©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Role of the Enzyme Calmodulin-binding Domain in Membrane Association and Phospholipid Inhibition of Endothelial Nitric Oxide Synthase (*)

Richard C. Venema (§) , Hassan S. Sayegh , Jean-Franois Arnal , David G. Harrison

From the (1)Division of Cardiology, Emory University School of Medicine, Atlanta, Georgia 30322

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Endothelial nitric oxide synthase (eNOS) is a calmodulin (CaM)-dependent, membrane-associated, myristoylated enzyme, which has an important role in regulation of vascular tone and platelet aggregation. In this study, wild-type and mutant forms of bovine eNOS were overexpressed in a baculovirus/Sf9 insect cell system and examined for interactions with membrane phospholipids. Purified wild-type eNOS binds to pure anionic phospholipid vesicles but not to neutral phospholipid vesicles, demonstrating that eNOS attachment to lipid bilayers requires electrostatic as well as hydrophobic interactions. Moreover, catalytic activity of the enzyme is potently inhibited by anionic phospholipids, notably phosphatidylserine (PS), but not by neutral phospholipids. eNOS activity is also significantly inhibited upon enzyme binding to biological membranes isolated from cultured cells. Binding of eNOS to PS vesicles prevents subsequent binding of the enzyme to CaM-Sepharose. Interactions of eNOS with PS are not affected by site-specific mutation of the myristic acid acceptor site in the enzyme. Deletional mutation of the eNOS CaM-binding domain, however, results in loss of binding capacity of the enzyme not only for CaM-Sepharose but also for PS vesicles. Furthermore, removal of the CaM-binding domain converts eNOS from a membrane to a cytosolic protein when the enzyme is expressed in Sf9 cells. These data demonstrate that electrostatic interactions between anionic membrane phospholipids and basic residues in the eNOS CaM-binding domain are important for enzyme membrane association. Membrane association can thus function to inhibit eNOS catalytic activity by interfering with the interaction of the enzyme with calmodulin.


INTRODUCTION

Nitric oxide is synthesized in diverse mammalian tissues by three different isoforms of nitric oxide synthase (NOS)()(1) . In blood vessels, endothelial NOS (eNOS) plays a key role in regulation of vascular tone and platelet aggregation. Neuronal NOS (nNOS) is involved in neurotransmission in the brain and peripheral nervous system, while the cytokine-inducible NOS (iNOS) is an important mediator of the tumoricidal and bactericidal actions of macrophages. Each of the three NOS isoforms is similar in structure and function and catalyzes the oxidation of L-arginine to produce NO and L-citrulline via a complex reaction involving NADPH, FAD, FMN, tetrahydrobiopterin, and a p450-type heme moiety(2) . Primary structures of each of the enzymes contain conserved consensus sequences for binding of heme, calmodulin (CaM), FMN, FAD, and NADPH (3). The presence of a putative CaM-binding domain in iNOS is intriguing, since iNOS (in contrast to eNOS and nNOS) is a Ca-independent enzyme. CaM appears to function as a subunit of iNOS, however, remaining tightly bound to the enzyme even in the absence of elevated intracellular Ca(4) .

eNOS is found predominately in membrane (rather than cytosolic) subcellular fractions of endothelial cells(5, 6) . Furthermore, since the distribution of eNOS activity in various membrane fractions closely resembles that of the plasma membrane marker, 5`-nucleotidase, it has been suggested that most, if not all, eNOS in endothelial cells is specifically associated with the plasma membrane(6) . Thus, it is speculated that membrane association of eNOS may have a role in coupling the enzyme's activation to cell surface receptors or to physical stimuli such as hemodynamic fluid shear stress. It has further been suggested (7, 8, 9, 10) that membrane association of eNOS is due to the presence of an N-myristoylation consensus sequence (MGXXXS), a sequence not present in either iNOS or nNOS. Site-directed mutagenesis of the myristic acid acceptor site (Gly-2) converts eNOS from a membrane to a cytosolic protein in transfected COS-7 cells(7, 8) .

Myristoylation may not be the only mechanism responsible for membrane attachment of eNOS. The Gibbs free energy for binding of a myristoylated peptide to a phospholipid vesicle is 8 kcal/mol, equivalent to an apparent K of 100 µM(11) . Myristoylation alone, therefore, is probably not sufficient for stably anchoring a protein to a cellular membrane(12) . Indeed, many myristoylated proteins are cytosolic(13) . Myristoylated proteins that are membrane-associated often interact with membranes, not only through hydrophobic interactions between protein and membrane fatty acyl chains, but also through interactions of positively charged protein residues with negatively charged membrane phospholipids. Furthermore, it appears that a substantial fraction of both neuronal NOS (14, 15) and macrophage-inducible NOS (16) is also localized to membranes. Since these enzymes lack an N-myristoylation site, it is clear that properties of NOS other than myristoylation can mediate enzyme membrane association. Therefore, in this study we have investigated in detail the interactions of the eNOS enzyme with phospholipids present in membranes and have determined the functional consequences of these interactions on enzyme catalytic activity.


