Calmodulin Promotes Dimerization of the Oxygenase Domain of Human Endothelial Nitric-oxide Synthase*

(Received for publication, September 20, 1996, and in revised form, February 11, 1997)

Gary R. Hellermann Dagger and Larry P. Solomonson Dagger §par

From the Dagger  Department of Biochemistry and Molecular Biology, College of Medicine, the § Institute for Biomolecular Science, and the  H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, Florida 33612

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

The active form of endothelial nitric-oxide synthase (eNOS) is a homodimer. The activity of the enzyme is regulated in vivo by calcium signaling involving the binding of calmodulin (CAM), which triggers the activation of eNOS. We have examined the possible role of calcium-mediated CAM binding in promoting dimerization of eNOS through the oxygenase domain of the enzyme. A recombinant form of the oxygenase domain of human eNOS was expressed in a prokaryotic expression system. This recombinant domain contains the catalytic cytochrome P-450 site for arginine oxidation by molecular oxygen as well as the binding sites for tetrahydrobiopterin and Ca2+-CAM but lacks the reductase domain and associated FAD, FMN, and NADPH binding sites. Binding of Ca2+-CAM caused an association of monomeric eNOS oxygenase domain as determined by changes in fluorescence, both intrinsic and extrinsic, and by gel filtration, chemical cross-linking, and particle-sizing. Dimerization of the domain was not dependent on the presence of the substrate, arginine, or the cofactor, tetrahydrobiopterin. A truncated form of the eNOS oxygenase domain lacking the Ca2+-CAM binding region did not undergo self-association to form dimers. These results show that the eNOS reductase domain is not required for Ca2+-CAM-induced dimerization of eNOS and suggest that this dimerization may be a primary event in the activation of eNOS by Ca2+.


INTRODUCTION

The family of nitric-oxide synthases (NOSs)1 can be divided into two classes: those that are synthesized constitutively and activated by the binding of Ca2+-CAM (endothelial and neuronal isoforms) and those that are induced through the action of specific cytokines and endotoxin and possess tightly bound Ca2+-CAM (macrophage isoform). Calmodulin (CAM) is a calcium-activated regulatory protein of 148 amino acids that binds to and regulates a number of enzymes known to respond to changes in intracellular calcium levels (1). As a calcium-triggered switch, Ca2+-CAM recognizes specific basic, amphiphilic alpha -helical domains in target proteins to which it binds with high affinity (2, 3). The constitutive nitric-oxide synthases type I (neuronal (nNOS)) and type III (endothelial (eNOS)) possess these amphiphilic, helical regions (4-6) and are expressed as inactive apoenzymes that only become functional upon binding Ca2+-CAM. The inducible NOS (iNOS, type II) differs markedly in that Ca2+-CAM is so tightly bound as to become virtually a subunit of the iNOS monomer, thus obviating the need for calcium as a switch to turn on activity (7-12). The activity of constitutive NOSs is regulated primarily by intracellular calcium levels through the binding of calcium-charged calmodulin. The mechanism(s) involved in this CAM-dependent activation are not known, but by analogy to other CAM-dependent enzymes could involve a conformational change, oligomerization, or both (14-16).

The active form of all isoforms of NOS appears to be the homodimer (8, 17-21). Another common structural feature of the three isoforms of NOS is a bidomain structure comprised of an N-terminal oxygenase domain and a C-terminal reductase domain connected by a Ca2+-CAM binding region, which presumably facilitates electron transfer between the reductase and oxygenase domains (9-11, 22-25). In the case of iNOS, subunit association is through the oxygenase domain and is dependent on BH4, arginine, and heme (8, 11-13, 18). Neither the reductase domain nor the CAM binding region are required for dimerization. Dimerization of nNOS also is promoted by BH4 and requires heme (20). Dimerization of eNOS, in contrast, does not appear to be influenced by BH4 (26), and both the N-terminal and C-terminal domains may be involved in assembly (21). Thus, the dimerization of eNOS may be unique and may reflect differences in regulation because oligomerization is a possible mode of regulation for the NOSs.

