ABSTRACT
Marine invertebrate embryos can concentrate amino acids
from dilute solution against gradients as steep as 10°. I
report here that Notoacmea incessa embryos can concentrate
leucine and methionine 200 and 400 times, respectively, over
ambient levels after 15 minutes incubation. Transport
increased at 3 hours post-fertilization with maximal rates for
the first 36 hours of development occurring by 12-18 hours
post-fertilization. A Lineweaver-Burk analysis of leucine
of 4.9 X 10-14
uptake revealed a Km of 1.262 uM and a Vmay
moles/embryo/15 minutes. This Ky value is comparable to that
observed for other marine invertebrate embryos, but the Vmay
value is 10-100 times lower. Approximately 703 of the leucine
transported by the embryo was incorporated into protein, as
compared to 153 for methionine. Several possible explanations
for the different rates of incorporation are presented.
INTRODUCTION
From the moment of fertilization until an organism has
developed a functional digestive system, one might assume that
the events of development must be fueled by the embryo's
endogenous reserves stored in the yolk of the egg. Manahan,
however, has reported that only 133 of a developing marine
invertebrate embryo's metabolic demands can be met by these
endogenous reserves (Manahan, 1988). Many studies (reviewed in
Stephens, 1988) have suggested that the developing embryo
supplies the remainder of its metabolic demand by concentrating
dissolved organic matter from the surrounding seawater. In
fact, marine invertebrate embryos can concentrate free amino
acids from seawater against gradients as steep as 10° to 10'.
As thermodynamically unfavorable as this would seem, Manahan
has calculated that the cost of transporting a mole of glycine
against such a concentration gradient would be less than 43 of
the potential benefit an organism could derive by the oxidation
of that same mole of glycine (Manahan, 1983).
Most of the present work, however, has concentrated on the
mechanics of the transport system itself and few have focused
attention on the changes which this system may undergo as an
organism passes through the early stages of development. This
study describes the pattern of amino acid transport during the
first 36 hours of development of the marine invertebrate
gastropod, Notoacmea incessa.
Uptake of leucine and methionine rapidly increased at 3
hours post-fertilization, reaching maximum concentrations of
200 and 400 times, respectively, over ambient. The Ky value of
leucine uptake was comparable to that observed for other marine
invertebrate embryos, but the Vmay
value was 10-100 times lower
than the other Vpax values. The bulk of the leucine taken into
the embryo was incorporated into protein, wheras for
methionine, most remained unincorporated.
MATERIALS AND METHODS
HANDLING OF GAMETES
Notoacmea incessa, commonly found grazing on the brown
algae Egregia menziesii, were collected from the intertidal in
the general vicinity of the Hopkins Marine Station, Pacific
Grove, California. Immediately following collection, the
limpets were placed in filtered seawater, originally at 150,
but allowed to come to room temperature over several hours to
induce spawning.
Eggs were fertilized in filtered seawater with penicillin
and streptomycin (each at 150 mg/1, hereafter referred to as
FSW+) by addition of dilute sperm. Approximately 1 hour after
fertilization, excess sperm was removed by washing the eggs
three times. Washing involved sedimentation of the embryos by
hand centrifugation, aspiration of the supernatant, and
resuspension in fresh FSW+.
Embryo concentrations were adjusted to 500/ml (0.053 y/v)
or 1000/ml (0.18 v/v). Direct embryo counts were performed on
multiple 50 ul aliquots from homogenous suspensions. Embryo
volume was calculated from measurements of egg diameter using a
stage micrometer and light microscope.
The developing embryos were incubated at 500/ml FSW+ at
150 in a beaker with stirring. FSW- was changed at 12 hour
intervals to maintain water quality.
MEASUREMENT OF AMINO ACID TRANSPORT
Embryos were pulsed with radioactive-labeled amino acid
for 15 minutes at 150. "H-leucine (stock specific activity of
52 Ci/mmol, diluted with cold leucine to 5.2 Ci/mmol) and 328-
methionine (stock specific activity of 1361 Ci/mmol, undiluted)
were added simultaneously to 4 ml of a 1000 embryo/ml
suspension to final concentrations of 1.0 x 10"°M for leucine
and 7.0 x 10"2 M for methionine. The embryos were kept in
suspension with gentle bubbling. At the end of the incubation
period, embryos were washed three times with cold FSW to
remove unassimilated labeled amino acid. Two ml of cold 102
trichloroacetic acid (TCA) with 1 mg/ml cold carrier leucine
was then added to the embryo pellet. Following extraction at
4C for 1 hour, the embryos were sedimented and 500 ul of
supernatant was added to 5 ml of Ecolume scintillation fluid
for determination of free amino acid content. The remainder of
the TCA supernatant was aspirated and the embryo pellet was
then washed three times with 2 ml cold TCA. The pellet was
dissolved in 2 ml 0.5 M-NaoH with 58 Triton X to obtain the
