Glycerol Affects 0, Consumption
Abstract
Measurements obtained by the direct method of Warburg,
definitely show that glycerol has an effect on the oxygen
consumption of a whole cell preparation of Anthopleura ele¬
gantissima. However, inconclusive evidence from the util¬
ization of U-140 glycerol has not shown whether glycerol is
directly metabolized or inductively used by A. elegantissima
cells. A comparison with other results for a similar investi¬
gation with A. xanthogrammica, shows that whole cell prepara¬
tions of both A. elegantissima and A. xanthogrammica react
in the same manner to glycerol.
Key Words
Anthozoa - Anthopleura elegantissima - 0, consumption
Glycerol - Respiration
Glycerol Affects 0, Consumption
Introduction
Sea anemones may have symbiotic dinoflagellates, termed
zooxanthellae, residing within their tissues. In Anthopleura
elegantissima, the symbionts are confined to the endodermal
layer (Trench 1971a). The amount of zooxanthellae may be
quantifiably measured and identified by the presence of the
chlorophyll pigments a and c, and the carotenoid peridenin
(Strain 1944, Jeffrey 1968).
The benefits which this symbiotic relationship can pro¬
duce are manyfold; however, only some aspects have been
studied. For example, previous studies have shown that the
zooxanthellae can release a substantial portion of soluble
organic products to their host animal. In vivo experiments
conducted by Trench (197la, b) have shown that approximately
508 of the 140 fixed by photosynthesis in the zooxanthellae
is translocated to the tissues of A. elegantissima. The
photosynthate obtained was determined by in vitro and in
vivo studies (Trench 197la, b) to be 140 glycerol, 140 glu¬
cose, 140 alanine and other 140 organic acids. Of these,
radioactive glucose was the major carbohydrate to be retained
by the zooxanthellae, whereas radioactive lipids and proteins
were found to be present in the host's tissues. Deacylation
of the lipid moiety yielded 140 glycerol while hydrolysis of
the protein yielded 140 amino acids.
Glycerol Affects 0, Consumption
Earlier experiments (Muscatine 1974, Taylor 1969) have
shown that symbiotic anemones starved in the dark, lose
weight at a faster rate than those starved in the light.
This implies that the algal's photosynthesis and subsequent
translocation of products to the host is sufficient to aid
the anemones in their nutritional requirements. Thus the
zooxanthellae's relationship with the sea anemones is most
likely one of a nutritional role.
The purpose of this paper is to investigate the utili¬
zation of glycerol in symbiotic A. elegantissima on the
basis of oxygen consumption. Since glucose is retained by
the zooxanthellae, it appears reasonable to assume that
glycerol, the only other major carbohydrate produced in the
photosynthate, should also be capable of affecting the rate
of oxygen consumption by the anemone's cells. If this is
correct, it remains to be seen whether glycerol is directly
metabolized or whether it has an inductive effect on a sys¬
tem which increases the amount of oxygen consumed.
Glycerol Affects 0, Consumption
Materials and Methods
1. Collection and Maintenance of Specimens
The sea anemone, Anthopleura elegantissima, containing
symbiotic algae, was collected from the intertidal zone at
Point Joe, Seventeen Mile Drive, Del Monte Forest, California.
Only clonal specimens were obtained. These were maintained
in a sea water aquaria under constant aeration on an approx¬
imate 12h light - 12h dark regime. Experiments were con¬
ducted within four weeks of collection.
2. Tissue Preparation
Three anemones from the same clone were taken for each
experimental run. These were then scissor minced to produce
a whole cell preparation. Verification by microscopic exam¬
ination, revealed that cells of approximately 7 in diameter
were interspersed with unicellular zooxanthellae, of an
average diameter of 12,. The zooxanthellae were observed to
be intercellular. This mince was then suspended with milli¬
pore filtered sea water and allowed to settle for five min¬
utes. Only the supernatant of cells and mucus was used
while the residue was returned to the mince. The whole
process was repeated until a suitable amount of the super¬
natant was obtained; this was then centrifuged and only
the pellet of whole cells was retained.
Glycerol Affects 0, Consumption
This pellet was washed with sea water and centrifuged
repeatedly for a total of three times. In all cases the
residue was recovered and diluted to become the stock solu¬
tion. A bath at 25°C was used to keep the solution agitated
and thus allow a thorough mixture with air. At appropriate
time intervals (48h, 72h, and 96h) the solution was removed,
washed with sea water and centrifuged -- this process was
repeated three times. After dilution, the residue became
the final working solution and upon removing a quantity
suitable for the day's experiment, the solution was then
returned to the bath until the next interval.
