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CALIFORNIA STATE UNIVERSITY, NORTHRIDGE
ISOLATION OF ACID PROTEASE FROM MURINE
TERATOCARCINOMA ASCITES FLUID
A thesis submitted in partial satisfaction of the
requirements for the degree of Master of Science in
Biology
by
Robert Clayton Steiner
January, 1984
Cal ifornfa State University, Northridge
January,
ii
1984
ACKNOW L EDG f·11ENTS
would
Ronald J.
first
like to especially thank my
Steiner and Margaret L.
the seeds that started a II
to sincerely thank Dr.
me
a chance
tvleyer
for
Thompson
work,
Dr.
and
this out.
Steven B.
being
Donald E.
his
my
expertise
scapegoat
Bianchi
and
Then
Carlson for
constantly
bloody thing.
ii i
wou I d II ke
the way,
with
when
Dr.
me~
James
Pepe
things
Edward G.
for their assistance while on my committee,
Kay L.
for planting
Oppenheimer for giving
advising me along
sharing
for
Steiner,
parents~
did
"T"
M.
not
Pollock
and finally
nagging me to finish this
TABLE OF CONTENTS
ABSTRACT
vi
INTRODUCTION
. ... .. . . ... . ... .. . . .. . . . . . . . . . .. .. ..
METHODS AND MATERIALS
•
•
•
•
•
•
•
•
•
•
•
•
0
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Solutions and Reagents
8
8
. . . . . . .. . . . .. . . . . .
. . . . .. .. .. .. . .. . . . . .
Teratocarsinoma Cel I Line
8
Protease Activity Assay
9
Gel Filtration Chromatography
...............
10
Affinity Chromatography
11
Polyacrylamide Gel Electrophoresis
12
RESULTS
. . . . . .. . . .. . . . . .. . . . . . . . .. . . . . . . .. . . . .. . .
14
Liquid Chromatography
14
Polyacrylamide Gel Electrophoresis
14
. .. . . . . . .. . . . . . . . . . . . . . . . .. . . . .
15
. . . . . . . . . . . . . . . . . . . . . . .. . . .. ... . . . . . . .
16
. . .. . . . . . . .. . . . . . . . . . . . . . . .. . . . . .. . .
32
Calculations
DISCUSS JON
BIBLIOGRAPHY
iv
LIST OF FIGURES AND TABLES
FIGURE
1.
Protease Activity Assay Standard Curve
for Tyrosine Released •••••••••••••••••••••• 20
2.
Elution Profile of Clarified Teratocarcinoma
Ascites Fluid Fractionated by Gel Filtration
Chromatography •••••••••••••••••••••••••••• • 22
3.
Protein Elution Profile of Acidified Ascites
Fluid Fractionated by Affinity
Chromatography •••••••••••••••••••••••••••• • 24
4.
Native Polyacrylamide Gel of Acidified
Ascites Fluid and Protein Peaks Eluted from
Affinity Chromatography Column ••••••••••••• 26
5.
SDS Polyacrylamide Gel of Active Protein
Peak Eluted from Affinity Chromatography
Column and Standard Molecular Weight
Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 28
v
TABLE
1.
Tabulation of Acid Protease Data from
Affinity Chromatography Column ••••••••••••• 30
vi
ABSTRACT
ISOLATION OF ACID PROTEASE FROM MURINE
TERATOCARCINOMA ASCITES FLUID
by
Robert Clayton Stetner
Master of Science In Biology
An
acid
protease
causing cell-cell
teratocarcinoma
characterized.
that
appears
disaggregation
was
partially
responsible
in a mouse
purified
for
ascites
and
The protease was purified from the mouse
ascites fluid by affinity chromatography on Pepstatin A
agarose beads.
The purified material yielded one broad
band on silver stained native polyacrylamide slab gels
and two bands on silver stained SDS, 8-mercaptoethanol,
slab gels, with approximate molecular weights of 66,000
and 20,000 Daltons.
The affinity purification method
yielded a 46 fold purification of the protease from the
ascites fluid.
