c
A STUDY OF INTRACELLULAR
FREE AMINO ACID POOLS IN TISSUES
OF METAIDIUMASENILE
Kim D. Suelzle
Hopkins Marine Station of Stanford University
Pacific Grove, California 93950
Running title: F.A.A. in Metridium Senile
F.A.A. in Metridium Senile
Abstract
Kim Suelzle
Abstract
1) Metridium Senile taken from natural environments show no sig¬
nificant differences beteen animals in free amino acid pool
concentrations.
2) Gastrodermal tissue of Metridium Senile has a significantly high¬
er free amino acid pool concentration than does Tenntacular tis¬
sue which has a higher concentration than either muscular or
ectodermal tissue which have similar concentrations.
3) Fed Metridium Senile show higher intracellular free amino acid
concentrations than do starved Metridium senile.
4) Free amino acid pool concentrations in Metridiun Senile vary
in direct proportion to the salinity of the surrounding medium.
F.A.A. in Metridium Senile
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Kim Suelzle
Introduction
High concentrations of unbound intracellular free amino
acids have been described in a wide variety of marine invert¬
ebrates. Concentrations are found to be much higher than either
vertabrates of terrestrial invertebrates, Awapara (1962). These
free amino acid, F.A.A., pools appear to have several functions.
Stephens (1968), Potts (1958), Shaw (1958). and Lange (1963,1964)
have shown the concentration and individual constituents of F.A.A.
pools in a variety of marine invertebrates change as a result of
osmotic stress which suggests a possible osmoregulatory role for
intracellular F.A.A. Shick (1975) has shown that the levels of in¬
tracellular F.A.A. in polyps of Aurelia Aurita Scyphistomae increase
with feeding which suggests the F.A.A. pool may serve as a tempor¬
ary intracellular storage form of Amino acids for use in synthesis
as well as catabolism. Within an individual organism different tis¬
sues have been foundtto have radicly different concentrations of
F.A.A. Schlicter (1974) showed significantly higher levels of F.A.A.
in the cells of the gasrodermal tissue than in the cells in the ecto¬
dermal tissue of the sea anemone, Anemonia Sulcata, which suggests
the pools may serve different functions in different parts of the
animal.
The study reported here was designed to assess the function
of the free amino acid pool of The sea anemone,Metridium Senile.
Four aspects have recieved particular attention. One deals with the
variation in F.A.A. pools between different animals in the natural
environment. Another deals with differences between F.A.A. pools of
four different tissuesof a same animal. Also studied were the ef
fects of feeding and osmotic stress on F.A.A. levels.
F.A.A. in Metridium Senile
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Kim Suelzle
Methods and Materials
All animals were collected from the floating docks in Mon¬
ter arbor. Montere California.
Results
F.A.A. levels in Natural Populations
Eight animals were blotted dry and weighed. The animals were
immediatl frozen. A one gram sample of each animal was homogenized
in a glass tube and teflon homogenizer with four milliliters of sev¬
enty per cent cold ethanol. The homogenate was allowed to stand for
twenty four hours at 5° (before centrifugation at 10,000 rpms to
remove ethanol insoluable materials. The supernatant was removed and
the pellet washed by resuspension in two milliliters of 70% ethanol
followed by centrifugation. The supernatants and their washings were
filtered through Whatman #1 paper. Six samples of each extract were
andlysed using the ninhydrin technique of Moore and Stein (1952).
Qualitative analysis of individual amino acids was accomplished by
two demensional ascending paper chromatography employing Butanol¬
glacial acetic acid-HoO, 100:40:60, followed by phenol saturated
with H9O with detection with 2% ninhydrin spray.
Most animals showed similar levels of F.A.A. pool concen¬
trations. There are no significant differences between any values
of F.A.A. in the animals of the sample with the exception of animal
A which is significantly different from all other animals. Student
t test performed,P .05. The mean value for the sample was 94.4 M.
Replicates from the same anemones showed no significant difference
in values and indicates a high degree of reproducablity of extraction
and measurment methods.