All the sea water used in this experiment had been pre¬
viously millipore (0.45) filtered. This filtering removed
any particulate food sources bigger than 0.45p from the med¬
ium. The repeated washings and centrifugation of the cell
suspension eliminated any soluble substances from the lysed
cells, thus removing any food sources which could be added
to the metabolite pool and so be used by the cells. These
washings allowed for a faster depletion rate of the prepara¬
tion.
Samples of 1 ml were withdrawn from the working solu-
tion for the following determinations: 1) protein by the
Lowry Method (Lowry 1951), 2) chlorophyll a and c using an
Glycerol Affects 0, Consumption
acetone extraction method (personal communication with J.
Phillips), and 3) dry weight.
3. Measurement of Oxygen Consumption
The direct method of Warburg (Dixon 1951, Umbreit 1972)
was used for all experiments. The volumes of the Warburg
vessels used, ranged from 11.2 ml to 16.9 ml. Three samples
of cell preparation were obtained from the working solution:
0.5, 1.0, and 1.5 ml; these were all brought to a constant
volume of 1.5 ml by the addition of sea water. Two dupli¬
cates were used at each volume. The working solution was
then returned to the depletion bath. In addition to the
above liquid volume, 0.2 ml of 208 KOH solution was added
to a filter paper wick in the vessels' centre well for the
absorption of CO.. Either 0.2 ml of 1 Molar or 0.2 of 14
mMolar glycerol was added to the side arm and used as the
substrate to be tested.
The gas phase was air for all experiments. The vessels
were shaken at a rate of 104/min with a stroke length of 1.5
cm at 25°C. After allowing 30 minutes for thermoequilibra¬
tion, readings were taken on the half hour until a linear
endogenous rate was obtained. Once this was acquired,
readings were taken every 15 minutes for four hours. Glycerol
was added to the main reaction vessel after the first four
Glycerol Affects O, Consumption
15 min readings. At this point the manometers were reset
and the interval was timed from the end of this process.
4. Utilization of U-14c Glycerol
In order to determine whether the anemone's cells
directly metabolized or inductively used glycerol, it was
necessary to use U-140 glycerol. A 50 C sample was obtained
from Ammersham/Searle. This was diluted with distilled water
until 2 050 counts was obtained, 2 x 10 ml was added to 0.2
ml of 14 mMolar of cold glycerol.
A 72 hour depletion was used and three 1.0 ml samples of
the cell preparation were taken. Beta-phenethylamine (0.07ml)
was used instead of KOH. After completing the experimental
run, the filter paper wick was removed and put into a scintil¬
lation vial. Upon the addition of Aguasol, the vials were
placed into the scintillation counter (Unilux II) and counted
for 1 minute after a 30 minute refrigeration period.
Glycerol Affects 0, Consumption
Results and Discussion
The investigation of the utilization of glycerol was
divided into two parts: 1) the qualitative demonstration,
and 2) the quantitative measure of the effects of glycerol.
1. Qualitative Demonstration
For this investigation, 0.2 ml of 1 Molar glycerol was
used in the side arm. The experiment was designed to show
the effects of an unlimited amount of glycerol on the basis
of oxygen consumption, the results, for which, are shown in
Table 1. The percentage increase in the rate of oxygen con¬
sumption after the addition of glycerol may be taken to
measure the amount that the anemone's reserves have been
depleted — it is an index of their depletion as shown in
Graph 1.
As can be seen from Table 1, samples taken at later
intervals show a greater increase in rate after the addition
of glycerol, than do those taken at a lesser interval. This
may be described in terms of depletion: the highest rate of
depletion appears to occur between 48 and 72 hours, while
the rate between 72 and 96 hours is one-half that of the
previous rate. If the change in rate is observed rather
than the percentage increase, it can be seen that the 48
and 72 hour samples have approximately the same increase in
Glycerol Affects O, Consumption
rate, whereas the 96 hour sample has more than twice that
increase. This again suggests that the 96 hour cell sus¬
pension is more devoid of reserves than the 48 and 72 hour
samples. A difference in density of the cell suspension
caused by errors in pipetting could perhaps explain the low
72 hour reading.
