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The polypeptide structure of Vitellogenin and Vitellin from the cockroach Leucophaea maderae.

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Archives of Insect Biochemistry and Physiology 3:577-591 (1986)
The Polypeptide Structure of Vitellogenin and
Vitellin From the Cockroach,
Leucophaea maderae
Victor J. Brookes
Department of Entomology, Oregon State University, Cowallis
Vitellogenin (Vg) synthesized by the fat body of Leucophaea maderae is made
u p of four polypeptides with molecular weights of 160,000, 105,000, 98,000,
and 57,000. Other polypeptides previously reported as part of Vg are
associated with other proteins. Vitellin (Vt), the yolk protein (UP) isolated
from mature oocytes and from newly formed oothecae, i s a protein with a
sedimentation coefficient of 28s and consists of three polypeptides with
molecular weights of 105,000, 85,000, and 57,000. During vitellogenesis, the
YP of developing oocytes contains both Vt and a 14s component. The 14s
component i s made up of four polypeptides with molecular weights of
105,000, 90,000, 85,000, and 57,000. The data suggest that 14s may not be a
discrete protein but rather a form in transition between Vg and Vt in which
the 98,000 dalton polypeptide i s converted t o the 85,000 dalton polypeptide
of Vt through a 90,000 dalton intermediate. The 160,000 dalton peptide of Vg
does not appear to be a part of Vt. Under alkaline conditions, both the 14s
component and Vt are reduced to a polypeptide with a lower sedimentation
rate i n sucrose gradients. When acid conditions are restored, a protein
resembling 14s i s obtained. This suggests that the YP is a loosely held
aggregate of similar or identical proteins with a molecular weight of about
250,000.
Key words: Leucophaea maderae, polypeptide structure, protein processing, yolk proteins
INTRODUCTION
During the development of the oocytes, the fat body of insects synthesizes
a protein, Vg", which is secreted into the hemolymph, recovered by the
*Abbreviations used: 2,5,diphenyloxazole, PPO; 2-mercaptoethanol, PME; polyacrylamidegel
electrophoresis, PAGE; sodium dodecylsulfate, SDS; tetramethylethylenediamine, temed; trichloroacetic acid, TCA; Tris(hydroxymethy1) aminomethane, Tris; vitellin, Vt; vitellogenin, Vg;
yolk protein, YP.
Acknowledgments: I wish to thank Dr. Renk Feyereisen for a critical reading of the manuscript. Dr. Barbara Triplett kindly supplied radioactive globin. The research was supported in
part by funds from the Research Office, Oregon State University.
Received September 20, 1985; accepted March 21,1986.
Address reprint requests to Victor J. Brookes, Department of Entomology, Oregon State
University, Corvallis, OR 97331.
0 1986 Alan R. Liss, Inc.
578
Brookes
oocytes, and deposited by them into membrane-enclosed vesicles [l-41. In
many insects, Vg is synthesized as a primary translate of one or two proteins
with a molecular weight of about 250,000 and then modified by the attachment of lipids, carbohydrates, and phosphorous, and by cleavage into two
or more polypeptides [5]. In the hemolymph, Vg is a protein with a molecular
weight of about 220,000 to 650,000, depending upon the species; in some
insects, further modification occurs during deposition [4]. In another group,
the primary translate of Vg is smaller, about 170,000, and no cleavage occurs
[5]. In still a third group, the primary translate consists of three proteins with
molecular weights of about 45,000 [6]. In all insects studied, the protein in
the hemolymph is an aggregate of the primary translates or their cleavage
products. Nothing is known about the mechanisms of cleavage of primary
translates that exist in the majority of insects but appear to have been lost in
the evolutionary development of some members of this group of animals.
Among species in which cleavage is extensive, the number of polypeptides
produced is not clear [7-121.
As a preliminary to a study of cleavage mechanisms, the polypeptide
structure of Vg and Vt of Leucophaea maderae has been reexamined. In this
species, processing occurs both in the fat body and in the follicles [11,12].
The results obtained are similar in some respects to those previously reported
[ll,13,141, but additional evidence suggests another interpretation.
