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Lipase Activity
in
Pagurus granosimanus (Stimpson, 1862)
(Arthropoda: Decapoda)
Daniel C. Cabrera
Hopkins Marine Station
Pacific Grove, California
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Examination of the gut contents of the hermit
crab Pagurus revealed both algae and many types of ani-
mal foodstuffs. In view of the varied diet of Pagurus,
it would seem probable that its digestive system contained
enzymes capable of hydrolyzing carbohydrates, proteins
and fats. Although the ability to hydrolyze fats had
been demonstrated in other Crustacea (2) (6), a study
remained to be done in Pagurus. The experiments here out-
lined are designed to determine lipase activity—its
distribution and optimal conditions, in the genus Pagurus
MATERIALS AND METHOD
Pagurus granosimanus, as the largest of the
species common to the intertidal zone surrounding the
Hopkins Marine Station, was selected as representative
of the genus. The crabs used were collected at nearby
Pinos Point and had an average wet weight of one gram.
Preparation of enzyme The hepatopancreas were removed
from forty P. granosimanus, dehydrated in acetone for
two hours and defatted in petroleum ether for an addi-
tional two hours. (In the case of the gut, sixty indi¬
viduals were necessary.) The dehydration and defatting
were carried out a 5°C. One gram of this residue was
ground to a fine powder in a mortar and extracted at
5°C in 100 ml. of 50% aqueous glycerol for twenty-four
hours. The filtered extract was used as the source of
enzyme.
Substrate preparation A water-soluble, polyoxyethylene
derivative of sorbitan mono-oleate, commercially known
as Tween 80 (Sigma Chemical Co., St. Louis, Missouri)
was used as substrate. The Tween 80 to be used as
substrate was diluted to 50% with water and titrated
with 17% phosphoric acid to the desired pH. All measure-
ments of hydrogen ion concentration were made with a
Beckman zeromatic pH meter.
Enzyme assay The procedure followed was essentially that
of Archibald (1). Three ml. of 50% neutralized Tween 80,
0.5 ml. of 0.2 M phosphate buffer, and 0.5 ml. of the
enzyme preparation were placed in a 15 ml. centrifuge
tube, thoroughly mixed with a glass stirring rod and
incubated for 1 hour. After incubation, 1.0 grams of
reagent-grade NapHøP04-H20 and 10 ml. of ether mixture
" 5:1) were added.
(diethyl ether, petroleum ether, b.p.
Simultaneously, a blank was prepared containing all of the
components of the assay mixture prepared immediately prior
to extraction with ether. After mixing and centrifugation
for 10 minutes at 4000 r.p.m., the ether layer containing
the released fatty acids was withdrawn and placed in a
30 ml. beaker. The ether was removed by evaporation after
the addition of O.5 ml. 2-methyl-2, 4-pentanediol (J.T.
Baker Chemical Co, Phillipsburg, New Jersey). The vis-
cous residue was then titrated with O.Ol N NaoH to the
original pH.
Lipase activity was estimated in terms of milli-
equivalents of acid produced per hour. Activity was
determined by the use of the following equation: A -
D - C / 100 T where D is ml. of 0.O1 N NaoH used in
titration of the reaction mixture, C is ml. of NaoH used
in titration of the blank, and T is the reaction time in
hours.
RESULTS
Direct measurement of the gut contents revealed
a decrease in hydrogen ion concentration from the fore-
gut (pH 5.5) to the hindgut and hepatopancreas (pH 7.4).
The gut exhibited a relatively negligible amount of li-
pase activity compared to that found in the hepatopan-
creas (0.16 milliequivalents of acid / hour / 0.05 gram
of dehydrated, defatted tissue at pH 7.2, 23.5 C). For
this reason, further experiments were carried out using
hepatopancreatic extracts only.
Figure 1 illustrates the finding that lipase
activity in P. granosimanus increases with increasing
pH to approximately 7.2.
It can be seen in Figure 2 that lipase activity
varies with temperature and that its optimum temperature
is approximately 35C.
The effects of variations in substrate concentra¬
tion upon lipase activity in P. granosimanus are presented
in Figure 3. Activity increases with increasing substrate
concentration to a maximum at approximately 2.16 grams
Tween / 0.05 grams of dehydrated, defatted tissue extract.
The relative concentration of substrate used in the above
studies was in excess of this amount.
The lipase preparation was labile, losing approx-
imately 60% of its activity after sitting for only two
hours at room temperature (23 C).
DISCUSSION
Although esterases which attack simple ester link-
ages are found in many animal tissues, lipases appear to
4.
be confined to the pancreas and intestinal tract (4). No
attempt was made to measure lipase activity in tissues
other than those associated with the digestive tract.
Digestive enzymes in Crustacea have been reported
to have their pH optima in the slightly acid ranges (7),
but it has been shown that with progressing purification
of enzyme preparations, optima tend to move from lower
to higher pH (3). In studies where lipase activity was
greatest in the acid range, the gut contents were also
slightly acid whereas some gut contents of P. granosi-
manus are slightly alkaline.
That the optimal temperature range for the lipase
preparation should exist at about 35 C is in approximate
agreement with that of 28 C found for glycerol-extracted
pancreatic lipase described by Schwartz (8). The obser-
vation of extreme temperature lability of lipase prepara-
tions has been noted before (see 9).
SUMMARY
Optimum pH, temperature and substrate concen-
tration were determined for the hepatopancreatic lipase
present in Pagurus granosimanus.
1. The pH optimum was 7.2.
2. An optimal temperature of approximately 35 C was
demonstrated.
3. Highest activity was reached at a substrate concen-
tration of 0.432 grams Tween per ml. of lipase ex-
tracted from 0.05 gram tissue.
Figure 1.
Determination of pH optimum of hepato-
pancreatic lipase from P. granosimanus.
Activity is expressed in milliequiva-
lents of acid produced in one hour by an
extract of 0.05 gram of dehydrated, de-
fatted tissue.
8

