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J Biol Chem, Vol. 273, Issue 49, 32416-32420, December 4, 1998
From the The susceptibility of purines to form a covalent
attachment with proteins upon exposure to UV irradiation was applied to
adenylyl cyclase by use of [32P]2'-d-3'-AMP, a
dead-end inhibitor that binds to the post-transition configuration of
the enzyme. [32P]2'-d-3'-AMP was synthesized
enzymatically. It and [ Mammalian adenylyl cyclases (ATP pyrophosphate-lyase (cyclizing);
EC 4.6.1.1) comprise a family of proteins with similar domain
organization. The enzyme contains a short and variable amino terminus
followed by a tandem repeat of transmembrane and cytosolic domains. The
cytosolic domains, referred to as C1 and C2,
are both required for catalysis (1). Truncated forms of C1
and C2 have been chimerically linked or separately
expressed and used in the development of soluble forms of the enzyme
(1-3). Both catalytic activity and regulatory properties can be
reconstituted by a simple mixture of the two cytosolic domains of the
enzyme after their independent synthesis in Escherichia coli
(4). When recombined in solution, the independently expressed truncated C1 domain from the type V adenylyl cyclase
(VC1) and the truncated C2 domain of type II
adenylyl cyclase (IIC2), together, demonstrate many of the
features of native or wild type enzyme, including stimulation by
Gs An intriguing aspect of adenylyl cyclases is their inhibition by a
class of compounds collectively referred to as P-site ligands. The
P-site is so designated because of its requirement for an intact purine
in the inhibitory ligand. Naturally occurring P-site ligands include,
for example Ado, 2'-dAdo, 3'-AMP, and 2'-d-3'-AMP (7), and the most
potent ligands in this class are 2'-deoxy- and 2',5'-dideoxyadenosine
3'-polyphosphates (8). Inhibition of adenylyl cyclases by this class of
compounds is either uncompetitive or noncompetitive with respect to
ATP, depending on reaction conditions (9). The nature of inhibition and
results from experiments with covalent ligands targeted specifically to
the P-site (10, 11) give rise to questions about the number,
specificity, and localization of nucleotide binding configurations on
the enzyme per se. In an approach to these questions, we
synthesized [32P]2'-d-3'-AMP and have used it for
photoaffinity labeling of VC1 and IIC2 to
evaluate distribution of nucleotide binding sites between domains of
mammalian adenylyl cyclase and to estimate specificity of nucleotide
binding configurations on the enzyme.
Materials--
TdNT buffer, alkaline phosphatase, and glycogen
were from Promega. Oligonucleotide primer (pdA)9 and
terminal nucleotidyltransferase were from Sigma, and
[ VC1 and IIC2 Domains of Adenylyl
Cyclase--
Recombinat VC1 and IIC2 were
expressed in E. coli and were purified as described (4).
Purified proteins were electrophoretically homogeneous with apparent
molecular masses of 30 and 26 kDa for VC1 and
IIC2, respectively.
Synthesis of
[32P]2'-d-3'-AMP--
[32P]2'-d-3'-dAMP
was prepared from enzymatic hydrolysis of 32P-labeled
poly(dA). 32P-Labeled poly(dA) was prepared by repetitive
additions of [32P]2'-d-5'-AMP moieties to the 3'-end of
(pdA)9 in a reaction catalyzed by terminal
nucleotidyltransferase. The reaction mixture contained TdNT buffer,
0.05 A260 units of (pdA)9, 35 units
of terminal nucleotidyltransferase, 1 to 2 mCi of
[ Adenylyl Cyclase Assay--
Adenylyl cyclase was assayed at
30 °C in a 10-min reaction in 100 µl of reaction mixture
containing 50 mM HEPES buffer, pH 7.5, 1 mM
MnCl2, 100 µM forskolin, 0.5 mM
[ Photoaffinity Labeling--
Labeling of adenylyl cyclase
subunits VC1 and IIC2 by
[ ATP Photolysis and Effects of Acetone--
Simple exposure of
5'-ATP in water to high intensity UV light was not sufficient to induce
photolysis (Fig. 1, panel B).