MATERIALS AND METHODS
Animals
Leucophaea maderae was reared as previously described [15]. Females were
mated on the eighth to tenth day after eclosion, and mating was established
by the presence of spermatophore in the bursa.
Chemicals
Reagents used for electrophoresis, including SDS, acrylamide, temed, and
molecular weight markers, were purchased from BioRad (Richmond, CA).
Tris buffer and ingredients for tissue culture were purchased from Sigma (St.
Louis, MO). Routine chemicals, including PME were Baker Analyzed Reagent. Radioactive chemicals were purchased from New England Nuclear
(Boston, MA), PPO from Packard Instruments (Downers Grove, IL), and
Triton N-101 from Rohm and Haas (Philadelphia, PA).
Purification of Yolk Proteins
The following definitions describe the various proteins associated with the
yolk:
1. YE' represents the proteins extracted from developing oocytes or from
newly formed oothecae and purified by dilution with water. Purified YP
consists of two components, 14s and 28s, which are obtained by centrifugation through a 10-30% sucrose gradient [16].
2. Vt is the mature yolk protein and is purified as described above.
Yolk Proteins From Leucophaea maderae
579
3. Vg is the protein secreted by the fat body during development of the
oocytes and is the precursor of YP. It was obtained from tissue cultures as
described below. The concentration of protein was estimated by absorption
at 280 nm as described [16].
Preparation of Antisera
Purified Vt was mixed with Freund’s adjuvant, and 10 mg of protein was
injected intramuscularly into the backs of sheep. One further injection of the
same amount without adjuvant was given after two weeks, and the sheep
were bled after a total of five weeks. In some experiments, the antiserum
was adsorbed with male hemolymph.
Cultivation of Fat Body and Analysis of Medium
Fat body from one insect was cultured for 4 or 5 h in 3 ml of a medium
described by Wyatt and Wyatt [17] and containing 4 pCi [14C]leucine. The
medium was removed and filtered through miracloth, and then centrifuged
for 15 min at 29,OOOg in a refrigerated centrifuge. In most experiments, the
medium from three to six cultures was pooled.
For the analysis of total protein secreted, 0.2 ml of medium was taken in
triplicate and diluted with an equal volume of 20% TCA. The samples were
refrigerated overnight and then centrifuged. The precipitate was washed
three times with 1ml of 5% TCA, and once each with ethanol, ethanol-ether
(1/1),and ether, dissolved in 0.2 ml 10% SDS and 0.01 ml58% NH40H [18]
and boiled for 3 min. The sample was made up to lml in sample buffer (0.125
M Tris, pH 8.8, and 0.1% @ME).Samples prepared in this way produced
bands on SDS gels identical to those obtained by precipitation of protein at
low ionic strength.
Samples of 0.2 ml were taken in triplicate and diluted with an equal
volume of antiserum, allowed to stand at room temperature for 2 h, and then
refrigerated for 24 h. In the earlier experiments, the samples were washed
three times with 0.15 M NaC1, but more recently with 0.4 M NaCl and then
three times with a solution described by Gellissen and Wyatt [19]. Precipitates were dissolved in sample buffer (0.125 M Tris, pH 8.8, 0.1% @ME,0.2%
SDS).
For determination of radioactivity, 0.2 ml of the SDS solutions were emulsified in 4 ml of counting solution (2 liters xylene, 1 liter Triton N-101, 14g
PPO) and counted in a Packard liquid scintillation counter, efficiency 90 to
95%.
Collection of Hemolymph
Hemolymph was collected by the method of Bohm [20], diluted five- to
tenfold with 0.4 M NaC1, and centrifuged for 15 min at 3,OOOg. Aliquots were
made up to 1ml in sample buffer and boiled for 3 min.
Polyacrylamide Gel Electrophoresis
SDS-PAGE was performed using the buffer system of Laemmli [21] at 10°C
and a constant voltage of 125 V. Slab gels were 9 cm long and 1 mm thick
Brookes
580
with a 4.5% stacking gel (pH 6.8). The gels were stained in 0.15% Coomassie
brilliant blue, 30% methanol, 0.6% acetic acid, and destained in 5% methanol, 10% acetic acid. For radiofluorography, the gels were immersed in
sodium salicylate [22], dried, and placed on Kodak X-OMAT AR film. Molecular weights were estimated by the method of Weber and Osborn [23].
