chemotactic movement toward a source of cAMP pulses but also ..... a relatively high concentration (0.5 M) of KCl was used to ..... of Phosphodiesterase-Rab bit.
J. Biochem.
Purification
and Characterization
Phosphodiesterase
of the Extracellular
of Dictyostelium
Toshiaki
Received
for publication,
phosphodiesterase
was
from
discoideum, I
and
enzyme
molecular
on
of
The
SDS
During
about
Km
in
growth
developmental
phase,
suggesting
enzyme
of the
of the
an two
pH
types
the
two
were
of
but
that
the
the
type ‡U
ratio
had
type
The an
II
similar
(2-4 ƒÊM).
however,
enzyme
were
accumulated
of activity
was
of
about
both
55,000 and 57,000, constituents.
enzyme
type
apparent
weight
analyses,
weights of of common
types
amounts,
found.
the
3.1.4.17]
Dictyostelium
and
molecular
gel electrophoresis
of
were 8.5
contrast,
apparent
[EC
culture
enzyme
at In
had
with molecular forms composed equal
phase
present
types
indicating in
culture
predominantly
of the
two
forms
in is under
control.
Rabbit I as
type ‡U)
daltons.
values
phase,
3•Œ:5•Œ-monophosphate
aggregation
DEAE-Sephacel
and
roughly
aggregation
and
67,000
polyacrylamide
the
University,
adenosine of
on
DEAE-Sephacel
supernatant
type
for
(type ‡T
produced the same bands that they are two different
the
of Science, Kyoto
absorbed
daltons.
Upon
and Ikuo TAKEUCHI
supernatant
types
not
weight
adsorbed 120,000
the
two
was
AMP
June 21, 1982
Extracellular purified
(1983)
discoideum1
NOCE, Koji OKAMOTO,
Department of Botany, Faculty Sakyo-ku, Kyoto, Kyoto 606
Cyclic
93, 3745
antiserum well
possess
as
prepared
membrane-bound
some
common
against
purified
enzyme,
type ‡U
indicating
enzyme that
cross-reacted
the
three
classes
with of
the
sequence.
Cells of the cellular slime mold, Dictyostelium discoideum, which grow as singular amoebae, form
of aggregation in that a cell not only exhibits chemotactic movement toward a source of cAMP
multicellular
pulses but transmitting
structures
upon
exhaustion
of a food
source and eventually differentiate into spores and stalk cells of a fruiting body, showing a series of distinct
morphogenetic
Cyclic
AMP
plays
(1, 2). Moreover, cAMP has been shown to promote the differentiation of both preaggregative and postaggregative cells (3-5). There is also accumulating evidence (6-9) to suggest that this organism requires cAMP at all stages of its devel opment.
changes. a central
role
in the process
1 This work was supported in part by Grants-in-Aid (Nos. 444003, 56108008) for Scientific Research from the Ministry of Education, Science and Culture of Japan. Abbreviations: Con A, concanavalin A; DTT, di thiothreitol.
Vol.
93, No.
1, 1983
also itself emits a pulse of cAMP, a chemotactic signal to the next cell
the
One of the important level of cAMP is
enzyme
37
produced
by
D.
elements which phosphodiesterase.
regulate The
discoideum
may
cells
be
38
T. NOCE,
classified into three groups depending on the loca tion: extracellular, membrane-bound and intra cellular soluble. Previous studies (4, 10) showed that the syntheses of extracellular and membranebound enzymes are regulated differently during the differentiation and dedifferentiation processes. They are also known to differ in properties such as Km value, molecular weight and sensitivity to a specific macromolecular inhibitor (11, 12) which is also secreted by D. discoideum cells (13). More-
with
by
centrifugation
over, mutants have been isolated (14) in which one type of the enzyme is produced in an abnor mally low or high amount. The extracellular enzyme itself has been further resolved into multiple forms with distinct properties (15-17), although a part of the multiplicity of the enzyme appears to be explainable by its association with a macromolecular inhibitor (18).