EXPERIMENTAL PROCEDURES

Materials

Spodoptera frugiperda Sf9 cells, baculovirus transfer vector pVL1393, and Baculogold viral DNA were purchased from Pharmingen (San Diego, CA). Grace's insect cell culture medium was obtained from Life Technologies, Inc. 2`,5`-ADP-Sepharose and CaM-Sepharose were obtained from Pharmacia Biotech Inc. Phospholipids were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). AG 50W-X8 cation exchange resin, electrophoresis reagents, and Bradford protein dye reagent came from Bio-Rad. L-[C]Arginine, [H]myristic acid, [H]palmitic acid, and ECL detection kit were purchased from Amersham Corp, and ENHANCE was purchased from DuPont NEN. Anti-eNOS antibody was a product of Transduction Laboratories (Lexington, KY), and Sequenase 2.0 was a product of U. S. Biochemical Corp. All other chemicals were obtained from Sigma.

Expression, Purification, and Characterization of eNOS

The cDNA sequence encoding bovine eNOS (17) was subcloned into the EcoRI site of the transfer vector pVL1393. Recombinant transfer vector was then cotransfected with Baculogold viral DNA into Sf9 insect cells, and a high titer recombinant viral stock was obtained and used for subsequent infection of Sf9 cells. Insect cells were infected at a multiplicity of infection of 5 in Grace's medium supplemented with hemin chloride (3 µg/ml). Three days after infection, cells were harvested and lysed on ice for 30 min in 50 mM Tris-HCl (pH 7.0) buffer containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride and 1 µg/ml each antipain, aprotinin, bestatin, leupeptin, soybean trypsin inhibitor, and pepstatin A), 0.1% 2-mercaptoethanol, and 1% Triton X-100 (Buffer A). Lysate was applied to a 2`,5`-ADP-Sepharose affinity column. The column was washed with 20 volumes of Buffer B (Buffer A containing 20% glycerol but without Triton X-100), 20 volumes of Buffer B containing 0.5 M NaCl and 2 mM EGTA, and an additional 20 volumes of Buffer B alone. eNOS was eluted from the column with 10 mM NADPH in Buffer B. Following chromatography on 2`,5`-ADP-Sepharose, eNOS protein was >95% pure as determined by Coomassie staining of purified proteins separated on SDS-polyacrylamide gels. Fatty acyl modifications of eNOS in Sf9 cells were detected by radiolabeling of cells with either [H]myristate or [H]palmitate. 5 h after addition of 1 mCi of either compound, Sf9 cells were harvested and eNOS was purified by 2`,5`-ADP-Sepharose. Purified proteins were separated on SDS-polyacrylamide gels, and gels were washed overnight in either 1 M Tris-HCl or 1 M hydroxylamine (pH 7.0). Radiolabel remaining in the gel was visualized by fluorography with ENHANCE.

Cosedimentation of eNOS with Phospholipid Vesicles and Biological Membranes

Phospholipids in chloroform solution were dried to a thin film, resuspended at a concentration of 5 mg/ml in 20 mM Tris-HCl (pH 7.5), and sonicated with a probe-type sonicator for 5 min. This method of phospholipid vesicle preparation yields a mixture of large unilamellar and multilamellar vesicles(18) . Vesicles were also prepared from biological membranes. Bovine aortic endothelial cells (BAEC) and HeLa cells were grown to confluence, harvested, and homogenized in 50 mM Tris-HCl buffer (pH 7.5) containing protease inhibitors and 0.1% 2-mercaptoethanol. Unbroken cells were pelleted at 1,000 g for 10 min. Membranes in the supernatant were then pelleted by centrifugation at 100,000 g for 30 min. The pellet was resuspended in buffer containing 1 M KCl and recentrifuged at 100,000 g. The final membrane pellet was resuspended in Buffer B and stored at -70 °C. Purified eNOS (1 µg) and either pure phospholipid vesicles (200 µM) or membrane vesicles (equivalent to 50 µg of protein) were mixed in a volume of 500 µl in Buffer B containing 100 mM NaCl. Mixtures were centrifuged at 100,000 g for 30 min at 35 °C. Pellets were resuspended in 500 µl Buffer B and analyzed with supernatants on SDS-polyacrylamide gels followed by Coomassie staining. For binding curves, PS vesicles (200 µM) were mixed with varying amounts of wild-type and (-myr)eNOS (1-36 µg) and binding was measured by Bio-Rad protein assay of pellets after centrifugation.

Phospholipid Vesicle and Membrane Vesicle Effects on eNOS Activity

Purified eNOS (1 µg) was mixed with varying amounts of pure phospholipid vesicles (0-500 µM), and activity was determined by the method of Bredt and Snyder(19) , which determines the rate of formation of L-[C]citrulline from L-[C]arginine (100 µM) in the presence of excess cofactors including Ca (2 mM), CaM (200 units), NADPH (1 mM), FAD (4 µM), FMN (4 µM), and tetrahydrobiopterin (40 µM). Product (L-[C]citrulline) was separated from substrate on Bio-Rad AG 50W-X8 cation exchange columns. Biological membrane effects on eNOS activity were determined by incubation of the enzyme (1 µg) with membranes (50 µg of protein) followed by centrifugation of the mixture at 100,000 g for 30 min and assay of arginine-to-citrulline conversion activity in the resuspended membrane pellet. In some enzyme activity determinations, the products that were detected were the stable degradation products of NO, quantitated by a chemiluminescence NO analyzer.

CaM-Sepharose Chromatography of eNOS

Binding capacities of various forms of eNOS for CaM was determined by CaM-Sepharose affinity chromatography. Purified eNOS (4 µg) was applied to a CaM-Sepharose column (1 ml), and the column was washed extensively with Buffer B containing 2 mM CaCl. Bound eNOS was specifically eluted from the column with Buffer B containing 2 mM EGTA.

