A Functional Domain of the
1 Subunit of Soluble
Guanylyl Cyclase Is Necessary for Activation of the Enzyme by Nitric
Oxide and YC-1 but Is Not Involved in Heme Binding*
Markus
Koglin and
Sönke
Behrends
From the Institut für Experimentelle und Klinische
Pharmakologie, Universität Hamburg, Martinistrasse 52, D-20246 Hamburg, Germany
Received for publication, December 13, 2002, and in revised form, January 16, 2003
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ABSTRACT |
Soluble guanylyl cyclase is a heterodimeric
enzyme consisting of an
1 and a
1
subunit and is an important target for endogenous nitric oxide and the
guanylyl cyclase modulator YC-1. The activation of the enzyme by both
substances is dependent on the presence of a prosthetic heme group. It
has been unclear whether this prosthetic heme group is sandwiched
between the
1 and
1 subunits or whether it exclusively binds to the
1 subunit. Here we analyze
progressive amino-terminal deletion mutants of the human
1 subunit after co-expression with the human
1 subunit in the baculovirus/Sf9 system.
Spectral, biochemical, and pharmacological analysis shows that the
first 259 amino acids of the
1 subunit can be deleted without loss of sensitivity to nitric oxide (NO) or YC-1 or loss of
heme binding of the respective enzyme complex with the
1
subunit. This is in contrast to previous data indicating that NO
sensitivity and a functional heme binding site requires full-length
amino termini of bovine
1 and
1 subunits.
Further deletion of the first 364 amino acids of the
1
subunit leads to an enzyme complex with preserved heme binding but loss
of sensitivity to NO or YC-1 despite induction of the typical spectral
shift by NO binding to the prosthetic heme group. We conclude that
1) the amino-terminal part of the
1 subunit is not
involved in heme binding and 2) amino acids 259-364 of the
1 subunit represent an important functional domain for
the transduction of the NO activation signal and likely represent the
target for NO-sensitizing substances like YC-1.
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INTRODUCTION |
Soluble guanylyl cyclase
(sGC)1 is an important target
for endogenous nitric oxide (NO), NO-releasing drugs like glyceryl
trinitrate, and novel substances like YC-1 or BAY 41-2272 that
sensitize the enzyme for activation by NO (1, 2). The enzyme has been purified from lung as a heterodimeric, heme-containing enzyme consisting of an
1 and a
1 subunit. After
cloning of the
1 and
1 cDNAs, two
other subunit cDNAs have been cloned by homology screening: the
2 subunit from rat kidney and the
2
subunit from human fetal brain (3, 4). Co-expression of the
1/
1 and
2/
1
cDNAs yielded NO-sensitive enzymes in expression systems (4), and
the
2/
1 heterodimeric enzyme has been
demonstrated on the protein level in human placenta by co-precipitation
experiments (5). We recently isolated a
2 cDNA
variant from rat kidney that shows NO-sensitive enzyme activity after
expression in Sf9 or HEK-293 cells in the absence of a second
subunit, most likely as
2/
2 homodimer
(6).
Most studies regarding the activation mechanism by binding of NO to the
prosthetic heme group have concentrated on the
1/
1 heterodimeric enzyme. Since the first
purification of this enzyme isoform it has been assumed that the enzyme
contains one prosthetic heme group per heterodimer (7). Before the
cDNA sequences of the two subunits were identified, it was proposed
that one subunit was regulatory and bound the heme and that the other
subunit was catalytic (8). However, analysis of the cDNA sequences
revealed that the two subunits show a high degree of homology both in
their amino-terminal and their carboxyl-terminal halves (9, 10). While
the carboxyl-terminal parts were assigned as being responsible for
catalysis based on homology to the related adenylyl cyclases, it seemed
plausible that both homologous amino-terminal regions of the
1 and
1 subunits participate in binding
of the prosthetic heme. This hypothesis was strengthened by findings
using amino-terminal deletion mutants of the bovine
1
and
1 subunits showing that NO sensitivity and a
functional heme binding site of sGC requires full-length amino termini
of both subunits (11, 12).
