BIOLOGY
OF
REPRODUCTION
31,
(1984)
1061-1071
Characterization
of (Ca2 + + Mg2 +) Adenosine
Triphosphatase
Activity
and Calcium Transport
in Boar Sperm Plasma Membrane
Vesicles and
Their Relation to Phosphorylation
of Plasma Membrane
Proteins
M. ASHRAF,
R. N. PETERSON’
Department
of
School
Southern
and
L. D. RUSSELL
Physiology
of Medicine
Illinois
Carbondale,
University
Illinois
62901
ABSTRACT
Properties
of (Ca2 + + Mg2 +) adenosine
triphosphatase
(ATPase)
in plasma
boar
epididymal
spermatozoa
are described.
Enzyme
activity
is optimum
at high
affinity
for Ca2+.
It is not inhibited
by the caimodulin
antagonist
trifluoperazine
inhibited
by low concentrations
of Ca2 .
Plasma
membrane
vesicles
obtained
by hypotonic
lysis of intact
sperm
Imixed
and
membranes
from
pH and has a high
(TFP),
but it is
inside-out
(IOV)
right
side-out
(ROV)
vesicles]
transport
45Ca2+
in the presence
of oxalate.
Similar
to the
Ca2 +stimulated
Mg ATPase
activity,
transport
is unaffected
by TFP,
but unlike
the ATPase,
transport
is at an optimum
rate near
neutral
pH and is completely
inhibited
by p-chloromercurphenylsulfonate
(pCMS).
When
plasma
membranes
are labeled in the presence
and absence
of Ca2+
and Mg2+
alkali and
with I’r32 P1 ATP, differences
hydroxylamine
suggest that
in the
intensity
of labeling
and lability
of bound
“P to
between
100-120K
may be related
to a
two polypeptides
TFP
at concentrations
transport
ATPase.
The
addition
of
which
stimulate
net Ca2
uptake
in
intact
cells
causes
intense
labeling
of a single neutrally
charged
protein
near 68K. These labeling
patterns
and
the
properties
of (Ca2+
+ Mg2+)
ATPase
indentify
particular
plasma
membrane
proteins
(PMPs)
from
the complex
surface
of these
cells that
may
be involved
in Ca2 +.dependent
functions
and support
the view that
calmodulin
is not directly
involved
in the regulation
of ATPdriven
Ca2 + effiux
from
boar spermatozoa.
INTRODUCTION
The
port
recent
have
which
significantly
to this
adenosine
transport.
triphosphatase
A (Ca2
be involved
in transport
has
been
ram and bull flagellar
membranes
Forrester,
1980a,
Breitbart
the
from
boar
plasma
has
been
has
plasma
been
identified
membranes
Peterson
1982).
mammalian
et
al.,
Calmodulin
sperm
et a!., 1981;
modulin-like
Mg2 )
to
sented
in
and
flux)
et
1983).
membrane
+
al.,
(PMV)
1983a;
has
(Jones
(Ashraf
et
Vijayasarathy
been
et al.,
Tash
and Means,
activity,
present
al.,
Bradley,
evidence
et
to
our
in
PM
described
(Fortes-
of
PM
intact
identified
in
1980;
Feinberg
sperm
with
tides
)2
are
Ca2+
+
boar
similar
and
by
regulated
1982)
and a calin seminal
plasma,
(Ca2
+
P1 ATP
identified
has
also
sperpre-
(but not
spermatozoa
we
had
not
Ca2 +timulated
Mg2-ATPases
In this
Mg2 )
report
ATPase
spermatozoa.
to ATP-driven
PMV,
does
is also
may
-
in
we present
is present
appear
labeling
described
which
been
The
enzyme
efflux
from
not
The
ef-
Mg2
active
calmodulin.
tn-
in dog
We have
uptake
in boar
but
a
highly
that
the
1983b)
preparations.
(on enzymes),
1982a;
et al.,
al.,
from
evidence
+
ram
by
Calmodulin
Ca2
distinguish
ATPase
in
that
of
is inhibited
been
has
caimodulin
and
play
to
of
be
PM
polypep-
a role
in
function.
MATERIALS
Accepted
Received
Reprint
Mg2 +) ATPase
+
which
1980).
involves
able
described and a Ca2 +ATPase
in boar
and
bull
sperm
(PM)
(TFP),
(Peterson
1983;
ATP-driven
vesicles
and
+
and
been associated
with Ca2 + transport
matozoa
(Skinner
et al., 1982).
identified
(Bradley
Breitbart
Rubinstein,
and
efflux
1981;
+
ter
(Ca2
PM,
fluoperazine
thought
(ATPase)
activates
epididymal
mechanism
and
control
of Ca2+
transin spermatozoa
is poorly
understood
but
developments
from
several
laboratories
described
enzymes
that may
contribute
AND
METHODS
Materials
August
20, 1984.
May 14, 1984.
requests.
ICN
1061
[-y-32P]ATP
Chemicals
(500
Ci/mmoi)
was
(Cleveland,
OH)
as
purchased
from
the
tniethylam-
ASHRAF
1062
monium
the
salt.
