Effect of Concanavalin A on cellular slime mold development
BIOCHEMICAL
Vol. 53, No. 4, 1973
EFFECT
OF CONCANAVALIN
PREMATURE
AND BIOPHYSICAL
A ON CELLULAR
APPEARANCE
RESEARCH COMMUNICATIONS
SLIME
OF MEMBRANE-BOUND
MOLD
CYCLIC
DEVELOPMENT:
AMP
PHOSPHODIESTERASE Martha Ulbrick Gillette and M. F. Filosa, Zoology Department, Scarborough College, University of Toronto, West Hill, Ontario, Canada. Received
June
29,
1973
Concanavalin A delays aggregation of slime mold amoebae, Summary: apparently by interfering with the cells' response to the chemotactic agent, cyclic AMP. Concanavalin A also induces the premature appearance in non-aggregating cells of a membrane-bound cyclic AMP phosphodiesterase normally found only at the time of aggregation. The appearance of this enzyme is not due to activation of an inactive form of the enzyme. After cells
a period
of
of
Dictyostelium
cellular
aggregates
body.
The
growth
as
discoideum
move
together
each
attraction
of
of
the
dispersed
Malchow
(2) have demonstrated --et al. of a membrane-bound cyclic dramatically
tion
and
returns
These
investigators
pates
both
that
in
very
Concanavalin
A (Con
of
A), in
For washed
all free
(Difco),
and
and
assayed
activity bound released
at
enzyme from
22'C
washed
whole
to cells
exposed the
at cells.
Copyright @ 19 73 by Academic Press, Ix. All rights of reproductiorl irr any form reserved.
aggregation the
amoebae
discoideum
(mPDE)
preceding
after
aggrega-
aggregation.
AMP and lectin
mold
partici-
the
chemotactic
here
a study
known
surfaces
(1).
the
phosphodiesterase
a plant
coli
incubated according
of
this
studies
Escherichia
AMP responsiveness,
a fruiting
(3-8),
to
showing
bind
to
affects
the
development.
AND METHODS
developmental of
D. just
cyclic
cell
slime
MATERIALS
by
We present
of
aggregation
into
levels
aggregation.
determinants
into
cells in
multi-
AMP phosphodiesterase
low of
amoebae, into
secreted
activity
that
detection cell
AMP
in
suggest
during
process
to
the
carbohydrate
cyclic
increases
then
response
to
solitary
differentiates
is
which
response
which
centres presence
in
vegetative
cells Konijn
B, in
the
the
cell
1159
To
Cyclic
includes and
cell
test by
were agar
for
cyclic
aging
at
4'C
AMP phosphodiesterase
which
surface whole
phase amoebae on 1% purified
sensitized
(9).
examined
log
light.
were
was
Total
late plated
soluble
both
membrane-
enzyme
phosphodiesterase
Vol. 53, No. 4, 1973
BIOCHEMICAL
Table
Con
1.
Effect of Dictyostelium
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A in delaying aggregation discoideum amoebae. Time
2.5 x 103 Con
23
22
100
27 28
24 27
200 300
42 38
30 32
30 31
400
53
34
33
21
20
20.5
21
21
22
5 x 1O-2
M
200 i.lg/ml + 2.5 x 1O-2
M
Stock cultures of slime with Escherichia coli B Hydrophobic 1% purified prepared; Con A (Sigma, necessary and the mixture dishes. Cells were grown dark. The amoebae were of cell suspension were 10~1 Hamilton microsyringe. duplicate or triplicate;
activity
was
determine
This
twice
particulate
internal
All
enzyme
allowed (Table
method
of
A on
slime
mold
When
late
log
phase
amoebae
is normal. tration and
develop, although The
extent
the
density
was
of
prepared as well
according the
the (2).
to
as
Malchow Protein
legends.
(10).
are
a striking
delay of
at
cell
development
morphology of
the
AND DISCUSSION
Con to
in
Lowry
at
surface
were
to
order
consisting
cells
assays
of
1),
fraction
described
in
localized
includes
modifications the
(2)
enzyme
freeze-thawed
RESULTS Effect
of
preparation
membranes. by
activity
a particulate
of
(2) with --et al. was determined
soluble
activity
addition,
g pellet
crude
for
specific
In x
cells/mm'
molds were maintained in association on buffered nutrient agar (1.1). agar (12) in slime mold saline was Grade IV) or a-MG (Sigma) was added where poured into sterile 35mm plastic Petri from spores on E. -coli at 22“C in the washed free of bacteria and 2~1 droplets applied to the hydrophobic agar with a Each cell density was prepared in the experiment was repeated four times.