No meaningful changes in the UV spectra of ATP were observed
after 60 min of irradiation. However, in the presence of 0.5% (v/v) acetone, UV light induced time-dependent photoactivation of
the adenine ring of 5'-ATP (Fig. 1, panel A). Half-time for
photolysis of 5'-ATP was approximately 10 min, and photolysis of
2'-d-3'-AMP in acetone followed a similar time course (not shown).
Consequently, in subsequent experiments, a 5-min exposure with either
nucleotide was used for protein labeling. These results are consistent
with the idea that energy is transferred from the UV-excited acetone molecules to the adenine ring.
Whereas low concentrations of acetone facilitated photoactivation of
5'-ATP or 2'-d-3'-AMP (above), these concentrations were essentially
without effect on adenylyl cyclase (Fig.
2). Acetone at 0.5% to 1% (v/v) did not
affect adenylyl cyclase appreciably, but, as expected, higher
concentrations inactivated the enzyme. The concentration eliciting a
50% reduction in activity of purified and recombined VC1
and IIC2 was approximately 3% acetone. By comparison, crude enzyme extracted from rat brain by simple detergent dispersion was unaffected by concentrations of acetone as high as 10% (not shown). Consequently, in subsequent experiments on photoaffinity labeling of VC1 and IIC2, 0.5% acetone was
used.
Photoaffinity Labeling of Adenylyl Cyclase Domains by
[ Photoaffinity Labeling of Adenylyl Cyclase Domains by
[32P]2'-d-3'-AMP--
Irradiation of adenylyl cyclase
cytosolic domains with UV light in the presence of 100 µM
[32P]2'-d-3'-AMP resulted in covalent modification of
both VC1 and IIC2, whether exposed individually
or in the form of a VC1·IIC2 complex (Fig.
4). The extent of covalent modification
of IIC2 was 2%, as determined by scintillation counting of
excised gels, and the ratio of 32P incorporated into
VC1 and IIC2 was 0.7.
Photoaffinity labeling of VC1, IIC2, or
VC1·IIC2 by 100 µM
[32P]2'-d-3'-AMP occurred in the presence of a 10-fold
molar excesses of either 5'-ATP (1 mM) or cAMP (1 mM), respectively, substrate and product of the cyclase
reaction (Fig. 4). Although both nucleotides suppressed incorporation
of 32P into VC1 ~ 50%, this was less evident
for incorporation of 32P into IIC2 but was
visually most evident when the enzyme was in the
VC1·IIC2 complex (Fig. 4, lanes
7-9). By comparison, a 10-fold molar excess of 3'-IMP (1 mM) afforded partial protection against 32P
incorporation into IIC2 but afforded no protection of
VC1 (Fig. 5, lanes
2 and 5). 3'-IMP gave partial protection of each
subunit when the VC1·IIC2 complex was
irradiated in the presence of [32P]2'-d-3'-AMP (Fig. 5,
lane 8). In striking contrast, at a concentration of only 50 µM, 2',5'-dd-3'-ATP resulted in essentially complete protection of both VC1 and IIC2, individually
or in complex, when labeled by 100 µM
[32P]2'-d-3'-AMP (Fig. 5, lanes 3, 6, and
9). Data from several experiments are summarized in Table
II.
The influence of Inorganic Pyrophosphate--
Data from both
dead-end inhibition kinetics and equilibrium binding studies with
3H-2'-dAdo to VC1·IIC2 in complex
with Gs Direct photocross-linking of nucleotides with proteins has been
widely used for identification of nucleotide binding sites (15,16).
However, protein labeling by adenosine derivatives is complicated by
instability of the glycoside bond of the UV-activated nucleoside (17) and low quantum yield of purine photoactivation (~3 × 10 Studies of adenylyl cyclases have included enzyme kinetics, reversibly
binding ligands, and covalent modifications of wild type and mutated
enzyme. Inhibition kinetics of adenylyl cyclase forward and reverse
reactions are consistent with an interaction of P-site ligands with the
post-transition state of the enzyme (E°) as dead-end
inhibitors (7, 9, 14, 23). By this mechanism, adenine nucleosides and
adenosine 3'-monophosphates interact with the cAMP binding site and
before metal·PPi leaving (6, 14 ,23). These analyses
would place both substrate 5'-ATP and P-site ligands at a common site
believed to be formed at the interface of the C1 and
C2 domains during the reaction cycle (6, 14, 24, 25).