Smith,
Trifluoperazine
& French
was purchased
(TFP)
Kline
Calmodulin
ton, TX). All
Sigma Chemical
Isolation
from
other
materials
Co. (St. Louis,
of Plasma
Sperm
of freshly
were
CAABCO
nitrogen
cavitation
centrifugation,
Co.
purchased
from
(PM)
from
and
also
et al., 1980a)
Transmission
the caudae
epididymides
sucrose
density
as previously
gradient
described
(Peterson
and as modified
from Gihis et al. (1978).
electron
microscopy
has
shown
that
isolated
on top of 1.0 M sucrose
in sucrose
these
PMV,
gradients,
are free
membranes
(Russell
from
contamination
by other
et al., 1984;
Kaplan
et al.,
Two-dimensional
polyacrylamide
(2-D
PAGE)
all
plasma
ejaculated
of
and
by
these
PMV
membrane
PM, except
originating
al.,
from
1983,
1984).
gel
has
proteins
those
accessory
times
(PMPs)
that can
gland
Membranes
in 0.9%
them
were
in
be accounted
fluids
(Russell
for
et
isolated
sperm
at -80#{176}C
as described
washed
in a lysis buffer
and disrupted
by
NaCI,
resuspended
0.5 mM CaCI3)
were
(1 mM Nal-1C03
and
25 strokes
in a Dounce
homogenizer
with
a tight-fitting
pestle. The lysate was then processed
as described
for
ROV. Membrane
vesicles (a mixed population
of ROV
and
IOV)
sucrose
were
isolated
on top
of 1.0 M sucrose
after
Transmission
electron
microscopy
showed
these
vesicles
to be free from
other
sperm
membranes.
PAGE
analysis
of these
vesicles
showed
them
to contain
the same
proteins
as
PM5 obtained
by nitrogen
cavitation.
The vesicles
were
resuspended
in buffer
containing
110 mM KCI and 50
mM Tris-HCI
pH 7.4 and stored
at -80#{176}C.
Seminal
plasma was isolated
from
freshly
collected
semen.
One
portion
of the seminal
plasma
was heat
denatured
as described
by
Forrester
and
Bradley
(1980)
and another
was dialyzed
(12,000
mol.
wt.
cutoff
NaCI,
MgCI4,
7.4.
Enzyme
PMs
density
centrifugation.
tubing)
once against buffer containing
103 mM
12.5 mM K3P04,
2.5 mM
K2HPO4,
3.0 mM
0.4
mM EDTA,
and 30 mM Tris-HCI,
pH
Assay
were
pelleted
in
isolated
buffer
from
sucrose
containing
either
gradients
and were
60 mM NaC1 and
Tris-HCI
(pH 7.4), or buffer
containing
75 mM
Tris-maleate
and 75 mM glycine (pH 8.5). The reaction
mixture
(200
MI) for (Ca2+
+ Mga)
ATPase
activity
contained
60
mM NaCI,
50 mM Tris-HCI,
4 mM
50
mM
MgCI3,
Cad3
3 mM
(at
free
ATP,
5 mM
concentrations
0.1
mM
ouabain,
and
PMP.
The
Mg2+ATPase
lar
buffer
containing
added
CaCI3.
The
effect
NaN,,
0.2
indicated
and
0.012
EGTA,
text),
between
0.1-0.150
mg of
was
determined
in a simi0.2
mM
EGTA
without
of pH on the
activity
of
(Ca3+ + Mg2+)
ATPase
was determined
mixture
containing
75 mM Tris-maleate,
cine,
5 mM NaN3,
4mM
MgCl2,
3 mM
ouabain,
mM
in the
mM CaC12.
in a reaction
75 mM glyATP,
0.1 mM
The Mg2 -ATPase
was
containing
The
reaction
0.2mM
EGTA
was started
after
a
absence
of PMs. Ca3 +activated
Mg’ +ATPase
was cal-
culated
as the increment
in P1 above
the Mg2 +ATpase
activity
upon
addition
of Ca2 + in the presence
of 4
mM
M8CI,.
Rates
were
linear
with
time
(up
to 90
mm)
and
with
protein
concentration
(50-150
Mg).
Since
it is not
clear
whether
more
than
one Ca3+.
a single
to contain
buffer
20-mm
incubation
at 37#{176}C
by adding
ATP and carried
out for 40 to 60 mm at 37#{176}C
or as indicated
in the text.
The
reaction
was terminated
by addition
of 20 M’ of
HgCI2
(100
mM), and 0.6 ml of a reagent
solution
for
inorganic
phosphorus
(P1) determination
as described
by Piper and Lovel (1981),
and immediately
centrifuged
at 12,000
X g for
10 mm
(Eppendorf).
All
ATPase
values
were
corrected
for
released
in the
ATPase
term
(Ca2 +
Ca3 +stimulated
present
stored
determined
in a similar
without
added CaCI2.
stimulated
sperm
1984).
electrophoresis
shown
used within
a week
of isolation.
Inside-out
vesicles
(IOV)
were
Lichtman
et al. (1981).
Briefly,
two
PA).
(Hous-
slaughtered
boars
as previously
described
et al., 1984).
Plasma
membrane
vesicles
(right
side-out
vesicles,
ROV)
were prepared
by
(Russell
(PMV)
as
from
a gift
(Philadelphia,
were
MO).
Membrane
isolated
was
Labs.
ET AL.
enzyme
Transport
is present
in the
PMs
used,
the
Mg2 +) ATPase
is used as a synonym
for
Mg ATPase
activity
and not to denote
+
activity.
Assay
The
uptake
of 45Ca2+
by IOV was carried
out in
an assay
medium
containing
110
mM
KCI,
20 mM
NaCI, 50 mM Tris-HC1
(pH 7.4),
5 mM NH4 oxalate,
5
mM NaN,,
4 mM MgCl,,
0.2
mM EGTA,
0.1 mM
ouabain,
and with
or without
3 mM ATP.
The reaction
mixture
(200 M’ containing
50 to 100 g protein)
was
incubated
for
5 mm
at 37#{176}Cand
the reaction
was
started
by adding
0.2 mM CaCI3
(10 MCi, 45Ca2+/ml).
At
appropriate
times,
the sample
was
vacuum-filtered
on
0.45-Mm
Millipore
filters
that
had
been
presoaked
in 250 mM KCI and
50 mM Tris-HCI
(pH
7.4) and washed once with 5 ml distilled
H, 0 at room
temperature.
