NON-PROTEIN AMINO ACIDS IN MUSCLE
AND BLOOD OF
MARINE AND FRESH WATER
CRUSTACEA
BY MERRILL N. CAMIEN,* H. SARLET, G. DUCHBTEAU,
AND M.
FLORKIN
(From the Laboratoires de Biochimie, Institut LBon Fredericq, Universitb de
Libge,
Likge, Belgium)
(Received for publication, August
1, 1951)
Muscle and other tissues of marine
invertebrates are very dilute with
respect to inorganic ions, but are
nevertheless in osmotic equilibrium with
the blood, in which ionic
concentrations are nearly the same as in sea
water (1). It has been postulated,
therefore, that these tissues contain
relatively high concentrations of
small organic molecules (l), and it is of
interest in this connection that
considerable quantities of glycine (2) and
taurine (3-5) have been found in
tissues of various marine invertebrates.
Fifteen amino acids have now been
determined in muscle of both marine
and fresh water
Crustacea.
EXPERIMENTAL
Live North Sea lobsters (Homurus
vulgaris) were obtained from local
markets. Live spider crabs (M&u
squin~do)~ were supplied by the Station
Biologique de Roscoff (Faculte des
Sciences de Paris). Chinese crabs
(Eriocheir sinemis) and crayfish
(Astucus JEuviutilis) were taken from the
fresh water aquarium of the
Institut. The animals were allowed to bleed
as rapidly and as completely as
possible from an amputated anterior leg.
Muscle samples (from abdomen of
lobsters, abdomen, and large chela of
crayfish, and legs and large chela
of crabs) were rapidly isolated, weighed,
and placed in boiling water for 5
minutes to inactivate proteolytic enzymes.
They were then homogenized and
treated with tungstic acid essentially
according to the procedure of
Schurr et al. (6). Serum samples were likewise
treated with tungstic acid, and all
the protein-free samples, unless
otherwise noted, were hydrolyzed by
refluxing 24 hours with 6 N
hydrochloric
acid before the amino acid
determinations were made.
Aspartic acid, arginine, glutamic
acid, glycine, isoleucine, leucine, lysine,
methionine, phenylalanine, and
valine were determined essentially as de-
* United States Fulbright Scholar.
Present address, Department of Chemistry,
University of California, Los
Angeles.
* The samples of Maia were carried
through the first stages of preparation at
Roscoff, protected with toluene,
and kept refrigerated until their arrival in the
authors’ laboratories. The authors
are indebted to Mr. C. Jeuniaux for the work
done at Roscoff and for
transporting the samples to Liege.
881
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882 AMINO ACIDS IN CRUSTACEA
scribed by Dunn et al. (7).
Tyrosine was determined by the same method
as that employed for arginine,
except that the tyrosine in the assay medium
was replaced by arginine (final
concentration, 19 mg. per cent). Alanine,
histidine, proline, and threonine
were determined essentially by the procedures
of Sauberlich and Baumann (8), Dunn
et al. (9, lo), and Henderson
and Snell (ll), respectively. The
reliability of these methods was controlled
by determining the amino acids in a
known mixture in each experiment,
and, in general, recoveries of 100
f 5 per cent were obtained.
DISCUSSION
Tungstic acid was added to the
samples in slight excess to insure complete
precipitation of the proteins. This
procedure seemed desirable since
it had been established by Schurr
et al. (6), and confirmed by experiments
in this laboratory (unpublished),
that such excesses of tungstate do not
interfere with the microbiological
assays. That significant amounts of
protein or other non-dialyxable
amino acid precursors did not remain in
the samples was demonstrated by
dialysis of a lobster muscle extract.
The total weight of amino acids
found in the dialysate was 96.8 per cent
of that present before dialysis
(Columns 1 and 2, Table I)?