These studies represent a first approach
to purification and characterization of an enzyme that
appears to be involved in altering the adhesiveness of a
specific mouse ascites tumor.
vii
INTRODUCTION
Teratocarcinoma (I lteral ly meaning malignant tumors
resembling a monster or malformed baby} are neoplasms most
often
derived
from
morphologically
similar
embryonal-type
cells
differentiated
They
1955).
pluripotent
to
be
both
(embryonal
adult-type
may
germ
cells
benign
or
cells
which
are
undifferentiated
carcinoma)
{teratoma),·
malignant
and
(Mel lcow,
and
their
composition can vary from being relatively simple to having
almost as many types of tissues as the individual bearing
them.
The
first
theory
about
the
cause
of
teratocarclnogenesis was proposed by Askanazy (1907) who,
after doing detailed histological studies on benign cystic
ovarian
teratomas,
somatic tissues of
concluded
the
\'/ell
differentiated
the teratoma developed
embryonic differentiation.
by means of
This embryonic differentiation
was thought to be either from a single multlpotent type of
cell
or from a group of eel Is composed of representatives
of each of the germ layers. Askanazy bel leved these eel Is
were derived from early embryonic primodia, could remain
dormant for
years,
and then cou I d commence gro\'1 I ng and
differentiating.
Budde (1926) appl led the principles of developmental
1
2
biology to the study of teratomas.
He belIeved teratomas
probably represented a misdirection of
the action of a
primary embryonic organizer, and that the tumors originated
In cells released from normal developmental control at the
primitive streak stage.
In contrast,
Jackson and Brues
( 1 9 4 1 ) f o u n d e v I d e n c e I n s u p p or t of As k a n a z y ' s t h eo ry wh en
they observed,
In a murIne ovar ran teratocarcInoma, more
mitoses occured
In the undifferentiated eel Is than In the
adult-appearing somatic tissues.
They concluded some of
the rapidly growing eel Is matured under a delicate control
mechanism that kept eel I division and maturation relatively
constant,
thus
allowing
both
differentiated
and
undifferentiated eel Is to persist.
More recent morphologic studies by Fekete and Ferlgno
{1952), on ovarian teratomas in mice, have shown transition
stages
between
undifferentiated
embryonal
eel Is through
Immature forms to adult eel Is exist in teratocarcinomas.
Extensive
studies
by
Stevens
(1962)
on
the
early
development of spontaneous teratocarcinomas have shown In
fetal
mice,
the
teratocarcinomas
undifferentiated embryonal
cells,
are
composed
and the tumors
more complex histological Jy as the animals age.
of
become
Both these
studies support the presently accepted theory regarding the
cause of teratocarclnogenesls:
adult-type
cells
In
All
of the
teratocarcinomas
are
immature and
derived
undifferentiated pluripotent embryonal cells.
from
3
"Embryoid Bodies" are one form of teratocarcinoma in
which the tumorous growths have structures morphologically
similar to early embryos.
Peyron et al.
(1936 and 1939)
first described them In human testicular teratocarcinomas.
They
found
the
embryoid
bodies
blastocyst-like" morphology
endodermal eel Is.
to
composed
have
of
a
"human
ectodermal
and
Since then, embryoid body teratomas have
been repeatedly observed In human testicular tumors of germ
cell origin 0•1elicow, 1940 and 1955; Ntcod, 1945; Friedman
and Moore,
1946;
Friedman, 1959; Teilum, 1950; Dixon and
Moore, 1952 and 1953; Masson, 1956; Simard., 1957; Gaillard,
1957
and
Marin-Pad! I Ia,
transplanted
1959,
Cabanne,
1958;
1965)
1957;
and
testicular
In
Evans,
mouse
1957;
metastatic
teratocarcinomas
and
and
(Stevens,
1958,
and 1960; Pierce and Dixon, 1959, and Pierce et al.,
1 960) •
Stevens (1959) first observed embryoid bodies of the
mouse In a retroperitoneal
origin.
metastatic tumor of testicular
These tumors were shown to have Inverted primary
germ layers composed of an outer layer of eel Is resembl fng
endoderm and an Inner layer resembling ectoderm.
a I so
shown
that
when
sub I i nes
of
It \'/as
transp I anted
teratocarcinomas were grafted into the peritoneal cavity of
a
mouse,
thousands
of
free-floating
embryoid
body
teratocarcinomas similar to mouse 5-6 day old embryos were
4
contained
in the peritoneal
Pierce et al.