Myosin (M, = 200,000) galactosidase (M, = 116,000), phosphorylase b
(M, = 92,500), bovine serum albumin (M, = 66,000) and ovalbumin
(M, = 45,000) were used as standards.
RESULTS
The Structure of Vitellogenin
Hernolymph polypeptides (Fig. 1A). Samples of hemolymph were
denatured in sample buffer and the polypeptides separated on SDS-PAGE.
The hemolymph of mated females with developing oocytes contains at least
18-20 polypeptides, many of which are also found in the hemolymph of
males and young virgin females with nonvitellogenic oocytes. Particularly
prominent are polypeptides in the molecular weight range of 78,000-85,000.
Polypeptides specific to oocyte development are found at positions
corresponding to molecular weights of 160,000 and 57,000 and between
105,000 and 92,000. Polypeptides of 105,000 and above 200,000 daltons are
common to all three types of hemolymph but are intensified in the
hemolymph of mated females.
Fat body polypeptides (Fig. lB,lC). Fat body was incubated in medium
containing [14C]leucine for 4 h. Protein was precipitated from the medium
with TCA, and the precipitate was dissolved in sample buffer and analyzed
-200
200-
-116
-
-
92
116105 92-
-
66
160
85 80 66 -
45
57 45
HEMOLYMPH PROTEINS
FAT BODY PROTEINS
Fig. 1. Electrophoretic patterns on S D S polyacrylamide gels of hemolymph proteins and
proteins synthesized by fat body. Hernolymph was collected and prepared as described in
Materials and Methods. Fat body was incubated in radioactive medium for 4 h, and proteins
precipitated with TCA and prepared as described in Materials and Methods. A and B) stained
with Coomassie brilliant blue; C) Fluorogram of gel shown in B. M = Mated female; V =
Virgin female; FB = Fat body proteins for comparison; Std = Standard.
Yolk Proteins From ieucophaea maderae
581
by SDS-PAGE. Polypeptides of proteins released into medium during
incubation of fat body from mated females showed a banding pattern similar
to that of hemolymph when stained with Coomassie Brilliant Blue.
Radiofluorographs of the gels showed that the principal proteins being
synthesized by fat bodies of males and virgin females were associated with
polypeptides larger than 200,000 and with three or four polypeptides in the
78,000 to 90,000 range. Fat bodies from mated females synthesized a
polypeptide with a molecular weight of 57,000, four others in the 94,000 to
190,000 range, and several larger than 200,000, one of which was common to
all three types of fat bodies but intensified in the medium of mated females.
Sucrose gradient separation of fat body proteins. After a 4 h incubation,
samples of medium containing [14C]leucine from fat bodies of males, virgin
females, and mated females were fractionated by sucrose density-gradient
centrifugation. Samples of the peaks were analyzed by SDS-PAGE and
radiofluorographed. The rate of synthesis by fat bodies of males and virgin
females was only a fraction of that of mated females. As shown in Figure 2,
media from all three stages separated into three or more peaks. Medium
from fat body from mated females contained a large peak (IIJ), which, in
D.P.M
2000c
10001
5001
250l
FRACTION NUMBER
Fig. 2. Sucrose density gradient fractionation of fat body incubation medium. Fat body was
incubated in radioactive media for 4 h, and 0.5 ml samples were centrifuged through a 1030% sucrose gradient for 16 h at 64,OOOg in a 27.1 Beckman rotor. Fractions of 1.2 ml were
collected precipitated with TCA, and prepared and counted as described in Materials and
Methods. (0-0,mated female; 0-0,virgin female *-*, male. Top of the gradient to the left.
582
Brookes
other runs, sedimented with the 14s component of yolk protein. This peak
contained a 57,000 dalton polypeptide, four other well defined polypeptides,
M, = 98,000, 105,000, 160,000, and one greater than 200,000 (Fig. 3, lane e).
