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/. Embryol. exp. Morph. Vol. 35, 3, pp. 507-519, 1976
Printed in Great Britain
507
Immunological studies on the
incorporation of radioactive leucine into sea urchin
embryonic proteins: extractability and
turn-over of newly synthesized proteins
By MONICA WESTIN 1
From the Wenner-Gren Institute, University of Stockholm
SUMMARY
This study was undertaken in order to investigate the turn-over of newly synthesized sea
urchin embryonic proteins, the distribution of these proteins in the cell and their changes in
solubility with time. Refined immunological methods providing unique possibilities for
studies of individual proteins were utilized. The proteins were obtained in two soluble fractions: a concentrate of proteins from the cytosol was obtained by homogenization in a
hypotonic medium and another fraction constituting membrane-associated proteins was
solubilized by detergent treatment of the residual pellet; 75 % of the total protein content in
early and 60 % in later developmental stages was thus solubilized. The proteins solubilized
by these mild methods retained their antigenicity and could be analyzed by immunodiffusion
methods.
The fate of newly synthesized embryonic proteins was studied by incorporation of a
radioactive amino acid using two different incubation procedures. The results of these studies
indicated that newly synthesized proteins changed their solubility properties with time.
Proteins were thus transfered from the soluble to the insoluble fraction. The findings that
more proteins became insoluble during development mentioned above also supports such an
interpretation.
The synthesis of individual proteins was studied by immunodiffusion and autoradiography.
Most of the antigens in the two soluble fractions were identical. Experiments with absorbed
antisera revealed that only two to four antigens were present exclusively in one of the two
soluble fractions. Three groups of antigens differing in regard to their metabolic activities
were distinguished. Some antigens were labeled after both procedures without substantially
increasing in concentration during development. Thus, their labeling was probably sustained
by turn-over. Other antigens were labeled only after one of the two incubation procedures
indicating large variations in regard to synthesizing rates of individual proteins.
INTRODUCTION
Protein synthesis as an expression of gene activity is suitably studied on sea
urchin embryonic material. It is well established that development and protein
synthesis are not directed by an immediate genetic control during the early
cleavage stages (Gross & Cousineau, 1964; Gross, Malkin & Mover, 1964;
1
Author's address: University of Stockholm, The Wenner-Gren Institute, Department of
Immunology, Fack, S-104 05 Stockholm, Sweden.
508
M. WESTIN
Denny & Tyler, 1964; Baltus, Quertier, Ficq & Brachet, 1965; Giudice, Mutolo
& Donatuti, 1968; Westin, 1969). Differentiation beyond the hatching blastula
stage, however, requires an intact genome (Gross et al. 1964; Giudice et al.
1968; Westin, 1969). The total protein content per egg or embryo is constant
during the whole development to the pluteus stage (Fry & Gross, 1970) and no
protein or nitrogen leaves the cells (Gustafsson & Hasselberg, 1951). Radioactive amino acids in the culture medium are readily taken up by the embryos,
increasing the cellular amino acid pool, and are utilized for protein synthesis.
The radioactivity thus incorporated does not leave the embryo (Fry & Gross,
1970). Fry and Gross also found that, although as much as 80 % of the added
radioactive leucine, when free in the pool, was converted into other amino acids
within an hour, 95 % of the radioactivity incorporated into proteins was still in
leucine. Thus, the newly synthesized proteins seemed to be very stable. The high
synthetic rate and the fact that there is no gain in protein content suggest that
storage proteins are degraded.
The high discriminating ability of the immunodiffusion methods was utilized
in this study for the investigation of individual sea urchin embryonic proteins.
These methods can, however, only be used for studies of proteins in solution.
Detergent treatment and enzymic digestion can often solubilize proteins bound
to membranes or in particles without destroying the antigenicity or enzyme
activity (Raftell & Blomberg, 1974).