For
purification,
At clear
the
present
whether
time,
all
the
however,
different
groups
phodiesterase and their subfractions one gene and are then modified different
forms
scribed
or
whether
on different
genes
it remains
each with
of
un phos
originate from to give rise to of them
different
is tran
regulation.
As an initial step to answer these questions, we aimed to purify and characterize the extracellular phosphodiesterases. We found two forms of the enzyme even under conditions where protein aggregation was avoided. Although their activ ities were differently controlled during develop ment, SDS gel electrophoresis and immunological analyses revealed that these forms contain common components. The present paper describes the results of these experiments.
20 mM
pended
AND
same
cells/ml.
The
cell
for
h
10-12
to
allow
and
Harvesting-Wild-type
coideum
NC-4
was
reported
here.
Cells
Escherichia rpm)
at
coli 21•Ž.
required,
200
ml)
was
diluted
2%
polypeptone
Under 4-6
these
used
B/r
in
When ml
of
in
were
all
the
grown
in
shaken
cultures
preculture 8
liters
and
bubbled
conditions,
the
(5-10 of
2% through
doubling
dis
experiments culture
suspension
largescale
the
into
D.
x 106 glycerol
21•Ž
with to
6.0),
then
a density
of
was shaking
or
develop
to
resus
1-2
then
x 107
incubated
air
an
Purification-Crude
by
natant
(NH4)3SO4
of
(19)
the
was
bubbling
aggregation
aggregation
the
instead
step
and
of
was
loaded
with
and
washed
(10 mM the
the
final
KCl,
was
buffer
0.2 mM
of 2 ml/min.
The
most
active
brane
on
to
The
concentrate
was
cm)
of Sephacryl
S-200
0.5
M KCl,
a flow
rate
As ConA
the
of
final
of
step
and gel
(150
with
gel.
at
were
20 mM
cells/
ml
8.5,
eluted
the
same
of
purification,
ml)
which
least
pH 50
ml
Tris-HCl
Gerisch
(20).
bed
and
Tris-HCl
air.
covered
time
was
a yield
(pH in
the
of more
buffer
had
of 0.3 7.5)
been
7.5).
were
applied
10 mg
of
ConA/
pre-equilibrated
The
column
was
M D-galactose, and
then
enzyme
in washed
1 M NaCl, eluted
activity
1% ƒ¿-methylmannoside than
the frac
5 ml)
with
I M NaCl, The
utilized to
volume:
(Pharmacia,
of 1% ƒ¿-methylmannoside,
with
been 0.2 mM
Suitable
procedure (gel
7.5.
(pH
we
according
filtration
4B
Tris-HCl,
had
pH
a column
ConA-Sepharose
20 mM
to
superfine
which
chromatography
the
top
and
applied
M Tris-HCl with
a
5 ml/h.
Eitle
from
40-105 ƒÊm)
0.1 and
of
affinity
the
size in
M
at
filter-mem
x 90 wet
0.5
pooled
ml.
preequilibrated
7.5,
PM-30
(2.6
(Pharmacia,
DTT),
volume)
were
AMICON
a DTT-
gradient
pH
total
of 40-
of starting
0.2 mM
M Tris-HCl ml
of
ml
a linear
fractions
an
ml
8.5,
of
size
3-5
a column
DTT,
20
with
(500
rate
concentrated
at
(0.1
DTT)
instead x 25 cm)
100
pH
10 mM
activation
particle
with
eluted
the
used
about
Tris-HCl
enzyme
DEAE-
that
(2.3 wet
Porter
the
in
column
min).
and
to
used
(Pharmacia,
5-10
except
was
A
sample
buffer
was
DEAE-Sephacel
150 ƒÊm) treated
up
super-
(obtained
for Chassy
step,
5 mM
DEAE-Sephacel
flow
of
pre
the
culture
rpm
followed
DEAE-cellulose.
to
3,000 method
was
from
phase
at
chromatography
DTT
then
enzyme
precipitation
essentially
cellulose
packed
When the cells reached a density of 4-6 x 108 cells/ml (late exponential or early stationary phase), they were harvested and washed free of bacteria
at
suspension
cells
Enzyme pared
with
h.
