BIOLOGY
OF
REPRODUCTION
16,
(1977)
377-384
Characterization
of Sea Urchin DAVID
Departments
of
Vanderbilt
Sperm
Adenylate
Cyclas&
L. GARBERS2
Pharmacology
University
and School
Nashville,
Physiology,
of
Tennessee
Medicine,
37232
ABSTRACT
(Lytechinus pictus) sperm adenylate cyclase was inhibited by the methylxanthines, 1-methyl-3-isobutylxanthine (MIX), caffeine and theophylline. MIX was the most potent inhibitor (I, = 1.4mM) while caffeine and theophyiline were about equal in potency (I, = 15 mM). A more detailed kinetic analysis with theophylline indicated that it was a linear competitive inhibitor Sea urchin
with
respect
to
MnATP. but not
30#{176}, and Mn2+, plots were linear these
data
which
also
both that
The Mg2
sperm or
as a function were
adenylate cyclase accelerated
of either
linear.
Thus,
free
the
Mn2+
was inactivated by temperatures this rate of denaturation. Double
or MnATP,
and
slope
and
sperm
adenylate cyclase conforms a Mn2+ regulatory site exist.
intercept an
enzyme
as
replots
of
model in these data
substrate site and Furthermore, is not a potent inhibitor of the enzyme. When the concentration of total was less than the concentration of total AT?, Ca2+ and Mg2+ were capable of activating the enzyme, but when the concentration of total Mn2+ exceeded that of ATP, both Ca2+ and Mg2+ acted as inhibitors. Various phosphorylated nudeosides (GDP, AD?, GTP, dGTP) inhibited the suggest
a MnATP free AT?
to
as low reciprocal
enzyme, pymvate,
but adenosine and guanosine were without effect. A number of carbohydrates (acetate, glucose), fatty acids, and fluoride also had little, if any, effect. Two polyamines, cadaverine (2mM) and putrescine (2 mM) caused a slight stimulation (‘\30 percent) of enzyme activity. Under a variety of conditions, solutions containing factors released from sea urchin eggs either had no effect or inhibited the sperm adenylate cyclase.
Hoskins, well-known many types
INTRODUCTION
Although adenylate 1971;
spermatozoa cyclase (Casillas
Gray,
1971;
1975;
Garbers
1976;
Towns
and
et
Tash,
1976; in the
slightly
regulation
increase
AMP
in
1970),
but if any,
little, hormones
monkey
on
al.,
the
1975a;
1976)
studies effects
sperm
adenylate
of
al.,
in spermatozoa plished by the phosphodiesterase
phosphodiWells and mechanisms
1971).
AMP
sea
accumulation
various direct
et
metab-
and
Hoskins,
1
obtained
mm
after
1976); this any substance,
However,
despite
sperm
cyclic
addition
had been on sperm AMP
accom-
or other et al., from
to increase sperm much as 100-fold
1975b, that
methylxanthines, marked effects
(Casillas
Accepted November 4, 1976. Received September 28. 1976. ‘This study was supported by NIH grants HD and HD 10254. 2lnvestigator for the Howard Hughes Medical tute.
substance(s)
eggs were shown AMP levels by as
Hardman, first time
since have shown a wide variety of cyclases
Recently,
within
cyclic
generally has been of methylxanthines inhibitors (Garbers
use
urchin
cyclic
Thyroxshown of
a in also
has been shown not to stimulate sperm adenylate cyclases (Casillas and Hoskins, 1971; Gray, 1971). Thus, the elevation of cyclic AMP levels
1973;
adenosine
remain unclear. were initially (Casillas
1970,
Gray
and
1976) the of cyclic
sperm
contain
Albagli,
(cyclic AMP) (Gray et al., 1976;
olism in the sperm cell ine and triiodothyronine to
and
Luck,
3’,S’-monophosphate esterase activity Garbers, involved
are known to and Hoskins,
Morton
Braun,
1971; Gray, 1971). Fluoride, activator of adenylate cyclase of broken cell preparations,
and
(Garbers
and
represented including
the the
shown cyclic
these
marked
levels,
solutions
to have such AMP levels. effects
on
containing
the egg factor(s) either had no effect or inhibited the adenylate cyclase of broken sperm cell preparations (Garbers and Hardman, 1976). These apparently inconsistent results could be
05797
due, about
Insti-
sea
377
at
least the
urchin
in part, properties or
other
to our of sperm.
