THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc.

Val. 261, No. 23, Issue of August 15, pp. 10844-10849,1986 Printed in U.S.A.

Physical and Chemical Properties of Microvitellogenin A PROTEIN FROM THE EGG OF THE TOBACCO HORNWORM MOTH, MANDUCA SEXTA*

(Received for publication, March 25, 1986)

John K. Kawooya, Ellie 0. Osir, and John H. Law$ From the Department of Biochemistry, Biological Sciences West, University of Arizona, Tucson, Arizona 85721

Microvitellogenin belongs to a new class of low mo- lecular weight female-specific proteins in insects. The protein is found in the hemolymph (blood) and egg of the tobacco hornworm, Manduca sexta. The isolation of microvitellogenin has been achieved by a combina- tion of gel permeation, cation-exchange, and adsorp- tion chromatographic steps. Microvitellogenin is syn- thesized by the fat body and appears in the hemolymph 17 days before adult emergence, or 16 days before the onset of egg development. The protein is sequestered from the hemolymph into the egg where it accumulates to a relatively high concentration. The proteins iso- lated from the hemolymph and the egg are identical in their molecular weight, amino acid compositions, isoelectric points, circular dichroic spectra, immuno- logical properties, and NH,-terminal amino acid se- quence. Thus, microvitellogenin does not seem to undergo any modifications before or after it is seques- tered in the egg. In solution, the protein exists in a monomeric form and has a secondary structure com- posed of approximately 38% a-helix, as estimated by CD analysis. The CD spectrum of microvitellogenin is unusual in that it has a strong positive band between 220 and 240 nm that may be due to contributions from the aromatic amino acid residues. Unlike the major egg yolk protein of insects, vitellogenin, microvitellogenin does not contain measurable carbohydrate or lipid, and has no immunological, chemical, or physical similari- ties to vitellogenin. The amino acid composition of microvitellogenin is low in cysteine, but is rich in aspartate. The sex specificity of the protein and its accumulation in the egg justifies the name microvitel- logenin, first given to an analogous protein in the egg of the giant silkmoth, Hyalophora cecropia.

The construction of a mature insect egg is a complex process that requires the assembly of various components produced from within and without the ovary. The components produced outside the ovary are transported to the oocyte and are taken up by a selective endocytotic process (Roth and Porter, 1964; Pan, 1971; and Telfer et al., 1981). Studies by Telfer (1954) on egg proteins of the silkmoth, Hyalophora cecropia, showed that a variety of proteins occur in the egg and that many of these are taken up from the hemolymph. The best character- ized of these proteins is the major phosphoglycolipoprotein vitellogenin (Engelmann, 1979; Hagedorn and Kunkel, 1979;

* This work was supported by Grant GM 29238 from the United States Public Health Service. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$To whom correspondence and requests for reprints should be addressed.

Kunkel and Nordin, 1985; Osir et al., 1986). In most insects, vitellogenin is synthesized by the fat body and transported through the hemolymph to the ovary; in Diptera, however, some vitellogenin is also synthesized by the ovary (Heubner et al., 1975; Jowett and Postlethwait, 1980).

In addition, the insect egg contains a number of other proteins that include the major hemolymph lipoprotein, li- pophorin (Chino et al., 1977), and in Manduca sexta, a blue biliprotein, insecticyanin (Cherbas, 1973). In pursuing the purification of an unrelated hemolymph protein (Kawooya et al., 1984), we observed a 31,000-dalton protein present in the hemolymph of females and in eggs, but absent in males (Kawooya and Law, 1983). Telfer and Kulakosky (1984) ob- served the presence of a similar protein in H. cecropia, which they named microvitellogenin. A preliminary account of the purification of microvitellogenin from the hemolymph of M. sexta has been published (Kawooya and Law, 1983).

In this paper, we describe the isolation of microvitellogenin from the egg of M. sexta and compare the chemical, physical, and immunological properties of the hemolymph and egg forms. We also report experiments that define the site and time course of microvitellogenin synthesis.