PCR-based Mutagenesis of eNOS

Mutations were introduced into the wild-type bovine eNOS cDNA sequence (17) by the splicing-by-overlap-extension technique(20) . In the (-myr) mutant, the glycine 2 codon (GGC) was mutated to an alanine codon (GCC). In the CaM mutant, 60 nucleotides (coding for eNOS residues 493-512) were deleted. Incorporation of the desired mutations was confirmed by dideoxy chain termination sequencing with Sequenase 2.0.

Subcellular Localization of Wild-type and Mutant Forms of eNOS Expressed in Sf9 Insect Cells

Sf9 cells were infected with recombinant baculovirus encoding either wild-type eNOS, (-myr)eNOS, or CaM eNOS. Three days after infection, cells were harvested and homogenized in 50 mM Tris-HCl buffer (pH 7.5) containing protease inhibitors and 0.1% 2-mercaptoethanol. Unbroken cells were pelleted at 1,000 g for 10 min and the first supernatant was then centrifuged at 100,000 g for 30 min to obtain particulate (pellet) and cytosolic (supernatant) fractions. Following resuspension of the pellet to the original volume of the homogenate, equal volumes of supernatant and pellet fractions were analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting with anti-eNOS antibody. Bound antibody was visualized with the ECL detection system and autoradiography. Relative amounts of eNOS in the different fractions was quantitated by densitometry of autoradiograms.


RESULTS AND DISCUSSION

Expression, Purification, and Characterization of Wild-type eNOS

The purpose of this investigation was to characterize the interactions between eNOS and membrane phospholipids and to determine the consequences of these interactions on eNOS activity. Comprehensive study of eNOS-phospholipid interactions, however, requires larger quantities of purified protein than can be routinely obtained from cultured endothelial cells(21) . Therefore, we developed a baculovirus/Sf9 insect cell system for overexpression of the bovine eNOS enzyme. Expression of heme-containing enzymes in a baculovirus system often requires hemin supplementation of the culture medium for expression of protein with high specific activity(22) . This requirement has recently been demonstrated for baculovirus overexpression of nNOS(23, 24) . In the present study, addition of 3 µg/ml hemin chloride to the insect cell culture medium resulted in production of eNOS enzyme having a 3.3-fold higher specific activity than enzyme produced in the absence of hemin supplementation. Expressed protein was purified to >95% homogeneity by affinity chromatography on 2`,5`-ADP-Sepharose(19) . As shown in , most properties of the purified overexpressed enzyme, including subunit molecular mass, k for L-arginine, Ca and cofactor dependence, and inhibition by substrate analog inhibitors were identical to those previously reported for the BAEC enzyme(21) . The specific enzyme activity (146 nmol of L-citrulline produced/mg/min) determined by L-arginine to L-citrulline conversion was 10-fold higher than that reported previously. Similar specific activity was also determined using measurements of NO and NO (quantitated by chemiluminescence after vanadium/HCl reduction). The explanation for the higher enzyme activity in the present study compared to that reported previously remains unclear. It is likely that the ability to purify the baculovirus-expressed enzyme in highly concentrated solution avoids the thermal denaturation and inactivation characteristic of more dilute enzyme(25) . In addition, purification by a single chromatography step allows for assay of purified protein within a few hours after beginning the purification procedure with less time for loss of activity.

eNOS expressed in Sf9 cells was similar to the BAEC enzyme in terms of its fatty acylation by myristate and palmitate(9, 26) . Sf9 cells were incubated with [H]myristic acid, eNOS was purified, and incorporation of radiolabel into the protein was analyzed by fluorography of proteins separated on SDS-polyacrylamide gels. [H]Myristate was incorporated into the protein and was stable to hydroxylamine treatment (data not shown), suggesting that the lipid is attached to the protein via an amide linkage. Sf9 cells were also labeled with [H]palmitate, and acylation was determined by the same protocol used to study myristoylation. Radioactive palmitate was also incorporated into eNOS but was quantitatively removed by hydroxylamine treatment (data not shown), suggesting that lipid attachment occurs through an ester bond.

Binding of eNOS to Phospholipid Vesicles

The interaction of eNOS with specific membrane phospholipids was assessed by cosedimentation analysis. Phospholipid vesicles (each containing identical dioleoyl fatty acyl side chains) were formed from each of the eight most abundant membrane phospholipids(27) . These included the anionic (acidic, negatively charged) lipids phosphatidylserine (PS), phosphatidic acid (PA), cardiolipin (CL), and phosphatidylinositol (PI) and the neutral (zwitterionic) lipids phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingomyelin (SM), and lysophosphatidylcholine (LPC). Vesicles (200 µM) were mixed with purified eNOS (1 µg) and centrifuged at 100,000 g. Supernatant and pellet fractions were analyzed by SDS-polyacrylamide gel electrophoresis and Coomassie staining. eNOS selectively bound to phospholipid vesicles formed from anionic phospholipids (Fig. 1A) but not to vesicles formed from neutral phospholipids (Fig. 1B). Cosedimentation represented specific binding rather than aggregation or precipitation, since all minor contaminating proteins in the preparation remained in the supernatant fraction. Binding was Ca-independent, since no change in sedimentation was observed in the presence of 2 mM EGTA. Selective binding of eNOS to negative lipids (but not to neutral lipids) clearly demonstrates that eNOS attachment to membrane lipids requires electrostatic interactions in addition to hydrophobic interactions. Thus, although myristoylation (7-10) and perhaps also palmitylation (26) may be required for eNOS membrane phospholipid binding, these modifications by themselves are not sufficient to completely account for the association. If fatty acylation alone were sufficient for binding, the enzyme should bind equally well to both neutral and negatively charged phospholipid vesicles.


