Table Of ContentENZYMES IN ONTOGENESIS &HOLINE ESTERASE
IN DEVELOPING MELANOPLUS DIPPERENTIALIS EGGS
by
Theodore Newton Tahmlsian
A dissertation submitted In partial fulfillment of the
requirements for the degree of Doctor of Philosophy,
In the Department of Zoology, In the Graduate
College of the State University of Iowa
July, 1942
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Table of Contents
Page
Acknowledgment------------------------------------------- ii
Introduction---------------------------------------------- 1
Material and Methods------------------------------------- 2
Results-------------------------------- ------- 9
Ontogenesis of the acetyl-choline hydrolyzing
enzyme------------------------------------------------- 9
Optimum pH---------------------------------------------12
Enzyme concentration vs. hydrolysis--------------------12
Optimum temperature--------------------- 13
Acetyl-choline In the eggs------------------------------13
Discussion------------------------------------------------- 14
Summary----------------------------------------------------- 19
Literature Cited------*------------------------------------ 20
Appendix (Tables and Figures)---------------------------- 23
A cknowl e dgrne nt
The author wishes to express his gratitude
to Professor J. H. Bodine who suggested this problem
and who helped in many ways In making this report
possible.
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ENZYMES IN ONTOGENESIS: CHOLINE ESTERASE -
IN DEVELOPING MELANOPLUS DIFFERENTIALIS EGGS
Introduction
Loewi (1921) discovered that vagus stimulation
of the heart released a substance into the perfusion
medium which would stimulate another frog heart* It was
soon found (Loewi and Navratil 1926a) that the physiologi
cal activity of the vagus substance was Identical with
that of acetyl-choline. The same authors (1926b) noted
that extracts from minced frog hearts would destroy the
activity of the vagus substance as well as that of acetyl
choline* They proved the enzymatic nature of the extract
and found that eserine would Inactivate this acetyl-choline
hydrolyzing substance. This observation was substantiated
by Btedman, Stedman, and Eason (1932) who proposed to call
the newly discovered enzyme chollne-esterase*
Since the neurophyslologlcal significance of
acetyl-choline and choline-eeterase was established much
literature has appeared on the various aspects of these
substances In invertebrate as well as in vertebrate ani
mals. There was some controversy as to the presence of
1* Aided by a grant from the Rockefeller Foundation for
research In cellular physiology.
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acetyl-choline and chollne-esterase in the Insects, but
recently Corteggl&nl and Serfaty (1939) showed that these
substancee were present In various Insects Including some
Orthoptera.
Despite the voluminous literature on the various
aspects of acetyl-choline and the enzyme that hydrolyzes
It, there appear to be but few articles on the ontogenetic
development of ehollne-esterase in vertebrate material
(Youngstrom 1958). In view of the many advantages of work
ing with grasshopper eggs (Bodlne 1935) this task was under
taken to study the ontogenetic development of chollne-ester
ase and to determine the presence of acetyl-choline In
Melanoplus differentlalls eggs*
In a major portion of this paper the acetyl
choline hydrolyzing enzyme will be referred to as such; or,
as enzyme until sufficient evidence for its likeness
to chollne-esterase can be brought forth.
Material and Methods
Grasshopper eggs (Melanoplus dlfferentlalis)
used in the following experiments were collected dally,
placed on moist sand in glass covered Jars either separa
ted or In the pods, and kept at 35° C. After such treat
ment they develop for 30 or 31 days and enter diapause
(Slifer 1931)* This developmental-block Is characterized
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by a drop in oxygen consumption (Bodine 1929), cessation
of mitotic activity, and arrested morphological develop
ment of the embryo (Slifer 1932). This block in develop
ment was broken by subjecting the eggs to 5° 0, for three
months (Bodine 1925)* Subsequently, at 25° 0., they
hatched on the 18th day (Slifer 1932).
Ten lots of eggs were used for the determina
tion of the ontogenetic development of the acetyl-choline
splitting enzyme* The eggs in each lot were of the same
age. Five lots each containing approximately 8,000 eggs
were used for the pre-diapause and diapause study, and
five each containing about 6,000 eggs were used for the
post-diapause one. Determination of the enzyme concentra
tion in a given series was completed before a second group
was assayed.