TCA-insoluble fraction (protein). 500 ul of this solution was
added to 5 ml of Ecolume scintillation fluid to assay for
incorporation of the labeled amino acid into protein.
Radioactivity measurements were made in a Beckman LS-1300
scintillation counter. Counting efficiencies were corrected
for differing quenches by the two solvents, TCA and NaoH/Triton
using external standards. The efficiencies observed were in
the range of 15-208 for 3H and 508 for 328.
SDS GEL ELECTROPHORESIS AND AUTORADIOGRAPHY
Samples were prepared by adding 100 ul of 2X SDS buffer
(200 mg sucrose, 40 mg sodium lauryl sulfate, and 40 ul beta¬
mercaptoethanol in 2 ml distilled water) to a pellet of 3000
embryos which had been pulsed for 145 minutes with 7 x 10"2 M
998-methionine. The samples were boiled for 10 minutes to
neutralize endogenous protease activity, sonicated with a cell
disrupter to homogenize the mixture, and frozen for storage.
The samples (15 ul) were run on a 108 SDS gel for 2 hours at a
constant current of 25 mA and allowed to expose X-ray film for
24 hours to produce the autoradiograph.
RESULTS
UPTAKE RATES
Figures 1 and 2 describe uptake of leucine and methionine
by N. incessa embryos from the environment during the first 36
hours of development. Transport occurs at a low level in the
unfertilized egg and declines as the egg deteriorates.
Fertilized eggs show increased transport activity beginning at
3 hours post-fertilization, continuing to increase for the next
9-15 hours to a level 4 times the unfertilized rates.
Transport rates then remained constant through the termination
of the experiment at 36 hours post-fertilization. The
approximate stages of development at each time point are given
in Table 1. Figure 3 shows that embryos concentrated leucine as
high as 200 times over ambient and methionine as high as 400
times over ambient during the 15 minute pulse. Figure 4 shows
the depletion of the available amino acid pool from the
environment as calculated from the moles of amino acid taken up
by the embryos. This depletion of the available pool may have
been large enough to alter significantly the ambient
concentrations of the amino acids, perhaps thereby affecting
uptake rates.
LEAKAGE OF AMINO ACIDS FROM EMBRYO
Although there was an apparent uptake of labeled amino
acid, it is possible that uptake involved both transport into
the embryo and diffusion out of the embryo down the steep
concentration gradient. If this were the case, then the
washing procedure with FSW- to remove the remaining labeled
amino acid would also wash out much of the amino acid which had
accumulated in the embryo. To test this, 6-hour embryos were
incubated in 1 x 10-6 M leucine with or without 2x 10"9
emetine HCl, a protein synthesis inhibitor, for 15 minutes at
150. After rapid washing to remove the remaining labeled amino
acid, the embryos were left in fresh FSW at 150 for varying
periods of time during which they could leak out accumulated
amino acid, if they do indeed leak.
The leakage of up to 308 of the transported leucine after
10 minutes in FSW as implied by Figure 5 indicates that leakage
from the embryo is substantial. The downward trend of Figure
5, however, is inconsistent and most likely due to experimental
error. As the rate of leakage, if it occurred, would depend
upon the concentration gradient, one would expect that if the
internal concentration of free amino acid were increased, the
leakage rate would show a corresponding increase. The presence
of a protein synthesis inhibitor would prevent amino acids from
incorporating into proteins, forcing them to remain in solution
and thereby increasing their internal concentration relative to
the control. The addition of emetine, a potent protein
synthesis inhibitor (843 inhibition observed), during the
pulse, however, failed to elicit increased leakage rates,
implying that leakage is not occurring to a significant extent
and that the transport system does not have to overcome rapid
diffusion of amino acids to the environment.
Km AND Vmax
Values for K and Vpay of transport were obtained for
leucine uptake only. Table 2 shows how the values obtained for
N. incessa compare to those obtained by Epel for the sea urchin
embryo, S. purpuratus, (Epel, 1972) and by Manahan for the
oyster embryo, C. gigas (Manahan, 1988). Note that depletion
of the available amino acid pool (Figure 5) may have
significantly decreased the ambient concentration of leucine,
consequently giving erroneous values for Ky and Vmay.
INCORPORATION
Figures 6 and 7 report the fate of the transported amino
acids, whether remaining free or becoming incorporated into
protein. Leucine was incorporated into protein to a much
greater extent (70.83 +/- 6.4, n=8) than was methionine (14.43
+/- 4.8, n=8). Note that the free amino acid fraction may
contain both intact amino acids and metabolized forms as well.
AUTORADIOGRAPH
An autoradiograph (Figure 8) was done to reveal any
patterns of protein synthesis which might be characteristic of
a particular developmental stage. Note that only proteins
synthesized during the radioactive pulse will appear as bands
