Structural Elements Contribute to the Calcium/Calmodulin Dependence on Enzyme Activation in Human Endothelial Nitric-oxide Synthase
2003; Elsevier BV; Volume: 278; Issue: 52 Linguagem: Inglês
10.1074/jbc.m305469200
ISSN1083-351X
Autores Tópico(s)Photosynthetic Processes and Mechanisms
ResumoTwo regions, located at residues 594–606/614–645 and residues 1165–1178, are present in the reductase domain of human endothelial nitric-oxide synthase (eNOS) but absent in its counterpart, inducible nitric-oxide synthase (iNOS). We previously demonstrated that removing residues 594–606/614–645 resulted in an enzyme (Δ45) containing an intrinsic calmodulin (CaM) purified from an Sf9/baculovirus expression system (Chen, P.-F., and Wu, K.K. (2000) J. Biol. Chem. 275, 13155–13163). Here we have further elucidated the differential requirement of Ca2+/CaM for enzyme activation between eNOS and iNOS by either deletion of residues 1165–1178 (Δ14) or combined deletions of residues 594–606/614–645 and 1165–1178 (Δ45/ Δ14) from eNOS to mimic iNOS. We measured the catalytic rates using purified proteins completely free of CaM. Steady-state analysis indicated that the Δ45 supported NO synthesis in the absence of CaM at 60% of the rate in its presence, consistent with our prior result that CaM-bound Δ45 retained 60% of its activity in the presence of 10 mm EGTA. Mutant Δ14 displayed a 1.5-fold reduction of EC50 for Ca2+/CaM-dependence in l-citrulline formation, and a 2–4-fold increase in the rates of NO synthesis, NADPH oxidation, and cytochrome c reduction relative to the wild type. The basal rates of double mutant Δ45/Δ14 in NO production, NADPH oxidation, and cytochrome c reduction were 3-fold greater than those of CaM-stimulated wild-type eNOS. Interestingly, all three activities of Δ45/ Δ14 were suppressed rather than enhanced by Ca2+/CaM, indicating a complete Ca2+/CaM independence for those reactions. The results suggest that the Ca2+/CaM-dependent catalytic activity of eNOS appears to be conferred mainly by these two structural elements, and the interdomain electron transfer from reductase to oxygenase domain does not require Ca2+/CaM when eNOS lacks these two segments. Two regions, located at residues 594–606/614–645 and residues 1165–1178, are present in the reductase domain of human endothelial nitric-oxide synthase (eNOS) but absent in its counterpart, inducible nitric-oxide synthase (iNOS). We previously demonstrated that removing residues 594–606/614–645 resulted in an enzyme (Δ45) containing an intrinsic calmodulin (CaM) purified from an Sf9/baculovirus expression system (Chen, P.-F., and Wu, K.K. (2000) J. Biol. Chem. 275, 13155–13163). Here we have further elucidated the differential requirement of Ca2+/CaM for enzyme activation between eNOS and iNOS by either deletion of residues 1165–1178 (Δ14) or combined deletions of residues 594–606/614–645 and 1165–1178 (Δ45/ Δ14) from eNOS to mimic iNOS. We measured the catalytic rates using purified proteins completely free of CaM. Steady-state analysis indicated that the Δ45 supported NO synthesis in the absence of CaM at 60% of the rate in its presence, consistent with our prior result that CaM-bound Δ45 retained 60% of its activity in the presence of 10 mm EGTA. Mutant Δ14 displayed a 1.5-fold reduction of EC50 for Ca2+/CaM-dependence in l-citrulline formation, and a 2–4-fold increase in the rates of NO synthesis, NADPH oxidation, and cytochrome c reduction relative to the wild type. The basal rates of double mutant Δ45/Δ14 in NO production, NADPH oxidation, and cytochrome c reduction were 3-fold greater than those of CaM-stimulated wild-type eNOS. Interestingly, all three activities of Δ45/ Δ14 were suppressed rather than enhanced by Ca2+/CaM, indicating a complete Ca2+/CaM independence for those reactions. The results suggest that the Ca2+/CaM-dependent catalytic activity of eNOS appears to be conferred mainly by these two structural elements, and the interdomain electron