In the present study, we used deletion mutagenesis to identify
functional regions that are responsible for NO-heme and YC-1-mediated activation of sGC. To our surprise we found that the deletion of the
first 259 amino acids of the human
1 subunit leads to an
enzyme with strong sensitivity toward the heme-dependent
activators NO and YC-1. Deletion of 364 amino acids of the
1 subunit leads to an enzyme complex that is insensitive
to the heme-dependent activators NO and YC-1 but shows
preserved heme binding with the typical shift in the spectral analysis
by NO binding to the prosthetic heme group. This indicates that the
amino-terminal part of the
1 subunit is not involved in
heme binding and that amino acids 259-364 of the
1
subunit represent an important functional domain for the transduction
of the NO activation signal and likely represent the target for
NO-sensitizing substances like YC-1.
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EXPERIMENTAL PROCEDURES |
Materials--
3-(5'-Hydroxymethyl-2'-furyl)-1-benzylindazole
(YC-1) was from Alexis Biochemicals (Lausen, Switzerland).
2,2-Diethyl-1-nitroso-oxyhydrazine (DEA/NO) and all other chemicals, in
the highest grade of purity, were obtained from Sigma. Products for
Sf9 cell culture were from Invitrogen.
Cloning of
1 Deletion Mutants and Generation of
Recombinant Baculovirus--
Cloning of the
1 subunit
(a kind gift of Dr. Georges Guellaën, Créteil; Ref. 13) and
the
1 subunit has been described previously (6, 14).
Cloning of diverse
1 deletion mutants was carried out by
digestion with different restriction endonucleases. For construction of
the
1
N364 mutant, a
BsmFI/HindIII fragment of full-length
1 was cloned using StuI/HindIII
into the pFASTBAC vector. Before cloning into pFASTBAC the
BsmFI 5'-ends were filled in with Taq polymerase
(Invitrogen). For construction of the
1
N259 mutant, a single nucleotide exchange was done with
the QuikChangeTM kit (Stratagene, La Jolla, CA) using the
following primer pair: P 216, 5'-GCG AGT TTG TGA ATC AGC CCT ACT
AGT TGT ACT CCG-3'; and P 217, 5'-CGG AGT ACA
ACT AGT AGG GCT GAT TCA CAA ACT CGC-3'. The modified
nucleotide is underlined. A SpeI/HindIII fragment of the mutated
1 full-length clone was then ligated
using SpeI/HindIII into the pFASTBAC
vector. Recombinant baculoviruses of respective subunits were generated
according to the BAC-TO-BACTM System (Invitrogen).
Sf9 Cell Culture, Expression of Recombinant Guanylyl
Cyclase Subunits, and Cytosol Preparation--
Sf9 cells were
cultured in Sf-900 II serum-free medium supplemented with 1%
penicillin/streptomycin and 10% fetal calf serum. Spinner cultures
were grown to a cell density of 3.0 × 106 cells/ml
and then diluted to 1.2 × 106 cells/ml for infection.
30 ml of cell solution were infected with multiplicities of infection
of 1. After 74 h cells were harvested and collected by
centrifugation (1500 × g for 10 min at 4 °C). All
following steps were performed at 4 °C or on ice. The cell pellet
was resuspended in 4 ml of homogenization buffer containing 50 mM TEA/HCl, pH 7.6, 0.2 µM benzamidine, 1 mM EDTA, pH 8.0, and freshly dissolved
dithiothreitol with a final concentration of 10 mM.
The cells were passed through a Sterican® needle
(0.45 × 25 mm, B. Braun, Melsungen, Germany) several times for
lysis. To remove complete cells and nuclei the solution was centrifuged
for 2 min at 800 × g. Cytosolic fractions were
obtained by a second centrifugation step for 30 min at 40,000 × g.