corrected
the
surface. 27000
of the (hr) 1.25 x lo4
30 30
0 pg/ml
50
Con A + u-MG
completion aggregate x 103
23
A
a-MG
of first 7.5
of
the
in the
delay
which
1160
plated
A agar
aggregation
resulting is
the
on Con
related cells
is fruiting to
are
Con
plated.
and
observed bodies A concen-
BIOCHEMICAL
Vol. 53, No. 4, 1973
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Figure 1A: Response of D. discoideum to 10w3M cyclic AMP. Vegetative cells were deposited on hydrophobic agar (12) in 0.1~1 droplets at a density of 3.7 x 103 cells/mm2. Cyclic AMP (Sigma) in saline was applied with a microsyringe in a 0.1~1 drop O.l-0.2mm from the edge of each droplet of sensitized amoebae. Movement of amoebae outside the perimeter of the drop was considered a response to cyclic AMP (9). The rate of response was scored for cells incubated on various concentrations of Con A:
l v
, Oug/ml; -
,
A --a, Figure Procedure
1B:
200ug/ml
+ 2.5
Response and symbols
aggregation of The
tested
for
cyclic
AMP.
are at Con
A/ml
When to very
there
inhibition that lowering
ability cells
high
levels
similar is
inhibition
increasing cyclic
cyclic
AMP
agar
cyclic
controls Con
10m6
be
of was to
that
amoebae
to
performed of
Con
in Con
response,
the Con A mediated concentration should
A
which
A were supplied
(10V3M), 1A).
the
cyclic
50-200ug/ml
A concentration.
1161
by
exogenously
AMP
AMP.
mediated
presence
(Fig. the
M cyclic
possibility
containing
of
with
AMP reverses the
the
respond of
to
the (9)
in
on
, 400ugfml;
to
response test
to
to
shown
AMP, the
cells
their
exposed a rate
cyclic Konijn
aggregation-competent
lOOpg/ml;
a-MG.
been
with
tested.
)(--.-,
discoideum Fig. 1A.
has
action
AMP was
x lo-'M
of D. as-in
interfering
50pg/ml;
, 300Ug/ml;+**~.-
+a-.
chemotactic be
,
200uglml;
Since might
m***.*-
of they
Above the
respond 2OOug
degree This
inhibition. result
Con
of suggests
in
If so, reduction
A
Vol. 53, No. 4,1973
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Time After Plating (min)
of Con A on phosphodiesterase activity of Figure 2: Effect crude particle fraction; two separate experiments. Abscissa: minutes after plating cells. Ordinate: pmol of cyclic AMP broken down per min per mg of Assays for mPDE were performed at 19'C using about protein. fipg, particle protein in 110~1 of a reaction mixture that included 200 pmol [3H]-cyclic AMP (20.7 Cilmmol Amersham Searle) in .05M Tris, pH 7.4 containing .02M MgS04(2). Under these conditions the protein concentration was rate-limiting and 5'AMP production was proportional to the protein concentration. The reaction was stopped with TCA, and the mixture chromatographed on paper to separate cyclic AMP and 5'AMP which were counted in a Beckman LS250 scintillation counter (2). For these experiments cells were plated at a density of 1 x lo4 cells/mm2 on agar containing: 0, l , agar v,
3.75
only;
0
x 10m2M
,+,
a-MG;
3OOug n
,A,
Con
A/ml;
300ug/ml
Con
A + 3.75
x
10m2M a-MG.
of
the
This
cyclic predicted
AMP
the
300
to
and
response 4OOug
cyclic
Effect
AMP
Con
Con
II.
are A on
lower was
discoideum
is
a dose A/ml
the
response dependent
of
A on
prevented
both by
inhibitor
concentrations found. depressed cyclic
aggregation of
membrane-bound
Con cyclic
1162
all
Con cyclic
Con
1B)
while
AMP
response
and
of
10B6M
by
(Fig.
the
addition
of
Using
manner
inhibition
complete. of Con
a competitive of
at
in
of in
essentially The effects
(a-FIG),
response
shift
concentrations is
AMP
the
A at
response
a-methyl-D-glucoside
A binding
(3)
(Fig.lA,lB).
AMP phosphodiesterase.
A.
BIOCHEMICAL
Vol. 53, No. 4, 1973
Since Con
has
A interacts
lectin
been
its
both
cell
these
reactions
(mPDE)
has
gating
are
been
(2),
it
changing
with
the
cell
surface.
cells
and
Within
than
is
measured
for
in
specific
activity
not
appear
membranes of
Con
A on
reduced of
by
Con
of
specific
been
the
with
that
requirement is
for
al.
