©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Endothelial Nitric-oxide Synthase
EXPRESSION IN ESCHERICHIA COLI, SPECTROSCOPIC CHARACTERIZATION, AND ROLE OF TETRAHYDROBIOPTERIN IN DIMER FORMATION (*)

(Received for publication, November 28, 1995; and in revised form, February 6, 1996)

Ignacio Rodríguez-Crespo (§) Nancy Counts Gerber Paul R. Ortiz de Montellano (¶)

From the Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, California 94143-0446

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Bovine endothelial nitric-oxide synthase (eNOS) expressed in Escherichia coli does not have the post-translational modifications found in the native enzyme and is free of tetrahydrobiopterin (BH(4)). In the presence of BH(4), eNOS has an absorption maximum at 400 nm that shifts to 395 nm when the substrate L-arginine is added. The low-spin component of the spectrum of the BH(4)-free protein is decreased by the addition of BH(4) without a corresponding increase in the high-spin component. Addition of BH(4) decreases the low-spin population of eNOS even in the presence of excess L-arginine. These results indicate that BH(4) directly modulates the heme environment. BH(4)-free eNOS is completely inactive, but catalytic activity is recovered when BH(4) (EC 200 nM) is added. The spectroscopically determined binding constants for L-arginine are 1.9 µM in the presence and 4.0 µM in the absence of BH(4). The BH(4)-supplemented enzyme has an activity of 90-120 nmol of citrullinebulletminbulletmg and K values of 3 and 14 µM for L-arginine and N-hydroxy-L-arginine, respectively. Of particular interest is the finding by SDS-polyacrylamide gel electrophoresis that BH(4)-free eNOS exists in a monomer-dimer equilibrium very similar to that observed with the BH(4)-reconstituted protein. Addition of BH(4) increases the percent of the dimer by only 5%. The results establish that BH(4) influences the heme environment and stabilizes the protein with respect to heme loss but is not required for dimer formation.


INTRODUCTION

Nitric oxide (NO), a major regulatory factor in the immune, nervous, and cardiovascular systems, mediates vasodilation, causes inhibition of platelet aggregation, has a role as an important effector molecule of the host defense system, and functions in neuronal transmission. Nitric-oxide synthases (NOS, (^1)EC 1.14.13.39) oxidize L-arginine in a process that consumes NADPH and produces stoichiometric amounts of L-citrulline and NO(1, 2, 3, 4, 5) . Two general categories of NOS are known, (a) constitutive enzymes that are regulated by Ca and CaM and (b) inducible enzymes with a tightly bound CaM that are not regulated by Ca. Both types of NOS are modular proteins in which a P-450-like heme domain is connected to a two-flavin domain by a linker peptide that displays a consensus calmodulin-binding sequence.

Studies carried out with bovine aortic endothelial cells first demonstrated the presence of nitric-oxide synthase activity that is principally associated with the particulate fraction and is both Ca- and CaM-dependent(6) . Affinity chromatography and gel filtration yield a purified protein that, after denaturation, migrates as a single band on SDS-PAGE with a molecular mass of 135 kDa(7) . As reported for the neuronal (8) and inducible (9, 10) isoforms, the endothelium-derived enzyme also requires BH(4) for full activity(7) . This eNOS normally accounts for both basal and stimulated NO synthesis throughout the vascular system.

Cloning of the human (11, 12) and bovine (13, 14, 15) eNOS shows that these proteins have approximately 60% sequence identity with nNOS and 50% with iNOS. The presence of a consensus sequence for N-terminal myristoylation, absent in the other two isoforms, accounts for the observation that more than 90% of the enzymatic activity remains associated with the particulate fraction(6) . Replacement of Gly-2, the myristic acid acceptor site, by an alanine converts eNOS into a cytosolic enzyme without detectably altering its enzymatic properties (16, 17) . eNOS can also be palmitoylated at cysteines 15 and 26 (18) in a process that may be dynamically regulated as part of a response to enzyme agonists such as bradykinin(19) . More recently, a specific interaction between recombinant eNOS and acidic phospholipids has been reported(20) . Surprisingly, deletion of the entire CaM-binding region of eNOS produces a cytosolic myristoylated protein no longer able to interact with lipids(20) .