on the autoradiograph. No unique pattern of bands was observed
for any of the time point samples. The overall darker
character of the gel lanes which contained fertilized embryo
samples confirms that fertilized embryos are indeed taking up
and incorporating exogenously-supplied amino acids to a much
greater extent than unfertilized eggs.
DISCUSSION
Embryos of N. incessa were pulsed with labeled leucine and
methionine and rates of uptake were observed to change during
the course of the first 36 hours of development. Over a 15
minute pulse, the embryos maximally concentrated leucine to 200
times over ambient and methionine to 400 times over ambient.
Leucine also showed considerable incorporation into protein,
while much of the methionine remained free.
The apparent ability of unfertilized eggs to concentrate
amino acids 50 to 100 times over ambient (Figures 1,2,3)
suggests that unfertilized eggs possess the same transport
system as fertilized eggs, but which is not activated to the
same extent. This proposition that increased transport of
amino acids results from the activation of a pre-existing
system rather than the de novo synthesis of one post-
fertilization is demonstrated much more directly by Epel (Epel,
1972), where activation of transport is shown to remain
unaffected by fertilization in the presence of protein
synthesis inhibitors.
Leucine and methionine transport is significantly
activated by 3 hours post-fertilization. Uptake rates increase
steadily through 12-18 hours, but level-off soon afterwards.
The leveling-off of uptake rates after 12 to 18 hours may
indicate that maximal transport has been achieved by that time,
but it is also possible that this is simply a consequence of
the depletion of the available amino acid pool to levels
sufficiently low to affect uptake rates adversely (Figure 4).
Adding amino acid in non-limiting amounts would resolve this
10
ambiguity.
The close agreement of the K value for N. incessa with
those for S. purpuratus and C. gigas suggests that a similar
type of membrane transporter molecule is involved, although the
particular amino acids used to determine the values were not
same in all three cases. The lower value of Vpay observed for
N. incessa as compared to S. purpuratus (Table 2), however,
may be due either to a lower density of transporters in the N.
incessa membrane or a smaller surface area available for
transport. Although the surface area of the N. incessa egg
(diameter 140 um), assuming a smooth sphere, is nearly 3.5
times larger than that of the S. purpuratus egg (diameter 75
um), microvilli as observed by Epel (Epel, 1977) on the surface
of the S. purpuratus egg could conceivably provide the smaller
egg with a greater surface area. As to the reason why N.
incessa might have either a lower density or a lesser surface
area for transport, perhaps N. incessa embryos do not need to
transport amino acids as vigorously as S. purpuratus or C.
gigas, since they must only survive for 4 days before they
begin feeding, wheras S. purpuratus and C. gigas must last for
over 30 days (Strathmann, 1987). Further studies are necessary
to confirm the Vmax
value observed for N. incessa and to
determine the nature of its deviance from other values, if any.
Berg has noted that exogenous valine is incorporated into
protein preferentially over endogenous stores (Berg, 1970).
While incorporation of endogenous leucine was not considered
here, the tendency of leucine transported from the environment
11
to be incorporated into protein appears to be consistent with
Berg's findings. Methionine's tendency, however, to remain
unincorporated contradicts the findings of Ilan and Ilan (Ilan
and Ilan, 1981), who reported that exogenously supplied
methionine is preferentially channeled into protein by S.
purpuratus embryos.
Methionine's tendency to remain unincorporated relative to
leucine could have several possible explanations. One
possibility is that methionine is simply not as ubiquitous in
N. incessa proteins as is leucine. A CRC table listing mole
percentages of amino acids in several common proteins indicated
that leucine typically comprises 10-158 of a protein, while
methionine typically comprises only 1-48 (CRC, 1970). Another
possibility is that although methionine, whose tRNA is coded
for by the start codon, is present in the leader sequence of
every peptide, N. incessa could cleave off the leader sequences
from its proteins more rapidly than other organisms, thereby
decreasing the frequency of its occurrence in the protein
fraction. Lastly, methionine could be prevented from being
incorporated into protein by its metabolism to other forms.
In
one particular metabolic pathway, methionine serves as a methyl
donor for the methylation of phosphatidyl ethanolamine to
produce phosphatidyl choline, a major cell membrane component
(Lehninger, 1970). As considerable cell membrane must be
synthesized during the early stages of development, it seems
quite plausible that the demand for phosphatidyl choline could
cause much of the exogenous methionine to be metabolized,
thereby precluding its incorporation into protein.
12
SUMMARY