F.A.A. in Metridium Senil
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Kim Suelzle
Chromatography revealed several ninhydrin positive spots
which corresponded with R values of standard solutions of: leu¬
cine, taurine, aspartic acid, valine, gluatamic acid, proline,
threonine, glycine, serine, and lysine. The following pairs of
am no acids could not be resolved chromatographicly: isoleucine
and leucine; proline and hydroxiproline; and glycine and serine.
The F.A.A. in different Tissue Fractions
The different tissue fractions were seperated during the course
of thawing frozen animals. The ectoderm was taken to be the first
layer on the column of the anemone to thaw. It is easily removed
with scalpel and tweezers. The mesoglea content of this sample is
Paction
not known. The tenntacles and oral discwas taken as the upper por¬
tion of a horizontal section through the column at the base of the
tenntacles and includes oral disc and mouth. Some mesentaries and
ectoderm were unavoidably included in this fraction. Muscular tis¬
was seperated from the mesentaries by scrapping the retractor mus¬
cles free fromm mesentary tissue. Gastrodermal tissue taken to be
exclusivly mesentaries. Tissue samples were dried in 100°C oven and
ground by mortar and pestal. Extraction same as above.
Table 2 presents results and shows significant differences
between F.A.A. levels of different tissues. Student t test shows
significant difference between all tissue fractions except between
ectoderm and muscular tissue which are similar. Gastrodermal tissue
shows the highest concentation of F.A.A. with tenntacles and oral
disc next highest. Ectoderm and muscular tissue had the lowest values,
conc
rat
F.A.A. in Metridium Senile
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Kim Suelzle
Table 3 gives the relationship of these concentrations relative to
the values for the gastrodermal fraction.
Chromatography did not reveal gross differences in the comp¬
osition of the F.A.A. pools in the different tissue fractions.
Effects of eeding and Starvation on the F.A.A. Pool
Ten animals were maintained for two weeks in running seawater
from which 90% of the particles 44and larger had been removed by
filtration. At the end of this period five of the animals were fed
2 ml of squid homogenate every three days. After three weeks of feed¬
ing F.A.A. levels were assessed in these five starved animals and
five fed animals. Extraction was carried out seventy two hours after
the last feeding.
The results are shown in table 4 and indicate F.A.A. levels
in tissues of fed animals were slightly higher than star animals.
However student t test performed on the data failed to show a sig¬
nificant difference, p.05(.10
The Effect of Salinity on F.A.A. Pools
Ten animals were cut in half and allowed to recuperate for
three days. All halves were reattached and opened during this period.
All animal halves were placed in 100%"instant ocean" for twenty four
hours to provide a common osmotic baseline.One half of an animal
was used as a control for the other half in subsequent experiments.
For four animals one half of the animal was placed in 100% instant
ocean and the other half placed in 125% instant ocean. For one ani¬
mal both halves were placed in 100% instant ocean. For four other
animals one half was placed in 100% instant ocean and one half in
F.A.A. in Metridium Senile
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Kim Suelzle
70% instant ocean. One animal one half was placed in 125% instant
ocean and one half in 70% instant ocean. All samples were kept at
14°C. After ten hours all animals were removed from water and dried
in 100°C oven to remove the influence of differences in intracellular
water concentrations. Extraction and analysis as before.
Results show that F.A.A. concentration varies in proportion
to the salinity. Increased salinity resulted in higher F.A.A. con¬
centrations. Lower salinities resulted in lower F.A.A. concentration.
Samples placed in 125% salinity had an average value of 25% higher
than samples in 100% salinity. Samples placed in 70% salinity had
an average of 20% lower concentration of F.A.A. than samples in 100%
salinity. Student t tests were performed on all pairs of values.
Allshowed significant difference, p2.05, except for two animals.
Dalas presnted on able
Discussion
Values for F.A.A. concentrations in animals taken from the
natural environment show surprizing consistancy. Shick (1975) noted
a variability of an order of magnitude in natural populations of
Aurelia Aurita polyps. I suspect that the lack of variability found
in this study is due more to my sampling technique than any inher¬
ent characteristic of the specie. Animals were taken from sample
sitesvery close in proximity thus providing a vec homogeneous en¬
vironment amoung sample animals. Samples taken from entirely differ¬
ent environments could be expected to have substantialy different
concentrations. To make any conclusions as to intra specie level
a much larger sample would have to be taken.