The functioning of calmodulin as the molecular switch for activation of eNOS has been known for some time but the potential role of Ca2+-CAM binding in homodimer formation has not been addressed. The bidomain structure of NOS permits the molecular dissection of the molecule into separate domains, which facilitates molecular studies aimed at establishing important structure-function relationships associated with the respective domains. To study the dimerization step of eNOS activation in relation to Ca2+-CAM binding, we utilized a prokaryotic expression system to generate the oxygenase domain of human eNOS, which contains the Ca2+-CAM regulatory domain, substrate and BH4 binding sites, and the catalytic heme binding site. Conformational changes in the recombinant oxygenase domain were observed as quenching or enhancement of fluorescence both from intrinsic sources (primarily tryptophan) and from extrinsically tagged (dansyl) CAM. Association of monomers separate from or concomitant with Ca2+-CAM binding was tested by gel filtration chromatography, chemical cross-linking followed by SDS-PAGE analysis, and light scatter particle sizing. Our results provide the first experimental evidence that Ca2+-CAM promotes dimerization of human eNOS through the oxygenase domain of the enzyme. A preliminary report of these findings has been published (27).


MATERIALS AND METHODS

Preparation of NTF Expression Clones

The human endothelial nitric-oxide synthase cDNA (28) used in this study was the kind gift of Dr. Kenneth D. Bloch (Harvard Medical School). An EcoRI-EagI fragment containing the N-terminal 1635 nucleotides of the coding region (NTF) was generated from this cDNA and included the catalytic and regulatory domains of the enzyme but not the flavoprotein reductase portion. The N-terminal oxygenase domain was cloned into pET23b (Novagen) to form the plasmid, pNTF, which was then transformed into the expression host, Escherichia coli BL21(DE3). The recombinant peptide was verified as eNOS using an eNOS-specific monoclonal antibody (gift of Dr. J. Pollock, Abbott Labs). A truncated form of the NTF cDNA consisting of the first 1430 nucleotides, which deleted the CAM-binding region, was also generated and transformed into E. coli BL21(DE3) as plasmid, pNTFcm-.

Purification of Recombinant eNOS Oxygenase Domain

Recombinant BL21(DE3)-pNTF cells were grown to an A600 of 0.5 to 0.6 in Luria broth, aminolevulinate was added to a final concentration of 0.2 mM, and expression was induced by the addition of isopropylthiogalactoside to a final concentration of 0.4 mM. Incubation was continued for 6-18 h, after which cells were harvested and eNOS oxygenase domain was purified by a method adapted from McMillan and Masters (22). Cell pellets were resuspended in 0.01 vol of S buffer composed of 20 mM Hepes (pH 7.4), 2 mM CaCl2, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 7 µM pepstatin A, 10 µM leupeptin, and 10% glycerol. Lysozyme was added to 1 mg/ml, and the suspension was allowed to stand on ice for 30 min. Cells were lysed by 3-6 min of pulsed sonication (Branson 450, 30 watts on 50% duty cycle) on ice, and the lysate was centrifuged for 60 min at 30,000 × g. The supernatant was mixed with 0.5 vol of calmodulin agarose (Sigma) containing 0.2 mg of calmodulin/ml of matrix equilibrated with S buffer. The supernatant and beads were mixed on a rotator at 4 °C for 2 h. The mixture was then poured into a column, washed with 50 column volumes of S buffer plus 0.2 M NaCl, and then washed with 50 column volumes of S buffer plus 0.5 M NaCl. Bound proteins were eluted in 20 ml of S buffer without CaCl2 and containing 5 mM EGTA. Peak protein fractions were pooled, concentrated by ultrafiltration (Amicon, 30k NMWCO), and further purified by Superose 12 gel filtration chromatography. Recombinant eNOS fractions were verified by Western blotting and concentrated by ultrafiltration. Protein concentrations were determined by the BCA reaction (Pierce) using bovine serum albumin as standard. Recombinant protein from BL21(DE3)pNTFcm- was expressed and purified in a similar manner except that a histidine-binding nickel column was used according to the manufacturer's instructions (Novagen) to bind the expressed, polyhistidine-tagged fusion protein.

