of the
Digestive Gland
rosobranch Mollusc Littorin
plana.
ger A. Pederse
Phillips
Hopkins Marine Station
Stanford Universi
Biology
June
otelytie Enzymes of the Digestive Gland
of the Prosobranch Wollusc Littorina planaxis
This paper describes an in vitro analysis and part
haracterization of the proteolytic enzymes of the digestiv
planaxis, a prosobranch mollusc.
The fe
gland of Littorina
studies which have been conducted on the digestive enzymes o
marine gastropods have revealed no prõteinases in the saliva
lands of the herbivorous prosobranchs (Freter and Graham, 1962
These organs appear to function principally in secreting mucus
which facilitates the passage of ingested food from the bucc
cavity to the stomach. There is also unanimity in the conclu
that no digestive enzymes are secreted, nor any food absorbed
in the intestine (Freter and Graham, 1962). The present studie.
nave been confined to the digestive gland, although the esophagi
also merits study.
his analysis is limited to the characterization of th
enzymes with respect to their activity at differing hydroge
ion concentrations, activatiwation by a reducing agent and
metallic cofactors, and purification by ammonium sulfate frac
lonation. It has been necessary to disregard other factors
concerned with the production and secretion of digestive enzyme
V the living organism. Digestive enzyme secretion in the gastr
Murex (Hirsch, 1931), Aplysia and Helix (Yonge, 1937), is
pods
known to be a rhythmic process, accelerated by the presence o.
food in the gut. In order to minimize the effedt of such variable
in the gut, snails
to decrease the numbers of microorganisms
starved in the laboratory for 12 hours
prior to dissection.
vere
imental
The surfaces from which the snails were selected are rer
sentative of the position of the Littoring planagis population
intertidal zone at Hopkins Marine Station, Cabrillo Point.
acific Grove, California. Snails came predominantly from surfaces

12 feet above mean low tide, and were sheltered from direet wave
tion, receiving splash only at high tide.
No-distinction wa
ade as to the sex of the animals
used in these studies.
paration of the ehayme extract from the digestive
gland involved collection, starvation and
issection of the sn.
escribed aboye.
The shells were
roken i
a Small vice and
shell muscie detached from the shell
remnants. The midgut
removed posterior to the left kidney
and contsined, in a

ion to the digestive diverticula, gonadal tissue, the stomach
ind stomach contents. In snails weighing 1.0-1.5 g., this
complement weighed 30-50 mg., or 3-4% of the total weight. All
preparative procedures following the weighing were carried out
in an ice bath to retard denaturation of the enzymes. Sufficient
digestive gland to give 10-15 mg. of tissue, or its equivalent
homogenate per milliliter of final reaction mixture was homogenized
in artificial sea water, ASW (MacLeod, et. al., 1954), with a
Thomas tissue grinder, (Cat. 44288-B, Philadelphia). Centrifugatior
fog 30 minutes in a Serval centrifuge, Model SS-1, to precipitate
cell fragments, followed by filtration through whatman 4l paper t
remove lipids, readied the tissue homogenate for use in the
reaction mixtures. Each reaction mixture contained 1 mi. o.
1% casein, obtained from non-fat milk solids by the method
Cohn (1930) and prepared according to Kunitz (1947) at ph
the casein substrate was added 2 ml. of enayne preparation
ind 8 ml. of the appropriate buffer, to give a final volume of
per resction mixture. The buffers used were Acetate, ph
5, Phosphate, pH 5,7-7.1, and Tris (hydroxymethyl)amino-
.
ethane
pH 7.3-8.7. All concentrations were 0.06-0.08M in the
action mixture. For determination of activation by metallic
ofactors, 1 ml. of buffer was replaced
by an equivalent volune
metal cation: Mn, Ni or Co, x 1
M in reaction mixture.
For determining the effect of a reducing agent, 1 ml. of buffer
was replaced by sodium cyanide, 1X
Min reaction mixture.
In enzyme controls lul. of buffer replaeed the casein substrate,
in substrate controls, 2 ml. of buffer replaced the enzyme pre-
ration.
Reaction mixtures were layered with toluene to pre-
vent the proliferation of bacteria and were incubated in
390. for the duration of the reaction.
ter bath at ?
The assay method to detect proteolysis was the Lowry mod-
ification of the Folin test (Lowry, 1951). At 4 hour intervals,
samples were withdrawn from the reaction mixtures and treated
ml. of 10% trichloroacteic acid, TCA, to stop the reaction
and precipitate the non-hydrolgzed protein. In order to provid
maximum denaturation and precipitation of the non-hydroljzed
rotein, assay samples were incubated in TCA at room temperature
for at least 12 hours before centrifugation. Following centri-
fugation for 15 minutes at 3000 r.p.m. in an International Refri-
gerated Centrifuge, Model PR-2, the amount of Lowry-positive
droljzed casein present in the TCA supernatant was determined
By comparisor
Klett-Summerson Photoelectric Colorimeter.
th a standard curve of known amounts of casein,
proteolytie
tivi
was determined in terms of micro
rams of casein digested
and
sue or its equivalent homogenate) in ea
of