In the present study the amount of extractable proteins obtained by different
procedures was determined. The embryos were incubated with a labeled amino
acid and the amount of radioactivity incorporated into different protein fractions was studied. Individual proteins were investigated by two-dimensional
(crossed) immunoelectrophoresis combined with autoradiography. In order to
study the turn-over of the newly synthesized protein species, two different
labeling procedures were applied. Some embryos were incubated with the
labeled amino acid shortly before freezing while others were allowed to develop
for different lengths of time after the isotope incubation.
MATERIAL AND METHODS
The sea urchin employed in this study was Paracentrotus lividus, collected at
Laboratoire Arago, Banyuls-sur-Mer, France.
Fertilization and rearing of the embryos were performed as previously described (Westin, Perlmann & Perlmann, 1967). The cultures always contained 106
eggs per 100 ml sea water and the temperature was kept at 18 °C. At this temperature the embryos hatched at around 12 h and were fully developed gastrulae
at 23 h. Streptomycin and penicillin were added to the cultures each at a concentration of 10/£g/ml sea water.
In vivo labeling of protein. Protein was labeled by [14C]-leucine (specific
activity 165 mCi/mM). 20/*Ci [14C]leucine per 100 ml culture was administered in
Turn-over of newly synthesized embryonic antigens
509
one dose at different developmental stages 30 min before freezing. In other
experiments the embryos were incubated with [14C]leucine for 2 h and then
allowed to develop for different lengths of time in unlabeled leucine, 2 mg/100 ml
culture, before freezing.
[14C]Leucine was purchased from the Radiochemical Centre, Amersham,
England.
Extraction of protein. The embryos were homogenized in 0-005 M Tris buffer,
pH 7-8, containing 0-01 M EDTA until all cells were broken. The suspension was
centrifuged at 105000 g for 60 min. The supernatant was collected and the
pellet was homogenized once more in the same buffer and centrifuged as
described. The pooled supernatants were designated sup I.
The following detergents were used for further extraction of the pellet: 1 %
DOC (sodium deoxycholate, Merck, Germany), 0-5 % Lubrol W (cetyl polyoxyethylene condensate, ICI, England) and 1 % Triton X-100 (Sigma, U.S.A.).
The pellet was homogenized in combinations of these detergents, incubated for
60 min at 0 °C and centrifuged as described. The pellet was extracted once more
by detergent. The two supernatants were pooled and called sup JJ. The residual
pellet was solubilized in 1 MNaOH. Protein concentrations in all extracts, were
determined according to Lowry, Rosebrough, Farr & Randall (1951) with
bovine serum albumin as standard.
Measurement of[uC]leucine incorporation into TCA-insoluble proteins. Protein
concentrations and radioactivity were always determined on aliquots from the
same diluted sample. Proteins in sup I and II were precipitated on Whatman 3M
chromatographic paper (Mans & Novelli, 1961) by ice-cold 10% TCA and
treated as described previously (Westin, 1969). The residual pellet was subjected
to the same procedure but in a tube. After the extraction the pellet was solubilized in I MNaOH and the protein concentration determined. Samples were
evaporated to dryness in glass vials for determination of radioactivity.
Aliquots of whole eggs or embryos were solubilized in 1 MNaOH and protein
concentration and radioactivity were measured as described.
The radioactivity was measured in a Beckman liquid scintillation counter
with 97% efficiency. The scintillation liquid was PBD (0-5% butylphenyl
biphenyloxadiazole in toluene).
Antisera production. Antisera against total homogenates of unfertilized eggs
(AS-E) and plutei (AS-P) were obtained by intramuscular injections of these
homogenates into rabbits. The antigens were emulsified in Freund's complete
adjuvant. For detailed description see Westin et al. (1967).