Fluorescence Measurements

All measurements were made at room temperature (21 °C) using a Jasco FP770 spectrofluorometer and a quartz cuvette with 3-mm path length. Extrinsic fluorescence was used to measure Ca2+-CAM binding to recombinant eNOS oxygenase domain. A dansylated derivative of calmodulin (Sigma) was used to provide the external fluor, and solutions were freshly prepared just before use. The extent of dansylation was calculated using the extinction coefficient, epsilon 320 = 3400 mol-1 cm-1 and found to be 0.8 mol dansyl group/mol CAM (29). Incubations were done for 10 min at room temperature in a 200-µl assay mixture of 20 mM Hepes (pH 7.4), 0.1 M NaCl, 0.1 mM CaCl2, 0.3 mM L-arginine, 10 µM BH4, and 1 µM dansyl-CAM. Longer incubation times did not produce any change in the reading. Excitation was at 345 nm with slit width of 10 nm, and emission was read at 480-500 nm with slit width of 5 nm. The recombinant eNOS oxygenase domain showed negligible emission under these conditions. Changes in fluorescent emission due to association with the recombinant protein were expressed as the percentage of change relative to dansyl-CAM alone after correcting for background fluorescence.

For measurement of intrinsic fluorescence changes, purified eNOS oxygenase domain was incubated in the same buffer as used for extrinsic measurements with the substitution of CAM for dansyl-CAM. In experiments to measure spontaneous association, incubation was allowed to continue for 30 min. EGTA was present at a final concentration of 5 mM to block CAM binding. For assaying Ca2+-CAM-mediated changes, incubation was for 10 min. The measurements were done using an excitation wavelength of 295 nm with a slit width of 10 nm and an emission wavelength of 330 nm at a slit width of 5 nm. Calibration was done on buffer blanks. Baseline fluorescence for monomer was determined by heating the samples 10 min at 55 °C. Calmodulin lacks tryptophan, and tyrosine does not contribute significantly to the intrinsic fluorescence under these conditions. Changes in fluorescence emission intensity are expressed as the percent of change relative to the monomer value.

Cross-linking and Electrophoresis

Recombinant eNOS oxygenase domain was incubated in assay mixture (see recipe under "Fluorescence Measurements") with or without calcium and sampled at various times or for 30 min with various concentrations of recombinant domain at room temperature. After incubation, the reactions were made 100 mM in phosphate buffer (pH 8.0), and cross-linking was done by incubating the samples for 1 h at room temperature with 0.1 mM dimethylsuberimidate (Pierce). Reaction was terminated by the addition of 1 M Tris-HCl (pH 6.7) to a final concentration of 1 mM. Samples were then analyzed by SDS-polyacrylamide gel electrophoresis. For protein visualization, gels were stained with Coomassie Brilliant Blue. In some experiments proteins were blotted to nitrocellulose filters, and the eNOS oxygenase domain was detected using a specific antibody and an alkaline phosphatase-based colorimetric method (Bio-Rad).

Gel Filtration

Aliquots of eNOS oxygenase domain were treated as described for cross-linking and analyzed by fast protein liquid chromatography using a Superose 12 sizing column (Pharmacia). The column was calibrated with protein molecular mass standards as follows: myosin, 205 kDa; beta -galactosidase, 116 kDa; phosphorylase B, 97 kDa; bovine serum albumin, 67 kDa; and, ovalbumin, 47 kDa.

Particle Sizing

An aliquot of eNOS oxygenase domain was incubated at a final concentration of 1 mg/ml in 20 mM Hepes (pH 7.4), 0.13 M NaCl, 1 mM CaCl2, 0.3 mM L-arginine, 10 µM BH4 with or without 150 nM CAM for 10 min at 21 °C. Samples were analyzed using a DynaPro 801 Molecular Sizing Instrument (Protein Solutions, Inc., Charlottesville, VA). Bovine serum albumin was run as a standard.


RESULTS

Structure and Purification of Recombinant eNOS Oxygenase Domain

The domain structure of human eNOS is shown in Fig. 1 with the cloned regions for NTF and NTFcm- indicated. The oxygenase portion of the enzyme contains the heme coordination site, the sites for L-arginine and BH4 binding, and the Ca2+-CAM regulatory region.


Fig. 1. Domain structure of eNOS. The regions included in the NTF and NTFcm- constructs are indicated.
[View Larger Version of this Image (15K GIF file)]

Purification of the recombinant eNOS oxygenase domain was based on the use of CAM affinity chromatography, an extensively used and very effective method for isolating CAM-binding proteins (29). The expressed peptide had a molecular mass of about 60 kDa as determined by SDS-PAGE analysis and Western blotting. The yield of purified recombinant protein was approximately 0.5 mg/liter of cell culture. CAM-agarose affinity chromatography provides a nearly single-step purification of the recombinant protein from the bulk of bacterial proteins, few of which bind to CAM because the organism does not produce this protein. The fast protein liquid chromatography-purified recombinant protein is >90% pure as judged by SDS-PAGE analysis and has a reduced carbon monoxide spectrum characteristic of a cytochrome P-450-type hemeprotein (data not shown).