Although numerous reports have been published describing the spectral, catalytic, and structural properties of purified nNOS and iNOS, the corresponding information on the endothelial isoform is limited. Both nNOS (21, 22, 23) and iNOS (24, 25) are homodimers, the dimer being the catalytically active form(23, 25) . These two isoforms contain an average of 1 FAD, 1 FMN(24, 25, 26, 27) , and 1 iron-protoporphyrin IX(28, 29, 30, 31) per subunit and have the spectroscopic properties of a cytochrome P-450 (31, 32) . This fact, together with sequence homologies with P-450 reductase (32) and to a very small extent with the heme-binding sequence of P-450 3A4(33) , suggests that NOS is a self-sufficient relative of the P-450 superfamily.

eNOS has been purified from endothelial cells (6, 7, 34) and heterologous expression has been achieved in COS cells(13, 15, 16, 17) , NIH3T3 cells(12) , and in baculovirus systems(35, 36, 37, 38) . However, only small amounts of protein have been obtained from these sources. We report here the first successful expression of catalytically active bovine eNOS in Escherichia coli. The recombinant enzyme displays spectral properties comparable with those reported for the neuronal and macrophage isoforms and is present in a monomer-dimer equilibrium with the dimer surviving in 0.1% SDS. Of particular interest is the fact that BH(4)-free eNOS, which has an absorbance spectrum that differs from that of the BH(4)-bound enzyme, retains the ability to dimerize even though it is catalytically inactive.


EXPERIMENTAL PROCEDURES

Materials

L-Arginine was obtained from Aldrich and 2`,5`-ADP-Sepharose from Sigma. Calmodulin-Sepharose 4B and Phast System products were from Pharmacia Biotech Inc. CaM, (6R)-5,6,7,8-tetrahydrobiopterin, and N^G-hydroxy-L-arginine were from Alexis Biochemicals (San Diego, CA). Ni-NTA-agarose resin was purchased from QIAGEN (Chatsworth, CA). Electrophoresis reagents, disposable mini-columns (catalog number 7311553), and Dowex 50W-X8 were from Bio-Rad. Monoclonal antibodies against NOS were from Transduction Laboratories (Lexington, KY). BioScint scintillation mixture was from Fisher. Restriction enzymes, Bacto-yeast extract, IPTG, and DH5alpha E. coli cells were purchased from Life Technologies, Inc. BL21 (DE3) pLys cells were from Novagen (Madison, WI). L-[2,3,4,5-^3H]Arginine and Western blotting detection reagents were purchased from Amersham. The cDNA clone for bovine eNOS was a gift from William C. Sessa (Yale University)(14) . The pCWori vector was a gift from Robert Fletterick (University of California, San Francisco)(39) .

Cloning

Different procedures were designed to subclone the bovine eNOS gene from pBluescript KS into the NdeI and XbaI sites of pCWori. The general cloning strategy consisted of the sequential introduction of an NdeI-KpnI fragment and a KpnI-XbaI fragment of the gene into the pCWori vector. The eNOS gene was initially double-digested with KpnI (unique site in the gene) and XbaI (in the polylinker of pBluescript close to the 3` end of the gene), and the excised fragment was ligated in a pCWori clone containing the putidaredoxin reductase gene. (^2)which had also been double-digested with KpnI and XbaI. This gene was chosen because it contained the three required unique sites (NdeI, KpnI, and XbaI) in the correct order. Subsequently, the polymerase chain reaction with a proof-reading enzyme (UItma polymerase; Perkin-Elmer) was used to amplify a region of the pBluescript-bovine eNOS gene between the initiator methionine and the unique KpnI site (2200 bp approximately). At this point, a new NdeI site was introduced at the 5` end of the gene, and Ala was introduced as the second amino acid, since this substitution improves heterologous cytochrome P-450 expression in bacterial systems(40) . This product was ligated into the pCW vector in which the KpnI-XbaI fragment had already been inserted. The resulting pCWeNOS gene was used to transform competent E. coli DH5alpha or BL21 (DE3) pLys cells.

