Ammonia excretion in A. elegantissima.
Peter T. Hetzler
INTRODUCTION.
Most marine invertebrates are ammonotelic, that is,
they excrete ammonia as the major end product of their pro¬
tein catabolism. Coelenterates appear to be no exception.
Delaunay (1934) states that Actinians excrete 52.7% of their
nitrogenous waste as ammonia, but through what pathways and
by what enzymes has not been elucidated. The findings pre¬
sented in this paper suggest that the sea anemone, A. elegan-
tissima (Brandt, 1835), is ammonotelic as expected, and that
two of the steps leading to ammonia excretion in this an¬
imal are catalyzed by enzymes commonly involved in amino acid
degredation.
MATERIALS.
All anemones were taken from the low intertidal zone
at the Hopkins Marine Station, Pacific Grove, California.
Large solitary animals, symbiotized by zooxanthellae, were
used as the source of all enzyme preparations. The symbi¬
otic and "aposymbiotic" anemones used in the feeding experi¬
ment were clonal and only members taken from the same two
clones were used. Animals selected as aposymbiotic, howev¬
er, were found to contain small populations of zooxanthellae.
METHODS.
Ammonia was measured by the method of Solorzano (1964)
except that all reagent additions were one half the volume,
and only 1 ml. of sample was used. Deammoniated water was
used as adiluent. 10 mls. of IN NaOH was added to 1 liter
of distilled water. The solution was brought to a
Peter T. Hetzler
Ammonia excretion in A. elegantissima.
boil, cooled to room temperature, and 10 mls. of IN HCI
was added.The method was used in the range from 0.6 to
Aug NH./ml. Measurements were made using a Klett-Sumerson
Photoelectric Colorimeter with a red filter against a re-
agent blank. The method was reproducible within 6%.
Enzyme preparations were obtained either by homo-
genization in deammoniated water or from imidazole buffer
extracts of acetone powders. Acetone powders were pre¬
pared as follows. 500 mls. of redistilled acetone kept at
0°C. was added to 50 mls. of anemone in a Waring Blendor,
macerated at high speed for 1 minute, and filtered through
Whatman No. 5 filter paper on a Buchner funnel with suc-
tion. The residue was washed twice with 500 ml. volumes
of chilled acetone on the filter. When the filter cake
had completely dried, it was pulverized with mortar and
pestle to give a fine power and stored at 0°0. 9 mls. of
O.5M Imidazole-HCl buffer, pH 7.2, was added to 0.2 grams
of this powder and the mixture was centrifuged in a re¬
frigerated International Centrifuge, PR-4. Essentially
no activity was associated with the sedimented pellet.
The resulting extract was purified by gel filtration us¬
ing Sephadex, G25.
Enzyme activity was assayed as follows. Glutamine
synthetase activity was measured in terms of the y-glu¬
tamyl transfer reaction as described by Rowe, et. al.
(1970). The release of ammonia from DL-alanine was mea-
sured using 0.235 umoles DL-alanine and  ketogluterate
at the same concentration; in,a total volume of 5 mls.
Peter T. Hetzler
Ammonia excretion in A. elegantissima.
of which 0.5 ml. was 1M potassium phosphate buffer, pH
7.3, and 1 ml. was enzyme solution. The volume of the mix¬
ture was then brought to 5 mls. with deammoniated water.
Appropriate controls were used in all assays. The mix¬
tures were incubated at 37°C. and sampled at intervals
for periods up to twolhours. Replicate samples were ta¬
ken at each sample time.
Four symbiotic and four "aposymbiotic" anemones
varying from 0.8 to 1.2 grams dry weight, which had been
starved for two months,were used in the feeding experiment.
Only two of each type were fed 0.07 grams, wet weight, of
squid. One fed and not fed of each type was placed in
the light and the dark in 80 mls. of Instant Ocean and
maintained at ambient seawater temperature, between 10.5
and 12°0. Samples of the water were taken for ammonia de-
termination at intervals for four hours after feeding.
RESULTS.
The results of the feeding experiment (Table 1 and
Figure 1) show that ammonia excretion is markedly in¬
creased after feeding. The levels of excreted ammonia were
decreased on prolonged incubation as can be seen in Fig¬
ure 1.
No evidence for the existence of glutamine synthe¬
tase was found in either type of enzyme preparation. How¬
ever, the L-alanine amidotransferase experiment did show
significant activity. The results appear in Table 2 and
in Figure 2. Note that the ammonia released is maximum at
15 minutes in all three enzyme assays. After correction
Ammonia excretion in A. elegantissima.