transfer from reductase to oxygenase domain does not require Ca2+/CaM when eNOS lacks these two segments. Nitric-oxide synthase (NOS) 1The abbreviations used are: NOSnitric-oxide synthaseH4B(6R)-5,6,7,8-tetrahydro-l-biopterinCaMcalmodulineNOSendothelial NOSWTeNOSwild-type eNOSiNOSinducible NOSnNOSneuronal NOScNOSconstitutively expressed NOSCPRNADPH-cytochrome P450 reductaseMOPS4-morpholinepropanesulfonic acid. catalyzes the synthesis of NO through a series of electron transfers from the C-terminal reductase domain, which harbors the FAD and FMN cofactors and the NADPH binding site, to the N-terminal oxygenase domain, which contains the heme catalytic center, the H4B cofactor, and the arginine binding sites (1Förstermann U. Schmidt H.H.W. H. H. W. Pollock J. S. Sheng H. Mitchell J. A. Warner T. D. Nakane M. Murad F. Biochem. Pharmacol. 1991; 42: 1849-1857Crossref PubMed Scopus (814) Google Scholar, 2Nathan C. FASEB J. 1992; 6: 3051-3064Crossref PubMed Scopus (4158) Google Scholar, 3McMillan K. Bredt D.S. Hirsch D.J. Snyder S.H. Clark J.E. Masters B.S. Proc. Natl. Acad. Sci. U. S. 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The NOS family comprises three isoforms that share domain structures, sequence homology, and catalytic properties (10Pollock J.S. Förstermann U. Mitchell J.A. Warner T.D. Harald H.H. Schmidt H.H.H.W. Nakane M. Murad F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10480-10484Crossref PubMed Scopus (899) Google Scholar, 11Bredt D.S. Hwang P.M. Glatt C.E. Lowenstein C. Reed R.R. Snyder S.H. Nature. 1991; 351: 714-718Crossref PubMed Scopus (2171) Google Scholar, 12Xie Q.W. Cho H.J. Calaycay J. Mumford R.A. Swiderek K.M. Lee T.D. Ding A. Troso T. Nathan C. Science. 1992; 256: 225-228Crossref PubMed Scopus (1740) Google Scholar). Despite these similarities, there are considerable differences among the NOS isoforms with respect to their cellular expressions, Ca2+-dependent CaM activation, and rate of electron transfer. Two isoforms, i.e. nNOS and eNOS, are constitutively expressed NOSs (cNOSs), but their expressed enzymes are latent until CaM binding is elicited by an elevated intracellular calcium level (13Bredt D.S. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 682-685Crossref PubMed Scopus (3125) Google Scholar). In contrast, iNOS is absent or expressed in low abundance at the resting state, and its expression is induced by cytokines and endotoxins. The expressed iNOS is catalytically active, thought to be due to its high affinity for CaM binding even at a basal level of intracellular calcium (14Cho H.J. Xie Q.W. Calaycay J. Mumford R.A. Swiderek K.M. Lee T.D. Nathan C. J. Exp. Med. 1992; 176: 599-604Crossref PubMed Scopus (562) Google Scholar). Among the three isoforms, eNOS has lower electron transfer rate and catalytic activity than nNOS and iNOS (15Nishida C.R. Ortiz de Montellano P.R. J. Biol. Chem. 1998; 273: 5566-5571Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), suggesting other control mechanisms for eNOS catalysis. nitric-oxide synthase (6R)-5,6,7,8-tetrahydro-l-biopterin calmodulin endothelial NOS wild-type eNOS inducible NOS neuronal NOS constitutively expressed NOS NADPH-cytochrome P450 reductase 4-morpholinepropanesulfonic acid. The x-ray crystallographic analysis of the oxygenase domains of NOS isoforms has revealed a striking degree of conservation at the active-site structure (16Crane B.R. Arvai A.S. Ghosh D.K. Wu C. Getzoff E.D. Stuehr D.J. Tainer J.A. Science. 