lack
adenylate
of information cyclase
from
GARBERS
378 MATERIALS
AND
METHODS
creatine
L. Pictus sea urchins were purchased from Pacific Bio-Marine, Venice, CA. The unlabeled nucleotides and fatty acids were obtained from Sigma or Boehringer and the tritiated [311-2]-ATP (25 Ci/nmole) from Schwartz/Mann. The metal chlorides were from Fisher, 1-methyl-3-isobutylxanthine from Aldrich, and theophylline and caffeine from Merck. Preparation
ence tions
pellet was resuspended in the TEA-DTT buffer and centrifuged again at 36,000 X g for 1 h. The resulting pellet was diluted to 0.01 percent based on the original wet weight with the TEA-OTT buffer, and then frozen in 5-mI aliquoss at -70#{176}.The enzyme was stable at -70#{176} for at least 3 months, but was not stable to repeated freezing and thawin& For this reason, enzyme not used after one thawing of a 5-mI aliquot was
of
of 1.0 mM ATP, adenylate cyclase were linear for at least 10 mm. Thus,
reacboth
could
effects on adenylate NaN3 (20 mM) nor
direct
Neither
mM)-creatine initial
(data
not
enzyme centrations the
shown).
general adenylate cyclase assay mixture con40 mM TEA at pH 7.9, 1.0 mM cyclic AMP, 20 mM sodium azide (NaN3) 0.5 mM ATP, 6.0 mM MnCI3, 333 ig sperm particulate protein, and 1.01.5 X 10 dpm of l HI AT? in a final volume of 0.25 ml. In some instances, 10 mM creatine phosphate and 300 Mg/rnl of creatine kinase replaced the NaN3. Enzyme reactions were linear for at least 20 mm at 23#{176}under these conditions, and were linear as a function of added protein between 67 and 660 g. Reactions were stopped by the addition of 2 ml of 0.5 N perchloric acid, and the cyclic AMP was then purified on acid alumina and neutral Dowex-50 (l1-form) columns (Jakobs et al., 1976). Of the 6 ml collected from the Dowex-50 column, 1 ml was used The
tained
for
the
determination of the remaining cyclic AMP (absorbance at 259 nm) while counted for cyclic E3H] AMP in a liquid counter. Cyclic nucleotide recoveries after were generally about 65 percent. Prior to the selection of the above tions, a number of potential constituents adenylate cydase assay mixture were
determine directly presented
whether affected in the
or not enzyme Results.
any activity.
of
kinase
or
Two
of ATP systems
percent
tion under
remained standard
at
NaN3
and
creatine
to
represent
preservation
when
of
With
the
initial
the assay
the
end of conditions. useful
ATP assay
the
et
al.,
1971),
The
1976);
the
some
instances
inhibit adenylWeinryb and of
the
primarily sperm
in
at low
cyclase
sea
hydrolysis the
homogenate
thus
adenylate
in
for
exists
of
are inhibi-
phosphodiesterase
responsible
Garbers,
the
sperm
methylxanthines
also have been reported to cyclase (Sheppard, 1970;
fraction
urchin
phosphodiesterase
(Robison
AMP
for
Degradation
they ate
cyclic
kinase
in sea
AMP
various
1971).
incubation Thus both
mixtures.
to be effective
sperm
system,
concentra-
systems
levels
of Cyclic
urchin
either
phosphate-creatine
cyclase
Michel,
either
ATP
tors
of soluble
(Wells
protein
activity,
and
concentrawhich
is par-
>
06
TI-4EOPHYLLINE
CArFINE
assay
40 20
2
FIG.
cyclase
Levels to preserve
conditions
when using phosphate-creatine
below).
of
appear
adenylate
obtained creatine
(see
80
scintillation purification
condiof the studied to the added agents These data are
NaN3, a nucleoside and 2) creatine
altered
these
4
6
8
0
12
[IN4I8lr0R1
designed
were studied: 1) phatase inhibitor
pg/mI)
under
Furthermore,
were the
system
over
tions,
conditions
cyclase activity. creatine phosphate
unlabeled the rest was
RESULTS
Maintenance
these
(300
rate
in
kinetics were studied at MnATP conranging from 20 pM to 2.0 mM, the
results NaN3
known
under
kinase
reaction
Although Assay
tested
and
In
pres-
be
system
for
same
system.
the
Prevention
discarded.
