From the Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284-7760
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ABSTRACT |
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A variety of monovalent anions and cations were
effective in stimulating both calcium ion/calmodulin
(Ca2+/CaM)-independent NADPH-cytochrome c
reductase activity of, and Ca2+/CaM-dependent
nitric oxide (NO·) synthesis by, neuronal nitric oxide synthase
(nNOS). The efficacy of the ions in stimulating both activities could
be correlated, in general, with their efficacy in precipitating or
stabilizing certain proteins, an order referred to as the Hofmeister
ion series. In the hemoglobin capture assay, used for measurement of
NO· production, apparent substrate inhibition by
L-arginine was almost completely reversed by the addition
of sodium perchlorate (NaClO4), one of the more effective
protein-destabilizing agents tested. Examination of this phenomenon by
the assay of L-arginine conversion to
L-citrulline revealed that the stimulatory effect of
NaClO4 on the reaction was observed only in the presence of
oxyhemoglobin or superoxide anion (generated by xanthine and xanthine
oxidase), both scavengers of NO·. Spectrophotometric examination
of nNOS revealed that the addition of NaClO4 and a
superoxide-generating system, but neither alone, prevented the increase
of heme absorption at 436 nm, which has been attributed to the nitrosyl
complex. The data are consistent with the release of autoinhibitory
NO· coordinated to the prosthetic group of nNOS, which, in
conjunction with an NO· scavenger, causes stimulation of the reaction.
The nitric-oxide synthases
(NOSs)1 comprise a family of
calmodulin (CaM)-dependent flavoheme enzymes that catalyze
the NADPH-dependent oxidation by molecular oxygen of
L-arginine to L-citrulline and nitric oxide
(NO·). The isoforms of NOS are grouped into three categories,
neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). The first two constitutive isoforms are collectively referred to as
cNOS. nNOS is a homodimer, which contains one molecule each of heme,
FAD, FMN, and tetrahydrobiopterin (BH4) per subunit (1-5). The binding sites for NADPH, FAD, and FMN are located in the
carboxyl-terminal half of nNOS, which exhibits sequence homology to
NADPH-cytochrome P-450 reductase and also contains FMN and FAD (6). The
amino-terminal half contains the binding sites for heme,
L-arginine, and BH4 (7). Electron transfer from
NADPH via the flavins is facilitated by the binding of
Ca2+-calmodulin (Ca2+/CaM) and
L-arginine (8, 9). Electron transfer to the artificial electron acceptor cytochrome c is stimulated by, but is not
totally dependent on, Ca2+/CaM (10, 11) and represents the
NADPH-cytochrome c reductase activity of nNOS.
A primary area of interest regarding the NOSs is the control of their
activities. cNOS requires Ca2+/CaM and is therefore
sensitive to levels of Ca2+ in the cell. On the other hand,
iNOS contains tightly bound Ca2+/CaM and is not sensitive
to cellular levels of Ca2+. cNOSs contain what appears to
be an autoinhibitory element, whereas iNOS does not (12). There is also
considerable evidence that cNOS and iNOS are feedback-inhibited by
NO· (13-18). Wang et al. (19) obtained optical and
resonance Raman scattering spectroscopic evidence for a ferrous
NO· complex of nNOS. These investigators found that arginine
stabilized the complex. Abu-Soud et al. (20) showed that the
nitrosyl complex was formed rapidly and experienced a relatively slow
O2-dependent turnover in which nitrate was a
product. In the steady state, most of the enzyme appeared to be
complexed in this fashion, dissociating upon the depletion of either
NADPH or L-arginine. An estimated Km for
oxygen (KmO2) of 38 µM
was determined for NADPH oxidation in the absence of
L-arginine. In the presence of L-arginine, a
value of KmO2 of about 350 µM was
reported (21). It has been suggested that the formation of nitrosyl NOS
and its slow recycling to free NOS enable the enzyme to synthesize
NO· over a broad range of O2 concentrations.
