Abstract
In the Monterey Bay, there are two types of Anthopleura elegantissima:
symbiotic and aposymbiotic. Symbiotic anemones have a relationship with unicellular
dinoflagellates. Algae are singly bound in membrane vacuoles within the endoderm of the
anemone. Aposymbiotic anemones do not have this association and probably do not have
the metabolic functions that are part of the symbiosis. Because of the differences in these
anemones, the hypothesis was established that there were protein differences between
aposymbiotic and symbiotic anemones.
Qualitatively, both membrane and soluble proteins in both anemones were
analogous. Quantitatively, however, disparate soluble protein quantities of sizes 106 kD
and 14 kD were found. The 14 kD protein was more prevalent in the symbiotic anemone
based on optical density readings taken from a scanned gel (0.292 vs. 0.057 Trace Optical
Density x mm); the 106 kD protein had a greater expression in the aposymbiotic
anemone (0.25 vs. 0.17). Further testing is needed to identify these proteins.
Please see Appendix for an explanation of band quantification.
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Introduction
Anthopleura elegantissima comes in two varieties in the Monterey Bay. White
aposymbiotic anemones are found on rocks that are not exposed to sunlight; brown
symbiotic anemones thrive in the high intertidal under the sun's rays.
The symbiotic anemones have a relationship with dinoflagellates. These are
brown, unicellular algae that give the anemones their characteristic color (Smith and
Douglas, 1987). The algae are found in membrane-bounded vacuoles within the
endodermal cells and are localized in regions of the anemone that receive the most
sunlight. These areas are the tentacles and the oral disk (Shick, 1991). Besides
dinoflagellates, there may also be green algae, or zoochorellae, that have a symbiotic
relationship with the anemones (Muscatine, 1971).
There are many examples of metabolic exchange between the symbiont and host.
On a macroscopic scale the nutritional contribution of the symbiotic algae to the host is
suggested by the fact that aposymbiotic anemones lose weight more quickly than do their
symbiotic counterparts when both are subjected to starvation under light. In addition,
symbiotic anemones gain weight more quickly when fed in light than in the dark and lose
weight more quickly when starved in the dark than in the light (Shick, 1991).
Many past studies have focused on the transport of photosynthate to the host.
Fixed carbon moving from the algae to the host has been demonstrated. Nitrogen can
move in the reverse direction from the host to the algae (Smith and Douglas, 1987). In
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addition, it has been shown that the majority of the metabolites released by the algae are
glycerol and fumarate (Trench, 1971).
Given the metabolic exchange between the algae and the symbiotic anemones and
the apparent lack of this exchange in aposymbiotic anemones, the question arises: what
protein differences are there between these two Anthropleura elegantissima?
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Materials and Methods
Aposymbiotic & symbiotic anemone collection and storage
Aposymbiotic sample collection involved scraping anemones off rocks below
Monterey Bay Aquarium. The symbiotic anemones were obtained from the high intertidal.
Sand and other debris were removed from the anemones. Aposymbiotic samples were
placed in salt water tanks exposed to artificial light. Symbiotic anemones were placed in
tanks outside under normal sunlight.
Protein extraction
The anemones were anesthetized in 1:1 volume 0.37 M MgCl: sea water. The
oral disk was removed from the anemones and the sample was homogenized in 1 ml buffer
on ice. The buffer consisted of 50 mM Tris at pH 7.4, 0.45 MNaCl, and 1 mM PMSF.
Homogenized solutions were transferred to Corex tubes and more buffer was added to
make the total volume of the samples 1.5 ml. These were spun for 2.5 min at 1000 rpm to
remove the algae. All spins were performed at 10 °C and whenever possible, samples
were placed on ice.
After the spin, the supernatant was decanted to microcentrifuge tubes and set
aside. To the remaining algal pellet in the symbiotic sample and to the empty
aposymbiotic tube, 2 ml of buffer was added. These were spun at 1000 rpm for 2 min.
The supernatant was transferred to microcentrifuge tubes.
All microcentrifuge tubes were spun for 20 min at 15000 rpm to separate the
soluble and membrane protein fractions. The supernatant from the first soft spin tube was
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decanted, this contained the soluble proteins. The supernatant from the second soft spin
tube was disposed. The membrane protein pellets in the microcentrifuge tubes were
resuspended and the solutions were transferred to two Corex tubes; one contained the
aposymbiotic sample and the other contained the symbiotic sample. Buffer was added to
make the solutions 10 ml. These tubes were spun for 20 minutes at 15000 rpm.
The supernatant from these tubes were disposed. The pellets were resuspended in
300 ul 2% SDS solution made from the above buffer. One scoop of glass beads was
added to both tubes and the tubes were vortexed to eliminate bubbles. The resulting fluid
was decanted and saved in a microcentrifuge tube. This sample contained the membrane
protein fraction. The final step was a 5 min spin at 15000 rpm to remove debris.
Bradford Assay
From both membrane and soluble protein samples from aposymbiotic and
symbiotic anemones, 10 ul and 100 ul fractions were taken. To the 10 ul fraction, 90 ul
of the 2% SDS buffer was added. The blank was 50 ul 1.0 N NaÖH.
Fifty ul 72% TCA was added to all tubes. These were vortexed for 5 min and
subsequently centrifuged for 10 min at 15000 rpm. 50 ul 1.0 N HCl was added to the
pellets. Fluid was transferred to large test tubes and 5 ml P-250 Coomassie protein
determination reagent was added. Large test tubes were immediately vortexed.
Beckman spectrophotometer readings were obtained using visible light at 595 7
utilizing the blanker as the calibration. The standard curve was produced by using 100 ul
aliquots of BSA in concentrations of 75, 100, 250, 500 ug/ml.
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Gel electrophoresis
Once the protein sample concentrations were ascertained, 5 ug of membrane and
soluble protein were respectively prepared from aposymbiotic and symbiotic protein
samples. To the 5 ug samples, 5 ug of 2X solubilizing buffer was added. Samples were
boiled for 5 min and subsequently spun at 15000 rpm for another 5 min.
The samples and prestained molecular weight Sigma standard were loaded and run
in a 10% polyacrylamide gel at a constant 25 mA. The gel was stained overnight in
Coomassie Blue and subsequently destained in 10% acetic acid and 10% isopropanol.
Gel Analysis
Gel analysis was performed using the Pharmacia IM Desk Top Scanner and
Pharmacia software. The gel was scanned into the computer as an image. Background
was appropriately subtracted from the defined lanes. Bands were then specified, matched,
and then compared on the basis of optical density and band size calculated from the
standard lane.
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Results