ATP-regeneration
either
systems
the
Sea urchin sperm (Lytechinus pictus) were collected and washed as described previously (Garbers et al., 1975a). The washed cells were diluted to 0.01 percent (wet weight/volume) in a solution containing 25 mM triethanolamine (TEA) and 2 mM dithiothreitol (DTT) at pH 7.6, and homogenized with an Ultraturrax (10 sec intervals, 10 times) at 0-4#{176}. The suspension was centrifuged at 36,000 X g for 1 h, and the resulting supernatant fluid was discarded. The
an
absence
(10 Enzyme
kinase,
the
ATP
levels
triphosphosphate-
1.
by
Inhibition
of
methylxanthines. mixtures contained
sea
6
4
IS
20
Ir,,M)
urchin
sperm
Adenylate 0.4 mM AT?
adenylate
cyclase reaction and 4.0 mM total 4j2 and were otherwise as described in Methods, except that various concentrations of methylxanthines were added as indicated. The control enzyme activity in these experiments was 9.1 pmol cyclic AMP formed/mix/mg protein.
SPERM
ADENYLATE
CYCLASE
Under
379
the
same
or theophylline Theophylline was
to
respect
10.1
mM.
tus
2. Linear competitive inhibition adenylate cyclase with respect
of sea urchin to MnATP by
sperm theophylline. Adenylate cydase reaction mixtures contained 4.0 mM free Mn and were otherwise as described in the text except that theophylline and AT? were varied as shown. Reactions were for 10 mm. at 23#{176}.The inset is a replot of the slopes of the primary double reciprocal plot.
ticulate, both
can
AMP
“cold
cyclic
AMP
reaction
estimated
was 1.4
mM
all 1);
or
absence most an
the
absence
did
and
these not
the
shown).
and
1-methyl-3-isobutylxan-
of
ATP
1.0
mM
concentration
inhibitor
2)
with
a
Ki
of
(1976)
labile,
rates
were
However,
We
370
sea not
that sperm
that
linear
possible
at
23#{176}the
product
was
that
S-purpura-
reaction
found
urchin
and
at
linear
of time
reported
cyclase
rates the
was
for
reaction
either
30#{176}or
was
quite
accumulation
possible
extremely
enzyme
enzyme
as
at least
were
adenylate
37#{176}.
stable,
a function
20 mm.
of
initial
However,
inhibitor,
(Fig.
mM. and
a cyclic
not
potent
caffeine
15 detail
conditions,
alter
inhibited adenylate this occurred in
the at
Under
mM)
(data
(MIX) (Fig.
presence MIX
trap”.
caffeine
thine activity
in inhibitors
(1.0
rate
phylline,
of
be
phosphodiesterase
al.
from
heat
and
for
competitive
MnATP
adenylate at
cyclase
FIG.
be a linear to
et
sperm
non-linear
I/MnATP
150
Stability
Gray 0
the
was approximately was studied in more
shown
with
Thermal
conditions,
theo-
cyclase either the
cyclic
AMP.
with of
an 0.4
mM.
3
S
60 PREINCUBATION
g
FIG.
the
FIG. 3. The effects of Ca2 and Mg2 on sea urchin sperm adenylate cydase at various Mn2+ concentrations. Reaction mixtures contained 0.5 mM AT? and were as described in Methods except that or jg2 (6.25 mM) was added to some of the reactions as shown.
and the total All reactions
Mn2+ concentration were for 15 mm.
was
varied
at 23#{176}.
rate
adenylate protein) no added mM (2+
4.
The
TIME
(mm)
AT
30#{176}
effects
of Mn2, Mg2 and Ca2’ on denaturation of sea urchin sperm cyclase. Sea urchin sperm particles (5.3 mg were preincubated at 30#{176} in the presence of metal, 1.8 mM Mn2, 1.8 mM Mg2 or 1.8 in a final volume of 0.55 ml. At the times
of thermal
indicated in the Fig., 50 td aliquots were removed from the preincubation mixture and assayed for adenylate cyclase activity as described in Methods. The basal adenylate 0-2#{176}) was 20.2
protein.
cyclase
pmol
activity
cyclic
AMP
(preincubation
formed/mm/mg
at
380
GARBERS
Metal
Requirement
The
sea
highly detectable
Thermal
urchin
sperm
dependent (rates
on less
Mn2+.stimulated observed mM not
Mg2, shown).
putrescine absence or
activity ceeded (putrescine tions when
the
(2
mM)
Mn2.