1998; 279: 2121-2126Crossref PubMed Scopus (626) Google Scholar, 17Raman C.S. Li H. Martasek P. Kral V. Masters B.S. Poulos T.L. Cell. 1998; 95: 939-950Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar, 18Fischmann T.O. Hruza A. Niu X.D. Fossetta J.D. Lunn C.A. Dolphin E. Prongay A.J. Reichert P. Lundell D.J. Narula S.K. Weber P.C. Nat. Struct. Biol. 1999; 6: 233-242Crossref PubMed Scopus (409) Google Scholar). The results from studies of chimeric enzymes in which the oxygenase domain was swapped to the reductase domain of another isoform suggested that divergence in the reductase domain rather than in the oxygenase domain accounted for the differences in Ca2+ sensitivity and the rate of electron transfer between the cNOSs and the iNOS (15Nishida C.R. Ortiz de Montellano P.R. J. Biol. Chem. 1998; 273: 5566-5571Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). An ∼50-amino acid fragment (Fig. 1) present in the FMN-subdomain of human eNOS (residues 594–645) and nNOS (residues 834–882), but absent in the corresponding part of iNOS, was proposed as an autoinhibitory element that impedes the electron transfer of cNOSs in the absence of CaM (19Ruan J. Xie Q.-w. Hutchinson N. Cho H. Wolfe G.C. Nathan C. J. Biol. Chem. 1996; 271: 22679-22686Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 20Salerno J.C. Harris D.E. Irizarry K. Patel B. Morales A.J. Smith S.M. Martasek P. Roman L.J. Masters B.S. Jones C.L. Weissman B.A. Lane P. Liu Q. Gross S.S. J. Biol. Chem. 1997; 272: 29769-29777Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). Deletion of this region rendered the mutant enzymes less dependent on Ca2+ concentration, with a faster rate of electron flow (21Nishida C.R. Ortiz de Montellano P.R. J. Biol. Chem. 1999; 274: 14692-14698Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 22Daff S. Sagami S. Shimizu T. J. Biol. Chem. 1999; 274: 30589-30595Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 23Montgomery H.J. Romanov V. Guillemette J.G. J. Biol. Chem. 2000; 275: 5052-5058Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). We previously characterized an eNOS mutant (24Chen P.-F. Wu K.K. J. Biol. Chem. 2000; 275: 13155-13163Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) in which residues 594–606 and 614–645 were deleted (Δ45eNOS) with preservation of residues 607–613 because it was conserved between the sequences of cNOSs and iNOS (Fig. 1). The Δ45eNOS contained an endogenous CaM bound to the protein isolated from an Sf9/baculoviral expression system. This mutant was completely CaM-independent as well as significantly Ca2+-independent in l-citrulline formation and exhibited a higher rate of cytochrome c reduction in a CaM-independent manner (24Chen P.-F. Wu K.K. J. Biol. Chem. 2000; 275: 13155-13163Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The results confirm that residues at 594–606 and 614–645 in eNOS control calcium sensitivity for CaM-dependent enzyme activation. However, because this mutant still requires Ca2+ to achieve a maximal catalytic activity, the calcium requirement for electron transfer and NO production may be controlled by other intramolecular mechanisms. Recent studies have implicated the C-terminal region of all NOS isoforms as an additional regulatory element in modulating electron transfer. The C terminus of cNOS contains a conserved serine (Ser1178 in human eNOS and Ser1417 in nNOS) with a kinase-dependent phosphorylation motif (RSRXX(S/T)) that has been noted in eNOS to be phosphorylated in response to a number of stimuli (25Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2232) Google Scholar, 26Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3047) Google Scholar, 27Dimmeler S. Dernbach E. Zeiher A.M. 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Phosphorylation has been shown to trigger eNOS activation at a lower Ca2+ concentration and to increase the rate of NO production by 2-fold in vivo (25Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2232) Google Scholar, 26Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3047) Google Scholar). Mutation of this serine to Asp in eNOS (33McCabe T.J. Fulton D. Roman L.J. Sessa W.C. J. Biol. Chem. 2000; 275: 6123-6128Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar) and nNOS (34Adak S. Santolini J. Tikunova S. Wang Q. Johnson J.D. Stuehr D.J. J. Biol. Chem. 2001; 276: 1244-1252Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), which mimics phosphorylation by