yolk platelet. The formation of an intcrmcdiatc, high molecular weight (about 90,000) phosphopro- tein during incubation of vitellogenin with chymotrypsin (Fig. 5E) is an indication that the phosphoproteins from the platelets, i.e. phosvitin and lipovitcllin subunit Lz, arc clustered together on one side of the vitellogcnin polypeptidc chain and arc not interrupted by the lipovitellin subunit LI.

M7e do not have direct evidence for a specific pi-oteolgtic en- zyme activity during the transformation of vitcllogenin into yolk platelet proteins. However, during a very short incubation of oocytes in medium containing [% ,321’]vitcllog~nin, an inter- mediate product with L,-like characteristics could be detected by chromatography of an extract of incubated oocytes on 'I'ILW-

cellulose (8). A considerable amount of material, labeled pre- dominantly with [3H]leucinc, could only be flu&cd off the column with a solution containing Triton X-100. The amount of this component was substantially reduced during longer incubation periods in unlabeled medium, and the 3H label then was found to elute primarily in the position of native lipovitellin. We propose that ow of the first steps after vitellogcnin uptake is the forma- tion of Ll. This subunit would initially not chromatograph as an LI-L2 complex (lipovitellill) since the formation of L, could bc rate-limiting.

WC have thus provided some evidence that vitellogenin COII- sists of two polypeptide chains of equal size and that conversion of the pcptide chains into the yolk platelet proteins, lipovitellin and phosvitin, occurs by specific protcolytic splitting in regions which are also susceptible to proteolytic attack by trgpsin and chymotrypsin. This relatively specific attack by several differ- ent types of enzymes is an indication that the tertiary structure of vitellogenin consists of esposcd sites which are susceptible to proteolytic attack and which serve as links to more inaccessible parts of the molecule. The same type of structure, a folded, coil- tinuous polypeptide chain, has been proposed for the immuno- globulins (39, 40) and hemocyanin (33), proteins that are highly susceptible to proteolytic attack. In addition, other proteins that contain high molecular weight polypeptide chains, such as certain viral proteins (41) and chicken vitellogenin (38), arc similarly susceptible to proteolytic degradation.

Acknowledgments-We are grateful to Mrs. Mary Welch and Dr. Fred Hartman for performing the amino acid analyses and to Dr. Dave Hoffman for providing us with a preparation of RNA polymerase from E. coli.

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E. Willem Bergink and Robin A. WallaceProteins, Lipovitellin and Phosvitin

Precursor-Product Relationship between Amphibian Vitellogenin and the Yolk

1974, 249:2897-2903.J. Biol. Chem. 

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