mouse originated
of
solid
There,
(1960)
by
composed
of
of
teratomas
ln
teratoma,
developed
yitro.
originated as small
superficially
by
a
I ayer
detached
further
work
vI seer a I
not
arise
arise
from
carcinoma)
germ
the
layers.
transplanted
concluded
yo I k
Stevens
sac
from
Further
probably
carcinoma
grew
have
led
teratomatous
and
became
as wei I
to
embryonal
bodies
rather,
stem eel Is of the tumor.
by
Pierce
and Stevens
and
(1960)
Dixon
(1959),
Pierce
et
and
into the
anterior
bodies,
undifferentiated
growths of
chamber
many
al.
have shown embryoid bodies are
grafted mouse embryoid
ectoderm
found
they
(embryonal
also able to further differentiate into various eel I
They
as
the
embryoid
germ ce I Is;
undifferentiated
lying
an explant overlaid
These studies,
the
cysts
testicular
these
whIch
(1962),
In mice,
dIrect I y
Studies
(1960),
a
the main growth.
by
embryoid
fluid.
showed new ~ouse embryoid
(1961)
They
ascites
aggregates of embryonal
understanding that,
do
typical
three
from
necrotic areas
lntc the
in a necrotic portion of
of
from
partially
into
the
work by Pierce and Varney
body
of
growths
developed
elements
(ascites fluid).
found the embryoid bodies of the
sloughing
intraperitoneal
granules
fluid
of
some composed of only
eel Is,
the
eye
different eel I
types.
subcutaneously
of
adult mice
and
and
types were obtained.
5
Pierce and Verney (1961) observed the development of cystic
embryoid bodies from explants of teratocarcinoma that were
maintained for as long as five months as organ cultures.
The
microscopic
structures
of
the
tumors
developed
subcutaneously from explants of embryoid bodies ln Yli£Q
for
sIx months were compared to those of
control
tumors
developed
subcutaneously
bodies that had never been ln vitro.
consisted
predominantly
dominant tissue
muscle.
brain
from
whereas
subcutaneously In mice.
after
two
the
tumors was striated
of these tumor strains
ability
embryoid
The control tumors
tissue,
In the experimental
Almost all
muscle-producing
of
a series of
or
lost their
three
New embryoid bodies
passages
we~e
observed
to develop In the tissue cultures long after they had lost
the pattern of the original explanted embryoid bodies.
In our laboratory, we have observed the spontaneous
transformation of a mouse
tumor
Into
a
intraperitoneal
embryoid
body
single
cell
passages
form
of
embryo!~
form
(Meyer
body teratocarcinoma
after
et
al.,
successive
1983).
The
the teratocarcInoma was a s I ow
growing tumor which normal Jy did not cause any significant
accumulation of ascites fluid while the single cell form
had a dramatic Increase In growth rate as wei I as ascites
fluid accumulation.
Morphological characterization of the
single cell
form by Stevens (1983) showed the cells were
not viseral
yolk sac cells though they
had an altered
6
morphology from the original embryoid body teratocarcinoma.
The factor or factors responsible for transforming
eel Is to a more mal lgnant state have been investigated by
many laboratories.
One thought is that proteases might be
Involved in the transformation or at least involved In the
release of a normal cell from contact Inhibition.
(1974)
Bosmann
(1972)
and Bosmann et al.
and Christman and Acs
(1974)
have reported finding elevated protease levels In
transformed chicken fibroblasts by Reus sarcoma virus (RSV)
and
Schmidt-Ruppin strain of
(1972)
RSV,
Schnebll
and Burger
in transformed mouse fibroblasts by Murine sarcoma
virus (MSV), and Hatcher et al.
(1976 and 1977)
In human
melanoma and transformed mouse epidermal eel Is.
Further evidence In support of this theory involves
treating normal
cells with proteases.
Burger (1970) and
Sefton and Rubin (1970) have shown trypsin, pronase, and
ficin,
at final
release
normal
concentrations between 0.007 and 0.0003%,
cells
from
contact
inhibition
growth.
Proteases may, therefore, release normal eel Is from contact
Inhibition of growth, a property that is characteristic of
transformed eel Is.