Figures 1-3 were the product of computer analysis. Figure 1 displays matching
bands which were defined on the computer for both membrane and soluble protein.
Matching bands were determined from the proximity and relative heights of the optical
density peaks in Figures 2 and 3.
Figure 2 shows a membrane protein comparison between symbiotic and
aposymbiotic samples. Every peak from the aposymbiotic graph is consistently higher
than those found in the symbiotic graph. Band numbers 1 through 6 in both lanes
correspond to matches. In addition, band numbers 9 and 12 from the membrane
symbiotic lane respectively correspond to the membrane aposymbiotic bands 12 and 15.
Figure 3 shows a soluble protein comparison. Matched bands for this figure are
also displayed in Figure 1. More bands were able to be defined for the symbiotic protein.
The most significant quantitative differences between symbiotic and aposymbiotic soluble
proteins were band matches 7 and 15 in Figure 1. These respectively correspond to 106
KD and 14 KD proteins as labeled in Figure 3.
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Discussion
Data analysis yielded evidence that there are some differences between proteins
found in the anemones. Qualitatively, membrane and soluble proteins were generally
analogous; quantitatively, however, soluble proteins showed differences in a 14 kD and
in a 106 KD protein.
The Bradford assay with BSA standards provided a reliable protein determination
curve with linear regression 0.998. Unfortunately, due to low volume of anemone
membrane protein sample, the membrane assays were performed only once and not in
duplicate. Despite the attempt to load equal quantities of protein, the aposymbiotic lane
in Figure 2 had consistently higher optical density readings than those in the symbiotic
lane. Qualitatively, however, many bands were able to be matched. Symbiotic bands
with optical density higher than 0.05 were used since readings below 0.05 fell too close to
background density. Accordingly, aposymbiotic bands with optical density lower than
0.10 were not used in qualitative analysis.
The soluble protein determination, however, provided a more reliable result. The
optical density readings were consistent throughout lanes 8 and 9 in Figure 3.
Quantitative as well as qualitative data could be used for soluble protein analysis.
Qualitatively, most symbiotic and aposymbiotic bands could be matched. The symbiotic
lane had more distinguishable bands. This may imply that symbiotic anemones have
different proteins than those found in aposymbiotic anemones, but it is more likely that
the missing proteins in the aposymbiotic lane did not resolve. The general optical density
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traces of both lanes were too consistent with each other to warrant an argument for the
lack of protein in the aposymbiotic anemone.
Qualitative analysis yielded some interesting implications. Match number 15 in
Figure 1 is a 14 KD protein. In Figure 3, this corresponds to band 18 in the symbiotic lane
and band 13 in the aposymbiotic lane. Ribulose bisphosphate carboxylase is an enzyme
that is found in chloroplasts. It has two subunits of size 56 kD and 14 kD (Stryer, 1988).
The presence of more 14 kD protein in the symbiotic lane implies that the algae were not
completely removed from the buffer during the soft spin. Given this, however, the 56 kD
protein should also have a higher optical density in this lane. In the 56 kD region of the
symbiotic lane, there is no band that has a notably higher optical density than the
corresponding band in the aposymbiotic lane. The identity of the 14 kD is left unknown;
cross referencing proteins of approximate size in an available 1968 CRC Handbook
yielded no satisfactory result.
In addition, the 40 kD soluble protein in Figure 1, which likely corresponds to
actin given experimental error, occurred in approximately two times greater quantity in
symbiotic anemones than in aposymbiotic anemones. This may imply that this protein
serves a greater metabolic role in symbiotic rather than aposymbiotic anemones.
Conversely, the matching 106 kD soluble protein found in Figure 1 raises another issue.
Apparently, the aposymbiotic anemone had a greater expression of a 106 kD protein than
in the symbiotic anemone. In Figure 3, this is seen in band 6 of the soluble symbiotic
protein and band 2 of the aposymbiotic protein. The identity of the protein is unknown,
but there may be a possible explanation for this phenomena. This protein may be
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expressed in greater quantities in aposymbiotic anemones due to specific needs of
metabolism without sunlight
With these findings, the next step is to rerun the experiment with greater protein
quantity and to perform the Bradford assay in duplicate or triplicate. In addition,
different lanes could be run in the gel. Control lanes could be both symbiotic anemone and
algal proteins, and algal proteins alone. In this manner, algal proteins could be
ascertained to determine their contribution of bands to the gel. A Western blot could also
be performed testing for the presence of Rubisco to see if band 18 in Figure 3 is indeed the
14 kD subunit. In addition, a higher percentage SDS gel could be run to examine in detail
the low molecular weight proteins. Finally, a two dimensional gel could be executed to
further resolve the proteins isoelectrically.
This study strongly suggests that there are noticeable protein differences between
aposymbiotic and symbiotic Anthopleura elegantissima. Further experiments will be
needed to determine the identity of these proteins and the reason for the disparity of
occurrence between the anemones.
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Literature Cited
Muscatine, L. (1971) Experiments on green algae coexistent with zooxanthellae in sea
anemones. Pacific Science, 25, 13-21.
Shick, J. M., 1991. A Functional Biology of Sea Anemones. Chapman & Hall.
Smith, D. C. and Douglas, A. E., 1987. The Biology of Symbiosis. Edward Arnold
Publishers.
Sober, H. A., 1968, CRC Handbook of Biochemistry. The Chemical Rubber Co.
Stryer, L., 1988. Biochemistry. W. H. Freeman and Company.
Trench, R. K. (1971) The physiology and biochemistry of zooxanthellae symbiotic with
marine coelenterates II. Liberation of fixed 14C by zooxanthellae in vitro. Proceedings
of the Royal Society. London., B, 177, 237-250.
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Appendix
Below is a schematic that explains how the computer quantifies bands. It was
taken from page 112 of the Pharmacia ImageMaster"" 1.0 Software manual.
In calculating band quantitation, the average optical density (O.D.) is first
determined across a row of pixels in a band. A specified band has N number of average
O.D. values. N is determined by band height. The wider apart the brackets, the more
average O.D. values will be included in the quantitation calculations.
The density trace is the average O.D. values that are plotted versus the width of
the band. The last step in calculating band quantitation is integrating under the curve to
the baseline, thereby giving units of O.D. x mm which is reported in the Abstract.
Band Quantification Method
Sample Width