To facilitate complete purification of the protein, a polyhistidine tag (6 histidines) was placed at the N terminus. Due to the presence of two BamHI sites in pCWeNOS, a BamHI and NdeI double-digestion was performed on a pCW-putidaredoxin plasmid,^2 and a small fragment of approximately 25 bp was excised. Two synthetic oligonucleotides of 43 and 45 bases were designed so that upon annealing and ligation between the BamHI and NdeI sites, the Shine-Delgarno region was reinserted with the additional 18 bp corresponding to the polyhistidine tag. A new BsmI site was also introduced between the BamHI and NdeI sites for screening purposes. The construct was designated as poly-His pCWPdX. An NdeI-XbaI double digestion was then performed, and the putidaredoxin gene was replaced by the bovine eNOS gene. This new construct was designated poly-His pCWeNOS.

Expression

Typically, 4 liters of cell culture were grown at a time in the presence of 200 µg/ml ampicillin in 2 times YT media with casein N-Z plus hydrolysate (Sigma) in place of tryptone. An overnight culture of 50 ml of cells of pCWeNOS was used to inoculate each of two 2.8-liter Fernbach flasks containing 2 liters of media. The cultures were grown to an A of 0.8 and induced by addition of 2 mM IPTG. The 4 liters of induced cell culture were equally divided among three 2.8-liter Fernbach flasks in order to increase aeration and grown at 22 °C at a rotation rate of 210 rpm for 16-20 h before the cells were harvested by centrifugation.

Purification

The purification sequence employing calmodulin-Agarose and 2`,5`-ADP-Sepharose columns was essentially the same as that previously reported for purification of nNOS and was typically carried out with the protein from 4 liters of culture(41) . The only significant difference from the previous protocol is that elution from the final 2`,5`-ADP-Sepharose column was done with Buffer B containing 25 mM 2`-AMP rather than NADPH. Due to the instability of eNOS, the purification was carried out in the presence of 10% glycerol within a period of 1 day, and the resultant protein was stored at -70 °C.

In the case of the poly-His tagged protein, an additional Ni-NTA column was used. The protein bound to the ADP resin was washed with Buffer C (Buffer B without BH(4) or BME), and after elution was loaded onto a 5-ml Ni-NTA-agarose column. The column was washed with 25 ml of Buffer C and eluted with Buffer C plus 200 mM imidazole. After elution of the purified protein, BH(4) was added to a final concentration of up to 10 µM before storage. When the activity of the protein eluted with imidazole was measured, a small calmodulin-agarose column was used to eliminate imidazole and to concentrate the sample prior to the assay.

The protein purification was performed at 4 °C, and no L-arginine was included in the buffers during the purification steps. When protein without BH(4) was desired, this cofactor was omitted from all the buffers, and the columns were thoroughly washed with buffers lacking the cofactor before loading the protein to remove any trace of pterin from previous purifications. The enzyme was stored in aliquots at -70 °C, and multiple freeze-thaw cycles were avoided. The desired quantities of eNOS for the various assays were routinely obtained by scraping the frozen protein.

Enzyme Assays

The activities of the protein preparations were determined by measuring either the production of NO using the conversion of HbO(2) to Met-Hb or the conversion of L-[^3H]arginine to L-[^3H]citrulline at 37 °C(41, 42) . The reduction of cytochrome c by eNOS was assayed at 37 °C in a 500-µl volume using an extinction coefficient of 21 mM cm at 550 nm. The reaction mixture containing 50 mM Hepes, pH 7.5, 100 µM EDTA, 100 µg/ml bovine serum albumin, 2 mM CaCl(2), 500 µM NADPH, 50 µM cytochrome c, 50 units of catalase, and 2-5 µg of eNOS was initiated by adding the cytochrome c and NADPH. When indicated, assays contained 50 µML-arginine and/or 10 µg/ml CaM. Both BH(4) and DTT were avoided when cytochrome c reduction was assayed to minimize the reduction caused by the buffer itself.

Protein Determination

Protein concentrations were determined by the Bradford assay (Bio-Rad) using bovine serum albumin as a standard. Concentrations of heme-containing NOS were determined from the absorption spectrum of the protein using the extinction coefficient = 100 mM cm for the ferric enzyme.

