Vol. 79, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATfONS
PHOSPHOLIPASE A INHIBITION
OF ACETYLCHOLINE RECEPTOR FUNCTION
IN ~-___ TORPEDO CALIFORNICA MEMBRANE VESICLES Terrence
J. Andreasen
Department University
Received
October
19,
and Mark
of Biochemistry of California,
G. McNamee
and Biophysics Davis, CA 95616
1977
SUMMARY. A protein isolated from Naja naja siamensis venom on the basis of its phospholipase A activity inhibits acetylcholine receptor function in postsynaptic membrane vesicles from Torpedo californica. Specifically, the phospholipase A prevents the large increase in sodium efflux that can normally be induced by carbamylcholine, a receptor agonist. The phospholipase A inhibition shows the following properties: 1) it occurs at concentrations 50 times lower than the concentrations required for inhibition by a-neurotoxins; 2) the phospholipase A has no effect on the binding properties of the receptor; 3) the inhibition is abolished by removal of calcium ions; and 4) some phospholipid hydrolysis accompanies inhibition. It is suggested that the phospholipase A acts enzymatically to uncouple ligand binding from ion permeability in the receptor containing membrane vesicles. The binding change
in the
progress component
of acetylcholine ion
ion
in electric
of postsynaptic
tissue
that
indentified
However,
permeability
AChR represents function
permeability
has been made in isolating,
pharmacologically
(1,2).
to the acetylcholine
is not
and it
a complete
following
functional
purification
binds
and antagonists
by which
results
(1).
ligand
binding
and other
of the nicotinic is
coupled
yet established
complex.
Efforts only
the protein
acetylcholine
is not
have been at best
in a
Considerable
and characterizing
specifically
the mechanism known,
membranes
purifying
agonists
receptor
that
AChR
to membrane the isolated
to reconstitute partially
AChR
successful
(3,4).
The discovery prepared
from
the biochemical
that
electric
membrane
tissue
of Torpedo
and biophysical
properties
Abbreviations AChR, acetylcholine 4-IN-maleimidolbenzyltrit chloride.
Copyright All rights
functional
0 1977 by Academic of reproducfion in any
vesicles
species
enriched
has made it
in AChR can be possible
of the AChR in a membrane
to study environ-
recepsor; PLA, phospholipase A (EC 3.1.1.4); [3~]~~~~~, Hlmethyl ammonium iodide; Carb, carbamylcholine
Press.
form
Inc. reserved.
958 ISSN
0006-291X
BIOCHEMICAL
Vol. 79, No. 3, 1977
ment
(5,6).
The Torpedo
is blocked
tant
by specific
cholinergic
venom a-neurotoxins.
treatment
of the membranes
phenomenon In this
protein
interactions
antagonists,
Increased with
paper,
ion
are reported. in the membranes
naja
agonists
This
permeability
increased
such as d-tubocurarine is also
agonists,
by
and
blocked
by pre-
a physiologically
impor-
membrane
of a
(7). effects
siamensis
on Torpedo venom,
The PLA is being that
to cholinergic
permeability
cholinergic
the inhibitory from Naja
respond
permeability.
known as desensitization
component
PLA activity,
vesicles
-Iin Na , K' and Ca-l-t ion
an increase
snake
membrane
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
might
isolated
used
be important
on the basis
to investigate for
function
of its
lipid-protein
AChR function.
MATERIALS AND METHODS Torpedo Membrane Vesicles. Live Torpedo californica were obtained from Pacific Biomarine (Venice, CA) and sacrificed immediately upon arrival. Membrane vesicles were routinely prepared from 60 g of fresh electric tissue as described by Hazelbauer and Changeux (4), except that a Brinkmann Polytron was used for homogenization. The procedure was occasionally scaled up to use 500 g of tissue. In this case, the first supernatant was centrifuged at 15,000 x g for 45 min and resuspended in 150 mls of vesicle dilution buffer (255 mM KCl, 1.5 mM NaP04, 4 mM CaC12 2 mM MgC12, p H 7.0) before the sucrose step gradient. Assay for AChR Function. The [3~]~~~ affinity labeling assay described by Karlin the binding of tritiated Naja naja siamensis a-neuro--et al. (8) and/or toxin (2) were used to determine the number of AChR binding sites in the isolated membranes. The sodium efflux assay described originally by Kasai and Changeux (9) for Electrophorus electricus membranes was used to determine the functional integrity of the vesicles. Vesicles (5-10 mg/ml) were incubated with 22NaCl (New England Nuclear; 50 uCi/ml) overnight at 4'. At time 0, the vesicles were diluted loo-fold into ice-cold dilution buffer. At time t, 1 ml aliquots were filtered through HAWP 2400 Millipore filters, washed three times with 3.5 mls of ice-cold buffer, dried, and counted in a toluene-based scintillation fluid. Counts retained on the filter correspond to 22Na+ trapped within the vesicles. Fractionation of Naja naja siamensis venom. Lyophilized venom (428 mg, Miami Serpentarium) was dissolved in 5 mls of 0.03 M ammonium acetate (pH 6.5), and purified by ion exchange chromatography on a 1.5 x 30 cm CM-25 Sephadex column. The column was eluted step-wise with increasing concentrations of ammonium acetate (0.03-1.5 M) and protein was monitored at 280 nm with an ISCO UV detector. All the protein peaks were analyzed for PLA activity. Most of the activity was associated with the peak that did not bind to the CM-25 column in the initial 0.03 M ammonium acetate elution. This crude fraction was further purified by two different procedures: 1. Boiling and gel filtration as described by Cremona and Kearney (10) or 2. anion exchange chromatography on QAE Sepahdex. The QAE Sephadex column was eluted with increasing NaCl concentrations in 0.05 M Tris pH 8.3 essentially as described by Bon and Changeux for Bungarus caeruleus venom (11). The main PLA activity emerged as a skewed peak at 0.1 M NaCl.