Banc
Height
Lane Trace
Average pixel's O.D.
Average O.D.
across Sample Width
re
Plot Lane or Show Band
Integrate under curve to baseline
O.D. x cm).
r banc
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Table 1
Results of the Bradford Assay
Protein type
Protein concentration (ug/u)
0.286
Aposymbiotic membrane
0.494
Aposymbiotic soluble
Symbiotic membrane
0.335
Symbiotic soluble
0.298
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Figure Legends
Figure 1. The average KD size in this figure refers to the mean of the calculated band
sizes in both the aposymbiotic and symbiotic lanes. Lane number corresponds to
the numbering system of lanes found in Figure 2 and Figure 3. The matched bands
are numbered but do not correspond to the numbering system of the bands found
in Figure 2 and Figure 3.
Figure 2. Relative sizes of the molecular weight standard bands are labeled on the side of
lanes 4 and 5. Bands are numbered in order from the highest molecular weight
bands to the lowest molecular weight. A higher optical density of a band
compared with another signifies a greater presence of protein for that
corresponding molecular weight.
Figure 3. Relative sizes of the molecular weight standard bands are labeled on the side of
lanes 8 and 9.
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Average KD
207-
189-
159
132
107
41
7
Membrane Protein
Symbiotic Aposymbiotic
4
—
16

Figure 1
Matching Bands
Soluble Protein
Symbiotic Aposymbiotic
9

Average KD



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141—

106
102—

83-
74-
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63-

40
—15
14
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Figure 2
Membrane Protein Comparison
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Figure 3
Soluble Protein Comparison
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