illiliter of reaction,mixture
Only readings appreci
gher than
he sum of the enzyme
and substrate dontrols
vidence of proteolytic activity
toward
purification of the proteases of th
fractionation of the proteins with ammonium sulfate wa
nducted.
A tissue homogenate was prepared as described above
1.6 g. of tissue, the digestive glands from 50 animals.
material was first predipitated by 1008 saturation with
)280, and centrifugation for 15 minutes on the high-speed
Serval 88-1. The precipitate was repeatedly extracted with
series of ammonium sulfate solutions decreasing from 100% se
ration by 10% increments. No concentration of enzyme wa
tempted.
Bacteria present in the stomach contents of starved and
feeding snails were examined for the presence of proteolytie
strains. The nutrient medium contained 1.58 agar and 0.18
peptone,in sea water, and was rendered opaque by finely divided,
enature
ovalbumin.
Incubation was at room
ture for one week
and
ussior
The proteinase activity with respect to ph is represented
Two activity peaks, having optima at ph 4.9-5.1
and at pH.5.5, suggest the presence of at least two distinct
enzymes with
slightly acidic pH optima.
Proteinases activ
in this range have been detected in many invertebråtes ar
are
Vaguel.
characterized as extracellular or intracellular kathepsins
icol
Weither the metallic ions nor the sodium cyanid
1960).
kedly
activated or inhibited
the activi
of the enzymes.
Fractionation with ammonium sulfate suggests additional
See Fig. 12.
proteolytic activity at a higher
pH, 8.3. This activity which
was detectable only following fractionation is evidently due
to the removal of one or more engyme inhibitors by the fractions
The slightly alkaline
precipitation with ammonium sulfate.
PE and deactivation by inhibitors indicate a trypsin-like char
r for this enzyme. On the basis of this finding it seems
ble to undertake fractienation and partial purification
enzyme preparations involving prosobranch digestive tissue
the hope of activating enzymes considered weak or deficient
results from crude tissue extracts.
The cultures of bacteris from the stomach showed large
lations capable of hydrolyzing denatured ovalbumin. These
observations suggest that the bacterial flora of the gut may
tion of proteins.
assist in the di
unna
An account is given of experiments designed to dete
oteinases in the digestive gland of the marine gastropod
Littorina
planaxis and to characterize these enzymes with respect
pH optima, activation by dibasic cations and a reducing agent
and ammonium sulfate fractionation.
Activity of a crude tissue extract at various hydrogen
on concentrations suggests the presence of at least two kathepsin
ike proteinases having pH optima at 4.9-5.1 and 5.5.
Neither the metallic cations Mn, Ni, Co, at 1 X.10-4
3.
agent NacN at 1 X 103 M markedly inhibits o.
nor the reducing
activates eithe
of the above.
artial purification by fractionation with ammonium
sulfate apparently removes an enzyme inhibitor from the 406
ives evidene of a trypsin-like
turated fraction, and
Similar purifications are suggested
8.
proteinase active at
for any further study on this, or related marine gastropods.
Several strains of bacteria present in large numbers
Mlanaxis may assist in the digestion
the stomach of Littorina
f protein.
Bibliography
Baldwin, E. 1963. Dynamics of Biochemistry, fourth ed., Cambri
University Press.
Cohn, E. J. 1930. Organic Syntheses. 10:16.
Colowick, S. P. and Kaplan, N.O, eds. 1953. Methods in Enzym
ology. Academic Press, Inc., New York. Vol. 1, pp.138-146
. 1955. Methods in Enzymology. Academic Press, Inc.
New York, vol. II, pp 30-35.
Freter and Graham, ed. 1962. British Prosobranch Molluscs,
Ray Society, Bartholemew Press, Dorking, England.
Hirsch, G. C. 1931. The theory of fields of restitution w
special reference to the phenomena of secretion. Biol.
6:88-131.
Kunitz, M. 1947. Jour. Gen. Physiol. 30:291.
Lowry, O.H., et. al. 1951. Protein measurements with the Folir
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MacLeod, R.A., Onofrey, E., and Norris, M.E. 1954. Nutrition and
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Manual
gy of Marine Animals, Interscienc
Nicol, J.A.C. 1960. The Biolo..
Publishers, New York. 275-279
rosser,C.L., ed., et. al.i Comparative Animal Physiolog
W. B. Saunders, Co. Philadelphia, 1950. pp.144-186.
amidt, ed. 1944. The Chemistry of the Amino Acids and Proteins.
Charles C. Thomas, Co., Baltimore, Maryland.
van Weel, P.B.. 1961. The comparative physiology of digestion
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Yonge, C.M. 1937. Evolution and adaptation in the digest
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Figure +
70
50
8 CASEIN
374.
40
30
20
Reaction Time : 8 hours
10
—
37 11 13 75 17 1.7 5. 5.3 5.5 5.7 5.9 c.] 6.3
Fiaure +2 Ammonium Sulfate frachimation
O CONTROLS HAUE B
Mlaesaien are PCasei
2EDACIED,
Keactien Time: Ghours
Reactign Lme: 10 heues
SAtCALN PH 1.3
215. 1  2. 2 n Sren 21 1 2 a1 5 1a 8. 3 Fns one
—
40
0.0%
12.7
11
26
70
0.07.
32
4.8
90.03
90.0%
80.8/
80.6%
70.0%
70.0%
60,0%
66.0%
5009
50.09.
17.3
O
10.0%
11.7
27
23
10.7
40.0%
35.5
18.
8.3
30.0%
233
O
33.7
3002.
0
20.0%
20.09
10.0%
10.0%