Confirmation of hexacoordinated NO· with the heme prosthetic
group of nNOS by EPR spectroscopy was reported by Migita et
al. (22), who also showed that L-arginine is bound
near the distal side of the heme prosthetic group in close proximity to
the bound NO·. The NO·-heme complex was stabilized by
L-arginine and analogs of L-arginine. Recently,
Abu-Soud et al. (23) have reported the results of studies in
which the rates of formation and decomposition of the nitrosyl iNOS
oxygenase domain dimer were measured. However, no conclusions were
drawn regarding the extent of autoinhibition of the full-length isoform
by NO·.
Previously, we had shown that 3-4 M urea simulated the
stimulatory effect of Ca2+/CaM on NADPH-cytochrome
c reductase activity of nNOS (24). Guanidinium chloride
(GmCl), which like urea is a protein denaturant, at 0.4-0.5
M, stimulated the reductase activity to essentially the
same extent. It was, therefore, of interest to determine whether other
reagents of lesser protein denaturing capability would elicit similar
changes in reductase activity and whether any of these reagents could
promote NO· synthesis in the absence of Ca2+/CaM. In
this work, we have found that neither urea nor GmCl is capable of
mimicking the stimulatory effect of Ca2+/CaM on NO·
synthesis by nNOS and that both reagents are actually inhibitory to the
reaction when added in the presence of Ca2+/CaM. However,
we have found that a variety of salts stimulated both the
NADPH-cytochrome c reductase and
Ca2+/CaM-dependent NO· synthase
activities of nNOS. Their efficacy in stimulating these activities
could be correlated with the ability of these salts to induce
structural changes in collagen and cold gelatin in dilute solution and
unfolding or destabilization of native ribonuclease. The background of
these investigations has been reviewed (25). Concentrations of salts
required to stimulate reductase activity were somewhat higher than
those required for optimal stimulation of NO· synthesis. Using
the hemoglobin capture assay of the NO·-producing activity of
NOS, it was found that sodium perchlorate (NaClO4), at 50 mM, brought about a 5-fold increase in NO· synthesis
at 100 µM L-arginine, the assay concentration
of this substrate. At very low arginine concentrations (below 1 µM), NaClO4 had no effect. However, at higher
arginine concentrations, reversal of the substrate inhibition by
NaClO4 was apparent. Use of the NOS assay in which
conversion of arginine to citrulline is measured revealed that
oxyhemoglobin (oxyHb) or superoxide anion (generated by the oxidation
of xanthine in the presence of xanthine oxidase) was required to obtain
optimal stimulation with NaClO4. Comparable results were
obtained when sodium chloride (NaCl) was used in place of
NaClO4. Spectrophotometric observation of the nitrosyl nNOS
complex, which is characterized by an absorption maximum at 436 nm,
revealed that its formation was completely inhibited by the addition of
50 mM NaClO4 and a superoxide-generating
system. Thus, it would appear that NaClO4, as well as other
salts, prevent the formation of, or destabilize, the nitrosyl
derivative in conjunction with reagents that react with NO·. CD
measurements of nNOS·Ca2+/CaM indicated that only small
changes in the secondary structure of the enzyme occurred in the
presence of 25-75 mM NaClO4.
Materials--
GmCl was obtained from Whitaker Corp. (Delaware
Water Gap, PA). Guanidinium thiocyanate was a product of Eastman Kodak.
NaClO4 was purchased from Aldrich. Horse heart cytochrome
c (type VI), HEPES, BisTris, NADPH, sodium nitrate, and
bovine brain CaM were obtained from Sigma. BH4 was a
product of Research Biochemicals International (Natick, MA). Other
compounds were of reagent grade and were purchased from reputable
commercial sources.
Enzymes--
nNOS was overexpressed in Escherichia
coli and purified according to Roman et al. (26). Highly purified
Cu/Zn superoxide dismutase (3,400 units/mg) was a generous gift from
Dr. John Crow (University of Alabama at Birmingham). Catalase (25,000 units/mg) and buttermilk xanthine oxidase (1.1 unit/mg) were purchased
from Sigma.