Absorption of antisera. Antiserum against eggs (AS-E) or plutei (AS-P) were
absorbed with the soluble (sup I and sup II) and insoluble protein fractions of
eggs or plutei. Nine mg soluble egg or pluteus proteins per ml antiserum were
added in three aliquots to each of AS-E and AS-P. Insoluble proteins of egg or
pluteus were homogenized in the antisera. Approximately 20 mg insoluble
proteins were added per ml antiserum. The mixtures were incubated at 37 °C
510
M. WESTIN
Table 1. Distribution of radioactivity in protein fractions from sea urchin eggs
or embryos labeled in vivo with [uC]-leucine (20 fiCi\100 ml culture)
(The cultures were incubated with the amino acid for 30 min before freezing.)
Final
age of
embryos
(h)
0
4
6
cpm/mg protein x 10~3
sup I
sup II
Radioactivity ('
pellet
\
Total
11
187
c
sup I
sup 11
pellet
8
102
23
18
44
33
46
4
14
37
148
33
155
185
102
16
38
46
129
287
462
155
398
404
85
203
256
16
39
45
16
35
49
16
28
63
79
16
45
18
70
304
382
206
17
37
39
46
20
22
24
27
30
36
42
48
80
84
91
113
74
75
86
65
295
267
265
291
257
257
280
286
212
394
207
394
431
220
525
261
270
169
233
160
190
226
215
180
% mean values
19
20
21
22
22
23
16
12
18
35
32
30
28
38
40
37
40
37
10
12
36
46
48
49
50
40
37
47
48
45
0, Unfertilized egg; 16, mesenchymeblastula; 24, gastrula; 48, pluteus.
sup I, water-soluble proteins; sup II, detergent-extractable but water-insoluble proteins;
pellet, proteins insoluble both by water and detergent extractions.
The percentage radioactivity in the three fractions were calculated on the following
figures of protein extractibility: 50 % of the proteins in sup I for stages 0-30 h and 35 % for
stages 42 and 48 h; 25 % of the proteins in sup II for all stages.
for 1 h and then over night at 0 °C. The antigen-antibody complexes were
pelleted by centrifugation for 1 h at 100000 g.
Two-dimensional (crossed) immunoelectrophoresis. Soluble antigens in sup I
and II were analyzed by the technique of Clarke & Freeman (1966, 1968) as
modified by Westin & Perlmann (1972).
Autoradiography. The washed and dried agarose plates were exposed to
llford X-ray film (Industrial G) (Morgan, Perlmann & Hultin, 1961) for 6 weeks.
RESULTS
The soluble protein fraction in sea urchin eggs and embryos. By homogenizing
the eggs or embryos in a hypotonic medium about 50 % of the egg proteins was
extracted (sup I). During development the proteins became somewhat less
extractable and in plutei about 35 % could be extracted by this procedure.
This easily extractable fraction probably contained a concentrate of proteins
Turn-over of newly synthesized embryonic antigens
511
Table 2. Distribution of radioactivity in protein fractions from sea-urchin
embryos labeled in vivo with [uC]-leucine (20 /iCi/lOO ml culture)
(The embryos were incubated for 2 h in the radioactive amino acid and then allowed
to develop for varying lengths of time in [12C]leucine (2 mg/100 ml culture.)
cpm/mg protein x 10~3
Final age
of embryos
(h)
sup I
sup II
(2-4) 7
(2-4) 10
(2-4) 14
(10-12) 15
(10-12) 18
(10-12) 21
(12-14) 17
(12-14) 20
(12-14) 23
42
44
45
37
38
33
36
34
35
108
102
106
91
92
73
89
77
68
pellet
Radioactivity (%)
Total
291
121
282
118
306
125
280
111
244
103
239
95
233
99
233
94
219
89
% mean values
sup I
sup II
pellet
18
19
18
17
19
18
18
18
19
18
22
22
21
20
22
19
23
20
19
21
60
59
61
63
59
63
59
62
62
61
Figures within parentheses indicate hours after fertilization when labeling was done. .12,
hatching blastula; 18, early gastrula; 23, gastrula; sup I, sup II and pellet were the same as in
Table 1. The percentage radioactivity was calculated as in Table 1.
from the cytosol. The pellet after this extraction was analyzed by electronmicroscopy and showed tightly packed membranes.