Peter T. Hetzler
for activity in the absence of substrate the maximum rate
of ammonia release is O.13 ug NH/minute for extracts pre¬
pared using 0.2 grams of acetone powder in 9 mls. of buffer.
Thereafter, the ammonia concentration decreases at a rate of
0.04 ug NHa/minute. The general features of the time course
of ammonia generation was observed in all assays with this
preparation. The total amount of ammonia released is equi¬
valent to one half the amount present in the substrate level
of DL-alanine used in the assay.

DISCUSSION.
The feeding experiment suggests that the endosymbiotic
zooxanthellae of A. elegantissima take up the ammonia released
by the animal. These results support the observations of
Trench (1971) who found in the coral, Porites divaricata,
that 50% more C alanine was synthetized by zooxanthellae
during photosynthetic assimilation of C carbonate when
NH,Cl was added to the medium. Futher replications of the
feeding experiments are necessary to asses the signifi¬
cance of ammonia assimilation by the zooxanthellae in A.
elegantissima. It must be remembered that the lighter colored
animals selected as "aposymbiotic" have been found to con¬
tain small populations of zooxanthellae.
The enzymatic catabolism of alanine in the extracts of
A. elegantissima resulted in only one half the amount of am-
monia available from the substrate level of DL-alanine which
suggests that the enzyme in question is specific for either
the Dor the L isomer. The much lower level of ammonia re-
Ammonia excretion in A. elegantissima.
5.
Peter T. Hetzler
leased in the absence of d ketoglutarate suggests that the
ammonia release is not due to a D or an L alanine oxidase,
but occurs via a transferase reaction. In all cases in¬
vestigated, the L amino acids are more susceptible to cat¬
abolism. Futhermore, there is no report in the literature
of a D-alanine amidotransferase. It is highly likely,
therefore, that the observed activity is caused by an L¬
alanine amidotransferase coupled with a glutamic dehy¬
drogenase.
The uptake of ammonia by another enzyme system with
synthesis of other amino acids might cause the sharp drop
in ammonia concentration after 15 minutes. Attempts at
identification of such other products by chromatography have
been unsuccessful to date. The source of the ammonia as¬
similating enzymes has not been elucidated. The relative
importance of the contributions of enzymes from the anem¬
one and zooxanthellae must still be assessed in charac¬
terizing this system.
SUMMARY.
1. Fed specimens of the sea anemone, Anthopleura el¬
egantissima, excrete more ammonia than starved
specimens, suggesting that A. elegantissima is
ammonotelic as expected.
2. Preliminary findings suggest that the endosym-
biotic zooxanthellae present in A. elegantissima
take up ammonia excreted by the animal.
3. Two steps leading to the excretion of ammonia in
Ammonia excretion in A. elegantissima.
Peter T. Hetzler
SUMMARY (continued).
A. elegantissima are catalyzed by an L-alanine
amidotransferase coupled with a glutamic de-
hydrogenase.
4. The level of ammonia released during the course
of the L-alanine amidotransferase reaction
reaches a maximum at 15 minutes at a rate of
0.13 ug NH/minute, and thereafter decreases
sharply at a rate of 0.04 ug NHa/minute. This
decrease can not be attributed to a glutamine
synthetase reaction.
ACKNOWLEDGEMENT
I would like to express my deepest gratitude to Dr.
John H. Phillips whose profound knowledge and patience
were invaluable during the course of this project. I
would also like to thank Phil Murphy for his advice and
assistance. Special thanks go to the students and the rest
of the faculty at the Hopkins Marine Station.
6.
Ammonia excretion in A. elegantissima.
Peter T. Hetzler
FIGURE CAPTIONS.
Figure 1.
THE FEEDING EXPERIMENT. Four specimens of
A. elegantissima were fed (0) and four
were not (9). The animals were fed O.07
grams, wet weight, of squid at time 0. The
fed anemones released markedly greater a-
mounts of ammonia than the starved anemones,
especially the "aposymbiotic"animal in the
dark. Note the decrease in ammonia concen¬
tration after 180 minutes in all fed anemones.
Figure 2.
Figure 2 shows the release of ammonia in the
acetone powder extracts of A. elegantissima.
Note that the ammonia concentration in all
three assays reaches a maximum level at 15
minutes and sharply decreases thereafter.