Enzyme Assays--
Superoxide dismutase and xanthine oxidase
were assayed as described by McCord and Fridovich (27).
NADPH-cytochrome c reductase assays were performed according
to the procedure of Masters et al. (28). Nitric oxide
formation was measured by the hemoglobin capture assay at 25 °C
following the procedures of Kelm et al. (29) and Stuehr
et al. (30). Measurements were made in a Shimadzu UV-2101 PC
(dual beam) scanning spectrophotometer. Assay samples were read against
blanks containing all assay components except nNOS.
L-Citrulline formation from L-arginine was
measured after separation of the amino acids using cation exchange
chromatography by the method of Bredt and Snyder (31), except that 5 µM BH4 was added to the assay solution, and
0.5 µCi/ml L-[14C]arginine was used in
place of L-[3H]arginine. Dilutions of nNOS
were made with 50 mM Tris-HCl (pH 7.5) containing 0.1 mM sodium EDTA, 100 mM NaCl, 0.1 mM
2-mercaptoethanol, and 10% glycerol.
Salt Solutions--
Salts were usually prepared as 2 M solutions in 50 mM HEPES/KOH (pH 7.6). When
necessary, the pH was adjusted with small volumes of 6 N
HCl or 10 N NaOH.
To understand better the effects of urea and GmCl on the
NADPH-cytochrome c reductase activity of nNOS, the effects
of other protein denaturants and nondenaturants were examined. The
results are shown in Fig. 1. As with urea
and GmCl (24), in most of the cases there was a concentration
dependence of activity, attaining an optimal level followed by a
decline. As expected (Fig. 1A), GmSCN was the most effective
on a molar basis, undoubtedly because of the destabilizing effects of
both guanidinium and thiocyanate ions (25). It is notable that the
stimulation at optimal GmSCN was equal to that of Ca2+/CaM.
As reported earlier (24), GmCl stimulation of NADPH-cytochrome c reductase activity at 0.4-0.5 M equaled that
of Ca2+/CaM. The remaining anions described in the figure
were added as their sodium salts. In Fig. 1A, the order of
effectiveness of the anions, as indicated by lower concentrations
required to reach optimal activity, was SCN
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
RESULTS
,
ClO4
,
NO3
, and 1/2
SO42
= CH3COO
. Although the magnitudes of the
stimulations by the halides were significantly lower than those of many
of the other anions (Fig. 1B), there was a clear order of
effectiveness: I
, Br
, Cl
and
F
. Taking these data together, the order of molar
effectiveness of the anions in stimulating NADPH-cytochrome
c reductase activity of nNOS was SCN
= I
> ClO4
> Br
> NO3
> Cl
> SO42
= F
= CH3COO
. This order is in
general agreement with the efficacy of these compounds as denaturants
of other proteins (25). Thus, for native collagen and ribonuclease, the
order observed for these anions was SCN
> I
> ClO4
> NO3
> Br
> Cl
> CH3COO
> SO42
.
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Fig. 1.
Effects of sodium salts on the
NADPH-cytochrome c reductase activity of nNOS.
Panel A, 2.5 nM nNOS was incubated in
50 mM (pH 7.6) HEPES/KOH with 40 µM
cytochrome c, 100 µM NADPH, and the sodium
salts of the ions, except for GmSCN, at the final concentrations
indicated. The concentrations of Na2SO4 were
those of the sodium ion. Panel B, sodium halides were tested
as described in panel A. The data in the figure are
representative of at least three experiments.
Monovalent cations, added as their chloride salts, gave less definitive stimulations than the anions tested, as shown in Fig. 2. For the most part, optimal stimulations required concentrations close to 1 M. NH4+ was the most stimulatory, an unexpected observation in view of its reported effect as a protein-stabilizing cation (25). Over a broad range of concentrations, Cs+, Na+, K+, and Rb+, in that order, gave stimulations of 5-3-fold, whereas Li+, at the same concentrations, was only weakly stimulatory. It is of interest that Li+ was more effective than the other four alkali metal cations in stabilizing collagen (25). In an overall sense, these results probably indicate that various salts exert their stimulatory effect by influencing the conformation of nNOS and that mere changes in ionic strength cannot explain the striking differences in the data.