Fluorescent Analysis of CAM Binding

Because the oxygenase domain alone is incapable of NO production, the "activity" of the recombinant eNOS oxygenase domain was defined in terms of its capacity to bind Ca2+-CAM and/or to undergo self-association. Binding of Ca2+-CAM to its target proteins or peptides may be determined with great sensitivity by using CAM to which a fluorescent moiety such as dansyl has been attached. Dansyl-CAM retains the physical and biological properties of native CAM, has virtually the same dissociation constants, and can be detected in the low nanomolar range (30). Fig. 2A shows spectra of dansyl-CAM under various conditions. Dansyl-CAM undergoes a large increase in fluorescent intensity and a blueshift from about 520-495 nm upon binding of calcium (compare spectra a and b). This can be explained by the known alteration in conformation of CAM when it binds calcium, which in this case results in a change in the environment of the dansyl moiety to a more hydrophobic state, increasing the quantum yield, and shifting the emission wavelength (30). Addition of eNOS oxygenase domain (Fig. 2A, spectra c-f) to Ca2+-dansyl-CAM results in a further increase in fluorescent intensity and shift in wavelength of maximum emission to about 475 nm associated with the binding of Ca2+-dansyl-CAM to the recombinant oxygenase domain. Omission of the substrate for NOS, arginine, had no apparent effect on fluorescent intensity, whereas omission of the cofactor, BH4, appeared to slightly reduce the level of fluorescent intensity (data not shown). Both arginine and BH4 were included in the incubation mixtures to stabilize the recombinant oxygenase domain.


Fig. 2. Fluorescence analysis of dansyl-CAM binding to recombinant eNOS oxygenase domain. A, fluorescence emission spectra of 0.1 µM dansyl-CAM. Fluorescent intensity is given in arbitrary units. Spectrum a, no additions; spectrum b, with 1 mM EGTA; spectrum c, with 0.01 µM recombinant domain; spectrum d, with 0.02 µM recombinant domain; spectrum e, with 0.05 µM recombinant domain; spectrum f, with 0.10 µM recombinant domain. B, titration of 1 µM dansyl-CAM with complete or truncated recombinant eNOS oxygenase domains. C, stoichiometry of dansyl-CAM (dCAM) binding to recombinant oxygenase domain. FE, fluorescence emission intensity.
[View Larger Version of this Image (27K GIF file)]

Fig. 2B shows titration of 0.1 µM dansyl-CAM with the recombinant oxygenase domain. The concentration that gave half-maximal change in fluorescence emission intensity was about 1 nM. The truncated recombinant domain, which lacks the CAM binding site, shows little or no enhancement of fluorescence emission intensity. The stoichiometry of binding of eNOS oxygenase domain to Ca2+-CAM is approximately 1:1 (Fig. 2C).

Measurements of changes in intrinsic fluorescence of recombinant eNOS oxygenase domain (Fig. 3), which under the parameters used reflect primarily tryptophan fluorescence, demonstrated a Ca2+-CAM-dependent quenching, suggesting a reorientation of the peptide to expose tryptophan residues to a more hydrophilic or charged environment. Some quenching also occurs after extended incubation (30 min) and at high (>100 nM) concentrations of recombinant oxygenase domain in the absence of Ca2+-CAM binding, which may be attributed to a spontaneous concentration- and time-dependent association of monomers. The omission of BH4 from the incubation mixture appears to slightly alter the extent of complex formation, but a transfer of fluorescent energy mediated by BH4 cannot be ruled out as a cause of the changed fluorescent intensity.