Mn26 ATP
activity
0.5
mM
Co2’, activity
presence
of
or
cadaverine
The
addition
increased
when total the total
of
Ca2, Ba2, Enzyme
of
Mg2+
enzyme
presence
in
cyclase
apparent
ATP
The
No the
preincubation
6
polyamines, (2 mM)
of
either
enzyme
concentrations concentration,
increase
enzyme
< [ATP1.
addition
of
This
the
rapidly
Mn2,
enzyme form
and that
of
of
assuming
that
changes
the
is less
not
during
assay
(Fig.
Mg2
or
denaturation
effect
by
to
but
rate
destabilizing
explained
inactivated
30#{176} prior
stable
(Fig.
Mn2 Mn2
binds
higher
4).
could
be to
conformation
to
4).
Ca2,
the
to
a
temperatures.
Ca26 activity
were less than but depressed
apparent
was
at
in the
when total Mn2+ concentrations extotal ATP (Fig. 3). The polyamines and cadaverine) at 2 mM concentra-
did not [Mn2Jt
The
enzyme
accelerated
was and
or Ni2 (data also was not the
Denaturation
was
Mn26 for activity. than 0.5 percent of
rate)
in the
detectable
adenylate
activity
Kinetics
With kinase
either the creatine ATP-regeneration
shown)
or
of
NaN3,
MnATP
intersecting sperm MnATP
and
(Fig. adenylate catalytic
phosphate-creatine system (data
reciprocal free
5a,
plots
Mn2+
b).
site
were
This
cyclase
as
indicates contains
and
a
separate
not
a function linear
and
that
the
both
a
Mn26-
C
C V
LI.
a. 4 ‘I 5-
U I’
0
a. >.
6 U
0 -J
Lu >
20 I/MnATP
2.0
30 (rnMi1
I /Mn(mM)1
FIG. 5. Double reciprocal plots showing the effects of M.n2 and MnATP on sea urchin sperm adenylate cydase activity. Reactions were as described in the text except that [ATPltotai and [Mn2i free were varied as shown in the Fig. Analyses of the kinetic data were as described by Gathers et al. (1974) and Gathers and Johnson (1975). Replots of the slopes and intercepts of the primary double reciprocal plot are shown as insets. Although these data were obtained using NaN3 as an AT?ase inhibitor, the sante type of linear and intersecting double reciprocal plots were obtained when using the creatine phosphate-creatine kinase ATP-regeneration system.
SPERM
regulatory
site
Secondary
plots
(Garbers
of
and
slopes
were linear; thus free act as a potent enzyme Johnson,
1975).
fined
according
to
sperm
adenylate
cyclase
Johnson,
and
kinetic
not appear (Garbers parameters,
(1963),
Cleland were
CYCLASE
1975).
intercepts
ATP does inhibitor
The
ADENYLATE
for
381
theophylline
also
nucleosides
to and
known
facieus
de-
1965,
1967),
the
drates
did
(Table
1).
as follows:
(Table
1),
but
inhibited
the
enzyme.
activator
of
adenylate
not
=
MnATP
=
O.9SmM;KM
sea
3.6mM
=
mM;
and
KMn
8.9
=
elevate
sperm
the
sperm
of
Other
Agents
Adenosine ranging
or
from
appreciable in
affect
either
the
TABLE
1. The
presence
guanosine
0.02 the
to
sea
concentrations 2
urchin
or
presence
effect
at
mM
of various
mM
did
sperm
not
enzyme
absence
of
compounds
4
of
Ca2,
Mg2
only
observed
tion
sperm
to
The were
NAD,
and of
solutions
tested
adenylate
acids
cyclase
cyclic
(arachidonic,
AMP
form
ed/mm/mg
Theophylline
phylline
...
74±3
72
±
4
68
± ±
1 2
71
Cyclic
liMP
2.0
80
IMP TM? XMP dGMP
2.0 2.0 2.0 2.0
76±6 74±1 69±4 71 ± 5
S-Ado-Met GD? AD? GTP dGTP Cadaverine
2.0 1.0 1.0 1.0 1.0 0.2 2.0
96±3
Putrescine Acetate Pyruvate Glucose Triton X-100
2.0
92
2.0 2.0 2.0 2%
76 83 86 66
Fluoride
8.0
86
KH2PO4 FRE
2.0
79
...
...
aReactions
as
described
listed and of
82 79 68 80 83 64
... ... ... ...
...
68 76 59 64
43
...
54±3 52±2 55±2
56
57 51 28 78 96
... ...