EXPERIMENTAL PROCEDURES’

RESULTS

Female Specificity-Evidence for the female specificity of microvitellogenin is presented in Fig. 1. Fig. 1A is an SDS2- polyacrylamide gel showing the presence of this protein in female hemolymph and the egg extract, but its absence in the male hemolymph. Further evidence of the sex specificity of microvitellogenin was derived from immunological studies. Antibodies raised against microvitellogenin reacted with fe- male hemolymph and egg extract, but not with male hemo- lymph (Fig. 1B). Similar results were obtained in an immu- nodiffusion experiment in which microvitellogenin antiserum was tested with female hemolymph, male hemolymph, and the egg extract. In a separate immunoblotting experiment, we observed that microvitellogenin antibodies reacted with a

Portions of this paper (including “Experimental Procedures” and Figs. 2, 3, 4, 7, and 9) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biolog- ical Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 86M-946, cite the authors, and include a check or money order for $6.40 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

* The abbreviations used are: SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PTH, phenylthiohydantoin; [e], mean residue ellipticity; BSA, bovine serum albumin; HPLC, high performance liquid chromatography; TEMED, N,N’-tetramethyl- ethylenediamine.

10844

Insect Microvitellogenin 10845 1 2 3 4 5 6

B 7 0

"

FIG. 1. A, SDS-polyacrylamide gel (4-15%) electrophoresis. 1, mo- lecular weight standards (Bio-Rad) are: myosin (Mr = 200,000), b- galactosidase (M, = 116,000), phosphorylase b (M, = 93,000), bovine serum albumin (M, = 67,000), ovalbumin (M, = 45,000), carbonic anhydrase (M, = 31,000), soybean trypsin inhibitor (M, = 21,500), lysozyme (Mr = 14,400); 2, isolated microvitellogenin (mVg) (50 pg); 3, whole hemolymph proteins from adult male moths (150 pg); 4, whole hemolymph proteins from adult female moths (130 pg); 5, buffer-soluble proteins from whole egg homogenate (150 pg). B, autoradiograph showing immunological reactivity of anti-microvitel- logenin with: buffer-soluble proteins from whole egg homogenate (lane 6) , whole hemolymph proteins from adult female moth (lane 7), and whole hemolymph proteins from the adult male moth (lane 8).

1 2 3 4 5 6 0

200.0k-

116.Ok- 93.0k-

67.0k-

45.0k -

-93.0k

- 67.0 k

- 45.0k

-31.0k

- 21.5k -14.4k

+ I FIG. 5. Summary of purification. SDS-PAGE (4-15%) showing

microvitellogenin at different stages of purification: 1, molecular weight markers; 2, buffer-soluble proteins from whole egg homog- enate; 3, sample after gel permeation chromatography; 4, sample after cation-exchange chromatography; 5, homogeneous preparation of mi- crovitellogenin (40 pg) after adsorption chromatography. V,, large vitellin apoprotein; V,, small vitellin apoprotein; mVg, microvitello- genin.

protein of similar electrophoretic properties in whole hemo- lymph of H. cecropia.

Purification-The purification steps of microvitellogenin shown in Figs. 2-4 are very similar to those used to isolate the protein from the female hemolymph (Kawooya and Law, 1983). The major difference is that the lectin affinity chro- matographic step was not required during the purification of the protein from the egg. The products at the various purifi- cation steps of the protein are shown in Fig. 5. The procedures described here yielded a homogeneous preparation of micro-

5 10 15

NH2-Ala-Pro-Thr-Ser-Asp-Asp-Ile-Tyr-Asn-Asn-Val-Val-Ile-Gly-Asp-

20 25 30

Ile-Asp-Gly-Ala-Val-Ala-Lys-Ser-Lys-Glu-Leu-Gln-Lys-Gln-Gly-

35 40 45

Lys-Gly-Asp-Ile-Ile-Thr-Glu-Ala-Val-Asn-Arg-Leu-Ile-Arg-Asp-

so 53

Ser-Gln-Arg-Asn-Thr-Met-Glu-Tyr-

FIG. 6. The covalent structure of microvitellogenin NH2- terminal segment. Details are given under "Experimental Proce- dures." The sequence shown was determined in a single automated Edman degradation run using intact microvitellogenin. Microvitel- logenin isolated from hemolymph was similarly analyzed for 20 cycles and gave an identical result.

