INTRODUCTION
It has been shown by Stephens and his co-workers
that many marine invertebrates can accumulate amino acids from
ambient seawater (Stephens, 1962, 1963, 1964, 1967, 1968; Stephens
and Schinske, 1961; Stephens and Virkar, 1966; North and Stephens,
1961; and Virkar, 1966). The present study further examines
some of the characteristics of amino acid uptake and utilization
in the cirratulid polychaete, Cirriformia spirabrancha. The
amino acid glycine was used for this study because it is the
most abundant free amino acid in seawater. Studies were made
to determine the rate of concentration, the rate of incorporation
into alcohol insoluble material, the total rate of uptake, and
the anatomical sites of amino acid uptake.
MATERIAL AND METHODS
Worms were collected from sandy areas just south
of Fisherman's Wharf, Monterey, California and stored in the
laboratory under simulated natural conditions until used,
usually between one and five days after collection. The worms
were washed free of external mucous and sand, blotted on filter
paper to remove excess water, weighed to the nearest 10 mg.
and placed in room temperature seawater (20-22°c). The wet
weight varied between.14 and.40 grams. Various concentrations
of glycine solution were made in artificial seawater by
dissolving C glycine (Sigma Chemical Corp.) in a constant
amount of glycine-UL-Ct (ICN, specific activity 76.0 m0/mM).
In a number of experiments, indicated in the figure legends,
glycine anhydride (NBC) was mistakenly used in place of the
18.
glycine. Each worm was placed in 10ml of one of these
solutions which ranged in concentration from .65x10-0 M/L
to 10-3 "/L. After a stipulated period of time, the worm
was washed for 30 seconds in seawater, homogenized in a tissue
homogenizer in 20 volumes of 80% ethanol, and then centrifuged
for 10 minutes in a clinical centrifuge. The supernatant
was saved while the pellet was washed again in 80% ethanol,
centrifuged and dissolved in 10 volumes of formic acid. O.lml
aliquots of the supernatant and dissolved pellet were then
placed on separate planchets, spread with 0.5ml formic acid,
evaporated under an infra-red lamp, and radioactivity measured
with a thin window Geiger-Muller detector. Radioactivity of
each glycine-seawater solution was also measured in this manner
after plating 0.lml of the solution with 0.5ml 80% ethanol and
evaporating in a drying oven. All samples were taken in
triplicate and corrections for background and quenching have
been included in the data presented.
RESULTS
The three major parameters of uptake are the
concentration of free glycine in the worm (alcohol-soluble
radioactivity), the rate of incorporation of glycine into
proteins and nucleic acids (alcohol-insoluble radioactivity),
and the total rate of uptake (total radioactivity). Figurell
shows that uptake of free glycine is linear for at least 60
minutes, and that rate of incorporation and total rate of
uptake are linear for the two hour period.
Since the worms differed in weight, it was necessary
to find the relation between wet weight and total rate of
uptake. When wet weight was plotted against total rate of
uptake. (figure 2), it was found that a linear relationship
existed.
The total rate of uptake increases as the external
glycine concentration increases, up to an external concentration
of 5 x10- "/L. At this concentration the rate of uptake is no
longer affected by an increase in external concentration
(figure 3). This uptake curve is characteristic of a transport
system.
Other experiments indicate that the gut is not
involved in uptake, since ligation of the head and tail
regions had no effect on uptake rate. This suggests the major
sites of uptake are through the body wall and tentacles.
Table 1 compares uptake of the body and tentacles
on a weight basis. In this experiment the worms were
incubated in C+ glycine for the indicated periods of time.
At the end of the incubation period the tentacles and body
were separated from each other and the uptake determined.
The results show that the tentacles are five times more
effective in uptake, on a weight basis, than is the body wall.
DISCUSSION
The uptake vs. wet weight relation in C. spirabrancha
can be expressed as Uptake - kW, where k is a constant and W
is wet weight. This means that uptake varies directly with
59
the volume of the worm, where one would expect uptake to
vary directly with the surface area. One explanation for
this is that surface area also varies directly with volume
or weight. This is not too hard to imagine if one considers
that this worm and its tentacles have a great capacity for
stretching. This means that it has a great surface area per
volume and also that a change in volume is mostly indicated
in a change in length which is proportional to surface area.
Thus it seems that surface area might indeed vary directly
with volume over the range of weights used in this study.
Stephens (1964) showed that the radioactivity of
the alcohol-soluble fraction of an extract of Clymenella
Torquata resided in glycine alone. This is probably true
in the present experiments also, especially since the incubation
periods were relatively short.