959
Vol. 79, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Complete details of the manuscript in preparation).
purification
will
be published
elsewhere.
(M. McNamee,
Assay for PLA Activity. 0.5 pmoles of purified egg phosphatidylcholine was dispersed in 0.3 mls of 10 mM TES--2.5 mM calcium acetate buffer, pH 7.0; 0.1 mls of diethyl ether was then added and at time 0, 10 pl of the sample to be assayed was added. After 5 min at 25O, a 25 pl aliquot was spotted on a silica TLC plate and developed in chloroform:methanolania:water (65:25:1:4). The plate was sprayed with a phospholipid spray (Supelco Phospray), and the results expressed as pmoles of phosphatidylcholine hydrolyzed per min per mg protein. Other Assays and Procedures. Protein was determined by the Lowry procedure (12). Slab SDS-polyacrylamide gels were prepared, run and stained according to the general procedures of Ames (13). RESULTS Characterization
of Torpedo
From 60 g of fresh tein
is
ated
a-neurotoxin
for
obtained.
pure
toxin
bound
is
1.
completely
(Fig. vesicles
Also, with
properties
Carb response cules
just
Inhibition
10
with
-4
M Carb
(Fig.
about
10% of the total
corresponding
is
pmole/mg
about
by [3H]MBTA
is
purified
of their
one-half
assumed
protein
the number
the vesicles
within
the
Naja naja
these
is
first
siamensis by prior
vesicles
electroplax
by toxin
of
minute
and
incubation retain
of the
the functional
(5).
until
receptor
shown in
a-neurotoxin
is stoichiometric;
the vesicles
fraction
isolated
to a MW of ~18,000 venom protein. about
occurs
Thus,
of available
lo-30%
is
AChR (2).
is blocked
(see METHODS) appears
gels
of 8,000
a value
of triti-
i.e.,
the number sites
a partial
of toxin
(Figure
mole-
2).
by PLA
PLA containing
amide
If
of AChR in intact
the number
procedures
pmoles
efflux
from
mg of pro-
800-2500
with
1B).
lo-30
of AChR is
in efflux
of AChR inhibition
containing
of 22 Na+ from
efflux
the Carb-induced
exceeds
activity
labeled
pellet
contain
by preincubation
of 22Na+ Efflux
different
vesicles
enhancement
can be elicited
The major
specific
isolated
characteristic
The extent
activity
of Carb on the
blocked
1A).
a membrane
as observed
The large
Vesicles
per mg protein.
of sites
exactly
The effect Figure
the
The number
sites,
tissue,
The specific
AChR (2),
as AChR.
Membrane
from rJ. 5
as a single daltons.
Similar
960
venom by two
band on SDS-polyacrylThis
In the PLA assay
150 Units/mg.
&.
specific
protein
system
accounts
used here,
activities
were
for its ob-
BIOCHEMICAL
Vol. 79, No. 3, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
B
A
~-ahnltrol k.ti3Wo-4 A-A3xlO-4i
M Carb Garb; Pre-lncubatm Toxin
with 80 1
-i
----_ L ---L,-
1 I i
-i
i I i I i i I
5
4 \
g
50-
i.
i
%
“.. .
0
-“.-.&
40-
i)
30 -
L.4
-.-. *. --‘--A 20 -
20
IO -
IO
L
0
,
I
I
2
b
11
3 4
11
5
6
1
0
”
8 9
7
11111lllI1 I 2 3 4
IO
1.
tained
for
PLA samples
Pre-incubation
within
increase
the filter
results 10 min
in passive 1 min after
decreases
(Fig.
Unlike by PL,A is not imately
obtained
of Torpedo
PLA (5 pg/ml)
+ Na efflux
slow
6
7
8 9
IO
(mid
Effect of Carb and Naja naja siamensis a-neurotoxin on "Na+ efflux from Torpedo membrane vesicles. The effect of Carb was measured by including 3 x 10-b M Carb in the dilution medium. (A) : Pre(180 ug/ml), or incubation of membrane (5 m /ml) with a-neurotoxin into (B) pre-incubation with lo- f M Carb for 30 min before dilution 3 x 10-4 M Carb completely blocks the Carb response.