(15)
on
Con
from
shown
that
is
to
for the
synthesis
one
of
which
were
increase of
15%
same in the
this
of
period. activity enzyme
the
specificity
response. amoebae the
near
in
the
with dark.
result than
A
depressed
Particles
A and
mPDE activity the
is D.
Con
Con
Sussman
incorporation
in
This A
stimulation
actinomycin on
at
enzyme.
simultaneously
rather
which
level
which occurs experiments.
A-mediated
This
effect is
mPDE in
Con
incubated
This
the
is
this
of
2). The
that
induce
[3H]-uridine
only the
hour
cells
concentration
showed
A alone
particulate
does
internal
particles
for
D (125ng/ml) that
this
cells
3 hours
notion due
have
critical
difference
cells
(Table
indicating
during
treatment
a marked
surface. and
the
four
mPDE activity
into
cell
A,
is
control
begins
cells
prematurely
actinomycin
75% at
prepared D for
by and
about
A may
the
examined.
Con
cells, and
of aggregation, used in these
RNA synthesis
demonstrated
(300ng/ml) --et by
Con
intact
a-MG,
A for
suggests
interaction activity
When
incorporated
is
observed at the time under the conditions
aggrega-
was
2).
treatment
whole
normally 12 hours
aggre-
its
300ng/ml
A-treated
first
of Con
of
fraction
(Fig.
to
activity
treated
of
affect
through
on
of
after
cell
A may
particulate
Con
sugar
to
Con
plating the
both
Both
response
preparations
transported
haptenic
A determinants
phosphodiesterase
only
is
agglutination
Con
phosphodiesterase
enzyme
controls
hour
activity
A binding
The have
of
enzyme
then
the
in
between
the
and
after
surface
one
that
this
particulate
that
the
until
suggests
AMP
that
of
hour
exhibit
results).
chemotactic
possible
mPDE
manner.
A and of
this
a-MG.
Accordingly,
of
greater
Con
the
activity
same
presence
cyclic in
crude
activity
times
by
is
the
one-half
specific
the
unpublished
implicated
amoebae
in
that
that
amoebae
the
inhibited
by whole
indicating
membrane-bound
tion of
molds
organisms
possible
differentiating
(Gillette,
the
other
is
fluorescein-labeled
A,
surface
Because
it
slime
and
with Con
for
surfaces, on
vegetative
unlabeled the
cell
affect
staining
with
well-established
with
exerts
Indeed, surface on
it
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
actinomycin
of
cells
kept
supports
presence activation
of
the Con of
A an
Vol. 53, No. 4, 1973
Table
2.
BIOCHEMICAL
Effect whole
of Con cells
AND BIOPHYSICAL
A on
Phosphodiesterase
Phosphodiesterase cells plated 1% agar Time after plating (minutes) 2.5
RESEARCH COMMUNICATIONS
activity
activity
on
(pmol.mg-'
cells plated Con A/ml agar
16.8
27.0
60
31.7
289.0
60
29.7(plus
ci-MG x 10-2M)
3.75
on
70.0(plus 3.75
form
of
untreated
assayed
for
there
was
the
increase
appears
the
of
of
1OmM glutathione that
above
response its
to normal
stimulation
of
the
by
enzyme,
extracellular required
at
cyclic
AMP,
of
and
secreted
aggregation.
cyclic
nucleotide Then,
1164
control
by
Con
values. A is
identical
aggregation. with
the
mPDE activity This
delay
Also,
cells.
A interferes
increases the
cycliccompletely
such
aggregation.
explain the
of
Con
which
millimolar
40% of
Con A, values.
against
inhibitor,
induced
time
at
hydrolyzing
trigger
the
of
effective
Five
to
enzyme that
increase
were
(2)
mPDE of
activity
in
the presence above control also
Con A. a competitive
the
indicate
mPDE might levels
to
the that
Enzyme broken
preparations
are
particulate
produced
data
to
the
suggest
normally
The cells'
with
reduces
results
prior
aggregation
treated
activity
particulate
cultures plated on collected Sijrensen's lo5 or
experiments
AMP phosphodiesterase
monophosphate,
inhibits
to
cyclic
during
cells
3',5'-inosine
the
and
in
activity in activity specific
in
of
normally
These
cells
phosphodiesterase no
Furthermore,
enzyme.