Filters
were
washed
rapidly
two
times
with
4 ml buffer
containing
250 mM sucrose
and 50
mM Tris-HCI.
uptake
was
was
corrected
filters
in the
Filters
for
Two
was
assay
carried
was
210
nmol);
the
and counted.
and
bound
45Ca2
the
to
+
uptake
PM
and
of ATP.
were
used
of experiments
out
at pH 7.4
100 Mi and
adding
nmol.mg.h
of PMs
methods
group
dried
as
radioactivity
absence
Phosphorylation
one
were
expressed
pmol
in a similar
except
that
the
reaction
of
to
PMs. In
of ROV
phosphorylate
phosphorylation
manner
as the
ATPase
the assay mixture
volume
at 37#{176}C
was initiated
by
[7-’2P]ATP
(6-9
reaction
was terminated
after
M1 of “SDS-stopping”
solution
X
iO
30
cpm/
sec by
adding
50
containing
9% sodium
dodecylsulfate
(SDS),
6% 3-mercaptoethano!, 15% glycerol,
4 mM EDTA,
20 mM Tnis-HCI
(pH
6.8)
and a small amount
of bromphenol
blue. Phosphorylation
of
membranes
(at
the
same
specific
activity
and concentration
of isotope)
was also carried
out
at 4#{176}C,essentially
as described
by Niggli
et al.
(1979).
The
reaction
buffer
was modified
to contain
0.04%
Triton
X-100.
Phosphorylation
was
stopped
after
30 sec by the addition
of cold
trichloroacetic
acid as described
by those authors.
In chase experiments,
1 mM unlabeled
ATP
was added
immediately
after the 30-sec labeling
period,
and after
30 sec the
reaction
was
stopped
as described
above.
Hydroxylamine
treatment
was
carried
Spitzer
et al. (1983).
For some
PMs
were
washed
in hypotonic
out
as described
by
experiments,
isolated
buffers
to increase
CALCIUM
permeability
Schulman
and
before
phosphorylation
Greengard
(1978).
BY
TRANSPORT
as
described
BOAR
SPERMATOZOA
RESULTS
by
Properties
Electrophoresis
In
(1-D
(10%
and
Autoradiography
some
PAGE)
experiments
discontinuous
acrylamide
gels) was
(Peterson
et
al.,
1983c).
one-dimensional
PAGE
system
of Laeinmii
(1970
used as previously
described
experiments
used
sperm)
more
a
phosphate-buffered
continuous
system
at pH 7.2 as
described
by Hames
and Rickwood
(1981).
In order
to
resolve
high
molecular
weight
proteins,
7.5%
acrylamide
was used
to prepare
these
gels. 2-D PAGE
nonequilibrium
pH
gradment
electrophoresis
(NEPHGE)
(O’Farrell
et al., 1977)
was also used.
The procedure
has also been described
in detail
elsewhere
(Peterson
et
al.,
1983c).
Following
electrophoresis,
the gels were
stained
with
0.03%
Coomassie
brilliant
blue
in 10%
acetic
acid,
25% isopropyl
alcohol,
and then
destained
first
in 10% acetic
acid,
10% isopropyl
alcohol,
and
then
in 10% acetic
acid.
The gels were
soaked
in 3%
glycerol
solution
for S mm before
drying
over
a slab
gel dryer
(LKB
Bromma
#2003)
under vacuum.
The
dried
gel was
placed
in close
contact
with
Kodak
X-omat
AR film at -80#{176}C for 2 to 8 days
and developed.
Other
used
and
trations
absence
-Stimulated
+
experiments
+
Mg2)
(obtained
Mg2
activity
of
of
Ca2+
2
+
-A TPase
which
attempted
to
ATPase
in PM (ROV)
from
gas cavitation
of
Ca2+
concentrations
physiological
pH.
Mg2 +ATPase
of 100
Stimulation
by Ca2+
at these
concen-
was
not
observed.
Ca2 + stimulated
the
+,
tM or
of
In the
hydroly-
sis of ATP but the apparent
Km for the
was high and maximum
rates of ATPase
were
observed
The
activity
only
of
calmodulin
in
tions
of
ing
EGTA,
free
our
Ca2
this
concentration
in the reaction
mixture
by
the
method
described
by Wolf
(1973)
using a TRS-80
computer.
Apparent
stability
constants
for Ca2 +.EGTA
at pH 7.4
and
8.5 were
calculated
as reported
by Fabiato
and Fabiato
(1979).
Total
Ca’ + concentrations
were determined
by atomic
absorption
spectroscopy
as described
by McDonald
et
al. (1977).
Protein
concentration
was determined
by
the
method
of Markwell
et al. (1978)
using
bovine
serum
albumin
as a standard.
creased
and
when
activity
optimum
unique;
was
for
we
concentra-
in buffers
contain-
Ca2-stimulated
the
was
pH
range
used
at
maximum
a PM
have
activity
observed
was
above
+
-ATPase
Mg2
the
optimum
was
by
1983a).
was identified
(Fig.
1). As shown
enzyme
activity
was maximum
at
of free Ca2+
near
2 .tM, but
concentration
physiological
lower
used
affinity
declined.
No activity
free Ca2+
concentration
Calcium-activated
Free
calcium
was
estimated
unaffected
et a!.,
when
were
enzyme
activity
concentrations.
was
(Peterson
study,
+
(mM)
enzyme
a higher
Mg2-ATPase
in the figure,
a concentration
above
at high
the
antagonists
However
Determinations
C
of Ca2
Previous
detect
(Ca2
preparations
the
Other
1063
was
in early
enzyme
reported
the
8 pM.
raised
pH
9.5;
at lower
rapidly
when
activity
in-
above
the
studies
(Fig.
