Bulletin of the Japanese Society of Scientific Fisheries Vol.26, No.
3,1960 317
STUDIES ON THE ORGANIC PHOSPHATES IN MUSCLE OF
AQUATIC ANIMALS-VIII. PREPARATION OF
INOSINIC ACID FROM SQUID MUSCLE
Tsuneyuki SAITO*, Minoru MATSUYOSHI**, Kenichi ARAI* and
Yasuzo ITO***
(Received Dec.15,1959)
In the muscle of vertebrates, as is known today, inosinicacid (IMP) isprincipally
formed from adenylic acid (AMP) by the action of 5'-adenylate deaminase which is
found commnon1y in vertebrate musc!es. In the previous papers1),2)it has been estab
lished that in squid muscle adenosine triphosphate (ATP), in the course of autolysis,
is dephosphorylated rapidly with a concomitant accumulation of AMP, while scarcely
any IMP is found. Furthermore the living muscle of squid was found to contain as
high levels of ATP as muscle of mammals. From these results it will be reasonable
to consider that the muscle is a good source of ATP, AMP or the products derived
from them. The present paper reports a study on the preparation of IMP from AMP
of squid muscle by the action of 5'-adenylate deaminase of fish muscle.
Experimental
Materials:
Freshly obtained mantle muscle of squid, Ommastrephes sloanipacificusSteenstrup,
was used. Fresh muscle of some fishes, mackere1, Scomber japonicus Houttuyn, or
carp, Cyprinus carpio Linne, was used as a source of 5'-adenylate deaminase.
Method of analysis:
Optical densitieswere measured with a Shimadzu Spectrophotometer, Model QB-50,
using 1-cm. cells. Identificationof the recrystallizedsodium inosinate was carried out
by determining the absorbance ratios, D250/D260 and D280/260,of the solution and by
determining the phosphate content by the method of Bartlett (modified method of
Fiske and Subbarow's)3).
Extraction:
0ne kg. of squid muscle and 50-100 g. of fish muscle were ground finely with 5
volumes of ice-cold water in a mechanical meat grinder. Incubation was carried out
in a refrigerator at O-5℃ with occasional stirring for 2-3 hrs. (under these conditions
the action of muscle nucleotidase or phosphatase is reduced markedly). To determine
the degree of formation of IMP , 10ml. of the reaction mixture were taken off several
*Fac . Fish., Hokkaido Univ., Hakodate, Japan.
**Hachinoheshokuhin Co . Ltd., Hachinohe, Japan.
***Kushiro Branch, Hokkaido Gakugei Univ . Kushiro, Japan.
318
times at regular intervals. The enzyme activity was stopped by the addition of 10 ml.
of 10 per cent perchloric acid and the mixtures were filtered. The ratios of absorption
at 250 mμper 260 mμof these filtrates were calculated. When these values reached
to about 1.2 the reaction was stopped by the addition of perchloric acid to make the
final concentration of 4 per cent;then after thorough stirring the mixture was left
overnight in a cold room. The mixture was filtered through three layers of gauze.
The residue was stirred in 4 per cent of perchloric acid solution to extract the residual
IMP. The combined extracts were clarified by filtration through filter paper. The
procedures for preparation of IMP from these extracts, which are shown in Fig.1,
Squid muscle 1.O kg.
Fish muscle O.1kg.
Water 3.O t.
ground in meat grinder.
held at O-5ｰC. for 2-3 hrs.
added with perchloric acid to make final
concentration of four percent and then filtered.
i iFi至
trate← ■ . .一.一.-一
一 … 一.一.・Precipitate
washed with 4/perchloric acid
neutralized with potassium hydroxide solution.
i_ iFilt
rate .一_.___.. _. '一
一 一 Precipitate
washed with water
added with 20/barium acetate solution filtered.
i.. iFilt
rate Precipitate
・dd・d with ac・ti・ ・cid t。 融 。 pH 5. (圭n・
・g・・i・ph・ ・ph・t・)
added with mercury acetate(20/in 2/acetic acid).
centrifuged
l.. lP
recipitate Supernatant
added with water
treated with hydrogensulfide gas.
ce獄trif犠ge(葦
_______Supernatant Preci
neutralizedwith barium carbonate to make pH 5.0-6.O
added with barium hydroxide to make pH 8.O and held at OｰC
Crystal
IMP-Ba(about 2.6g.)
added with calculatedamounts of sodium sulfatesolution.