Studies done by Meyer et al. (1983) with the single
ce I I form of the embryo f d body teratocarcInoma have shown
an acid protease {possibly the carboxypeptidase Cathepsin
7
D)
in
the
ascites
surface may
of
play
fluid
a multlcel lular
results
sIng I e
ce I I
tumor
suggest
may
protease on the cell
that
the
beads)
fluid-mediated
clusters
in
Oppenheimer
activity
which
bee au se
and
adhesiveness
presence
was
quickly
In
the
of
the
res-tored
by
colleagues
CPeps"tatln-A
bound
A preven-ts
ascites
Pep stat in
of
teratocarcinoma
has
unpublished
shown
a
high
In sonicates of washed single eel I
work
eel I
by
specific
teratocarcinoma
indicates the eel Is themselves may contribute to the
This
study
protease found
the
In the ascites fluid.
describes
attempts
to
purIfy
"the
acl d
In the ascites fluid of the single eel I
teratocarcinoma
characterization
tumor.
Into a single eel I
the
Additional
protease activity found
of
of
activity
disaggregation
culture.
cell
beads derivattzed wi"th a specific
protease
and
tumor
This was concluded after i t
adhesiveness
Inhibitor
the
in the transformation
from
surface.
eel Is with
aga rose
loss
resu It
Incubation of
of
on
embryoid body tumor
The
observed
possibly
a significant role
form.
was
and
of
the
and
should
enzyme
and
lead
fts
to
form
fur-ther
effects
on
the
& MATERIALS
METHODS
Solutions and Reagents-- Hanks'
CHBSS)
was
prepared
0.06g KH 2 Po 4 ,
NaCI,
and
0.35g
0.01g
as follows:
phenol
distil led water,
red
and
were
1.42 g Na2HP04 x 7H20,
(When
the
these
values,
buffer
glycine
and
distilled
were
the
pH
the
NaCI
noted
as
sulfate
In 1.0
to
in
dissolved
in 0.1
and
the
of
Dulbecco's
0.20g KH 2 Po 4 ,
adjustedto
pH
from
Electrode
Trts
and
14.4g
distilled water,
Electrophoresis
0.002g
7.4.
differed
text.)
3.02g
20.0ml
liter of
pH
liter of
8.3.
(SDS),
8-mercaptoethanol,
I iter
were dissolved in 1.0
and/or
follows:
dissolved
adjusted
and
solution was prepared as follows:
dodecyl
1.0
was prepared as follows:
and 8.0g NaCI
are
prepared
in
l.Og g I ucose,
0.10g MgCI 2 x 6H 2 0,
water,
they
7H20,
dissolved
(DPBS)
concentration of
was
solutions
0.40g KCI,
the pH adjusted to 7.4.
0.20g KCI,
of
0.04g CaCI2,
0.09g Na2HP04 x
phosphate buffered sal fne
liter
salts
0.10g MgCI 2 x 6H 2 0, 0.01g MgS0 4 x 7H 2 0, 8.0g
NaHC03,
0.10g CaCI 2 ,
balanced
sample
1.5g Trls, 4.0g sodium
glycerol,
bromphenol
distilled water,
lO.Oml
blue
were
and the pH was
adjusted to 6.8.
Teratoma
Cell
form
ascItes
teratocarcInoma
In
from
of
obtaI ned
Line--
1981
An embryoid
Dr.
Leroy
8
body
(OTT
(now
single cell)
6050-2568)
Stevens of
was
the Jackson
9
Laboratory
by
(Bar Harbor,
intraperitoneal
passage
(average weight 34g)
depending
on
bloating.
the
how
r·llfce
contents
placed
In
for
I nternatl ona I
was
only
the peritoneal
I lne was maintained
male
allowed to grow
conical
for
by cervical
glass
minutes
129/J
at
cavity
Centrifuge
accumulation of
x
abdominal
dislocation,
was
centrifuge
180
removed,
tubes,
gravity
in
{rotor
#221);
ascites
fluid
cavity was rinsed with
(supernatant)
mice
1-3 weeks,
began to show
peritoneal
C I In i ca I
a small
young
sacrifled
their
5
in
the mice
were
15.0ml
centrifuged
and
soon
of
ME.). The eel I
a minimal
and
an
If
there
present,
amount of
HBSS.