Purification of sGC--
All purification steps were performed
at 4 °C. The cell pellet from 1800 ml of cell solution infected with
the respective subunits was homogenized with a cell disruption bomb
(Parr, Moline, IL) at 60 bars for 1 h in 180 ml of 50 mM TEA/HCl, pH 8.0 containing 10 mM
dithiothreitol, 1 mM benzamidine, 10 µg/ml
phenylmethylsulfonyl fluoride, and 900 µl of protease inhibitor
mixture (Sigma). The homogenate was centrifuged at 40,000 × g for 30 min, and 180 ml of supernatant were collected. All
chromatographic steps were performed on a FPLC system (Amersham
Biosciences). The protease inhibitor benzamidine (1 mM),
dithiothreitol (10 mM), and phenylmethylsulfonyl fluoride
(10 µg/ml) were used in all chromatographic steps. The supernatant
was immediately applied to a Q-Sepharose column (20-ml volume) at 2 ml/min. Ion exchange buffer A contained 50 mM TEA/HCl, pH
8.0. Ion exchange buffer B contained 5 mM potassium
phosphate (pH 7.2). Ion exchange buffer C was prepared by adding 1 M NaCl to buffer B. The column was washed at 3 ml/min with
buffer A, buffer B, and 8% buffer C until
A280 was stable. A linear gradient from 8% C to
30% C for 828 ml was used to elute sGC. The sGC-containing fractions
were pooled by determining sGC activity at basal or NO-stimulated
conditions after each column. The pooled fractions (104 ml) were
diluted with 104 ml of 5 mM potassium phosphate (pH 7.2)
and applied immediately to a ceramic hydroxyapatite column (Bio-Rad, 5 ml volume) at 1.5 ml/min. Hydroxyapatite buffer A contained 5 mM potassium phosphate (pH 7.2), and hydroxyapatite buffer
B contained 400 mM potassium phosphate (pH 6.6). The column was then washed with 10% B until the A280 was
stable. The enzyme was eluted with a linear gradient running from 10%
B to 50% B for 180 ml. The sGC-containing fractions (44 ml) were again
pooled and applied immediately to a blue Sepharose column (Amersham
Biosciences, 5 ml volume) at 1.5 ml/min. Blue Sepharose buffer A
contained 5 mM potassium phosphate (pH 7.2), and blue
Sepharose buffer B was prepared by adding 1 M NaCl to
buffer A. The column was then washed with 10% buffer B until the
A280 was stable. The enzyme was eluted with a
linear gradient running from 10% B to 100% B. The sGC-containing
fractions (18 ml) were again pooled and concentrated in centrifugal
devices with a 50-kDa cut-off (Millipore, Bedford, MA) to 1.5 ml. The
enzyme was then loaded on a Superdex 200 column (Amersham Biosciences,
60 × 2.6 cm) and eluted overnight with 50 mM TEA/HCl,
pH 8.0 containing 250 mM NaCl at 0.15 ml/min. Fractions with the highest sGC activity were pooled and concentrated as described
above to a final volume of ~200 µl. For spectroscopic measurements
100 µl of purified enzyme were used. Purified enzyme was diluted with
50 mM TEA/HCl, pH 8.0 containing 250 mM NaCl and stored with 10% (v/v) glycerol at
80 °C.
Determination of Protein Concentration and Guanylyl Cyclase
Activity Assay--
Protein concentrations were determined by the
method of Bradford using bovine plasma gamma globulin (Protein Assay
Standard I, Bio-Rad) as standard. sGC activity of Sf9 cytosol
(approximately 40 µg of protein per assay tube) or purified protein
(50 ng of protein per assay tube) was determined by incubation for 10 min at 37 °C in the presence of 1 mM cGMP, 0.5 mM [32P]GTP (about 0.2 µCi), 3 mM MgCl2, 50 mM TEA/HCl, pH 7.4, 0.25 g/liter creatine kinase, 5 mM creatine phosphate, and
1 mM 3-isobutyl-1-methylxanthine in a total volume of 0.1 ml as described by Schultz and Böhme (15). Reactions were started
by the addition of protein and incubation at 37 °C. All experiments
were stopped by ZnCO3 precipitation, and purification of
the enzyme-formed cGMP was performed as described previously (15).