Kinetics analyses involve evaluations that include both ligand binding
and enzyme conformational changes, whereas direct measurements of
ligand binding assess bimolecular interactions, albeit with a set of
possible enzyme configurations. Because substrate and inhibitor share a
common binding site, although of different configurations, one might
expect some competition between P-site ligands and substrate. However,
this was not observed in equilibrium binding studies with
[3H]2'-dAdo nor with
[3H]5'-AP(CH2)PP (6,14). 2'-d-3'-dAMP neither
displaced nor competed with binding. Binding of these ligands with this
chimeric truncated construct of the C1·C2
complex required not only the formation of an active tertiary structure
but also catalysis. Because conformational states of an enzyme are in
equilibrium and a certain percentage of the enzyme will be in the
different configurations, reversible binding techniques may not have
been able to detect associations that might be observable with labeled
covalent probes.
The availability of [32P]2'-d-3'-AMP, a more potent and
easily detected ligand than [3H]2'-dAdo, allowed these
interactions to be addressed by a different technique. The data
presented here clearly show that by use of direct photo cross-linking,
both [ The reduced labeling by [32P]2'-d-3'-AMP noted upon
association of VC1 with IIC2, particularly with
regard to effects of competing ligands (Figs. 4-6), suggests either
that interaction of the cytosolic domains affects access of ligand to
those sites to which covalent linkages are formed or that the
association forms a preferential configuration to which the ligands
have differential access. That is, association of C1 and
C2 domains changes during the catalytic cycle, exhibiting
more than one conformational state. These are represented minimally by
two configurations, one that is a catalytically competent configuration
and one that occurs as a result of P-site-mediated inhibition. These
two configurations are in equilibrium, but the equilibrium between them
is not rapid. Differential protection by competing ligands of
cross-linking with [ The presence of multiple conformation states is also supported by the
facts (i) that diffusion of 2'-d-3'-ATP into crystals of
IC1·IIC2·Gs Taken together the data strongly argue that the site of covalent
cross-linking by [32P]2'-d-3'-AMP has the same
characteristics as that through which P-site-mediated inhibition of
native adenylyl cyclases occurs. It is selective for an intact adenine
moiety, and it recognizes the ribosyl 3'-polyphosphate moiety (cf.
Refs. 7, 8, 28, 29). Effective inhibition by or binding of adenine
nucleosides or adenine nucleoside-3'-monophosphates requires the
presence of PPi, whereas inhibition by the
adenosine-3'-tri- or 3'-tetraphosphates (7, 28) does not.
P-site-directed ligand does not compete with 5'-ATP for binding with
adenylyl cyclase (9), and P-site-targeted covalent modification occurs
in the presence of 5'-ATP (11). The catalytically competent form of the
enzyme exists in two conformational states, one with which substrate,
metal-5'-ATP interacts, and a post-transition state in which product is
released and with which P-site ligands interact. It is this latter form
that [32P]2'-d-3'-AMP covalently modifies and the
labeling of which is enhanced by pyrophosphate. Each cytosolic domain
evidently contains elements of a nucleotide binding site, but an
effective catalytic cleft must form only when they associate. Because
the two states of the enzyme are not in rapid equilibrium, the
opportunity is provided for independent binding of ligands to substrate
and P-site configurations of the enzyme.
*
This research was supported by National Institutes of Health
Grant DK38828 (to R. A. J.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence and reprint requests should be
addressed. Tel.: 516-444-3040; Fax: 516-444-3432; E-mail:
rjohnson{at}ccmail.sunysb.edu.
The abbreviations used are:
5'-ATP, adenosine
5'-triphosphate; 2'-d-3'AMP, 2'-deoxyadenosine 3'-monophosphate; 3'-IMP, inosine 3'-monophosphate; 2', 5'-dd-3'-ATP,
2',5'-dideoxy-3'-ATP; 5'-AP(OH2)PP, adenosine
5'-( 2
An important technical aspects of these studies
is that inorganic pyrophosphate and Mn2+, which
we used in all experiments as our activating ligand, form a complex.