whole
Inhibitors mPDE
3OOug
o-MG x 10-2M)
Late log phase amoebae were collected from shaking (13, 14), washed free of bacteria with saline, and 1% purified agar at 1 x lo4 cells/mm2. Cells were from plates at the times indicated and washed with phosphate, pH 6.0. For enzyme assays either 8.3 x 1.7 x 106 cells in 160~1 of phosphate buffer containing 150pmol of [3H]-cyclic AMP were incubated at 19'C. activity is expressed as number of pmol of substrate down per min per mg of protein.
which
.min-
4.7
30
inactive
of
premature
in
aggregation
AMP,
reduces
below the threshold if no other part
if
of
the
'1
BIOCHEMICAL
Vol. 53, No. 4,1973
aggregation time
machinery
would
level.
be The
in
lectin
appears than
activation
Since
cr-MG
significantly
by
lectin of
on the
to
Con
this
to
enhances
mPDE that on
are
of
its
of
have
shown
Phaseolus found
lymphocyte for of
that
level that
of
after
the
cyclic
to
examine
lowered
is
synthesis
cyclic
AMP
would to
linked
to
the
of
the
a
for
these
results
from in
(PHA),
elevate In
view Con
of
as
apparently
of
--et al. lectin
a (17)
have also inhibit
findings
A stimulated
activity
by
have
AMP levels these
have
lymphocytes They
cyclic
and
the
the
membrane-
binding Smith
controls.
to
activation
(16).
incubation
results
protein
program that result
of
our
a foreign
to
levels
which
development
surface
phosphodiesterase
A is This
turn
cell
PHA
surface. Con
with
thought
transformation.
cell
the
binding
by binding
lymphocyte
which
of
by
aggregation.
mold of
than
synthesis
molecules.
effect
whether
on
AMP
enzyme
development
temporal
is
the
membrane.
attachment
6 hours
compounds
value
in
slime
studies
the
of
effective
of
about
effect
phytohemagglutinin
of
increased the
to
vulgaris
a lower
be
lectins
of its
an
mPDE activity
the
on
involved
for
in
that
we believe
Activation of
time
the
of
normal
which
changes
as well
variety
the
period to
enzyme
increase
of
during
of
that
presence
moieties
into
Furthermore,
mitogens.
question
surface
in
the
brought
produces
standpoint
significance
the
machinery
amoebae
PDE.
likely
incorporation
the
the
very
at
cell
interest
surface bound
the
in
existing
concluded
a longer up
stimulation
produced a factor
metabolic
From
is
A, build
a true
be
RESEARCH COMMUNICATIONS
to
previously reduces
can
activity
receptor
and
to
raise
such
internal enzyme
due
it
Con
AMP
mPDE activity
carbohydrate
a factor
by
cyclic
of
A,
results
mimicking imply
be
enzyme
lectin These
affected for
increase
rather produced
is
required
AND BIOPHYSICAL
it
would
lymphocytes a possible required
cause for
activation. ACKNOWLEDGEMENT of
This Canada
work with
was supported a predoctoral
by the National fellowship to
Research M.U.G.
Council
REFERENCES 1. 2.
Barkley, Malchow, Eur.
D. S., Science, D., Nagele, B., 2, J. Biochem.
165, 1133 (1969). Schwartz, H., and 136 (1972).
1165
Gerisch,
G.,
Vol. 53, No. 4,1973
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
BIOCHEMICAL
AND BIOPHYSICAL
Agrawal,
RESEARCH COMMUNICATIONS
B. B, L., and Goldstein, I. J., Biochem. Biophys. Acta 147, 262 (1967). Nicholson, G. L. and Singer, S. J., Proc. Nat. Acad. Sci. USA, 942 (1971). 68, Moscona, A. A., Science, 3, 905, (1971). Weiser, M. M., Science, 177, 525 (1972). Inbar, M. and Sachs, L., Proc. Nat. Acad. Sci. USA, 63, 1418, (1969). Burger, M. M. and Martin, G. S., Nature New Biology 237, 9, (1972). Konijn, T. M., Experientia, 15, 367 (1970). Lowry, 0. H., Rosebrough, N. J., Parr, A. L., Randall, R. J., J. Biol. Chem. 193, 265 (1951). Bonner, J. T., The Cellular Slime Molds, Princeton, 1967. Konijn, T. M. and Raper, K. B., Develop. Biol. 3, 725 (1961). Gerisch, G., Naturwiss. 46, 654 (1959). Hohl, H. R., and Raper, K. B., J. Bacterial. 85, 191 (1963). Sussman, M., Loomis, W. F., Ashworth, J. M., and Sussman, R. R., Biochem. Biophys. Res. Comm. 26, 353 (1967). Greaves, M., and Janossy, G., Transplant. Rev. 11, 87 (1972). Smith, J., Steiner, A., and Parker, C., J. Clin. Invest. 50, 442 (1971).