2. Quantitative Demonstration
In these experiments, 0.2 ml of 14 mMolar glycerol was
used. From a balanced equation of the oxidations of glycerol
into CO, and H,O, it was calculated that this amount of
glycerol gave slightly more than 20 l of oxygen consumption.
The purpose of having only 20 pl instead of an unlimited
amount of glycerol was to show that the rate of oxygen con¬
sumption returns to its original endogenous value upon the
utilization of the 14 mMolar glycerol.
The actual results from a 96 hour depletion bath show
exactly what was expected. A linear endogenous rate is
observed initially, followed by an increase in rate upon
the addition of glycerol for a certain period of time, and
finally a return to its previous endogenous value. Graph 2
shows the results of this experiment; it should be noted
that this graph only shows the four hour portion of the
whole ten hour graph. Table 2 shows the rates of oxygen
10
Glycerol AffectsO Consumption
11
consumption obtained before, during, and after glycerol
utilization, the percentage increase of rate, and the amount
of oxygen consumed during this utilization.
The rates obtained in this experiment are approximately
triple the rates found in Table 1 for 96 hours. It is not
known why there is such an increase, future experiments
should clarify this.
As can be seen from Table 2, the rate of utilization of
glycerol increases along with the increasing concentration
of tissue sample. It is interesting to note that the percen¬
tage increase in rate during glycerol utilization increases
by approximately 1003 increments for each concentration and
that the amount of oxygen consumed also increases by 5yl each
time. More experiments are necessary before any further
analysis of these figures is possible.
One aspect which was not expected to occur is the time
lag from the addition of glycerol to the beginning of its
utilization. The lag ranges from 45 minutes for both the
0.5 and 1.5 ml samples to 60 minutes for the 1.0 ml sample.
It would seem that this period is too long to account for
the diffusion of glycerol into the cells. A more suitable
assumption would be that glycerol has an inductive effect.
The time lag would then represent the amount of time required
for the induction to occur and somehow cause an increase in
Glycerol Affects 0, Consumption
12
the amount of oxygen consumed.
The results from the use of 14C glycerol show that
approximately 108 of the glycerol is oxidized to form 140-co..
However, this figure is known to be inaccurate because the
14C carbonate ions were not driven from the suspension med¬
ium. The number of counts (background counts subtracted)
obtained for 0.2 ml of 14 mMolar glycerol/U-14C glycerol
was 2050 whereas the value for Beta-phenethylamine was 190.
This experiment neither confirms nor denies the assumption
that glycerol is directly metabolized, further experiments
are needed to clarify this.
Glycerol Affects 0, Consumption
13
Conclusion
From qualitative experiments, it was determined that a
96 hour depletion bath was a suitable period of time to
devoid a cell preparation of A. elegantissima of most of its
reserves. The depletion became apparent from the observation
of a linear endogenous rate. This result was also obtained
by DiRaimondo (1975) in a similar investigation on A. xantho-
grammica. Comparing the mean endogenous rate of both Antho¬
pleura, it was found that after 96 hours both consumed oxygen
at much the same rate. The rate of oxygen consumption for a
cell suspension of A. elegantissima was 13.8 1/mg protein/h
while that of A. xanthogrammica was 12.8 l/mg protein/h.
After these figures have been standardized for the amount of
zooxanthellae present (measured by the amount of chlorophll
a), they become 3.9 and 4.3 l/mg protein/h/mg chlorophyll a
respectively. Thus from these experiments it appears that
whole cell preparations of A. xanthogrammica and A. elegantis¬
sima respire at nearly identical rates.
Glycerol has an effect on the consumption of oxygen
when measured by the Warburg respirometer on a cell suspen¬
sion of A. elegantissima. This suggests that the glycerol
produced by the zooxanthellae which is translocated to the
host's tissues could also be used in respiration, rather than
just as a subsidiary storage molecule such as lipid. However,
Glycerol Affects 0, Consumption
this does not suggest whether glycerol has an inductive
effect, as possibly shown by the time lag on the graph, or
whether it is directly metabolized. If it was directly
metabolized, glycerol would require phosphorylation and
oxidation to form dihydroxyacetone dehydrogenase. This
would then be enzymatically converted to glyceraldehyde
phosphate -- a metabolite which can enter the second
stage of glycolysis to produce ATP and indirectly consume
oxygen. Radioactive studies with U-14C glycerol did not
conclusively prove that glycerol was directly metabolized
by a cell preparation of A. elegantissima.