SDS-PAGE

Electrophoresis was carried out using a Pharmacia Phast Gel system. Homogenous 7.5% gels were used with SDS buffer strips and were stained with Coomassie Blue. Western blotting was carried out using the Phast Transfer system, and the protein was detected using a commercial anti-iNOS antibody (Transduction Laboratories; elicited against the reductase domain of the protein and known to cross-react with the endothelial isoform (38) by enhanced chemiluminescence). When the monomer-dimer equilibrium was studied by SDS-PAGE, the Mini Protean II system (Bio-Rad) was used, following a modification of a published protocol(22) . Five ml of running gel (7.5% acrylamide, 0.2% bisacrylamide, 370 mM Tris, 0.1% SDS, 0.28% TEMED, 0.2 µg/µl ammonium persulfate, pH 8.8) and two ml of stacking gel (4% acrylamide, 0.1% bisacrylamide, 125 mM Tris, 0.1% SDS, 0.4% TEMED, 0.2 µg/µl ammonium persulfate, pH 6.8) were prepared. The gel was run in 25 mM Tris buffer, pH 8.3, containing 192 mM glycine and 0.1% SDS. The electrophoresis tank was placed in an ice-bath inside a cold box, and the separation was performed at a constant voltage of 185 V. The gels were stained with Coomassie Blue. Quantitation was done by scanning densitometry using a Hoefer Scientific Instruments GS300 instrument. The scanning speed was 6 cm/min. The results were analyzed with the GS 371 software. The base line was subtracted from the calculated areas.

Calculation of the EC Value for BH(4)

The EC for BH(4) was calculated measuring the transformation of L-[^3H]arginine to L-[^3H]citrulline at different BH(4) concentrations with 50 µM BH(4) as a control of maximal activity. A 4-ml solution of 50 µML-arginine, 200 µM NADPH, 1 mM CaCl(2), and 3.75 µCi/ml [^3H]-arginine in 100 mM Hepes, pH 7.5, was prepared. From this solution, 2 aliquots of 100 µl each were loaded onto 1 ml of Dowex 50W-X8 columns (Na-form) (negative control), and 2 aliquots of 100 µl each were directly measured for ^3H counts. The remaining 3600 µl was divided equally among 6 tubes and equilibrated at 37 °C before addition of 5-10 µg of eNOS and the desired amount of BH(4) (typically 100-500 nM range). The appropriate BH(4) dilution was prepared from a stock solution of 10 mM BH(4) in 100 mM DTT. The reaction was immediately initiated by addition of 1 µM CaM and was quenched after 1, 3, or 5 min by transferring aliquots to a 5 mM EGTA solution and boiling for 1 min. The mixtures were then loaded onto the Dowex column and washed with 2.5 ml of distilled water. Both the sample-loading eluate and the wash volume were directly collected in scintillation vials. BioScint mixture (15 ml) was added to each sample, and the radioactivity was quantitated by liquid scintillation. All the points were done in duplicate, and the reaction mixtures were kept at 37 °C during the entire experiment.

Difference Spectrophotometry

eNOS (100-300 µg/ml, in the absence of CaM) in 50 mM Hepes, pH 7.5, 10% glycerol (total volume of 0.5 ml) was titrated with a 100 µML-arginine solution in the 0.2-2 µM range (1-10 µl). The substrate was added with a 10-µl syringe, and the solution was carefully mixed to avoid protein precipitation as this increases the optical dispersion. After addition of arginine, the absorbance spectrum was immediately recorded. The maximal dilution error was 2%. Spectra were recorded in 1-cm masked cuvettes at 15 °C in a dual-beam Cary 1E spectrophotometer connected to a Lauda circulating-water bath. The negative increment in 415 nm absorbance was determined, and the spectral binding constants (K(s)) were determined from the x-intercept of a double-reciprocal plot of this increment versus the arginine concentration.


RESULTS

Expression of Bovine eNOS in E. coli

pCWori was recently used in this laboratory to express catalytically active nNOS in E. coli(41) . In the present work, cloning of an eNOS gene into pCWori, transformation of DH5alpha E. coli cells with the resulting vector, and growth of the cells at 22 °C resulted in expression of functional eNOS as determined by Western blotting and activity assays. Expression of the protein without the polyhistidine tag yielded 10-15 mg of purified protein from 4 liters of cell culture. Purification on a calmodulin-Sepharose 4B column followed by a 2`,5`-ADP-Sepharose column provided protein that was at least 85% pure as judged by SDS-PAGE. Most of the studies described here have been performed with protein without the polyhistidine tag. When a higher purity of the sample was desired, the poly-His pCWeNOS gene was used to transform BL21 (DE3) pLys E. coli cells, and a third affinity column (Ni-NTA) was used during the purification. The final yield was then 5-10 mg of highly purified protein from 4 liters of cell culture (Table 1). When the E. coli lysate was loaded onto the calmodulin-Sepharose resin and the bound protein was thoroughly washed with buffer, NOS was obtained in which the only contaminants appeared to be proteolytic eNOS cleavage products. Subsequent utilization of the ADP-Sepharose and Ni-NTA columns, which select for affinity sites at opposite ends of the protein, provides the highly purified full-length protein (Fig. 1).