Filtrate Precipitate
added with about 5 volumes of ethanol
Crystal
IMP一
Fig. 1. Preparation of sodium inosinate from squid muscle
.
are divided into three steps. The firstis removal of inorganic phosphate as barium
phosphate, the second is precipitationof IMP as mercuric salt and the last is isolation
of barium inosinate. The details of these procedures were already described by J
.
Marmur et al4).
319
Results
The yields of barium inosinate from squid muscle, which are shown in Tabie 1,
Tabte 1. Yields of barium inosinate from squid muscle.
Test
No. Sq。 、 Muscled(9'。 、
。h. IMP-BtS)・IMP-Bag/kgofSquidMuscle P・rity{/)*
1 1,180 1181) 3,097 2,624
2 . 2.500 2502) 6,604 2,640 84.5
3770772)2,0502,66086.4
Mea繊value 2,642 85.5
* Determined by ionexchange chromatography .
1) Carp
2) Marckerel
were better than that from rabbit4): the former was about 2.6 g. per kg. of muscle
while the latter 1-1.5g. From these results it can be seen that the activity of 5'
- adenylate deaminase of the muscle of these fishes was almost the same. This was
also ascertained on that of saury and of horse-mackerel. The barium salt, thus
obtained, was recrystallized from water. The sodium salt was prepared from the
barium salt by satisfactory removal of barium ion by the addition of calculated amount
of sodium sulfate. The sodium salt can be purified by recrystallization from dilute
aqueous solution with ethanol. The maximum absorption of the recrystallized sodium
salt is at 249 mμih phosphate buffer of. pH 7.0. The absorbance ratiGs, D250/260 and
D280/D260, are shown in Table 2. These are the same as the values shown by Kalcker.
Table 2. Absorbance ratios of sodium inosinate.
DzSOIDzao Dzso/D2so
Nucleotide
pH 2.O pH 6.O pH 2.O pH 6.O
Samgle畠 1.58 1.67 0)20 0,24
Standardb I 1.58 ( 1.68 ( 0.20 1 0.24
a Recrystallized Na-inosinate prepared from squid muscle .
b H . M. Kalckar; J. Biol. Chem. 176, 429(1949).
The phosphorus content of the anhydrous sodium salt is as follows:
Test number 1 2
Sample 4.94 mg. 6.33 mg.
found 385γ 494γ
P (%) found 7.79 7.80
P(%) calculated 7.89
From these results it can be concluded that IMP, by using these methods, may be
prepared from squid muscle at fairly good yield as compared with that prepared from
other animals.
320
Summary
By using the 5'-adenylate deaminase of some fish muscles IlvlPwas prepared from
squid muscle at fairly good yield.
Acknowledgment
We are extremely grateful to Miss T. Tanaka for her assistance with experiments
on ionexchange chromatography. The financialsupport of this work by the Fisheries
Agency is gratefully acknowledged.
References
1) Saito,T., Arai, K., and Tanaka, T: Nature 181, 1127 (1958).
2) Saito,T., Arai, K., and Tanaka, T: Bull. Fac. Fish.,Hokkaido Univ. 9 , 121 (1958).
3) Bartettt,G. R: J. Biol.Chem., 234,466 (1959).
4)Marmur, J.,Schlenk,F., and Overland, R. N:Arch. Biochem. Biophys.,34, 209 (1951) .