2.0 20.0 were
protein No Theo-
GM?
various compounds with theophylline tus eggs.
arachi-
activitya.
4mM
+
Cyclic
Theophylline
the inhibi-
was
tration mM
0.02 0.20 2.0 0.02 0.2 2.0
Guanosine
the GTP,
shown).
of fatty
Pmol
None Adenosine
in
instances
factors
Concen-
Compound
and inhibit
of
in all
the
known
(Garbers
concentrations
effect
A number
on sea urchin
from
are
shown
cyclase.
various
not
levels
also
enzyme
released
were
factors
and
(data
mM
AMP
1976),
the
carbohythe
or all of which
adenylate
containing Effects
affect
cyclic
1975b,
Hayaishi,
other
factor(s)
some
a lique-
and
various
appreciably
eggs,
Hardman,
mM.
(Hirata as
containing
urchin
to 2.3
well
Pyruvate,
Brevibacterium
cyclase as
Solutions KMnATP
the
phosphorylated
±
4
94
±
±
2 5 2
81 73 84
±
5
72
±
5
88
±
2
...
±
35 78 41
... ...
in
Methods
above were 2 observations
added
([ATPI
total
to the reaction
without
theophylline.
=
0.4
mixtures. FRE
mM;
1Mn2I
Data represents
total
are means factors
4.0
mM)
except
of 4 observations released from
that
the
(± SEM) S. purpura-
382
GARB
dic, n-caproic, palmitoleic, myristic, and above
fatty
were
tested
activity; 10%
effects
effects
inhibition. to
on
(0.25
ml
sulfoxide, cyclase
which activity.
final
the
vol.)
acids
5
il
inhibit
to were
of
dimethyl
adenylate
obtained
cles
(see
bath
for
during
Methods)
10
were
in the
In
also
in
the
the
parti-
a boiling
tested
of
some
supernatant of
heated
and
effects
tightly or that
sperm.
first
preparation was
mm
No
fluid
were bound not eluted,
do not exist experiments,
fractions.
mixture
basal
or that activators column and were
activators additional fluid
effect
reaction
in
not
no
fatty
cyclase
did
cyclase
from
of
adenylate
also
adenylate
ranged All
the
the
at 24 j.zM concentrations
for the
added
filtration to the
palmitic, 11, 14-eicosadienoic, oleic, 1 1,14,17-eicosatrienoic, all of the methyl esters of acids)
ERS
on
water
the
heated
column
supernatant
observed.
DISCUSSION
Detergent-Dispersion The and
enzyme
applied
The
and was
to
peak
nation
of
(#7)
near other
resulted
6).
Thus,
separated
it from
Filtration
solubiized
BioGel
with
A0.5
detergent-dispersed
single
Gel
m
void
fractions
essentially appears the
that
WX
(Fig.
migrated
volume. with
Lubrol
columns
activity the
The
The
peak
the
in additive activators
adenylate
6).
as a recombi-
fraction
by
with the consistent a MnATP site exist
substrate (Garbers of
enzyme
gel
by
model.
a
ATP.
This
with
earlier
and
tion
by
the
model
brain 8
suggested
by
and
in
Hammes
The
FRACTION
(Lin
et
al.,
inhibi-
activator
site
agree
with
detergent-dispersed
with
and
the
Johnson,
model
(1976)
proposed
with
respect
to
site.
mechanisms
involved
in
cyclase factors
the
regulation
remain associated
unclear. with
eggs, although causing marked elevations in the sperm cyclic AMP levels (Garbers and Hardman, 1975b, 1976) appear only to inhibit adenylate
(9.1-mi)
FIG. 6. Gel filtration of the Lubrol WX-dispersed sea urchin sperm adenylate cyclase on a BioGel A0.5m column (2.6 cm X 34 cm). Sea urchin sperm particles were prepared as described in Methods, then resus-
in a solution containing 25 mM amine, 1mM dithiothreitol, and 1% Lubrol 7.6. The suspension was allowed to set on mm. and then centrifuged at 36,000 g for
cardiac
potent
(Garbers
Rodbell
free agree
adrenal
liver
metal
the
of the sperm adenylate Solutions containing
2
pended
for
general
and
the
essentially
cyclase
a metal-activator 4
or
no
does
not
1974),
where
and
but
by
for
1975)
cyclases,
adenylate
1975),
6
models
ATP
evidence
does
(deHaen,
the above
no
model
Rodbell,
free
the
(K1