Figure 1: Cosedimentation analysis of eNOS binding to phospholipid vesicles. Purified wild-type eNOS (1 µg) was mixed with 200 µM each of phospholipid vesicles formed from PS, PA, CL, PI, PC, PE, SM, or LPC. After centrifugation at 100,000 g, supernatant (S) and pellet (P) fractions were analyzed on Coomassie-stained SDS-polyacrylamide gels. Only the relevant portion of the gel is shown. A, anionic phospholipids; B, neutral phospholipids. Similar results were obtained in three separate experiments.



Effect of Phospholipids on eNOS Activity

Phospholipid vesicles were also tested for their effects on eNOS catalytic activity. Anionic phospholipids (PS, PA, CL, and PI) inhibited enzyme activity with IC values of approximately of 6, 50, 45, and 350 µM, respectively (Fig. 2B). Neutral phospholipids (PC, PE, SM, and LPC) were completely without effect (Fig. 2A). Inhibition appears to be correlated with binding, since PS, PA, and CL vesicles demonstrated complete inhibition and complete binding while PI showed partial inhibition and partial binding. PS inhibition also appears to depend on arrangement of the phospholipids into a bilayer, since inhibition was completely reversed by subsequent addition of the detergents Triton X-100 (0.1%) and CHAPS (2 mM). The fact that full activity of PS-inhibited eNOS could be restored by subsequent addition of detergent demonstrates that inhibition is not simply a result of PS-promoted denaturation of the enzyme. If inhibition were due to denaturation, it would not be reversible. In order to confirm that the observed inhibition was not an artifact of baculovirus expression, eNOS was purified from BAEC(21) . eNOS purified from BAEC was also potently inhibited by PS. Furthermore, solubilization of BAEC particulate fractions with Triton X-100 resulted in an approximately 4-fold increase in eNOS activity (specific activity was increased from 0.218 nmol/mg/min to 0.881 nmol/mg/min) confirming the observation previously made by Pollock et al.(21) , who found a >4-fold increase in BAEC particulate eNOS activity following CHAPS solubilization. Detergent solubilization of particulate fractions apparently releases the enzyme from inhibitory interactions with anionic phospholipids. PS inhibition of eNOS probably has physiological significance, since the enzyme functions in endothelial cells in close association with the PS-rich inner leaflet of the plasma membrane. The inner leaflet of the endothelial cell plasma membrane may in fact have a PS content that approaches 50% of total inner leaflet phospholipid. This conclusion follows from the following two observations. First, whereas PS represents only 5% of total endothelial cell phospholipid (28), it is found in mammalian cells almost exclusively in plasma membranes, which comprise 20% of the total cellular membranes. Intracellular membranes, representing the other 80% of cellular membranes, are almost completely devoid of PS(29) . Endothelial plasma membranes, therefore, may have a total PS content of up to 25%. Furthermore, endothelial cells and other vascular cells such as erythrocytes and platelets possess a lipid membrane asymmetry with all of the PS being segregated in the inner leaflet(30) . Aminophospholipid translocases (``flippases'') maintain an antithrombogenic surface on vascular cells by selectively transporting PS from the outer to the inner leaflet, which could thus consist of as much as 50% PS. Membrane association of eNOS, therefore, may hold the enzyme in an (at least partially) inactive state.


Figure 2: Effect of phospholipids on eNOS activity. Purified wild-type eNOS (1 µg) was mixed with 0, 10, 50, 100, 200, and 500 µM phospholipid vesicles formed from PC, PE, SM, LPC, PS, PA, CL, or PI. eNOS activity was then determined by monitoring the rate of conversion of L-arginine to L-citrulline. A, neutral phospholipids; B, anionic phospholipids. Results shown are representative of three experiments.



PS Interactions of eNOS Lacking the N-Myristoylation Site

Lack of binding of eNOS to neutral phospholipid vesicles suggests that fatty acid modifications (such as N-myristoylation) are not sufficient for attachment of the protein to lipid bilayers. Binding to anionic phospholipid vesicles, however, may require both electrostatic interactions and hydrophobic insertion of myristate into the membrane interior. In order to further define the role of eNOS N-myristoylation in binding to (and inhibition by) anionic phospholipids, a mutant of eNOS lacking the N-myristoylation site was expressed in the baculovirus system. PCR-based mutagenesis was used to replace the glycine 2 codon (GGC) with an alanine codon (GCC). The (-myr) mutant protein was expressed in Sf9 cells and purified by 2`,5`-ADP-Sepharose chromatography. Sf9 cells expressing either wild-type or mutant protein were treated with [H]myristate. Radiolabel was incorporated only into wild-type protein. No labeling was detected for the (-myr) mutant (data not shown). Specific activities of purified wild-type and mutant proteins were determined and found to be identical, indicating that myristoylation has no influence on activity of the isolated protein. Both proteins were also inhibited to the same extent by PS vesicles. To determine the apparent affinity of wild-type and (-myr)eNOS for PS vesicles, binding of varying amounts of the protein (1-36 µg) to 200 µM PS vesicles was quantitated and analyzed by the method of Scatchard(31) . Saturable binding curves (Fig. 3, panelsA and C) and apparent dissociation constants (K values) determined by Scatchard analysis (Fig. 3, panelsB and D) were very similar for the two proteins (Fig. 3). K values for binding of wild-type and (-myr)eNOS were 1.0 and 1.5 µM, respectively, indicating that myristoylation is not required for eNOS binding to pure PS vesicles. Binding of eNOS to biological membrane vesicles prepared from BAEC, CHO cells, and COS-7 cells, however, has been shown previously to be dependent on enzyme myristate modification(10) . The present study suggests that the requirement for a hydrophobic component in the phospholipid interaction becomes less important when the percent anionic lipid in the phospholipid vesicle is increased to 100% of the total.