F.A.A. in Metridium Senile
page 7
Kim Suelzle
The differences in the levels of F.A.A. in specific tissues
are in accordance with several previously published reports. Schlicter
(1974) found a much higher concentration,by anorder of magnitude,
in gastrodermal tissue as compared to ectodermal tissue in the sea
anemone ,Anemonia Sulcata.Ferguson (1967) found similar differences
in tissue concentrations in the starfish, Henricia Sanquiolenta.
A possible explaination tissue differences may be related to
the digestive and absorbtive function of the gastrodermal tissue.
Ectodermal tissue must rely heavily on diffusion of F.A.A. from the
gastrodermis. Indications are thar this rate of diffusion is quite
low. The result is that there are fewer F.A.A. available to the ecto¬
dermal cell to incorporate into their F.A.A. pools.
Another possible explaination for the low levels of F.A.A.
in E to dermal tissues relates to osmotic regulation. The ecto¬
dermal tissue may be more suseptable to osmotic stress than Gastro¬
dermal tissue. Experiments have shown that movement of F.A.A. out
of the cells in response to osmotic stress may result in considerable
Ke
loss of F.A.A. to the environment.loweconcentrations in the most
stressed tissues could prevent any large scale loss of F.A.A. to the
environment.
in the anemone nemen sata
Schlicter (1974) showed thatthe majority of uptake of dissol¬
ved amino acids from the surrounding medium takes place in the cells
of the ectoderm. Lower concentrations of intracellular F.A.A. in
these tissues lowers the concentration gradient for transport of ex¬
tracellular F.A.A. into the cells.
F.A.A. in Metridium Senile
Page8
Kim Suelzle
The magnitude of the differences between F.A.A. concentrations
between starved and fed animals is somewhat lower than found in sim¬
ilar studies with other marine invert brates. Shick (1975) found
differencs between F.A.A. concentrations in starved and fed animals
was approximately fivefold. A possible explaination for the low
values observed in this study maybe related to the time period be
tween the last feeding and the time of extraction. Shicks values re¬
present values for animals extacted within twentyfour hours of the
last feeding. Studies have shown that there is an initial increase
in F.A.A. concentration within twentyfour hours of feeding, with with
the concentration dropping to a lower level after this time.By ex¬
tracting seventy two hours after final feeding it appears what is
being measured is the increase in this lower baseline level.
The salinity data indicates the response of the F.A.A. pool
to osmotic stress. STephens (1969) observed a similar response in
the brittle star, Ophiactis sp.Changes in the concentrations of
individual amino acids can not be detected by the methods employed
in this study. Actual concentrations of individual amino acids are
known to vary as a result of environmental conditions while the tot¬
al ninhydrin positive composition of the pool remains constant. Tau¬
rine and glycine have been shown to be moved out of the cell prefer¬
entially to other constituents of the pool in response to osmotic
stress. This situation could prove beneficial to the organism in
that more important structural and metabolic amino acids could
be retained in the tissue while the more expendable amino acids
i.e. taurine and glycine could fullfl the osmoregulatory functions,
page 9
in Metridium Senile
F.A
Kim Suelzle
Chromatoraphy revealed a very large concentration of taurine in
F.A.A. pool of Metridium Senile.
Summary
1) Metridium Senile taken from natural environmental conditions
show no significant differences in the F.A.A. pool concentration
between different animals
2) Gastrodermal tissue of Metridium Senile has a significantly high¬
er F.A.A. pool concentration than does tissue from the tenntac
which contains significantly concentrations than either muscular
tissue or ectodermal tissue which have similar concentrations.
Fed Metridium Senile show higher concentrations of intracellular
F.A.A. than starved M.S.
4) F.A.A. pool concentration varies in proportion the salinity
of the surrounding medium.
0
F.A.A. in Metridium Senile
Page 10
Kim Suelzle
Acknowldgements
I would sincerly like to thank the faculty and staff of Hopkins
Marine Station for making this Quarter such a wonderful experience.
