THE SOURCES OF AMYLASE
IN THE DIGESTIVE TRACT
OF PAGURUS SAUELIS
(Arthropoda: Decapoda)
Philip Sullivan
Hopkins Marine Station
of Stanford University
Pacific Grove, California
June 5, 1965
An examination of the gut contents and feeding habits
of Pagurus samuelis (Stimpson, 1857) revealed that their
diet contains a wide variety of animal and plant matter,
no doubt containing a significant amount of starch and
possibly glycogen. A preliminary iodine test on starch
substrate incubated with hepatopancreas extract showed that
this organ does, indeed, contain a powerful amylase. An
investigation of the literature revealed that very little
work has been done on the origin of carbohydrases in decapods.
Wolvekampo)found glands in the wall of the gut which he be-
lieved to produce mucus and "perhaps amylase." Waterman (2)
concludes that the secretion of digestive enzymes takes
place "almost exclusively in the hepatopancreas." The pur-
pose of this investigation was to locate the origin of amylase
in the digestive tract, at the same time providing an indica-
tion of possible sites of origin of other carbohydrases for
another investigation being done at the same time. In addition,
the properties of the amylase present in the hepatopancreas
were investigated.
MATERIALS AND METHODS
Crabs were taken from the intertidal zones of China
Point, the site of Hopkins Marine Station, and Point Pinos
at Pacific Grove. They were kept in aquaria with fresh sea-
water until needed.
For the purpose of this investigation, the digestive
tract was divided into five segments: foregut, extending
from mouth to juncture of anterior midgut diverticula: midgut,
from anterior midgut diverticula to posterior midgut diver-
ticulum; hindgut, from posterior midgut diverticulum through
the anus; hepatopancreas; and, anterior midgut diverticula.
Before testing, the gut segments were split and thouroughly
washed in distilled water to eliminate the amylase activity
of the particulate contents as well as the amylase secreted
elsewhere and transported to that portion of the gut. The
tissues were then ground in an homogenizing tube and assayed
immediately.
The method of assay, also described by waksman(3),
basically that of Wohlgemuth. One ml of.1% soluble starch
(Baker and Adamson) was incubated with different dilutions
of enzyme extracts for thirty minutes at room temperature
(21922°0.). Two drops of iodine solution (Io.2%; KI.4%)
and 3ml of Hø0 were added. The reaction mixture containing
the lowest enzyme concentration that gave no blue color with
iodine was taken as the end-point of the assay. The expression
of enzmye concentrations in tissues is in mg of extrafcted
tissue required to hydrolyze 1 mg of starch in 30 minutes.
All tests were run in duplicate with starch solutions buffered
at pH 5.4 (acetate) and pH 7.4 (tris-maleate).
RESULTS
The results of the investigation on the sources of
amylase are presented in Table 1. By far the most important
organ in the secretion of amylase appears to be the hepato-
pancreas. It has over twenty time the activity of the stomach
and forty times the activity of the hind and midguts.
The optimum pH for the amylase present in the hepato-
pancreas was determined in citrate-phosphate buffers varying
by 0.4 of a pH unit over the range from 5.8 to 7.8. One
ml of 11% starch was incubated for 30 minutes with varying amounts
enzyme extract at each pH. and the end-points determined as
before. The results of this test are presented in Table 2.
The optimum temperature for the amylase present in the
hepatopancreas was determined by the same method. Here, the reaction
mixtures were buffered at the optimum pH (7.0)and were incubated
at five temperatures from 10° to 40°0. Since reaction mixtures
at each temperature showed the same end-point, colorimeter readings
were taken on those reaction mixtures which still showed
blue staining. From these readings, the reaction rates appeared
to increase with increasing temperture up to 35°, decreasing
thereafter.
4
The final hydrolysis prooducts were determined by in-
cubating 2ml of 1% starch with.lg of enzyme extract at 1506
for 10 hours. Analysis was made by paper chromatography after
the method of Jeanes(5) using a pyridine, butanol, water
solvent in the ratio of 3:2:1.5 over a period of 20 hours.
The developing spray reagent was that quoted by Gordon (6)
as CD-1. Two test spots were made in addition to one control
spot each of maltose, glucose, and a mixture of maltose and
glucose. While the control spots showed good separation of
maltose and glucose, the test spots showed a high content of
glucose and only the faintest trace of maltose. This result
would strongly indicate the presence of a maltase powerful
enough to break down the maltose as fast as it is produced
by the amylase.
An investigation of the presence of a ß-amylase was made
using .1% concentrations of amylose (Nutritional Biochemical
Corp.) and amylopection (Calbiochem). Two samples of enzyme
extract from the same source were used, one sample being
