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MUCHMORE, ANDREW V. (Hopkins Marine Station of Stanford University,
Pacific Grove, California). Laminarinase and Hexokinase Activity
during Embryonic Development of Acmaea scutum (Mollusca:
Gastropoda: Prosobranchia). The Veliger
Activity of embryonic carbohydrases, hexokinase,
glucose-6-phosphate dehydrogenase, and isocitrate dehydrogenase
were analyzed fluorometrically in the unfertilized eggs,
trochophores, and veligers of the limpet Acmaea scutum.
and socae
were
Glucose-6-phosphate,dehydrogenase activity, constant at
all stages. Hexokinase activity increased almost three-fold
between the trochophore and veliger stage. No maltase,
galactosidase, or amylase activity was found at any stage.
A laminarinase-type enzyme, capable of degrading algal laminarin
to free glucose, was found in all embryonic stages. This
enzyme may be involved in digestion of laminarin when the
embryo becomes a feeding larvae, or may be involved in
intracellular breakdown of 8,1-3 linked glycoproteins during
embryogenesis.-Author.
PLEASE DO NOT TYPE BELOW THIS LINE
1
LAMINARINASE AND HEXOKINASE ACTIVITY DURING
EMBRYONIC
DEVELOPMENT OF ACMAEA
(MOLLUSCA, GASTROPODA)1;2
Andrew V. Muchmore
Hopkins Marine Station of Stanford University
Pacific Grove, California
Andrew V. Muchmor
This report describes some enzyme activities related
to carbohydrate metabolism during embryonic development of
the gastropod, Acmaea scutum (Escholtz, 1833). Data are
presented on activities of enzymes releasing free glucose
from various polysaccharides, on hexokinase (an enzyme com-
mon to both the pentose shunt and glycolysis), glucose-é¬
phosphate dehydrogenase (an enzyne of the pentose shunt),
and isocitrate dehydrogenase (an enzyme of the citric acid
eyele).
MATERTALS AND METION
Obtaining and culturing gametes:
Adult A. scutum were collected from an area on
Cypress Point in Pebble Beach, California. The animals
were removed from their shell, and the dorsal body wall
ruptured near the posterior end of the animal, with care
taken not to puncture the nearby digestive gland. Parts
of the ovaries from five females were removed with a
forceps, agitated in alkaline sea water to remove the
mature eggs, and then treated as described below to remove
the chorion. Sperm were obtained by agitating the testes
in 25 ml of normal filtered sea water, and filtering the
suspension through a double layer of cheesecloth.
Andrew V. Muchmore
Removal of the chorion by the alkaline sea water
treatment of Wolfson (1907) was necessary to obtain ferti-
lization and normal development. For A. scutum, best
results were obtained by 90 minutes incubation in alkaline
sea water (2.5 ml of O.IN NaOH in 100 ml of filtered sea
water, final phi 9.2). The alkaline sea water was then
removed, the eggs resuspended in filtered sea water, and
1 ml of sperm suspension added. After two hours the eggs
were agitated, allowed to settle, and the supernatant
water containing most of the sperm removed and filtered
sea water added. After about 36 hours, the embryos were
gently centrifuged, and resuspended in freshly filtered
sea water. Above operations were all performed at 15-17 C
Enzyme preparation:
Assays of one large group of eggs were made before
fertilization, 24 hours after fertilization at the troco-
phore stage, and 48 hours after fertilization at the
veliger stage. In initial experiments, enzyme instabilit
was noted, which suggested the possibility of degradative
enzymes (such as proteases). Therefore, in some experi-
ments 6-9 milligrams ovomucoid prepared by the method of
Fredricq and Deutsch (1949) were added to one half of the
samples as a specific trypsin inhibitor.
Andrew V. Muchmore
The eggs, trocophores, or veligers were divided into
two parts and centrifuged. One sample was suspended in 2.5
ml buffer (0.05M TEA, pH7.6), the other in 2 mi buffer-
0.5 ml of ovomucoid solution (also suspended in buffer),
and both samples then disrupted in a Dounce honogenizer at
0 degrees centigrade. The volumes of embryos used ranged
from 0.05 - 0.1 ml packed cells per 2.5 ml. All preparations
were assayed for enzyme activity at 26 degrees centigrade.
