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INTRODUCTION |
Two types of adenylyl cyclase
(AC)1 are ubiquitously
expressed in mammalian cells, a well characterized gene family of
transmembrane ACs (tmACs) and the recently discovered "soluble" AC
(sAC). The tmACs are plasma membrane bound, and their activities are
regulated by G proteins in response to extracellular stimuli such as
neurotransmitters and hormones (reviewed in Ref. 1). In contrast, sAC
is associated with various intracellular organelles, including
mitochondria, centrioles, mitotic spindle, mid-bodies, and nuclei (2).
sAC activity is modulated by bicarbonate (3) and, as shown in this report, by Ca2+; regulation by these intracellular
signaling molecules suggests that sAC mediates
cAMP-dependent responses to intrinsic cellular changes (4,
5).
The catalytic mechanism of tmACs has been determined from biochemical
and crystallographic studies. tmACs convert ATP to cAMP using two-metal
catalysis where one ion acts as a free metal and the other coordinates
ATP in the active site (6, 7). Its activators, G
s
subunit or forskolin, stimulate tmACs by allosteric modulation of the
active site (8, 9). More than 25 years ago, when soluble AC activity
was first discovered, it was predicted to be molecularly distinct from
tmACs because its activity appeared to be dependent on the presence of
the divalent cation, Mn2+, and it was insensitive to
forskolin and G protein regulation (10-12). These differential
properties enabled purification (13) and cloning of sAC from rat testis
(14). The sAC gene is indeed molecularly distinct from tmACs; it
possesses no transmembrane domains, and its catalytic domains are more
closely related to those of cyanobacterial ACs than to those from other
eukaryotic ACs. The purified soluble AC exhibited ~10-fold lower
affinity for substrate ATP relative to tmACs (tmAC
Km for ATP-Mn2+ is ~ 100 µM, whereas purified rat testis sAC Km
for ATP-Mn2+ is ~ 1 mM), and the
activity of the heterologously expressed, cloned sAC gene product is
insensitive to forskolin or G proteins (14).
Soluble AC, as the predominant, if not only, source of cAMP in
sperm, was predicted to be responsible for the cAMP changes induced by seminal and oviductal fluids (and mimicked by in
vitro fertilization (IVF) media) necessary for fertilization of an
egg, capacitation, hyperactivated motility, and acrosome reaction
(15-18). Two essential components of defined IVF media are bicarbonate and calcium, and we previously demonstrated that sAC is directly stimulated by physiological levels of the bicarbonate anion (3). The
role of calcium in IVF media is less clear because of contradicting reports detailing Ca2+ modulation of sperm cyclase
(19-21). We performed kinetic analyses on purified recombinant 48-kDa
truncated human sAC protein (sACt) fused to GST. This
truncated protein corresponds to a splice variant of the sAC gene (22)
that consists almost exclusively of the sAC catalytic domains and
corresponds to the native isoform originally purified from testis
cytosol (14). We identify a synergistic interaction between bicarbonate
and calcium ions, where bicarbonate functions to increase the
Vmax of the enzyme whereas calcium increases its
affinity for substrate ATP-Mg2+.
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EXPERIMENTAL PROCEDURES |
Chemicals--
ATP, chlorpromazine, and LaCl3 were
purchased from Sigma; all tissue culture reagents were from Invitrogen
and [
32P]ATP and [3H]cAMP were from
PerkinElmer Life Sciences.
Cloning of Human GST·sAC--
Human sACt was
subcloned using gateway cloning technology (Invitrogen) into a
baculovirus expression vector utilizing the polyhedron promoter to
generate an N-terminal glutathione S-transferase (GST)
fusion protein. Recombinant GST·human
sACt-expressing baculovirus was produced in adherent SF9
cells (Bac-to-Bac baculovirus expression systems, Invitrogen), and
identity of the resultant fusion protein was confirmed by Western
blotting and enzymatic activity.