1166
Vol. 53, No. 4, 1973
EFFECT
OF CONCANAVALIN
PREMATURE
AND BIOPHYSICAL
A ON CELLULAR
APPEARANCE
RESEARCH COMMUNICATIONS
SLIME
OF MEMBRANE-BOUND
MOLD
CYCLIC
DEVELOPMENT:
AMP
PHOSPHODIESTERASE Martha Ulbrick Gillette and M. F. Filosa, Zoology Department, Scarborough College, University of Toronto, West Hill, Ontario, Canada. Received
June
29,
1973
Concanavalin A delays aggregation of slime mold amoebae, Summary: apparently by interfering with the cells' response to the chemotactic agent, cyclic AMP. Concanavalin A also induces the premature appearance in non-aggregating cells of a membrane-bound cyclic AMP phosphodiesterase normally found only at the time of aggregation. The appearance of this enzyme is not due to activation of an inactive form of the enzyme. After cells
a period
of
of
Dictyostelium
cellular
aggregates
body.
The
growth
as
discoideum
move
together
each
attraction
of
of
the
dispersed
Malchow
(2) have demonstrated --et al. of a membrane-bound cyclic dramatically
tion
and
returns
These
investigators
pates
both
that
in
very
Concanavalin
A (Con
of
A), in
For washed
all free
(Difco),
and
and
assayed
activity bound released
at
enzyme from
22'C
washed
whole
to cells
exposed the
at cells.
Copyright @ 19 73 by Academic Press, Ix. All rights of reproductiorl irr any form reserved.
aggregation the
amoebae
discoideum
(mPDE)
preceding
after
aggrega-
aggregation.
AMP and lectin
mold
partici-
the
chemotactic
here
a study
known
surfaces
(1).
the
phosphodiesterase
a plant
coli
incubated according
of
this
studies
Escherichia
AMP responsiveness,
a fruiting
(3-8),
to
showing
bind
to
affects
the
development.
AND METHODS
developmental of
D. just
cyclic
cell
slime
MATERIALS
by
We present
of
aggregation
into
levels
aggregation.
determinants
into
cells in
multi-
AMP phosphodiesterase
low of
amoebae, into
secreted
activity
that
detection cell
AMP
in
suggest
during
process
to
the
carbohydrate
cyclic
increases
then
response
to
solitary
differentiates
is
which
response
which
centres presence
in
vegetative
cells Konijn
B, in
the
the
cell
1159
To
Cyclic
includes and
cell
test by
were agar
for
cyclic
aging
at
4'C
AMP phosphodiesterase
which
surface whole
phase amoebae on 1% purified
sensitized
(9).
examined
log
light.
were
was
Total
late plated
soluble
both
membrane-
enzyme
phosphodiesterase
Vol. 53, No. 4, 1973
BIOCHEMICAL
Table
Con
1.
Effect of Dictyostelium
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A in delaying aggregation discoideum amoebae. Time
2.5 x 103 Con
23
22
100
27 28
24 27
200 300
42 38
30 32
30 31
400
53
34
33
21
20
20.5
21
21
22
5 x 1O-2
M
200 i.lg/ml + 2.5 x 1O-2
M
Stock cultures of slime with Escherichia coli B Hydrophobic 1% purified prepared; Con A (Sigma, necessary and the mixture dishes. Cells were grown dark. The amoebae were of cell suspension were 10~1 Hamilton microsyringe. duplicate or triplicate;
activity
was
determine
This
twice
particulate
internal
All
enzyme
allowed (Table
method
of
A on
slime
mold
When
late
log
phase
amoebae
is normal. tration and
develop, although The
extent
the
density
was
of
prepared as well
according the
the (2).
to
as
Malchow Protein
legends.
(10).
are
a striking
delay of
at
cell
development
morphology of
the
AND DISCUSSION
Con to
in
Lowry
at
surface
were
to
order
consisting
cells
assays
of
1),
fraction
described
in
localized
includes
modifications the
(2)
enzyme
freeze-thawed
RESULTS Effect
of
preparation
membranes. by
activity
a particulate
of
(2) with --et al. was determined
soluble
activity
addition,
g pellet
crude
for
specific
In x
cells/mm'
molds were maintained in association on buffered nutrient agar (1.1). agar (12) in slime mold saline was Grade IV) or a-MG (Sigma) was added where poured into sterile 35mm plastic Petri from spores on E. -coli at 22“C in the washed free of bacteria and 2~1 droplets applied to the hydrophobic agar with a Each cell density was prepared in the experiment was repeated four times.