Faint bands were also present in the upper part of the gels and at the 78,000
position. Fat bodies from males and virgin females also produced a protein
with a peak (IV) in this region of the gradient. As shown in the
radiofluorogram (Fig. 3, lane f), Peak IV from males was predominantly
labeled in the 78,000 dalton polypeptide. Peak I1 of mated females contained
two polypeptides with molecular weights greater than 200,000 and two of
about 80,000 (Fig. 3, lane c; the bands are very faint). Similar bands were
found in the peaks from the media from males and virgin females sedimenting
near the top of the gradient (not shown in Fig. 3). Peak I1 from virgin females
contained a band located between the 160,000 and 116,000 positions
(not shown). Fractions from the remaining peaks were not visible in
radiofluorograms.
Variations in polypeptide synthesis during development of the oocytes. Fat
body was taken from females at various times during the development of the
oocytes and cultured for 4 h in radioactive medium. Samples with about
equal numbers of counts (3,500-4,000; except g, 2,500 cpm) were analyzed
by SDS-PAGE. The rate of synthesis at the time of mating was low but rose
shortly after mating and reached a peak at about the time the oocytes reached
half maximal length. This rate was maintained until just before ovulation,
a
b
c
d
e
f
-205
---160
-116
-
-. 1 0 5
92- 98
- 87 68
-
- 66
-
57
- 45
- 29
Fig. 3. Electrophoretic analysis of sucrose gradient peaks of Figure 2. Samples were separated on SDS polyacrylamide gels and fluorographed. a) TCA precipitate, and b) precipitate
obtained by five-fold dilution with water, are from samples of incubation medium of fat body
from mated females taken before centrifugation; c fraction 3 (Peak I) mated female; d) fraction
7 (Peak I l l ) mated female; e) fraction 9 (Peak 111) mated female; f) fraction 11 (Peak IV) male.
Yolk Proteins From Leucophaea maderae
583
when it dropped to the premated rate. Figure 4A shows the polypeptide
pattern of proteins synthesized by fat body at 6 periods during the 15 days
of oocyte maturation. At the time of mating, the principal peptides produced
were those in the 78,000 to 84,000 dalton range (lane a). Traces of others,
including those associated with Vg, were also present, but the bands were
very faint. Two days after mating, when the oocytes were 1.6 mm long,
polypeptides associated with Vg were obvious, and those in the 78,000 to
84,000 range were diminished. When the oocytes reached one-half maximal
length (2.8 mm), the principal peptides synthesized were those associated
with Vg (lane c). The synthesis of a very large polypeptide (M,-250,000)
was just visible at this time, but it was pronounced in fat body of females
with large oocytes (lanes d,e). When the oocyte reached near maximum size,
Vg was apparently the major protein produced, and the large polypeptide
was no longer present (lane f). Fat bodies taken from females 24 h later and
just before ovulation only synthesized peptides in the 78,000 to 84,000 range
(lane g).
Antibody precipitation of vitellogenin. Antiserum prepared against purified
YP was used to precipitate protein from medium of fat body of mated
females. The precipitate was washed in 0.15 M NaCl or more extensively, as
described in Materials and Methods. As shown in Figure 4B, the antiserum
precipitated proteins that separated into four well defined polypeptides,
M, = 160,000, 105,000, 98,000, and 57,000 during SDS-PAGE. When the
precipitates were washed with saline alone, traces of other polypeptides,
a
b
c
A
d
e
f
a
g
b
c
d
a
'.I60-
-1 60-
-1 05T
9 8-
-1 05-98-
B
b
c
d
e
f
9
C
Fig. 4. Electrophoretic analysis on SDS polyacrylamide gels of fat body incubation media.
Figure is of fluorograms of the gels. Fat body was incubated for 4 h. A) Fat body from females
during various stages of oocyte development. Oocyte length: a) 1.1 mm (0 time); b) 1.6 mm;
c) 2.4 mm, d) 4.0 mm; e) 4.5 mm; f) 5.0 mm; g) 5.0 mm) (ovulation) 3,500-4,000 cpm each lane
except g, which was 2,500. B) Antibody precipitation of media proteins. a) and d) washed
three times with 0.15 M NaCI; b) and c) washed with 0.4M NaCl and according to Gellissen
and Wyatt [I91 approximately 3,000 cpm each. C) Solubility of proteins synthesized by fat
body. Fat body from eight incubations were pooled. Samples were taken for total protein
analysis (TCA) and the remainder diluted with five volumes H20 and stored on ice for 48 h.