Hence, in such experiments PPi must be added immediately before the reaction is initiated. Otherwise precipitates may form, and
the effects of PPi will not be evident.
Direct Photoaffinity Labeling of Individual Cytosolic Domains of
Adenylyl Cyclase by [32P]2'-deoxy-3'-AMP and
[
-32P]5'-ATP*
,¶
Department of Physiology and Biophysics,
Health Sciences Center, State University of New York, Stony Brook, New
York 11794-8661 and § Department of Pharmacology University
of Texas Southwestern Medical Center, Dallas, Texas 75235
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-32P]5'-ATP were used for
direct photocross-linking to individually expressed cytosolic domains
of adenylyl cyclase. Both the C1 domain of the type V
isozyme (VC1) and the C2 domain of the type II
isozyme (IIC2) were labeled, whether alone or combined,
upon photolysis of [32P]2'-d-3'-AMP in the presence of
acetone. Labeling of VC1 and IIC2 was greatly
enhanced in the presence of PPi, was almost completely suppressed by 50 µM 2',5'-dideoxy-3'-ATP, the most potent
reported P-site inhibitor of adenylyl cyclases, but was partially
suppressed by 1 mM 3'-IMP, a ligand that does not inhibit
the enzyme via the P-site. Neither 3':5'-cAMP nor 5'-ATP had a major
effect on labeling by [32P]2'-d-3'-AMP. Direct
cross-linking of VC1 with [-32P]5'-ATP was
substantially suppressed by 2',5'-dideoxy-3'-ATP and partially
suppressed by 2'-d-3'-AMP, whereas cross-linking of IIC2
was less affected by the 3'-triphosphate. The data imply that either
cytosolic domain can interact directly with either substrate or P-site
ligand and that subunit interaction modifies the susceptibility of each
domain to UV-induced covalent modification by either
[-32P]5'-ATP or [32P]2'-d-3'-AMP.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
and forskolin and inhibition by
2'-d-3'-AMP1 and 2'-dAdo (5,
6). Thus, these preparations are well suited for studies aimed at
identification of domains involved in specific aspects of regulation of
this important enzyme family.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-32P]2'-d-5'-ATP (3000 Ci/mmol) was from
International Chemical and Nuclear Corp. Calf spleen phosphodiesterase
(2 units/mg) was from Boehringer Mannhem. Photolysis was conducted in a
Rayonet mini-reactor from Southern New England Ultraviolet Company.
-32P]2'-d-5'-ATP (3000 Ci/mmol) that had been
previously lyophilized in a volume of 100 µl. After incubation at
30 °C for 4 h or overnight, the mixture was treated for 5 min
with 1 unit of alkaline phosphatase to remove residual 2'-d-5'-ATP and
to dephosphorylate the 5'-end of the oligonucleotide. Without this
treatment, overall yields of [32P]2'-d-3'-AMP were poor
and variable. Alkaline phosphatase was inactivated by placing the
reaction tube in boiling water for 5 min. 32P-Labeled
poly(dA) was isolated by precipitation with 70% ethanol in 0.3 M sodium acetate buffer, pH 5, in the presence of 1 µg of
glycogen. [32P]2'-d-3'-AMP was released upon reaction of
32P-labeled poly(dA) with 0.2 units of calf spleen
phosphodiesterase (2 units/mg) for 4 h at 30 °C in 100 µl of
reaction mixture containing 50 mM sodium acetate buffer, pH
5. The sample was deproteinated by extraction with phenol/chloroform
(1/1, v/v), and [32P]2'-d-3'-AMP was isolated by reverse
phase column chromatography on an C18 Ultrasphere column
(4.6 × 250 mm from Beckman). Elution was with a linear gradient
of 50 mM triethylammonium bicarbonate, pH 7.5, to 50%
methanol at 1 ml/min. Fractions containing
[32P]2'-d-3'-AMP were pooled, and triethylammonium
bicarbonate was removed by repetitive evaporation from methanol. The
purified [32P]2'-d-3'-AMP gave an estimated specific
activity of 1 to 2 × 1016 cpm/mol, as determined by
Cherenkov radiation.