Basal enzyme activity measurements were performed in the absence of NO
or YC-1. NO-stimulated measurements were performed in the presence of
the NO donor DEA/NO, and NO/YC-1-stimulated enzyme activity
measurements were performed in the presence of both DEA/NO and YC-1 in
variable concentrations. YC-1 was dissolved in 25% (v/v)
Me2SO so that the final Me2SO concentration in
the enzyme assay did not exceed 2.5% (v/v). At this concentration no
effects of Me2SO on enzyme activity were observed. DEA/NO
was dissolved in 10 mM NaOH, which also did not affect the
enzyme activity.
Generation of the
1-1200 and the
1-89 Antibodies, SDS-PAGE, and Immunoblotting--
The
1-1200 antibody was raised against two peptides
(EP012493, H2N-FTPRSREELPPNFP-COOH; and EP012494,
H2N-CFQKKDVEDGNANFLGKASGID-COOH) of the carboxyl-terminal
domain of the human
1 subunit, and the
1-89 antibody was raised against the carboxyl-terminal
peptide (EP990255, H2N-CSRKNTGTEETKQDDD-COOH). Antibodies
were coupled by an additional cysteine to keyhole limpet hemocyanin.
Rabbits were immunized on days 0, 14, 28, and 56 and were finally bled on day 80. Successful antigen response was estimated by enzyme-linked immunosorbent assay. For monitoring the purity of enzyme preparations and for the determination of apparent molecular masses of the purified
enzyme, SDS-polyacrylamide gel electrophoresis was performed in 10%
slab gels, and proteins were stained with Coomassie Blue G-250.
For immunoblotting, protein fractions were subjected to 10% SDS-PAGE
and then transferred electrophoretically to a nitrocellulose membrane.
The membrane was reversibly stained with Ponceau S, and unspecific
binding sites were saturated by immersing the membrane for 1 h in
TBST buffer (10 mM Tris/HCl, pH 8.0, 150 mM
NaCl, 0.05% Tween 20) containing 5% nonfat dry milk. The membranes
were incubated for 1.5 h in TBST buffer containing
1-1200 and
1-89 in a 1:1000 dilution and
0.5% dry milk. Negative control reactions were run in the presence of
synthetic peptides used for immunization in different
combinations (5 µg/ml). The membranes were washed three times for 10 min with TBST and subsequently incubated for 1 h with horseradish
peroxidase-labeled anti-rabbit IgG antibodies (diluted 1:4000, Sigma).
After three washes with TBST the membranes were processed with the ECL
Western blotting detection system according to the recommendations of
the manufacturer (Amersham Biosciences).
Statistical Analysis--
All results were controlled for their
statistical significance by one-way analysis of variance followed by a
Newman-Keuls post test. A value of p < 0.05 was
considered to be statistically significant.
 |
RESULTS |
To determine the function of the amino-terminal part of the
1 subunit a series of recombinants containing
progressive deletions of the amino-terminal sequences of
1 were constructed and expressed in Sf9 cells
together with the dimerizing subunit
1. On Western immunoblots using an antibody directed against a carboxyl-terminal sequence of the
1 subunit, full-length
1
and
1 deletion mutants (
1
N259 and
1
N364) exhibited
molecular masses corresponding to those predicted from their deduced
amino acid sequences (Fig. 1). Expression
levels of the full-length
1,
1 deletion
mutants, and
1 were very similar (see Fig. 1). Guanylyl
cyclase activity was measured in the respective cytosols from
Sf9 cells under basal conditions, activation with NO, and
activation with the combination of NO and YC-1 (Fig.
2). Guanylyl cyclase activity was similar under all experimental conditions for
1 and the
1 deletion mutant
1
N259.
The deletion mutant
1
N364 showed a
complete loss of sensitivity toward NO or YC-1 and a slight decrease of
guanylyl cyclase activity under basal conditions (see Fig. 2).

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Fig. 1.
Characterization of expression of the
1 subunits by Western blot analysis in
cytosolic fractions of Sf9 cells infected with the respective
variants of the 1 subunits.
For Western blot analysis an antibody was raised against the
carboxyl-terminal domain of the 1 subunit.
1 (AK 1200) and 1 (AK 6889) antibody was
used in a 1:1000 dilution. All lanes were loaded with 40 µg of
protein of the cytosolic fractions.