The precipitate was recovered and dissolved in 0.4 M NaCI. The recovery was about 90%.
Aliquots were diluted to the concentration shown, and the precipitates that formed were
dissolved in sample buffer. Approximately 5,000 cpm placed in each lane except e, 1,500 cpm.
a) 0.036 M; b) 0.065 M; c) 0.079 M; d) 0.1 M; e) 0.132 M; f) total protein (TCA); g) H20precipitate,
original sample.
584
Brookes
both larger and smaller than the four, were also present (lanes a,d). These
other polypeptides were removed by more extensive washing (lanes b,c). In
Ouchterlony immunodiffusion tests, the antiserum reacted with a single
component of hemolymph of mated females but not of males, and with
samples of medium incubated with fat body of mated females (data not
shown).
Effect of ionic strength on protein solubility. Fat bodies were dissected from
eight females late in the development of oocytes (4-5 mm long) and cultured
individually for 4 h in radioactive medium. The medium was pooled and
samples taken to measure total labeled protein by TCA precipitation. The
remainder was diluted with five volumes of water. The resulting precipitate
was recovered and dissolved in 0.4 M NaC1. Ten ml more water were added
to the supernatant, which produced an additional precipitate that was
recovered and dissolved with the first. The total recovery of labeled protein
by dilution was approximately the same as the recovery by precipitation with
TCA, and the polypeptide pattern was similar. To determine if the proteins
of the medium could be fractionated on the basis of ionic strength, aliquots
of the NaCl solution were diluted with various volumes of water, the
precipitates were recovered, and the radioactivity counted. Recovery
depended on the ionic strength as shown in Table 1. Samples of about equal
counts (with the exception of the 0.132 M solution) were separated by SDSPAGE and radiofluorographed (Fig. 4C). The pattern does not vary with the
concentration of NaC1. Each concentration contains polypeptides corresponding to Mr = 57,000, 98,000, 105,000, 160,000, and a major and minor
band of higher molecular weight. Several peptides found in the original
sample were not present, however, suggesting that a second precipitation by
dilution produces some purification.
Proteolysis of Vg. The possibility that proteolytic digestion of Vg may be
occurring after secretion into the medium was tested in three ways: a) fat
body was incubated in labeled medium for 1 h, then removed, and the
medium incubated for an additional 4 h; b) fat body was incubated for 4 h,
removed, and the medium incubated for 16 h at 30°C; c) globin, synthesized
in a cell-free system to obtain a high specific activity, was incubated with
TABLE 1. Effect of NaCl Concentrationon the
Solubility of Vitellogenin*
Final concentration
NaCl (M)
0.132
0.100
0.078
0.065
0.036
Amount precipitated
(% total activity)
0.07
23.0
64.0
81.0
90.0
*Samples of purified Vg in 0.4 M NaCl were
titrated with various amounts of water.
Precipitates were recovered by centrifugation,
dissolved in sample buffer, and counted. The
recovery is expressed as the percentage (%) of
total activity precipitated by TCA.
Yolk Proteins From feucophaea maderae
585
unlabeled medium after a 4 h incubation with fat body. Aliquots of the
medium in all three tests were precipitated with TCA or by dilution with
water and analyzed by SDS-PAGE. In none of the experiments was there
any evidence of proteolysis. Incubation of the medium after removal of the
fat body does not result in the disappearance of polypeptides normally found
after such incubations nor in the appearance of new polypeptides. The globin
protein remains intact even after 4 h of incubation with medium (data not
shown).