Expression and Purification of GST·sAC Fusion
Protein--
Hi-Five cells, grown to a density of ~1.0 × 106 cells per ml, were infected with GST·human
sACt baculovirus. After 48 h, cells were pelleted,
resuspended in lysis buffer (phosphate-buffered saline with 1 mM EDTA, pH 7.4, 1 mM dithiothreitol, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM
phenylmethylsulfonyl fluoride), and lysed by sonication. The cell
lysate was cleared by centrifugation at 17,500 × g for
60 min at 4 °C, and the supernatant was applied to a
glutathione-Sepharose 4B column (Amersham Biosciences). The GST·human
sACt fusion protein was eluted with glutathione elution buffer (10 mM reduced glutathione, 50 mM
Tris-HCl, pH 8.0, 10 µg/ml aprotinin, 10 µg/ml leupeptin).
GST·human sACt was further purified by gel filtration
over Superdex 200 HR 10/30 column (Amersham Biosciences);
sAC-containing fractions were stored in 50% glycerol at
20 °C.
Coomassie Blue-stained SDS-PAGE reveals one predominant band
corresponding to GST·human sACt and a minor contaminant
corresponding to GST alone (Fig. 1, inset). Multiple
independent assays comparing the GST·sACt fusion protein
with cleaved and re-purified sACt confirmed that the GST
fusion does not affect enzymatic activity or kinetic parameters of sAC
(data not shown).
Cyclase Assay--
Cyclase assays were performed in 100 µl of
total reaction volume using ~100 ng of purified GST·human
sACt fusion protein in the presence of 50 mM
Tris-HCl, pH 7.5, substrate [
-32P]ATP, and either
MnCl2, MgCl2, and/or CaCl2 as
indicated. Reactions were incubated at 30 °C for 30 min unless
otherwise noted and were stopped by adding 200 µl of 2% SDS.
[32P]cAMP generated by the reaction was recovered using
the two-column method (23, 24). For kinetic analysis, sAC activity was
assayed as a function of varying ATP-Mn2+ or
ATP-Mg2+ in the presence of excess MnCl2,
MgCl2, or CaCl2. ATP was preincubated with
MnCl2 or MgCl2, and serial dilutions were
prepared; assays were started by addition of sAC protein. Kinetic
analyses were performed using the program EnzymeKinetics v 1.11 (Trinity Software, Plymouth, NH).
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RESULTS |
ATP-Mn2+-dependent Activity of
sAC--
Adenylyl cyclases require a divalent cation for catalytic
activity. Historically, soluble adenylyl cyclase activity found in
testis has been assayed in the presence of Mn2+ (10);
little or no in vitro activity was detected when
Mg2+, Ca2+, or Co2+ was used as the
sole divalent (19, 25, 26). The Km for
ATP-Mn2+ of soluble AC activity from testis cytosol has
been reported to be 1-2 mM (11, 21, 27). We now
demonstrate that recombinant human sAC has similar activity, displaying
a Km of 0.8 mM ATP-Mn2+
(Fig. 1).

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Fig. 1.
Activity of sAC in the presence of
ATP-Mn2+. Soluble adenylyl cyclase activity was
measured as a function of substrate ATP-Mn2+ in the
presence of excess MnCl2 (10 mM) for 5 min.
Km = 0.8 mM ATP-Mn2+ was
determined using non-linear regression analysis. Graph is
representative of three independent experiments. Inset,
Coomassie Blue-stained 10% SDS-PAGE demonstrating the purity of
GST·human sAC fusion protein used in these studies.
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Bicarbonate Releases Substrate Inhibition by ATP-Mg2+
and Increases the Vmax of sAC--
We recently described
direct stimulation of rat sAC by the bicarbonate anion (3). Bicarbonate
activation of sAC is thought to be at the center of
fertilization-related processes that occur in all mammalian sperm, and
we now confirm that purified human sAC is also stimulated by
bicarbonate (Fig. 2A). Similar
to rat sAC, in the presence of 10 mM ATP human sAC was
stimulated up to 30-fold with a half-maximal effect (EC50)
of ~11 mM NaHCO3. Bicarbonate stimulates sAC
activity in the presence of magnesium, and because manganese is not
found at the millimolar concentrations necessary to support sAC
activity inside cells, magnesium represents the more
physiologically relevant cation. Therefore, all subsequent experiments
were performed using Mg2+-ATP as substrate.