N. incessa embryos concentrated leucine and methionine in
15 minutes to levels as great as 200 and 400 times,
respectively, over ambient concentrations. This concentrating
ability did not become activated until 3 hours post-
fertilization. The apparent concentrating ability of
unfertilized eggs, albeit 4 times lower than maximal fertilized
rates, suggests that the transport system is present in the
unfertilized egg, simply awaiting activation upon
fertilization. Leucine appears to be channeled primarily into
protein while methionine seems to remain largely
unincorporated. The lower level of methionine incorporation
may be due to its infrequent occurrence in common proteins, its
cleavage from proteins with the leader peptide, or its
metabolism in other biosynthetic pathways.
ACKNOWLEDGEMENTS
I would like to acknowledge the help and guidance of
Professor David Epel, Dr. Robert Swezey, and Denis Larochelle,
without which this project would have been impossible.
LITERATURE CITED
Berg, W.E. Further Studies on the Kinetics of
Incorporation of Valine into the Sea Urchin Embryo.
Experimental Cell Research 60 (1970) 210-217.
Handbook of Biochemistry, Selected
Chemical Rubber Company.
Data for Molecular Biology. Cleveland: The Chemical
Rubber Co. (1970) pp. C281-C293.
D. Activation of an Na"-Dependent Amino Acid
Epel,
Transport System Upon Fertilization of Sea Urchin Eggs.
Experimental Cell Research 72 (1972) 74-89.
The Program of Fertilization.
Scientific
Epel
D.
American Vol. 237, No. 5 (November 1977) 128-138.
J. and J. Ilan. Preferential Channeling of Exogenously
Ilan
Supplied Methionine into Protein by Sea Urchin Embryos.
Journal of Biological Chemistry 256 (1981) 2830-2834.
The Molecular Basis of Cell
Lehninger, A.L. Biochemistry:
Structure and Function. New York: Worth Publishers
(1970) 194-195,523,524.
Manahan, D.T. et al. Simultaneous Determination of Net
Uptake of 16 Amino Acids by a Marine Bivalve.
American Journal of Physiology 244 (1983) R832-R838.
Manahan, D. Shape and Form During Animal Development:
Adaptations to the Chemical Environment. Lecture
delivered 5/27/88 at Hopkins Marine Station,
Pacific Grove, CA.
Stephens, G.C. Epidermal Amino Acid Transport in Marine
Invertebrates. Biochimica et Biophysica Acta
947 (1988) 113-138.
Strathmann, M.F. Reproduction and Development of Marine
Invertebrates of the Northern Pacific Coast. Seattle:
University of Washington Press (1987) pp. 224,233,328,528.
TABLE/FIGURE LEGENDS
Table 1. Approximate stages of N. incessa development
observed at time-course experiment time points. Embryos
incubated at 15c.
Table 2. Comparison of Kn and Vmay
values for amino
acid transport in three different marine invertebrates.
Particular amino acids used to determine each of these sets of
values may not have been the same.
Figure 1. Leakage of labeled-leucine from pulsed embryos
into surrounding filtered seawater. Embryos pulsed in the
presence of 2 X 10
M emetine (protein synthesis inhibitor)
did not show increased leakage over control.
Figure 2. Concentration of leucine and methionine in
developing N. incessa embryos reaches several hundred times
ambient concentration.
Ambient leucine concentration = 1x 10°M
3 M.
Ambient methionine concentration = 7x 10
Figure 3. Leucine uptake rate by N. incessa embryos
increases dramatically post-fertilization. Ambient leucine
concentration = 1x 10
M.
Figure 4. Methionine uptake rate by N. incessa embryos
increases dramatically post-fertilization. Ambient methionine
concentration = 7x 10
Figure 5. Approximately 703 of leucine transported into
developing N. incessa embryos is incorporated into protein.
Figure 6. Approximately 153 of methionine transported into
developing N. incessa embryos is incorporated into protein.
Figure 7. Developing N.incessa embryos deplete a closed
environment of available amino acids by as much as 503,
significantly lowering ambient concentrations and,
consequently, altering uptake rates. Initial ambient
concentrations:
leucine = 1x 10
methionine - 7x 10
Values calculated from embryo uptake rates -- measurements were
NOT taken for amino acid content of seawater.
Figure 8. Autoradiograph of SDS Gels.
Upper gel contains successive time points of developing embryos
(1,3,6,12,18,24,36,48 hours post-fertilization).
Lower gel contains successive time points of unfertilized eggs
(0,1,3,6,12 hours old).
Samples in both gels for time point 1 hour post-fertilization
were incorrectly processed. Time points 0,1,3 hours actually
probably represent 4 hours post-fertilization.
TIME IN HOURS
—---——
12
18
24
36
TABLE 1
STAGE OF DEVELOPMENT
—---——
Unfertilized egg
2-cells
4-,8-,16-cells
Solid ball of cells
Oval to muffin-shaped with semi-
active, sparse band of cilia.
Muffin-shaped with denser band of
cilia.
Trocophore larvae: muffin-shaped with
band of cilia about crest and tuft of
cilia at apex. Very active/motile.
Early veliger larvae: shell
beginning to form.
TABLE
ay in moles/emb/15 min
Ki
1.262 uM 4.922 x 10-14
N. incessa
2-hour embryo
3 X 10-13
purpuratus 1.4 - 2.0 uM
3-hour embryo
4 X 10-12
gigas
1 - 4 uM
larvae
3.5 —
3 -
2.5
1.5
5
0 +
FIGURE 1
AMINO ACID UPTAKE