(pH
buffer
I. TAKEUCHI
phase.
to Growth
at
the
and
phosphate
the
tions
METHODS
Na-K
in
method
MATERIALS
K. OKAMOTO,
25
20 mM was
fraction
rein
85%.
Assay of Phosphodiesterase-The activity was assayed with [3H]cAMP (45 Ci/mmol, Radiochem ical Centre, Amersham) as described previously (10).
The reaction
mixture
contained
8.88 pmol J. Biochem.
EXTRACELLULAR of
[3H]cAMP
unit
of
in
a
activity
total
was
hydrolyzed 1 under
PHOSPHODIESTERASE
nmol
the
of
conditions
Gel
out
acrylamide with
0.1%
racetic For
each
and of were
was
8.5,
4•Ž
at
25•Ž
(18). In our experiments, a 5-10 fold increase activity was achieved by this treatment.
The
blue
R-250
destained
mashed
(21)
7
8.5,
stained
One
in
50%
trichlo
tion
7%
acetic
in
the
1 mM
in 400
with
were
in
into
When jected
gels
activity
sliced
and
incubated
the
activity
assay.
acid.
gel,
segments
Id of 20 mM
DTT
before
gel conditions
Davis
9.5.
enzyme
0.2 mM
I h at
pH
then
gels slice
pH
at
detection
stained
min
that
Electrophoresis-Disc
to
Coomassie
acid
per
the
at
the
to two
peaks
during
(peak
former
was
even
when
applied
that
the
of
high
to
gel and a 10% acrylamide running gel. The gels were stained by the silver staining method de scribed by Switzer et al. (23).
was
type
Freund's
(Difco-Bacto)
the
adjuvant
back
weeks,
of and
rabbits
II
emulsified
three
blood
was
in
was
times
drawn
at
7 days
after
of the
2
third
Immunoprecipitation CL-4B
protein
(Pharmacia,
A/ml-gel)
immune 0.1 M A
25 ƒÊl
pH
aliquot
was
of
A-beads
with
added
maining (3,000
in rpm,
the
test
sample
pl
of diluted for
suspension whole
rpm).
was
2 h
was The
after
for
used
1 mg/ml buffer
at mg
from
no
even
when
it
activity
tivation
was
in
the
treated indicate
into
peaks
I is not
of one
enzyme,
was
two
different
entities,
BSA.
and
type ‡U,
respectively.
in
as
other
a broad
hand,
the
due
which
to
peak en
fraction, for
the
in
in band
crude
DTT that
flow-
only
flowthrough
and ‡U
but
the
appeared
with
results
was profile
chromatography.
DTT-activated
These
chromatography
elution
enzyme the
crude
DTT
a second
resolution
of
DEAE-Sephacel
an
incomplete
reflects
the
are
designated
ac
existence type
of I
solu
serum.
incubated enzyme
2 mg
adsorbent
in
(40
supernatant
2 min)
an
gel;
solution
mt KCl,
mixture
the (26
as
buffer 0.5
25
the
and
rotation
the
with
of
protein
7.5,
of
mixed
incubation
was
utilized The
ml
On
derived
gave
activity
A-Sepha
I g=3.5
was
complexes. Tris-HCl
tion
Assay-Protein
peak ‡U
by due
If
observed
activity
shown).
zyme
time.
injection.
rose
not
‡Ufraction
into
intervals
of the
caused
by
different
was
and
DEAE-
probably
fraction.