Figure 3: Binding of wild-type eNOS and (-myr)eNOS to PS vesicles. PanelsA and C, wild-type and (-myr)eNOS was incubated with PS vesicles and binding was determined as described under ``Experimental Procedures.'' PanelsB and D, binding data plotted according to Scatchard where protein bound is expressed as pmol of eNOS bound/nmol of phosphatidylserine. Results are representative of two experiments.



Effects of PS Binding on eNOS-CaM Interactions

PS binding of eNOS appears to result in enzyme inhibition. Because eNOS is a CaM-dependent enzyme, we have considered the possibility that eNOS may be regulated similarly to several other proteins that are regulated by both CaM and PS. These include the brain proteins, neuromodulin(32) , neurogranin(33) , calcineurin(34) , and NAP-22(35) , as well as intestinal brush border myosin I (36) and the ubiquitously expressed MARCKS (myristoylated alanine-rich protein kinase C substrate) protein(37) . All of these proteins are similar in that they possess a CaM-binding domain containing clusters of basic residues, which also bind anionic phospholipids, notably PS, in the plasma membrane. CaM-binding domains in proteins are typically 20 residues in length and conform to the Baa-helical (basic amphiphilic -helix) model with hydrophobic residues on one side of the helix and positively charged amino acids on the opposing face(38) . Bovine eNOS contains such a sequence at positions 493-512 of the polypeptide chain(17) . We have investigated the hypothesis that binding of PS to this domain in eNOS interferes with subsequent CaM binding. eNOS (4 µg) was preincubated with 500 µM of either PS vesicles or PC vesicles (as a control) for 10 min at 37 °C and then subjected to affinity chromatography on CaM-Sepharose. eNOS preincubated with PC vesicles bound to CaM-Sepharose in the presence of Ca and was specifically eluted with EGTA (Fig. 4A). In contrast, eNOS preincubated with PS vesicles did not bind to CaM-Sepharose and came through the column in the flow-through fractions (Fig. 4B). Loss of eNOS binding to CaM-Sepharose in these experiments appears to be due to binding of PS to the eNOS CaM-binding domain, rather than due to an effect of PS to significantly alter eNOS three-dimensional structure. This interpretation is supported by experiments in which PS preincubation was found to have no effect on subsequent binding of eNOS to 2`,5`-ADP-Sepharose. Binding of eNOS to 2`,5`-ADP (an NADPH analog) would probably not occur with a denatured eNOS enzyme. PS bound to eNOS did not appear to be displaced by CaM under these conditions. A similar inability of CaM to displace previously bound PS was also observed in the cosedimentation experiments. Three-fold molar excess concentrations of CaM (relative to eNOS) when added after preincubation with PS (4200-fold molar excess relative to eNOS) did not inhibit cosedimentation of eNOS with PS vesicles, nor was any CaM detected in the pellet fraction following sedimentation (data not shown). However, when eNOS was first incubated with a 3-fold molar excess of Ca and CaM (10 min at 37 °C), subsequent cosedimentation with PS vesicles was reduced by approximately 70%. PS and CaM thus appear to compete for binding to the same site in the wild-type eNOS enzyme. Taken together, these data suggest that PS may inhibit eNOS activity by binding to the CaM-binding domain of the protein, thus preventing the calmodulin-protein interaction that is required for activation of the enzyme.


Figure 4: CaM-Sepharose chromatography of eNOS following preincubation with PC or PS vesicles. Purified wild-type eNOS (4 µg) was mixed with 500 µM PC or PS vesicles and preincubated for 10 min at 37 °C. Mixtures were applied to CaM-Sepharose columns and four fractions (1 ml each) were eluted with buffer containing 2 mM Ca (flow-through). Four additional fractions (1 ml each) were then eluted with buffer containing 2 mM EGTA. Fractions were vacuum-evaporated down to 50 µl and proteins were analyzed on SDS-polyacrylamide gels with Coomassie staining. A, eNOS preincubated with PC vesicles; B, eNOS preincubated with PS vesicles. Equivalent results were obtained in three experiments.