The Structure of Vitellin
The effect of pH. Purified YP in 0.4 M NaCl was dialyzed overnight against
0.15 M Tris buffer, pH 8.8, and 0.4 M NaC1. Half the dialyzate was dialyzed
again, this time against 0.15 M Tris, pH 6.8, and 0.4 M NaC1. Samples of the
two dialyzates were analyzed by density-gradient centrifugation with samples
of the original solution that had not been dialyzed. As shown in Figure 5,
undialyzed YP separates into two fractions, 14s and 28s, as has been
repeatedly demonstrated [11,16]. In slightly alkaline solution (pH 8.8), only
one fraction with a lower sedimentation was obtained. Acidification by
redialysis aggregated the protein to a component that sedimented at the
same rate as 14s. No fraction comparable to 28s was found. Alkaline
conditions did not alter the polypeptide pattern of 14s or 28s. When these
components were purified and individually subjected to alkaline and acidic
conditions as described above, the polypeptide pattern after SDS-PAGE was
still characteristic of the component (data not shown, but patterns are
described in Fig. 6).
Polypeptide structure of 14s and 28s. The 28s component contains three
subunits (M, = 105,000, 85,000, and 57,000). The 14s component contains the
same polypeptides and a fourth one that migrated to a position just below
that of the 92,500 standard (Fig. 6A). Also shown is a radiofluorogram of a
partially purified fraction of Vg. Superposition of the exposed X-ray film over
the Coomassie brilliant blue-stained gel showed that 105,000 and 57,000
dalton polypeptides of Vg are identical to those of the 14s and 28s
components, but the 98,000 dalton polypeptide is slightly larger than the
fourth component of 14s.
DISCUSSION
During periods of vitellogenesis, the hemolymph of mated females contains proteins whose polypeptides separate into at least 18 to 20 bands of
SDS-PAGE. Although at least three of these polypeptides are not obvious in
the hemolymph of males and young virgin females with nonvitellogenic
oocytes, many others are common to all three types, and some are intensified
in the hemolymph of mated females (Fig. 1).Because of the large numbers
of peptides, those specific to females are difficult to identify in the hemolymph. Although all these proteins are probably synthesized by fat body at
some stage, the rates of synthesis and turnover probably vary, so that many
proteins are carried over to the adult from earlier stages.
586
Brookes
pH 6.8
pH 8.8
FRACTION
NUMBER
Fig. 5. The effect of pH on the structure of yolk protein. YP was purified as previously
described. Samples were dialyzed overnight against 0.15 M tris buffer pH 8.8, 0.4 M NaCI,
and then redialyzed against 0.15 M tris buffer pH 6.8, 0.4 M NaCI, and then analyzed by
sucrose density gradient centrifugation as described in Figure 2. Optical density of fractions
was measured at 280 nm.
The principal proteins synthesized by the fat body of adult males and
early virgin females contain high molecular weight polypeptides greater than
160,000 daltons and several in the 78,000 to 90,000-daltonrange. Polypeptides
of equivalent molecular weight are synthesized by fat body of mated females,
but in addition, four others appear to be specific. All the data presented here
strongly suggest that these four polypeptides with molecular weights of
160,000, 105,000, 98,000, and 57,000 are part of the Vg molecule. A large
polypeptide (M, - 250,000) is present in many fractions where Vg is expected and is most easily identified in Figure 4C.A polypeptide of this size
has been described as a subunit of Vg of L. maderue [ll] that is processed
during passage through the hemolymph. My data suggest that it is a polypeptide of another protein and not a subunit of Vg for the following reasons:
Yolk Proteins From Leucophaea maderae
V g 14s
28s
587
V g 14s 28s
-200-1 6 0 -105-
- 98 -90
- 85
-57
A
B
j
-
_I
Fig. 6 . Comparison of the polypeptide structure of vitellogenin and yolk protein by SDSPAGE. Vg was purified by precipitation at low ionic strength as described in Figure 3. The 14s
and 28s components were obtained from sucrose gradients [16].
1. It was present in protein precipitated with antiserum to Vt but was
removed by extensive washing in 0.4 M NaCl and detergent (Fig. 4B). In
previous studies the precipitate was washed only in saline solutions [HI.