The low ammonia concentration levels in the
absence ofaketoglutarate suggests that the
ammonia release is not due to an alanine ami¬
no oxidase reaction but to an L-alanine ami¬
dotransferase reaction coupled with a glu¬
tamic dehydrogenase reaction.
0
Ammonia excretion in A. elegantissima.
Peter T. Hetzler
TABLE CAPTIONS.
Table 1. The results of the feeding experiment, expressed
in ug NH3/ml.water/gram (dry weight) of anemone.
Table 2. The results of the acetone powder assays, ex¬
pressed in ug NH3 / ml. of sample.
SAMPLE
Enzyme
Alanine
Ketoglutarate
Enzyme
Enzyme
Alanine
Alanine
Ammonia excretion in A. elegantissima.
Peter T. Hetzler
TABLE 2
Sample Times in Minutes
—
O 15 30 60 00 120
3.4
2.24
— — —
— — — —
O.50
2.2
2.08
2.22
2.08
2.06
O.41
2.04
0.45
1.98
1.66
1.68
3.31
o.41
O.85
O.91
1.O1
O.87
O.84
1.10
O.50
O.51
0.62
O.50
O.63
0.4
0.45
0.46 0.47
O.41
0.33
ANEMONE
—
Fed
'Aposymbiotic
(Dark)
Fed
Aposymbiotic
(Light)
Fed
Symbiotic
(Dark)
Fed
Symbiotic
(Light)
Starved
Jymbiotic
(Dark)
Starved
Aposymbiotic
(Dark)
Starved
Symbiotic
(Light)
Starved
"Aposymbiotic'
(Light)
Ammonia excretion in A. elegantissima.
Peter T. Hetzler
TABLE 1
Sample Times in Minutes
60
90
120
180 240
—
1.08
1.64 1.96
3.46 4.12 2.81
1.26
1.35 1.48
1.88 2.18 1.78
1.07 1.14
1.00
1.44 1.68 1.23
1.66
1.29 1.36
1.74 2.42 1.80
O.96
O.84 1.08
1.11 1.09 1.06
O.89 0.94
O.96 0.99 0.97
1.01
1.32 1.25
1.24 1.19 1.21
1.13
1.48
1.48 1.50
1.41 1.38 1.29
Dry
Weight
rams
1.30
O.85
O.95
1.00
1.24
1.00
1.14
0.84
ug NH,/mI.
3 5
1.5
1.0



0

120
180
OSYABIO
VOSgOTC (LIGNT)
SYMBIOTICOLIGAT
BTC (Rk)
—sorie
(DARK)
or c
()
BSYMBIOTIC
(LIGH7)
gc
(LIGMT)
240
FIGURE
ug NH3/
3.5
3.0
FIGURE 2
60
ENZYME
A KETOGLUTARATE
ALANINE
— ENZYME
ALANINE TEN2V
ALAUINE
120
Ammonia excretion in A. elegantissima.
Peter T. Hetzler
BIBLIOGRAPHY
1. Delaunay, H. Review of nitrogen excretion. Ann. Physico¬
chim. biol. 10: 695-724 (1934).
2. Przylecki, St. J. L'excretion ammonicale chez les in¬
vertebres dans les conditions normales et experimentales.
Arch. int. Physiol. 20: 45-51. (1922).
3. Rowe, W. Bruce; Ronzio, Robert A.; Wellner P.; Wellner,
Varia; Meister, Alton. Methods in Enzymology. v. XVII.
Tabor and Tabor (eds.). Academic Press, New York (1970).
900-910.
Solorzano, ucia. Limno. Oceanography. 14: 799-801 (1972).
5. Trench, R. K. The physiology and biochemistry of zoo¬
xanthellae symbiotic with marine coelenterates. Three
Papers in Proc. Roy. Soc. B. 177: 225-264 (1971).
General Biochemistry.
1. Harper, Harold A. A Review of Physiological Chemist:
Lange Medical Publications. Los Altos, Ca. 303-349 (1971).
2. Hochachka, Peter W. Strategies of Biochemical Adaption.
Saunders, Philadelphia (1973).
3. Lehninger, Albert L. Biochemistry. Worth Publishers, New
York. (1970).
4. Prosser, Ladd C. (ed.). Comparative Animal Physiology.
W. B. Saunders Co. Philadelphia. 187-208. (1950).