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Fig. 2.
Guanylyl cyclase activity in the respective
cytosolic fractions of Sf9 infected cells. Guanylyl cyclase
activity was measured under basal conditions (only 3 mM
Mg2+, black columns), in the presence of 100 µM DEA/NO (white columns), or in the presence
of additional 100 µM YC-1 (gray columns). The
columns represent means ± S.E. of at least four independent
experiments.
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Concentration response experiments were done using the NO donor DEA/NO
(Fig. 3). The EC50 values for
DEA/NO showed no significant differences and were 421 ± 69 nM for full-length
1 and 585 ± 246 nM for
1
N259.
1
N364 was NO-insensitive.
Concentration-response curves for YC-1 were performed both in the
absence (Fig. 4A) and presence
(Fig. 4B) of DEA/NO (100 µM). The
EC50 values for YC-1 showed no significant differences for
full-length
1 and
1
N259 and were 25 ± 7 and 19 ± 6 µM in the absence
and 0.89 ± 0.05 and 1.94 ± 0.64 µM in the
presence of DEA/NO, respectively.
1
N364 was YC-1-insensitive (see Fig. 4A).

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Fig. 3.
Concentration-dependent effect of
DEA/NO on guanylyl cyclase activity in Sf9 cell cytosol after
infection with the respective 1
subunits in combination with
1. Dose-response curves of
1 (closed circles), 1
N259 (open circles), and 1
N364 (closed squares) were measured in a
range of 0.1 nM-100 µM DEA/NO. All values
show the result of at least three independent experiments performed in
duplicate.
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Fig. 4.
Concentration-dependent effect of
YC-1 on guanylyl cyclase activity in Sf9 cell cytosol after
infection with the respective 1
subunits in combination with
1. The results show the effect of
YC-1 in a range between 0.1 and 300 µM YC-1. A
shows dose-response curves for 1 (closed
circles), 1 N259 (open
circles), and 1 N364 (closed
squares) for basal guanylyl cyclase activity, whereas B
shows DEA/NO (100 µM)-stimulated curves. Enzymatic
activity of 1 N364 was measured only for
the highest YC-1 concentration at basal conditions. All points of
YC-1-dependent curves represent means (±S.E.) of at least
three independent experiments performed in duplicate.
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All mutant and wild type enzymes were purified to apparent homogeneity.
Coomassie Blue-stained SDS-PAGE analyses are shown in Fig.
5. Spectroscopic analysis of the purified
wild type enzyme revealed absorption maxima at 432 nm in the absence
and 400 nm in the presence of the NO donor DEA/NO (100 µM) (Fig. 6A).
Analysis of the purified
1
N259/
1 enzyme revealed an almost
identical spectrum with very similar absorption maxima (431 nm and 399 nm, respectively; see Fig. 6B). Purified
1
N364/
1 enzyme showed absorption maxima at
432 nm in the absence and 399 nm in the presence of the NO donor DEA/NO
(Fig. 6C). Although these maxima were almost identical to
the wild type enzyme, the ratio of the absorption at 432 nm to 280 nm
was lower, indicating lower heme content (see Fig. 6C).
During the purification of the wild type and the
1
N259/
1 enzyme, fractions from each column
were pooled by determining sGC activity at basal and NO-stimulated
conditions. For the NO-insensitive
1
N364/
1 enzyme fractions could only be
tested for basal enzyme activity after each column. Thus we selected
for heme-containing, NO-sensitive enzyme in the case of wild type and
1
N259/
1 enzyme. To
control whether this effect accounts for the lower amount of heme in
1
N364/
1 enzyme, we
purified
1
N259/
1 enzyme
and assayed the fractions after each column only for basal enzyme activity. This resulted also in a significantly lower ratio of the
absorption at 432 nm to 280 nm but absorption maxima very similar to
those obtained before (432 nm in the absence and 399 nm in the
presence of DEA/NO).

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Fig. 5.
SDS-PAGE analysis of purified sGC
variants. 1 µg of purified enzyme was electrophoresed by 10%
SDS-PAGE and stained with Coomassie Blue. On the right side
of each panel a low molecular mass standard (LMW) is
shown.