-32P]5'-ATP (2 × 1013 cpm/mol by
Cherenkov radiation), and 50 nM VC1 and 50 nM IIC2. The reaction was started by the
addition of [-32P]5'-ATP and was terminated by the
addition of zinc acetate and sodium carbonate. [32P]cAMP
was purified by sequential chromatography on Dowex 50 and alumina as
described previously (12). Acetone was included in the assay mixture as
indicated at 0.5% v/v to 10% v/v.
-32P]5'-ATP or [32P]2'-d-3'-AMP was
achieved with a 5-min exposure to 254 nm UV light at room temperature
in a Rayonet mini-reactor. The 25-µl reaction volume contained 50 mM HEPES, pH 7.5, 5 mM MnCl2, 100 µM forskolin, 0.5% acetone, 100 µM [-32P]5'-ATP (25 Ci/mmol) or 100 µM [32P]2'-d-3'-AMP (1 to 2 × 1016 cpm/mmol, by Cherenkov radiation), 5 µM
of VC1, and/or 5 µM of IIC2. The
reaction was initiated by the addition of VC1 and/or IIC2 to the rest of the reaction mixture at 0 °C. The
resulting mixtures were transferred immediately to a Parafilm support
in the mini-reactor with 254-nm lamps on. After irradiation, the reaction mixture was transferred to a tube containing 0.1% SDS, 0.1 M dithiothreitol, and 5% glycerol, and this was placed in a boiling water bath for 3 min. Proteins and unreacted nucleotides were
separated on an 11% polyacrylamide SDS gel. Protein was visualized by
silver staining as described (13). The dried gel was exposed to a
phosphoimager screen for 3 to 5 h, and protein labeling was quantified by PhosphorImager and ImageQuant software (from Molecular Dynamics). Alternatively, dried gels were exposed at 
65 °C to X-Omat imaging film (from Kodak) for 12 h with an intensifying screen. To quantify the incorporation of 32P-labeled ligand
into proteins, bands corresponding to VC1 and IIC2 were cut from dried gels and counted in a liquid
scintillation counter.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
UV-spectra of 5'-ATP irradiated by a 254-nm
lamp. Panel A, irradiation of 0.1 mM 5'-ATP in
the presence of 0.5% (v/v) acetone for 0, 10, 20, 30, and 40 min at
room temperature. Panel B, irradiation of 0.1 mM
5'-ATP in the absence of acetone at room temperature for 0 or 60 min.
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Fig. 2.
Effect of acetone on adenylyl cyclase
activity. Adenylyl cyclase activity in the absence of acetone was
taken as 100%. Adenylyl cyclase activity of
VC1·IIC2 complex was assayed in the presence
and absence of acetone as described under "Experimental
Procedures."
-32P]5'-ATP--
Irradiation of individual adenylyl
cyclase cytosolic domains with UV light in the presence of
[-32P]5'-ATP (100 µM) resulted in
covalent modification of both VC1 and IIC2
(Fig. 3). 32P-Labeled ligand
was incorporated into 5% of VC1, as determined by excision
of gel slices and counting in a scintillation counter. By this method
the extent of labeling of VC1 was found to be 3.5 times
greater than that of IIC2. The addition of 1 mM
2'-d-3'-AMP or 50 µM 2',5'-dd-3'-ATP resulted in
protection of both VC1 and IIC2 domains from
covalent labeling by [-32P]5'-ATP (Fig. 3).
Efficiencies of VC1 or IIC2 protection by
2'-d-3'-AMP and 2',5'-dd-3'-ATP were estimated by PhosphorImager
techniques. Because isotope decay events (cpm) are directly
proportional to arbitrary PhosphorImager units (over 5 orders of
magnitude), the ratio of densities of any two bands will directly
reflect the ratio of isotope incorporated into the respective proteins.
Consequently, the relative protecting effect of a 3'-nucleotide ligand
is simply measured by comparison of band densities when the
density with no added ligand is taken as 100% for each cytosolic
domain. A summary of such data obtained from two experiments is given
in Table I. It was not possible to
attempt photoaffinity labeling of a mixture of VC1 and
IIC2 by [-32P]5'-ATP per se.
