From the Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446
Received for publication, February 19, 2001, and in revised form, March 16, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To clarify the role of the autoinhibitory insert
in the endothelial (eNOS) and neuronal (nNOS) nitric-oxide synthases,
the insert was excised from nNOS and chimeras with its reductase
domain; the eNOS and nNOS inserts were swapped and put into the
normally insertless inducible (iNOS) isoform and chimeras with the iNOS reductase domain; and an RRKRK sequence in the insert suggested by
earlier peptide studies to be important (Salerno, J. C.,
Harris, D. E., Irizarry, K., Patel, B., Morales, A. J.,
Smith, S. M., Martasek, P., Roman, L. J., Masters, B. S., Jones, C. L., Weissman, B. A., Lane, P., Liu, Q., and
Gross, S. S. (1997) J. Biol. Chem. 272, 29769-29777) was mutated. Insertless nNOS required calmodulin (CaM)
for normal NOS activity, but the Ca2+ requirement for this
activity was relaxed. Furthermore, insert deletion enhanced CaM-free
electron transfer within nNOS and chimeras with the nNOS reductase,
emphasizing the involvement of the insert in modulating electron
transfer. Swapping the nNOS and eNOS inserts gave proteins with normal
NOS activities, and the nNOS insert acted normally in raising the
Ca2+ dependence when placed in eNOS. Insertion of the eNOS
insert into iNOS and chimeras with the iNOS reductase domain
significantly lowered NOS activity, consistent with inhibition of
electron transfer by the insert. Mutation of the eNOS RRKRK to an AAAAA
sequence did not alter the eNOS Ca2+ dependence but
marginally inhibited electron transfer. The salt dependence suggests
that the insert modulates electron transfer within the reductase domain
prior to the heme/reductase interface. The results clarify the role of
the reductase insert in modulating the Ca2+ requirement,
electron transfer rate, and overall activity of nNOS and eNOS.
The three major isoforms of nitric-oxide synthases differ
biochemically and biologically in multiple ways (1-9). One of these differences, a dependence of activity on reversible calmodulin (CaM)1 binding, distinguishes
the constitutive neuronal (nNOS) (10) and endothelial (eNOS) (11)
isoforms from the inducible (iNOS) isoform (12). A second significant
difference is provided by the level of catalytic activity, in that eNOS
has a relatively low activity of ~30 nmol of NO/min/nmol of NOS,
whereas nNOS and iNOS have higher activities of ~100 and 150 nmol of
NO/min/nmol, respectively.
The differences in catalytic activity derive from the relative
abilities of the reductase domains to transfer electrons to the heme, a
process strictly controlled by CaM activation in the wild-type enzymes
(13). A peptide insert of about 50 amino acids in nNOS and eNOS but not
iNOS is evident upon alignment and comparison of the sequences of the
three isoforms (14). We have shown in earlier work that this insert
helps to lower the rates of electron transfer in eNOS in both the
CaM-bound and -unbound states (15). Thus, removal of the insert from
wild-type eNOS or from NOS chimeras in which the native flavin domain
of nNOS has been replaced by the eNOS flavin domain leads to proteins
that are hyperactive in both overall NO synthesizing activity and in
electron transfer from the CaM-bound or -unbound protein to electron
acceptors such as cytochrome c and ferricyanide.
Conflicting results have been obtained concerning the effect of the
autoinhibitory domain on the CaM dependence of activity. In accord with
our earlier results on eNOS, Guillemette and co-workers (16) found that
nNOS mutants in which the peptide insert was deleted still required CaM
for NOS activity. In contrast, Shimizu and co-workers (17) reported
that removal of the nNOS insert yielded a protein that retained 25% of
its activity in the absence of CaM, although this activity was <10%
that of wild-type nNOS.
We sought to clarify the role of the nNOS insert by excising it from
both wild-type nNOS and NOS chimeras in which the reductase domain of
other NOS isoforms was replaced by the nNOS reductase domain. The
chimera mutants provide a larger sampling of data points and make it
possible to determine the effects of the nNOS insert in various protein
contexts. This diversity of NOS mutants was further increased by
creating NOS proteins that varied solely in the nature of the insert in
the reductase domain. In these latter variants, the eNOS and nNOS
inserts in the parent proteins were exchanged, and the eNOS insert was
added to insertless NOS proteins bearing the iNOS reductase domain,
including both wild-type iNOS and a chimera in which the reductase
domain of eNOS had been replaced by that from iNOS. Finally, we sought
to determine whether a highly basic region in the insert was the
primary molecular determinant of the autoinhibitory properties of the
eNOS insert, as proposed by Masters and co-workers (14) based on
peptide inhibition studies.
We have found that the nNOS insert acts similarly to the eNOS insert in
raising the Ca2+ requirement for activity. When placed in
the context of eNOS, the nNOS insert can substitute for the native one
in this role as well as in maintaining the lower eNOS activity. The
eNOS insert, however, does not exert the same full effects in nNOS as
it does in its native context. Thus, autoinhibition of Ca2+
activation is decreased, thereby lowering the Ca2+
requirement; and activity levels are only partially lowered by the
presence of the eNOS insert in nNOS. Finally, our results show that the
five consecutive basic residues RRKRK implicated by the peptide studies
as key features of the eNOS autoinhibitory insert are not actually
required, as an insert with all these residues replaced by alanines is
still able to fully autoinhibit CaM activation and to fully depress the
activity level. The results are discussed in the context of possible
structural requirements for the insert.
Materials
HEPES and agarose were from Fisher. DNA manipulations were done
using enzymes, buffers, and reagents from New England Biolabs and
purification kits from Qiagen, which also supplied the
nickel-nitrilotriacetic acid resin. Oligonucleotide primers were
synthesized, and the DNA sequenced, by the Biomolecular Resource Center
(University of California, San Francisco). All other materials were
from Sigma.
General PCR Cycling
A Progene thermocycler from Techne (Cambridge, UK) was employed.
Mutagenesis was accomplished via standard overlap extension PCR
techniques, utilizing as the template previously constructed NOS
chimeras (18) that possessed engineered NheI splice sites at
amino acids 760-761, 538-539, and 527-528 in nNOS, iNOS, and eNOS,
respectively. 5' end primers annealed before or at the NheI splice site, whereas 3' end primers annealed after preexisting unique
sites (AatII in nNOS, BsrGI in iNOS, and
KpnI in eNOS).