1
J

—
N
1
0
0
G
J
0
C
Figure 2.
Determination of temperature optimum for
hepatopancreatic lipase from P. granosi¬
manus. Activity is expressed in milli-
equivalents of acid produced per hour by
an extract of 0.05 grams of dehydrated,
defatted tissue at pH 7.0.
—


S
—
2
D

0
0
8
0
0

QL
00
Figure 3.
Determination of optimal substrate con-
centration of hepatopancreatic lipase of
ranosimanus. Activity is expressed in
P.
milliequivalents of acid produced in one
hour by an extract of 0.05 gram of dehy-
drated, defatted tissue at pH 7.0, 23 C.
oo
7C



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REFERENCES
R.M. Archibald "Determination of Lipase Activity"
Journal of Biological Chemistry, V. 165 (1946) pp. 443-48
Arthur D. Hasler "Physiology of Digestion in Plank-
ton Crustacea" Biological Bulletin, 68, pp.208-14.
3. Colowick, S.P. and N.O. Kaplan, ed. Methods i
Enzymology Vol. I, Academic Press Inc. (New York,
1955) 835 pp.
Fatty Acids and Their Derivatives
Ralston, A.W.
John Wiley and Sons, Inc. (New Tork, 1946) 986 pp.
Sumner, James B. and Karl Myrback eds. The Enzymes
Vol. I, Academic Press Inc. (New York, 1950) 724 pp.
Yonge, C.M. "The mechanism of feeding, digestion,
and assimilation in Nephrops norvegius" Brit. Jour.
of Experimental Biology 1: pp.343-389.
Waterman, T.H. ed. The Physiology of Crustacea Vol. I,
Academic Press, Inc. (New fork, 1960) 670 pp.
Schwartz, B. "The Effect of Temperature on the Rate of
Hydrolysis of Triglycerides by Pancreatic Lipase" in
The Journal of General Physiology 27: pp.113-118.
Sizer, I.W. "Effects of Temperature on Enzyme Kinetics"
in Advances in Enzymology, V.3, (New York, 1943) p.35