DEAE-Sephacel
and
column,
not
was
activated
a
activity
fraction,
vicinity
(data
complete
injected
on no
through the
purified
obtained
Essentially
this
been
column,
on
was
1).
gradient
a second
but
of
not
salt
retained to
column
value had
the
not
pH
frac
buffer
by
flowthough
the
pI
applied
phosphodiesterase
the
over-charging
sub
(Fig.
starting
obtained
in
at
flow-through
the
was
The
indicating
was
observed
the
with
II)
Sephacel
which
the
in
washing
other
were
elution.
to
of
activity
un-
least
enzyme chromatography
appeared
enzyme
200 ƒÊg
of
(peak ‡T)
SDS slab gel electrophoresis was performed as described by Laemmli (22) with a 4.5% stacking
Immunizations-About
DTT-activated
DEAE-Sephacel
and
Tris-HCl;
for
39
- diesterase, the inhibitor was inactivated and dis sociated from the enzyme by treatment with DTT
One
amount
non-denaturing
according
gels
20 pl.
the
cAMP
under
carried
of
as
employed.
Polyacrylamide electrophoresis was
volume
defined
OF D. discoideum
After
22•Ž,
50 ƒÊl
beads/ml) for activity
1 h re
centrifugation
assayed.
Protein Determination-Protein was determined by the method of Lowry et al. (24) with BSA as a standard. RESULTS
Purification of Extracellular cAMP Phospho diesterases-The culture supernatant was collected after 10-12 h of starvation, when relatively tight agglomerates had formed. Since the supernatant contained both free and inhibitor-bound phospho
Vol. 93, No.
1, 1983
Fig. 1. Chromatography on DEAE-Sephacel of crude extracellular phosphodiesterase obtained after DTTactivation. About 100 mg protein was applied to the column. Elution was carried out as described in "MA TERIALS AND METHODS." The gradient elution was started at the fraction No. 9. Fractions of 14ml were collected and assayed for enzyme activity.
40
T. NOCE,
This view was substantiated by the following experiment, in which the two fractions were sub
from
jected to Sephacryl S-200 column chromatography. For this experiment, a solution which contained a relatively high concentration (0.5 M) of KCl was used to avoid possible protein aggregation. As shown in Fig. 2, the type I and II enzymes each displayed an essentially single peak at positions corresponding to ca. 67,000 and 120,000 daltons, respectively.
gave
The
gel
affinity as
filtration
shown
in
Table
600-900-fold mately
for
500 ƒÊg
tions from
step
chromatography.
of
the
4 liters
1,
in
both each
type
an
types (as
was
followed
This
procedure
overall of
protein)
of
culture
supernatant.
of the
linear values
regions
gels. double of 4 and
of
Both
the
the
reciprocal 2 ƒÊM,
1. TAKEUCHI
non-denaturing type
I and plots
poly II
(Fig.
enzymes 4)
with
respectively.
ConA
purification
enzymes
Km
peak
and
resulted,
enzyme.
I and ‡U
Characterization
by
the
acrylamide
K. OKAMOTO,
of
Approxi the
final
was
frac
obtained
of Two Extracellular
Phos
phodiesterases-The purity of the two types of phosphodiesterase after ConA affinity chromatog raphy was examined by the use of polyacrylamide gel electrophoresis under non-denaturing condi tions. Two gels were run simultaneously, one being stained with Coomassie blue R-250, and the other sliced and assayed for activity assay. In the case of the type I enzyme, Coomassie blue staining gave one major band (R f 0.20) plus diffuse multiple bands near the top of the gel (Fig. 3), and the activity coincided with the major band. On the other hand, the type II enzyme displayed a single band (R f 0.25) both by Coomassie blue staining and activity assay (Fig. 3). In both cases, more than 80% of the activity applied to the gels was recovered in the major bands. Km
values
for
cAMP
the two
phosphodiesterases
TABLE
I.