Figure
22
5
TIME
TIME(min)
with
-L‘.-.4
af
3B),
(12 mg/ml; blockage
(Fig.
After
3A).
of the Carb-induced
with
into
due to non-specific a-neurotoxin
33 AChR binding
961
sites.
times,
Carb-free
buffer
in
there
retained
is a on
gradually
disruption.
the blockage
of 5 ugfml,
sites) increase
of counts
vesicle
inhibition,
At a concentration per
incubation
the number
of the membranes
presumably
~10 uM a-neurotoxin
longer
efflux. , i . e .,
dilution
stoichiometric.
1 PL,A molecule
memhranes
(see below).
in complete
"Na'
the situation
commercially
there
of "Na+ is approx-
BIOCHEMICAL
Vol. 79, No. 3, 1977
I
Figure
2.
ever
available
including
PLA from
associated
I
naja
with
PIA
I
This
(Sigma)
I
shows three
PLA also
(Sigma)
blocks
does -not block
There
at MW 21,000.
the PLA proteins
under
and intensity
of Torpedo
gels,
the
bands
on How-
even at Crotalus
proteolytic
conditions
membrane
bands
22Na+ efflux
is no detectable the assay
major
22Na+ efflux.
on SDS-polyacrylamide
used
actihere,
as
on SDS-polyacryl-
Sites
The PLA has no apparent
effect
on the binding
of a-neurotoxin
is
not
altered
vesicles
with
0.5-50
treatment
not
site
altered sequence for
binding of Torpedo
The extent
).lg/ml.
action ing
band
of PLA on AChR Binding
taneous
also
I
gels.
Effect
tent
I
as 100 ug/ml;
by the position
amide
Naja
adamanteus
as high
PLA shows one major
judged
I 2
one at MN 18,@00.
Crotalus
concentrations
vity
I
Concentration dependence of a-neurotoxin inhibition. Torpedo membranes were pre-incubated with various concentrations of a-neurotoxin (O-27 PM) for 30 min before dilution into vesicle dilution buffer with and without 2.5 x 10s4 M Garb. The difference after 1 min in 22Na+ counts between the plus Carb and minus Carb samples is used as a quantitative measure of the Carb response (no difference = 100% inhibition; the difference in the absence of a-neurotoxin = 0% inhibition). The concentration of a-neurotoxin binding sites was 4 uM.
A commercially SDS gels
I 051
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
by PLA. (8),
and specificity
MBTA would
by either PLA,
over
of'affinity
The MBTA assay
so even slight
properties
involves
decreases
be detected. 962
of AChR.
pre-incubation a concentration labeling
or simulrange
of
by [3HJMBTA is
a kinetically-controlled
in the affinity
The ex-
re-
of the AChR hind-
BIOCHEMICAL
Vol. 79, No. 3, 1977
I
I
20 INCUBATION
Figure
3.
Inhibition
Increase time.
PLA is dependent
are resuspended
branes, Lipid
the
free
I
I
I
I
30
40
50
60
TIME
phospholipids PLA concentrations
is
enzymatic
is
containing
however,
with
the
of Torpedo
these
but still
this blocks
response
of 5 pg/ml
was monitored
(a concentration
963
under
con-
have
containing
conditions, response
cannot
the response.
a normal be If mem-
can be restored.
Membrane Vesicles by PLA 22 Na+ efflux experiments, the effect
vesicles
Vesicles
in buffers
Under
normal
of 10 mM CaC12 and
to 10 -6 M in the EGTA-containing
of the carbamylcholine
in Torpedo
Under
4 mM CaC12.
resuspended
< 10 -g M.
increased
of incubation
activity.
in the presence
from the vesicles,
The a-neurotoxin,
PLA inhibition
In parallel
for
buffer
as a function
i-lCa
are prepared
[Ca*]
i-l- concentration Ca
Hydrolysis
ions
into
can be elicited
by PLA.
the free
22 Na + efflux
in 0.1 M EGTA and then
so that
Carb response
membranes
and diluted
been prepared
inhibited
I
20
INCUBATION
by PLA Requires
on calcium
the Torpedo
Ca*-EGTA
(mln)
in passive
of AChR Function
ditions,
I IO
54001
40
30 TIME
Vesicles were pre-incubated with PLA (5 pg/ml). At the indicated times, 20 pl aliquots of the incubation solution were diluted lOOfold with vesicle buffer (k2.5 x 10-4 M Carb) and assayed as described in Figure 2. 22 + of Carb induced A: Inhibition Na efflux as a function of incubation time. B:
also
I
I
IO
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of PLA on the
identical conditions. At 22 Na + efflux is completely at which
Vol. 79, No. 3, 1977
blocked), amine
BIOCHEMICAL
there
during
is
detectable
the first
phosphatidylcholine hydrolyzed
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
lipid
hydrolysis
10 min of treatment. can be detected,
before
At longer
but
all
any phosphatidylcholine
+ICa , there
sence of free
(by TLC) of phosphatidylethanolsome hydrolysis
of
of the phosphatidylethanolamine
hydrolysis
is no detectable
times
lipid
is
observable.
is In the ab-
hydrolysis.