The ascites fluid
50.0ml
polycarbonate centrifuge tubes and centrifuged
ten minutes at 12000 x gravity,
Superspeed Centrifuge
ascites
fluid
(rotor
was transferred to
for
at 4oc in a Sorval I RC2-B
type SS-34).
The clarified
was
Its
(supernatant)
removed,
volume
measured,
and stored at 4°C If used the same day or frozen
at
for
-20°C
(pellet)
were
use
at
washed
a
future
three
date.
times
with
wash the eel Is were centrifuged for
gravity
#221)
then
in
and
an
the
diluted
International
Activity
determIned
HBSS;
with
HBSS
for
usIng
Assay-the
The
assay
cells
after
Centrifuge
supernatant was dIscarded.
1:1
tumor
each
five minutes at 180 x
Cl inlcal
(rotor
The ce I Is were
reinjection
129/J mice to maintain the tumor
Protease
The
Into young
I ine.
proteolytic
method
activity
descr l bed
by
was
Anson
10
(1938) with the following modifications:
sodium citrate
buffer at pH 2.0 was used Instead of sodium acetate buffer
at
pH 5.0,
cysteine was eliminated,
and samples were
incubated at 37°C on a rotary shaker for 4.5 hours Instead
of 90 minutes to determine low levels of enzyme activity.
A tyrosine release standard curve was prepared using the
assay
volumes with
known concentrations of
L-tyrosine
{Sigma Chemical Co., St. Louis, MO.) as the sample (see
figure 1).
Specific activity was defined as mg tyrosine
released/ml/hour/mg protein.
Gel Filtration Chromatography-- Sephadex G-150, fine mesh
{ Ph arm a c i a , Swe de n ) wa s s u s pe n de d r n DPBS , 0 • 1 fv} . i n Na C I at
pH 7.4 (the running buffer).
A Bfo-Rad Econoline column
2.5cm x 75.0cm (Bio-Rad Co-. Richmond, CA.) was packed
with the solvated Sephadex G-150 to a height of 70.0cm and
cooled down to 4°C, the temperature at which all column
work was carried out.
A 5.0ml sample of the clarified
ascites fluid was then carefully layered on the top of the
gel and allowed to enter the column.
Once the sample had
entered the gel, the column was eluted with the running
buffer at 1 x gravity and a flov1
rate of 15.0ml/hr.
Fractions of 5.0ml were collected using a Golden Retriever
Pup
Model
1100
fraction
col
lector
{Instrumentation
Specfalties Coot Lincoln, NE.); an Industrial Fracto-scan
inllne UV monitor Model #3-5100 (Buchler lnst.,
Lee,
NJ.)
was
used to determine
the
Inc., Fort
proteIn e I uti on
11
profile of
the column.
The
protein content of each
fraction was then determined using the method described by
Lowry., et a!.
( 1951), protein peak fractions were pooled,_
and the proteolytic activity was measured using the method
described above (see Protease Activity Assay).
Affinity Chromatography-- Pepstatin A bound agarose beads
purchased from Pierce Chemical
packed
Co.
(Rockford,
IL.} were
Into a 0.7cm x 9.0cm Bto-Rad Econolfne column
CBio-Rad Co.,
Richmond,
CA.) to a height of 8.0cm and
equilibrated with PBS, 0.9tJI In NaCI
at pH 5.0 (loading
buffer).
NaCI was added to 2.0ml of the clarified ascites
fluid
a
to
Initial
final
concentration of 0.9t.IJ
(assuming an
NaCI concentration of 9.0% or 0.1M.IJ) and the pH
adjusted to 5.0.
This acidified ascites fluid was then
centrifuged In an International ClInical Centrifuge (rotor
#221) at 180 x gravity to remove the precipitate formed
and carefully loaded to the top of the gel bed.
After the
sample had entered the gel., the column was eluted with the
loading buffer at
x gravity and a flow rate of 10.0
ml/hr; fractions of 5.0ml were collected and the protein
elution profile was determined as described above (see Gel
Filtration Chromatography).
When no more protein was
eluted
determined
from
the
column
(as
by
the
UV
monitoring) PBS, 0.9M In NaCI at pH 8.4 (eluting buffer),
was appl led to the column to free any bound proteins.