TABLE I Amino acid composition of microvitellogenin

Data were obtained from analysis of duplicate samples hydrolyzed using 6 N HCl in vacuo for 24,48, and 72 h

Isolated from blood"

Isolated from enz Amino acid

Aspartic acid and aspar- agine

Threonine Serine Glutamic acid and glu-

Proline Glycine Alanine Valine Methionine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Tryptophan' Cysteine' Total

tamine

residws/31,000 daltons 43

11 20 26

8 23 23 20 7

21 12 11 7

15 20 5 1

290 -

43

11 20 26

8 22 23 19 8

21 11 11 7

15 20 5 1

288 -

Kawooya and Law (1983). * Duplicate samples were hydrolyzed for 24 h in 3 N mercaptoeth-

Duplicate samples were performic acid-oxidized followed by hy- anesulfonic acid in vacuo a t 110 "C.

drolysis for 24 h in 6 N HCl in vacuo a t 110 "C.

vitellogenin as shown by SDS-PAGE (Figs. lA and 5), by the NH2-terminal amino acid sequence (Fig. 6), and by isoelectric focusing (Fig. 7).

Chemical and Physical Properties-Microvitellogenin iso- lated from the egg and that purified from the hemolymph had an identical molecular weight of M, = 31,000. On SDS-PAGE, the protein co-migrated with the carbonic anhydrase molec- ular weight standard, M, = 31,000. A molecular weight of M, = 31,000 for microvitellogenin was also obtained when the protein was chromatographed on a calibrated Sephadex G-75 column. Sedimentation equilibrium measurements of the pro- tein, using a i, of 0.712 cm3/g yielded a molecular weight, M, = 30,500 2 500. Microvitellogenin is not glycosylated since it gave a negative reaction when assayed by the phenol-sulfuric acid procedure (Dubois et al., 1956). The protein also did not bind to concanavalin A-Sepharose. Extraction of the protein by the Bligh and Dyer (1959) method, followed by thin layer chromatographic analysis, showed the absence of lipids in microvitellogenin. Table I shows the amino acid composition of microvitellogenin isolated from the egg and that previously

10846 Insect Microvitellogenin

purified from the hemolymph. It is evident that the compo- sitions of the two preparations are identical. The protein is rich in aspartate, but poor in cysteine. The two preparations also have identical NHp-terminal amino acid sequences (Fig. 6). The secondary structure of microvitellogenin in solution was determined by CD measurements (Fig. 8). The spectrum is dominated by two bands. The most prominent band is negative and has a [e] minimum at 207.5 nm. The second band has a positive [e] maximum at 232 nm. The secondary structure of microvitellogenin was estimated to contain 38% a-helix. On isoelectric focusing gels, microvitellogenin mi- grated to the same isoelectric point as the myoglobin PI marker, with PI 7.3 (Fig. 7). These results were reproducible in three different runs, using different microvitellogenin prep- arations.

Site of Synthesis-The insect fat body is the site of synthe- sis of most hemolymph proteins, including vitellogenin (En- gelmann, 1979; Hagedorn and Kunkel, 1979; Telfer et al., 1981). In the present study, in vitro incubation of the fat body tissue with [35S]methionine led to the incorporation of the label into microvitellogenin (Fig. 9). The product of synthesis was identified as microvitellogenin since it could be immu- noprecipitated using rabbit antibodies raised against the iso- lated protein. In similar experiments with isolated follicles (developing ovum and associated cells), no incorporation of [35S]methionine into microvitellogenin was observed. These experiments define the fat body as the site of synthesis of this

-40 L I I I I 1 200 220 240

Wavelength, (nm) I

FIG. 8. Circular dichroic spectrum of microvitellogenin. CD measurements of a 0.12 mg/ml protein solution in 0.01 M K,HPO,, 0.2 M KCl, pH 7.0, were performed in a 1-cm pathlength quartz cell a t 25 "C using a Cary 60 spectropolarimeter.

protein. Microvitellogenin occurs in the hemolymph at an estimated concentration of 5.83 f 0.13 pg/mg ( n = 5) of total protein and 66.51 .+ 0.15 pg/mg (n = 5) of the total buffer- soluble egg protein.