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Because it was clear that many salts were able to mimic the stimulating effect of Ca2+/CaM on nNOS-catalyzed NADPH-cytochrome c reductase, it was important to determine whether the presence of these reagents could affect NO· synthesis by nNOS in the absence of Ca2+/CaM. When urea was tested in the hemoglobin capture assay or in the citrulline assay, it was found not to substitute for Ca2+/CaM. In fact, in the presence of Ca2+/CaM, 2 M urea completely inhibited the enzyme, as measured by both the hemoglobin capture and citrulline assay methods. Likewise, in the hemoglobin capture assay, GmCl failed to stimulate NO· synthesis in the absence of Ca2+/CaM, and, at 0.1-0.2 M, it completely inhibited the reaction in the presence of Ca2+/CaM. This was not surprising because guanidinium ion would be expected to antagonize the binding of L-arginine to the enzyme. Sorrentino et al. (32) have shown that GmCl and other guanidinium compounds are inhibitory to both cNOS and iNOS. In the present study, other compounds, including NaClO4, NaNO3, and NaCl, failed to substitute for Ca2+/CaM in NO· synthesis.
In the course of using the hemoglobin capture method in the assay of
NO· synthesis by nNOS, it was found that several of the
compounds used in the study of the NADPH-cytochrome c
reductase activity of nNOS actually stimulated
Ca2+/CaM-dependent NO· synthesis. In
this study, GmSCN was not used because of its inhibitory effect on the
hemoglobin capture assay of NO· synthesis. NaSCN, as well as
GmSCN, brought about a rapid and nonenzymatic change in the absorption
spectrum of oxyHb in the presence of assay solution
components,2 which precluded
their use in the assay. However, the order of effectiveness in the
stimulation of NO· synthesis by nNOS was
ClO4 > I
= Br
> NO3
> CH3COO
= Cl
(Fig.
3). With the exception of the positioning
of I
and Br
, the order of efficacy, as
anticipated from the protein-stabilizing and -destabilizing properties
of this group of anions, was essentially maintained (see above
discussion of Fig. 1). The data show that ClO4
brought about a 5-fold
stimulation of the reaction at a concentration of 50 mM.
The effects of I
and Br
were practically
superimposable, whereas the effect of
NO3
was distinguishable from that of
either acetate ion or Cl
. F
was not tested
because of its likely interaction at high concentrations with
Ca2+.
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All of the alkali metal cations stimulated the hemoglobin capture assay without demonstrating differences in their effective concentrations, as shown in Fig. 4. In this respect, the results are similar to those obtained with these cations in the NADPH-cytochrome c reductase reaction (Fig. 2). Again, Li+ was the least stimulatory, which is consistent with its reported protein-stabilizing effects (25).
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All of the salts tested did not behave predictably. Thus, as shown in
Fig. 5, Na2SO4
produced significant stimulation of NO· production over a broad
concentration range (0.05-0.5 M
SO42), in stark contrast to its lack
of effect on NADPH-cytochrome c reductase activity (Fig. 1).
In addition, NH4Cl and
(NH4)2SO4, both stabilizing salts
(25), were unexpectedly effective at relatively low ammonium ion
concentrations (optimal at 0.1-0.2 M), although
(NH4)2SO4 was significantly less
stimulatory. In the NADPH-cytochrome c reductase assay,
NH4+ was the most stimulatory cation
tested (Fig. 2). It is possible that the stimulatory effect of
NH4+/NH3 may represent a
phenomenon unrelated to stabilization or destabilization of the enzyme
protein.