Unless otherwise noted, the standard PCR cycling parameters were 25 cycles of 1 min at each temperature (94 °C melting, 60 °C
annealing, and 72 °C extension) followed by a 10-min extension, using ~0.2 ng/µl template, 0.5 µM primers, 400 µM dNTPs, and 0.02 units/µl Vent® polymerase that
possesses 3' to 5' proofreading activity. For extension of two
overlapping PCR products, 5 cycles were performed without the
appropriate end primers, followed by addition of primers and 20 cycles
of amplification, and the final 10-min 72 °C extension. For
syntheses of overlapping PCR products from megaprimers, a pair of
primers (megaprimer and corresponding paired primer) were usually
sufficient to give the desired amplification, except in the case of
E/I(Einsert), in which three primers were necessary (the
megaprimer, its paired primer, and the same-strand primer which
produced the megaprimer). The cycling parameters in this case were the
same as for overlap extension reactions.
Construction of nNOS Insert Deletion Mutants
pCWori//I/N pCWori//nNOS pCWori//E/N Construction of Insert-swapped Mutants--
These mutants were
constructed in the same way as the insert deletion mutants, except that
the primers analogous to primers 2 and 3 above were "megaprimers"
made via PCR amplification and were composed of the entire insert
sequence plus small flanking regions corresponding to 6 and 3 amino
acids at the 5' and 3' termini, respectively. The megaprimers,
which were synthesized using primers 5 and 6 (GGTGGTGACC AGCACCTTTG G
and CAGAAGTGGG GGTACGCC) plus template cDNA nNOS or eNOS,
anneal to sequences adjacent to the insert sequence that are virtually
identical in nucleotide sequence (identical in protein sequence)
between eNOS and nNOS isoforms.
pCWori//nNOS(Einsert)--
Amplification of
template pCWori//I/N using megaprimer(Einsert) and primer 7 (TAATACTAGT TCCATTCTAC TACTACCAGA) produced fragment C, whereas
megaprimer and primer 8 (TAATTCTAGA TTAGGAGCTG AAAACCTCA) produced fragment D. Overlap extension produced product CD, which was subcloned into pCWori//nNOS pCWori//eNOS(Ninsert)--
Amplification of template
pCWori//I/E using megaprimer(Ninsert) and primer 7 produced fragment E,
whereas megaprimer and primer 9 (CCCGGAGGTC GACGAC) produced fragment
F. Overlap extension gave product EF, which was subcloned into
pCWori//E/E pCWori//E/I(Einsert)--
The sequence designated
the eNOS insert sequence (55 amino acids
593MEMSG ... ALGTL) was inserted into
iNOS, replacing the analogous 10-amino acid sequence,
604FMLRELNHTF. Megaprimer(eNOS) was synthesized from
template pBluescript/eNOS cDNA and primers 10 (GGCAGACTCT
GAAGAAATCT CTGATGGAGA TGTCGGGGCC) and 11 (CAAGGCCAAA CACAGCATAC
CTGAGGGTCC CCAGGG). The "outer" ends of the resulting
megaprimer were identical to the iNOS sequences "outside" the
insert, whereas the interior of the megaprimer was identical to the
eNOS insert (Fig. 1).
Amplification of template pCWori//E/I using
megaprimer(Einsert) and primers 11 and 12 (CCAGACCCCT
GGAAAACTAG TGCGACCAAG GGCGC) produced fragment G, while megaprimer and
primers 10 and 13 (CCCATGTTGC ATTGGAAGTG AAG) produced fragment H. Overlap extension gave product GH, which was subcloned into pCWori//E/I
via the NheI and BsrGI unique sites.
pCWori//iNOS(Einsert)--
Replacement of
the NdeI/NheI eNOS heme domain gene
fragment with that of iNOS resulted in
pCWori//I/I(Einsert), hereafter referred to as
"iNOS(Einsert)," which has a conservative T539S mutation due to the NheI splice site.
Construction of 5Ala Mutant eNOS
RRKRK located in the bovine eNOS insert was mutated to five
consecutive alanine residues. Mutagenesis was accomplished by standard
overlap extension PCR mutagenesis using mutagenesis primers 13 (GGGCCGCAGC GGCCGCAGAG TCCAGCAACA CAGACAGC, forward) and 14 (CTGCGGCCGC
TGCGGCCCAG GAGGACACCA GCGG, reverse), end primers 12 and 9, and
template pHis//E/E.
Protein Expression and Purification
Conditions were as reported previously (19, 20). Proteins were
purified to >95% purity (as judged by SDS-polyacrylamide gel
electrophoresis) via affinity chromatography on nickel-nitrilotriacetic acid-agarose and 2',5'-ADP agarose.
To address the goals of this study, we constructed a series of
mutant proteins by deleting or swapping the insert in the wild-type NOS
isoforms as well as in chimeras derived from them. The constructs are
shown schematically in Fig. 2.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
The NheI site and another unique
site (AatII) downstream of the insert were used to
subclone the insert-deleted gene fragment. Specifically, primers
1 and 2 (5'-TAATACTAGT TCCATTCTAC TACTACCAGA and GGGGTCCAGT
ACTCTCCATTA AAGCACAGCC GAATTTCTCCC CG) and template pCWori//I/N were
used to produce fragment A (25 standard cycles, except the first 5 cycles were at 55 °C annealing). Primers 3 and 4 (AGTACTGGAC
CCCTGGCCAA TGTGAGG and TAATTCTAGA TTAGGAGCTG AAAACCTCA) were used
to produce fragment B using identical cycling parameters. Equimolar
amounts (1 nM) of fragments A and B, plus 500-fold excess
primers 1 and 4 and the same cycling parameters produced product AB,
which was subcloned via NheI and AatII into chimera pCWori//I/N, yielding deletion chimera pCWori//I/N
.
--
Subcloned from pCWori//N/E into
pCWori//I/N
, the NdeI/NheI nNOS gene fragment
corresponding to the heme- and CaM-binding domain of nNOS replaced that
of iNOS, yielding pCWori/N/N
, hereafter referred to as "nNOS
," which differs from the true wild-type insert deletion mutant by a
T761S mutation resulting from introduction of the NheI
chimeric splice site. As serine, the native amino acid in eNOS, is
highly homologous and represents a conservative mutation, we believe
that this mutant is indistinguishable from the exact nNOS
insert
deletion mutant.
--
This mutant was produced by subcloning of
the NdeI-NheI eNOS heme domain gene fragment from
pCWori//E/N (18) into pCWori//N/N
.
via the NheI and
AatII unique sites.
(15) via the NheI and KpnI unique sites.
View larger version (12K):
[in a new window]
Fig. 1.