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Fig. 2.
Bicarbonate relieves ATP-Mg2+
substrate inhibition and increases enzyme velocity. A,
human sAC activity was assayed in the presence of indicated
concentrations of NaHCO3 with 10 mM ATP and 40 mM MgCl2. Values represent averages of
triplicate determinations, with error bars indicating S.D.
from the mean. B, sAC activity was measured as a function of
substrate ATP-Mg2+ for 30 min in the presence of excess
MgCl2 (20 mM) and increasing concentrations of
NaHCO3, ( ) 0 mM HCO ;
( ) 15 mM HCO ; ( ) 50 mM
HCO . All determinations are representative of at
least two independent experiments. Kinetic curves were generated by the
EnzymeKinetics computer program using data points not reflecting
substrate inhibition. C, sAC activity was measured as a
function of substrate ATP-Mg2+ for 30 min in the presence
of excess MgCl2 (80 mM) and NaHCO3
(80 mM).
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In the absence of bicarbonate, activity of recombinant human sAC was
inhibited at high ATP-Mg2+ concentrations. This substrate
inhibition was relieved by the addition of bicarbonate (Fig.
2B). At 0 mM NaHCO3, the onset of inhibition is at [ATP-Mg2+] > 6 mM; at 15 mM NaHCO3 the onset is shifted to
[ATP-Mg2+] > 13 mM and, at 50 mM
NaHCO3, substrate inhibition is virtually abated. The data
indicate that, in addition to relieving the inhibition observed at high
substrate concentrations, bicarbonate was not altering the apparent
Km for ATP-Mg2+; rather it was
increasing the Vmax of sAC (Fig. 2B).
These 2-fold effects contribute to the observed 30-fold stimulation of
activity (Fig. 2A). Similar effects were observed with
purified recombinant rat sAC; bicarbonate increased the
Vmax of the enzyme and abated substrate
inhibition (data not shown).
Previously published studies of crude soluble AC activity revealed an
elevated Km for ATP-Mg2+ (12-16
mM) relative to ATP-Mn2+ (1-2 mM)
(21). We were unable to determine a true Km for
ATP-Mg2+ in the presence of NaHCO3 because even
at the highest NaHCO3 (80 mM) and substrate (up
to 30 mM ATP-Mg2+) concentrations used, sAC
activity did not plateau (Fig. 2C). We could only conclude
from non-linear regression analysis and Eadie-Hofstee plots that the
apparent Km for ATP-Mg2+ was greater
than 10 mM, consistent with published reports using crude
soluble AC activity (21).
Calcium Synergizes with Bicarbonate and Activates sAC by Decreasing
Its Km for ATP-Mg2+--
Like bicarbonate,
CaCl2 (1.7-3 mM) is an essential component of
in vitro fertilization media, and Ca2+ has been
implicated along with bicarbonate in activation of sperm cyclases (12,
15, 16, 19). We added CaCl2 to bicarbonate-stimulated sAC
and found enzymatic activity increased synergistically (Fig. 3A). CaCl2
activation was dose-dependent with an EC50
~750 µM (Fig. 3B). Interestingly, the dose
response to NaHCO3 was unaffected by calcium; the
EC50 for NaHCO3 remained ~11 mM
in the presence or absence of CaCl2 (Fig.
3C).

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Fig. 3.
Synergistic activation of adenylyl cyclase by
CaCl2 and NaHCO3. A, sAC
activity was assayed in the presence of CaCl2 and/or
NaHCO3 as indicated. All reactions contained 2.5 mM ATP and 5 mM MgCl2. Values
represent averages of duplicate determinations and are representative
of at least two independent experiments. B, sAC activity was
assayed as a function of increasing CaCl2 concentration in
the presence of 2.5 mM ATP, 5 mM
MgCl2, and 40 mM NaHCO3.