corrected
the
surface. 27000
of the (hr) 1.25 x lo4
30 30
0 pg/ml
50
Con A + u-MG
completion aggregate x 103
23
A
a-MG
of first 7.5
of
the
in the
delay
which
1160
plated
A agar
aggregation
resulting is
the
on Con
related cells
is fruiting to
are
Con
plated.
and
observed bodies A concen-
BIOCHEMICAL
Vol. 53, No. 4, 1973
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Figure 1A: Response of D. discoideum to 10w3M cyclic AMP. Vegetative cells were deposited on hydrophobic agar (12) in 0.1~1 droplets at a density of 3.7 x 103 cells/mm2. Cyclic AMP (Sigma) in saline was applied with a microsyringe in a 0.1~1 drop O.l-0.2mm from the edge of each droplet of sensitized amoebae. Movement of amoebae outside the perimeter of the drop was considered a response to cyclic AMP (9). The rate of response was scored for cells incubated on various concentrations of Con A:
l v
, Oug/ml; -
,
A --a, Figure Procedure
1B:
200ug/ml
+ 2.5
Response and symbols
aggregation of The
tested
for
cyclic
AMP.
are at Con
A/ml
When to very
there
inhibition that lowering
ability cells
high
levels
similar is
inhibition
increasing cyclic
cyclic
AMP
agar
cyclic
controls Con
10m6
be
of was to
that
amoebae
to
performed of
Con
in Con
response,
the Con A mediated concentration should
A
which
A were supplied
(10V3M), 1A).
the
cyclic
50-200ug/ml
A concentration.
1161
by
exogenously
AMP
AMP.
mediated
presence
(Fig. the
M cyclic
possibility
containing
of
with
AMP reverses the
the
respond of
to
the (9)
in
on
, 400ugfml;
to
response test
to
to
shown
AMP, the
cells
their
exposed a rate
cyclic Konijn
aggregation-competent
lOOpg/ml;
a-MG.
been
with
tested.
)(--.-,
discoideum Fig. 1A.
has
action
AMP was
x lo-'M
of D. as-in
interfering
50pg/ml;
, 300Ug/ml;+**~.-
+a-.
chemotactic be
,
200uglml;
Since might
m***.*-
of they
Above the
respond 2OOug
degree This
inhibition. result
Con
of suggests
in
If so, reduction
A
Vol. 53, No. 4,1973
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Time After Plating (min)
of Con A on phosphodiesterase activity of Figure 2: Effect crude particle fraction; two separate experiments. Abscissa: minutes after plating cells. Ordinate: pmol of cyclic AMP broken down per min per mg of Assays for mPDE were performed at 19'C using about protein. fipg, particle protein in 110~1 of a reaction mixture that included 200 pmol [3H]-cyclic AMP (20.7 Cilmmol Amersham Searle) in .05M Tris, pH 7.4 containing .02M MgS04(2). Under these conditions the protein concentration was rate-limiting and 5'AMP production was proportional to the protein concentration. The reaction was stopped with TCA, and the mixture chromatographed on paper to separate cyclic AMP and 5'AMP which were counted in a Beckman LS250 scintillation counter (2). For these experiments cells were plated at a density of 1 x lo4 cells/mm2 on agar containing: 0, l , agar v,
3.75
only;
0
x 10m2M
,+,
a-MG;
3OOug n
,A,
Con
A/ml;
300ug/ml
Con
A + 3.75
x
10m2M a-MG.
of
the
This
cyclic predicted
AMP
the
300
to
and
response 4OOug
cyclic
Effect
AMP
Con
Con
II.
are A on
lower was
discoideum
is
a dose A/ml
the
response dependent
of
A on
prevented
both by
inhibitor
concentrations found. depressed cyclic
aggregation of
membrane-bound
Con cyclic
1162
all
Con cyclic
Con
1B)
while
AMP
response
and
of
10B6M
by
(Fig.
the
addition
of
Using
manner
inhibition
complete. of Con
a competitive of
at
in
of in
essentially The effects
(a-FIG),
response
shift
concentrations is
AMP
the
A at
response
a-methyl-D-glucoside
A binding
(3)
(Fig.lA,lB).
AMP phosphodiesterase.
A.
BIOCHEMICAL
Vol. 53, No. 4, 1973
Since Con
has
A interacts
lectin
been
its
both
cell
these
reactions
(mPDE)
has
gating
are
been
(2),
it
changing
with
the
cell
surface.
cells
and
Within
than
is
measured
for
in
specific
activity
not
appear
membranes of
Con
A on
reduced of
by
Con
of
specific
been
the
with
that
requirement is
for
al.