Fig. 3. Changes in intrinsic fluorescence of recombinant eNOS oxygenase domain as a function of concentration. Incubations were done for 30 min at room temperature in an assay mix containing 1 µM CAM with or without 0.1 mM CaCl2 or 10 µM BH4 (solid line, +BH4; dashed line, -BH4). The change in fluorescent intensity is expressed relative to that obtained from monomer.
[View Larger Version of this Image (21K GIF file)]

Determination of Dimerization by Gel Filtration and Light Scatter Particle Sizing

Activation of eNOS is known to involve both Ca2+-CAM binding and homodimerization of the enzyme (21). To characterize the Ca2+-CAM-eNOS oxygenase domain complex implicit in our dansyl-CAM fluorescence enhancement results, we examined the association by gel filtration (Fig. 4) and particle sizing (Table I). In Fig. 4, aliquots of eNOS oxygenase domain were incubated with CAM in the presence (panel B) or the absence (panel C) of Ca2+ and then chromatographed on a size exclusion column equilibrated with the appropriate buffer. Peaks corresponding to both monomeric and dimeric forms were observed in the presence of Ca2+, indicating some instability of the complex under these conditions. In the absence of Ca2+ (+EGTA), there was a minor peak corresponding to a dimer, which was presumably due to the concentration- and time-dependent association of monomers observed in the absence of Ca2+. The elution volume of this minor peak corresponds to a lower molecular weight than the major peak observed in the presence of Ca2+ due to the absence of bound CAM. Fig. 4A gives the sizes of protein standards run under the same conditions. A standard curve of molecular mass versus retention was plotted by linear regression, and the sizes of eNOS oxygenase domain monomer and dimer were calculated as approximately 55 and 120 kDa, respectively.


Fig. 4. Gel filtration molecular sizing of recombinant eNOS oxygenase domain. Aliquots of recombinant domain were incubated for 30 min in complete reaction mixture with or without CaCl2 then analyzed by fast protein liquid chromatography on a sizing column of Superose 12. The column was calibrated with protein standards as described under "Materials and Methods." A, protein size standards. B, with CaCl2. C, with EGTA.
[View Larger Version of this Image (20K GIF file)]

Table I. Quaternary structure of recombinant eNOS oxygenase domain


Condition Apparent massa

kDa
Controlb 149  ± 11c
Minus BH4 150  ± 29
Minus CAM 80  ± 10
Bovine serum albumin standard 79  ± 3

a Apparent molecular masses were determined at 23 °C with a DynaPro 801 Molecular Sizing Instrument.
b The control mixture contained 1 mg/ml of recombinant eNOS oxygenase domain in Hepes (pH 7.4) containing arginine, BH4, CaCl2, calmodulin, and dithiothreitol.
c 95% confidence limits.

To further test the self-association of eNOS oxygenase domain monomers as a function of Ca2+-CAM binding, we incubated the recombinant domain in the presence and the absence of Ca2+-CAM and measured complex size with a laser light scatter particle-sizing apparatus. The results, which are shown in Table I, indicate a complex of 149 kDa formed when Ca2+-CAM was present. This complex did not form in the absence of CAM but did form in the absence of BH4. The size of the complex would suggest association of two recombinant domains with two Ca2+-CAMs. This would agree with the 1:1 stoichiometry result seen previously.

SDS-PAGE Analysis of Cross-linked Domains

To determine whether the eNOS oxygenase domain undergoes self-association in the absence of calmodulin binding, increasing concentrations of recombinant domain were incubated in assay mixture without calcium (with 5 mM EGTA) and then subjected to chemical cross-linking and SDS-PAGE analysis. Fig. 5A shows that spontaneous association can occur under these conditions but requires relatively high concentrations and longer times. Panel B compares rates of dimerization with and without binding of Ca2+-CAM. Whereas dimerization in the presence of Ca2+-CAM is essentially complete within 5 min, a substantial amount of monomer remained in the assay without calcium even after 60 min. Although some rearrangement in peptide conformation seems likely as a result of dimerization alone, it cannot be determined from these data exactly what the respective contributions of Ca2+-CAM binding and dimerization are to the molecular switch that turns on NOS activity. It seems clear, however, that Ca2+-CAM binding serves to promote association of eNOS subunits. The apparent spontaneous concentration- and time-dependent association of recombinant eNOS oxygenase domain monomers in the absence of Ca2+ may suggest that dimerization alone is not sufficient for the activation of eNOS.