Time Course of Appearance of Microvitellogenin in Hemo- lymph-Samples of hemolymph from animals at different stages of development showed that microvitellogenin first appeared in the hemolymph 17 days before adult emergence. From this day on, the protein was found in the hemolymph of both pupal and adult stages.

DISCUSSION

Most studies on insect egg proteins have centered on vitel- lin, which comprises more than one-half of the buffer-soluble proteins in the egg (Engelmann, 1979; Hagedorn and Kunkel, 1979; Kunkel and Nordin, 1985). This protein is derived from, or in many cases is virtually identical with, a hemolymph precursor, vitellogenin (Pan, 1971). Vitellogenin is synthe- sized in the insect fat body cells, secreted into the hemolymph, and carried to the ovaries, where it is rapidly taken up by endocytosis (Telfer et al., 1981). Vitellogenin has been gen- erally considered as the only female-specific protein of insect hemolymph. Comparison of adult male and female hemo- lymph by gel electrophoresis usually allows easy identification of vitellogenin or its component apoproteins because of their restriction to the female hemolymph.

Recently, additional female-specific hemolymph proteins have been observed. In the larval hemolymph of Lepidoptera, proteins that are either exclusive to the female (Tojo et al., 1980) or predominant in the female (Ryan et al., 1985) have been reported. These proteins are apparently stored to provide an amino acid pool that is used later for the construction of adult structures. However, these proteins in intact form do not appear to contribute significantly to the structural com- ponents of the insect eggs.

Telfer et al. (1981) noted that several hemolymph proteins, not necessarily restricted to female insects, are also found in the eggs. In M. sexta, some of these proteins that have been identified include insecticyanin (Cherbas, 1973), arylphorin (Osir et al., 1986), and lipophorin? The discovery of microvi- tellogenin in M. sexta (Kawooya and Law, 1983) was the by- product of an investigation of apolipophorin-111. While puri- fying the latter protein from hemolymph, we observed that a contaminating protein of the same apparent molecular size was present in female hemolymph, but not in the hemolymph of male animals (Kawooya and Law, 1983; Kawooya et al., 1984). The two proteins separated readily on SDS-polyacryl- amide gels, since the apolipophorin-I11 has a molecular weight approximately only one-half that of microvitellogenin, al- though its asymmetry causes it to behave as a much larger molecule on gel filtration columns (Kawooya et al., 1986).

Microvitellogenin is a female-specific protein found in both hemolymph and eggs. Since the egg and hemolymph forms are identical by several criteria including immunodiffusion, amino acid composition, chromatographic and electrophoretic behavior, isoelectric focusing, CD spectra, and NH2-terminal amino acid sequence, there is no need to use a second term, microvitellin, for the egg form. Microvitellogenin was earlier identified by Telfer et al. (1981) as a component of H . cecropia eggs and was termed reluctin. The current name was proposed when the relationship between the hemolymph form and that in the egg was realized (Telfer and Kulakosky, 1984). As antibodies raised to the M. sexta protein cross-react with a protein of similar properties in H . cecropia hemolymph, we

J. K. Kawooya, E. 0. Osir, and J. H. Law, unpublished observa- tions.

Insect Microvitellogenin 10847

believe that H. cecropia microvitellogenin is immunologically closely related to the protein of M. serta.

Microvitellogenin is a quantitatively important component of the egg (the high concentration of microvitellogenin, 6% of the buffer-soluble proteins, may be greater than that of vitel- lin on a molar basis). Its concentration in the egg suggests that it is sequestered from the hemolymph by an active selective process, just as is vitellogenin (Osir and Law, 1986). Microvitellogenin has clearly been established as a second type of adult female-specific hemolymph protein, synthesized by the fat body, and transported to the ovary where it is sequestered. In other respects, it bears no resemblance to vitellogenin, as it contains neither lipid nor carbohydrate, and consists of a single relatively small polypeptide (M, = 31,000). It is probably not phosphorylated, since we have shown that the two apoproteins of vitellogenin were the only proteins significantly labeled when 32P-labeled inorganic phosphate was injected into adult females (Osir et al., 1986). Further- more, microvitellogenin and vitellogenin share no antigenic determinants.