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Two multivalent cations and other multivalent anions were also studied with regard to their stimulatory/inhibitory effects on NO· formation. Sodium phosphate (between 150 and 250 mM) stimulated NO· formation some 3-4-fold. Sodium tartrate (between 100 and 200 mM) stimulated about 2.5-fold. Sodium citrate was stimulatory at low levels (2-fold at 25 mM) but inhibitory above 50 mM, probably because of its ability to chelate Ca2+. CaCl2 and MgCl2 were slightly stimulatory (10-30%) at low concentrations (<4 mM and < l mM, respectively) but inhibitory at higher concentrations.
Further experiments were performed with NaClO4 because this reagent was effective at relatively low concentration (see Fig. 3) and did not interfere with either the hemoglobin capture assay or, as will be discussed shortly, the citrulline assay.
Km values for L-arginine of 1.5 µM (31), 2.2 µM (33), and 2.8 µM (26) have been reported for nNOS. In the experiment described in Fig. 3, the L-arginine concentration was 100 µM. It was therefore of interest to examine the effect of NaClO4 over a broad range of L-arginine concentrations. For this purpose, the nNOS preparation was subjected to removal of residual L-arginine introduced during the purification procedure by the use of spin columns of Sephadex G-50 (34). The results of this study are described in Fig. 6. Substrate inhibition of NO· synthesis by L-arginine is quite apparent in the data (Fig. 6A). The first evidence of inhibition appeared at about 5 µM L-arginine. The addition of 50 mM NaClO4 substantially increased NOS activity over most of the concentration range studied. At 100 µM, the concentration of L-arginine used routinely in the assay, a stimulation of more than 5-fold was observed. Examination of the kinetics at low L-arginine concentrations (Fig. 6B) reveals stimulation by NaClO4 at concentrations in the 1-2 µM L-arginine range. It was at concentrations below 1 µM L-arginine that NO· synthesis was not stimulated by NaClO4. It thus appears that NO· synthesis by nNOS was extensively inhibited by its substrate L-arginine and that 50 mM NaClO4 almost completely reversed the inhibition. This impression is supported by the data in Fig. 6C, a double reciprocal plot of the data in Fig. 6A. In the presence or absence of NaClO4, the data can be extrapolated to give the same Vmax and a Km value of approximately 5 µM for L-arginine. However, substrate inhibition was obvious in the absence of NaClO4 but much less apparent in its presence.
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Further study of the effect of NaClO4 was pursued by use of the citrulline assay. This method involves incubation of nNOS in the presence of necessary cofactors with substrate L-[14C]arginine and subsequent ion exchange chromatography to separate substrate and product (31). Up to 300 mM NaClO4 or NaCl in the incubation solution could be tolerated in the subsequent ion exchange chromatography on Dowex 50-Na+ columns. The concentrations of the components in the assay solution were the same as those described for the hemoglobin capture assay except that oxyHb was omitted and the L-arginine concentration was 20 µM instead of 100 µM as employed in the capture assay. As shown in Table I (Experiment 1), 50 mM NaClO4 alone had no effect on the reaction. However, the addition of oxyHb with NaClO4 stimulated the reaction some 3-4-fold. Using this assay method, Rogers and Ignarro (13) have shown that the addition of 30 µM oxyHb linearized the kinetics of cerebellar NOS beyond 5 min, ostensibly by trapping inhibitory NO·. It thus appeared likely that NaClO4 brought about release of inhibitory NO· from the heme prosthetic group of nNOS and that the released NO· was then scavenged by the heme group of oxyHb. In this process, NO· is oxidized to nitrate (35), which is not inhibitory to NOS. Because NO· is known to react with superoxide to produce peroxynitrite (36), xanthine/xanthine oxidase, which generates superoxide anion, was used in place of oxyHb. As shown in Table I (Experiment 3), this resulted in a near 3-fold stimulation in the presence of NaClO4. This effect was negated by superoxide dismutase in the presence of catalase. Qualitatively comparable data were obtained when superoxide anion generation by the autoxidation of BH4 (37) at 100 µM (38), instead of the normal assay concentration of 5 µM, was performed in the presence of 50 mM NaClO4 (data not shown).