Mutagenesis strategy for insert
swapping. Schematic diagram illustrating the location of primers
used to construct mutants bearing swapped or added inserts. PCR primer
pairs 5 and 6 were used to create megaprimers for construction of
eNOS(Ninsert) and nNOS(Einsert), whereas
pairs 10 and 11 were used to produce E/I(Einsert) and
iNOS(Einsert). (
) indicates 3' direction of a primer
(anti)sense sequence; 10u(d) indicates up(down)stream
sequence portion of primer 10 sequence; // indicates primer sequence
continues; ... indicates intervening sequence omitted for
brevity; - - - indicates gaps inserted for alignment.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (36K):
[in a new window]
Fig. 2.
Schematic diagram of the mutant NOS
proteins. nNOS sequences are shown in black, iNOS in
white, and eNOS in gray. Loops
represent the targeted autoinhibitory inserts, and
cross-hatching indicates the absence of the insert, as in
iNOS. The arrow indicates the location of the DNA splice
site used to produce chimeras of the oxygenase (O) and
reductase (R) domains. In all the mutants, the CaM-binding
sequence (C), approximately in the middle of the sequences,
is from the same isoform as the oxygenase domain. Figures are not to
scale.
nNOS Insert Deletion Proteins
Protein Expression and Purification--
All the expressed and
purified proteins exhibited similar stabilities and yields (~2-4
mg/liter). I/N, like native iNOS, required coexpression with CaM to
prevent significant proteolysis (19, 21, 22), presumably due to a
folding or protection requirement for CaM. Therefore, I/N and
iNOS(Einsert) also were expressed in the presence of CaM.
None of the other proteins required CaM for expression and purification.
Deletion of the eNOS autoinhibitory sequence was shown to affect the Ca2+ dependence of eNOS, decreasing the free Ca2+ concentration required for the expression of NOS activity in the presence of CaM (15, 23). The same eNOS deletion also resulted in enhanced NO production as well as enhanced electron transfer from both CaM-free and CaM-bound eNOS to external oxidants such as ferric cytochrome c and ferricyanide (15). The rate enhancements were seen not only upon deletion of the insert from wild-type eNOS, but also upon its deletion from the chimeric NOS proteins N/E and I/E, which were constructed by replacing the reductase domains of nNOS and iNOS, respectively, with that of eNOS.
Ca2+ Dependence of Activity--
We likewise examined
the effect of nNOS deletions on the Ca2+ requirement for
NOS activity. In the presence of a 4-fold excess of CaM, the nNOS
mutant lacking the insert, nNOS, exhibited a lower Ca2+
requirement compared with parent nNOS (Fig.
3A, compare
to
), as
was seen for the eNOS
analog (Fig. 3A, compare
to
), and an approximate 15-fold lowering of the apparent
EC50 for Ca2+ from 300 to 20 nM.
Unexpectedly, E/N, a chimeric protein with eNOS oxygenase and
CaM-binding domains but an nNOS reductase domain, similarly required
less Ca2+ for NOS activity (EC50 ~70
nM) despite the fact that it possesses the autoinhibitory
insert. The corresponding deletion E/N
did not exhibit a lower
EC50 for Ca2+ (Fig. 3B, compare
to
).
|
The alternative approach is to prepare the I/N chimeras in which the
insertless iNOS reductase is replaced by the insert-bearing nNOS
reductase domain. We reported previously (18) that this replacement led
to an altered Ca2+ dependence that was intermediate between
that of the wild-type constitutive isoforms and iNOS, for which
coexpression with CaM was required to obtain full-length, active
protein. Approximately 50% activity was retained in 2.5 mM
EGTA, compared with full activity for iNOS (Fig. 3B, compare
to ×). We were interested in whether this disruption in the
Ca2+ independence of iNOS could be explained by the
introduction of the eNOS or nNOS insert which accompanied swapping of
the reductase domains. Deletion of this insert produced I/N
whose
Ca2+ dependence was nearly identical to that of I/N (Fig.
3B). Thus, the deletion did not produce the defining iNOS
property of retaining NOS activity even at very low (<0.1
nM) free Ca2+ concentrations.
NO Synthesis and Cytochrome c Reduction-- Deletion of the eNOS insert yielded an enzyme with enhanced NOS and reductase activities (15). Thus, the insert appears to be at least partially responsible for attenuating the reductase, and hence overall, activity of eNOS, both of which are intrinsically lower than those of nNOS and iNOS.
The nNOS reductase alone (24) and full-length nNOS (10, 25-31)
typically exhibit 2-5 times higher activities than eNOS (31-42).
Therefore, whereas the insert may help to lower activities in eNOS, it
must not do so in nNOS. To confirm this, we compared the activities of
proteins bearing the insert-deleted nNOS reductase, i.e. we
compared the nNOS, E/N
, and I/N
deletion mutants with wild-type nNOS and the E/N and I/N chimeras.
As reported by us and by others (15, 43, 44), the activity of both NOS
and NADPH-cytochrome P450 reductase is significantly influenced by the
ionic strength of the assay solution. For an increase in ionic strength
from 25 to 125 mM, nNOS and eNOS show increases in overall
activity of 10 and 30%, respectively, whereas iNOS shows a decrease of
20% (see Fig. 4). Because activities vary with the NOS isoform, and for other reasons soon to become apparent, assays were done both as typically reported in the literature but also in the presence of 100 mM KCl. The salt
enhancement is nearly maximal with this KCl content (43).
|
Overall Activity--
Removing the insert from eNOS resulted in
eNOS, an enzyme that exhibited a roughly 2-fold increase in overall
NO synthesis (15). The N/E and I/E chimeras also exhibited higher
activities upon deletion of the insert to give the N/E
and I/E
insert-deleted chimeras.
Similar removal of the insert from nNOS gave nNOS, which in the
KCl-free assay had an activity approximately half that of wild-type
nNOS (Fig. 4, 36 ± 2 versus 75 ± 2 min
1), a decrease not unlike that reported by
Shimizu and co-workers (17) (30%) and Guillemette and co-workers (16)
(20%)). However, we found virtually no difference in nNOS and nNOS
activities in the presence of 100 mM KCl. In fact, if
anything, nNOS
exhibited a slightly higher activity (94 ± 2 versus 85 ± 5 min
1). The
same behavior was seen when the E/N and I/N chimeras bearing the nNOS
reductase were compared with the corresponding insert-deleted proteins,
although the "rescue" by KCl was incomplete in these cases.
Nevertheless, 81 and 85% of the activity was retained with the E/N
and I/N
chimeras, respectively, in KCl.
Cytochrome c Reduction-- For the previously constructed NOS chimeras, the overall activity reflected the activity of the reductase (18). We therefore examined the reductase activity to see if this characteristic was preserved in the deletion mutants of proteins with an nNOS reductase domain.