That microvitellogenin is synthesized in the fat body and not in the ovaries is shown by the incorporation of [35S] methionine into microvitellogenin by fat body in vitro, but not by follicles in vitro. The active synthesis of radiolabeled microvitellogenin by adult fat body confirms that the synthe- sis of this protein continues actively in the adult moths, although synthesis begins early in the pupal stage, as shown by immunological detection in pupal hemolymph. This con- trasts with vitellogenin, which is detectable only 4 days before adult eclosion, and then only in small amounts. Substantial synthesis of vitellogenin begins only 1 day before eclosion (Imboden and Law, 1983).

The molecular weight of microvitellogenin was established by gel electrophoresis under native or denaturing conditions, as well as by gel permeation chromatography and by analytical ultracentrifugation. Thus, the molecule appears to be a mon- omeric globular protein. Fig. 8 shows the CD spectrum of microvitellogenin. At 222 nm, the mean residue ellipticity was -2471 k 230 ( n = 3), and the a-helicity was estimated to be 38%. An interesting feature of the CD spectrum, however, is the strongly positive band with a maximum at 232 nm. A maximum of this type has been observed in only a few other proteins and is usually attributed to disulfide bonds or to the interaction of the aromatic residues with the peptide backbone (Woody, 1978). As microvitellogenin appears to have only 1 cysteine, disulfide bonds cannot be responsible for this band. We therefore suggest that it may be due to the orientation of aromatic residues.

While the exact function of this second vitellogenic protein in the insect eggs remains a matter for conjecture, a knowledge of its chemical and physical properties, its tissue of origin, and the time course of its synthesis should pave the way to detailed studies on its biological role.

Acknowledgments-We thank Dr. Michael Wells for providing use of the analytical ultracentrifuge, Dr. Michael Cusanovich for discus- sion of CD results, Dr. Norbert Haunerland for isoelectric focusing, Pamela Keim for sequence analysis, Richard Apothaker for densitom- etry, and Mary Gonzales for expert care of the experimental animals.

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Continued on next page.

10848 Insect Microvitellogenin

Physical and Chemical P roper t i es o f P i c rov i te l l ogen in Supplementary Pa te r ia l TO

A P ro te in fm1 the Eo0 of the Tobacco Hornwom Poth. Vanduc; sexta

John K. Kawooya. E l l i e 0. Os i r and John P. Law

Experimental Procedures

O e p a r 5 G T j f A g r i c u l t i h T g o Nn The eggs were al lowed to hatch and the larvae'vere ra ised ind iv idual ly (Rel l 'and Joichi.. 19761 on a high wheat germ die; l n e i n e c k e e t c . , l"P01.

Animals--Eggs of M. sexta wew supplied by Or. J . P. Reinecke and Dr. J. Buckner U. S .

day a f t e r emergence were used. Indiv idual animals were anesthesized by c h i l l i n g a t 4'C fo r 20 min ami t h e i r abdomens were cu t Open t o expose the eggs. The eggs Yere removed fmm the

Another dry f i l t e r paper was placed on top o f the eggs and was moved by hand several times abdomens by means o f a spatula and wew spread onto m i s t Whatman No. 3 f i l t e r papers

i n c i r c u l a r motion. The eggs were t rans fer red to a f r e s h m o i s t f i l t e r paper and the cleaning pmcess repeated. This procedure ensured the removal of m s t of the f a t body t issue from the eggs. The eggs were then transferred i n 50 m l p las t i c cu l tu re tubes and were washed f ive t imer wi th de ion ized water to remve any remaining f a t body t issue and hemolymph prote ins.

Processing of the Eggs--Approximately 20 adu l t female animals i n t h e t h i r d o r f ou r th

i n phosphate buffered saline I P R S I : 0.10 P sodium phosphate. 0.15 r Narl. 0.05 P E0TP and Extraction of Buffer Soluble Proteins frnm the Eqgs--The egos were homogenized a t 6.C

0.02 percent l w l v ) sodium azide. pH 7.0) made i n 0.05 V glutath ione and 0.07 P protease i n h i b i t n r diisopropylphosphorofluoridate. The eoo homogenate was centr i fuged 115.000 rpm

m l by rwans of a YP 10 h n i c o n u l t r a f i l t r a t i o n membrane 1Arnicon Carp., Lexington. PA). 10 min, 4'r). The supernatant solution was pooled and the volume reduced to appmximately '5