By detergent treatment another protein fraction was brought into solution
(sup II). The quantitatively best results were obtained by a mixture of 1 % DOC
(sodium deoxycholate) and 0-5 % Lubrol W (cetyl polyoxyethylene condensate).
A further 20-25 % protein could be solubilized by this treatment. Essentially
the same figures were obtained when different developmental stages were
extracted. The detergent extractable fraction represented proteins bound in
membranes and granules. An electronmicroscopical study of the detergenttreated suspension only revealed minor membrane fragments. All detergent
extractions described below were done with the DOC-Lubrol mixture.
In vivo incorporation of leucine into the protein fractions. Radioactive leucine
was administered to cultures of sea urchin embryos for 30 min before freezing.
The specific radioactivity was determined in the three protein fractions specified
above. As the percentage protein in each fraction was known, the distribution
of radioactivity in the different fractions could be calculated. Only about 20 %
of the radioactivity was found in the easily extractable fraction (sup I) although
this fraction constituted 50 % of the protein content (Table 1). About 35 % more
radioactivity could be extracted by detergents, but a substantial amount, about
45 % of the radioactivity, was found in proteins non-extractable by the methods
used. These figures were obtained both with unfertilized eggs and developmental
stages (Table 1).
512
M. WESTIN
Table 3. Number of immunoprecipitates seen in autoradiograms when watersoluble protein extracts (/) of 4 h embryos or 48 h plutei or detergent extracts {II)
of the same developmental stages were reacted with absorbed antisera
(The antigens were obtained from embryos, which had incorporated radioactive
leucine in vivo.)
AS-E
A
Extract
4hl
4hII
abs e I
abs e 11
abs e pellet
0
1
1
0
2
2
AS-P
A
Extract
abs pi I
abs pi II*
abs pi pellet
48 h i
48hII
0
1
3
2
4
4
AS-E: antiserum against whole homogenates of unfertilized eggs absorbed with watersoluble extract (el) detergent extract (e II) or insoluble pellet of eggs (e pellet); AS-P:
antiserum against whole homogenates of plutei absorbed with three fractions of plutei
(pi I, pi II and pi pellet).
* The antiserum was not completely absorbed. It developed, however, fewer radioactive
precipitates when reacted with the extract used for absorption than with the other extract.
In order to study the turn-over of newly synthesized proteins another labeling
procedure was used. Embryos were labeled by incubation for 2 h with [14C]leucine,
and were subsequently transfered to sea water containing an excess of unlabeled
leucine. The development was interrupted after different lengths of time (see
Table 2). The distribution of the radioactivity between the three protein fractions was determined and compared with that of the short-term incorporation
described above (Tables 1, 2). The water-soluble proteins contained the same
percentage of radioactivity under both incorporation conditions, namely about
20 % of the total activity (Tables 1, 2). The detergent-extracted proteins, however, contained much less radioactivity after the long-term incorporation. This
activity constituted only 21 % (range 19-23 %) of the total as compared to
37 % (range 30-45 %) after short-term incorporation. The insoluble proteins
contained correspondingly more radioactivity 61 % (range 59-63 %) as compared to 45 % (range 33-50 %). The radioactivity per mg protein in the combined fractions changed somewhat from experiment to experiment as can be
seen in the Tables 1 and 2. This may be due to slight differences in the experimental conditions or the condition of the embryos. The long-term experiments
showed somewhat lower total radioactivities, than the short-term experiments.
It was, however, quite clear that the radioactivity was differently distributed in
the two types of experiments.
Turn-over of newly synthesized embryonic antigens
513
Fig. 1. A photograph of an autoradiogram obtained from a two-dimensional
(crossed) immunoelectrophoresis plate. 48 II: a detergent extract of 48 h plutei;
AS-P abs pi I: antiserum against plutei absorbed with a water-soluble extract of
plutei.