heated at 57° for 20 minutes. Four ma of each enzyme
preparation (heated and unheated) was incubated at room
temperature with 2 ml of each substrate (amylose and amylo-
pectin). The mixtures were buffered at pH 6.8. One-tenth
ml aliquots were taken at 20-minute intervals and tested accord-
ing to the Somogyi(7) method for the production of reducing
sugars, and a rate of sugar production was determined. The
difference in susceptibility of amylose and amylopectin to
hydrolysis by the amylase in the hepatopancreaslis presented
in Fig. 1.
DISCUSSION
Although the hepatopancreas is by far the strongest
producer of amylase, the foregut, containing the esophagus
and stomach, does show an activity twice as great as that of
the muscle, which probably owes its activity to the presence
of amylase in the blood and hemolymph. Amylase in the blood
and hemolymph of another decapod, Astacus, was demonstrated
by Damboviceanu(8). The hind and midguts also showed activity
corresponding to this base level. The even lower activity
of the diverticula may be due to that organ's small size and
smooth surface which allow a more efficient washing as compared
to the other bulkier, more irregular organs tested, which
probably keep a larger portion of their original fluids.
In the determination of the pH optimum, the use of tris-
maleate buffer in place of citrate-phosphate buffer showed
no significant difference in amylase activity.
The finding, in the final hydrdysis products, of glucose
and very little maltose corresponds with the findings of
Hourani (9) of maltse activity in the hepatopancresa and weak
maltase activity in the foregut.
While £-amylase is generally considered to be solely
a plant enzyme, evidence for its presence in other decapods
is given by Yonge(10). The sensitivity of O-amylase and the
resistence of Ø-amykse to heating is discussed by Waksman(11).
The differences in activity of heated and unheated preparations
of hepatopancreas extract, and the differences in susceptibility
of amylose and amylopectin to hydrolysis suggest the possible
presence of a ß-amylase in this organ of P. smmuelis.
SUMMARY
The hepatopancreas was found to be the most important
producer of amylase in Pagurus samuelis.
2. The foregut, though much weaker, was also shown to have
significant amylase activity.
3.
The other portions of the digestive system had activities
equal to or lower than muscle tissue. This activity is
probably the result of amylase in the hemolymph and blood.
4. Under laboratory conditions, the pH optimum was found to
be 7.0.
5. Under the same conditions, the temperature optimum was found
to be near 35°0.
Some evidence for the possible presence of aß-amylase
was found.
REFERENCES
Wolvekamp, H.P., 1947, Sur la presence de deux amylases
1.
dans les suc gastrique des decapods. Actualites Biochim,,
10:19-21. noted in Waterman, p. 298.
2.
Waterman, T.H., Physiology of Crustacea, Academic Press,
New York, 1960, p. 298.
Waksman and Davidson, Enzymes: Properties, Distribution,
Methods, and Applications, Williams and WIlliams, Baltimore,
1926. p. 159.
4. Wohlgemuth, J., Ueber eine neue Methode zu quantitativen
Bestimmun des diastatischen Fermentes. Biochem. Z., 1908,
9:1-9. noted in Waksman and Davidson, p. 159
5. Jeanes, Wise, and Dimler Improved techniques in paper
chromatography of carbohydrates. Analytical Chemistry,
23:3, March, 1951.
6. Gordon, Thornburg, and Werum, Rapid paper chromatography
of carbohydrates and related compounds. Analytical Chemistry,
28:5, May, 1956.
Somogyi, M., 1945, Determination of blood sugars. J. of
Biol. Chem., 160, pp. 69-73.
and 1952, Notes on sugar dtermination. J. of Biol.
Chem., 195, pp. 19-23.
8. Damboviceany, A., 1932, Composition chemique et physio-
chemique du liquide cavitaire chez les crustaces decapodes,
Arch. Romanes Pathol. Expel. Microbiol., 5:239-309. noted
In Waterman, p. 152.
9. Hourani,B., Carbohydrase activity in the intestinal
tract of Pagurus. samuelis. unpublished article, Hopkins
Marine Station, Pacific Grove, Ca ifornia.
10. Yonge, C.N. 1924, The mechanism of feeding digestion,
Brit. J. Exptl.
and assimilation in Nephrops norvegicus.
Biol., 1:343-389. nõted in Waterman, p.
289.
11. Waksman and Davidson, p. 159.
TABLE 1
CONCENTRATION OF AMYLASE IN TISSUES OF P. SAMUELIS
HEPATO-
HIND AND
ANTERIOR
MUSCLE
FOREGUT
DIVERTIC
PANGREAS
MIDGUTS
—
85
150
300
153
pH 5.4
153
85
150
300
pH 7.4
mg of extracted tissue required to hydrolyze
1 mg of starch in 30 minutes.
—
50
TABLE 2
DH OPTIMUM OF HEPATOPANCREAS AMYLASE
6.6
77.
6.
5.8
5.4
L

25
12
12
12
25
50
—
ing of extracted hepatopancreas required to
hydrolyze 1 mg of starch in 30 minutes.
7.8
—
25
175 h
1965
Pagarus
O
FIG.GI
RATE OF HYDROLYSIS OF AMYLOSE AND AMYLOPECTIN
BY HEATED AND UNHEATED HEPATOPANCREAS EXTRACT
200
150
roncentråtion
ofgreduding
sugars iniquot

aliquotsiof
reaction
100
mixture
mg/10 ml
.

50

40 min.
60 min.
20 min.
amylopectin + unheated enz.
amylose +unheated enz.
amylopectin + heated enz.............
amylose + heated enz.- -
incubated a 57°0. for 20 minutes before
introduction of substrate
297