LEUGNE

—
—
10
HOURS POST-FERTILIZATON
UNFERTILIZED
D FERTUZED
4.5 —
3.5 —
2.5 -
1.5 -
0.5
O -
FIGURE 2

AMINO ACID UPTAKE
METHIONINE





HOURS POST-FERTILIZATON
UNFERTILIZED
D FERTLZED
450 -
400 -
350 -
300 -
250
200 -
150 -
100
0 +
FIGURE 3
AMINO ACID CONCENTRATION



g
30
HOURS POST-FERTILIZATOON
METHIONNE
LEUGNE
FIGURE 4
DEPLETION FROM ENVIRONMENT
AMINO AC
CALCULATED FROM EMBRYO UPTAKE
100—

so
80 -
gd
70 -
60 -

50 -

40 -
30 -
20 -
10 -
0 +
10
HOURS POST-FERTIUZATON
METHIONNE
LEUGNE
40
FIGURE 5
LEAKAGE OF LABELED-LEUCINE
FROM 6-HOUR EMBRYOS


90 -

70 -
50 -
40 -
30 -
20 -
—
10
12 14 16 18 20
2 4 6
0
MINUTE
TIME
EMETINE-TREATED
D CONTROL
3.2 -
2.8 —
2.6 —
2.4 -
2.2
1.8 -
1.6 -
0.8 -
0.6 -
0.4 -
0.2
0
FIGURE 6
AMINO ACID UPTAKE/INCORPORATION
LEUGNE





30
HOURS POST-FERTILIZATON
X TOTAL
D PROTEIN
FREE
4.5 -
3.5
2.5 -
1.5
0.5 -

FIGURE 7
AMINO ACID UPTAKE/INCORPORATION
METHIONINE





30
10
HOURS POST-FERTILIZATON
X TOTAL
H PROTEIN
FREE
205 KD -
116.
97.4-
205 KD
116
97.4
66
44
FIGURE 8
3 6 12 18 24 36 48
01 3 6 12