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• W2049032587 abstract "Like other nitric-oxide synthase (NOS) enzymes, neuronal NOS (nNOS) turnover and activity are regulated by the ubiquitous protein chaperone hsp90. We have shown previously that nNOS expressed in Sf9 cells where endogenous heme levels are low is activated from the apo- to the holo-enzyme by addition of exogenous heme to the culture medium, and this activation is inhibited by radicicol, a specific inhibitor of hsp90 (Billecke, S. S., Bender, A. T., Kanelakis, K. C., Murphy, P. J. M., Lowe, E. R., Kamada, Y., Pratt, W. B., and Osawa, Y. (2002) J. Biol. Chem. 278, 15465–15468). In this work, we examine heme binding by apo-nNOS to form the active enzyme in a cell-free system. We show that cytosol from Sf9 cells facilitates heme-dependent apo-nNOS activation by promoting functional heme insertion into the enzyme. Sf9 cytosol also converts the glucocorticoid receptor (GR) to a state where the hydrophobic ligand binding cleft is open to access by steroid. Both cell-free heme activation of purified nNOS and activation of steroid binding activity of the immunopurified GR are inhibited by radicicol treatment of Sf9 cells prior to cytosol preparation, and addition of purified hsp90 to cytosol partially overcomes this inhibition. Although there is an hsp90-dependent machinery in Sf9 cytosol that facilitates heme binding by apo-nNOS, it is clearly different from the machinery that facilitates steroid binding by the GR. hsp90 regulation of apo-nNOS heme activation is very dynamic and requires higher concentrations of radicicol for its inhibition, whereas GR steroid binding is determined by assembly of stable GR·hsp90 heterocomplexes that are formed by a purified five-chaperone machinery that does not activate apo-nNOS. Like other nitric-oxide synthase (NOS) enzymes, neuronal NOS (nNOS) turnover and activity are regulated by the ubiquitous protein chaperone hsp90. We have shown previously that nNOS expressed in Sf9 cells where endogenous heme levels are low is activated from the apo- to the holo-enzyme by addition of exogenous heme to the culture medium, and this activation is inhibited by radicicol, a specific inhibitor of hsp90 (Billecke, S. S., Bender, A. T., Kanelakis, K. C., Murphy, P. J. M., Lowe, E. R., Kamada, Y., Pratt, W. B., and Osawa, Y. (2002) J. Biol. Chem. 278, 15465–15468). In this work, we examine heme binding by apo-nNOS to form the active enzyme in a cell-free system. We show that cytosol from Sf9 cells facilitates heme-dependent apo-nNOS activation by promoting functional heme insertion into the enzyme. Sf9 cytosol also converts the glucocorticoid receptor (GR) to a state where the hydrophobic ligand binding cleft is open to access by steroid. Both cell-free heme activation of purified nNOS and activation of steroid binding activity of the immunopurified GR are inhibited by radicicol treatment of Sf9 cells prior to cytosol preparation, and addition of purified hsp90 to cytosol partially overcomes this inhibition. Although there is an hsp90-dependent machinery in Sf9 cytosol that facilitates heme binding by apo-nNOS, it is clearly different from the machinery that facilitates steroid binding by the GR. hsp90 regulation of apo-nNOS heme activation is very dynamic and requires higher concentrations of radicicol for its inhibition, whereas GR steroid binding is determined by assembly of stable GR·hsp90 heterocomplexes that are formed by a purified five-chaperone machinery that does not activate apo-nNOS. hsp90 1The abbreviations used are: hsp, heat shock protein; NOS, nitricoxide synthase; nNOS, neuronal NOS; eNOS, endothelial NOS; GR, glucocorticoid receptor; Hop, hsp70/hsp90 organizing protein; apo-nNOS, heme-deficient, inactive nNOS monomer; holo-nNOS, heme-complexed, active nNOS dimer; LBD, ligand binding