introducing a negative charge, also causes a faster electron flow through enzymes. By sequence comparison with cytochrome P450 reductase (CPR), Roman et al. (35Roman L.J. Miller R.T. de La Garza M.A. Kim J.J. Masters B.S. J. Biol. Chem. 2000; 275: 21914-21919Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 36Roman L.J. Martasek P. Miller R.T. Harris D.E. de La Garza M.A. Shea T.M. Masters B.S. J. Biol. Chem. 2000; 275: 29225-29232Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) proposed that the 21–42-amino acid C-terminal extension tail present in all NOSs but absent in CPR was involved in modulating electron transfer. Their experimental data demonstrated that deletion of the entire C-tail from rat nNOS (33 residues) or bovine eNOS (42 residues) greatly increased electron transfer into and between flavins in the absence of CaM. Paradoxically, their cytochrome c reductase activities were suppressed rather than enhanced by exogenously added CaM, and their CaM-induced NO synthesis activities were only 50% that of CaM-bound wild-type enzymes (36Roman L.J. Martasek P. Miller R.T. Harris D.E. de La Garza M.A. Shea T.M. Masters B.S. J. Biol. Chem. 2000; 275: 29225-29232Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). In contrast, Lane and Gross (37Lane P. Gross S.S. J. Biol. Chem. 2002; 277: 19087-19094Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) did not delete the entire C-terminal tail but instead partially removed the Ser1179 and the subsequent 26 C-terminal amino acids from bovine eNOS (Δ27). This mutant exhibited a 5-fold reduction in EC50 for calcium and a 2–4-fold increase in maximal catalytic activities. Both reductase and oxygenase activities of Δ27 were enhanced 3-fold by exogenously added CaM (37Lane P. Gross S.S. J. Biol. Chem. 2002; 277: 19087-19094Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). These findings underscore a complex control process of the C-terminal tail, especially with respect to the influence of CaM on electron transfer. Sequence alignment reveals that a segment at the proximal C-terminal tail is conserved in the sequences of human eNOS (residues 1165–1178) and nNOS (residues 1404–1417) but absent in iNOS. Besides the autoinhibitory loop of residues 594–606/614–645, this segment indicates another, more obvious dissimilarity between cNOSs and iNOS. We postulated that this conserved region in conjunction with the autoinhibitory loop might confer the dramatic differences in Ca+2 sensitivity and the rate of electron flux between eNOS and iNOS. To test this hypothesis, we assessed the changes in electron transfer and NO synthesis caused by deletion of eNOS sequence (residues 1165–1178, Δ14eNOS). We were particularly interested in learning whether combined deletions of Δ594–606/Δ614–645 and Δ1165–1178 would yield an eNOS mutant protein resembling iNOS in terms of the CaM requirement for reductase and oxygenase activities. Wild-type and mutant eNOSs expressed in an Sf9/baculovirus system were purified by adding an adequate amount of chelators to remove calcium, and the reductase and NO synthesis activities were measured. All of the purified proteins were free of the endogenous CaM. The results showed that a combined Δ45/Δ14 deletion mutant had a significant increase in the rates of cytochrome c reduction, NADPH oxidation, and NO synthesis in a completely Ca2+/CaM-independent manner. Materials—l-[2,3,4,5-3H]Arginine (58 Ci/mmol), the ECL detection kit, and 2′,5′-ADP-Sepharose were products of Amersham Biosciences. (6R)-5,6,7,8,-Tetrahydro-l-biopterin (H4B) was obtained from Research Biochemical International. AG 50W-X8, cation exchange resin, Bradford protein dye reagent, and electrophoretic chemicals were products of Bio-Rad. Spodoptera frugiperda (Sf9) cells, baculovirus transfer vector (pVL1392) and BaculoGold viral DNA were obtained from BD Pharmingen. Grace's insect cell culture medium was purchased from Invitrogen. NADPH, CaM (catalog no. P-1431), anti-CaM monoclonal antibody, and other reagents were obtained from Sigma. Constructs—Fig. 1 shows the alignment of the reductase domains among three human NOS isoforms (38Janssens S.P. Shimouchi A. Quertermous T. Bloch D.B. Bloch K.D. J. Biol. Chem. 1992; 267: 14519-14522Abstract Full Text PDF PubMed Google Scholar, 39Nakane M. Schmidt H.H. Pollock J.S. Förstermann U. Murad F. FEBS Lett. 1993; 316: 175-180Crossref PubMed Scopus (474) Google Scholar, 40Geller D.A. Lowenstein C.J. Shapiro R.A. Nussler A.K. Di Silvio M. Wang S.C. Nakayama D.K. Simmons R.L. Snyder S.H. Billiar T.