2. Its rate of synthesis was not consistent with that of the four polypeptides
I associate with Vg. During vitellogenesis, the relative proportion of proteins
synthesized by the fat body changed. At the time of mating, and later when
vitellogenesis was complete, the principal proteins synthesized contained
polypeptides smaller than 90,000 daltons. Polypeptides larger than 200,000,
found during the early stages of vitellogenesis, reached a peak before the
oocytes were maximum size and were not present or were present in lesser
amounts during the final phase (Fig. 4A).
3. Precipitation at low ionic strength, a method also used in previous work
[ll], results in only a partial purification of Vg (Fig. 4C).
4. The high molecular weight polypeptides appear to be stable, and no
evidence was obtained for a conversion to polypeptides of Vg.
Gellissen and Wyatt [19] found that the principal proteins synthesized
during vitellogenesis in the locust were Vg and a diacylglycerol binding
protein, lipophorin. They describe lipophorin as a high molecular weight
protein with many properties similar to Vg. The rate of synthesis of Vg in
locusts increases during vitellogenesis, but that of lipophorin remains more
or less constant. Lipophorin now has been identified in a number of insects
588
Brookes
[24] including Peripluneta umericunu [25], and characterized as an aggregate of
two polypeptides with molecular weights of 240,000 to 250,000 and 78,000 to
85,000. Lipophorin has not been described in L. maderue, but such a protein
is likely to be present and represented by at least some of the nonvitellogenic
polypeptides described here, particularly those with molecular weights comparable to the ones associated with lipophorin in other species. This is
indicated by a recent experiment in which hemolymph from vitellogenic
females was subjected to density gradient centrifugation in KBr as described
by Ryan et al. [24]. A fraction was obtained with a density expected for
lipophorin, which contained two major polypeptides with M, = 250,000. In
the present work, polypeptides with comparable molecular weights present
something of an enigma, because they appear in several fractions after
centrifugation of fat body incubation medium in a sucrose gradient (Fig. 3).
Lipophorin, if it exists in L. maderue, may consist of a family of lipoproteins
differing in density, depending on the amount of lipid attached.
Koeppe and Ofengand [ll]isolated Vg by sucrose density-gradient centrifugation and by immunoprecipitation. They identified seven polypeptides
associated with this protein and designated them as: a (M, = 260,000); fi
(M, = 179,000); the four polypeptides of YP, A, B, C, and D (see below); and
one, E, with a molecular weight intermediate between A and D
(M, = 108,000). From the evidence presented in this paper and discussed
above, I suggest that the polypeptides a,B, and D are not related to Vg. The
polypeptide P is probably equivalent to the one I identlfy as 160,000 daltons,
and E to the one I find to be 98,000 daltons. The only other study of the
structure of Vg of L. maderue was done by Engelmann [12]. Vg was immunoprecipitated from preparations of microsomes and from hemolymph and
found to be made up of three polypeptides (M, = 96,000, 82,000, and 52,000).
He also found that the three were present in vitellin. The data are presented
in the form of scans, and I cannot explain the discrepancy.
Koeppe and Ofengand [ll]and Masler and Ofengand [14] described the
polypeptide structure of YP of L. maderue. They identified four polypeptides
designated A (M, = 118,000), B (M, = 87,000), C (M, = agreement in that
the 28s component contains three polypeptides, A, B, and C; and four
polypeptides, A, B, C, and D, are found in 14s. One difference between my
results and the earlier ones concerns the molecular weights of these polypeptides. The difference arises because of a discrepancy in the designation of the
molecular weight of the standards, P galactosidase and phosphorylase b.
Koeppe and Ofengand used 130,000 and 100,000, whereas the supplier of the
standards, BioRad, gives 116,000 and 92,500 as the molecular weight.
Koeppe and Ofengand [ll] suggested a ratio of polypeptides to conform
to the molecular weights established by Dejmal and Brookes ([16];
14s = lA:lB:2C:2D; 28s = lA:3B:2C). They further suggested that polypeptide B of 28s is derived from one unit of B and two units of D from the 14s
component. Masler and Ofengand [14] attempted to support this hypothesis
by an experiment in which the 28s and 14s components were disaggregated
in urea and the polypeptides separated by ion exchange chromatography.