The
protein content of each fraction was then determined using
12
the method described by Lowry, et al. (1951), protein peak
fractions were pooled, and the proteolytic activities were
determined
as
descrIbed
above
(see
Protease Act l vI ty
Assay).
E I ectrophores f s--
Polyacrylamide
Gel
polyacrylamide
(Bio-Rad
Laboratories,
Ten
pecent
Richmond,
CA.)
running gels were prepared using the method described by
Laemml i (1970); the gels were poured Into a 1.5mm x 16.0cm
Bio-Rad
Protean
(Richmond,
double
slab
.electrophoresis
eel I
CA.) and allowed to polymerize for one hour.
After the gels had polymerized, a three percent stacking
gel,
prepared as described by Laemmlf (1970), was poured
on top of the running gel and allowed to polymerize.
All
samples to be electrophoresed, including clarified ascites
f I u I d and
t he
prot e I n peaks
e I uted
agarose column for the native gel
containing
the
proteolytic
f rom
Peps tat I n A
and the protein peak
activity
(bound
peak)
and
Bio-Rad SDS-SAGE Low Molecular Weight Standard Proteins
{Richmond,
CA.)
for
the SDS gel,
were dialyzed at 4oc
against two 2.0 liter changes of half strength electrode
buffer C0.013M In Tris and 0.096M in glycine at pH 8.3).
Samples were then prepared for electrophoresis by diluting
the
dialyzed
solution;
samples
native
electrophoresis
1:1
gel
sample
with
samples
electrophoresis
were
solution
diluted
without
SDS
sample
with
and
B-merca ptoeth a no I whi I e SDS ge I samp I es were comp I ete I y
13
dissociated
m t nutes.
loaded
by
immersing them
Appropr tate
Into
the
electrophoresed
volumes
wei Is
with
#3-1 014A
(Buch I er
constant
current
in
a
of
of
Buchler
20mA
D.C.
the
gel
power
Lee,
were
and
supply
NJ.)
track r ng
1.5
model
at
dye
a
had
entered the running
gel,
Increased to 40mA.
When the tracking dye had migrated to
about
turned
l.Ocm
off
at which
for
samp I es
stacking
Fort
unti I
water
d i I uted
the
Instruments~
of
boi I tng
from the bottom of the
and
Silver Stain Kit
the
gels
were
(Richmond~
point the current was
gels~
stained
CA.)
the current was
with
the Bto-Rad
and then dried.
\'
RESULTS
Liquid
Chromatography--
The
isolation
of
the
acid
protease, possibly the carboxypeptidase Cathepsin D, from
teratocarcinoma OTT 6050-2568 ascites fluid was attemped
by first chromatographlng the clarified ascites fluid on
Sephadex G-150; the protein elution profile consisted of
two large overlapping
peaks (with the proteolytic
pro~ein
activity residing between them) and a smal I protein peak
with
no protease activity (see figure 2).
proved
to
be
Inadequate
in
resolving
the
components of the clarified ascites fluid,
chromatography
with
Pepstatin
Pepstatin A speclfical
~y
This method
A agarose
protein
so affinity
beads,
as
binds to Cathepsin D, was tried.
When the ascites fluid was acidified to pH 5.0 (the pH of
the loading buffer), there was a slight globular protein
precipitate
formed
centrifugation.
The
which
was
protein
~as!
ly
elution
removed
profile of
by
the
acidified ascites fluid on the Pepstatin A agarose column
consisted of one large unbound protein peak and one smal I
bound protein peak with most of the proteolytic activity
residing In the bound protein peak (see figure 3).
Electrophoresis-- When the acidified ascites fluid and
both
the
affinity
protein
peaks
off
the
Pepstati n A agarose
column were electrophoresed on a ten percent
14
.
15
native polyacrylamide gel,
the banding pattern for the
acidified ascites fluid gave ten to fifteen bands; the
unbound protein peak fractions gave six to ten bands, and
the bound protein peak gave one distinct band (see figure
4).
Finally,
when
the
protein
peak
containing
the
proteolytic activity {the bound peak) was electrophoresed
on
a
ten
standard
percent SDS
weight
poI yacry I am I de ge I agaInst the
proteins,
It gave two
bands,
one at
approximaely 20,000 Daltons and the other at approximately
66,000 Daltons (see figure 5).