Figure 1: SDS-PAGE analysis of the purified eNOS isolated from E. coli. Lane A, molecular mass standards (bovine serum albumin, 66 kDa; phosphorylase b, 97.4 kDa; beta-galactosidase, 116 kDa; myosin, 205 kDa); lane B, crude cell lysate; lane C, sample after calmodulin-Sepharose column; lane D, sample after the 2`,5`-ADP-Sepharose column; lane E, final sample after the Ni-NTA-agarose column. Approximately 1.5 µg of protein was loaded per lane.



Characterization of eNOS Expressed in E. coli

The spectra of the purified, recombinant ferric protein purified in the presence of BH(4) under various conditions are shown in Fig. 2. In the absence of substrate, the enzyme has an absorbance maximum at 400 nm. Upon addition of L-arginine (50 µM), the absorbance maximum shifts to 395 nm, in agreement with an increase in the heme high-spin component. The difference spectrum for the L-arginine-bound minus L-arginine-free ferric protein clearly shows the shift from a low-spin to a high-spin iron state (Fig. 2B). Reduction of the protein with dithionite produces the ferrous state with a maximum at 415 nm (Fig. 2A, trace 3). The reduced, CO-bound difference spectrum has an absorbance maximum at 445 nm characteristic of a thiolate-ligated iron (Fig. 2C).


Figure 2: Absorbance spectra of eNOS under different conditions: A, absorbance spectrum of purified ferric eNOS in the absence (trace 1) and presence (trace 2) of 50 µM L-arginine, and absorbance spectrum of the dithionite-reduced eNOS (trace 3); B, difference spectrum obtained by subtracting trace 1 from trace 2 in panel A; C, ferrous CO difference spectrum. The samples were in 50 mM Hepes, pH 7.5, containing 10 µM BH(4).



Difference spectrophotometry was used to calculate the spectral binding constant K(s) for the protein purified in the presence and absence of BH(4) (Fig. 2B). The BH(4)-bound protein has a spectral binding constant of 1.9 µM for L-arginine that increases to 4.0 µM in the absence of the cofactor. Hence, the pterin moiety facilitates the binding of the substrate 2-fold.

The recombinant protein gave similar activity values with both of the assays used in this study, with a V(max) at 37 °C of 95-120 nmol NO minmg eNOS and K(m) values of 3 and 14 µM for L-arginine and N-hydroxy-L-arginine, respectively. No significant activity changes were observed when superoxide dismutase, FAD, or FMN were added to the reaction mixture. Unlike previous results with nNOS(23, 41) , addition of high concentrations of L-arginine (up to 100 µM) in the absence of high concentrations of BH(4) does not result in enzyme inhibition by NO. The rate of reduction of cytochrome c at 37 °C by eNOS was 3-5 µmolminmg, with approximately a 7-fold decrease in the reduction rate in the absence of CaM. The presence of L-arginine in the cytochrome c reduction assay did not detectably alter these rates.

Effect of BH(4) on the Catalytic and Spectroscopic Properties of eNOS

Elimination of the cofactor from all of the buffers and columns yields purified enzyme completely devoid of BH(4). Quantitation of the pterin content of the purified protein shows that it contains less than 0.1% of BH(4), (^3)in accord with earlier studies which showed that BH(4) is not present in E. coli lysates(44) . When the cofactor was excluded from the purification buffers, NO production was not detected by the oxyhemoglobin method unless BH(4) was added to the assay system whether L-arginine or N-OH-arginine was used as the substrate (Table 2). Approximately 90% of the maximum activity was immediately recovered with both substrates when BH(4) was included in the assay buffer (10 µM final concentration). When eNOS was purified in the presence of the cofactor but assayed in the absence of BH(4), the initial rate of NO production was approximately 65% of its maximal value. The cytochrome c reduction rate for the protein purified in the presence or absence of BH(4) was about 6.5 µmolminmg, with a 6-fold decrease in the absence of CaM. The protein devoid of BH(4) was denatured more rapidly by freeze-thawing than the protein supplemented with BH(4).