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Fig. 6.
Spectroscopic analysis of purified guanylyl
cyclase enzyme complexes. Spectroscopic analysis shows relative
absorption values at basal (solid line) or NO-stimulated
(100 µM DEA/NO, dotted line) conditions.
A and B show the spectra of wild type enzyme
1/ 1 (A, 0.27 µg/µl
protein) and the deletion mutant 1
N259/ 1 (B, 0.28 µg/µl
protein). Both spectra were the result of a purification controlled
with NO-stimulated guanylyl cyclase activity for pooling after each
column (see "Experimental Procedures"). C and
D show spectra of the deletion mutants 1
N364/ 1 (C, 0.87 µg/µl
protein) and 1 N259/ 1
(D, 0.33 µg/µl protein), respectively. The spectra were
the results of a purification controlled with basal guanylyl cyclase
activity for pooling after each column (see "Experimental
Procedures").
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To compare the kinetic properties of the purified enzyme complexes,
cGMP formation was determined in the presence of increasing GTP
concentrations. A Lineweaver-Burk plot of the data revealed apparent
Km values that showed no significant differences between full-length
1 (134 ± 19 µM),
1
N259 (119 ± 13 µM), and
1
N364 (163 ± 12 µM) (Fig. 7).
Vmax values showed no significant differences
and were 145 ± 16 nmol of cGMP/min × mg for full-length
1, 104 ± 15 nmol of cGMP/min × mg for
1
N259, and 98 ± 15 nmol of
cGMP/min × mg for
1
N364.

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Fig. 7.
Double reciprocal Lineweaver-Burk plot
for substrate dependence of the respective purified enzyme complexes
under basal conditions. Substrate dependence was measured in a
range from 0.01 to 2 mM GTP for the respective
1 subunits (see figure key) in the presence
of Mg2+. Data represent means of at least three independent
experiments performed in duplicate (±S.E.).
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Guanylyl cyclase activity of the purified enzymes was measured under
basal conditions and activation with NO to investigate the status of
the enzymes (Fig. 8). Guanylyl cyclase
activity was not significantly different under all experimental
conditions for
1 and the
1 deletion
mutant
1
N259. In the presence of the NO
donor DEA/NO (100 µM) enzyme activity was increased by 242-fold for
1 and 252-fold for
1
N259. Analysis of the purified deletion mutant
1
N364 confirmed the complete loss of
sensitivity toward NO and demonstrated a slight decrease of guanylyl
cyclase activity under basal conditions (see Fig. 8).

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Fig. 8.
Guanylyl cyclase activity of the respective
purified guanylyl cyclase enzyme complexes. Guanylyl cyclase
activity was measured under basal conditions (only 3 mM
Mg2+, black columns) and in the presence of 100 µM DEA/NO (white columns). The
columns represent means ± S.E. of at least three
independent experiments.
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Concentration-response curves for YC-1 were performed both in the
absence (Fig. 9A) and presence
(Fig. 9B) of a submaximally active DEA/NO concentration (100 nM). The EC50 values for YC-1 showed
significant differences between full-length
1 and
1
N259 and were 51 ± 7 and 30 ± 5 µM (p < 0.05) in the absence and
2.62 ± 0.26 and 4.53 ± 0.71 µM
(p < 0.05) in the presence of DEA/NO (100 nM), respectively.
1
N364 was YC-1-insensitive (see Fig. 9A).
Vmax values showed significant differences only
in the absence of DEA/NO and were 9717 ± 216 nmol of
cGMP/min × mg for full-length
1 and 5745 ± 420 nmol of cGMP/min × mg for
1
N259 (p < 0.001) at basal conditions
and 24,259 ± 3237 nmol of cGMP/min × mg for full-length
1 and 17,369 ± 3308 nmol of cGMP/min × mg
for
1
N259 in the presence of 100 nM DEA/NO.

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Fig. 9.
Concentration-dependent effect of
YC-1 on purified guanylyl cyclase activity. The results show the
effect of YC-1 in a range between 0.1 and 200 µM YC-1.