DISCUSSION The results lated
demonstrate
that
of its
PLA activity,
on the basis
in Torpedo
californica
than
the concentration
fect
on the binding
in the absence
vesicles
required
for
result
from
the enzymic
could
result
in the disruption
tions
necessary
for
proper
meability.
Bartels
cottonmouth
moccasin
eel
electroplax
tributed
However,
ethanolamine
fatty
is
acids
(14)
venom inhibited
hydrolysis in
described
be detected.
the
important
or the loss
It
event
of the
This
remains and if
intact
it
here
hydrolysis
the
phospholipid
interacion
PLA fraction
from
was not
low
at-
concentrations
of phosphatidylif
resulting that
per-
of isolated
inhibition
at the
ef-
hydrolysis
to be established is
inhibitory
depolarization
none was detected
the experiments
the
lipid-protein
a crude
Carb induced
(10 ugfml).
since
that
to AChR mediated
that
lower
is abolished
Phospholipid
binding
observed the
effect
important
of ligand
permeability
The PLA has no ef-
suggest
of the PLA.
iso-
10 to 50 times
inhibitory
results
venom,
ion
inhibition.
of functionally
and Rosenberg
could
hydrolysis
activity
at low concentrations
to lipid
used;
These
coupling
siamensis
at a concentration
a-neurotoxin
ions.
na&
AChR mediated
of AChR, and the
Cact
fects
from Naja
inhibits
membrane
properties
of free
a protein
specific
lysolipids
accounts
for
lipid and
the
inhi-
bition. It
is
interesting
that
22 Na+ efflux
from
bungarotoxin
isolated
be attributed failure of lipid
Torpedo.
to its
This
from
(16).
all
PLA proteins
type
Bungarus
PLA activity
of C. adamanteus specificity
not
of result
bring
has been
multicinctus. (15),
but
PLA to inhibit
not
964
inhibition
reported
all
purification
PLA's
of
for
Most of the toxic
may indicate
In the original
about
the effects
are neurotoxic.
a membrane of Baja
can The
dependent naja
g-
degree
siamensis
Vol. 79, No. 3, 1977
venom,
Karlsson
BIOCHEMICAL
reported
in a mouse bio-assay now being
(17).
marmorata
caeruleus vesicles.
Experiments
activity. cificity
PLA containing
fractions
of the purified
lipid
(11)
reported
hydrolysis,
that
venom (ceruleotoxin) Their
purified
are now in progress
of the PLA protein,
between
The toxicity
Bon and Changeux
from Bungarus
Torpedo
the crude
were
protein
non-toxic
used here
is
investigated.
Recently, lated
that
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
its
molecular
lipid-protein
protein
a toxic inhibited fraction
to determine properties, interactions
protein
fraction
22Na+ efflux also
isofrom
showed PLA
more precisely
the spe-
and the relationships and AChR function.
ACKNOWLEDGEMENTS The expert This
research
technical is
supported
assistance
of Ms. Crystal
by USPHS Grant
Chan is
greatly
appreciated.
NS13050.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
Rang, H. P. (1974) Quart. Rev. Biophys. 7, 283-399. Karlin, A., McNamee, M. G., Weill, C. L., and Valderrama, R. (1976) Methods in Receptor Research, Vol. I, M. Blecher, ed., pp. l-35, M. Dekker, New York. Michaelson, D. M., and Raftery, M. A. (1974) Proc. Natl. Acad. Sci. USA 71, 4768-4772. Hazelbauer, G. H., and Changeux, J.-P. (1974) Proc. Natl. Acad. Sci. USA 71, 1479-1483. Popot, J. L., Sugiyama, H., and Changeux, J.-P. (1976) J. Mol. Biol. 106, 469-483. Changeux, J.-P., and Cohen, J. B. (1975) Annu. Rev. Pharmacol. 2, 83-103. Sugiyama, H., Popot, J.-L., and Changeux, J.-P (1976) J. Mol. Biol. 106, 485-496. Karlin, A., McNamee, M. G., and Cowbum, D. A. (1976) Anal. Biochem. 76, 442-451. Kasai, M., and Changeux, J.-P. (1971) J. Membrane Biol. 5, l-23. Cremona, T., and Kearney, E. B. (1964) J. Biol. Chem. 239, 2328-2334. Bon, C., and Changeux, J.-P. (1977) Eur. J. Biochem. 3, 31-42;43-51. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. Ames, G. F.-L. (1974) J. Biol. Chem. 249, 634-644. Bartels, E., and Rosenberg, P. (1972) J. Neurochem. 19, 1251-1265. Howard, B. D., and Truog, R. (1977) Biochemistry 16, 122-125. Zwaal, R. F. A., Roelofsen, B., Comfurius, P., and Van Deenan, L. L. M. (1975) Biochim. Biophys. Acta 406, 83-96. Karlsson, E., Arnberg, H., and Eaker, D. (1971) Eur. J. Biochem. 1, l-16.