Pur i f icat ion o f Hicrovitellogenin--Picrovitellogenin from the eqgs was i so la ted hy gel

described i n the appropriate f igure legends. The protPin was assayed accardino t o Lowry e t - al . 1Io51). Pur i t y O f the p ro te in was ascertained by SDS-PAGE isoelectr ic focusing, mf iy t l - terminal ar ino acid sequence. E lect rophoret ic procedures. m;lecular weight est imation by gel chromatography and amino acid analyses Y e w perfonned as described prev ious ly 1Kawooya and Law. 1oP31.

c i r ~ ~ l a r Oichroisv--The tD spectrum of microvitellogenin was measured a t wavelengths of between 700 and zoo nn using a Cary 60 spectropolarimeter. The instrument was ca l i b ra ted w i th a 1 mg/ml s o l u t i o n o f dl')-ln-canphor-rulfonic a c i d a t 7W nm. The measurercents were performed i n a 1 cm pathlength quartz cel l using a 0.12 mglml p r o t e i n s o l u t i o n a t 25'C. The p ro te in was d isso lved i n a 0.01 P K2HP6q. 0.2 P K C ] , pH 7.0. The mean residue P l l i p t i c i t i e s were ca lcu la ted accord ing to Fasman 11063). The percentage .-helix was ca lcu la ted accord ing to Por r ise t t g dl. (10731.

automate an egra a t on an an individu:lE& dgriva:iv:s wL:ed"subse$%?; %$zed w i th the Reckman 11n HPLt system f i t t e d w i t h a C-IP reversed phase Column. The column was e lu ted w i th a l inear gradient Consistino O f A) 10 percent acetoni tv i le wi th 0.02 P sodium acetate and 8 ) 100 percent ace ton i t r i l e . The e luates were monitored by a 33BPA Hevlet t Packard in teprator .

Oeteminat ion o f the N- terminal amino ac id sequencp--picroviteltogenin was subjected to using the Beckman POOP sequencer. The

complete adjuvant and was in t ramuscu lar ly admin is te red in to the h ind l imbs o f the New Production of Antibodies--Elicrovitellagenin 11.0 mg) was emulsif ied i n 1 a1 Freund's

Zealand V h i t e r a b b i t (0 .5 ~ l / l i r nb1 . A f te r f ou r weeks a booster dose (0.5 mo) was

major artery on the ear and Processed a s previously described 1Shapim 9 c.. 1~84) . administered, and a f t e r another two weeks blood was col lected f rom the animbl thrnuqh the

polys yrene p a es i l e r Laboratories IN) using 1 percent agarAre 1Seakern HE) i n PRS. Imunodiffusion--Oouble radial imunodiffusion (Ouchterlony IP6P) was perfomed On

The &le5 t: :e t b t t e d vew appl ied io the we l ls made in t he agamre ge l ;mi yew allowed

another 12 h i n d i s t i l l e d water i n o r d e r t o remove any vnreacted proteins. The gel vas a i r t o d i f f use f o r ahout 17 h a t ram temperature. Tbe qel vas soaked i n PRS f o r 12 h and

Mol. d r i e d a t ?om temperature and s ta ined w i th romars ie Rr i l l i an t P lue F"25P (Pierce, Pockford,

whole e m ymp an on bu f fe r so lub le p ro teKf im ' the e;g homogenite. The p ro te ins were separated by SOS-PAGE on 4-15 percent gradient s lab ge ls , and then e lec t rophore t i ca l l y t r a n s f e r r e d t o n i t r o c e l l u l o s e paper 1 M i l l i p o r e Bedford MA) fo r 24 h r a t 16'C The n i t rocel lu lose paper was f i r s t incubated wi th k t ibodie; against microv i te l1og;n ln . fo l lwed

det -om., 1984). by Sta h lococcus aureus protein A labe led w i th 125I and subsequent autoradiographic

Inyn~blo;t in~-- lmunoblott ing (Towbin e t a l . 1979. Burnet te 1981) was p e r f o m d on

solub e n ow an g on c s reng u e r r owevcr upon l y o p h i l i z a t i o n t h e Quant i ta t ive Assays o f Egg and Hemolymph Mic rov i te l logen in- - M ic rov i te l logen in was