Calculations-- By employing the isolation scheme described
above (see Methods and Materials), a 46 fold purification
of proteins was achieved using the affinity chromatography
method (see table 1).
DISCUSSION
The
role
transformation
of
and
proteolytic
possibly
ma I I gnant state has
enzymes
in
IntrIgued
In
malignant
maintenance
of
the
InvestIgators for over a
decade (Bosmann, 1972; Bosmann et al., 1974; Burger, 1970;
Christman and Acs, 1974; Hatcher et al., 1976 and 1977;
Schnebll and Burger,
1972; Sefton and Rubin,
Unkeless et al., 1973).
1970; and
Our intet-est in this particular
area of research originated when we observed a spontaneous
transformation
of
a
multicellular
teratocarcInoma to a sIng I e ce I I form.
embryoid
body
The embryoId body
form rs a slow growing tumor which normally does not cause
any significant accumulation of ascites fluid, while the
single cell
form tends to be a rapidly growing tumor and
ls associated with a rather large accumulation of ascites
fluid.
the
These observations led us. to initiate studies on
possible
role
of
ascites
fluid
Influence
on
teratocarcinoma eel I characteristics (Meyer et al., 1983}.
This study focuses on isolating the acid protease found in
the ascites fluid associated with the single eel I form of
the tumor.
The work by Meyer et a I. ( 1983)
in this I aboratory
has shown that the ascites fluid from the tumor has a
protease activity with an optimum pH
16
In the range of
17
2.0-3.0 and Is Inhibited by both diazoacetyi-DL-norleucine
methyl
ester (DAN)
identification
of
Cathepsin D.
Cathepsin
Barrett.,
its
and Pepstatln A,
the
enzyme
as
which suggests the
the
carboxypeptidase
With the knowledge that Pepstatin A binds
D in
a
stoichiometric
fashion
(Knight
and
1976), and assuming the enzyme Is Cathepsin D,
concentra-~lon
In ascites fluid was determined from
Pepstatin A titration curves.
The equimolar concentration
was found to be 9.6 x 10-6 mM, which corresponds to 0.36ng
of Cathepsin D per mg of protein in the ascites fluid.
The affinity
chromatography method
used
tn this
study yielded a 46 fold purification of protease from the
ascites fluid.
indicates
The SDS polyacrylamide gel electrophoresis
the molecular
weights of
the two sub-units
making up the acid protease are approximately 66,000 and
20,000 Daltons, which is not consistent with the accepted
molecular weights of Cathepsin D
Seyer (1978)
s~b-unlts.
Whitaker and
have shown that when Cathepsin D,
isolated
from bovine brain by affinity chromatography on Pepstatin
A sepharose,
was
polyacrylamide
e I ectrophoresed
gels,
approximate molecular
13,000 Daltons.
there
were
weights of
on
ten
three
48,000,
percent
bands
SDS
with
31,000,
and
At this point, an amino acid composition
would be necessary to confirm whether the acid protease
found In the teratocarcinoma ascites fluid is Cathepsin D
or another acid protease.
18
The
question
of
whether
the
acid
protease
Is
synthesized by the tumor or by the mouse In response to
the tumor Is still under Investigation.
et al.
Studies by Meyer
have shown the specific activity of the
(1983)
enzyme does not fluctuate significantly during the growth
of the tumor In the mouse; this suggests that the enzyme
is
produced
at
a
rate
Though
accumulation.
constant
lt
fs
wIth
generally
per I tone a I ascites f I u l d accumu I at ron
of I ymphatl c
the
mouse
ducts~
ascItes
f I uf d
accepted
that
is due to b I ockage
hence the enzymes are synthesized by
lymphatic
tissue,
recent
results
fn
our
laboratory Indicate the tumor eel Is themselves have a high
proteolytic specific activity.
The studies also show that
when slngte eel Is from ttssue culture are Incubated with
Pepstattn A agarose beads, there Is a clustering of eel Is
around the beads,
Jndtcattng there are receptor sites on
the eel Is surface for Pepstatln A•.
