Download Lymphocyte Membrane Enzymes. II. Cyclic 3`,5

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Lymphocyte
II.
Cyclic
Membrane
3’,5’-Adenosine
Monophosphatase
Unstimulated
Human
Small
Nuclear
By
This
study
enzyme
phatase
was
S.
undertaken
portance
ulation,
Cyclic
in mediating
hence
localization
is thought
may
histochemical
of action
employed
that
was
the pliosphodiesterase,
resulting
in a lead
then
INCREASINGLY,
enzymes
portaiice
lead
the
in
early
to ivuiphocyte
small
lymphocytes
I)OSSSS
while
if!
is
it
that
from
In
may
the
monophosphate
work
in
indicates
It
where
does
at some
other
on the
indicates
cyclic
not
that
AMP
necessarily
point
in the
pathway,
and
the
be the site
mediating
is
mean
is the site
this may
lymphocyte
including
the cell
nuclear
membrane
where
cyclic
AMP
from
intracellular
effects
AMP
may
The
it
and
events,
to serve
of
the
be
an
freshly
enzyme
value
stimulation
nucleus.
stimulatory
is known
with
of the
state.
ascertain
the
cyclic
nature
resting
the
AMP)
is concerned
the
surface
to the
intracellular
that
site
localized
which
BEING
CONCENTRATED
on the
membranes,
as they
may
be of imrelated
to antigen
recognition,
that
study
of
(cyclic
particularly
This
ascertain
along
variety
the
to be
precipitate
the prestimulation
be possible
to
lymphocyte
found
This is subsulfide.
The
is degraded.
hydrolyzed,
to
occur
events
a wide
et
stimulation.
human
chemical
be
ATTENTION
IS
with
lymphocyte
events,
immediately
associated
is
stimulation
al.#{176} membrane,
by
may only
degraded
phosphate
was
membranes,
action.
as lead
that
the nuclear
membrane
where
cyclic
AMP is effective;
may
Shanta
is
enzyme
destroyed.
AMP
technique
of
KENNEDY
nuclear
this
of cyclic
substrate
ANIP
A.
at the site of enzyme
sequently
visualized
stimenzyme
AMPase,
point
to the site
in the cell. The
Cyclic
the
its intracell-
cyclic
on
Lymphocyte
LESLEY
AND
be of im-
lymphocyte
of the
to modulate
concentration,
COULSON
monophoshuman
small
AMP
Located
Membranes
to localize
cyclic
3’,5’-adenosine
in unstimulated
lymphocytes.
that
ular
ALAN
Enzymes.
of such
related
data
pathway
cyclic
they
information
what
bio-
that
leads
3’,5’-adenosine
as a second-echelon
peptide
hormones.1’2
intermediary
separated
systems
hormone,
Recent
in thymocyte
and
from
the
Department
of Surgery,
Stanford
University
School
of Medicine,
Stanford,
Calif.
Submitted
April
20, 1971;
revised
May 20, 1971;
accepted
May 22, 1971.
This study
was carried
out in the Department
of Pathology,
Guy’s
Hospital,
London,
England,
and was supported
by the University
of London
Laura
de Saliceto
Studentship,
the Peel Medical
Research
Trust,
and the British
Medical
Association
Ernest
Hart Award.
ALAN
S. CouLsoN,
NI.A., NIB.,
B. Cim.,
PH.D.,
L.R.C.P.,
M.R.C.S.:
Senior
Research
Fellow,
Department
of Surgery,
Stanford
University
School
of Medicine,
Stanford,
Calif.;
Leverhulme
Research
Fellow.
LE5LEY
A. KENNEDY:
Tissue-typing
Immunologist,
Departnients
of Immunology
and Surgery,
Guy’s
Hospital,
London,
England.
BLOOD,
\OL.
38,
No. 4
(OCTOBER),
1971
485
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486
COULSON
lymphocyte
stimulation,
hence
the phosphodiesterase,
in this way,
probably
cyclic
serves
the
it was
of
interest
3’,5’-AMPase,
to modulate
to
that
the
attempt
destroys
effect
AND
the
KENNEDY
localization
of
cyclic
AMP
and thus,
of cyclic
AMP
within
cell.