C, sAC activity was assayed as in Fig. 2A in the
presence ( ) or absence ( ) of 10 mM CaCl2.
D, sAC activity was assayed as a function of substrate
ATP-Mg2+ for 30 min in the presence of excess
CaCl2 (10 mM) and NaHCO3 (40 mM). Km = 0.9 mM for
ATP-Mg2+ was determined by non-linear regression analysis.
E, sAC activity was assayed as a function of substrate
[ATP-Mg2+] for 30 min in the absence of any added
modulator ( ) or in the presence of 10 mM
CaCl2 ( ), 40 mM NaHCO3 ( ), or
10 mM CaCl2 and 40 mM
NaHCO3 ( ). Kinetic curves were generated by the
EnzymeKinetics computer program using data points not reflecting
substrate inhibition.
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As shown above, bicarbonate stimulated sAC activity by alleviating ATP
inhibition and by increasing Vmax with little
effect on apparent Km. In contrast, kinetic analysis
revealed that calcium had no effect on ATP inhibition and little effect on Vmax (Fig. 3E) but stimulated sAC
activity by decreasing its apparent Km for
ATP-Mg2+ (Fig. 3D). Addition of
CaCl2 causes a dramatic shift in the apparent Km of sAC for ATP-Mg2+ from greater than
10 mM (Fig. 2B) to less than 1 mM
(Fig. 3D). Overlaying the kinetic curves (Fig.
3E) illustrates how the effects of NaHCO3 and
CaCl2 differ; NaHCO3 activates sAC by
increasing Vmax, whereas CaCl2
increases its affinity for substrate.
The Calcium Effect on sAC Is Direct and Independent of
Calmodulin--
Coomassie Blue staining of purified human sAC did not
reveal any substantial contaminating proteins (Fig. 1,
inset), suggesting that calmodulin was not mediating the
effect of calcium on sAC. To confirm that calmodulin was not involved,
we found that adding exogenous bovine calmodulin did not have any
affect on Ca2+-dependent sAC activity (data not
shown) and inhibiting any potential calmodulin contamination using two
independent calmodulin inhibitors, chlorpromazine or LaCl3,
had no effect on calcium-stimulated sAC activity (Fig.
4). Therefore, we conclude that the
effect of CaCl2 on sAC activity is because of direct
binding of Ca2+, making sAC the first mammalian adenylyl
cyclase to be stimulated by calcium directly. Specific tmAC isoforms
are known to be stimulated by Ca2+, but they require
calmodulin for the modulatory effect (23, 28-34).

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Fig. 4.
Effect of calmodulin inhibitors. sAC
activity was measured in the presence of 2.5 mM ATP, 5 mM CaCl2, 5 mM MgCl2,
and 40 mM NaHCO3 and the indicated
concentrations of chlorpromazine ( ) or LaCl3 ( ). Data
points represent averages of duplicate determinations and are
representative of at least two independent experiments.
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DISCUSSION |
Soluble AC activity was first characterized in sperm and testis in
the presence of MnCl2 (10). Activity had been described in
the presence of other divalents, such as Co2+,
Ca2+, and Mg2+, but
Mn2+-dependent activity was always
significantly higher (19, 25, 26). We purified the 48-kDa isoform of
human sAC fused to GST and confirmed that its Km for
ATP-Mn2+ (~0.8 mM) was indistinguishable from
the value obtained for sAC activity purified from rat testis (14) and
matched the values reported for soluble cAMP-producing
activity in testis and sperm from a variety of mammals (11, 21, 27).
These data confirm that the cloned sAC gene is responsible for the
activities described in testis and sperm, and the kinetic parameters
determined here correlate well with values established using native
enzyme. However, it should be remembered that the kinetic analyses
described in this study were performed on a heterologously expressed
and purified enzyme; the in vivo properties of native sAC
may differ because of post-translational modifications or interactions
with regulatory proteins.