In order to calculate an EC value for the cofactor, increasing concentrations of BH(4) were added, and the conversion of L-[^3H]arginine to L-[^3H]citrulline was measured after incubation for 1, 3, and 5 min. BH(4) is able to rescue enzyme activity with an EC of 200 nM using the data from the 1-min reaction time point. The assay was not linear at longer time points, particularly in incubations with low BH(4) concentrations.

BH(4) appears to directly modulate the heme environment of eNOS. The absorbance spectrum of the BH(4)-free protein is depicted in Fig. 3. In the absence of the cofactor, there is an elevated low-spin population of the heme (Fig. 3, trace 1) that immediately decreases on addition of BH(4) without a clear increase in the high-spin population (trace 2), unlike the addition of L-arginine (trace 3) which produces a clear shift from low to high spin. As shown in Fig. 4, these effects are additive, since the sequential addition of L-arginine and BH(4) produces a more profound decrease in the low-spin population than that caused by L-arginine alone. Conversely, sequential addition of BH(4) and L-arginine produces a decrease in the low-spin population when BH(4) alone is added followed by a low- to high-spin conversion when the substrate is also added.


Figure 3: Effect of BH(4) on the spectroscopic properties of recombinant eNOS. Absorbance spectra of eNOS purified in a complete absence of BH(4) (spectrum 1) plus 40 µM BH(4) (spectrum 2). Spectrum 3 shows the effect of 100 µML-arginine on spectrum 2. The samples were in 50 mM Hepes, pH 7.5, 50 mM NaCl, 10% glycerol.




Figure 4: Heme modulation induced by BH(4) and by L-arginine. Panel A, difference spectra obtained on a 200 µg/ml eNOS solution upon the sequential addition of 100 µML-arginine (spectrum 1) and 40 µM BH(4) (spectrum 2). Panel B, difference spectra obtained on a 200 µg/ml eNOS solution upon the sequential addition of 40 µM BH(4) (spectrum 1) and 100 µML-arginine (spectrum 2). The samples were in 50 mM Hepes, pH 7.5, 50 mM NaCl, 10% glycerol.



Quaternary Structure of the Recombinant eNOS

SDS-PAGE has been employed to characterize the monomer-dimer equilibrium of eNOS (Fig. 5). (^4)Experiments were carried out with 7.5% acrylamide, 0.1% SDS gels with the usual boiled proteins and undenatured beta-amylase, a tetrameric protein of 200 kDa, as molecular mass markers. When eNOS (3 µg) was boiled for 5 min in the presence of loading buffer with 2% SDS, the expected 135-kDa monomer band was observed (Fig. 5, lane C). However, if the protein was loaded onto the gel without previous boiling, three different bands were observed (Fig. 5, lane D), one of which comigrated with the denatured monomer. The high molecular weight band observed under these conditions corresponds to a higher molecular weight than that of beta-amylase and is assigned to eNOS dimers that are stable in the presence of the detergent. The band that migrated at a lower molecular weight than the SDS-denatured monomer was assigned to at least partially folded monomers. Heating of the eNOS sample at 45 °C for 15 min (but not boiling) resulted in the disappearance of this band due to its conversion to denatured monomer. The dimer, however, was not denatured by these conditions, which indicates that the dimeric form is relatively thermostable. The presence of 5% BME in the SDS-PAGE loading buffer ruled out the possibility of a monomer-monomer interaction mediated by disulfide bridges. When eNOS purified in the complete absence of BH(4) was analyzed under the same conditions (Fig. 5, lane E), a very similar migration pattern was observed. Densitometric quantitation of SDS-PAGE gels indicates that, in the absence of BH(4), purified eNOS migrates 50% as the dimer, 20% as the monomer, and 30% as the ``denatured'' monomer (Table 3). Addition of up to 2.5 mM BH(4) to the purified enzyme only slightly increases the proportion of the dimer in the mixture (Table 3). These results demonstrate that the dimerization of eNOS does not depend critically on the presence of BH(4).