The
histochemical
Smears
of
AMP,
and
cells
to
this
form
exogenous
also
was
verted
to
visualized
black
by
The
the
The
lead
microscopically
in
in
sulfide
MATERIALS
Lq;nplzocyte
human
described
carbonyl
third
of
Gelatine
rotated
allowed
lymphocytes
to prevent
to make
the slides
subsequently
a
that
medium
in
form
later
lead
then
the
stage.
The
presumptive
AND
METHODS
et al.#{176}
cyclic
3’,5’-
within
hydrolyzed
form
of
black
site
snake
which
staining
of
the
by
precipitated
phosphate,
the
Shanta
located
radical
of
of
containing
was
phosphate
marked
on
phosphodiesterase
the
the
at
and
based
a medium
compound
liberated
medium,
in
the
latter
supplied
atrox.7
in
was
incubated
degraded
5’-AMPase
present
employed
were
5’-AMP.
Crotalu9
from
technique
lymphocytes
the
venom
with
lead
was
con-
could
enzyme
be
activity.
Separation
small
lymphocytes
were
separated
from
healthy
donors
using
the technique
by Coulson,
Corner,
and Coombs.8
Defibrinated
blood
was mixed
with
100 mg
iron particles
( Fine Dyestuffs
and
Chemicals,
Manchester,
England
and with one
its volume
of 3%
gelatin
in phosphate-buffered
saline
(Batch
277,
Glue
and
Research
Association
Birmingham
England
. The
blood-gelatin-iron
mixture
was
on a blood
cell suspension
mixer
for 30 mm
at 37#{176}C,then
decanted
and
to sediment
for a further
30 mm
at 37#{176}C.The
supematant
suspension
of
was
diluted
with
an equal
volume
of 199 medium
(Weilcome,
lot M3413)
the gelatin
from gelling,
and centrifuged
to produce
the pellet
of cells used
the smears.
The time that elapsed
from
the initial
venipuncture
to the fixing of
in acetone
was 2 hr. The slides
on which
the smears
were
made,
and which
came
in
contact
with
the incubation
media,
were
scrupulously
cleaned.
)
)
Fixation
and
Histochemistry
Lymphocyte
smears
were air dried
within
a period
of 5 sec and then plunged
into 60%
distilled
water at 20#{176}C
for 120 ± 5 sec. The slides were washed
gently
for 3 mm
in tap water
and then in two changes
of glass distilled
water
for a total of 4 mm.
Test slides
were
incubated
in a medium
containing
the following
constituents:
cyclic
3’,5’-adenosine
monophosphate
( 1.44
mM,
Sigma,
lot 60C-1620),
Tns-maleate
buffer
(pH 7.6, 50 mM),
magnesium
chloride
( 10 mM), lead acetate
(2 mM),
and Crotalus
atrox
venom
( 1 mg in 10 ml, Sigma,
lot 78B-1460).6
When
the medium
was
made
up in this
way, the final pH was 7.30, and it had to be adjusted
to 7.60 with Tris base.
Incubation
was continued
for 90 ± 5 mm at 37#{176}C
in disposable
Petri dishes.
At the end of incubation
the pH of the medium
was still 7.60.
The molarity
of the incubation medium
was
unavoidably reduced slightly by the residual
dampness
of the lymphocyte
smear.
After
incubation,
the slides
were
washed
carefully
by gentle
agitation
in five changes
of glass
distilled
water
for a total of 25 mm. Any significant
curtailment
of this washing
led to nonspecific
background
staining
on the slide. The next stage in the staining
procedure
was
immersion
of the slides
in freshly
prepared
ammonium
sulfide
solution
(1 % weight!
volume
in distilled
water),
followed
by three
washes
in distilled
water
totaling
9 mm.
Finally
the slides
were mounted
in glycerol jelly.