F.A.A. in Metridium Senile
Page 11
Kim Suelzle
Bibliography
Awapara J.(1962) Free amino acids in invertebrates; a comparative study of
their distribution and metabolism. In Amino Acid Pools (edited by
HoldenJ.T) pp 158-175. Elsevier, Amsterdam
Ferguson, J.C. (1971) Uptake and release of free amino acids in starfish.
Bio. Bull. 141:122-129
Greenstien and Winitz (1961) Chemistry of Amino acids vol.2 Wiley
Heftman, Erich (1969) Chromatography Sec. Ed. Von nostrand Reinhold Company
Johanes, R:E. (1969) Are dissolved organics an energy source for marine in¬
vertebrates? Comp. Biochem. Physiol. 29: 283-288
Kittredge, J. §. (1962) Free amino acids in Marine invertebrates. In
Amino Acid Pools (Edited by Holden J. T.) pp 176-186
Mopre and Stein (1952) Colorometric determination of amino acids. J.
Biol. Chem. 176:3026
Schlicter D. (1974) The influence of physical and chemical factors on
the uptake by Actinians of amino acids dissolved in sea water.
Mar. Bio. 25:279-290
Shick M.J. (1975) Uptake and utilizeation of dissolved organic s by Aurelia
.A.A. in Metridium Senile
Page 12
Kim Suelzle
aurita syphistomae: temperature effects on the uptake process; nut¬
ritional role of dissolved Amino Acids. Bio. Buli. 149:117-140
Stephens (1969) Uptake of organic molecules in Marine invertebrates
Comp. Biochem. Physiol. 10: 191-202
Zweig, Gunter (1971) Paper Chromatograph
and electrophoresis. Vol. 2
Acedemic press.
F.A.A. in Metridium Senile
Page 13
Kim Suelzle
Table Captions
1) Concentrations of intracellular F.A.A.inidividuals of naturally occur¬
ing populations of Metridium Senile
2) Concentrations of intracellular F.A.A. in tissue fractions of Metrid¬
ium Senile.
3) Percentages of Intracellular F.A.A. in tissue fractions of Metridium
Senile relative to levels in gastrodermal tissue.
umS
4) Concentrations of intracellular F.A.A. in Metr
as a function
of feeding and starvation.
lium Senile as a function
5) Concentrations of intracellular F.A.A. in
of salinity.
Animal
Mean
* Replicates of animal B
+ Replicates of animal C
able 1
Concentrations
ngrm wet weight)
65 - 2.8
96 - 16.6
95 - 14.6
90 - 20.1
81 - 15.5
104 - 19.23
104 - 12.0
110 - 15.23
104 - 8.8
117 - 16.2
94.4 - 21.8
Page 14
Gastroderm
Muscular
Tenntacles
Ectoderm
Page 15
Table2
Animal
2
—
105416
67424 174412
15844.2 14449.9
6147.6 2444 63.645.6 63.644.7 81414
92115
4543
81125
10846.6 12048.7
7046.1 361.79
57114
30410 6847.3
c
n Metridium Senile
Kim Suelzle
Gastroderm
100
100
Muscular
42
Tenntacles
87
78
Ectoderm
54
Table 3
100
36
62
39
100
40
75
44
5
100
56
56
page 16
100
49
71
42
0
Kim Suel zle
.A.A. in Metridium Senile
Table
FED
Animal
concentrations
M/gm. wet wt.)
63.215.43
51.2 17.08
73.6+6.34
49.6 12.94
10117.88
67.72121.0
Mean
Page 17
NON-FED
concentrations
M/gm. wet wt.)
54.4+1.63
37.0+4.07
50.4 f2.51
73.6117.67
57.6 111.33
54.60 +13.2
Kim Suelzle
Page 18
F.A.A. in Metridium Senile
Table 5
Salinity
125%
100%
Animal
70%
CONCENTRATIONS
CONCENTRATIONS CONCENTRATIONS
Mgm. wet wt.) M/gm. wet wt.) M/gm. wet wt.)
88 113.4
74 18.63
59 17.0
60 f3.9
6819.9
32 15.7
9919.8
6812.2
108111.9
11318.9
86-8.6
5614.5
60 16.73
5617.5
5613.2
7614.3
88 15.5
102 14.3
8417.2
68 16.1
10