PPOtJn k?%a11Y :":ll:b;e i n t ies, ::f:e:: b;t ~ead i l y ' d i sso l ved i n 0.05 M Godim phosphate buffer containing 6 M guanidinium hydr&hlor ide pH 7 0. I n t h i s assay, 500 Of m ic rov i te l l ogen in was d i s s o l v e d i n t h e above b u f f e r t o a h n a l ;oncentration o f 1 mglml. Buf fe r so lub le p ro te ins from the egg e x t r a c t and whole hemolymph p ro te ins were assayed according t o Lowry e t a l . 119511 us ing RSA as the standard. All p r o t e i n s i n t h i s assay were S o l u b i l i z e d i n 0 . 0 5 T i t a s S i u m phosphate. 6 M guanidinium hydrochloride. pH 7.0.

ug). and 5 samples of p r o t e i n s I n t h e egg homogenate 14.3-34.3 ug) were subjected t o Six samples o f m i c r o v i t e l l o g e n i n 10.28-2.28 ug). 5 samples of whole hemolymph (49-390

SOS-PAGE on a 4-15 percent gradient s lab ge l . The separated proteins were e lec t rophore t ica l l y t rans fer red to n i t roce l lu lose paper as descr ibed above The paper was soaked i n T-PBS 110 nN sodium phosphate 0 09 percent sodium chloride, pH i.5. 0.05 percent l v I v 1 T w e n 201 and incubated f o r 30 mi; w i th rabb i t an t i bod ies ra i sed aga ins t mic rov i te l logen in . M ic rov i te l logen ln bands were revealed on t h e n i t r o c e l l u l o s e paper

Bur l ingam. CA. " according t o HSU e t a l . 11981a.bl. The reagents were purchased from Vector Laboratories,

scann ing dens i tometer IKontes Sc ien t i f i c lns t rmnts NJ1 w l t h t h e s e t t i n g a t a s ing le beam The S t a i n i n g i n t e n s i t y of m lc rov i te l l ogen in bands vas measured by the Kontes Model 300

concentrat ion versus peak area was constructed. The amount o f e ic rov i te l logen in i n the ref lectance mode. The area under each peak was i n te i ra ted . and a standard curve of protein

buffer soluble egg Prote ins and i n whole hemolymph Were subsequently deduced from the curve.

Synthesis by the Fat Body--The abdominal f a t body t issue o f two females o f M. sexta. 12 h a f t e r emergence. was remved and r i nsed I n lep idopteran sa l ine 10.005 M K 2 P O q . 7 J . F KCl, 0.004 M NaCl. 0.015 M MgC12. 0.004 M CaC12, pH 6.5 conta in ing 1 percent sucrose)

reac t i on v c l con ta in ing 100 u l o f t h e s a l i n e and 100 uCi o f 3%-methionine l s p e c i f i c 1Jungreis e t c., 1973). The t i ssue was r i n s e d t w i c e i n t h e s a l i n e and t rans fe r red t o a

a c t i v i t y = w)O Ci/mnol. Amersham) where It was incubated for 3 h a t 27'C. The t i ssue was then iso la ted from the medium by cen t r i f uga t ion 110.000 x g fo r 3 min1. r i n s e d t h r e e t i m s i n the sa l ine and then homgenized i n 1 nl of saline. The h m g e n a t e was centr i fuged and

S. aureus c e l l s 1Calbiochem-Behrlng Co.. La Jo l l a . CAI t o r e m v e labeled products tha t b ind the supernatant s o l u t i o n t r e a t e d w i t h r a b b i t s e r m , followed by an incubat ion wi th Pansorbin - ""=""'lV in the ant ibody DreCiDi ta te. The o r e c i a i t a t e Was co l l ec ted bv cen t r i f uga t ion . and the superna tan t so lu t i on t rea te i with antibody r a i s e d ~ a ~ a 6 s t microv i te l logenin. fo l lowed by another t reatment wi th S. aureus ce l l s (Harn i sh e t a l . . 1982). The second p r e c i p i t a t e was c o l l e c t e d as d e s c r i ~ d b a n d d i s s o l v e d b y b r l i n g f o 7 5 min I n 50 u l of SOS-PAGE sample t rea tment bu f fe r (Laeml i . 19701. The sample was then subjected t o SOS-PAGE and e lec t rophore t ica l l y t rans fer red on to n i t roce l lu lose paper as described above. The labe led p ro te in bands were v i sua l i zed by autoradiography.