965
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATfONS
PHOSPHOLIPASE A INHIBITION
OF ACETYLCHOLINE RECEPTOR FUNCTION
IN ~-___ TORPEDO CALIFORNICA MEMBRANE VESICLES Terrence
J. Andreasen
Department University
Received
October
19,
and Mark
of Biochemistry of California,
G. McNamee
and Biophysics Davis, CA 95616
1977
SUMMARY. A protein isolated from Naja naja siamensis venom on the basis of its phospholipase A activity inhibits acetylcholine receptor function in postsynaptic membrane vesicles from Torpedo californica. Specifically, the phospholipase A prevents the large increase in sodium efflux that can normally be induced by carbamylcholine, a receptor agonist. The phospholipase A inhibition shows the following properties: 1) it occurs at concentrations 50 times lower than the concentrations required for inhibition by a-neurotoxins; 2) the phospholipase A has no effect on the binding properties of the receptor; 3) the inhibition is abolished by removal of calcium ions; and 4) some phospholipid hydrolysis accompanies inhibition. It is suggested that the phospholipase A acts enzymatically to uncouple ligand binding from ion permeability in the receptor containing membrane vesicles. The binding change
in the
progress component
of acetylcholine ion
ion
in electric
of postsynaptic
tissue
that
indentified
However,
permeability
AChR represents function
permeability
has been made in isolating,
pharmacologically
(1,2).
to the acetylcholine
is not
and it
a complete
following
functional
purification
binds
and antagonists
by which
results
(1).
ligand
binding
and other
of the nicotinic is
coupled
yet established
complex.
Efforts only
the protein
acetylcholine
is not
have been at best
in a
Considerable
and characterizing
specifically
the mechanism known,
membranes
purifying
agonists
receptor
that
AChR
to membrane the isolated
to reconstitute partially
AChR
successful
(3,4).
The discovery prepared
from
the biochemical
that
electric
membrane
tissue
of Torpedo
and biophysical
properties
Abbreviations AChR, acetylcholine 4-IN-maleimidolbenzyltrit chloride.
Copyright All rights
functional
0 1977 by Academic of reproducfion in any
vesicles
species
enriched
has made it
in AChR can be possible
of the AChR in a membrane
to study environ-
recepsor; PLA, phospholipase A (EC 3.1.1.4); [3~]~~~~~, Hlmethyl ammonium iodide; Carb, carbamylcholine
Press.
form
Inc. reserved.
958 ISSN
0006-291X
BIOCHEMICAL
Vol. 79, No. 3, 1977
ment
(5,6).
The Torpedo
is blocked
tant
by specific
cholinergic
venom a-neurotoxins.
treatment
of the membranes
phenomenon In this
protein
interactions
antagonists,
Increased with
paper,
ion
are reported. in the membranes
naja
agonists
This
permeability
increased
such as d-tubocurarine is also
agonists,
by
and
blocked
by pre-
a physiologically
impor-
membrane
of a
(7). effects
siamensis
on Torpedo venom,
The PLA is being that
to cholinergic
permeability
cholinergic
the inhibitory from Naja
respond
permeability.
known as desensitization
component
PLA activity,
vesicles
-Iin Na , K' and Ca-l-t ion
an increase
snake
membrane
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
might
isolated
used
be important
on the basis
to investigate for
function
of its
lipid-protein
AChR function.
MATERIALS AND METHODS Torpedo Membrane Vesicles. Live Torpedo californica were obtained from Pacific Biomarine (Venice, CA) and sacrificed immediately upon arrival. Membrane vesicles were routinely prepared from 60 g of fresh electric tissue as described by Hazelbauer and Changeux (4), except that a Brinkmann Polytron was used for homogenization. The procedure was occasionally scaled up to use 500 g of tissue. In this case, the first supernatant was centrifuged at 15,000 x g for 45 min and resuspended in 150 mls of vesicle dilution buffer (255 mM KCl, 1.5 mM NaP04, 4 mM CaC12 2 mM MgC12, p H 7.0) before the sucrose step gradient. Assay for AChR Function. The [3~]~~~ affinity labeling assay described by Karlin the binding of tritiated Naja naja siamensis a-neuro--et al. (8) and/or toxin (2) were used to determine the number of AChR binding sites in the isolated membranes. The sodium efflux assay described originally by Kasai and Changeux (9) for Electrophorus electricus membranes was used to determine the functional integrity of the vesicles. Vesicles (5-10 mg/ml) were incubated with 22NaCl (New England Nuclear; 50 uCi/ml) overnight at 4'. At time 0, the vesicles were diluted loo-fold into ice-cold dilution buffer. At time t, 1 ml aliquots were filtered through HAWP 2400 Millipore filters, washed three times with 3.5 mls of ice-cold buffer, dried, and counted in a toluene-based scintillation fluid. Counts retained on the filter correspond to 22Na+ trapped within the vesicles. Fractionation of Naja naja siamensis venom. Lyophilized venom (428 mg, Miami Serpentarium) was dissolved in 5 mls of 0.03 M ammonium acetate (pH 6.5), and purified by ion exchange chromatography on a 1.5 x 30 cm CM-25 Sephadex column. The column was eluted step-wise with increasing concentrations of ammonium acetate (0.03-1.5 M) and protein was monitored at 280 nm with an ISCO UV detector. All the protein peaks were analyzed for PLA activity. Most of the activity was associated with the peak that did not bind to the CM-25 column in the initial 0.03 M ammonium acetate elution. This crude fraction was further purified by two different procedures: 1. Boiling and gel filtration as described by Cremona and Kearney (10) or 2. anion exchange chromatography on QAE Sepahdex. The QAE Sephadex column was eluted with increasing NaCl concentrations in 0.05 M Tris pH 8.3 essentially as described by Bon and Changeux for Bungarus caeruleus venom (11). The main PLA activity emerged as a skewed peak at 0.1 M NaCl.