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3491-3495Crossref PubMed Scopus (814) Google Scholar) and CPR (41Shephard E.A. Palmer C.N. Segall H.J. Phillips I.R. Arch. Biochem. Biophys. 1992; 294: 168-172Crossref PubMed Scopus (48) Google Scholar). A mutant (Δ45) with deletion of residues 594–606/614–645 from human eNOS (highlighted by a bold font) was previously characterized (24Chen P.-F. Wu K.K. J. Biol. Chem. 2000; 275: 13155-13163Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). A 14-amino acid insert near the C terminus of human eNOS (residues 1165–1178) represents another notable difference between eNOS and iNOS. Two mutants with either deletion of residues 1165–1178 (Δ14) or a combined deletion of residues 594–606/614–645 and 1165–1178 (Δ45/Δ14) were generated by PCR. Two pairs of primers were used for the generation of Δ14 and Δ45/Δ14: 5′-3385CTGCGGCGATGTTACCATGGCAA-3′ and 5′-3578GACATTTTCGGGTTAACGCTGCGCACC-3′; 5′-3560CGCATACGCACGTTAACCTTTTCCTTGCAG-3′, and 5′-3757GCACCACCTCTAGAGGGGAGG-3′. The wild-type eNOS (WTeNOS) cDNA was used as the template for Δ14 construct, and the Δ45 cDNA was used as the template for Δ45/Δ14 construct. All primers were synthesized by Genosys Inc. (Woodlands, TX). Sequences of mutant cDNAs at junctional regions were confirmed by DNA sequencing at the core facility of the University of Texas Medical School at Houston. Expression and Determination of Nitrate/Nitrite in Sf9 Culture Medium—The cDNAs of WTeNOS and deletion mutants (Δ45, Δ14, and Δ45/Δ14) were inserted into the EcoRI site of pVL1392 transfer vector, which was used to generate recombinant viruses in an Sf9/baculovirus system. The nitrate/nitrite accumulation in culture medium was measured using a colorimetric assay kit from Cayman Chemical Co. Ten million Sf9 cells were seeded in each T75 culture flask, which was individually infected with 2 multiplicities of infection of recombinant viruses of WTeNOS and each mutant. Because of naturally low heme biosynthetic capability in the Sf9 cells, heme chloride (4 μg/ml) was added into the culture medium at 48 h postinfection to enrich heme content for the expressed protein. The amount of nitrite/nitrate in the culture medium was determined at 72 h postinfection by adding Griess reagent. The absorbance at 540 nm was recorded by using a Dynatech MR5000 microplate reader, and NO2-/NO3- was quantified using NaNO3 as standard. Purification of CaM-free Proteins of WTeNOS and Deletion Mutants—To generate the CaM-free mutant proteins, cells were harvested at 72 h postinfection, washed twice with calcium-free phosphate-buffered saline, pH 7.2, and resuspended in Buffer A (25 mm Tris-HCl, pH 7.5, 0.2 mm dithiothreitol, 1 mm EDTA, 1 mm EGTA, 1 μm pepstatin A, 1 μm leupeptin, 1 μm antipain, 1 mm phenylmethylsulfonyl fluoride, and 10% glycerol). Cells were sonicated four times for 30 s and then centrifuged at 15,000 × g for 60 min at 4 °C. The supernatant was loaded onto a 2′,5′-ADP-Sepharose affinity column (1.5 × 5 cm) pre-equilibrated with Buffer A. The column was washed with 20 column volumes of Buffer A, and then with 10 column volumes of Buffer B (25 mm Tris-HCl, pH 7.5, 0.2 mm dithiothreitol, 0.1 mm EDTA, 0.1 mm EGTA, 0.5 M NaCl, and 10% glycerol). The protein was eluted with Buffer B containing 20 mm 2′-AMP and concentrated by Centriprep-30 (Amicon). The concentrated protein was applied onto a gel filtration