Mgl+ATPase
pH. A high
in boar sperm
previously
2)
pH
is not
that
1600
0
I-
0.
0)
E
I-
1400
-c
0
E
C
I-
1200
>
>
4-
0
2
8
Free
FIG.
1. Effect of added Ca’
on Mg’-ATPase
was assayed
at pH 7.4 in the presence
of Caa+
means
± SEM
of three
replications.
and
16
Calcium
30
45
(MM)
of boar sperm plasma membranes
200 pM EGTA
to give the free
(PMs). The enzyme
activity
Ca2+
indicated.
Values
are
1064
ASHRAF
ET AL.
rates
maximum
(Peterson
1600
a!.,
1400
1200
1973).
(Ca2
+
where
the
1000
guished
ATPase
-c
800
increasing
The
600
pM
centrations
of
decrease
10.5
11.0
pH
FIG.
2.
(Ca’
+
pH dependence
Mg’)
ATPase
of
(0-0).
Mg2+ATPase
(._.)
Mg’
ATPase was
measured
in the presence
of 4 mM MgCI2
Ca’ . Each point
represents
the means
±
easily
(Ca2
increase
were
,
Mg2 )
in rate
Ca2
were
pM)
difficult
Mg2-ATPase
in the reaction
+
free
at pH 8.5
respectively
made
distin-
+
.
5.0
(Fig.
Km at
conby
and
the
by
(Ca2+
ATPase
+
was
Mg2+)
not
Table
detected
1
of
at
free
in these
shows
ATPase
(ExLeriment
(Ca2
+
Ca2+
below
1
preparations.
1)
Mg2 )
that
the
from
ROV
0.8-
0.7-
0.6C
0.50
6
5
0.4-
4
-C
0.3-
V
3
2
0.2-
o1-
7Z2
0.15
0.20
025
0.30
0.35
0.40
045
0.00
I
I
I
I
1
2
3
4
FREE
FIG.
5
6
(pM)
3. Effect
of Ca2+
concentration
of the activity
of (Ca’
+ Mg2+)
ATPase
at pH 8.5. ATPase
activity
in buffer
containing
75 mM Tris-maleate
and 75 mM glycine
as described
in Materials
and Methods.
represents
net Ca2+.activated
ATPase,
i.e., the difference
between
ATPase
in the presence
and
of Ca’ . The inset
shows
the best-fitting
linear
plot which
yields
an apparent
Km of 5.0 pM. Each
point
the mean
± SEM of three
replications.
was assayed
The ordinate
absence
represents
CALCIUM
a
rate at relatively low
(Fig.
1). Similar
kinetic
obtained
from
PMV
isolated
by
and
significant
stimulation
of
activity
replications.
Ca2
of
pH
a high
to estimate
accurately
especially
at low (<1
activity
re-
Ca2
concentration
properties
were
hypotonic
lysis
pM
± 12 MM free
SEM of three
at
of
and Vmax
n mol.P.mg.h,
specific
9.0
have
parameters
was
The
Km
3). Measurements
physiological
pH,
high
calcium
concentrations
apparent
and 735
pH
others
kinetic
activity
from
Mg2 -ATPase.
showed
a sigmoidal
with
C
a.
of
sperm
in
above
for
Ca2 + -stimulated
and guinea
pig sperm
Barnett,
1967;
Gordon,
3 describes
ATPase
effect
or
and
high
Figure
Mg2)
cyclase
at
1980b)
pH
optima
ATPase
in human,
rabbit
membranes
(Gordon
and
I
and
adenyl
occur
et
ported
E
for
preparations
PM
CALCIUM
TABLE
membrane
1. The effect
ATPase.
of
TRANSPORT
trifluoperazine
(TFP)
BY
BOAR
thiol-group
SPERMATOZOA
inhibitors
and
1065
seminal
plasma
nmoi.P1.mg.h
ATPase
Experiment
Mg2 +
1 (pH
(Ca2
+
(Ca2+
+
Mg2+)
Mg2+)
+
pCMS
Mg2 +
Mg2+
(Ca2
(Ca’
Experiment
Mg’ +
Mg’
(50
+
Mg)
+
Mg2+)
3 (pH
+
pCMS
+
(50
1231
176
176
pM)
+
1223
1041
±
22
22’
97 ± 17
69 ± 20
pCMS
8.5)
(50
MM)
(50 pM)
(25
918
±
420
±
30
13
25’
45
355
±
20
1071
1191
1226
±
368
±
323
221
±
pM)
+
+
+
+
SEM
±
of three
unaffected
by
trifluoperazine
is similar
to
(50
pg/mI)
calmodulin
at
antagonist
physiological
observations
pH.
previously
made
This
cavitation,
obtained
with
by),
the
but
by
in the presence
of
(Peterson
et a!.,
2 and 3) shows
sulfonate
(pCMS;
ROV
were
then
the
50
pM)
obtained
and
quercetin
(25
inhibited the Mg2-ATPase,
had marked
effects
at these
(Ca2
organic
enzyme
experiments
Table
+
Mg2 )
+
mercurial
has
significantly
but neither
concentrations
drug
on
ATPase.
The
inability
to block
the
Ca2 +activated
relevance
to
described
1 (Experiment
the
of the
phosphorylation
below.