A shows dose-response curves for 1
(closed circles), 1 N259
(open circles), and 1 N364
(closed squares) for basal guanylyl cyclase activity,
whereas B shows DEA/NO (100 nM)-stimulated
curves. Enzymatic activity of 1 N364 was
measured only for the highest YC-1 concentration at basal conditions.
All points of YC-1-dependent curves represent means
(±S.E.) of five independent experiments performed in duplicate.
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DISCUSSION |
Previous studies have used amino-terminal deletion mutants of
bovine NO-sensitive guanylyl cyclase subunits to map functional regions
of this enzyme family (11). The deletion of only 131 amino-terminal
amino acids of the
1 subunit and co-expression of this
deletion mutant with the
1 subunit in the baculovirus system led to a 10-fold reduction in enzyme activity in co-infected Sf9 cytosol versus the respective
1
full-length construct and an almost complete loss of NO sensitivity
(11). By contrast, in our study there was no significant decline in
basal enzyme activity and stimulation by NO after deletion of 259 amino
acids of the
1 subunit. It is conceivable that our
approach of using endogenously occurring methionines rather than newly
introduced methionines as start codons poses less risk of unwanted
conformational changes resulting in lower enzyme activity. It is also
possible that sequence differences between the bovine and human
1 subunits might explain the discrepancies. Subsequent
to the study by Wedel and co-workers (11), the same group analyzed the
purified
1
N131/
1 enzyme
complex and showed a loss of affinity in binding of the prosthetic heme
group, which binds NO (12). Based on the findings of this study, it has
been suggested that heme binding to sGC requires the presence of both
subunits (
1 and
1) in full-length and
that both homologous amino-terminal regions of the
1 and
1 subunits participate in binding of the prosthetic heme
(12). Expression of only the amino-terminal part of the
1 subunit expressed in Escherichia coli
indicated that the amino terminus of the
1 subunit
(
1-(1-385)) is sufficient for heme binding with
preserved binding of NO (16). As pointed out by the authors of the
latter study (16), this result does not rule out an in
vivo heme binding site involving residues contributed by both the
and
subunits as suggested by Foerster et al. (12).
Given the degree of homology in the amino terminus of the subunits, it
is possible that the
1-(1-385) homodimer is an in
vitro outcome of expression in the absence of the
1
subunit and that the second
1 subunit provides crucial
amino acids for heme binding that would come from the
1
subunit in vivo (16). The results of the current study argue against an in vivo heme binding site involving
amino-terminal residues contributed by both the
and
subunits as
suggested by Foerster et al. (12) and a model where the heme
in sGC is sandwiched between the amino-terminal parts of the two
subunits (16). The results of the current study rather indicate that the amino-terminal region of the
1 subunit is not
involved in heme binding. In fact, we could fully reproduce the
findings by Zhao and Marletta (16) that the amino-terminal part of the
1 subunit can be expressed as a soluble, nitric oxide-,
and heme-binding protein in E. coli.2 In contrast, the
expression of longer constructs of the
1 subunit including the catalytic domain or co-expression with the
1 subunit in E. coli led to insoluble protein
in the form of inclusion bodies under different experimental
conditions.2 These findings should encourage
approaches to solve the structure of the heme binding domain of sGC by
focusing on the expression of the amino-terminal part of the
1 subunit.
We find in the current study that deletion of 259 amino acids of the
1 subunit leaves the enzyme functionally intact but that
deletion of the first 364 amino acids leads to an enzyme complex with
preserved heme binding but loss of sensitivity to NO or YC-1. NO still
binds to the enzyme variant lacking 364 amino acids since it induces
the typical spectral shift of the prosthetic heme group. This indicates
that amino acids 259- 364 of the
1 subunit are either
directly important for the transduction of the activation signal by NO
or that the deleted region is indirectly involved in the mediation of
the NO effect, e.g. by the stabilization of another domain
of the enzyme. The cysteine 238 and cysteine 243 region in the
1 subunit has been mapped as the likely binding site of
the YC-1-related substance BAY 41-2272 using photoaffinity labeling
(2). We show in the current study that both cysteines including an
additional 14 amino-terminal residues can be deleted without loss of
YC-1 sensitivity. Given the data by Stasch and colleagues (2), we think
that it is likely that the region adjacent to cysteine 238 and cysteine
243 (from amino acid 259 to 364) of the
1 subunit
represents the binding site of YC-1. The possibility that YC-1 still
binds to the
1
N364 mutant enzyme complex
and that the activation signal is not transduced within the enzyme
directly or indirectly as discussed for NO is also a plausible
explanation of our results.