959
Vol. 79, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Complete details of the manuscript in preparation).
purification
will
be published
elsewhere.
(M. McNamee,
Assay for PLA Activity. 0.5 pmoles of purified egg phosphatidylcholine was dispersed in 0.3 mls of 10 mM TES--2.5 mM calcium acetate buffer, pH 7.0; 0.1 mls of diethyl ether was then added and at time 0, 10 pl of the sample to be assayed was added. After 5 min at 25O, a 25 pl aliquot was spotted on a silica TLC plate and developed in chloroform:methanolania:water (65:25:1:4). The plate was sprayed with a phospholipid spray (Supelco Phospray), and the results expressed as pmoles of phosphatidylcholine hydrolyzed per min per mg protein. Other Assays and Procedures. Protein was determined by the Lowry procedure (12). Slab SDS-polyacrylamide gels were prepared, run and stained according to the general procedures of Ames (13). RESULTS Characterization
of Torpedo
From 60 g of fresh tein
is
ated
a-neurotoxin
for
obtained.
pure
toxin
bound
is
1.
completely
(Fig. vesicles
Also, with
properties
Carb response cules
just
Inhibition
10
with
-4
M Carb
(Fig.
about
10% of the total
corresponding
is
pmole/mg
about
by [3H]MBTA
is
purified
of their
one-half
assumed
protein
the number
the vesicles
within
the
Naja naja
these
is
first
siamensis by prior
vesicles
electroplax
by toxin
of
minute
and
incubation retain
of the
the functional
(5).
until
receptor
shown in
a-neurotoxin
is stoichiometric;
the vesicles
fraction
isolated
to a MW of ~18,000 venom protein. about
occurs
Thus,
of available
lo-30%
is
AChR (2).
is blocked
(see METHODS) appears
gels
of 8,000
a value
of triti-
i.e.,
the number sites
a partial
of toxin
(Figure
mole-
2).
by PLA
PLA containing
amide
If
of AChR in intact
the number
procedures
pmoles
efflux
from
mg of pro-
800-2500
with
1B).
lo-30
of AChR is
in efflux
of AChR inhibition
containing
of 22 Na+ from
efflux
the Carb-induced
exceeds
activity
labeled
pellet
contain
by preincubation
of 22Na+ Efflux
different
vesicles
enhancement
can be elicited
The major
specific
isolated
characteristic
The extent
activity
of Carb on the
blocked
1A).
a membrane
as observed
The large
Vesicles
per mg protein.
of sites
exactly
The effect Figure
the
The number
sites,
tissue,
The specific
AChR (2),
as AChR.
Membrane
from rJ. 5
as a single daltons.
Similar
960
venom by two
band on SDS-polyacrylThis
In the PLA assay
150 Units/mg.
&.
specific
protein
system
accounts
used here,
activities
were
for its ob-
BIOCHEMICAL
Vol. 79, No. 3, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
B
A
~-ahnltrol k.ti3Wo-4 A-A3xlO-4i
M Carb Garb; Pre-lncubatm Toxin
with 80 1
-i
----_ L ---L,-
1 I i
-i
i I i I i i I
5
4 \
g
50-
i.
i
%
“.. .
0
-“.-.&
40-
i)
30 -
L.4
-.-. *. --‘--A 20 -
20
IO -
IO
L
0
,
I
I
2
b
11
3 4
11
5
6
1
0
”
8 9
7
11111lllI1 I 2 3 4
IO
1.
tained
for
PLA samples
Pre-incubation
within
increase
the filter
results 10 min
in passive 1 min after
decreases
(Fig.
Unlike by PL,A is not imately
obtained
of Torpedo
PLA (5 pg/ml)
+ Na efflux
slow
6
7
8 9
IO
(mid
Effect of Carb and Naja naja siamensis a-neurotoxin on "Na+ efflux from Torpedo membrane vesicles. The effect of Carb was measured by including 3 x 10-b M Carb in the dilution medium. (A) : Pre(180 ug/ml), or incubation of membrane (5 m /ml) with a-neurotoxin into (B) pre-incubation with lo- f M Carb for 30 min before dilution 3 x 10-4 M Carb completely blocks the Carb response.