In some
preliminary
experiments
different
methods
of fixation
were
tried,
including
100%
methanol
and 70%
ethanol
is distilled
water.
acetone
in
Control
With
AMP
every
staining
run the following
controls
was omitted;
(2) exogenous
5’-nucleotidare
were
was
employed:
omitted
and
(1) the
fluoride
substrate
was
cyclic
added
to
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LYMPhOCYTE
inhibit
MEMBRANE
endogenous
5’-nucleotidase;
cyclic
AMPase.
water
was
substituted
ingredients
of
other
was
In
omitted,
20
mM;
practical
(3)
terms,
or
when
fluoride
addition,
the
solution
smears
cyclic
AMP
solution,
affected.
was
were
ammnophylline
the
for the cyclic
AMP
the medium
was not
sodium
in
487
ENZYMES
so that
When
substituted
to
preincubated
in
was
added
was
omitted,
the
the
an
inhibitor
1 ml
of
of
distilled
final concentration
of the
exogenous
5’-nucleotidase
produce
100
as
a
mM
final
sodium
concentration
fluoride
of
dissolved
in 50 mM Tris buffer
(pH 7.8 ) for 30 ± 3 mm at 37#{176}C.The aminophyllmne
control
studies
involved
preincubation
of the slide in 100 mM ammnophyllmne
B.P. ( British
Pharmacopoeia)
for 30 mm
at
37#{176}C,followed
by incubation
in a medium
identical
to the normal
test
medium
but with
added
aminophylline
B.P. to a final concentration
of 20 mM.
Further
control
studies
were
carried
out on two additional
donors,
including
heat
inactivation
of the enzyme,
inhibition
with formahin,
and variation
of the pH of the incubation
medium.
In practical
terms,
heat
inactivation
was achieved
by warming
the separated
lymphocytes
to 60#{176}Cfor 120
±
5 sec prior
to centrifugation
and
fixation.
Formalin
treatment
involved
immersion
of the slides for 10 mm in formahin
B.P. at room temperature
between
the acetone
fixation
stage
and the wash
in distilled
water.
Variation
in the pH
range
for the final incubation
media
was achieved
by making
up the test medium
to the
following
different
pH values:
6.0, 6.5, 6.8, 7.0, 7.6, 7.8, and 8.0.
RESULTS
Lymphocyte
The
Separation,
leukocytes
contained
the
in
more
lymphocytes
the
in
the
film
showed
and
70%
compared
small
branes
in the
were
smear
sharply
deposits
closely
showed
the
was
where,
of the
procedure
outnumbered
taken
on the
smear
the
to examine
Leishman
was
conversely,
film,
avoided
the
tail
beof
the
lymphocytes.
lymphocyte
to incubation
smears,
with
distorted
density
inferior
yielded
cells
Care
together;
considerably
fixed
the
after
of
final
which
were
substrate.
airThe
treatment,
and
this
positive
staining.
histochemical
Methanol
preparations
slides.
AMPase
found
of all
with
prior
in
Cyclic
was
lymphocytes
product
was
and no black
and
similarly
staining
smear
head
sedimentation
red
smears.
The
of the
were
done
in acetone
final
of the
closely
spreading
acetone
of the
Positive
tail
too
reduction
to the
Localization
the
classical.
swollen
marked
ethanol
was
the
although
6: 1 in the
crowded
trials
fixed
appeared
there
roughly
before
excessive
Preliminary
dried
but
not
cells
ratio
were
from
lymphocytes,
appeared
cells
Morphology
and
supernatant
99%
just
morphology
cause
the
than
lymphocytes
their
Fixation,
to be
10 of the
defined
were
localized
different
on
the
donors
nuclear
membrane
employed.
The
with
no extension
into the nucleus
seen elsewhere
in the cell (Fig.
1).
examined
and
no staining.
were
consistently
negative.
in the
final
reaction
or cytoplasm,
The cell mem-
The
erythrocytes
Controls
In
was
all
added,
the
controls
the
where
lymphocyte
cyclic
nuclear
AMP
was
membranes
omitted
were
or
where
unstained.
18 control
experiments
where
exogenous
5’-nucleotidase
fluoride
added,
the nuclear
membrane
appeared
slightly
plasm
very
slightly
gray.
On the
other
16 occasions
and
was
gray
during
aminophylline
In
two
out
of
omitted
and
and the cytothe prelimi-
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488
COULSON
AND
KENNEDY
1
-
-
.4
.
1:
o
‘is
#{149}
-
2
I
A
90
Fig.
1.-Freshly
separated
small
lymphocytes
from
different
donors
showing
localization
of cyclic
3’,5’-AMPase
on nuclear
membranes.