Figure 5: SDS-PAGE analysis of the monomer-dimer equilibrium. A 7.5% acrylamide, 0.1% SDS gel was used to determine the quaternary associations of eNOS. Lane A, SDS-boiled molecular mass standards (bovine serum albumin, 66 kDa; phosphorylase b, 97.4 kDa; beta-galactosidase, 116 kDa); lane B, unboiled beta-amylase standard (200 kDa); lane C, SDS-boiled eNOS purified in the presence of BH(4); lane D, unboiled eNOS purified in the presence of BH(4); lane E, unboiled eNOS purified in the absence of BH(4). Approximately 3 µg of protein were loaded per lane. The gel was stained with Coomassie Blue.






DISCUSSION

Successful expression of eNOS in E. coli and purification of the recombinant enzyme (Table 1) for the first time provide ready access to the large amounts of pure protein required for physical characterization. Although eNOS has recently been expressed in baculovirus systems(35, 36, 37, 38) , expression in E. coli is simpler and provides higher amounts of functional protein. Heterologous expression of eNOS is particularly useful because eNOS is currently the least readily available of the three types of NOS. Furthermore, the protein obtained by bacterial expression is devoid of BH(4) because bacteria do not produce BH(4)(44) . Roman et al.(46) have reported that nNOS expressed in E. coli contains 10% of the theoretical content of pterin. The reason for this discrepancy in the BH(4) content of enzymes expressed in E. coli is unclear but might reflect contamination if the enzyme is purified on columns also used to purify the BH(4)-supplemented protein. The BH(4) requirements of the protein can be satisfied by adding the cofactor (see below), but the protein purified in the absence of added BH(4) provides an excellent source of enzyme for studies of the role of BH(4) in catalysis.^3

The terminal amino acid in the mature eNOS expressed in mammalian cells is a glycine due to post-translational processing of the terminal methionine. Myristoylation of this N-terminal glycine leads to association of eNOS with the membrane. Myristoylation does not occur, however, and the protein does not associate with the membrane when the glycine is replaced by an alanine(16, 17) . The glycine in the protein studied here has been replaced by an alanine due to the observation that the expression of P-450 enzymes in E. coli is improved when the amino acid adjacent to the terminal methionine is an alanine (40) . The eNOS expressed in E. coli is thus a cytosolic protein not only because E. coli lacks N-myristoyltransferase activity (45) but also because the glycine that would be myristoylated in mammalian cells has been replaced by an alanine.

In order to facilitate the preparation of highly purified eNOS for crystallographic studies, a second form of the protein has been expressed with six histidine residues attached to the N terminus. The protein with the poly-His domain is indistinguishable from the recombinant protein in terms of both spectroscopic and catalytic properties.

Due to the unavailability of eNOS there is little published spectroscopic data on this form of the enzyme. Spectra of the ferric protein have recently been reported(36, 37) , but no spectroscopic data are available on the other oxidation states of the protein or its interactions with cofactors or ligands. As shown in Fig. 2, the BH(4)-reconstituted ferric protein has a Soret maximum at 400 nm, the ferrous protein a maximum at 415 nm, and the ferrous-CO complex a P-450-like maximum at 445 nm. The absorbance maximum of the ferric protein shifts from 400 to 395 nm on addition of arginine, indicating that substrate binding triggers a low- to high-spin transition of the prosthetic heme iron atom. Binding of BH(4) to the BH(4)-deficient enzyme in the absence of arginine decreases the amount of the low-spin state without detectably increasing the high-spin component (Fig. 3). These results suggest that BH(4) directly modulates the heme environment of eNOS. A different conclusion is suggested for nNOS by the report that nNOS has the same spectrum whether BH(4) is bound or not(47) . However, we find that nNOS expressed in E. coli and purified in the complete absence of BH(4) exhibits a substantial low-spin component that is even more pronounced than that observed with eNOS. (^5)

The specific activity of the BH(4)-supplemented recombinant protein at 37 °C is 95-120 nmol minmg, as measured by both NO and citrulline production, and the K(m) values for arginine and N-OH-arginine are 3 and 14 µM, respectively. These values compare well with those reported in the literature for the protein isolated from mammalian cells, a K(m) of 1-5 µM and a specific activity of 150 nmol minmg(20) . Older reports describe activities in the 1-5 nmol minmg range, and a specific activity of 900 nmol min mg has also been reported (34) , but this latter value is abnormally large and is suspect. No catalytic activity was observed in the absence of added BH(4), in accordance with the well established requirement for BH(4) for catalytic turnover of nitric-oxide synthases. The EC for rescue of the catalytic activity of eNOS by BH(4) is 200 nM, a value consistent with that for the enzyme isolated from mammalian cells(34) . In contrast to the findings with nNOS(23, 41) , high concentrations of arginine in the absence of high concentrations of BH(4) do not result in enzyme inhibition.