Purification
were
determined
which
of extracellular
had
been
with eluted
Fig. 2. Elution profiles of two types of extracellular phosphodiesterase on Sephacryl S-200. Samples con taining about 10 mg protein (3 ml each) were applied to the column. The effluent was monitored at 280 nm and 3 ml fractions were collected and assayed. A, phos phodiesterase type I; B, type II. The positions of marker BSA (m.w. 6.7 x 104)and aldolase (m.w. 15.8 x 104) are indicated by arrows.
phosphodiesterase.
J. Biochem.
EXTRACELLULAR
PHOSPHODIESTERASE
OF
D. discoideum
41
Enzymes
eluted
acrylamide
gels
acrylamide
gel
molecular
weights
in
Fig.
5, the
molecular and
the
naturing)
of
affinity tein
gel
phosphodiesterases
chromatography. per
Gels
tube
were
arrows
(0.5 x stained
indicate
enzyme,
which
ALS
AND
electrophoresis
5 cm) with
obtained
A
sample
was
used
assayed
of the
for
1,
both
common
polypeptides
some
faint
bands
ConA
band
in
pro
with
the
the
enzyme
with
the
gel
enzyme
that
the
peptides, adjacent
original
This
contain
two
weights
is
not
I enzyme
con
since to
the
a
the
gel.
gel filtration,
of
polypeptide
non-denaturing
of
with
activity
were
results
bands
type
contaminating observed
with 57,000
55,000.
every
has
seen
bands
molecular
Whether
It is possible
tains
of
be
and
two and
the
can
three
57,000
types
57,000.
determine
55,000, gave
of
SDS-poly
As
gave
enzyme
that
on
to
subunits.
53,000,
poly
by
few
major
Together
present
results
electrophoresis.
blue
R-250.
peak
as described
METHODS."
by
of 50-100 ƒÊg
Coomassie
the positions was
(non-de
of
indicates
known.
Polyacrylamide
the
I enzyme
type ‡U
separated
3.
of
type
and
analyzed
electrophoresis
weights
55,000
non-denaturing
then
weights
molecular
Fig.
from
were
activity
The of the
in "MATERI
phosphodiesterase
type ‡T;‡U
, type ‡U.
Fig.
5.
terns
Polyacrylamide of
naturating
Kinetic .
the at
behavior
Activities
non-denaturing various
of
of
concentrations
of
eluted gels
cAMP. •œ,
were
from
measured type ‡T; •Z,
4%
for
and
5
ml
and
at
(22).
as
The
positions
between
Pharmacia of the
Tris
to
the
to
of two
electrophoresis the
enzyme
markers samples.‡T,
HCl
with
were
Co to
in
1001d
pH
6.8,
of
and
were
then
SDS
method
of
weight
markers by
SDS-buffer type ‡T; ‡U,
kit in
the
type ‡U.
gel
Laemmli
obtained
calibration
incu boiled
polyacrylamide the
molecular runs)
with
2-mercaptoethanol,
extracts
10%
Non-de
stained
mashed
10%
The
pat
It.
corresponding
out,
SDS,
according
and
bands
cut
4•Ž.
subjected
electrophoresis
electrophoretic I
were
the
125 mM
overnight
ment
1, 1983
containing glycerol
cated
93, No.
and
solution
a
Vol.
gels
R-250
were
types ‡T and ‡Ubated
phosphodiesterases
polyacrylamide
type ‡U.
phosphodiesterase
blue
phosphodiesterases
20% 4.
gel types
polyacrylamide
omassie
Fig.
SDS
phosphodiesterase
(indi the
after same
use
of
treat way
42
suggest that the type I enzyme (Mr=67,000) is a monomeric form while the type 11 enzyme (Mr= 120,000) consists of two polypeptides. Developmental Changes in Activity of Two Extracellular Phosphodiesterases and the Effect of cAMP-It is known that the starvation of D. discoideum cells induces an accumulation of phos phodiesterase in the medium and that exogeneous cAMP added either in a single shot or in pulses (3, 4) greatly enhances this accumulation. Further studies (3, 4, 25, 26) indicate that the enhancement is due not only to the cessation of the synthesis of phosphodiesterase inhibitor, but also to the promotion of enzyme synthesis per se.