4) shows
that
in
(Forrester
purified
and
of45
Bradley,
Ca2
ATP-dependent
observed
in pure
sperm
ram
by
tail
dialyzed
membranes
1980).
was
of
approximately
± 99
89
67
33
±
176
Ca2
of 4SCa2+
prepared
by
was not
nitrogen
the
4SCa2+
that
+
concentrations
by
quercetin.
transport
were
of
pCMS
Thus,
and
Ca2
others
were
transport
and
When
presence
however,
by
partially
inhibited
some
not
total
low
properties
Mg2
shared,
the
activity,
not
TFP.
inhibited
+ild
certain
how
were related.
a pH
was
antagonist
and
while
(Table
had
and
of
ATPase
and pH
pH
completely
was
a rate
Mg2)
transport
affected
by the calmodulin
Unlike
(Ca2 + + Mg2 +) ATPase
transport
(from
with
physiological
and
transport
nmol.mg.h
(Ca2
shows
IOV
populations,
concentrations
+
2 also
of
compares
for
at
of
on
it is assumed
vesicle
17
This
dependent
If
amounts
rate
3).
same
2).
about
nmol.mg.h
Table
and
in these
maximum
was
at the
rapid
(Table
equal
present
Experiment
1).
ATP
Phosphorylation
PMV
uptake
ROV
that
optimum
seminal plasma (SP) or heat-treated SP did not
stimulate enzyme
activity at protein concentrations
as high
as 50 pg/ml.
SP has been shown
to
contain
ca!modulin-like
activity and has been
suggested
to modulate
(Ca2+
+ Mg2+)
ATPase
activity
44
±66
when
4SCa2+
was added
to PMV
hypotonic
lysis (mixed
ROy,
transport
presence
Ca2 +ATPase
or with ATPase
both
high Mg2+
and Ca2+
1983a).
Table
1 (Experiments
that
p-choloromercuriphenyl
pM)
6
replications.
the
(TFP)
±
310±
dialyzed
seminal
piasma
(SP)
heat-denatured
SP (50 pg/mI)
Mg2 )
Mg’ ) + dialyzed SP
Mg3+)
+ heat-denatured
SP
+
P<0.05.
Uptake
±
Mg2
+ pCMS
(50 MM)
Mg’)
+ quercetin (25 MM)
4 (pH 8.5)
+
Mean
the
±
598
+ +
(Ca2+
(Ca2+
Experiment
Mg’ +
Mg2
Mg2
(Ca’ +
(Ca2 +
(Ca2+
proteins
±21
± 10
± 10
895
MM)
+ quercetin
Mg2 )
(Ca2
plasma
7.4)
+ TFP
2 (pH 7.4)
Experiment
was
sperm
on boar
and
of
+ATPase
it is not
ATPase
yet
activities
of PMs
PM5
(ROV)
of
Ca2+
were
and
phosphorylated
Mg2
with
labeled
in
ASHRAF
1066
TABLE
of pH and various drugs on the AT?of 48Ca2 + by sperm plasma mem-
2. Effect
dependent
uptake
brane vesicles.
1
Experiment
2
Control
TFP (25
aMCSfl
6.0
±
0.95
1.10
remove
9.32
±
9.42
a
±
1.00
2.42
8.68
±
1.14
±
0.54’
±
0.72’
±
replications;
rates were
calcu-
from the difference
in 4SCa2+ uptake
in the presand absence
of ATP as described
in Materials
and Methods.
For each experiment,
uptake
in the absence of ATP (triplicate
assay) showed
no significant
difference
among treatments.
Except for pCMS-treated
vesicles
(Experiment
3), uptake
by vesicles
treated
with
ATP
was significantly
higher
than
vesicles
incubated
in the absence
of ATP by at least P<0.05.Experiments
2-3
were carried
out at pH 7.4.
*
compared
ATP
and
discontinuous
separated
system
approximately
tides
absence
labeled
weight
within
(Lane
region
the
42K,
suggesting
was
When
Mg2
4)
the
30-42K
labeled
was
not
noted
This
may
above
(Ca2
(50
that
+
+
Mg2 )
not
while
the band
the experiment.
this
concentration
Mg2-ATPase
ATPase
that
the
polypeptides
was
gels.
Other
in the
activity
region
nearer
responsible
of
may
be
the
same
PMPs
labeled
near
We
of
but
(Table
or
42K
have
pCMS
not
1).
to 42K
for some
as a
near
below
another
PMP
or absence
of TFP
distinguished
in
the
such specific
phosphorylain the presence
of TFP
our
observation
or
high
pH
phosphate
transport
reduced
separation
Ca2+
system
(8.8)
into
intact
gel,
as
have
those
been
intensity.
out
using
at
running
lower
When
a
continuous
pH
(7.2)
(see
Methods),
high molecular
weight
label
and were easily visualized
presence
of Ca2
and
micro-
retained
In
6).
at
study,
such
may
labeling
maintained
and
in the
ATPases,
carried
TFP,
this
proteins
shown
in
by a PAGE
system
bonds,
in
was
Materials
in
net
influx
of
et a!., 1983a).
contained
alkali-labile
present
in
that
used
Since
the phosphorylated
4 and 5 were separated
(Fig.
the
of
(NEPHGE)
band
was resolved
protein
migrated
easily
to
rapid
(Peterson
proteins
and
PAGE
conceniration
the
visualized,
+
increase
labeled
polypeptides
were
2-D
autoradiograph,
but when
was
placed
over Coomassie-
related
induces
sperm
gel
to
phosphorylation
slightly
presence
shall
discuss,
a polypeptide
we
that
no
2-D gels
from
which
it was prepared,
of the labeled
PMPs
could
be identified
stained
proteins,
indicating
that
these
were minor
in concentration
in the PM.
were
to
and
2-D
not
autoradiograph
labeled
weight
were
which
Mg2
Ca2
intense
labeled
This
in the
1-D
visualized
proteins
5).
and
molar
added
was
an
and
Figs.
calmodu-
4B).