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Because Na+ and Cl+ have been described as close to the "null point" between denaturing and nondenaturing ions (39), the effect of 300 mM NaCl on the citrulline assay was also studied. The results were consistent with those observed with NaClO4 (Table I, Experiment 2). Thus, NaCl had some effect in the absence of oxyHb, but, when added with the latter, a 2-3-fold stimulation was observed.
The question of whether NaClO4 or NaCl acted by interfering with nitrosyl nNOS formation or by dissociating nitrosyl nNOS was approached by delaying addition of NaClO4 or NaCl to the assay solution, as shown in Fig. 7. Whether NaClO4 was added at zero time or up to 2 min after the complete assay solution was constituted, stimulation of NOS activity was significantly greater than that observed in the absence of the salt. The same result was observed with 300 mM NaCl.
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Direct evidence that the presence of perchlorate and a superoxide-generating system prevented formation of the nitrosyl nNOS complex, monitored at 436 nm, is shown in Fig. 8. In agreement with Abu-Soud et al. (20), nitrosyl nNOS was formed rapidly and decayed rapidly as NADPH and L-arginine were consumed (panel A). The addition of 50 mM NaClO4 alone (panel B) or of a superoxide-generating system alone (panel C) had no effect on the formation of the complex or its disappearance. However, in the presence of both (panel D), nitrosyl nNOS formation was totally inhibited.
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The possibility that the effects of the various anions were exerted
through conformational changes in the protein was addressed through
examination of the nNOS·Ca2+/CaM complex by circular
dichroism, using a Jasco model J-720 spectropolarimeter (data not
shown). In the presence of 25, 50, and 75 mM
NaClO4, only minor changes in the CD spectrum of 1 µM nNOS were observed. These results are consistent with
the induction of local, rather than global, changes by the perturbant.
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DISCUSSION |
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The phenomena in which both protein denaturants and nondenaturants
stimulate the NADPH-cytochrome c reductase activity of nNOS
in the absence of Ca2+/CaM and NO· synthesis in the
presence of Ca2+/CaM present interesting questions with
regard to the relationship between the structure and function of nNOS.
The ability of a given compound to destabilize the native structure of
other proteins and the efficacy of that compound in stimulating the
reactions catalyzed by nNOS initially suggested that significant
conformational changes in the enzyme protein might be involved. The
data described in this manuscript have provided evidence that several
of the compounds tested, in addition to urea and GmCl (24), bring about increases in NADPH-cytochrome c reductase activity which are
equivalent to those induced by Ca2+/CaM. With practically
all of the compounds used, there was a peak concentration at which
optimal activity was observed. Thus, it was possible to make
comparisons of the efficacies of these compounds on a molar basis. It
was of interest that, by this criterion, the stimulatory effectiveness
of each ion tested could usually be correlated with its relative
position in the Hofmeister ion series, which originally defined the
relative efficacy of certain salts/ions to precipitate proteins from
whole chicken egg white (39, 40). Theoretical treatments of the
Hofmeister effect have been put forth for the solubility in aqueous
solution of amino acids, synthetic peptides, and other model compounds,
as a function of the concentrations of various salts (39, 41-44). Interestingly, a correlation could be drawn between these anions and
cations and their ability to effect transitional changes in the
structures of proteins, synthetic polymers, and DNA (25, 41). Thus,
with respect to proteins, the more effective a salt was in
precipitating a macromolecule, the more it stabilized its "native"
structure. In the present study, for the most part, sodium salts of
anions of interest and chloride salts of cations of interest were
employed. The rationale for this was that, with regard to their
stabilizing/destabilizing properties, Na+ and Cl have
been found to be relatively neutral ions (39). With regard to nNOS
(Figs. 1 and 3), it appears that the destabilizing anions were more
effective in stimulating both NADPH-cytochrome c reductase
activity and NO synthesis by nNOS. This was clear-cut in the case of
the reductase activity (Fig. 1), which was stimulated most effectively
by GmSCN and with decreasing molar efficacy by the sodium salts of
SCN
= I
,
ClO4
, Br
,
NO3
, and Cl
, and 1/2
SO42
= F
= acetate ion.