Antigens in the water-soluble and the detergent-extracted fractions assayed by
two-dimensional {crossed) immunoelectrophoresis. Antisera against unfertilized
eggs (AS-E) and against plutei (AS-P) were used. As these antisera were produced by injecting whole homogenates of the two developmental stages they
contained antibodies against antigens in all three fractions studied. The twodimensional immunoelectrophoresis plates showed a very complex system of
precipitates, about 25 different lines being distinguishable. When, in addition,
the invisible but radioactive precipitates were revealed by autoradiography, an
accurate comparison between different combinations of fractions and antisera
became very difficult. Most soluble antigens appeared to be the same in the two
soluble fractions of the same stage as judged from the wet electrophoresis
plates or from the autoradiograms.
In order to reveal possible differences in the antigen set-up between the
fractions, the antisera were absorbed. Thus, antiserum against unfertilized eggs
(AS-E) was absorbed with either the water-soluble fraction (I), the detergentextracted fraction (II) or the insoluble pellet of eggs. Antiserum against plutei
was absorbed in the same way with the three plutei fractions. These absorbed
antisera were then reacted with extracts of embryos which had incorporated
radioactive leucine in vivo. Only antisera absorbed with the insoluble fractions
developed visible immunoprecipitates. In the autoradiographs, however, even
antisera absorbed with the soluble fractions showed precipitation lines. The
antigenic differences between the two soluble fractions were remarkably small.
One to two antigens appeared to be specific for each of these fractions both in
514
M. W E S T I N
AS-P
+
AS-P
AS-P
(c)
Fig. 2. Photographs of six autoradiograms obtained from two-dimensional (crossed)
immunoelectrophoresis plates, (a-c) water-soluble extracts of embryos 8,16 and 24 h
old, respectively, labeled in vivo by 14C-leucine for 30 min before freezing, (d-f)
extracts of embryos 7,18 and 23 h old in vivo labeled by 14C-leucine at, respectively,
2-4, 10-12, and 12-14 h after fertilization and subsequently incubated in [12C]leucine. AS-P: antiserum against plutei. Arrows indicate immunoprecipitates only
labeled after one of the two labeling procedures.
Turn-over of newly synthesized embryonic antigens
515
early stages and in plutei (Table 3 and Fig. 1). The insoluble fractions seemed,
however, to lack additional antigens which were found in the soluble fractions.
The antisera absorbed with these fractions still contained antibodies against
two to four antigens in sup I and sup II (Table 3). It was, however, impossible
to test if the absorption with the insoluble fraction was exhaustive, as this
fraction cannot be assayed by immunodiffusion techniques.
Comparison of individual radioactive antigens obtained from embryos subjected to two different leucine incorporation procedures. Soluble extracts from
embryos labeled with [14C]-leucine for 30 min before freezing and embryos
labeled for 2 h and then left to develop for varying lengths of time were analyzed
by two-dimensional immunoelectrophoresis and autoradiography. Although
the visible precipitates were identical after the two incorporation procedures,
the autoradiograms showed distinctly different patterns of labeled precipitates.
Some antigens which were clearly radioactive after short-term labeling were not
labeled at all after the long-term labeling. More conspicuous were those precipitates which were heavily radioactive after long-term incorporation, but
which were not at all labeled after short-term incorporation (Fig. 2). Some
antigens were labeled in both types of experiments.
In the long-term experiments embryos were labeled early after fertilization
(2-4 h), just before hatching (10-12 h) and after hatching (12-14 h). The development was interrupted 5, 8 or 11-12 h later (see Table 2). In all these cases three
antigens, which were invisible in the agarose and in the autoradiograms after
short-term incorporation, were heavily labeled. Both the water soluble and
detergent extracted protein fractions seemed to contain these antigens. The
short-term incubation procedure did not label these antigens in any developmental stage from egg to plutei.