Whereas deletion of the insert did not significantly affect overall NOS
activity in any of the three proteins with an nNOS reductase domain
(Fig. 4), the CaM-bound cytochrome c reductase activity
(Fig. 5) gave varied results. In the
absence of KCl (Fig. 5A), nNOS (72%) and E/N
(76%)
were slightly lower than the corresponding nNOS and E/N parent
proteins, whereas I/N
(139%) was slightly higher than I/N. When the
assays were performed in the presence of KCl (Fig. 5B), the
effect of insert deletion was abolished for nNOS
(91%) but was
slightly enhanced for E/N
(54%) and I/N
(162%).
|
The cytochrome c reduction activity of CaM-free nNOS was
4-fold greater than that of CaM-free nNOS (2300 ± 200 versus 590 ± 20 min
1) in the
absence of salt but was only 2.5-fold greater in 100 mM KCl
(4400 ± 1600 versus 1620 ± 40 min
1). When CaM was added to the assay,
nNOS
and nNOS had similar activities, both of which were higher at
higher ionic strength (Fig. 5; nNOS, 9800 ± 500 and 13,000 ± 360 min
1 versus nNOS
,
7100 ± 1100 and 11,800 ± 600 min
1). E/N and E/N
showed a similar trend
in the CaM-free state; the deletion increased reductase activity
relative to the parent (1030 ± 80 versus 360 ± 11 min
1), and this difference was magnified
in the presence of KCl (1800 ± 100 versus 500 ± 20 min
1) due to a greater increase in the
CaM-free rate observed with the deletion mutant at higher ionic strength.
True CaM-free rates (exogenous CaM-free assay results are shown in Fig.
5) for I/N and I/N could not be measured because CaM coexpression is
required to express active I/N and I/N
. Nevertheless, whereas
removal of the insert from nNOS and the E/N chimera produced deletion
mutants with lower activity than the parents proteins, removal of the
insert from I/N gave I/N
with enhanced activity in both the presence
(7900 ± 200 versus 4700 ± 100 min
1) or absence (5290 ± 90 versus 3800 ± 200 min
1) of
KCl. This was true despite the fact that the insert-deleted proteins
had a somewhat lower NO-synthesizing activity (Fig. 4).
Thus, removal of the nNOS insert from nNOS and E/N
, the two
proteins based on constitutive NOS isoforms (nNOS or E/N), resulted in
a higher CaM-free rate relative to the CaM-bound rate regardless of KCl
concentration, consistent with the results seen for the eNOS deletions.
Insert-swapped Proteins
Deletion analysis provides the opportunity to study the function of the nNOS autoinhibitory insert in the context of either native nNOS or eNOS. To extend this approach, we put the insert in the structural context of a non-native "host" NOS. By swapping the inserts of eNOS and nNOS, or by introducing the eNOS insert into iNOS or an iNOS reductase-bearing chimera, it should be possible to retain or eliminate potential "pairing" sites outside the insert and thereby implicate or rule out such external sites in the proper function of the autoinhibitory insert. This method refines our earlier approach of swapping the entire reductase domain and focuses attention on the specific interactions of the insert.
Protein Expression and Purification-- The four "insert-swapped" proteins, eNOS(Ninsert), nNOS(Einsert), iNOS(Einsert), and E/I(Einsert), were expressed and purified identically as the parental native NOSs and chimera, producing similar yield and purity. iNOS(Einsert) was coexpressed with CaM.
Ca2+ Dependence of Activity--
When the eNOS
autoinhibitory insert was introduced into nNOS to give
nNOS(Einsert), the resulting protein exhibited a lower Ca2+ requirement (Fig.
6A). For this simple loop
swap, EC50(Ca2+) dropped from 200 nM for nNOS to ~50 nM. However, introducing the eNOS insert into the insertless iNOS had no significant effect. Similarly, the EC50(Ca2+) for the
E/I(Einsert) protein did not differ significantly from that
of the insertless E/I. Thus, introducing the eNOS insert into either
insertless iNOS or E/I does not increase the Ca2+
dependence, whereas replacement of the nNOS by the eNOS insert decreases that dependence. In fact the insert swap exhibits an EC50(Ca2+) very similar to that of the eNOS
from which the insert has been removed (~40 versus ~20
nM; compare of Fig. 3A and gray
of Fig. 6A). Finally, eNOS(Ninsert), in
which the eNOS insert was replaced by that of nNOS, exhibited
Ca2+ requirements identical to those of nNOS (Fig.
6B). That is the EC50(Ca2+) was
slightly higher than that for eNOS itself. The nNOS loop thus appears
to function in the context of the eNOS protein and evokes the
Ca2+-dependent behavior characteristic of eNOS
and nNOS.
|
Nitric Oxide Synthesis--
The insert-swapped proteins,
nNOS(Einsert) and eNOS(Ninsert), had overall
NOS activities similar to those of their parental protein (Fig.
7). Inclusion of 100 mM KCl
increased the NOS activity by less than 40% for
eNOS(Ninsert) but 70% for nNOS(Einsert). In
the previous converse experiment, in which the insert was deleted from
eNOS, the deletion enhanced the overall activity 2-fold (15). As shown
here, inserting the eNOS loop into iNOS and the E/I chimera significantly lowers the overall activity (Fig.
8). The iNOS(Einsert) activity was 32 and 36% of iNOS activity, respectively, in the absence
and presence of KCl, and the E/I(Einsert) activity was 40 and 25% of the E/I activity under the corresponding conditions.
|
|
Cytochrome c Reduction--
In contrast to the similar NO
synthesizing activities of nNOS and nNOS(Einsert) (Fig. 7),
replacement of the nNOS with the eNOS insert decreases the rate of
cytochrome c reduction by 50% in both the presence (Fig.
9A) and absence (Fig.
9B) of KCl. In contrast to insert deletion, the insert
exchange did not alter the CaM-free cytochrome c reduction
rate with respect to the CaM-bound rate (Fig. 9). Similarly, replacing
the eNOS with the nNOS insert yielded eNOS(Ninsert) without
significantly affecting the low eNOS rate, in agreement with the
results for the NO synthesizing activity (Fig. 7).
|
Introducing the eNOS loop into iNOS and the E/I chimera gave the same
trends for the CaM-bound cytochrome c reduction activity as
observed for the overall NOS activity. The attenuating effect of the
eNOS loop on cytochrome c reduction was similar for
iNOS(Einsert) and E/I(Einsert) in the absence
(21 and 25%, respectively) or presence (42 and 25%, respectively) of
KCl (Fig. 10B). The effect on the CaM-free rates was similar.
|
We showed previously that the chimeric E/I and N/I proteins, both of
which have the iNOS reductase domain, exhibit high reductase activity
even in the absence of CaM, consistent with what was observed for the
iNOS reductase expressed as a separate polypeptide without the
oxygenase and CaM-binding domains (45). The CaM independence of
cytochrome c reduction by E/I was retained in E/I(Einsert). Thus, whereas deletion of the eNOS insert
from eNOS increased the reduction rate (Fig. 10), insertion of the eNOS
loop into iNOS did not lower the high CaM-free rate. The CaM-free rate for E/I(Einsert) was identical (KCl, Fig.