Smears
of lymphocytes
were
incubated
in a medium
containing
cyclic
AMP,
exogenous
5’-nucleotidase
and
lead
ions;
lead
phosphate
deposited
at the site of enzyme
activity
is converted
to
black
lead
sulfide
by ammonium
sulfide
treatment.
X 1750.
In Fig.
1A the two
small
black
granules
to the right
of the lymphocyte
and the two small
circles
below
it are artifacts.
The
eight
erythrocytes
discernible
in the picture
are unstained.
nary
trials,
the
Treatment
tion
of
either
the
and
membrane
formahin
enzyme
8.0
6.8
nuclear
with
or
activity.
7.0,
below
the
density
there
was
and
was
of
no
unstained.
heat
When
inactivation
the pH of
the
staining
nuclear
resulted
in complete
the incubation
medium
was
considerably
membrane
inhibiwas
reduced;
at
pH
staining.
DIscussIoN
The
inhibitory
I)ility
partures
viev
AMP.
the
an
of the
at
sulfide
deposit
3’,5’-AMP
the
iron
left
high
magnifications
against
sulfide
the
recognizable.
Sutherland’
has
the
second
stage
at
a site
distant
procedure
employed,
the
related
out,
the
from
the
not
AMP
first
occur,
5’-nucleotidase,
cyclic
3’,5’-
of
and
the
known
of
Stage.
sharply
final
the
lead
cyclic
Confusion
residual
any
it
the
investigated
carbonyl
because
has
lymphoma
is
cells
of
of
shape
of NK/Ly
the
cyclic
of
degradation
traces
characteristic
zone
of the
de-
because
persistently
diffuseness
and
did
enzyme,
perinuclear
pointed
of
product
separation
A
in the
case
of
the
to
support
meaningfulness
was
lack
suscepti-
and
employed
the
in the
This
reaction
of
technique
product
the
cells
strongly
degradation
questioned
reaction
membrane.
of the
medium
the
However,
final
occurring
lead
from
demonstrated1#{176}
As
argues
incubation
has
and
heating
histochemical
he
product.
nuclear
substrate
between
the
the
the
the
aminophylline
prior
initiates
and
final
experiments,
localized
of
process
criticized
and
to both
pH
procedure,
localization
clearly
optimal
Pearse#{176} has
these
formaldehyde
procedure
enzymatic
a multistep
in
of
staining
from
that
is
effect
of the
with
particles
the
was
previously
been
cells.
to
play
the
role
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
LYMPhOCYTE
of
a
MEMBRANE
secondary
situations.
to be
the
intracellular
The
the
result
of
of
degrades
does
not
hormone-like
intracellular
synthesis
that
489
ENZYMES
content
a balance
cyclic
it.
and
point
of
within
the lymphocyte.
is at the cell membrane
brane
the
and
nucleus
The
and
glutinin-stimulated
of
stimulate
cyclic
that
indicate
thyroid
Small
cyclic
AMP
an earlier
nucleoside
observation17
diphosphatase
spectively
with
of
early
cyclic
to
the
noted
on
and
to
nuclear
best
membrane
for
membrane
be
to
as
will
with
mediate
the
the
playing
site
any
suggestive.
suppress
but
of
Studies
growth
for the
AMP
described
absence
where
is merely
cyclic
3’,5’-AMP
the
site
in
Various
phytohemag-
addition
that
some
phytohemagglutinin,
rat
thymic
their
will
lymphocytes
mitogenic
hormone.1214
paucity
of
of
part
effect
of
enzyme
para-
content,16
of enzyme
present,
particularly
that associated
with
important
role in early
triggering
events
following
thus be of immunologic
significance.
In this context
the increase
in lymphocyte
during
transformation
may
synthesis
AMP
ascribe
in
and
however,
the small
amount
membranes,
may play
an
antigen
recognition,16
and
case
nuclear
evidence
appears
bradykinin,
are
the
effects
AMP
transformation.
hormone,
lymphocytes
that
3’,5’-AMPase
AMP
mem-
transformation,
AMP,
lymphocyte
to
of
appears
cyclase
cyclic
as the
variety
It is possible
that the site where
cyclic
or some
other
site between
the cell
cyclic
lymphocyte
concentrations
AMPase
wide
moment
adenyl
enzyme
structure
the
that
a
any
cyclic
can
found
at
enzyme
cyclic
available
activation
have
in
AMP
modulator
latter
that
presently
lymphocyte
investigators5
the
the
to the
is effective
is effective
degradation.
in small
cyclic
between
AMP,
Localization
necessarily
substance
of
and
its
them
release
modulator
a
definite
membrane-associated
be
correlated
of lymphokines.18’1#{176}
enzyme
place
cyclic
in
the
M.