68K.
pH
labels
Ca2t
weight
molecular
+ depen-
Ca2
molecular
region
intenseof
higher
containing
inhibited
suggested
contain
most
intensely
pM)
lower
and withmolecu-
absence
molecular
the
in
induced
near
neutral
which
lost
PMPs
labeled,
highest
specifically
only
weakly,
affected
in
significantly
the
that
medium
(Lane
some
was
more
more
pCMS
phosphorylation
within
of
arrow)
was
of polypep-
but
remained
PMP(s)
group
band
ih
visualized
number
(Lane
2).
In the
the PMP(s)
of lowest
3),
dent.
the
+
decreased,
was
the
weight
were
using
the
(1970),
(Fig. 4). Most intensely
labeled
of 3-4
PMPs
with
molecular
region
of 30-42K
(Lane
1).
(near
ly labeled
however,
SDS-PAGE
of
Laemmli
bound
(Fig.
that
this
PM?
(Fig.
As
present
of
these
labeled
proteins
patterns
were
the same
in hypotonic
medium
to
on
Labeling
washed
showed
single
tion
rate.
bands
of Mg2
the 30-42K
in this
group,
lar
by
15
autoradiographs
were
a group
weights
in the
In the
to control
activity
orTFP
(25 pM) in the phosphorylation
containing
Ca2+
and
Mg2+
had
weakly
stained
none
with
PMPs
ATPase
Inclusion
effect
TFP
PMP
the
Mg2)
+
membranes.
permeability
lated
ence
P<0.05
(Ca2
(Lanes
5-6).
in membranes
±
4.06
-1.10
of three
SEM
±
these
significant
3
quercetin
(50 pM)
pCMS
(50 pM)
the
21.30
26.04
4.10
pM)
Experiment
Control
of
in
lin(5
pg)
medium
nmoi.4Ca’.mg.h
Experiment
pH 6.7
pH 7.1
pH 8.5
ET AL.
(Fig.
6, lane
1)
five
migrating
distinct
with
bands
molecular
masses
of 117 (I),
105 (II),
75
(III),
58 (IV)
and 49 (V) K. The high molecular
weight
bands
(I and
II) were
barely
visible
after
addition
of
EGTA
(500
pM)
(Fig.
6,
Lane
2).
Both
bands
were
sensitive
Lane
3)
and
Lane
4).
Bands
dependent
with
to
unlabeled
ATP
hydroxylamine
IV and
but
were
unlabeled
V appeared
not
or
AT?
chase
treatment
to
(Fig.
6,
(Fig.
6,
be
affected
by
by
hydroxylamine
Ca2
+
a chase
treatment.
The
strong
character
Bands
dependence
of the
I and
on Ca2
+
protein-phosphate
II (Fig.
6,
Lane
1)
and
the
acyl
linkage
suggested
of
their
CALCIUM
TRANSPORT
PMP 4
:t:3
.5.
-
BY
BOAR
SPERMATOZOA
..pi
.6
.
j
1067
..2
.
..
.
P1W
3.
4
.5.6
.
-
-
#{149}‘.
.
*
4
,
...
,
FIG.
described
4. Autoradiographs
in Materials
and
of
32P4abeled
PM proteins
separated
Reaction
tubes
contained
210
Methods.
by SDS-PAGE.
pmol
PJATP
[733
The assays
(6-9
were
X 10’
conducted
as
cpm/nmol)
in
p1 buffer.
Proteins
were
separated
by the discontinuous
SDS-PAGE
system
of Laemmli
(1970).
Lane 1, 4
MgCi,;
Lane 2,4mM
CaCI,; Lane 3, 4mM
MgCl,
and 200pM
CaCI, (2 pM free Ca’);
Lanes
4-6,
same
as
Lane 3 plus 50 pM pCMS,
5 pg calmodulin,
or 25 pM trifluoperazine,
respectively.
A) Membranes
are isolated
from sucrose
gradients
and used directly.
B) Membranes
isolated
from
sucrose
gradients
were
suspended
in 5 mM
Tris buffer,
pH 7.4 containing
0.5 mM EGTA
at 0#{176}C
for 30 mm and pelieted
(100,000
X g, 30 mm);
this hypotonic
wash
was repeated
but without
EGTA.
The band
just below
the 42-K marker,
indicated
by a bold arrow,
is
the same band that migrates
near 50 K in Fig. 6.
100
mM
+
P4EHGE
-
+ NEPHGE
.
.
-
3
68Kd
-
-:
...
-
-
.
A
FIG.
teins.
The
5. Autoradiographs
phosphorylation
illustrating
reaction
jected
to 2-D PAGE
as described
ring the reaction
mixture
into
cipitated
stopping”
protein
solution
was
for
the
was
under
1 ml ice
effect
carried
of
out
the phosphorylation
of PM proPM proteins
were treated
and subexcept
that
the reaction
was stopped
by transferacid and incubated
for 20 mm at 4#{176}C,
The pre(pH 7.4) and then
solubiized
in the 50 p1 “SDSTFP (20 pM).
trifluoperazine
as in Lane
Materials
and Methods
cold
20% trichloracetic
washed
2 times with 50 mM Tris-HCI
2-D PAGE.
A) Absence of TFP; B) plus
(TFP)
3, Fig. 4. The
on
ASHRAF
1068
ET AL.
has
been
PM
(Bradley
identified
al.,
1982b;
a
Ca2
described
105 Kdi
II
75K-u1
ia
and
(this
has
58Kd--’
boar
major
mechanism
been
able
AT?
intact
has
al.,
1982a;
Peterson
difficulty
in
ATPase
phorylation
with
as described
hydroxylamine
under
after
Materials
and
phos-
Methods.
in
ability
ty
in
bull
Rufo
Sydney,
been
to
sperm
PM
to
3-fold
However,
relationship
of
to
a
Ca2
molecular
to
the
phosphorylated
transport
weihts
other
of
Ca2
these
Moreover,
proteins
transport
the
were
ATPases
similar
IV
and
substrates
for
From
inspection
Band
V
near
42K
weights
in
(Penniston,
6
was
Fig.