When VC1 and IIC2 are combined at
concentrations sufficient to allow complex formation,
[-32P]5'-ATP is rapidly and completely converted to
[32P]cAMP.
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Fig. 3.
Photoaffinity labeling of VC1 and
IIC2 cytosolic domains with
[ -32P]5'-ATP. VC1 and
IIC2 (0.125 nmol of each) were individually exposed to UV
irradiation in the presence of 0.1 mM
[-32P]5'-ATP and subsequently separated
electrophoretically from unbound ligand as described under
"Experimental Procedures." Lanes contained
VC1 (1), VC1 irradiated in the
presence of 1 mM 2'-d-3'-AMP (2),
VC1 irradiated in the presence of 50 µM
2',5'-dd-3'-ATP (3), IIC2 (4),
IIC2 irradiated in the presence of 1 mM
2'-d-3'AMP (5), and IIC2 irradiated in the
presence of 50 µM 2',5'-dd-3'-ATP (6) .
Effect of 2'-d-3'-AMP and 2',5'-dd-3'-ATP on photoaffinity labeling
of VC1 and IIC2 by [-32P]5'-ATP
-32P]5'-ATP and separated by polyacrylamide gel
electrophoresis as described under "Experimental Procedures."
Densities of labeled bands were measured with ImageQuant software on
data obtained from a PhophorImager. Values are densities of the
respectives bands with the indicated additions of 2'-d-3'-AMP and
2',5'-dd-3'-ATP relative to densities in their absence and are averages
from two experiments.
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Fig. 4.
Photoaffinity labeling of VC1,
IIC2, and VC1·IIC2 with
[32P]2'-d-3'-AMP;effects of cAMP and 5'-ATP.
VC1 and IIC2 were exposed to UV irradiation
individually and in complex (VC1·IIC2) in the
presence of 100 µM [32P]2'-d-3'-AMP and
subsequently separated electrophoretically from unbound ligand as
described under "Experimental Procedures." Lanes
contained 0.125 nmol of VC1 (1); 0.125 nmol of
VC1 irradiated in the presence of 1 mM cAMP
(2); 0.125 nmol of VC1 irradiated in the
presence of 1 mM 5'-ATP (3); 0.125 nmol of
IIC2 (4); 0.125 nmol of IIC2
irradiated in the presence of 1 mM cAMP (5);
0.125 nmol of IIC2 irradiated in the presence of 1 mM 5'-ATP (6); complex of
VC1·IIC2 cytosolic domains, each 0.125 nmol
(7); complex of VC1·IIC2 cytosolic
domains, each 0.125 nmol, irradiated in the presence of 1 mM cAMP (8); and complex of
VC1·IIC2 cytosolic domains, each 0.125 nmol,
irradiated in the presence of 1 mM 5'-ATP
(9).
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Fig. 5.
Photoaffinity modification of individual
VC1, IIC2, and
VC1·IIC2 with [32P]2'-d-3'-AMP;
effects of 3'-IMP and 2',5'-dd-3'-ATP. VC1 and
IIC2 were exposed to UV irradiation individually and in
complex (VC1·IIC2) in the presence of 0.1 mM [32P]2'-d-3'-AMP and subsequently
separated electrophoretically from unbound ligand as described under
"Experimental Procedures." Lanes contained 0.125 nmol of
VC1 (1); 0.125 nmol of VC1 labeled
with [32P]2'-d-3'-AMP in the presence of 1 mM
3'-IMP (2); 0.125 nmol of VC1 labeled with
[32P]2'-d-3'-AMP in the presence of 50 µM
2',5'-dd-3'-ATP (3); 0.125 nmol of IIC2
(4); 0.125 nmol of IIC2 irradiated in the
presence of 1 mM 3'-IMP (5); 0.125 nmol of
IIC2 irradiated in the presence of 50 µM
2',5'-dd-3'-ATP (6); complex of
VC1·IIC2 cytosolic domains, each 0.125 nmol
(7); complex of VC1·IC2 cytosolic
domains, each 0.125 nmol, labeled in presence of 1 mM
3'-IMP (8); and complex of
VC1·IIC2 cytosolic domains, each 0.125 nmol,
labeled in presence of 50 µM 2',5'-dd-3'-ATP
(9).