B.,
retro-
However,
AMPase,
in the
it is still
lymphocyte
too
stimulation
pathway.
REFERENCES
1.
role
Sutherland,
of
cyclic
E.
ANIP.
W.:
JAMA
On
the biological
214:1281,
1970.
2. Robison,
G. A., Butcher,
R. W., and
Sutherland,
E. W.: Cyclic
AMP.
Ann. Rev.
Biochem.
37:149,
1968.
3. MacNianus,
J. P., and Whitfield,
J. F.:
Stimulation
of DNA
synthesis
and mitotic
activity
of thymic
lymphocytes
by cyclic
adenosine
3’,5’-monophosphate.
Exp.
Cell
Res. 58:188,
1969.
4. Hirschhorn, R., Grossman,
J., and Weissniann,
C.: Effect of cyclic 3’,S’-adenosine
monophosphate
and
theophyllmne
on
lym-
phocyte
transformation.
Proc. Soc. Exp. Biol.
Med.
133:1361,
1970.
5. Rigby,
P. C., and Ryan,
W. L.: The
effect
of cyclic
AMP and related
compounds
on human
lymphocyte
transformation (HLT)
stimulated by
phytohemagglutinmn
(PHA).
Rev. Eur.
Etud.
Clin.
Biol.
15:774,
1970.
6. Shanta, T. R., Woods,
W. D., Waitz-
man,
and
chemmcal
method
3’,S’-nucleotide
chemie
7:177,
Bourne,
C.
H.:
for localization
phospho-diesterase.
Histo-
of
cyclic
Histo-
1966.
7. Richards,
G. M., Do Vair,
C., and
Laskowski, M., Sr.: Comparison
of the levels
of
phosphodiesterase,
monophosphatases
Biochemistry
endonuclease,
mn several
(Wash.)
4:501,
snake
and
venoms.
1965.
8. Coulson,
A. S., Cumer,
B. W.,
and
Coombs,
R. R. A.: Macrophage-like
properties of some
guinea-pig
transformed
cells.
mt. Arch. Allerg. 32:264,
1967.
9. Pearse,
A. C. E.: Histochemistry,
Theoretical
and Applied.
London,
Churchill,
1968,
p. 526.
10. Kurnatowski,
A.,
and
Willighagen,
R. C.: Cytochemistry
of the NK!Ly
lymphoma.
Nature
(London)
198:1211,
1963.
11. Butcher,
R. W., and Sutherland,
E.
W.: Adenosine
3’,5’-phosphate
in biological
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490
COULSON
materials.
I. Purification
and properties of
cyclic 3’,5’-nucleotide
phosphodiesterase
and
use of this enzyme to characterize
adenosine
3’,5’-phosphate
in human
urine.
J. Biol.
Chein. 237:1244, 1962.
12. Whitfield, J. F., NlacManus,
J. P., and
Rixon,
R. H.: The
possible
mediation
by
cyclic
ANIP of
parathyroid
hormone-induced
stimulation
of mitotic
activity
and
deoxyribonucleic
acid synthesis
in rat thymic
lymphocytes.
J. Cell. Physiol.
75:213,
1970.
13. -,
-,
and Gillan,
D. J.: Cyclic AMP
mediation of bradykinmn-mnduced
stimulation
of mitotic
activity
and DNA synthesis
in thymocytes.
Proc.
Soc. Exp.
Biol.
Med.
133:
1270,
1970.
14. MacManus,
J. P., and Whitfield,
J. F.:
Mediation
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1971 38: 485-490
Lymphocyte Membrane Enzymes. II. Cyclic 3',5'-Adenosine
Monophosphatase Located on Unstimulated Human Small Lymphocyte
Nuclear Membranes
ALAN S. COULSON and LESLEY A. KENNEDY
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