PS Interactions of eNOS Lacking the Putative CaM-binding Domain

Loss of eNOS interaction with CaM-Sepharose following preincubation with PS suggests that PS and CaM bind to the same site on the enzyme. To further investigate this hypothesis, we created a deletion mutant of eNOS by PCR-based mutagenesis in which the putative CaM-binding domain (TRKKTFKEVANAVKISASLM, residues 493-512) was removed. The mutant protein (designated CaM eNOS) was expressed in the baculovirus system and purified by 2`,5`-ADP-Sepharose affinity chromatography. CaM eNOS comigrated with wild-type eNOS on SDS-polyacrylamide gels and reacted with anti-eNOS antibody on Western blots. The CaM mutant, however, had no capacity to catalyze the conversion of L-arginine to L-citrulline. Moreover, deletional mutation resulted in a complete loss of CaM-binding capacity of the enzyme. Wild-type and CaM eNOS were subjected to affinity chromatography on CaM-Sepharose. Wild-type eNOS bound to CaM-Sepharose in the presence of Ca and was specifically eluted with EGTA (Fig. 5A). CaM eNOS, on the other hand, did not bind to CaM-Sepharose and came through the column in the flow-through fractions (Fig. 5B). Residues 493-512 of eNOS, previously identified as a CaM-binding domain solely on the basis of sequence analysis, thus appear to represent a true CaM-binding site. Furthermore, this domain also functions as a PS-binding site. Cosedimentation analysis of wild-type and CaM eNOS showed that only the wild-type enzyme (and not the mutant) cosedimented with PS vesicles (Fig. 6). Loss of binding of CaM eNOS to PS vesicles (similar to the loss of PS binding that occurred with wild-type eNOS after preincubation with Ca/CaM) was probably not due to conformational changes in the protein, which could result from a deletional mutation. If major changes in protein folding had occurred, the enzyme would probably have lost its binding capacity for 2`,5`-ADP-Sepharose as well.


Figure 5: CaM-Sepharose chromatography of wild-type eNOS and CaM eNOS. Purified wild-type eNOS or CaM eNOS (4 µg each) were applied to CaM-Sepharose columns and four fractions (1 ml each) were eluted with buffer containing 2 mM Ca (flow-through). Four additional fractions (1 ml each) were then eluted with buffer containing 2 mM EGTA. Fractions were vacuum-evaporated down to 50 µl and proteins were analyzed on SDS-polyacrylamide gels with Coomassie staining. A, wild-type eNOS; B, CaM eNOS. Results are representative of three experiments.




Figure 6: Cosedimentation analysis of binding of wild-type eNOS and CaM eNOS to PS vesicles. Purified wild-type eNOS or CaM eNOS (1 µg each) were mixed with 200 µM PS vesicles. After centrifugation at 100,000 g, supernatant (S) and pellet (P) fractions were analyzed on Coomassie-stained SDS-polyacrylamide gels. Only the relevant portions of the gel is shown. Similar results were obtained in three experiments.



Interactions of eNOS with Biological Membranes

Association of eNOS with anionic phospholipids appears to be mediated, at least in part, through electrostatic interactions between negative charges in phospholipid polar head groups and positively charged amino acids in the eNOS CaM-binding domain. Among the residues in bovine eNOS that may contribute to this interaction are Arg-494, Lys-495, Lys-496, Lys-499, and Lys-506. To determine whether the eNOS CaM-binding domain also has a role in enzyme membrane association in intact cells, wild-type eNOS, (-myr)eNOS, and CaM eNOS were expressed in Sf9 cells and the subcellular localization of expressed proteins was determined. Cells were infected under identical conditions used to produce enzyme for purification. Three days after infection, cells were harvested and cytosolic and membrane subcellular fractions were prepared. Fractions were analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting in order to quantitate the relative amounts of expressed protein in the two subcellular fractions. As shown in Fig. 7, wild-type eNOS was found almost entirely in membrane fractions (91 ± 3%, mean ± S.D., n = 3) rather than cytosolic subcellular fractions (9 ± 3%) of Sf9 cells. In contrast, (-myr)eNOS was found almost entirely in cytosolic fractions (88 ± 4%) rather than particulate fractions (12 ± 4%). Identical results were also obtained when subcellular localization was quantitated by assay of enzyme activity in soluble and detergent-solubilized particulate fractions (data not shown). Similar subcellular distribution of these two forms of eNOS has been demonstrated previously in transfected COS-7 cells(7, 8) . The present findings strongly suggest that the eNOS enzyme expressed in Sf9 cells is fully myristoylated. If only a subfraction of the enzyme were myristoylated, wild-type eNOS would not be restricted to the particulate fraction. Furthermore, myristoylation by itself is probably not sufficient for eNOS membrane association. The CaM-binding domain of the enzyme appears to also be required for stable membrane anchoring. Thus, CaM eNOS (which presumably is fully myristoylated) was found predominately in cytosolic (65 ± 9%) rather than particulate (35 ± 9%) subcellular fractions of Sf9 cells expressing the mutant enzyme. Loss of membrane association of CaM eNOS in Sf9 cells is very likely due to loss of binding affinity of the deletional mutant for anionic membrane phospholipids. An alternative explanation of these results is that insect cells may lack a docking protein, which exists in mammalian endothelial cells, that interacts with the eNOS CaM-binding domain. This is very unlikely, however, since it has been recently demonstrated by Busconi and Michel (10) that eNOS binding to mammalian cell membranes does not involve a docking or receptor protein. Treatment of biological membranes prepared from BAEC, CHO cells, and COS-7 cells with either heat denaturation or trypsinization thus has no effect on subsequent ability of treated membranes to bind the wild-type eNOS enzyme(10) .