10A) or slightly higher (+KCl, Fig. 10B) than the
CaM-bound rate, as observed for the E/I parent.
Finally, the effect of salt on the cytochrome c reduction
rates depends on the identity of the reductase domain. The proteins with the iNOS reductase domain (iNOS, iNOS(Einsert), E/I, and E/I(Einsert) in Fig. 10, and N/I in our previous study (18)) exhibit substantially decreased activities in the presence of 100 mM KCl, whereas the proteins with eNOS and nNOS reductase domains (eNOS, eNOS, nNOS, nNOS
, nNOS(Einsert), and
eNOS(Ninsert), Figs. 5 and 9, and N/E and N/E
in our
previous work (15)) all show enhanced activity.
RRKRK AAAAA (5Ala) eNOS Mutant
Early studies indicated that a pentapeptide based on a stretch of
five basic residues within the eNOS autoinhibitory insert was an
effective inhibitor of nNOS (14). However, as this eNOS-derived pentapeptide was a less effective inhibitor of eNOS itself, it was not
clear whether the inhibition arose from a specific functional relationship between this basic region and CaM binding or was simply
the result of a non-functionally relevant interaction. To clarify the
role of the basic region, we converted the RRKRK amino acid sequence of
the eNOS autoinhibitory insert into AAAAA. The Ca2+
dependence of nitric oxide synthesis by the mutant enzyme was unaffected by this gross mutation (Fig.
11). Although cytochrome c
reduction was lower than for wild type (Fig. 10), the nitric oxide
synthesizing activity was somewhat higher than that of wild-type eNOS
(Fig. 8) but did not rise to the level seen upon removal of the entire
insert.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The reductase domains of the nitric-oxide synthases employ their flavin prosthetic groups to provide electrons from NADPH to the P450-like heme domains. Maximal electron transfer to the heme groups depends on the binding of CaM (13), which occurs reversibly in the constitutive eNOS and nNOS isoforms. In the inducible isoform, CaM is essentially irreversibly bound, and NO synthesis occurs at negligible Ca2+ levels. Comparison of the primary sequences of the three isoforms reveals that eNOS and nNOS possess a 38-42-amino acid insert within their FMN-binding domain that is not present in either iNOS or NADPH-cytochrome P450 reductase. Thus, the presence or absence of the insert coincides, respectively, with either reversible (eNOS and nNOS) or irreversible (iNOS) CaM binding.
We demonstrated previously (15) that removal of the insert from eNOS lowered the Ca2+ requirement for the synthesis of nitric oxide from an EC50 of 150 to 20 nM. Furthermore, a decrease in the Ca2+ requirement for NOS activity was also observed upon removal of the insert from chimeras in which the nNOS and iNOS heme domains were linked with the eNOS reductase domain. Somewhat to our surprise, removal of the insert also increased electron transfer to cytochrome c by more than 2-fold. Greatly increased electron transfer (>200-fold) to cytochrome c has recently been observed in C-terminal truncations of iNOS (46); however, the increased cytochrome c reducing ability was not reflected in a significantly increased ability to synthesize NO. The authors speculated that NO release may have become rate-limiting in these truncated iNOS proteins, but the fact that we and other laboratories have observed higher NO synthesis rates by the wild-type enzyme suggests that another explanation is likely. In any case, the enhanced electron transfer observed upon deletion of the insert from eNOS and chimeras bearing the eNOS reductase domain does translate into an increase in overall NOS activity, leading us to conclude that the insert is at least partially responsible for the lower NO synthesizing activity of eNOS relative to iNOS and nNOS.
As a similar insert is also present in nNOS, which has a higher
intrinsic NO synthesizing activity than eNOS, we examined the effect of
removing the nNOS insert, both in terms of the Ca2+
requirement and its consequences for electron transfer. Two other laboratories have reported (16, 17) removal of the insert from nNOS. In
contrast to the report of Guillemette and co-workers (16), nNOS did
not require coexpression with CaM and could be purified in the same way
as wild-type nNOS. In addition to the discrepancy concerning the
instability of the CaM-free protein, Guillemette and co-workers (16)
also reported an activity for their
nNOS protein (8 nmol
NO·min
1·nmol
1)
that was considerably lower than that of our nNOS
protein (36 nmol
NO·min
1·nmol
1).
The deletion reported by Guillemette and co-workers (16) (
nNOS)
differs from ours by only one amino acid, and it is surprising that
this small difference has such a drastic effect on the stability of the
CaM-free protein. We believe that the similar activity and similar
stability of our CaM-free nNOS
and nNOS proteins reflect a
preservation of the local tertiary structure near the mutation, making
conclusions about the mode of action of the mutation valid.
All of our proteins required CaM for significant NO synthesis (>5%
wild type), consistent with the eNOS deletions described by us
previously. In contrast, Shimizu and co-workers (17) reported that
deletion of 40 or 42 of the insert residues produced 40 and
42,
respectively, that retained 30% of the maximum activity in the absence
of Ca2+/CaM, the maximum activity being 22-30% that of
the wild-type enzyme. Under similar salt-free conditions, Guillemette
and co-workers (16) observed a decrease in the maximum activity to 20%
that of the wild-type enzyme, but CaM was required for activity, and we
observed a decrease to 50% of wild-type activity, again with CaM being
required for NO synthesis (Fig. 4). However, the activity we observed
was increased in 100 mM KCl to 111% that of the wild-type protein. Deletion of the insert from nNOS and chimeras with the nNOS
reductase domain yields proteins that are more sensitive to salt than
those obtained by deletion of the insert from eNOS or chimeras with the
eNOS reductase domain (Fig. 4). For example, the activity of E/N
without added KCl is 50% of the activity with KCl. I/N
shows a more
modest difference of 77%. In contrast, eNOS
(Fig. 4) exhibits a
smaller KCl dependence. The iNOS reductase activity is negatively
modulated by salt, as seen by comparing the activities of iNOS,
iNOS(Einsert), N/I, E/I, and E/I(Einsert).