Protecting effect of cAMP, PPi, 3'-IMP, 5'-ATP, and
2',5'-dd-3'-ATP on photoaffinity labeling of VC1 and
IIC2 by [32P]-2'-d-3'-AMP
suggested that inorganic pyrophosphate should
enhance or be required for binding of P-site ligands (14).
Consequently, the effect of both adenylyl cyclase products, cAMP and
PPi, alone and in combination, were evaluated for their
possible effects on photoaffinity cross-linking with [32P]2'-d-3'-AMP (Fig.
6).2
As predicted, PPi (1 mM) substantially enhanced
labeling of VC1, IIC2, or
VC1·IIC2 by [32P]2'-d-3'-AMP
(Table II), and cAMP afforded protection that was compatible with its
effect in the absence of PPi.
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Fig. 6.
Pyrophosphate-enhanced photoaffinity labeling
of VC1, IIC2, and
VC1·IIC2 with
[32P]2-d-3'-AMP. VC1 and
IIC2 were exposed to UV irradiation individually and in
complex (VC1·IIC2) in the presence of 0.1 mM [32P]2'-d-3'-AMP and subsequently
separated electrophoretically from unbound ligand as described under
"Experimental Procedures." Lanes contained 0.125 nmol of
VC1 (1); 0.125 nmol of VC1 labeled
with [32P]2'-d-3'-AMP in the presence of 1 mM
PPi (2); 0.125 nmol of VC1 labeled
with [32P]2'-d-3'-AMP in the presence of 1 mM
PPi and 1 mM cAMP (3); 0.125 nmol of
IIC2 (4); 0.125 nmol of IIC2
irradiated in the presence of 1 mM PPi
(5); 0.125 nmol of IIC2 irradiated in the
presence of 1 mM PPi and 1 mM cAMP
(6); complex of VC1·IIC2 cytosolic
domains, each 0.125 nmol (7); complex of
VC1·IIC2 cytosolic domains, each 0.125 nmol,
labeled in presence of 1 mM PPi (8);
complex of VC1·IIC2 cytosolic domains, each
0.125 nmol, labeled in presence of 1 mM PPi and
1 mM cAMP (9).
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
4) (18). This is somewhat circumvented by
the use of a chromophore to allow indirect activation of adenine by
facilitating energy transfer from UV light to the adenine ring (19).
Acetone has been used successfully in this capacity as a sensitizer
(19), and site-specific modification of nucleotide binding sites in proteins has been demonstrated with 5'-ATP and acetone (20-22). The
effect of acetone to enhance adenine nucleotide photolysis was also
clearly evident here (Fig. 1). Even so, covalent modification in such
experiments may not exceed a few percent of the protein, limited due
either to UV-induced cleavage of the glycosidic bond of adenosine, in
our case causing a loss of the reporter 32P label, or to
nonspecific interactions of the labeled ligand with buffer components.