T . NOCE,
K. OKAMOTO,
and
L TAKEUCHI
It is of interest to know how the synthesis of the two types of extracellular phosphodiesterase is regulated in response to such environmental changes. Crude extracellular enzymes were pre pared by ammonium sulfate precipitation from the supernatants of the exponential growth phase and the aggregation phase (8 h-starved, 5 mM cAMP added at 3 h) cultures. After activation by DTT, samples were applied to a DEAE-Sephacel column. In this experiment, a 0.5 M KCl solution containing Tris buffer and DTT was used for elution instead of a linear salt gradient. As shown in Fig. 6B, the activity ratio of type II to I increased 2-3-fold when the development progressed from the growth phase to the aggregation phase. It is also evident in Fig. 6C that a shot of cAMP at 3 h caused about a 10-fold increase in this ratio. These
Fig. 6. DEAE-Sephacel chromatography of the extracellular phosphodiesterases from the cultures at various developmental stages. A, from the growth phase (1 x 105cells/ml) culture (500 ml); B, from the aggregation phase (8 h starved) culture (50 ml); C, from the cAMPtreated aggregation phase (8h starved, 5 mM cAMP added at 3 h) culture (50 m1). Extracellular enzyme was concentrated by 70% (NH4)2SO4 precipitation, treated with 10mM DTT and applied to a DEAE-Sephacel column. The column was washed with 50 ml of the starting buffer and eluted with the final buffer, containing 0.I M Tris-HCl, pH 7.5, 0.5 M KCl, and 0.2 mM DTT. Fractions of 5 ml were collected.
Fig.
7.
Specificity
of
phosphodiesterase Rabbit at
serum
various
was
beads
incubated for
,
type ‡U
type ‡U
with
the
enzyme
activity.
an
(1.04
beef
heart 0.5
mixture
The
supernatant
For
details,
units)+immune serum; •¢,
serum
Co.,
addition the
enzyme +-non-immune
enzyme+immune
Chemical
incubated
centrifuged.
enzyme
extracellular
immune
After
enzyme
between its
suspension,
and
sayed
reaction and
dilutions.
Sepharose
added); • ,
the
type ‡U
cAMP
units)+immune
(no
protein phosphodiesterase
serum. solution
of
protein was
Afurther
was see
the
as text. •~
serum; •œ, type ‡U A-Sepharose (Sigma
serum.
J. Biochem.
EXTRACELLULAR results
indicate
lated
in
the
PHOSPHODIESTERASE that
much
aggregation
fractions
and
we
was
prepared
(27)
from
by
and
results
77%,
types in
the
of
phase
by
extracellular
which
much
the
means
ConA
than
that
the
was
hardly
the 55%,
that
the
observation
complexes
protein
A-Sepharose
type ‡U
reaction
with
fraction
without
enzyme.
rose. the
Protein enzyme
observed ‡U enzyme
or
By
enzyme
the
D.
of
other As
serum
reacted
diesterase
type
diesterase
(solubilized
same that logical
extent all
these
I
immune beef
of
with
as with
the
enzymes
a small A-Sepha
itself
inhibit
reaction
was
and
type
the
serum
and
immunological the
type ‡U
phosphodiesterase in
Fig.
extracellular
membrane-bound and
all the
heart.
between
illustrated
and
by No
method,
classes
discoideum.
mune
this
only
serum
examined
the
by
protein not
from
was
7 depicts
but
of
the
the onto
removed
shown).
between
use
and
was
non-immune
the
Essentially
serum,
phosphodiesterase
cross-reactivity
adsorbed
Figure
did
esti of
from
been
addition
(not
were
activity
reaction.