Table I shows a comparative summary of
our results and those of the Shimizu and Guillemette laboratories. It
is surprising that the modest differences in mutations result in such
significant differences in the CaM and Ca2+ requirements of
the mutant proteins. It is theoretically possible but, as the authors
pointed out, unlikely that CaM from the yeast expression system was
carried through the purification procedure in the Shimizu experiments
because the protein was purified in the presence of 1 mM
EGTA and was also bound to CaM-Sepharose. The difference in CaM
dependence between their experiments and ours is thus probably real. As
we deleted Met828-Glu869, whereas Shimizu and
co-workers (17) deleted Pro831-Ser870, the two
mutant proteins differed by a total of only two amino acids.
Nevertheless, it is possible that the structural difference inherent in
these two additional amino acids enables slow leakage of electrons from
the reductase to the heme in the latter protein. The much higher
sensitivity of nNOS than the other mutant constructs to ionic
strength suggests, in fact, that nNOS
is structurally relatively
unstable and is perhaps poised on a conformational knife edge. If so, a
small difference in the amino acids that remain after the insert is
deleted, or even in the purification and assay conditions, might be
sufficient to trigger significant differences in the quaternary
structures and properties of the mutant proteins.
|
Comparisons of the three wild-type isoforms and the eNOS and nNOS
proteins without the reductase domain insert has been informative, but
the comparison has been extended here to a more diverse pool of
proteins to amplify and strengthen the attendant conclusions. This has
been done by examining the effect of deleting the insert from chimeric
NOS proteins, by swapping the inserts between the constitutive nNOS and
eNOS proteins, and by introducing the eNOS insert into proteins with
the insertless iNOS reductase domain.
This diversification has clarified the source of the differing salt effect on the NOS activities. In a study of salt effects on nNOS, Masters and co-workers (44) concluded that salts elicited dissociation of the autoinhibitory product NO, leading to the observed enhanced activity. In an earlier rigorous examination of the effects of many salts on all three NOS isoforms, Mayer and co-workers (43) observed a general correlation with the Hofmeister series and trends for the three wild-type isoforms similar to those found here for KCl, i.e. activation of eNOS and nNOS and inhibition of iNOS by increasing salt. Mayer and co-workers (43) attributed the salt effects to "aspecific changes in protein solvation" and ruled out specific binding, alteration in CaM binding, and dissociation of the NOS dimers. They concluded that "salts mainly affect the interdomain electron transfer, but we cannot rule out an additional effect on the preceding electron transfer steps."
In the present study, we have shown that the nature of the
salt dependence is directly linked to the identity of the reductase domain regardless of the heme- and CaM-binding domains. Thus, I/N,
I/N, E/N, E/N
, eNOS(Ninsert),
nNOS(Einsert), nNOS and nNOS
in this paper, and N/E,
N/E
, I/E, I/E
, eNOS, and eNOS
in our previous paper (15), all
exhibited enhanced activity with 100 mM KCl, whereas N/I,
E/I, E/I(Einsert), iNOS, and iNOS(Einsert) were
inhibited by 100 mM KCl. Thus, it is likely that the
salts studied by Mayer and co-workers (43) alter electron transfer within the reductase domain, but irrespective of the insert,
as the effects are independent of the nature of the interdomain region.
The Ca2+ requirement for activation of nNOS was lowered
significantly by deletion of the insert (EC50 = 300 20 nM). Surprisingly, the E/N chimera was activated by a lower
Ca2+ concentration than eNOS itself (250 versus
70 nM) even though the nNOS reductase has a similar insert.
Deletion of the insert from the chimera did not alter further the
Ca2+ requirement. Since reductase structural elements are
required to bring about the complete Ca2+ independence of
iNOS (18, 47, 48), it is likely that reductase elements in addition to
the autoinhibitory loop play a role in CaM binding and that the
interactions between CaM and these elements have been perturbed in the
E/N chimera. Thus, the interactions involved in normal CaM binding have
been disrupted by the exchange of reductase domains in the chimera.
This might also explain the finding that the slight loss of
Ca2+ dependence is identical for I/N and I/N
, as this
suggests that the exchange of reductase domains induces the alteration,
independent of whether the autoinhibitory insert is present or not.
Our previous findings with chimeras incorporating the eNOS reductase
with or without the insert might lead one to conclude that the effects
of the insert on the Ca2+/CaM dependence and electron
transfer rates were directly related. Thus, deletion of the eNOS insert
without exception enhanced both electron transfer and NO synthesis and
lowered the Ca2+ requirement. However, the present results
on the proteins with exchanged autoinhibitory inserts suggest that the
Ca2+ dependence and electron transfer effects are discrete
phenomena. For example, replacing the insert in nNOS with that from
eNOS yields nNOS(Einsert) whose Ca2+ dependence
is lower and yet whose activity is nearly as high (KCl) or higher
(+KCl) than that of nNOS. One possible explanation for this difference
is a lack of binding requirements in the nNOS parent that is required
by the eNOS insert. Regardless of whether this is the correct
explanation, it is clear that one effect does not necessarily follow
the other.
Support for the idea that the Ca2+ dependence but not electron transfer effects of the insert require the participation of additional protein elements comes from the fact that the reductase and NO synthesizing activities of both iNOS(Einsert) and E/I(Einsert) are decreased relative to those of the parent iNOS and E/I proteins, respectively. In iNOS(Einsert), no nNOS or eNOS structural features other than those within the insert are present, which suggests that the mere presence of the loop is sufficient to inhibit electron transfer. This could occur by a mechanism as simple as disruption of a key contact within the electron transport pathway. Since electron transfer to cytochrome c is decreased in addition to the NO synthesizing activity, inhibition occurs prior to the step in which the electron is transferred from the FMN to either the heme domain or to cytochrome c. Inhibition therefore occurs upstream of the CaM-binding site. In E/I(Einsert), the heme and CaM-binding domains are complementary to the insert, yet the Ca2+ dependence is the same as for the E/I parent. This suggests that the effects of the insert on CaM binding depend on structural elements of the reductase in addition to those of the insert. It is unlikely that the structure of the reductase/heme electron transfer interface has been altered in the chimera, or that alterations have occurred in any required insert/heme domain interactions, as the catalytic activities of E/I are as high as those of iNOS. This finding is inconsistent with disruption of the quaternary structure. Further indication that the insert lowers electron transfer irrespective of CaM binding is seen in the rates of reduction of cytochrome c by E/I(Einsert) in the absence of CaM. The iNOS reductase has been shown to have CaM-free reduction rates that are as high as the CaM-bound rates and as high as the rate supported by cytochrome P450 reductase (45). As a result, E/I and N/I have CaM-free cytochrome c reduction rates as high as those of iNOS itself. Although addition of the eNOS insert in E/I(Einsert) did not alter the Ca2+ dependence relative to that of E/I, the reductase and NO synthesizing activities were reduced regardless of whether CaM was bound or not. Thus, the inhibitory effect of the insert is complete within the reductase domain and is independent of CaM.