14
Glycerol Affects 0, Consumption
I wish to thank Drs. F. Fuhrman and J. Phillips for
their advice on the procedures of this experiment and for
their encouragement, Mrs. G. Fuhrman for her suggestions
on the Warburg vessel "washing technique", Mr. Murphy for
his aid in the use of the radioactive glycerol, and Dr. R.
Burnett for his critical assessment of this paper. Special
thanks go to my Warburg partner, Chuck DiRaimondo, for his
help in making the "Warburg Technique" less of a mystery and
in making this spring quarter more of an enjoyable one.
15
Glycerol Affects O, Consumption
16
References
DiRaimondo, C.V., 1975. An Investigation of the Effects of
Glycerol on the Oxygen Consumption by the Sea Anemone
Anthopleura xanthogrammica, Unpublished Research Paper,
Hopkins Marine Station of Stanford University.
Dixon, M., 1951. Manometric Methods 3rd. ed., Cambridge
University Press.
Jeffrey, S.S., and Haxo, F.T., 1968. Photosynthetic Pigments
of Symbiotic Dinoflagellates (Zooxanthellae) from Corals
and Clams, Biol. Bull. 135, 149
Lowry, O.H., 1951. Protein Measurements with Folin Phenol
Reagent, J. Biol. Chem. 193: 265-275
Muscatine, L., 1974. Coelenterate Biology: Reviews and New
Perspectives, Academic Press, New York. He cites:
Muscatine, L., Some Aspects of the Relationship Between
a Sea Anemone and its Symbiotic Algae, Ph.D. Thesis,
University of California, Berkeley.
Strain, H., Manning, W.M., and Hardin, G., 1944. Xantho¬
grammica and Carotenes of Diatoms, Brown Algae, Dino¬
flagellates and Sea Anemones, Biol. Bull. 86: 169-191
Taylor, D.L., 1969. The Nutritional Relationship of Anemonia
sulcata (Pennant) and its Dinoflagellate Symbiont, J.
Cell. Sci. 4: 751-762
Glycerol Affects O, Consumption
17
Trench, R.K., 1971a. The Physiology and Biochemistry of
Zooxanthellae Symbiotic with Marine Coelenterates.
I. The Association of Photosynthetic Products of Zoo¬
xanthellae by Two Marine Coelenterates, Proc. Roy. Soc.
Lond. B177, 225
Trench, R.K., 1971b. The Physiology and Biochemistry of
Zooxanthellae Symbiotic with Marine Coelenterates.
II. Liberation of Fixed 17c by Zooxanthellae in vitro.,
Proc. Roy. Soc. Lond., B177, 237
Umbreit, W.W., Burris, R.H., and Stauffer, J.F. 1972. Mano¬
metric and Biochemical Techniques, 5th ed., Burgess
Publ. Co., Minneapolis.
Glycerol Affects O, Consumption
Graph Captions
1. Relative Depletion -- percentage increase in rate of
oxygen consumption upon addition of an unlimited
amount of glycerol -- plotted against time for sym¬
biotic A. elegantissima.
Cell suspension of symbiotic A. elegantissima after
a 96 hour depletion bath to show the increase in
rate of oxygen consumption upon addition of glycerol
and a return to the endogenous rate after glycerol
utilization. This graph represents an expanded
portion of the whole 10 hour graph. The numbers
at the end of the line segments indicate the 0.5,
1.0 and 1.5 ml samples. The other numbers stand
for the amount of oxygen consumed in l.
Table Captions
1. Percentage increase in rates of oxygen consumption
after addition of unlimited glycerol to a 1.0 ml
sample as compared to numbers of hours in depletion
bath for symbiotic A. elegantissima.
Cell suspension of symbiotic A. elegantissima after
a 96 hour depletion bath showing the different rates
of oxygen consumption, the percentage increase of the
rate during utilization of glycerol compared to be¬
Slycerol Affects O, Consumption


—
fore utilization, and the amount of oxygen consumed
during utilization.
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Glycerol Affects 0, Consumption
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Gylcerol Affects 0 Consumption
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Glycerol Affects O Consumption
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Glycerol Affects O, Consumption
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