A-Sepharose
between
cAMP
had
immune
activity
immune
centrifugation
activity
the
found
Brachet's
reactions
beads.
the
the and
enzyme
immune
enzyme
phosphodiesterase as those
immune
by
to we
with
Dicou
the
puri
phosphodiesterase
reaction
which
of the
same
way
the
assaying separated
specificity
Since
with
(28)), by
immune
the
by
been
chromatography
of extracellular
agreement
extracellular had
homogeneity.
inhibited
(in
against which
affinity
90%
activity
mated
amounts
were
more
Phosphodiesterase-Rab
prepared type ‡U,
supernatant
(like
type ‡U)
soluble
in
al.
h-starved).
DEAE-Sephacel
respectively,
activity
et
the
of
was
through
serum
station
(12
that
the
enzyme
Malchow
cells
showed
of intracellular
changed of
intracellular
phosphodiesterase 95%,
in
(2 x 106 cells/ml),
shown)
fied into
changes
method
adsorbed
and
resolved
antiserum
phosphodiesterase
chromatography,
The
the
bit
intracellular
developmental
phase
(not
activity
the
43
Iminunoreactivity
type ‡Tin cultures.
is similarly
aggregation
extracellular
two
that
fraction.
growth
phase
of
found
the
of each
is accumu the
cAMP-treated
DEAE-Sephacel
examined
activity
The
we
on
enzyme than
and
phosphodiesterase
two
ary
type ‡U
amounts
phase
Moreover, soluble
the
larger
OF D . discoideum
unsolubilized)
type ‡U share
of
8,
the
im
phosphophosphoto
enzyme,
indicating
a common
immuno
the
determinant.
DISCUSSION
Fig.
8.
other
Reactivity
cellular
type ‡U
(•Z,•œ)
or
esterase, 1%. ‡U
of
antitype ‡U
phosphodiesterases
either
Emulgen
in
(1.02
D.
units)
109P
(10)
serum and
the
"MATERIALS
starved
cells
as
was
was
AND
phosphodiesterase described
1, 1983
or
by
with
with
antitype
nonimmune
measured
described
Membrane-
prepared
Malchow
serum as
METHODS." was
units)
phosphodi
solubilized
mixed
or
reactivity
bound
Vol. 93, No.
(•¢, •£),
(•~,•Z,• ,•¢,)
(1.12
units)
(• )
with Extra-
type ‡T
(0.91
unsolubilized
serum
discoideum.
(•~),
membranebound
enzyme
(•œ,•£),
enzyme
of
from et
al.
(27).
12-h
The results presented in this paper indicate that the supernatant of D. discoideum aggregation phase culture contained two forms of cAMP phosphodiesterase with different apparent molecular weights (67,000 and 120,000 daltons) and different degrees of adsorption on DEAE-Sephacel. The existence of multiple forms of the enzyme partially purified from D. discoideum has been reported by several workers (15-17); these forms differs in apparent molecular weight, isoelectric point or Km for cAMP. A form with the lowest molec ular weight of 60,000-65,000 daltons and the highest isoelectric point of about 8, a commonly found form in the previous reports, seems to correspond to the type I enzyme of the present
44
T. NOCE,
study.
Other
forms,
however,
are
difficult
to
match. In as
spite
of
estimated
showed
the
by
that
both
of
55,000
possible
that
these
product
and
the
weights
is a
result
different
of
D.
by tide
purpureum,
the
reacted data
on
turing
of
their
that
type‡U
of
their or
case
I
these
enzyme
is
the
and
by
we
the
findings
strongly
the
or
type
I
addition
of
extracellular
phodiesterase from an axenic strain (Ax-3) of D. discoideum. They found two forms of the enzyme as we did: a monomer with a molecular weight of 55,000 and pl of 7.5-9 and oligomers with molecular weights of 150,000-200,000 and
connection
showed
type‡U
aggregation
two
forms since
observed
in Riedel
two
et
The
unlikely
to
(32)
the
enzyme
development: during
interconversion occur
in
developmental
intracellular a!.
of
the
predominantly
phase.
a similar the
types
during
accumulated
is
medium,
the
differently
enzyme
the
well.
that
regulated
culture
change
soluble reported
of the the
the
was
fraction
as
presence
of
addition,
continuous
however,
by
of may
amount
of not
but
the
also
the
enzyme
means
is
possible
contains
extracellular
that same but
some play only
extracellular
the
cAMP
a the
role
whereas
in
the by
that
of
enhanced.