Additional support for the view that the reductase domain provides
binding elements that help to determine the Ca2+ dependence
comes from the fact that the only proteins that show lowered
Ca2+ requirements are those in which the eNOS or nNOS
insert is present within its constitutive reductase domain. The
converse, however, is not necessarily true; for example,
eNOS(Ninsert) and E/N have parent-like Ca2+
requirements (Fig. 6). Nevertheless, eNOS
, nNOS
, N/E
, and nNOS(Einsert) have lower EC50(Ca2+)
values than the respective parent proteins, whereas
iNOS(Einsert) and E/I(Einsert) do not.
Interestingly, in eNOS(Ninsert), the nNOS insert appears to
function normally when placed in eNOS, in that Ca2+
requirements are high and activity levels are eNOS-like. On the other
hand, the eNOS insert alters Ca2+ requirements when placed
in nNOS, yet the activity levels are those of wild-type nNOS. One might
expect that nNOS possesses those elements required for the
Ca2+ dependence. It is to be expected that nNOS can
accommodate the eNOS loop without a decrease in its normally high
activities if, as appears likely, reductase elements external to the
loop control the electron transfer.
A clearer view is emerging concerning the residues within the insert
that are important for its effects. The portion of the eNOS loop
responsible for autoinhibition was previously hypothesized from
inhibitory peptide results to be the RRKRK sequence (14), although a
comparable basic sequence is lacking in nNOS. However, we have shown
here that mutation of the RRKRK sequence of eNOS to an AAAAA sequence
yields a protein with an unchanged Ca2+ dependence and only
a modestly elevated activity. The previously observed inhibition by
RRKRK-containing peptides probably occurred via a mechanism distinct
from that involved in autoinhibition of CaM binding by the insert in
the reductase domain. For example, Chen and Wu (23) examined various
eNOS deletions and also concluded that the more C-terminal portion of
the loop, which encompassed the RRKRK sequence, was not essential, in
contrast to the N-terminal portion of the sequence (residues 594-612).
Studies of rat nNOS and CaM kinases in vitro (49) and
in vivo (50) by Watanabe and co-workers identified
Ser847 (aligns with Ser617 of bovine eNOS) as
the site of phosphorylation. Phosphorylation of this site decreases CaM
binding and attenuates activity. Thus, rather than the basic region,
the residues at or near the phosphorylated serine appear to be the most
important in autoinhibition of Ca2+/CaM binding. The most
straightforward explanation is that these residues directly mediate
binding to CaM or to intramolecular sites within NOS that affect CaM
binding and contacts with NOS.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM25515.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: School of Pharmacy,
University of California, San Francisco, CA 94143-0446. Tel.: 415-476-2903; Fax: 415-502-4728; E-mail: ortiz@cgl.ucsf.edu.
Published, JBC Papers in Press, March 22, 2001, DOI 10.1074/jbc.M101548200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
CaM, Ca2+-dependent calmodulin;
nNOS/eNOS/iNOS, neuronal/endothelial/inducible nitric-oxide synthases;
E/N, chimera
of the heme- and CaM-binding domain of eNOS with the nNOS reductase
domain mutated to lack the autoinhibitory insert;
E/I(Einsert), chimera E/I in which the autoinhibitory
insert of eNOS was inserted;
PCR, polymerase chain reaction.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Kubes, P., and McCafferty, D. M. (2000) Am. J. Med. 109, 150-158[CrossRef][Medline] [Order article via Infotrieve] |
2. | Menshikova, E. B., Zenkov, N. K., and Reutov, V. P. (2000) Biochemistry (Mosc.) 65, 409-426[Medline] [Order article via Infotrieve] |
3. | Stuehr, D. J. (1999) Biochim. Biophys. Acta 1411, 217-230[Medline] [Order article via Infotrieve] |
4. | Bredt, D. S. (1999) Free Radic. Res. 31, 577-596[Medline] [Order article via Infotrieve] |
5. | Kibbe, M., Billiar, T., and Tzeng, E. (1999) Cardiovasc. Res. 43, 650-657[CrossRef][Medline] [Order article via Infotrieve] |
6. | Andrew, P. J., and Mayer, B. (1999) Cardiovasc. Res. 43, 521-531[CrossRef][Medline] [Order article via Infotrieve] |
7. | Papapetropoulos, A., Rudic, R. D., and Sessa, W. C. (1999) Cardiovasc. Res. 43, 509-520[CrossRef][Medline] [Order article via Infotrieve] |
8. | Marletta, M. A., Hurshman, A. R., and Rusche, K. M. (1998) Curr. Opin. Chem. Biol. 2, 656-663[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Michel, T.,
and Feron, O.
(1997)
J. Clin. Invest.
100,
2146-2152 |
10. | Bredt, D. S., and Snyder, S. H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 682-685[Abstract] |
11. | Busse, R., and Mülsch, A. (1990) FEBS Lett. 265, 133-136[CrossRef][Medline] [Order article via Infotrieve] |
12. | Cho, H. J., Xie, Q. W., Calaycay, J., Mumford, R. A., Swiderek, K. M., Lee, T. D., and Nathan, C. (1992) J. Exp. Med. 176, 599-604[Abstract] |
13. | Abu-Soud, H. M., and Stuehr, D. J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10769-10772[Abstract] |
14. |
Salerno, J. C.,
Harris, D. E.,
Irizarry, K.,
Patel, B.,
Morales, A. J.,
Smith, S. M.,
Martasek, P.,
Roman, L. J.,
Masters, B. S.,
Jones, C. L.,
Weissman, B. A.,
Lane, P.,
Liu, Q.,
and Gross, S. S.
(1997)
J. Biol. Chem.
272,
29769-29777 |
15. |
Nishida, C. R.,
and Ortiz de Montellano, P. R.
(1999)
J. Biol. Chem.
274,
14692-14698 |
16. |
Montgomery, H. J.,
Romanov, V.,
and Guillemette, J. G.
(2000)
J. Biol. Chem.
275,
5052-5058 |
17. |
Daff, S.,
Sagami, I.,
and Shimizu, T.
(1999)
J. Biol. Chem.
274,
30589-30595 |
18. |
Nishida, C. R.,
and Ortiz de Montellano, P. R.