Figure
22
5
TIME
TIME(min)
with
-L‘.-.4
af
3B),
(12 mg/ml; blockage
(Fig.
After
3A).
of the Carb-induced
with
into
due to non-specific a-neurotoxin
33 AChR binding
961
sites.
times,
Carb-free
buffer
in
there
retained
is a on
gradually
disruption.
the blockage
of 5 ugfml,
sites) increase
of counts
vesicle
inhibition,
At a concentration per
incubation
the number
of the membranes
presumably
~10 uM a-neurotoxin
longer
efflux. , i . e .,
dilution
stoichiometric.
1 PL,A molecule
memhranes
(see below).
in complete
"Na'
the situation
commercially
there
of "Na+ is approx-
BIOCHEMICAL
Vol. 79, No. 3, 1977
I
Figure
2.
ever
available
including
PLA from
associated
I
naja
with
PIA
I
This
(Sigma)
I
shows three
PLA also
(Sigma)
blocks
does -not block
There
at MW 21,000.
the PLA proteins
under
and intensity
of Torpedo
gels,
the
bands
on How-
even at Crotalus
proteolytic
conditions
membrane
bands
22Na+ efflux
is no detectable the assay
major
22Na+ efflux.
on SDS-polyacrylamide
used
actihere,
as
on SDS-polyacryl-
Sites
The PLA has no apparent
effect
on the binding
of a-neurotoxin
is
not
altered
vesicles
with
0.5-50
treatment
not
site
altered sequence for
binding of Torpedo
The extent
).lg/ml.
action ing
band
of PLA on AChR Binding
taneous
also
I
gels.
Effect
tent
I
as 100 ug/ml;
by the position
amide
Naja
adamanteus
as high
PLA shows one major
judged
I 2
one at MN 18,@00.
Crotalus
concentrations
vity
I
Concentration dependence of a-neurotoxin inhibition. Torpedo membranes were pre-incubated with various concentrations of a-neurotoxin (O-27 PM) for 30 min before dilution into vesicle dilution buffer with and without 2.5 x 10s4 M Garb. The difference after 1 min in 22Na+ counts between the plus Carb and minus Carb samples is used as a quantitative measure of the Carb response (no difference = 100% inhibition; the difference in the absence of a-neurotoxin = 0% inhibition). The concentration of a-neurotoxin binding sites was 4 uM.
A commercially SDS gels
I 051
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
by PLA. (8),
and specificity
MBTA would
by either PLA,
over
of'affinity
The MBTA assay
so even slight
properties
involves
decreases
be detected. 962
of AChR.
pre-incubation a concentration labeling
or simulrange
of
by [3HJMBTA is
a kinetically-controlled
in the affinity
The ex-
re-
of the AChR hind-
BIOCHEMICAL
Vol. 79, No. 3, 1977
I
I
20 INCUBATION
Figure
3.
Inhibition
Increase time.
PLA is dependent
are resuspended
branes, Lipid
the
free
I
I
I
I
30
40
50
60
TIME
phospholipids PLA concentrations
is
enzymatic
is
containing
however,
with
the
of Torpedo
these
but still
this blocks
response
of 5 pg/ml
was monitored
(a concentration
963
under
con-
have
containing
conditions, response
cannot
the response.
a normal be If mem-
can be restored.
Membrane Vesicles by PLA 22 Na+ efflux experiments, the effect
vesicles
Vesicles
in buffers
Under
normal
of 10 mM CaC12 and
to 10 -6 M in the EGTA-containing
of the carbamylcholine
in Torpedo
Under
4 mM CaC12.
resuspended
< 10 -g M.
increased
of incubation
activity.
in the presence
from the vesicles,
The a-neurotoxin,
PLA inhibition
In parallel
for
buffer
as a function
i-lCa
are prepared
[Ca*]
i-l- concentration Ca
Hydrolysis
ions
into
can be elicited
by PLA.
the free
22 Na + efflux
in 0.1 M EGTA and then
so that
Carb response
membranes
and diluted
been prepared
inhibited
I
20
INCUBATION
by PLA Requires
on calcium
the Torpedo
Ca*-EGTA
(mln)
in passive
of AChR Function
ditions,
I IO
54001
40
30 TIME
Vesicles were pre-incubated with PLA (5 pg/ml). At the indicated times, 20 pl aliquots of the incubation solution were diluted lOOfold with vesicle buffer (k2.5 x 10-4 M Carb) and assayed as described in Figure 2. 22 + of Carb induced A: Inhibition Na efflux as a function of incubation time. B:
also
I
I
IO
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of PLA on the
identical conditions. At 22 Na + efflux is completely at which
Vol. 79, No. 3, 1977
blocked), amine
BIOCHEMICAL
there
during
is
detectable
the first
phosphatidylcholine hydrolyzed
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
lipid
hydrolysis
10 min of treatment. can be detected,
before
At longer
but
all
any phosphatidylcholine
+ICa , there
sence of free
(by TLC) of phosphatidylethanolsome hydrolysis
of
of the phosphatidylethanolamine
hydrolysis
is no detectable
times
lipid
is
observable.
is In the ab-
hydrolysis.