chromatography (1 × 120 cm, Ultrogel AcA34) and eluted with a buffer containing 25 mm Tris-HCl, pH 7.5, 100 mm NaCl, 0.1 mm dithiothreitol, and 10% glycerol. SDS-PAGE and Immunoblotting—Protein concentration was estimated using an extinction coefficient of Soret absorption peak = 100 mm-1 cm-1 for the NOS proteins (15Nishida C.R. Ortiz de Montellano P.R. J. Biol. Chem. 1998; 273: 5566-5571Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) and also was determined by the method of Bradford (42Bradford M.M Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar). SDS-PAGE was performed on a 7.5% slab gel according to the Laemmli procedure (43Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207233) Google Scholar) and stained by Coomassie Blue R250. For CaM immunoblot, the purified protein (15 μg) was subjected to SDS-PAGE in a 15% gel under reducing condition and then transferred to a polyvinylidene difluoride membrane. The membrane was blotted by monoclonal antibody raised against CaM (Sigma, catalog no. C-7055). Goat anti-mouse IgG-horseradish peroxidase conjugate was used as secondary antibody detected by the ECL method (Amersham Biosciences). Ca2+-dependent Measurement—To measure NOS activity at different free Ca2+ concentrations, a 100 mm stock of Ca2+-EGTA (Molecular Probes, Inc.) was used to obtain the desired free Ca2+ solution as calculated according to manufacturer's procedure using the Kd value of (Ca2+-EGTA) = 107.9 nm at 37 °Cin10mm MOPS, pH 7.2, and 100 mm KCl. The conversion of l-[3H]arginine to l-[3H]citrulline was measured as described by Bredt and Snyder (13Bredt D.S. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 682-685Crossref PubMed Scopus (3125) Google Scholar) with slight modifications. The reaction mixture (100 μl) containing 10 mm MOPS, pH 7.2, 100 mm KCL, 0.3 μm calmodulin, 100 μm β-NADPH, 100 μm H4B, 50 μml-arginine, 1 μCi of l-[3H]arginine, 10% glycerol, and various concentrations of free Ca2+ was incubated with 50–100 nm enzyme at 37 °C for 5 min. CaM-dependent Measurement—CaM-dependent NOS activity was tested at 37 °C for 5 min by measuring l-[3H]citrulline formation in a mixture containing 10 mm MOPS, pH 7.2, 100 mm KCl, 39 μm free Ca2+ (prepared from 100 mm stock of Ca2+-EGTA, Molecular Probes, Inc.), 100 μm β-NADPH, 100 μm H4B, 50 μml-arginine, 1 μCi of l-[3H]arginine, 10% glycerol, 50–100 nm enzyme, and various concentrations of CaM. Steady-state Catalysis—NO synthesis, NADPH oxidation, cytochrome c reduction, and ferricyanide oxidation were determined by measuring the optical absorbance change using a Shimadzu-2501 PC equipped with a temperature controller. A 10-mm light path cuvette was used unless indicated otherwise. The rate of NO formation was quantified from the NO-mediated conversion of oxyhemoglobin to methemoglobin by monitoring the absorbance increase at 401 nm using an extinction coefficient of 38 mm-1 cm-1 (44Murphy M.E. Noack E. Methods Enzymol. 1994; 233: 240-250Crossref PubMed Scopus (347) Google Scholar). Assays were carried out at 37 °C in the absence and presence of 0.5 μm calmodulin and 300 μm CaCl2 with a mixture (500 μl) containing 25 mm Tris-HCl, pH 7.5, 0.2 mm EDTA, 10 μm oxyhemoglobin, 100 μm β-NADPH, 100 μm H4B,1mml-arginine, 10% glycerol, and 50–100 nm enzymes. NADPH oxidation was measured as the decrease in absorbance at 340 nm using an extinction coefficient of 6.22 mm-1 cm-1. Assays were performed at 37 °C in the absence or presence of 0.5 μm calmodulin and 300 μm CaCl2 with a buffer containing 25 mm Tris-HCl, pH 7.5, 0.2 mm EDTA, 10% glycerol, 100 μm NADPH, 1 mml-arginine, and 50–100 nm enzymes. Cytochrome c reductase activity was determined at 37 °C in a reaction mixture (500 μl) containing 25 mm Tris-HCl, pH 7.5, 100 mm NaCl, 10% glycerol, 100 μm cytochrome c, 100 μm NADPH, and 30 nm enzyme with or without 0.5 μm calmodulin and 100 μm CaCl2. The reduced cytochrome c was monitored with the absorbance increase at 550 nm and quantified