(1998)
J. Biol. Chem.
273,
5566-5571 |
19. | Hühmer, A. F., Nishida, C. R., Ortiz de Montellano, P. R., and Schoneich, C. (1997) Chem. Res. Toxicol. 10, 618-626[CrossRef][Medline] [Order article via Infotrieve] |
20. | Gerber, N. C., Nishida, C. R., and Ortiz de Montellano, P. R. (1997) Arch. Biochem. Biophys. 343, 249-253[CrossRef][Medline] [Order article via Infotrieve] |
21. | Fossetta, J. D., Niu, X. D., Lunn, C. A., Zavodny, P. J., Narula, S. K., and Lundell, D. (1996) FEBS Lett. 379, 135-138[CrossRef][Medline] [Order article via Infotrieve] |
22. | Wu, C., Zhang, J., Abu-Soud, H., Ghosh, D. K., and Stuehr, D. J. (1996) Biochem. Biophys. Res. Commun. 222, 439-444[CrossRef][Medline] [Order article via Infotrieve] |
23. |
Chen, P. F.,
and Wu, K. K.
(2000)
J. Biol. Chem.
275,
13155-13163 |
24. | McMillan, K., and Masters, B. S. S. (1995) Biochemistry 34, 3686-3693[Medline] [Order article via Infotrieve] |
25. | Mayer, B., John, M., and Bohme, E. (1990) FEBS Lett. 277, 215-219[CrossRef][Medline] [Order article via Infotrieve] |
26. | Förstermann, U., Schmidt, H. H., Pollock, J. S., Sheng, H., Mitchell, J. A., Warner, T. D., Nakane, M., and Murad, F. (1991) Biochem. Pharmacol. 42, 1849-1857[CrossRef][Medline] [Order article via Infotrieve] |
27. |
Stuehr, D. J.,
and Ikeda-Saito, M.
(1992)
J. Biol. Chem.
267,
20547-20550 |
28. | Richards, M. K., and Marletta, M. A. (1994) Biochemistry 33, 14723-14732[Medline] [Order article via Infotrieve] |
29. |
Gerber, N. C.,
and Ortiz de Montellano, P. R.
(1995)
J. Biol. Chem.
270,
17791-17796 |
30. | Riveros-Moreno, V., Heffernan, B., Torres, B., Chubb, A., Charles, I., and Moncada, S. (1995) Eur. J. Biochem. 230, 52-57[Abstract] |
31. | Nakane, M., Pollock, J. S., Klinghofer, V., Basha, F., Marsden, P. A., Hokari, A., Ogura, T., Esumi, H., and Carter, G. W. (1995) Biochem. Biophys. Res. Commun. 206, 511-517[CrossRef][Medline] [Order article via Infotrieve] |
32. | Förstermann, U., Pollock, J. S., Schmidt, H. H., Heller, M., and Murad, F. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1788-1792[Abstract] |
33. | Nishida, K., Harrison, D. G., Navas, J. P., Fisher, A. A., Dockery, S. P., Uematsu, M., Nerem, R. M., Alexander, R. W., and Murphy, T. J. (1992) J. Clin. Invest. 90, 2092-2096[Medline] [Order article via Infotrieve] |
34. |
Janssens, S. P.,
Shimouchi, A.,
Quertermous, T.,
Bloch, D. B.,
and Bloch, K. D.
(1992)
J. Biol. Chem.
267,
14519-14522 |
35. |
Janssens, S. P.,
Shimouchi, A.,
Quertermous, T.,
Bloch, D. B.,
and Bloch, K. D.
(1992)
J. Biol. Chem.
267,
14519-14522 |
36. | Sessa, W. C., Barber, C. M., and Lynch, K. R. (1993) Circ. Res. 72, 921-924[Abstract] |
37. | Wolff, D. J., Lubeskie, A., and Umansky, S. (1994) Arch. Biochem. Biophys. 314, 360-366[CrossRef][Medline] [Order article via Infotrieve] |
38. | Garvey, E. P., Tuttle, J. V., Covington, K., Merrill, B. M., Wood, E. R., Baylis, S. A., and Charles, I. G. (1994) Arch. Biochem. Biophys. 311, 235-241[CrossRef][Medline] [Order article via Infotrieve] |
39. |
Busconi, L.,
and Michel, T.
(1993)
J. Biol. Chem.
268,
8410-8413 |
40. |
Chen, P. F.,
Tsai, A. L.,
and Wu, K. K.
(1994)
J. Biol. Chem.
269,
25062-25066 |
41. | Seo, H. G., Fujii, J., Soejima, H., Niikawa, N., and Taniguchi, N. (1995) Biochem. Biophys. Res. Commun. 208, 10-18[CrossRef][Medline] [Order article via Infotrieve] |
42. |
Rodríguez-Crespo, I.,
Gerber, N. C.,
and Ortiz de Montellano, P. R.
(1996)
J. Biol. Chem.
271,
11462-11467 |
43. | Schrammel, A., Gorren, A. C. F., Stuehr, D. J., Schmidt, K., and Mayer, B. (1998) Biochim. Biophys. Acta 1387, 257-263[Medline] [Order article via Infotrieve] |
44. |
Nishimura, J. S.,
Narayanasami, R.,
Miller, R. T.,
Roman, L. J.,
Panda, S.,
and Masters, B. S.
(1999)
J. Biol. Chem.
274,
5399-5406 |
45. | Rafferty, S., and Malech, H. L. (1996) Biochem. Biophys. Res. Commun. 220, 1002-1007[CrossRef][Medline] [Order article via Infotrieve] |
46. |
Roman, L. J.,
Miller, R. T.,
de La Garza, M. A.,
Kim, J. J.,
and Masters, B. S. S.
(2000)
J. Biol. Chem.
275,
21914-21919 |
47. |
Ruan, J.,
Xie, Q.,
Hutchinson, N.,
Cho, H.,
Wolfe, G. C.,
and Nathan, C.
(1996)
J. Biol. Chem.
271,
22679-22686 |
48. |
Venema, R. C.,
Sayegh, H. S.,
Kent, J. D.,
and Harrison, D. G.
(1996)
J. Biol. Chem.
271,
6435-6440 |
49. |
Hayashi, Y.,
Nishio, M.,
Naito, Y.,
Yokokura, H.,
Nimura, Y.,
Hidaka, H.,
and Watanabe, Y.
(1999)
J. Biol. Chem.
274,
20597-20602 |
50. |
Komeima, K.,
Hayashi, Y.,
Naito, Y.,
and Watanabe, Y.
(2000)
J. Biol. Chem.
275,
28139-28143 |
51. | Tsien, R., and Pozzan, T. (1989) Methods Enzymol. 172, 230-262[Medline] [Order article via Infotrieve] |