(15)
on
Con
from
shown
that
is
to
for the
synthesis
one
of
which
were
increase of
15%
same in the
this
of
period. activity enzyme
the
specificity
response. amoebae the
near
in
the
with dark.
result than
A
depressed
Particles
A and
mPDE activity the
is D.
Con
Con
Sussman
incorporation
in
This A
stimulation
actinomycin on
at
enzyme.
simultaneously
rather
which
level
which occurs experiments.
A-mediated
This
effect is
mPDE in
Con
incubated
This
the
is
this
of
2). The
that
induce
[3H]-uridine
only the
hour
cells
concentration
showed
A alone
particulate
does
internal
particles
for
D (125ng/ml) that
this
cells
3 hours
notion due
have
critical
difference
cells
(Table
indicating
during
treatment
a marked
surface. and
the
four
mPDE activity
into
cell
A,
is
control
begins
cells
prematurely
actinomycin
75% at
prepared D for
by and
about
A may
the
examined.
Con
cells, and
of aggregation, used in these
RNA synthesis
demonstrated
(300ng/ml) --et by
Con
intact
a-MG,
A for
suggests
interaction activity
When
incorporated
is
observed at the time under the conditions
aggrega-
was
2).
treatment
whole
normally 12 hours
aggre-
its
300ng/ml
A-treated
first
of Con
of
fraction
(Fig.
to
activity
treated
of
affect
through
on
of
after
cell
A may
particulate
Con
sugar
to
Con
plating the
both
Both
response
preparations
transported
haptenic
A determinants
phosphodiesterase
only
is
agglutination
Con
phosphodiesterase
enzyme
controls
hour
activity
A binding
The have
of
enzyme
then
the
in
between
the
and
after
surface
one
that
this
particulate
that
the
until
suggests
AMP
that
of
hour
exhibit
results).
chemotactic
possible
mPDE
manner.
A and of
this
a-MG.
Accordingly,
of
greater
Con
the
activity
same
presence
cyclic in
crude
activity
times
by
is
the
one-half
specific
the
unpublished
implicated
amoebae
in
that
that
amoebae
the
inhibited
by whole
indicating
membrane-bound
tion of
molds
organisms
possible
differentiating
(Gillette,
the
other
is
fluorescein-labeled
A,
surface
Because
it
slime
and
with Con
for
surfaces, on
vegetative
unlabeled the
cell
affect
staining
with
well-established
with
exerts
Indeed, surface on
it
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
actinomycin
of
cells
kept
supports
presence activation
of
the Con of
A an
Vol. 53, No. 4, 1973
Table
2.
BIOCHEMICAL
Effect whole
of Con cells
AND BIOPHYSICAL
A on
Phosphodiesterase
Phosphodiesterase cells plated 1% agar Time after plating (minutes) 2.5
RESEARCH COMMUNICATIONS
activity
activity
on
(pmol.mg-'
cells plated Con A/ml agar
16.8
27.0
60
31.7
289.0
60
29.7(plus
ci-MG x 10-2M)
3.75
on
70.0(plus 3.75
form
of
untreated
assayed
for
there
was
the
increase
appears
the
of
of
1OmM glutathione that
above
response its
to normal
stimulation
of
the
by
enzyme,
extracellular required
at
cyclic
AMP,
of
and
secreted
aggregation.
cyclic
nucleotide Then,
1164
control
by
Con
values. A is
identical
aggregation. with
the
mPDE activity This
delay
Also,
cells.
A interferes
increases the
cycliccompletely
such
aggregation.
explain the
of
Con
which
millimolar
40% of
Con A, values.
against
inhibitor,
induced
time
at
hydrolyzing
trigger
the
of
effective
Five
to
enzyme that
increase
were
(2)
mPDE of
activity
in
the presence above control also
Con A. a competitive
the
indicate
mPDE might levels
to
the that
Enzyme broken
preparations
are
particulate
produced
data
to
the
suggest
normally
The cells'
with
reduces
results
prior
aggregation
treated
activity
particulate
cultures plated on collected Sijrensen's lo5 or
experiments
AMP phosphodiesterase
monophosphate,
inhibits
to
cyclic
during
cells
3',5'-inosine
the
and
in
activity in activity specific
in
of
normally
These
cells
phosphodiesterase no
Furthermore,
enzyme.
whole
Inhibitors mPDE
3OOug
o-MG x 10-2M)
Late log phase amoebae were collected from shaking (13, 14), washed free of bacteria with saline, and 1% purified agar at 1 x lo4 cells/mm2. Cells were from plates at the times indicated and washed with phosphate, pH 6.0. For enzyme assays either 8.3 x 1.7 x 106 cells in 160~1 of phosphate buffer containing 150pmol of [3H]-cyclic AMP were incubated at 19'C. activity is expressed as number of pmol of substrate down per min per mg of protein.