Preparation of the material for experimentation
was as follows: 200 eggs were selected daily from the lot;
they were rinsed in tap water, sterilized for ten minutes
in 70$ ethanol, washed with several changes of re-distilled
water, then rinsed In glycine-NaOH buffer mixture (described
below), and ground to a brei in a glass mortar. Periodically
the brei was examined under a compound microscope (430 X.)
and seldom a cell was detected. The brei was transferred
to a 15 c.o. graduated centrifuge tube and diluted with the
glycine-NaOH buffer solution to the 10 c.o. mark. An "In
ternational Clinical Centrifuge * was used, which was
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operated at 2,000 r. p. m. for 10 minutes.
In preliminary experiments the "A," "B," and
*CW layers (Bodine and Allen 1938) were separately tested
for the enzyme. All of the enzyme was found In the
layer. The HA W and HC ,f layers were discarded to facili
tate the colorimetric titrations. The "BM layer was then
diluted so that each cubic centimeter represented active
extract obtained from 20 eggs. Two c.c. of the extract
were transferred into a wide mouth Erlenmeyer flask and
diluted to 15 c.c. with the same buffer solution. To
this was added 10 c.c. of freshly prepared 1$ acetyl-choline
in the buffer solution which was made each day within
thirty minutes of use. The enzyme was allowed to act on
the substrate for one hour at 25° ± 0,1° C. after which
the enzymatic hydrolysis was Inhibited with eserine (CKLIck
1938). Colorimetric titration was carried out to pH 6.5
with brom-thymol blue Indicator, The titrations were made
with 0.05 N HC1 using an injection burette graduated to
0.05 c.c. Controls for this experiment consisted of the
following: 1. heated enzyme plus substrate, 2. substrate
alone, and 3. enzyme extract alone. The amount of hydroly
sis In 1 and 2 was of equal magnitude due to the autohy
dro lysis of the substrate.
According to Clark et. al. (1938) the amount of
acetyl-choline hydrolyzed at a given time Is directly pro
portional to the choline—esterase concentration of the
mixture. The concentration of the acetyl-choline hydro
lyzing enzyme in the ontogenetic development of the grass
hopper egg is determined indirectly by the amount of acetyl
choline hydrolyzed per unit time. The products of acetyl
choline hydrolysis are acetic acid and choline. In the
glycine-NaOH buffer mixture the acetic acid formed Is
neutralized by the base In the system. The degree of the
neutralization is determined by titrating the remainder of
the base of the buffer system to a predetermined H con
centration (pH 6.5 with brom-thymol blue Indicator). Know
ing the amount of 0.05N HG1 required to titrate the base in
a given volume of the buffer mixture to pH 6.5 on can deter
mine the degree of autbhy&rolysls In the substrate buffer
mixture. In the enzyme substrate buffer mixture the total
hydrolysis Is obtained, and the enzymatic hydrolysis is
determined in terms of c.c. of HC1 by the difference be
tween total- and autohydrolysis.
Known amounts of acetic acid were Introduced
into 25 c.c. of glydne-MaOH buffer solution and titrated
to pH 6.5 with 0.05N HC1 in order to determine the error
in titrating. This procedure was also helpful for the
determination of the least change in the H concentration
detectable with the apparatus used. The results are shown
in Table III.
The H concentration of the glycine-NaOH buffer
was corrected to pH 8.5 4 0.02 with the aid of a Leeds &
Northrup glass elctrode pH meter. The constituent® of the
buffer solution were 90 c.c. of 1M glycine, 10 c.c. of 1M
HaGH, 9.94 grams of NaCl, 150 c.c. of glycerine, and enough
re-distilled water to make one liter. The NaCl in the
buffer solution was necessary to keep the proteins in the
"B** layer from precipitating (Carlson 1941), The glycer
ine was used to facilitate the extraction of the acetyl
choline splitting enzyme from cells that may not be broken
up during the process of grinding (Gilck 1938), This
buffer was autoclaved Immediately after preparation at 15
lbs, for ten minutes, and kept sterile by autoclavlng every
other day thereafter.
The amount of enzyme In the embryo alone was
determined (see X marks In figure I.). At various stages
of development embryos were dissected out of the eggs,
cleaned of adhering yolk, placed In a glass stoppered
bottle with some buffer solution, and disintegrated by
shaking manually. This mixture was made to volume so that
each c.c, represented 20 embryos. This was used Instead of
the layer, and the rest of the procedure was the same as
described for the eggs above.
A lot of two day old eggs was divided into two
groups. One group was irradiated^ at 1000 Roentgen units
and the other used as a control. Using the titration method
1. The author Is Indebted to Mr. J. p. Goodrich of this
department for the X irradiation.