There was little discrimination on the part of the alkali metal
cations, except for Li+, which, consistent with its
protein-stabilizing properties, stimulated the least strongly (Fig. 2).
The stimulation by NH4+ was not expected
(see discussion of Fig. 5 below).
With regard to NO synthesis, the field of anions was reduced somewhat
because GmSCN was an inhibitor of NO synthesis and, like NaSCN,
interfered with the hemoglobin capture assay. However, the order of
decreasing effectiveness in stimulating the NO synthesis reaction of
ClO4, I
= Br
, NO3
, and acetate
ion = Cl
was consistent with the decreasing
destabilizing properties of these ions (Fig. 3).
SO42
exhibited a broad range of
stimulatory capacity (Fig. 5). Phosphate, which at pH 7.6 is nearly a
dianion, was more effective in stimulating nNOS activity than
SO42
(data not shown). The reverse
would have been predicted from the precipitating capacity of these ions
with respect to bovine ribonuclease (25). As mentioned earlier, both
MgCl2 and CaCl2 were inhibitory at relatively
low concentrations. It is thus clear that a more extensive study would
be required to develop insight into the mechanisms whereby divalent
cations and anions affect nNOS activity.
It is difficult to discern any distinguishing features regarding the effects of the alkali metal cations on NO synthesis because their molar efficacies were essentially the same (Fig. 4). However, as in the case of the reductase activity, Li+ was the least stimulatory of these ions (Fig. 2). NH4+, in the form of NH4Cl and (NH4)2SO4, exhibited molar efficacies that were significantly lower than what was expected on the basis of the protein-stabilizing properties of these salts (Fig. 5). Thus, ammonium ion or ammonia may have a specific stimulatory effect on nNOS.
It is of interest that the concentrations required to attain the optimal effect with each compound tested were, in general, greater for the NADPH-cytochrome c reductase reaction than for NO· synthesis, e.g. the optimal concentrations of NaClO4 and NaNO3 for reductase activity were approximately 7-8 times and approximately 3 times that for NO· synthesis, respectively. In the case of reductase activity, the role of the reagent was to replace the requirement for Ca2+/CaM. However, with NO· synthesis, the effect was only observed in the presence of Ca2+/CaM. From the literature and the results presented here, it may be possible to rationalize how the salts affect Ca2+/CaM-dependent NO· synthesis. It is possible that the salts relieve the autoinhibition of NOS by NO· (13-18). Abu-Soud et al. (20) have presented evidence for the rapid and reversible formation (<2 s) of a six-coordinate ferrous-nitrosyl complex, involving 70-90% of nNOS upon mixing of the enzyme with substrates. Thus, when substrate (L-arginine or NADPH) is depleted, the nitrosyl form of the enzyme can undergo dissociation to release NO· and form free NOS (20). Based primarily on the inhibition of NADPH oxidation in the presence of L-arginine, it was estimated that the enzyme was 90% inhibited during the assay. It has been proposed that, in this state, compared with NADPH oxidation in the absence of L-arginine, the enzyme is able to generate NO· over a broader range of O2 concentrations (21).