As found previously for nNOS, reduction of cytochrome c by the flavoprotein domain of eNOS is stimulated 7-fold by the addition of CaM and Ca, which indicates that electron flow from the flavins to even an exogenous electron acceptor is modulated by CaM. The reduction of cytochrome c was not detectably influenced by the presence or absence of BH(4), essentially the same rates and the same dependence on CaM were obtained with the BH(4)-free and BH(4)-supplemented enzymes.

Studies carried out with iNOS suggest that BH(4) is required for formation of the functional dimer of this protein(48, 49) . In the absence of BH(4), iNOS reportedly does not dimerize. As shown here, BH(4) is not required for dimerization of eNOS (Fig. 5, Table 3). Nondenaturing electrophoretic analysis indicates that the eNOS monomer-dimer equilibrium is similar for the protein purified in the absence and presence of BH(4) despite the fact that the protein purified in the absence of BH(4) is catalytically inactive and is less stable. Indeed, it is of some interest that the dimeric enzyme, whether formed in the presence or absence of BH(4), is stable to electrophoresis in the presence of 0.1% SDS (Fig. 5). Studies of porcine nNOS indicate that the formation of a 2% SDS-resistant dimer is synergistically promoted in that system by BH(4) and arginine(22) . Our results with eNOS differ from those with porcine nNOS in that the extent of dimer formation in the absence of BH(4) is only slightly increased by the addition of BH(4) (Table 3) or L-arginine. The dimerization requirements for dimerization of eNOS therefore appear to differ from those for dimerization of iNOS and nNOS.


FOOTNOTES

*
This work was supported by Grant GM25515, with support for Core facilities used in the study by Liver Center Core Grant 5 P30 DK26743, from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 415-476-2903; Fax: 415-502-4728; ortiz{at}cgl.ucsf.edu.

§
Recipient of a postdoctoral fellowship from the Spanish ``Ministerio de Investigación y Ciencia.''

(^1)
The abbreviations used are: NOS, nitric-oxide synthase; eNOS, endothelial NOS; nNOS, neuronal NOS; iNOS, inducible macrophage NOS; BH(4), (6R)-5,6,7,8-tetrahydrobiopterin; heme, iron protoporphyrin IX regardless of the oxidation or ligation state; CaM, Ca-dependent calmodulin; Hb, methemoglobin; HbO(2), oxyhemoglobin; IPTG, isopropyl beta-D-thiagalactopyranoside; DTT, dithiothreitol; BME, 2-mercaptoethanol; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; NTA, nitrilotriacetic acid; bp, base pair(s); TEMED, N,N,N`,N`-tetramethylethylenediamine.

(^2)
J. De Voss and P. R. Ortiz de Montellano, unpublished results.

(^3)
Determinations of the pterin content by Dr. Shel Milstien (Laboratory of Cell Biology, National Institutes of Health) using previously reported methodology (43) show that neither BH(4) nor any other pterin is bound to the enzyme expressed in E. coli when it is purified in the absence of added BH(4).

(^4)
Analytical ultracentrifugation studies carried out in collaboration with Dr. Germán Rivas (Centro de Investigaciones Biologicas, Madrid) indicate that the protein in solution is in a dimer-monomer equilibrium (unpublished results).

(^5)
N. C. Gerber, I. Rodríguez-Crespo, and P. R. Ortiz de Montellano, unpublished results.


ACKNOWLEDGEMENTS

We thank William C. Sessa (Yale University) for the bovine eNOS clone, Robert Fletterick (University of California, San Francisco), for the pCWori vector, and Shel Milstien (Laboratory of Cell Biology, National Institute of Mental Health) for quantitation of the BH(4) content of eNOS expressed in E. coli.


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