-32P]5'-ATP and [32P]2'-d-3'-AMP
labeled both VC1 and IIC2 (Figs. 3-6),
indicating that both ligands can interact with either VC1
or IIC2. This is consistent with the high level of sequence
homology between C1 and C2 (26) in all
mammalian adenylyl cyclases and is not inconsistent with the idea that
the two cytosolic domains are required for catalysis and
P-site-mediated inhibition (1-6). Because photoactivation induces
covalent cross-linking through the adenine ring, the fact that both
VC1 and IIC2 are labeled by
[32P]2'-d-3'-AMP also in the catalytically competent
complex of VC1·IIC2 implies contact of the
adenine ring of this ligand with both VC1 and
IIC2. Because cross-linking results in an irreversible
linkage, it will select for and lock in those enzyme configurations
with which the ligands interact. This is quite different from the
results one can obtain from either enzyme kinetics or reversible
binding studies. It was therefore not surprising that a measurable but weak competition between 5'-ATP and 2'-d-3'-AMP was noted. Both bind to
the enzyme under similar conditions. Either will displace binding of
the other, but only partially (Tables I and II), and that at 10-fold
greater concentrations of the competing ligand. These observations are
consistent with the steady-state kinetic behavior of the soluble enzyme
(6, 9, 14) but not inconsistent with the lack of competition of
2'-d-3'-AMP in the equilibrium binding studies with
[3H]5'-AP(CH2)PP, which suggested that
2'-d-3'-AMP binds after 5'-ATP but before the return of the enzyme to
its initial state. Shown here is that [32P]2'-d-3'-AMP
can bind to either free enzyme (E + I 
E·I) or to the
post-transition state of the enzyme in the absence of pyrophosphate (E° + I E°·I). PPi, which
shifts the equilibrium of enzyme to a state that accepts P-site
ligands, enhanced labeling of the individual domains 3- to 5-fold and
labeling of the VC1·IIC2 complex 7- to 9-fold (Table II). This effect of PPi is fully consistent with the
proposed model for inhibition by P-site ligands (6, 14, 23), and it is
consistent with the effect of PPi to enhance binding of [3H]2'-dAdo to the VC1·IIC2
complex (14). By comparison, cAMP afforded less protection of
VC1 and IIC2 from labeling by
[32P]2'-d-3'-AMP than might have been expected (Figs. 4
and 6 and Table II). This may be explained by the low apparent affinity of cAMP for adenylyl cyclase (Km cAMP ~16
mM in the reverse reaction). The use of cAMP concentrations
sufficient to interact with the enzyme effectively, e.g. 10 mM, could result in a "quenching" effect, absorbing UV
light used to activate the nucleotides. Overall the cross-linking
observed with both [32P]2'-d-3'-AMP and
[-32P]5'-ATP to both both VC1 and
IIC2 and the weak competitive behavior are consistent with
reaction kinetics and with the idea that the two cytosolic domains are
required for catalysis and P-site-mediated inhibition (1-6, 14, 24,
25).
-32P]5'-ATP or
[32P]2'-d-3'-AMP would result from these two distinct
enzyme configurations with which these nucleotides interact.
resulted in
structure disordering and (ii) that formation of crystals with
2'-d-3'-ATP was unsuccessful (25). The
VC1·IIC2 complex with 2'-d-3'-AMP and
PPi is not well ordered, and Tesmer et al. (25)
suggest that this is because of incomplete occupancy or incomplete
formation of the binding pocket because of constraints imposed by the
crystalline lattice. Whereas the locus of 2'-d-3'-AMP and
PPi in structures of VC1·IIC2 are
clear and suggestions have been made as to the locus for 5'-ATP in
models derived from these (27), no crystal structure of a
C1·C2·ATP complex is available, whether
with 5'-ATP, 3'-ATP, or a derivative of either (24-26). The rank order
for inhibitory potency of P-site ligands (2'-dAdo < 2'-d-3'-AMP < 2'-d-3'-ADP < 2'-d-3'-ATP < 2'-d-3'-A4P) (7,28), the effect of PPi to enhance binding
of [3H]2'-dAdo (14), and the effect of PPi to
enhance covalent cross-linking of 2'-d-3'-AMP (Fig. 6 and Table II)
clearly demonstrate the important contribution of 3'-(
, 
, and
)-phosphates to the association of these ligands with adenylyl
cyclase. Thus it was not surprising that the relatively low
concentration of 50 µM 2',5'-dd-3'-ATP caused substantial
protection against labeling of VC1 or IIC2 by
100 µM [32P]2'-d-3'-AMP and caused
virtually complete protection against labeling of the catalytically
competent VC1·IIC2 complex (Fig. 5 and Table
II). This contrasts sharply with the much less effective protection
afforded by 10-fold greater concentration of either 5'-ATP or 3'-IMP
(each 1 mM; Figs. 4 and 5). In the
VC1·IIC2 complex 3'-IMP afforded only
50-60% protection of IIC2 and only 30% protection of
VC1 and afforded no protection of VC1 alone
(Fig. 5 and Table II).
FOOTNOTES
,-methylene) triphosphate; 2'-d-3'-A4P, 2'-deoxyadenosine
3'-tetraphosphate. TdNT buffer, buffer for terminal
nucleotidyltransferase assay.
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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