The total rate of uptake and the rate of incorporation
of alcohol-insoluble material are linear for at least 120
minutes, whereas the rate of concentration of free glycine
slows down with time. There are two explanations for the
rate of concentration slowing down with time. One is that
the enzyme system may not be capable of increasing the
concentration gradient any further and thus the rate of
concentration will level off. The other is that the worm
has probably markedly depleted the external concentration of
glycine over the two hour period and this low external
concentration may be a limiting factor for the transport
system. This latter explanation seems quite plausible since
the worm was in only lOml of solution for two hours.
The total rate of uptake vs. concentration relation
or C. spirabrancha is similar to those of other marine
invertebrates studied by Stephens and his co-workers.
Stephens (1968) gives the typical ranges of Vmax and Km
for the organisms he has studied. Vmax (maximum rate of
uptake) ranges between 10-7 and 10-5 moles/g per hour and
Km (concentration at which a half maximal rate of uptake
occurs) ranges between 10-4 and 10-5 M/L. Calculations
derived from the data of figure 3, depicted in figure 4,
show that C. spirabrancha has a Vmax of 2.4 x10-6 M/g per hour
and a Km of 1.2x10-4 "/L, both well within the ranges given
by Stephens.
6
SUMMARY
Studies were made on amino acid uptake into alcohol
soluble and alcohol-insoluble products by the polychaete,
Cirriformia spirabrancha. A radioactive tracer method was
used employing glycine-UL-C+. The uptake vs. wet weight
relationship was linear, and can be expressed as Uptake - kW.
The body wall and tentacles were the major areas of uptake,
with the tentacles having a rate five times as great as the
body on a weight basis. Uptake vs. external concentration
was also studied. The Vmax was 2.4 x 10-6 M/g per hour and
the Km was 1.2 X10-4 M/L.
ACKNOWLEDGEMENTS
would like to extend my gratitude to Dr. D. Epel
and the faculty and staff of Hopkins Marine Station for
making this project possible. This work was supported in
part by the Undergraduate Participation Program of the
National Science Foundation Grant GY-4369.
BIBLIOGRAPHY
North, B. B., and G. C. Stephens. 1967. Uptake and
assimilation of amino acids by Platymonas. Biol. Bull.
133:391-400.
Stephens, G. C. 1962 Uptake of organic material by
aquatic invertebrates. I. Uptake of glucose by the solitary
coral, Fungia scutaria. Biol. Bull. 123:648-659.
Stephens, G. C. 1963. Uptake of organic material by
aquatic invertebrates. II. Accumulation of amino acids
by the bamboo worm, Clymenella torquata. Comp. Biochem.
Physiol. 10:191-202.
Stephens, G. C. 1964. Uptake of organic material by
aquatic invertebrates. III. Uptake of glycine by brackish
water annelids. Biol. Bull. 126:150-162.
Stephens, G. C. 1967. Dissolved organic material as a
nutritional source for marine and estuarine invertebrates.
p. 367-373. In G. H. Lauff, (ed.), Estuaries. AAAS.
Washington, D.C.
Stephens, G. C. 1968. Dissolved organic matter as a
potential source of nutrition for marine organisms.
Amer. Zool. 8:95-106.
Stephens, G. C., and R.A. Schinske. 1961. Uptake of
amino acids by marine invertebrates. Limnol. Oceanog.
6:175-181.
Stephens, G.C., and R. A. Virkar. 1966. Uptake of organic
material by aquatic invertebrates. IV. The influence of
salinity on the uptake of amino acids by the brittle star,
O
9.
Ophiactis arenosa. Biol. Bull. 131:172-185.
Virkar, R. A. 1966. The role of free amino acids in
the adaptation to reduced salinity in the sipunculid
Golfingia gouldii. Comp. Biochem. Physiol. 18:617-626.
C
FIGURE LEGENDS
Figure 1: Curve 1 gives the total rate of glycine uptake
over a two hour period; curve 2 shows the rate of incorporation
of free glycine into alcohol-insoluble material; curve 3 shows
the concentration of free glycine in the worm. External
glycine concentration is .65 X10-° M/L with glycine anhydride
at a concentration of .5 X10-6 M/L.
Figure 2: The rate of uptake (in counts per minute per worm)
vs. the wet weight of the worm in grams. External glycine
concentration .65 X10-° M/L with glycine anhydride at a
concentration of ,5 X10-6 M/L. Incubation time was 15 min.
Figure 3: The effect of external concentration vs. internal
concentration in moles per liter. External concentration varied
from .65 x10 ° to 10-2 M/L. Incubation time was 15 min.
Figure 4: The reciprocal of external concentration vs. the
reciprocal of internal concentration (M/L/15 min.). The slope
of the line indicates Km and the intercept indicates Vmax.
This figure was calculated from figure 3.
Table 1: A comparison of rate of concentration on a weight
basis of glycine between the tentacles and body over a 15
minute period. The numbers indicate 10-
/9 . The external
glycine concentration was .65 X10° M/L with glycine anhydride
at a concentration of .5X10-0 M/L.
.
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