Fluorometric assays:
Because of the small amounts of embryos obtainable,
sensitive fluorometric techniques, based upon the absorption
of TrNI at 366 mu and its fluorescence at wavelengths greater
than 420 mu were utilized to measure enzyme activity. Using
incubation mixtures described below, glucose-6-phosphate
dehydrogenase (C6PDh) and isocitrate dehydrogenase activity
were determined by direct measurement of TPNH formation;
hexokinase (HK) activity was measured by coupling its prod¬
uct, glucose-6-r04, with excess G6PDh, to yield TPNI; and
carbohydrases releasing free glucose measured by coupling
glucose production to HK and CoPDh in the presence of excess
ATP.
1) Glucose-6-phosphate dehydrogenase: The 2 mi
reaction mix contained, per ml, 43 umoles TEA buffer (pl 7.6)
Andrew V. Muchnore
0.25 umoles TPN, 5 jmoles Ngcla, and 4 jumoles glucose-6-
phosphate (G-6-P).
(2) Hexokinase: Same as (1), except 10 jmoles/mi
glucose were substituted for G-6-P, plus 2 umoles/ mi ATP,
and O.5 ugms/ml CöPDh.
3) Carbohydrase: Same as (2), plus 2.5 ugms/ ml IK,
and in place of glucose were substituted either maltose
(1 pmole/ml), lactose (1 umole/ml), laminarin or starch
(0.05%, final concentration). Negligible glucose was
present in the starch, laminarin, or cell extract. Ap-
preciable amounts, however, were found in the maltose and
lactose. To eliminate this contribution, the reaction mix
(minus cell extract) was incubated with sufficient substrate
to remove all free glucose, and the reaction then initiated
with cell extract.
(4) Isocitrate dehydrogenase: Same as (1), except
2 umoles/ml isocitric acid was substituted for the G-6-P.
Other assays!
Unfertilized and veliger stages were also assayed
for carbohydrases by measuring reducing sugars released,
using the method of Somogyi as modified by Nelson (1944
As a further check, amylase activity was also measured
qualitatively by adding iodine to samples of extract and
starch at five-minute intervals, and using decolorization
Andrew V. Muchmore
of the blue iodine-starch complex as an indicator of amylase
activity.
The fluorometric technique was capable of detecting
the production of as little as 6 x 10-11 moles glucose/min.
The reducing sugar test of Somogyi as modified by Nelson
could detect about 3 x 10-8 moles of reducing sugar.
Protein was measured spectrophotometrically by both
the method of Warburg and Christian (1941) and Lowry et. al.
(1951). Protein content was assumed to reflect enzyme
content, since the observed rates were closely related to
enzyme concentration (a twofold increase in extract approx¬
imating a twofold increase in rate). The activities are
therefore expressed as moles TPNI formed per minute per
milligram protein.
Commercial sources of enzymes and substrates:
Hexokinase and glucose-6-phosphate dehydrogenase (Boehringer
and Sohne), isocitrate, glucose, TFN, and ATP (Sigma Chemical
Company), lactose (Nutritional Biochemical Corporation), and
maltose (Difco Laboratories). The laminarin and starch were
prepared by Dr. John Phillips of Hopkins Marine Station.
RESULTS AND DISCUSSION:
Enzyme activities determined fluorometrically, are
isted in Table 1. The lack of amylase activity in unferti-
lized eggs and in veligers was also supported qualitatively
Andrew V. Muchmore
by the lack of decolorization of the iodine-starch complex,
and quantitatively by the reducing sugar test. This latter
test indicated that, if amylase were present, it produces
less than 30 x 10-9 moles reducing sugar per hour per mom
protein.
Enzyme extracts assayed for carbohydrase activit,
were prepared from embryos obtained from different females.
For comparison of enzyme activity during development, eggs
of five females were pooled, and enzyme activity was deter.
mined in the unfertilized egg, trocophore, and veliger
stages. The rates of enzyme activity of these various
stages are shown in Table I and Fig. I. Table I shows that
by the veliger stage, regardless of previous activity, the
embryos homogenized in ovomucoid show a greater activity
than the extracts prepared without this trypsin-inhibiting
compound. This could indicate the development of a trypsin¬
like protease. This conclusion is also supported by Fig. II,
which shows that storage of IK at 0°c, without ovomucoid,
results in considerable loss of activity, and that this loss
does not occur with ovomucoid present. Similar stabilization
of activity was also observed with laminarinase activity.
Isocitrate dehydrogenase activity in the unfertilized eggs
(Table I) shows anomalous behavior in ovomucoid which is
unlike the activity of HK and zr. The reasons for this are
unclear.
360
Andrew V. Muchmore
Perhaps the most interesting discovery is that the
eggs and early embryonic stages of A. scutum possess an
enzyme (s) capable of releasing glucose from laminarin. The
acwt
rate of this enzyme, however, was non-linear under the pres-
ent assay conditions, and therefore no comparison of activity
was attempted.