Figure 7: Subcellular distribution of wild-type eNOS, (-myr)eNOS, and CaM eNOS expressed in Sf9 cells. Sf9 cells were infected with recombinant baculovirus encoding either wild-type eNOS, (-myr)eNOS, or CaM eNOS. Cytosol and membrane fractions from infected cells were prepared as described under ``Experimental Procedures.'' Relative amounts of eNOS protein in fractions was quantitated by densitometry of Western blots. Results shown are mean ± S.D. from three separate infections.



The functional effects of eNOS binding to biological membranes was also determined. Wild-type enzyme was incubated with membrane vesicles (heat-denatured to eliminate endogenous eNOS activity) prepared from BAEC and HeLa cells. Membranes were sedimented at 100,000 g, and activity of the cosedimented (bound) enzyme was determined. eNOS binding to BAEC and HeLa cell membranes reduced catalytic activity by 71 ± 9% and 63 ± 11% (mean ± S.D., n = 3), respectively. Membrane association thus serves to significantly (although not completely) inhibit eNOS activity. Partial (rather than complete) inhibition is not surprising, since it is known that BAEC particulate fractions do have significant enzyme activity, even in the absence of detergent solubilization. Neutral lipids in membranes may thus diminish the inhibitory effects of anionic lipids, as has been observed previously for other phospholipid-regulated enzymes. The inhibitory effect of PI on actin severing activity of adseverin, for example, is diminished by PC and PE(39) . Similarly, PS activation of protein kinase C (perhaps the best known of the phospholipid-regulated enzymes) is markedly diminished by PC and SM (40). To examine the role of neutral phospholipids in modulating the effects of anionic phospholipids on eNOS activity, mixed phospholipid vesicles were formed from PC and PS in which the PS content was varied from 0% to 100%. As shown in Fig. 8, inhibition of eNOS activity by these vesicles depended on the percent anionic lipid in the vesicle, with half-maximal inhibition occurring at approximately 40% anionic phospholipid.


Figure 8: Effect of PC:PS mixed phospholipid vesicles on eNOS activity. Purified wild-type eNOS (1 µg) was mixed with phospholipid vesicles (200 µM) formed from mixtures of PC and PS. eNOS activity was determined by assay of the rate of conversion of L-arginine to L-citrulline. Results are representative of three experiments.



Potential Role of Phospholipids in Regulation of eNOS in Endothelial Cells

eNOS-phospholipid interactions may help to explain a number of previous observations. For example, bradykinin activation of eNOS in BAEC is associated with translocation of the enzyme from membrane to cytosol(41) . It is conceivable that translocation contributes to eNOS activation by releasing the enzyme from inhibition by anionic membrane phospholipids. Activation of eNOS also occurs in endothelial cells in response to acute increases in fluid shear stress(42) . The mechanism of physiochemical signal transduction in this circumstance is not known, but could involve shear-induced eNOS subcellular translocation or shear-induced changes in membrane fluidity (43) with resultant effects on eNOS-phospholipid interactions. High affinity of eNOS for PS could also function to specifically target the enzyme to the plasma membrane (rather than to intracellular membranes), since PS is found almost exclusively in cells in the plasma membrane(29) .

  
Table: Properties of purified bovine eNOS overexpressed in a baculovirus system



FOOTNOTES

*
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 all correspondence should be addressed: Division of Cardiology, P. O. Drawer LL, Emory University School of Medicine, Atlanta, GA 30322. Tel.: 404-727-3713; Fax: 404-727-3330.

The abbreviations used are: NOS, nitric oxide synthase; eNOS, endothelial nitric oxide synthase; nNOS, neuronal nitric oxide synthase; iNOS, inducible nitric oxide synthase; (-myr)eNOS, non-myristoylated eNOS; CaM, calmodulin; BAEC, bovine aortic endothelial cell(s); PS, phosphatidylserine; PA, phosphatidic acid; CL, cardiolipin; PI, phosphatidylinositol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; SM, sphingomyelin; LPC, lysophosphatidylcholine; PCR, polymerase chain reaction; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.