It
membrane-bound
catalytic
connecting
(11) of
unaffected
greatly
is linked
a much
200,000
synthesis
(4),
the
and
of
is almost
enzyme
as
non-linear
(12)
weight
enzyme
enzyme
We
molecular
well
considerably
having
plots
bound
the
differ in
Lineweaver-Burk
In
enzyme as
membrane-bound to
enzyme
(12).
antiserum
enzyme
is known
membrane-
During the preparation of this manuscript, we learned that Orlow et al. (30) had independently made similar studies on extracellular cAMP phos
were
in
the
type‡U
Solubilized
extracellular
500,000
is,
gel electrophoresis showed that the high molecular weight enzyme was dissociated by 6 M urea to the monomeric form, as identified by pI measurement and tryptic mapping. Immunological identity of the two forms was also shown by the use of antiserum prepared against the monomeric enzyme. They concluded that extracellular phosphodies terase of Ax-3 was a single species and the high molecular weight form was produced by binding to uncharacterized acidic material.
I. TAKEUCHI
that
membrane-bound
apparent
enzyme.
pI of about 5. Small discrepancies in apparent molecular weight may be explained by differences in the strains and/or the conditions of gel filtra tion, because the enzyme shows a strong tendency to aggregate at low salt concentrations (31). SDS
revealed
extracellular
I enzyme.
the
higher
suggest of
type
kinetics
non-dena
form
with
from
showed
with
under
work
against
phosphodiesterase pep-
cross-
Together
present
reacted
enzyme
enzyme
dimeric
The produced
substantiated
weights
a
is
and
only one molecular species (60,000-65,000 daltons) of extracellular phosphodiesterase in a mutant strain (agg 50) of D. diseoideum, and this seems to be equivalent to the type I enzyme of the present study. These facts indicate that the rate of syn thesis of each form is developmentally regulated.
gene
molecular
work,
enzyme.
It
glycosylation
was
type‡U
molecular
common
same
of
present
against
conditions,
the
contents
In the
type
are
possibility
carbohydrate
analysis
daltons.
processing the
weight
gel
shared
57,000
In
antiserum with
enzyme
variation
(29).
molecular SDS
subunits
this
of
mapping
that
of and
degrees.
analyses
in
filtration,
types
subunits
to
difference
gel
K. OKAMOTO,
units to
as
the
membrane
protein.
Such
regulation
of
membrane-bound
the
a the
enzyme
phosphodiesterase.
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1, 1983
45
24. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 25. Klein, C. (1975) J. Biol. Chem. 250, 7134-7138 26. Klein, C. & Darmon, M. (1975) Biochem. Biophys. Res. Common. 67, 440-447 27. Malchow, D., Nagele, B., Schwary, H., & Gerisch, G. (1972) Eur. J. Biochem. 28, 136-142 28. Dicou, E. & Brachet, P. (1980) Eur. J. Biochem. 109, 507-514 29. Tsang, A.S. & Coukell, M.B. (1979) Eur. J. Biochem. 95, 419-425 30. Orlow, S.J., Shapiro, R.I., Franke, J., & Kessin, H. (1981) J. Biol. Cheat. 256, 7620-7627 31. Tsang, A.S. & Coukell, M.B. (1979) Eur. J. Biochem. 95,407-417 32. Riedel, V., Malchow, D., Gerisch, G., & Nagele, B. (1972) Biochem. Biophys. Res. Commun. 46, 279-287