The results presented in this report suggest that NaClO4, NaCl, and probably other salts act by dissociating NO· from the nitrosyl form of NOS. However, both NaClO4 and NaCl were without significant stimulating effect in the absence of oxyHb or a superoxide anion-generating system (xanthine/xanthine oxidase), reagents that can be considered as traps of NO·. Therefore, the data are consistent with the ability of the salt to dissociate the ferrous nitrosyl complex of nNOS. Because the equilibrium of NO· binding would appear to favor the binding of NO· to give the inhibitory complex, the perturbation introduced by the appropriate salt could expose free NO· to either oxyHb or superoxide anion produced by the xanthine oxidase reaction or the autoxidation of BH4. The net effect would be to shift the equilibrium between nitrosyl nNOS and free nNOS, favoring the latter and causing enzyme activity to rise dramatically. The fact that the time of the addition of either NaClO4 or NaCl is not critical may indicate that either nitrosyl nNOS undergoes turnover once formed and that either reagent prevents reformation of the complex or these reagents act directly on the complex to cause its dissociation. The observation of the quenching of nitrosyl nNOS absorption at 436 nm in the presence of NaClO4 and a superoxide-generating system (Fig. 8) provides direct evidence for this interpretation. That the quenching appeared to be total would favor the argument that the stimulation by NaClO4 of nNOS activity in the hemoglobin capture assay reflected a complete reversal of autoinhibition by NO· of nNOS activity.
It would seem paradoxical that denaturing agents would elicit increases in the biological activity of a protein. However, in the circumstances studied here, it is clear that, as the perturbing compound/ion is increased in concentration, there is a decrease, sometimes sharp, following the stimulation, indicating that disruption of the active structure has been attained. It was therefore of interest to determine whether large conformational changes were involved in the stimulatory effects of the salts, particularly those that were very stimulatory. The CD experiment would appear to rule out changes that are global in magnitude and to favor changes that are more localized in nature. In this regard, a possible explanation comes from the observations of Gregor et al. (45). These investigators showed a selectivity of various anions for a quaternary base ammonium anion exchange resin. The order of selectivity involved a ranking that was very similar to the Hofmeister series described earlier. Thus, the more selective anion would displace a less selective anion in an anion-quaternary ammonium resin ion pair. By analogy, in the nitrosyl nNOS complex, an ion pair may be destabilized by perchlorate ion. The labilized nitrosyl nNOS would then be likely to break down, especially in the presence of an NO· trap, such as oxyHb or superoxide anion. It is also conceivable that the binding of a natural, albeit yet to be identified, activator/ligand of nNOS could also act by disrupting an inhibitory ion pair, yielding a significantly more active enzyme molecule.
After this manuscript was submitted for publication, a report appeared
in which effects of various Hofmeister salts onNO· synthesis by
nNOS, eNOS, and iNOS were described (46). Although the results
regarding stimulation of nNOS activity through the addition of the
salts were similar to those observed by us, the experiments were
conducted using the citrulline forming assay, in the absence of
hemoglobin. No inference was made concerning the importance of a nitric
oxide scavenging system or the prevention of nitrosyl nNOS formation in
the stimulation of citrulline synthesis in this publication (46).
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ACKNOWLEDGEMENTS |
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We are indebted to Drs. Magnus Hook and Steven La Brenz of the Center for Matrix Biology, IBT, Texas A&M University, Houston, TX, for the use of the Jasco J-720 spectropolarimeter and for invaluable assistance in conducting the experiments. We thank Dr. Steven La Brenz for calling Ref. 45 to our attention.
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FOOTNOTES |
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* This research was supported in part by National Institutes of Health Grants HL 30050 and GM 52419 and Robert A. Welch Foundation Grant AQ-1192 (to B. S. S. M.).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: Dept. of Biochemistry,
University of Texas Health Science Center at San Antonio, 7703 Floyd
Curl Dr., San Antonio, TX 78284-7760. Tel.: 210-567-6985; Fax:
210-567-6984; E-mail: nishimura{at}uthscsa.edu.
§ Supported by National Heart and Lung Cardiovascular Training Grant (HL 07350-19).
2 J. S. Nishimura and B. S. S. Masters, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: NOS, nitric-oxide synthase (n, neuronal; e, endothelial; c, constitutive; i, inducible); CaM, calmodulin; NO·, nitric oxide; BH4, (6R)-5,6,7,8-tetrahydro-L-biopterin; Ca2+/CaM, calcium ion-calmodulin; GmCl, guanidinium chloride; oxyHb, oxyhemoglobin; BisTris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane.
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REFERENCES |
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