REFERENCES
  1. Nathan, C., and Xie, Q.(1994) Cell78, 915-918 [Medline] [Order article via Infotrieve]
  2. Marletta, M. A.(1993) J. Biol. Chem.268, 12231-12234 [Free Full Text]
  3. Sessa, W. C.(1994) J. Vasc. Res.31, 131-143 [Medline] [Order article via Infotrieve]
  4. 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]
  5. Förstermann, U., Pollock, J. S., Schmidt, H. H. H. W., Heller, M., and Murad, F.(1991) Proc. Natl. Acad. Sci. U. S. A.88, 1788-1792 [Abstract]
  6. Hecker, M., Mülsch, A., Bassenge, E., Förstermann, U., and Busse, R.(1994) Biochem. J.299, 247-252 [Medline] [Order article via Infotrieve]
  7. Busconi, L., and Michel, T.(1993) J. Biol. Chem.268, 8410-8413 [Abstract/Free Full Text]
  8. Sessa, W. C., Barber, C. M., and Lynch, K. R.(1993) Circ. Res.72, 921-924 [Abstract]
  9. Liu, J., and Sessa, W. C.(1994) J. Biol. Chem.269, 11691-11694 [Abstract/Free Full Text]
  10. Busconi, L., and Michel, T.(1994) J. Biol. Chem.269, 25016-25020 [Abstract/Free Full Text]
  11. Peitzsch, R. M., and McLaughlin, S.(1993) Biochemistry32, 10436-10443 [Medline] [Order article via Infotrieve]
  12. Resh, M. D.(1994) Cell76, 411-413 [Medline] [Order article via Infotrieve]
  13. Towler, D. A., Gordon, J. I., Adams, S. P., and Glaser, L.(1988) Annu. Rev. Biochem.57, 69-99 [CrossRef][Medline] [Order article via Infotrieve]
  14. Hiki, K., Hattori, R., Kawai, C., and Yui, Y.(1992) J. Biochem. (Tokyo) 111, 556-558 [Abstract]
  15. Hecker, M., Mülsch, A., and Busse, R.(1994) J. Neurochem.62, 1524-1529 [Medline] [Order article via Infotrieve]
  16. Vodovotz, Y., Russell, D., Xie, Q. W., Bogdan, C., and Nathan, C. (1995) J. Immunol.154, 2914-2925 [Abstract/Free Full Text]
  17. 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]
  18. Szoka, F., Jr., and Papahadjopoulos, D.(1980) Annu. Rev. Biophys. Bioeng.9, 1-29 [Medline] [Order article via Infotrieve]
  19. Bredt, D. S., and Snyder, S. H.(1990) Proc. Natl. Acad. Sci. U. S. A.87, 682-685 [Abstract]
  20. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Gene (Amst.) 77, 51-59 [CrossRef][Medline] [Order article via Infotrieve]
  21. Pollock, J. S., Förstermann, U., Mitchell, J. A., Warner, T. D., Schmidt, H. H. H. W., Nakane, M., and Murad, F.(1991) Proc. Natl. Acad. Sci U. S. A.88, 10480-10484 [Abstract]
  22. Asseffa, A., Smith, S. J., Nagata, K., Gillette, J., Gelboin, H. V., and Gonzalez, F. J.(1989) Arch. Biochem. Biophys.274, 481-490 [Medline] [Order article via Infotrieve]
  23. Richards, M. K., and Marletta, M. A.(1994) Biochemistry33, 14723-14732 [Medline] [Order article via Infotrieve]
  24. Harteneck, C., Klatt, P., Schmidt, K., and Mayer, B.(1994) Biochem. J.304, 683-686 [Medline] [Order article via Infotrieve]
  25. Stuehr, D. J., and Griffith, O. W.(1992) Adv. Enzymol. Relat. Areas Mol. Biol.65, 287-346 [Medline] [Order article via Infotrieve]
  26. Robinson, L. J., Busconi, L., and Michel, T.(1995) J. Biol. Chem.270, 995-998 [Abstract/Free Full Text]
  27. Esko, J. D., and Raetz, C. R. H.(1983) in The Enzymes (Boyer, P. D., ed) 3rd Ed., pp. 207-253, Academic Press, New York
  28. Wey, H. E., Jakubowski, J. A., and Deykin, D.(1986) Biochim. Biophys. Acta878, 380-386 [Medline] [Order article via Infotrieve]
  29. Allan, D., and Kallen, K. J.(1993) Prog. Lipid Res.32, 195-219 [CrossRef][Medline] [Order article via Infotrieve]
  30. Devaux, P. F.(1991) Biochemistry30, 1163-1173 [Medline] [Order article via Infotrieve]
  31. Scatchard, G.(1949) Ann. N. Y. Acad. Sci.51, 660-672
  32. Houbre, D., Duportail, G., Deloulme, J. C., and Baudier, J.(1991) J. Biol. Chem.266, 7121-7131 [Abstract/Free Full Text]
  33. Baudier, J., Deloulme, J. C., Van Dorsselaer, A., Black, D., and Matthes, H. W. D.(1991) J. Biol. Chem.266, 229-237 [Abstract/Free Full Text]
  34. Politino, M., and King, M. M.(1987) J. Biol. Chem.262, 10109-10113 [Abstract/Free Full Text]
  35. Maekawa, S., Maekawa, M., Hattori, S., and Nakamura, S.(1993) J. Biol. Chem.268, 13703-13709 [Abstract/Free Full Text]
  36. Swanljung-Collins, H., and Collins, J. H.(1992) J. Biol. Chem.267, 3445-3454 [Abstract/Free Full Text]
  37. Kim, J., Shishido, T., Jiang, X., Aderem, A., and McLaughlin, S.(1994) J. Biol. Chem.269, 28214-28219 [Abstract/Free Full Text]
  38. O'Neil, K. T., and DeGrado, W. F.(1990) Trends Biochem. Sci.15, 59-64 [CrossRef][Medline] [Order article via Infotrieve]
  39. Maekawa, S., and Sakai, H.(1990) J. Biol. Chem.265, 10940-10942 [Abstract/Free Full Text]
  40. Kaibuchi, K., Takai, Y., and Nishizuka, Y.(1981) J. Biol. Chem.256, 7146-7149 [Abstract/Free Full Text]
  41. Michel, T., Li, G. K., and Busconi, L.(1993) Proc. Natl. Acad. Sci. U. S. A.90, 6252-6256 [Abstract]
  42. Kuchan, M. J., and Frangos, J. A.(1994) Am. J. Physiol.266, C628-C636
  43. Berthiaume, F., and Frangos, J. A.(1994) Biochim. Biophys. Acta1191, 209-218 [Medline] [Order article via Infotrieve]

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