The protein-free samples were
regularly hydrolyzed to avoid ambiguities
resulting from the variable
activities of “combined amino acids” which
might otherwise be present. In
lobster (other species were not tested)
muscle extract, however, it
appeared that, except for glutamic acid, aspartic
acid, histidine, and tyrosine, free
amino acids were predominantly
present, since essentially the same
values were found before and after
hydrolysis (Columns 1 and 3, Table
I). It may also be inferred from this
observation that proteolysis was
avoided prior to removal of the proteins
from the samples, since
proteolysis, especially in a short period of time,
would be expected to yield peptides
generally lower in activity3 than free
amino acids. That tyrosine may have
been present in peptide combination,
however, was indicated by the
higher value found for this amino
acid before hydrolysis, since Dunn
and McClure (12) have observed that
peptides are generally more
active for Lactobacillus casei, the tyrosine test
organism, than are free amino
acids. The lower values found for glutamic
acid and aspartic acid before
hydrolysis were as would be expected if these
amino acids were present largely
either as their amides4 or as peptides.
* Glycine appeared to be 13 per
cent lower and tyrosine 13 per cent higher in the
dialyzed sample. Since none of the
other amino acid values differed by as much as
10 per cent after dialysis, it seems likely that
these variations originated in ‘the
assays, and did not result from the
dialysis.
3 The activity of partially
hydrolyzed proteins in replacing free amino acids for
Lactobacillus arabinosus, Lactobacillus
fermenti, and Leuconostoc mesenteroides (representative
assay organisms) is proportional to
the degree of hydrolysis (12).
4 Glutamine itself is nearly as
active as glutamic acid (13), but it is converted to
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CAMIEN, SARLET, DUCHATEAU, AND
FLORKIN 883
TABLE I
Non-Protein Amino Acids in
Crustacean Muscle and
Blood Serum
Amino acid
Glycine .........
Proline .........
Arginine ........
Glutamic acid. ..
Alanine. ........
Aspartic acid.
..
Lysine ..........
Threonine .......
Valine ..........
Isoleucine.
......
Leucine .........
Histidine .......
Methionine .....
Phenylalanine ...
Tyrosine ........
Total. . . .
R
--
.1
--
.3
-
:egu1ar a&
-__
(1) (2)
m. w.
025 892
728 707
778 830
267 267
133 133
12 13
23 25
8.6 8.
22 22
9.3 8.
7.7 8.
11 12
5.4 5.
1.6 1.
--
‘031.6 2933.
--
1
0
8
-_
12
-
---
(3) (4) (5)
.
0;
. mg.
2 3.5
728 6 0.9
778 1.6 0.2
44 3.5 0.5
133 8.7 1.3
4.6 7.0 1.0
24 2.1 0.3
9.1 0.0 0.0
21 0.0 0.0
8.9 4.2 0.6
9.7 3.5 0.5
12 0.0 0.0
5.2 0.2 0.0
11 3.3 0.5
---
!813.5 64.1
9.3
In muscle per 100 gm. intracellular
water:
t-5) (7) (8) (9)
?nY ?nY ?nY
?nY
to.1 (13.5-22.3) 14.5 8.9
5.2
.0.6 (7.9-12.8) 0.9 3.0
1.6
6.4 (3.S 7.2) 4.8 1.7
0.8
3.4 (2.5 6.7) 2.9 3.5
3.5
2.3 (0.9- 3.1) 2.2 5.0
0.8
0.2 (O.l- 0.5) 0.3 0.6
0.8
0.3 (O.l- 0.5) 0.1 0.3
0.7
0.1 (O.l- 0.3) 0.3 0.3
0.4
0.3 (O.l- 0.7) 0.3 0.2
0.3
0.2 (O.l- 0.2) 0.2 0.1
0.2
0.1 (O.O- 0.3) 0.2 0.1
0.3
0.1 (O.O- 0.1) 0.0 0.1
0.3
0.1 (O.l- 0.2) 0.2 0.1
0.1
0.1 (O.O- 0.1) 0.0 0.0
0.1
0.0, (O.o- 0.0) 0.0 0.0
0.1
---
k4.2 26.9 23.9 15.2
* Muscle pooled from eight animals
was deproteinized as described in the text,
and one aliquot of the filtrate was
hydrolyzed according to the regular procedure.