The role of the enbryonic laminarinase in the egg and
early trocophore stages is difficult to visualize, since
neither of these are feeding stages. The veliger, however,
does feed, and could certainly utilize a minarinase-type
enzyme for digestion of the large amount of algal laminarin
found near the surf zone in small fragments of brown algae.
If the veligers are degrading laminarin to free glucose in
their natural environment the sharp increase in HK activity
(Fig. 1), also found at this stage, could result from free
glucose made available by laminarinase. ZF activity,
however, appears to remain relatively constant during
development.
Another possibility, which is presently being investi
gated, is that the observed laminarinase activity actually
reflects 81,3 glucanase activity involved in the breakdown
of cellular glyco-proteins.
Andrew V. Muchmore
SUNMMARY:
Activity of embryonic carbohydrases, hexokinase,
glucose-6-phosphate dehydrogenase, and isocitrate dehydro¬
genase were analyzed fluorometrically in the unfertilized
eggs, trocophores, and veligers of the limpet Acmaea scutum.
Glucose-6-phosphate dehydrogenase and isocitrate dehydro-
genase activity was constant at all stages. Hexokinase
activity increased almost threefold between the trocophore
and veliger stage. No maltase, galactosidase, or amylase
activity was detectable at any stage. A laminarinase-type
enzyme, capable of degrading algal laminarin to free glucose
was found in all embryonic stages. The role of laminarinase
in unfertilized eggs is puzzling since the substrate for
this enzyme is not available until feeding begins at the
veliger stage.
Andrew V. Muchmor
ACKNOULEDGEMENTS
The author is deeply indebted for help received from
Dr. David Epel and Miss Sharon Proctor, both of Hopkins
Marine Station. This work was made possible by Grant G1806
from the Undergraduate Research Participation Program of
the National Science Foundation.
Andrew V. Muchmore
FOOTNOTES
Page 1,
1) Permanent address:
2) The following abbreviations will be used:
HK - hexokinase, G6PDh - glucose-6-phosphate dehydrogenase
TEA - tri-ethanol amine buffer, TPN - triphospho pyridine
nucleotide (NADP), and G-6-P - glucose-6-phosphate.
- 12 -
Andrew V. Muchmore

LITER
TURE CITED
Fredricg, E. and H. F. Deutsch. 1949.
Studies on Ovomucoid,
Journ. Biol. Chem. 181:499-510.
Lowry, Oliver H., N.J. Rosebrough, A.L. Farr, and R.J. Randall.
1951. Protein Measurement with the Folin Phenol
Reagent. Journ. Biological Chemistry 193:265-275.
Nelson, Norton
1944. A photometric Adaptation of the Somogyi method for
the determination of glucose. Journal Biological
Chemistry 153: 375-380.
Warburg, O. and W. Christian. 1941. Protein F
timation by
Biochem. sch.
O:384.
Ultraviolet Absorption, B.,
Wolfsohn, Julian Mast. 1907. The Causation of Maturation
in the Eggs of Limpets by Chemical Means, Biol. Bull.,
3:345-350.
364
Andrew V. Muchmore
FIGURE LEGENDS:
Figure 1 - Activity of hexokinase during development. Activity
expressed as 10-11 moles TPNH/minute/milligram
protein.
Figure 2 - Inactivation of trocophore-stage hexokinase during
storage at 0°C. Activity expressed as 10-11 moles
TPIN/minute/milligram protein. The top line is with
ovomucoid, the bottom without ovomucoid.
36
1

TAOCHOPORE VEUIGER
EGtr
S7A6E
(171)

TIME (HeS.)
366
ndvend fjuehnde
713 2
Table I
Enzyme Activities at Various Embryonic Stages
trocophore same
same
egg
Stage
extract after
extract after extract
extract
24 hours
time -O
time - 0 24 hours
Enzyme
Hexokinase
3.8
3.8
3.7
3.5
with mucoid
0.8
3.5
2.2
with out mucoid
Laminarinase
5 to 30
104
with mucoid
with out mucoid
Maltase
with mucoid
ith out mucoid
Galactosidase
with mucoid
with out mucoid
Amylase
with mucoid
with out mucoid
Isocitrate dehydrogenase
35
43
with mucoid
with out mucoid
G-6-P dehydrogenase
with mucoid
36
with out mucoid
moles TPNH/minute/milligram protein
Enzyme rates expressed as 10
The table also shows enzyme activity after "aging" of extract at O degrees
centigrade for 24 hours in the presence and absence of ovomucoid.
veliger
extract
3.7
56
28
J