4.
the
could
be attributed
content
different
of
buffers
to
the
same
band
to
kinases.
patterns,
that
the
labels
molecular
PMs
+
PMs
by
(Gordon
and
addition
to
in
each
present
in
guinea
inhibited
by
organic
ATPase
ological
polyacryla4
and
6
and
used.
pH
activity
the
Figs.
is
Ca2
tions,
and
the
may
estimate
for
1983)]
is
easily
when
assays
enzyme
has
ATPases
on
of
guinea
pig
methods
Gordon,
1973).
optimum
of
(pH
these
pig
by
not
n-ethyl-
has
described
it
which
affinity
the
was
nor
have
In
of
9.0)
species,
sperm
distinguish
higher
also
least
membranes
and
we
and properties
relevance.
The
for Ca2 + is
Ca2 +.ATPase
than
help
at
at
concentrations.
+
mercurials
enzyme
have
described
1967;
enzyme
to
different
activity
activity
acrosomal
high
of
may
enzyme
enzyme
properties
in
1983)
immunocytochemical
the
1982;
(Penniston,
sperm
Barnett,
differed, these variations
gels
outer
rabbit
al.,
University
minimum
low
in-
activi-
specific
+activated
the
The
DISCUSSION
Ca2
on
maleimide.
activities
potentially
associated
with
transport have now
been
identified
in the
of mammalian
sperm:
Ca2 +/Na+
exchange
Several
Ca2
and
sperm
corresponded
Although
of these bands
mide
the
likely
Ca2 +dependent
protein
of Coomassie-stained
Fig.
in
V very
localized
human
1983).
Bands
by
a
enzyme
differentiated from Mg2 +ATPase
are carried
out
at high
H.
The
properties
similar
to Ca2
-dependent
intermediates
ATPase.
the
the
et
nmol-mg.h
et
Mg2-
Goh,
low
the
previous
perhaps
this
Enzyme
is inhibited
and
and
Jane
very
[600
with
Our
dissertation,
than
cells
IOV
efflux
(Ashraf
(Vijayasarathy
pH.
lower
other
into
boar
1983a).
PM,
(Dr.
its
physiological
on
Ca2+
identify
1984)
and
efflux
influx
the
al.,
the
ram
Ca2-stimulated
others
in
On
associated
sperm
doctoral
due
and
identifying
et a!.,
Na+.
ATP-driven
in
a
have
dependent
1983).
et
boar
of
is not
we
plasmolyzed
observed
a!.,
exchange
or
apparently
been
et
energy-dependent
sperm
PM,
al.
in uncapac-
since
transport,
+
a!.,
and
et
antiport
Ca2+
ROV
report),
in
treated
et
Ca2-loaded
(this
were
Ca2
Ashraf
significant
from
show
(Breitbart
from
branes
lOV
sperm
Breit-
least
+/Na+
with
been
1980b;
Peterson
at
transport
observe
loaded
hand,
bull
FIG. 6. Autoradiograph
of PM proteins
phosphorylated at 4#{176}C
and separated by SDS-PAGE
buffered at
pH 7.2. Phosphorylation
at a specific
activity
and concentration
of ATP
indicated
in Fig. 4 was carried
out
as described
under
Materials
and Methods:
Lane
1, 13
pM MgCI3
and 100 pM CaCi,;
Lane
2, 13 pM MgCl2
and 500 pM EGTA;
Lane 3, same as Lane
1 except
reaction
was
carried
out an additional
30 sec in presence
of 1 mM ATP;
Lane
4, same
as Lane
1 except
mem-
to
cells
other
Ca2
for
same
Ca2 +ATPase
and boar
PM
bull
that,
et
the
has
and
1982;
view
sperm,
in
ATPase
al.,
sperm
Ashraf
Forrester,
1983)
is our
bull
and
Mg2 +independent
described
in
et
It
intact
1984)
Rubinstein,
itated
not
49Kd1
a!.,
and
(Vijayasarathy
and
1980a;
+stimulated
report).
also
been
1983b).
&
et
(Bradley
barr
117 Kd-’-
boar
Forrester,
Rufo
species,
I
in ram,
and
from
Mg2-
may
have physiof the enzyme(s)
than
a Mg2 + -independent
present
in these
prepara-
be
of
pH
where
the
difference
and
the
Ca2
+stimulated
higher
at
5.0
pM
physiological
made
between
enzyme
pH
at
Mg2
alkaline
+ATPase
were
easily
CALCIUM
distinguished.
Most
ATPases
TRANSPORT
associated
range.
activity
at
While
the
affinity
physiological
for
Ca2
is
which
lin.
apparently
Such
have
regulators
been
ATPase
of
rat
)
has
it
of
activity
of
pH
Since
near
by
and
the
and
tinct
domains
ferent,
the
PM
also
Ca2
membranes.
may
Mg2+)
ATPase
(Breitbart
solving
enzymes
this
studies
described
and
several
PMPs,
Ca2+,
and
that
isolating
lated
the
properties
Mg2)
ATPase
in
Mg2
+
in
common
activity
we
the
ATPase.
be
plasma
of
these
ton,
to
active
proteins
are
ATPase
1983).
shown
to
molecular
consideration
the
less
than
These
one
that
of
any
proteins
stability
have
been
chain
with
100-150K.