DISCUSSION
To be able to survey as much of the proteins as possible different solubilization procedures have been tried. As earlier reported (Westin et ah 1967) and
also found by others (Perlmann & Couffer-Kaltenbach, 1964; Ellis, 1966) 50 %
of the proteins were easily extracted from early stages simply by homogenization
in a hypotonic medium. Older stages were somewhat less easy to extract. After
this extraction proteins more or less tightly bound to membranes and particles
remained unextracted. Some of these proteins were extractable by detergent
treatment without destroying the antigenicity. Membranes may also be extracted
by changing the pH or by enzyme digestion (Raftell & Blomberg, 1974).
According to Raftell and Blomberg the best extraction of rat liver membranes
was obtained by detergent treatment. The detergent treatment of sea urchin
material did not destroy or change the antigenicity or various enzyme activities
of the proteins as these parameters were tested in the water-soluble extract in the
presence of DOC and Lubrol (Westin, 1975a). In all 60-75 % of the total proteins and 40-55 % of the radioactivity after in vivo incorporation with a labeled
516
M. WESTIN
amino acid were soluble and available for immunological assay. Test-tube
experiments suggest that the antisera at hand precipitated roughly 30 % of the
protein (Westin, unpublished) and 50 % of the protein-associated radioactivity
of the water-soluble fractions (Westin et al. 1967). Corresponding figures on the
detergent-soluble fraction are not available. Nevertheless, these data suggest
that a quantitatively important part of the newly synthesized proteins may be
analyzed by immunodiffusion.
Incorporation of [14C]leucine was chosen for the studies of protein synthesis.
Leucine is present in the cellular pool at a low and constant level and is readily
taken up from the surrounding medium, resulting in a slight expansion of the
pool (Fry & Gross, 1970). Some amino acids are less readily taken up in presence
of other amino acids (Mitchison & Cummins, 1966; Tyler, Piatigorsky &
Ozaki, 1966) and a mixture may therefore give less incorporated radioactivity
than expected. According to Mitchison & Cummins (1966) the uptake rate does
not fluctuate after the first cleavages, but stay at a constant high level. Thus, the
acid-insoluble label was considered to reflect protein synthesis and the results
from different developmental stages were very likely comparable.
Almost half of the radioactivity appeared in the insoluble proteins at all
developmental stages. After short-term incubation with [14C]leucine nearly 20 %
of the radioactivity was recovered in the water soluble proteins. Almost 40 %
of the label could further be extracted by detergent. When the embryos were
kept in [14C]leucine for 2 h and were then chased by unlabeled leucine for
different lengths of time, the water-soluble proteins still contained 20 % of the
radioactivity but the detergent-extracted proteins had lost nearly half of the
radioactivity to the gain of the insoluble fraction. This was found in all developmental stages from blastula to fully developed gastrula. These findings suggest
that the two soluble fractions either differed in their protein composition or,
alternatively, the same proteins may be present but that these proteins varied in
their metabolic activities in the two fractions. The immunological analysis was
undertaken in order to throw some light on this question.
Two-dimensional immunoelectrophoresis as such and in combination with
autoradiography revealed the presence of a large number (> 25) of distinct
antigens in the soluble fractions. The water-soluble and the detergent-extracted
protein fractions appeared to contain largely the same antigens. This was also
brought out by absorption experiments in which antisera were absorbed with
one fraction and tested against the other. Under these conditions, no precipitates appeared in the wet immunodiffusion plates when the two soluble fractions
were used for absorption. Moreover, most of the antigens present in the soluble
fractions were also present in the insoluble fraction since absorption with the
latter abolished most of the precipitates usually seen in the wet agarose plates.
The much more sensitive autoradiographic method revealed that the two
soluble extracts contained at least two to four antigens seemingly absent in the
insoluble fraction. In addition, at least one or two antigens seemed to be
Turn-over of newly synthesized embryonic antigens
517
specific for each of the soluble fractions. The actual number of 'fractionspecific' antigens was most certainly higher as appeared from the absorption
experiments, since the degree of mutual contamination of the three fractions
with each other is unknown and unspecific co-precipitation of antibodies cannot
be excluded entirely. That additional 'fraction-specific' antigens exist may also
be demonstrated by other methods. Thus, the two soluble extracts contain
different sets of enzyme active antigens (Westin, 1975a).