In the present study, sGC in Sf9 cytosol is activated 25-fold,
while the purified enzyme is activated 80-fold. The -fold
stimulation by YC-1 in the literature varies from 7-fold (17), 10-fold
(18), and 14-fold (19) up to close to 100-fold (20). YC-1 activation of
sGC is to a very large degree heme-dependent (17, 21), and
thus -fold stimulation of YC-1 is a function of the heme content of the
enzyme similar to the heme-dependent activator NO. The result that stimulation factors by YC-1 are significantly higher for
the purified enzymes than in Sf9 cytosol can be explained by a
higher percentage of heme-containing versus heme-free enzyme that may also form by expression in Sf9 cells. Since we have
pooled the fractions during our purification protocol according to the determination of sGC activity at basal and NO-stimulated conditions, we
have purified selectively heme-containing enzyme.
The purified
1
N364 enzyme complex
contained less heme than the wild type enzyme or the
1
N259 enzyme complex purified under regular conditions.
Because of the lack of NO sensitivity, the
1
N364 enzyme complex had to be purified by controlling fractions after each column for basal guanylyl cyclase activity only.
In the case of the wild type enzyme and the
1
N259 enzyme complex we have pooled the fractions during
our purification protocol according to the determination of guanylyl
cyclase activity at basal and NO-stimulated conditions and have thus
purified selectively heme-containing enzyme. Attempts to establish a
purification protocol with wild type enzyme that would be applicable to
all three enzyme variants by pooling fractions after each column
according to their absorbance at 430 nm in the spectrophotometer were
not successful. Especially at the crucial initial stages of the
purification protocol the measurement of the absorbance at 430 nm gave
no reliable results with respect to sGC-containing fractions when
compared with NO-activated guanylyl cyclase activity measurements. To
find out whether the relatively low heme content of the
1
N364 enzyme complex was due to the
impossibility of pooling fractions according to NO-activated guanylyl cyclase activity, we purified the
1
N259 enzyme under the same conditions. This resulted in
an enzyme preparation with a similarly reduced heme content. While this
argues in favor of the hypothesis that the different purification
procedure is responsible for the relatively low heme content, we cannot
rule out the possibility that the
1
N364
mutant enzyme complex shows reduced heme binding affinity.
In summary, we show that 1) the amino-terminal part of the
1 subunit is not involved in heme binding and 2) amino
acids 259-364 of the
1 subunit represent an important
functional domain for the transduction of the NO activation signal and
likely represent the target for NO-sensitizing substances like
YC-1.
 |
ACKNOWLEDGEMENTS |
The expert technical assistance of Jutta
Starbatty, Jenny Behrens, and Alexandra Zielinski is gratefully acknowledged.
 |
FOOTNOTES |
*
This study was supported by Deutsche Forschungsgemeinschaft
Grant BE 1865/2-1 and the Forschungsförderungsfond Hamburg.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 should be addressed: University clinic
Eppendorf, Pharmakologisches Institut, Martinistrasse 52, D-20246 Hamburg, Germany. Tel.: 49-40-42803-2055; Fax: 49-40-42803-4876; E-mail: behrends@plexus.uke.uni-hamburg.de.
Published, JBC Papers in Press, January 30, 2003, DOI 10.1074/jbc.M212740200
2
S. Behrends, unpublished data.
 |
ABBREVIATIONS |
The abbreviations used are:
sGC, soluble
guanylyl cyclase;
DEA/NO, 2,2-diethyl-1-nitroso-oxyhydrazine;
TEA, triethanolamine.
 |
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Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.