which
.min-
4.7
30
inactive
of
premature
in
aggregation
AMP,
reduces
below the threshold if no other part
if
of
the
'1
BIOCHEMICAL
Vol. 53, No. 4,1973
aggregation time
machinery
would
level.
be The
in
lectin
appears than
activation
Since
cr-MG
significantly
by
lectin of
on the
to
Con
this
to
enhances
mPDE that on
are
of
its
of
have
shown
Phaseolus found
lymphocyte for of
that
level that
of
after
the
cyclic
to
examine
lowered
is
synthesis
cyclic
AMP
would to
linked
to
the
of
the
a
for
these
results
from in
(PHA),
elevate In
view Con
of
as
apparently
of
--et al. lectin
a (17)
have also inhibit
findings
A stimulated
activity
by
have
AMP levels these
have
lymphocytes They
cyclic
and
the
the
membrane-
binding Smith
controls.
to
activation
(16).
incubation
results
protein
program that result
of
our
a foreign
to
levels
which
development
surface
phosphodiesterase
A is This
turn
cell
PHA
surface. Con
with
thought
transformation.
cell
the
binding
by binding
lymphocyte
which
of
by
aggregation.
mold of
than
synthesis
molecules.
effect
whether
on
AMP
enzyme
development
temporal
is
the
membrane.
attachment
6 hours
compounds
value
in
slime
studies
the
of
effective
of
about
effect
phytohemagglutinin
of
increased the
to
vulgaris
a lower
be
lectins
of its
an
mPDE activity
the
on
involved
for
in
that
we believe
Activation of
time
the
of
normal
which
changes
as well
variety
the
period to
enzyme
increase
of
during
of
that
presence
moieties
into
Furthermore,
mitogens.
question
surface
in
the
brought
produces
standpoint
significance
the
machinery
amoebae
PDE.
likely
incorporation
the
the
very
at
cell
interest
surface bound
the
in
existing
concluded
a longer up
stimulation
produced a factor
metabolic
From
is
A, build
a true
be
RESEARCH COMMUNICATIONS
to
previously reduces
can
activity
receptor
and
to
raise
such
internal enzyme
due
it
Con
AMP
mPDE activity
carbohydrate
a factor
by
cyclic
of
A,
results
mimicking imply
be
enzyme
lectin These
affected for
increase
rather produced
is
required
AND BIOPHYSICAL
it
would
lymphocytes a possible required
cause for
activation. ACKNOWLEDGEMENT of
This Canada
work with
was supported a predoctoral
by the National fellowship to
Research M.U.G.
Council
REFERENCES 1. 2.
Barkley, Malchow, Eur.
D. S., Science, D., Nagele, B., 2, J. Biochem.
165, 1133 (1969). Schwartz, H., and 136 (1972).
1165
Gerisch,
G.,
Vol. 53, No. 4,1973
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
BIOCHEMICAL
AND BIOPHYSICAL
Agrawal,
RESEARCH COMMUNICATIONS
B. B, L., and Goldstein, I. J., Biochem. Biophys. Acta 147, 262 (1967). Nicholson, G. L. and Singer, S. J., Proc. Nat. Acad. Sci. USA, 942 (1971). 68, Moscona, A. A., Science, 3, 905, (1971). Weiser, M. M., Science, 177, 525 (1972). Inbar, M. and Sachs, L., Proc. Nat. Acad. Sci. USA, 63, 1418, (1969). Burger, M. M. and Martin, G. S., Nature New Biology 237, 9, (1972). Konijn, T. M., Experientia, 15, 367 (1970). Lowry, 0. H., Rosebrough, N. J., Parr, A. L., Randall, R. J., J. Biol. Chem. 193, 265 (1951). Bonner, J. T., The Cellular Slime Molds, Princeton, 1967. Konijn, T. M. and Raper, K. B., Develop. Biol. 3, 725 (1961). Gerisch, G., Naturwiss. 46, 654 (1959). Hohl, H. R., and Raper, K. B., J. Bacterial. 85, 191 (1963). Sussman, M., Loomis, W. F., Ashworth, J. M., and Sussman, R. R., Biochem. Biophys. Res. Comm. 26, 353 (1967). Greaves, M., and Janossy, G., Transplant. Rev. 11, 87 (1972). Smith, J., Steiner, A., and Parker, C., J. Clin. Invest. 50, 442 (1971).
1166