On the basis of these findings it may be assumed that a sizeable part of the
total radioactivity found in the different fractions reflects the presence therein of
the same individual proteins but in different metabolic conditions. This holds
true for the entire developmental period investigated. In essence, this implies a
change in solubility properties during embryogenesis of many newly synthesized
protein molecules. In fact, the insoluble protein fraction increases during
development. Also, the radioactivity of this fraction grows during prolonged
incorporation while that of the detergent-soluble fraction decreases correspondingly. Taken together these facts suggest a transfer of newly synthesized stable
protein molecules from the detergent-soluble fraction to the insoluble fraction.
It must be emphasized that autoradiography of immunodiffusion plates only
permits qualitative analysis of the antigenic fractions of the extracts. Therefore
the changes in radioactivity of the detergent-soluble fraction seen after application of different labeling procedures could not be traced back to the labeling
pattern of individual antigens seen in the autoradiographs. Two-dimensional
immunoelectrophoresis permits semi-quantitative estimates to be done of the
relative concentrations of individual antigens present in different extracts
(Clarke & Freeman, 1968). This is done by comparing the areas under the precipitation arcs, when extracts from different stages are reacted with aliquots of
the same antiserum. An increase in concentration by a factor of two will double
this area (Westin, 19756). On the basis of the labeling patterns seen after either
short-term [14C]leucine incorporation or long-term incorporation, and the
relative antigen concentrations in extracts from different developmental stages,
three groups of antigens could be distinguished.
(1) Some antigens were labeled both after short-term and after long-term
incorporation. Their relative concentrations in the extracts did not seem to
increase during development. The labeling could thus only be sustained by a
turn-over. These interpretations seemingly contradict the results of Fry & Gross
(1970). According to these authors, leucine is rapidly converted into other
amino acids when free in the cellular pool. Yet, the protein-bound radioactivity
is largely found in leucine. Thus, their results indicate that no turn-over takes
place.
(2) Other antigens were only labeled after short-term incorporation. These
antigens might have had a rapid turn-over, gradually losing their radioactivity
to stable proteins and proteins with slower synthesizing rates. At the time when
development was interrupted after long-term incorporation the radioactivity
33
EMB
35
518
M. WESTIN
may have been too diluted for sufficient labeling of these particular antigens.
Their relative concentrations did not seem to change much during the interval
studied.
(3) A group of antigens were labeled exclusively after long-term incorporation. These antigens were probably present at relatively low concentrations as
they never formed visible precipitates in the wet agarose plates. They appeared
to be heavily labeled, as judged from the blackening of the autoradiograms, 5 h
after the beginning of the incorporation. These antigens had probably a slower
rate of synthesis than the rest of the labeled antigens. Although these antigens
were synthesized at all stages studied, no increase in relative concentration in
the extracts could be detected.
While observation of labeling patterns in immunodiffusion plates only permit
some preliminary conclusions as to the synthesis and turn-over of individual
proteins it clearly demonstrates the great analytical potential of the immunological approach. Monospecific antisera against several individual antigens have
been produced. Such* antisera will be used to estimate absolute amounts and
specific radioactivity of these antigens throughout development. In addition,
they will be used to establish ultrastructural localization of these antigens in
eggs and different developmental stages (Westin, in preparation).
This investigation was supported by grants from the Swedish Natural Science Research
Council (B 2032-045). The author is indebted to the staff at Laboratoire Arago, Banyuls-surMer, France, for the supply of sea urchin material and working facilities. The author also
wishes to thank Professor Peter Perlmann for valuable discussion and criticism and Professor
Tore Hultin for reading the manuscript and good advice. The excellent technical assistance
of Miss Margareta Pihl is gratefully acknowledged.
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GROSS, P. R. & COUSINEAU, G. H. (1964). Macromolecule synthesis and the influence of
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GUSTAFSSON,
{Received 13 November 1975)
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