DISCUSSION The results lated
demonstrate
that
of its
PLA activity,
on the basis
in Torpedo
californica
than
the concentration
fect
on the binding
in the absence
vesicles
required
for
result
from
the enzymic
could
result
in the disruption
tions
necessary
for
proper
meability.
Bartels
cottonmouth
moccasin
eel
electroplax
tributed
However,
ethanolamine
fatty
is
acids
(14)
venom inhibited
hydrolysis in
described
be detected.
the
important
or the loss
It
event
of the
This
remains and if
intact
it
here
hydrolysis
the
phospholipid
interacion
PLA fraction
from
was not
low
at-
concentrations
of phosphatidylif
resulting that
per-
of isolated
inhibition
at the
ef-
hydrolysis
to be established is
inhibitory
depolarization
none was detected
the experiments
the
lipid-protein
a crude
Carb induced
(10 ugfml).
since
that
to AChR mediated
that
lower
is abolished
Phospholipid
binding
observed the
effect
important
of ligand
permeability
The PLA has no ef-
suggest
of the PLA.
iso-
10 to 50 times
inhibitory
results
venom,
ion
inhibition.
of functionally
and Rosenberg
could
hydrolysis
activity
at low concentrations
to lipid
used;
These
coupling
siamensis
at a concentration
a-neurotoxin
ions.
na&
AChR mediated
of AChR, and the
Cact
fects
from Naja
inhibits
membrane
properties
of free
a protein
specific
lysolipids
accounts
for
lipid and
the
inhi-
bition. It
is
interesting
that
22 Na+ efflux
from
bungarotoxin
isolated
be attributed failure of lipid
Torpedo.
to its
This
from
(16).
all
PLA proteins
type
Bungarus
PLA activity
of C. adamanteus specificity
not
of result
bring
has been
multicinctus. (15),
but
PLA to inhibit
not
964
inhibition
reported
all
purification
PLA's
of
for
Most of the toxic
may indicate
In the original
about
the effects
are neurotoxic.
a membrane of Baja
can The
dependent naja
g-
degree
siamensis
Vol. 79, No. 3, 1977
venom,
Karlsson
BIOCHEMICAL
reported
in a mouse bio-assay now being
(17).
marmorata
caeruleus vesicles.
Experiments
activity. cificity
PLA containing
fractions
of the purified
lipid
(11)
reported
hydrolysis,
that
venom (ceruleotoxin) Their
purified
are now in progress
of the PLA protein,
between
The toxicity
Bon and Changeux
from Bungarus
Torpedo
the crude
were
protein
non-toxic
used here
is
investigated.
Recently, lated
that
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
its
molecular
lipid-protein
protein
a toxic inhibited fraction
to determine properties, interactions
protein
fraction
22Na+ efflux also
isofrom
showed PLA
more precisely
the spe-
and the relationships and AChR function.
ACKNOWLEDGEMENTS The expert This
research
technical is
supported
assistance
of Ms. Crystal
by USPHS Grant
Chan is
greatly
appreciated.
NS13050.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
Rang, H. P. (1974) Quart. Rev. Biophys. 7, 283-399. Karlin, A., McNamee, M. G., Weill, C. L., and Valderrama, R. (1976) Methods in Receptor Research, Vol. I, M. Blecher, ed., pp. l-35, M. Dekker, New York. Michaelson, D. M., and Raftery, M. A. (1974) Proc. Natl. Acad. Sci. USA 71, 4768-4772. Hazelbauer, G. H., and Changeux, J.-P. (1974) Proc. Natl. Acad. Sci. USA 71, 1479-1483. Popot, J. L., Sugiyama, H., and Changeux, J.-P. (1976) J. Mol. Biol. 106, 469-483. Changeux, J.-P., and Cohen, J. B. (1975) Annu. Rev. Pharmacol. 2, 83-103. Sugiyama, H., Popot, J.-L., and Changeux, J.-P (1976) J. Mol. Biol. 106, 485-496. Karlin, A., McNamee, M. G., and Cowbum, D. A. (1976) Anal. Biochem. 76, 442-451. Kasai, M., and Changeux, J.-P. (1971) J. Membrane Biol. 5, l-23. Cremona, T., and Kearney, E. B. (1964) J. Biol. Chem. 239, 2328-2334. Bon, C., and Changeux, J.-P. (1977) Eur. J. Biochem. 3, 31-42;43-51. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. Ames, G. F.-L. (1974) J. Biol. Chem. 249, 634-644. Bartels, E., and Rosenberg, P. (1972) J. Neurochem. 19, 1251-1265. Howard, B. D., and Truog, R. (1977) Biochemistry 16, 122-125. Zwaal, R. F. A., Roelofsen, B., Comfurius, P., and Van Deenan, L. L. M. (1975) Biochim. Biophys. Acta 406, 83-96. Karlsson, E., Arnberg, H., and Eaker, D. (1971) Eur. J. Biochem. 1, l-16.
965