Soluble AC appears to be ubiquitously expressed (2, 35), and cells
throughout the body do not possess concentrations of Mn2+
necessary to support sAC activity. We recently demonstrated that the
ATP-Mg2+-dependent activity of purified rat sAC
was stimulated by bicarbonate. Among mammalian adenylyl cyclases,
bicarbonate regulation was unique to sAC; tmACs were unaffected by
NaHCO3 addition (3). Here we demonstrate that the human
ortholog of sAC is also responsive to bicarbonate stimulation. The
half-maximal effect (~11 mM NaHCO3) is
slightly lower for human sAC than the EC50 reported (25 mM NaHCO3) for rat sAC (3). Although both
EC50 for bicarbonate are within the physiological range
(24-26 mM in most extracellular fluids and 10-15
mM inside cells), the difference might reflect species
variation, or it could reflect differences between the two
preparations; rat sAC was purified as a His6·sAC fusion
protein (3), whereas this study uses a GST fusion protein.
Bicarbonate can increase sAC activity in two ways; besides increasing
enzyme velocity, bicarbonate also relieves the substrate inhibition
observed at high ATP-Mg2+ concentrations. Therefore, the
low (<5 mM) bicarbonate concentration in the epididymis
(36, 37), where sperm are stored awaiting ejaculation, would further
limit sAC activity in resting spermatozoa by allowing substrate
inhibition. Upon ejaculation, the higher HCO
(25 mM) found in seminal and prostatic fluids (which induces
sperm motility and initiates capacitation) would lead to large, rapid
increases in cAMP because of the increased Vmax
of sAC and relief from substrate inhibition.
Calcium (1.7-3 mM) is required in IVF media for
capacitation, hyperactivated motility, and the acrosome reaction in
sperm (15, 38). It is thought that Ca2+ is able to enter
sperm via voltage-dependent and cyclic
nucleotide gated calcium channels (39) as well as the putative cation
channel CatSper (40). Previous reports demonstrated that
detergent-dispersed adenylyl cyclase from guinea pig sperm membranes
was activated by CaCl2 (0.1-1 mM) in the
presence of 5 mM MgCl2 (19), and adenylyl
cyclase from human epididymal sperm membranes was activated by 50 mM CaCl2 and 50 mM
NaHCO3 (12). However, the molecular identity of the AC in
these preparations remained unclear. In this report we confirm that
calcium directly stimulates sAC activity and that calcium functions
independently from calmodulin to increase the affinity of sAC for its
substrate ATP-Mg2+.
Regulation by calcium, which is capable of supporting catalytic
activity in the absence of Mg2+ (19, 25, 26), suggests that
sAC, like tmACs, utilizes two metals in its active site; however, the
active center of sAC would function best with different metals. It is
possible that Ca2+, which lowers the apparent
Km for substrate ATP-Mg2+, would be
better at coordinating ATP, while Mg2+ would serve as the
catalytic metal.
The EC50 for CaCl2 stimulation appears to be
high (~750 µM). However, it is important to keep in
mind that CaCl2 concentrations reported in this study do
not reflect free Ca2+ concentrations. sAC activity would be
responsive to the transiently elevated Ca2+ concentrations
found during acrosome reaction and sperm motility, or sAC could be
located near the pore of Ca2+ channels, such as the cyclic
nucleotide gated ion channels (39), where it could mediate the cAMP
regulation of channel opening. A microdomain consisting of sAC and
cyclic nucleotide gated calcium channels could explain observed
cAMP/calcium oscillations in cells (as proposed in Ref. 33). Because of
the synergy between calcium and bicarbonate, even small intracellular
changes in calcium or subtle changes in intracellular pH and/or carbon
dioxide, which will be in equilibrium with bicarbonate, will result in
significant changes of cellular cAMP.