They found that urea divided each component into three fractions, one of
which eluted with the void volume and was much more pronounced in 28s
Yolk Proteins From Leucopbaea maderae
589
than in 14s. Analysis by SDS-PAGE indicated that the fraction eluted with
the void volume was principally polypeptide B, whereas the other two
fractions contained all the polypeptides. Masler and Ofengand interpret
these results as indicating the presence of two structural forms of polypeptide
B, thus supporting the hypothesis of Koeppe and Ofengand. Their results
(Figure 3 in reference [14]) clearly show, however, that all three fractions
contain several polypeptides, indicating that urea only partially degraded the
protein into a complex mixture.
What has been overlooked in the earlier studies is that both 14s and 28s
are no more than loosely bound aggregates that are easily disrupted under
mild alkaline conditions. The polypeptide pattern of the alkaline fraction
depends on its source, 14s or 28s. Sedimentation in sucrose gradients indicates that, regardless of the source, the fraction consists of a single protein
that reverts to a 14s-like structure when acid conditions are restored. The
relative position in sucrose gradients indicates that the molecular weight
must be about half of 14s, M, = 260,000 (Fig. 5, and reference [ll]). The
structure based on the polypeptide ratio of Koeppe and Ofengand cannot be
divided into a smaller unit.
The sum of the molecular weights of the three polypeptides of 28s is about
250,000 and when multiplied by six, comes to within 10% of the measuredmolecular weight of this component [16]. The four polypeptides of 14s add
up to about 350,000 and when multiplied by two, exceed the molecular
weight; however, 14s may not be a discrete protein. During maturation of
the oocytes, the yolk may be expected to consist of the final yolk protein, Vt,
varying amounts of Vg, and transitional forms produced as the result of
processing. During purification of YP,much of the Vg would be lost because
it is much more soluble at low ionic strength, but it should be present in the
supernatant after YP has been recovered. I suggest a scheme (Fig. 6) for the
conversion of the 98,000-dalton polypeptide to the 85,000 polypeptide with
the intermediate formation of a 95,000 polypeptide. The 14s is thus a transitional form, so that the molecular weight cannot be estimated on the basis of
the polypeptides. The polypeptide pattern of 14s and 28s displayed by SDSPAGE always consist of more bands than A, B, C, and D associated with
these proteins, as is clearly seen in the figures of Masler and Ofengand [14].
I have many gels that are identical. These minor bands are represented by
dotted lines in the scheme shown in Figure 6. In 14s, the minor band is
equivalent to the 98,000- and the 90,000-dalton components. I have seen
these bands in the polypeptide pattern of YP isolated from the freshly formed
ootheca, in which only the 28s component is present, although only in overloaded gels in which they are very faint. According to my scheme, the
evidence of processing is also seen in the 28s component and continues even
after the egg is mature and fertilized. I have found no evidence that the
160,000-dalton polypeptide of Vg is a part of Vt, but peptide maps have yet
to be made.
Harnish and White [lo] have divided insects into three groups based on
the structure of Vt. The dictyopteran and orthopteran insects fall into Group
I, with at least one large and one small polypeptide in the molecule. Processing of the precursor, Vg, occurs shortly after synthesis in the fat body, and,
590
Brookes
in some insects, further processing may occur at the time of deposition. The
actual number of polypeptides produced is controversial, because different
workers working with the same insect obtain different results. Part of the
problem stems from the presence of proteins in the hemolymph and synthesized by the fat body in vitro with characteristics similar to those of Vg and
Vt. Some of these proteins precipitate nonspecifically in the presence of
antiserum, which is otherwise specific to Vg or Vt. Proteases may be present
that cleave the proteins during preparation. Although I could find no evidence for proteolytic activity in the experiments designed to test for this, I
cannot exclude the possibility that a domain of Vg is proteolytic as it occurs
in the nerve growth factor [26]. Denaturing the protein with SDS may expose
susceptible regions to this domain resulting in cleavage.
The validity of the model in Figure 6 is being tested by peptide mapping.
I am also trying to identify the translation products that are the precursors to
Vg and to isolate Vg in pure form.
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