using Δξ of 21 mm-1 cm-1. Ferricyanide reduction was carried out in a reaction mixture similar to that described for the cytochrome c assay except that 3.2 mm ferricyanide and a 5-mm-light path cuvette were used. The amount of reduced ferricyanide was quantified using Δξ of 1.02 mm-1 cm-1 at 420 nm. Nitrite/Nitrate Accumulation in Sf9 Culture Medium— Studies have shown that cells expressing iNOS spontaneously produce a large quantity of NO· measured as NO2-/NO3- in their culture medium at a resting state of Ca2+, whereas cells expressing cNOSs produces only a trace amount of nitrate in the absence of agonists (19Ruan J. Xie Q.-w. Hutchinson N. Cho H. Wolfe G.C. Nathan C. J. Biol. Chem. 1996; 271: 22679-22686Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). To determine whether the deletion mutants behaved like iNOS, the constructs of wild-type and mutant eNOSs were expressed in Sf9 cells under identical conditions for 72 h. Western blot with anti-eNOS antibody was used to estimate the amount of protein expressed and showed that both Δ45 and Δ45/Δ14 were expressed at a level approximately half that of wild-type and Δ14eNOS (data not shown). Even with this constraint, the total amount of NO2-/NO3- from cells expressing Δ45 and Δ45/Δ14 was higher than that from cells expressing Δ14 (81, 37, and 62 μm for Δ45, Δ14, and Δ45/Δ14, respectively), whereas that from cells expressing wild-type eNOS was barely detectable (Fig. 2). The results suggest that all mutants with either a single deletion or a combined deletion of residues 594–606/614–645 and 1165–1178 are active at the basal level of intracellular Ca2+ but that Δ45 and Δ45/Δ14 are more sensitive to Ca2+ than Δ14. Purification of CaM-free Proteins—To obtain a CaM-free protein, the wild-type and mutant eNOSs were expressed in Sf9 cells and purified by the presence of an adequate amount of EGTA and EDTA. The purified enzymes were shown to be near homogeneity with an appropriate molecular mass in Coomassie-stained SDS-PAGE (Fig. 3A.). All mutants displayed the absorbance spectra identical to that of WTeNOS (45Chen P.-F. Tsai A.-L. Berka V. Wu K.K. J. Biol. Chem. 1997; 272: 6114-6118Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), indicating that the deletions did not perturb enzyme structure (data not shown). To further determine whether the WTeNOS and mutant enzymes contained an endogenous CaM, the purified proteins were subjected to SDS-PAGE followed by immunoblot with anti-CaM monoclonal antibody. None of the proteins purified in the presence of EGTA and EDTA contained an intrinsic CaM (Fig. 3B, lanes 1–4), whereas a Δ45 protein prepared in the absence of chelators and run parallel in SDS-PAGE had a detectable CaM (Fig. 3B, lane 5). Requirement for Ca2+ and CaM in l-Citrulline Formation— Dependence of l-citrulline formation on CaM was titrated by adding increasing concentrations of CaM (Sigma, catalog no. P-1431) along with 39 μm free Ca2+ and other cofactors in the reaction mixtures. CaM concentration response curves for wild-type and mutant eNOS are shown in Fig. 4A. The CaM-free Δ45 exhibited a constitutive activity in the absence of CaM, which was increased by increasing CaM concentration, whereas WTeNOS and Δ14 had a very low basal activity and required about 85 and 58 nm CaM, respectively, to reach half-maximal activity (EC50). Interestingly, Δ45/Δ14 was active in the absence of CaM, and its activity was reduced by about 25% in the presence of saturating Ca2+/CaM. Dependence on [Ca2+] was similarly titrated in the presence of 300 nm CaM, and the response curves are shown in Fig. 4B. The desired concentration of free Ca2+ was obtained by adding varied ratios of K-EGTA and Ca2+-EGTA as described previously (24Chen P.-F. Wu K.K. J. Biol. Chem. 2000; 275: 13155-13163Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). In the absence of Ca2+, Δ45eNOS had a considerable level of activity, which wa
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