""""YSrI I , L a , .

F i . 2. Gel enneation c h r m t o r a h --TO Buf fer so lub le prote ins frm M o l e egg hokgena te we!e appl ied on t o a &%ex& c o l m n 12.5 x 120 cm). The column was e lu ted w i t h PBS a t t h e r a t e of 15 ml/hr. The e lua te was co l l ec ted I n 4 ml fractions. Open c i r c les , absorbance a t 280 w; c losed c i rc les . absorbance a t 230 nm. 3: SOS-PAGE 14-15 percent) Of se lected f ract ions from the gel permeation colmn.

i -x""" -

- ? V I "

F i . 3. Cation exchan e c h m t o r a h --To Hicrov i te l logen in enr iched f rac t ions frm the

d ia lyzed against 0.01 M sodium succinate, pH 5.4. The p r o t e i n s o l u t i o n was a p p l i e d t o a w i s p y i i i k r a f i l t r a t i o n u s i n g VP 10 Amicon membrane. and

SP-Sephadex C-25 c a t i o n exchange column. H ic rov i te l l ogen in was e lu ted frm the column using a 0.1 M NaCl so lut ion. pH 7.0 (arrow). Open c i r c l e r 280 nm; c losed c i rc les . 230 nn

column. absorbance. -: SDS-PAGE (4-15 percent) o f se iected f ract ions f rom the cat ion exchange

I B . , . , , , , ,320

1 40

Insect Microvitellogenin 10849

Fig. 7. Isoelectric point detemination--When microvitellogenin was subjected t o isoelectric focusing using 5.000 Vh on an ultrathin 5 percent acrylamide gel slab. the protein comigrated w i t h the myoglobin (horse1 PI marker, with a PI 7.3. 1 and 3. Isoelectric point markers. 2. Microvitellogenin 115 us). The isoelectric point markers

myoglohin (horse. 7.3); myoglobin (whale, P.3); cytochrome c (10.7). are: amyloglucosidase 13.51; albumin 14.71: 0-lactoglobulin 1 5 . 3 ) ; ovalbumin ( 5 . 9 ) ;

Fi . 4. Adsor tion chrmato ra h --To Enriched fractions of microvitellogenin from the ca! on exc ang! step were c ~ m e ~ $ a t ~ t o 2 ml by u l t ra f i l t ra t ion The protein solution was dia:yred aghainst 0.03 M sodium phosphate pH 6 8 applied to a hydmxylapatite column I1 x 20

Microvitellogenin was eluted f rm the colmn a t about 0.14 M s o i i m p6ospha;e Open cm). and eluted w i t h a sodim phosphate linea; &adient (0.03-0 05 M) pH E 8.

c i rc les 280 nm; closed circles 230 nn' triangles sodium phosphate gradient: Bottm SOS-PAGE (4-15 percent) showing*homogen;ous sample; of microvitellogenin ImVg) fromthe

0.22 M sodium phosphate. adsorption chromatography column. Apolipophorin 111 fApoLp-111) eluted from the column a t

I I

Fi . 9. S nthesis of microvitello enin b f a t bod --When isolated fat body tissue frm 12 h ad:lt femayes was incubated w i t h JBS-meth);onine in'saline, pH 6.5 a t 27'C for 3 h, a number of proteins includinu mlcrovitelloaenin were svnthesized bv the tissue. Microvitellogenin was imnun6precipitated k i n g specif?c rabbit aniiserm and~Pansorbin 5 .

Daoer and the Drotein bands Visualized bv autoradiooraohv. The fioure t h w C an -cells. The sample was then subjected to SDS-PAGE. transferred to nitrocellulose-

autoradiograph of I . [l4C1-mthylated &tern standbd; i,imrshamI~-Z: i&io&ecioitated microvitellogenin.