Other results by Meyer et al. (1983) have shown that
ascites
fluid
causes
disaggregation
of
clusters
of
teratocarcinoma single eel Is grown in tissue culture.
ce II s
rema f n aggregated when
Pepstati n-A
conjunction with ascites fluid;
fs
added
The
rn
Pepstatrn A alone also
causes no disaggregation. These results suggest the acid
protease found
f n teratocarcinoma ascItes
Important in causing eel I disaggregation.
f I u t d may be
19
In
first
approach
protease
be
summary,
found
Involved
tumor cell
in
line.
to
the
studies
purifying
reported
and
In teratocarcinoma
Increasing
here
provide
characterizing
ascites
the malignancy
an
a
acid
fluid that may
of
a
specific
20
Frgure 1
Tyrosine
assay.
release
standard
curve
for
protease
activity
21
\
1\
1\
0
<!>
0
.
h
\,
\
~\
1\
\
LLJ
z
......
V)
0
,.,
I
0:::
>-
1-
1\,
\
0
N
•
0
~
\
1\
0
0
0
•
0
.
m
(X)
0
0
•
0
".
0
0
.
<0
0
WUQ99 0
0
.
lC)
0
0
.
"""
0
3JN~8~0SS~
0
.
tt)
0
0
N
•
0
0
0
.
0
0
.
00
0
0
.
22
Figure 2
Clarified ascites fluid dated from a Sephadex G-150 gel
filtration column.
(-G-)
=
mg/m I proteIn x 0 o 1
(--) = mg/ml tyrosine released by pooled fractions
to 60-75ml, 75-90ml, 90-110ml, 110-130ml,
130-165ml, and 165-185ml.
23
r-----------------------------------------------1rr
0
0
('!
0
ro
0
lO
0
..q-
0
N
0
0
"'¢
4
0
1.()
t<)
.
0
0
.
.o
t<)
{ LW/6w)
1.()
.
N
0
"13~
·~Al ~
0
N
0
( T "0
l()
.
,....
X
•
0
LWf6w)
0
,....
0
•
NI310~d
1.()
.
0
0
0
0
0
.
24
Figure 3
Acidified
ascites
fluid
eluted
from
a
Pepstatin
A
agarose affinity column.
(-tr-}
= mg/ml protein x 0.1
(~)
= mg/ml tyrosine released by pooled fractions
0-15ml, 15-35ml, and 35-45 mi.
C--)
= pH of eluting buffer
25
0
.
•
CXJ
::c
0.
0
0
.
N
0
0
.
If)
0
0
0
.
~
tl)
0
0
•
( LW/ LW)
"13~ ·~Al ~
0
N
0
~
(1"0 X LW/fiill)
-.
0
0
NI310~d
0
0
0
•
0
.
26
Figure 4
Native
fluid
polyacrylamide
(C)
and
unbound
gel
showing
acidified
pooled peak
fractions,
(8) and bound pooled peak fractions, 35-45ml,
from a Pepstatin A agarose affinity column.
ascites
15-35ml,
(A) eluted
27
28
Figure 5
SDS
polyacrylamide
gel
showing
Bfo-Rad
Molecular Weight Standard Proteins,
*
(B)
SDS - PAGE
Low
I 1st
and bound pooled peak fractions ,
35-45ml,
eluted
from a Pepstatin A agarose affinity column.
*=Phosphorylase B (92,500), BSA (66,200), Ovalbumin
(45,000}, Carbonic Anhydrase (31,000), Soybean
Trypsin Inhibitor (31,000), and Lysozyme (14,400).
29
:,~···
·-· :.·
.
~--
--
A
B
,.
30
Table 1
Tabulations of total protein, tyrosine release, specific
activity,
and
fluid
pooled
and
percent
peak
recovery
fractions
of
of
acidified
ascites
acidified
ascites
fluid eluted from a Pepstatln A agarose affinity column.
Protein
= mg
Tyrosine Release= mg/ml
Specific Activity= mg tyrosine/ml/hr/mg protein
Percent Recovery = Sp. Act. Bound Peak/Sp. Act.
Ascites Fluid
31
TOTAL
PROTEIN
TYROSINE
RELEASE
SPECIFIC
ACTIVITY
FOLD
PUR IF.
Acidified
Ascites Fluid
33.245
0.080
0.00054
N.A.
Unbound
Peak
28.125
0.050
0.00040
N.A.
1. 250
0.138
0.02462
46
SMWLE
C0-15ml)
Bound
Peak
(35-45ml)
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