A second aliquot was subjected to
dialysis through cellulose dialyzer tubing before
hydrolysis, and a third aliquot was
used untreated.
t Blood serum was pooled from eight
animals. Chloride was determined in the
muscle and serum samples from each
animal (serum chloride concentration was
assumed to be the same as in sea
water for Maia), and the extracellular water was
calculated, assuming the
intracellular chloride concentration to be negligible and
the composition of the
extracellular. phase to be identical with that of the blood
serum.
$ Muscles from twelve lobsters were
used, and the average values are given with
the ranges in parentheses. One
spider crab, twelve Chinese crabs, and nine crayfish
were used, and the muscle samples
were pooled for each species. Intracellular
water = difference between total
moisture and extracellular water (calculated as
described above). The intracellular
water, in gm. per 100 gm. of fresh muscle,
averaged 61.6, 55.4, 67.1, and
69.4, respectively, for Homarus, Maia, Eriocheir, and
Astacus.
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884 AMINO ACIDS IN CRUSTACEA
The concentrations of amino acid in
blood serum of Homarus (Column
4, Table I), Eriocheir,6 and
Astaczd were much lower than those in the
muscle, and calculated values for
extracellular amino acids in Homarus
muscle (Column 5, Table I) were
almost entirely negligible in comparison
with the totals of extracellular
and intracellular amino acids (Column 1,
Table I). It may be inferred,
therefore, that the non-protein amino acids
in Crustacean muscle are
essentially intracellular.
Glycine, proline, arginine,
glutamic acid, and alanine were the most
abundant of the amino acids in
Crustacean muscle, and their molar concentrations
(Columns 6 to 9, Table I) appeared
to be sufficient to produce
considerable osmotic pressure. The
concentrations of total amino acids
were greater in the marine species
than in the fresh water forms, and it is
of interest that these values were
nearly equal to those for chloride in the
blood serum of these species.”
Etiocheir should probably be grouped with
the salt water organisms in this
connection since it is euryhaline with an
internal medium characteristically
like that of a marine form, regardless
of its external environment. That
the amino acid concentrations in
Homarus were considerably greater
than those in Maia and Eriocheir
seems to indicate that substances
other than amino acids are less important
for the regulation of osmotic
pressure in the former than in the latter
animals. The relatively low
concentrations of amino acid in Astacus appear
to be in accord with the low
osmotic pressure of its external environment.
It seems likely, therefore, that
amino acids, particularly glycine,
proline, arginine, glutamic acid,
and alanine, may be important substances
for the regulation of osmotic
pressure in Crustacean muscles. Analogous
studies of other marine and fresh
water invertebrates are in progress.
SUMMARY
Fifteen amino acids were determined
in cleproteinizecl muscle and blood
of Astacus Jluviatilis, Eriocheir
sinensis, Maia squinado, and Homarus vulgaris.
High concentrations of amino acids,
particularly glycine, proline,
arginine, glutamic acid, and
alanine, were found intracellularly in muscle,
and these concentrations were
higher in marine species than in fresh water
forms. Only relatively low
concentrations of amino acids were found in
the blood. It was concluded that
certain amino acids are probably iminactive
pyrrolidonecarboxylic acid when
autoclaved, and asparagine is much less
active than is aspartic acid
(14).
6 Serum amino acid concentration
was too low to permit accurate determination
with the available amount of
sample. A serum sample from
Maia was not obtained.
6 The millimoles of chloride per
100 gm. of serum were
45,26, and 21, respectively,
for Homarus, Eriocheir, and Astacus. The serum chloride concentration for Maia
is assumed to be the same as in sea water
(approximately the same as for Homarus).
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CAMIEN, SARLET, DUCHfiTEAU, AND
FLORKIN 885
portant substances for the
regulation of osmotic pressure in Crustacean
muscle.
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متولد 1362 کرمانشاه و مقیم تهران هستم تحصیلات مقاطع کاردانی، کارشناسی و کارشناسی ارشد را به ترتیب در دانشگاههای لرستان(تکنولوژی تولیدات دامی)، شهید با هنر کرمان(مهندسی تولیدات دامی)و کردستان(ژنتیک و اصلاح نژاد دام)گذرانده ام.