of the
Ca2
(Pennis-
Peuch
This
“40-50K
boar
plasma
other
Ca2
+
muscle
is
+transport
Mg2)
neutrophils
ATPase
of
sarcoplasmic
and
regulated
1979;
mem-
to calmodulin
heart
a!.,
to
spermatozoa.
sperm
(Ca2
not
and,
of calmodulin
ATPase
transport
in
the
the
by
Chiesi
to
presence
Since
with
PM
phosphorylation
TFP
corpus
calmodulin
and
is
Carafoli,
the
play
protein
a role
interacts
1982),
1983a),
in
the
is involved
that
at
Ca2+
only
in
that
a
the
a concentra-
influx
suggest
that
physiological
and
regulation
be
it is possible
no
observations
net
to
nonspecifically
occurs
induces
a!.,
phosphoryremains
phosphorylated
phosphorylation
et
PMP
TFP
has
But
PMP
the
of
(Roufogalis,
that
the
1982a)
in
are
are
the
If
a!.,
anthat
not
Iwasa et a!., 1982; Verma
and Penniston,
Ochs
and Reed,
1984).
The relationship
Ca2
may
et
efflux
liver,
et
calmodulin
previously
ROV
is also
evidence
boar
as
rat
(Peterson
+
Mg2)
which
intense
weight
+
such
tion
+
the
muscle,
single
in these
molecular
polypeptide
between
the
pH
enzy-
phosphorylation
no
There
significance.
+stimulated
high
Ca2
of
skeletal
lated
pH
weakly
of
the
reported
from
(Ashraf
ATPases
of
remain
in
by
have
efflux
find
of
established.
high
characterized
transport
weights
and
far
Ca2
at
But
thus
contain
the
of
can
unique.
+
inhib-
PMPs
assay
exception,
We
(Ca2
1982;
1981;
inhibits
We
these
a direct
affected
by TFP
we
regulation
(Le
concentra-
PMPs
affected
+
phos-
weakly
These
most
membranes.
transport
low
conditions
related
+
for
not
tagonist
TFP.
ATP-dependent
luteum
The
significantly
only
under
(Ca2
remains
of
which
the
proteins.
isolate
2-D PAGE
in order
to raise antiThis will permit
their
reisolation
notable
was
reticulum,
have
described.
one
PMPs
not
Ca2
and
with
have
presence
but
Mg2 tATPase
alone
PMP(s)
a drug
of
are
acyiphos-
activity.
brane
of
of
chromatography
Insensitivity
The
presence
of
Mg2)
may
the
presence
phosphorylated
and
in
re-
SDS-PAGE
properties
transport
to
detergents
date,
identified
presence
pCMS,
+
have
+
these
activity.
the
Mg2-ATPase
(Ca2
isolate
in
in
of
to
report
weight
phorylated
its
+
approach
their
this
(Ca2
hydroxy-
by
lability
of
after
diminished
These
attempting
or
with
markedly
alkaline
immunoaffinity
With
in these
transport
to
currently
is
also
re-
ATP
separated
the
closely
proteins
treated
conditions.
intermediates
are
more
phosphorylation
also
are
PM
molecu-
unlabeled
are
+de
labeling intensity
the
is
but
Ca2
in these
of these
with
PMPs
for
properties
Their
when
make
100-120K
Labeling
with
phate
of
to
intensely
molecular
tions
be
phosphorylated
several
low
An
would
alkaline
dif-
permit
a start
toward
identifying
such
proteins.
Several
PMPs
and
“105-120K)
are phosphory-
more
in
1983).
the
matic
difficulties
PMs
when
under
denaturing
+ATPases
the
have
chased
chases
proteins,
present
in the
region
is
by
dis-
relating
sperm
reconstitute
(‘\‘40-50K
than
a!.,
activ-
kinases
Ca2 + dependent.
is not observed
ATP
transport
substrates
to transport.
by preparative
bodies
to them.
pH
be present
in
ram
problem
a
in
functionally
explain
weight
related
cold
are
Proteins
consistent
with
Mg2
others
in
et
be
may
may
by
+
differentiated
+-stimulated
This
+
morphologically
with distinct properties
encountered
(Ca2
ATPase
highly
shows
that
several
1981).
determined
on
are
protein
Labeling
physiological
mercurial
pendent
membranes
This contrasts the
pCMS.
they
lamine.
consistent
optimum
that
labeled
Mg2+)
+
preparations,
as
are
inhibitors)
al.,
PM
entirely
spermatozoa
cells
boar
to
they
action
total
Transport
pH
a
abolished
et
and
that
by calmodu(Ca2+
the
in
not
is
effects
ity.
(Lotersztajn
function.
IOV
and
liver
are
transport
the
1069
alkali
suggests
lar
high,
may be
of as
transport
and
for
properties
ATPase
however,
controlled
(activators
described
Several
Mg2
is not
to
preparations.
ATPase
specific
activity
is low. This conceivably
due to the absence
in these
preparations
yet
unidentified
regulators
for
this
SPERMATOZOA
it unlikely
values
pH
physi-
of
BOAR
PMPs
with
Ca2 + extrusion
pumps
have apparent
Km
in the submicromolar
range
at physiological
(Penniston,
1983)
and pH optima
in the
ological
BY
into
cells
this
PMP
that
of
transport.
calmodulin-depen-
dent transport, then it may
bind
to calmoduiindependent
proteins. Experiments
to identify
PMPs
that
interact
with
labeled
calmodulin
are
in progress.
1070
ASHRAF
Gordon,
ACKNOWLEDGMENTS
This
from
the
study
was supported
NIH. The authors
assistance
daram.
of
Mr.
Wallace
a grant, HD 10947,
acknowledge
the able
by
P.
Hunt
for
Dr.
K.
S. Sun-
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