Fig. 5. Characteristics of dimerization. A, SDS-PAGE analysis showing effect of increasing recombinant eNOS oxygenase concentration on monomer association. Recombinant domain was incubated for 30 min in assay mixture lacking CaCl2. Lane 1, 0.2 µM recombinant domain; lane 2, 0.4 µM recombinant domain; lane 3, 1.0 µM recombinant domain; lane 4, 2.0 µM recombinant domain; lane 5, 5.0 µM recombinant domain. The amount of recombinant domain in each reaction was constant, and the volume was reduced by ultrafiltration prior to cross-linking and loading onto the gel. B, SDS-PAGE analysis of the time course of dimerization without (lanes 1-5) or with (lanes 6-10) CaCl2. Lane 1, 0 min; lane 2, 10 min; lane 3, 20 min; lane 4, 30 min; lane 5, 60 min; lane 6, 0 min; lane 7, 1 min; lane 8, 2 min; lane 9, 5 min; and lane 10, 10 min. The concentration of recombinant domain and CAM in the reaction mixture was 2 and 3 µM, respectively, and samples were subjected to cross-linking before electrophoresis. m, monomer; d, dimer.
[View Larger Version of this Image (61K GIF file)]


DISCUSSION

The activation of a number of enzymes is dependent on either oligomerization of the enzyme or on binding of an allosteric effector molecule such as calmodulin. In some cases, such as myosin light chain kinase (7, 14), erythrocyte Ca2+-ATPase (14-16), and the NOSs, both CAM-binding and oligomerization may be involved in enzyme activation. The role of calmodulin in promoting dimerization of eNOS may be unique to this isoform of NOS and could be a part of the signal transduction pathway that regulates NO production in endothelial cells. As pointed out by Lee et al. (21), the NOS isoforms share many features but have different physiological roles and appear to be regulated by different mechanisms. These authors suggested that oligomerization may be a possible mechanism for the modulation of NOS activity based on their results and those of others indicating that the active form of all NOS isoforms is a homodimer and that dimer formation is influenced by substrates and cofactors (8, 11-13, 17-21). Ghosh and Stuehr (11) showed that the N-terminal oxygenase domain of iNOS participates in dimer formation and that this association is dependent on the cofactor, tetrahydrobiopterin. This association did not require calmodulin. The dimerization of nNOS also appears to occur through the oxygenase domain, requires heme, and is stabilized by BH4 and arginine (20). A possible requirement for calmodulin in the dimerization of nNOS was not addressed. Rodriguez-Crespo et al. (26), using recombinant bovine eNOS, reported that tetrahydrobiopterin was required for activity but did not appear to affect the extent of dimerization as determined by SDS-PAGE. An elegant study by Lee et al. (21) demonstrated that oligomerization of eNOS is important for eNOS activity in vivo. Their results suggested that both the N-terminal and C-terminal domains are involved in eNOS dimerization. However, the constructs used to produce the truncated N-terminal and C-terminal eNOS mutants contained overlapping sequences (residues 512-732 in the eNOS sequence), making interpretation of their results difficult. Some of this shared sequence (residues 512-545) is also contained in the recombinant eNOS oxygenase domain used in the present study.

Our results clearly demonstrate that the binding of calmodulin to a recombinant oxygenase domain of human eNOS promotes dimerization and that the C-terminal reductase domain is not required for dimer formation. Our results also show that the substrate, arginine, and the cofactor, tetrahydrobiopterin, are not required for eNOS dimer formation, in agreement with the the results of Rodriguez-Crespo et al. (26), but our results do suggest that tetrahydrobiopterin may stabilize the dimeric state of eNOS. These results are in contrast to the results reported by Stuehr's group, which demonstrated an essential role for tetrahydrobiopterin in the dimerization of iNOS (11). Thus, these two isoforms of NOS clearly differ in this regard. Interestingly, iNOS activity is not regulated by calcium signaling because calmodulin remains tightly bound to the enzyme (continuously activated). Because the calcium-dependent binding of calmodulin is required for the activation of eNOS, calmodulin-induced dimerization may play an important role in the modulation of the activity of this key enzyme in vasoregulation.


FOOTNOTES

*   This work was supported by Grants GIA 9501378 and GSF/1A from the American Heart Association, Florida Affiliate.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.
par    To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of South Florida College of Medicine, 12901 Bruce B. Downs Blvd., MDC Box 7, Tampa, FL 33612-4799. Tel.: 813-974-9558; Fax: 813-974-5798; E-mail: lsolomon{at}com1.med.usf.edu.
1   The abbreviations used are: NOS, nitric-oxide synthase; eNOS, the constitutive endothelial isoform of NOS; nNOS, the constitutive neuronal isoform of NOS; iNOS, the inducible isoform of NOS; CAM, calmodulin; BH4, 5,6,7,8-tetrahydrobiopterin; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl; PAGE, polyacrylamide gel electrophoresis.

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