domain; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid. has been shown to regulate over 100 signal transduction pathways by controlling the function, trafficking, and turnover of a variety of signaling proteins (reviewed in Ref. 1Pratt W.B. Toft D.O. Exp. Biol. Med. 2003; 228: 111-133Crossref PubMed Scopus (1266) Google Scholar). The regulation is achieved through the ATP-dependent assembly of signaling protein·hsp90 heterocomplexes by the multi-protein hsp90/hsp70-based chaperone machinery (1Pratt W.B. Toft D.O. Exp. Biol. Med. 2003; 228: 111-133Crossref PubMed Scopus (1266) Google Scholar). The association of hsp90 with its “client proteins” is not determined by specific amino acid motifs or secondary modifications (e.g. phosphorylation, acetylation, etc.) in the client protein, and it has not been clear how hsp90 could associate with and regulate such a wide variety of proteins regardless of their structure or sequence. How the chaperone machinery recognizes and interacts with its signaling protein clients is a fundamental mechanistic problem. In cell-free assays, the two essential components of the machinery, hsp90 and hsp70, have been shown to function individually as chaperones that bind to exposed hydrophobic amino acids in denatured proteins and through rounds of binding and release to facilitate protein refolding (2Hartl F.U. Hayer-Hartl M. Science. 2002; 295: 1852-1858Crossref PubMed Scopus (2792) Google Scholar). However, studies with steroid receptors suggest that this is not the way hsp90 and hsp70 function when they act together as part of an integrated chaperone machinery. hsp90 binds to the ligand binding domain (LBD) of steroid receptors, and the ligand binding activity of some steroid receptors and the aryl hydrocarbon receptor is absolutely hsp90-dependent (1Pratt W.B. Toft D.O. Exp. Biol. Med. 2003; 228: 111-133Crossref PubMed Scopus (1266) Google Scholar). Thus, when the glucocorticoid receptor (GR) is stripped of its associated hsp90, it immediately loses its ability to bind steroid, and steroid binding activity is regenerated when GR·hsp90 heterocomplexes are reformed by the hsp90/hsp70-based multichaperone machinery (3Morishima Y. Murphy P.J.M. Li D.P. Sanchez E.R. Pratt W.B. J. Biol. Chem. 2000; 275: 18054-18060Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 4Murphy P.J.M. Kanelakis K.C. Galigniana M.D. Morishima Y. Pratt W.B. J. Biol. Chem. 2001; 276: 30092-30098Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Steroid ligands bind deep in a hydrophobic cleft that appears to be collapsed in the absence of ligand, such that the receptor must change its conformation to allow entry of the ligand (5Gee A.C. Katzenellenbogen J.A. Mol. Endocrinol. 2001; 15: 421-428Crossref PubMed Scopus (56) Google Scholar). The chaperone machinery carries out the ATP-dependent opening of the steroid binding cleft in the GR LBD such that it can be accessed by steroid, and it promotes conformational changes that increase the sensitivity of the GR LBD to attack by thiolderivatizing agents and trypsin (6Stancato L.F. Silverstein A.M. Gitler C. Groner B. Pratt W.B. J. Biol. Chem. 1996; 271: 8831-8836Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 7Simons S.S. Sistare F.D. Chakraborti P.K. J. Biol. Chem. 1989; 264: 14493-14497Abstract Full Text PDF PubMed Google Scholar, 8Modarress K.J. Opoku J. Xu M. Sarlis N.J. Simons S.S. J. Biol. Chem. 1997; 272: 23986-23994Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). In contrast to the chaperoning action of hsp90 and hsp70 on denatured proteins, there is no evidence that the chaperone machinery acts on a GR that is in any way denatured or in a so-called “nearly native” state. Rather, the machinery acts on a GR where the hydrophobic steroid binding cleft appears to be collapsed such that the LBD is in a native, minimal energy conformation. The initial step in GR·hsp90 heterocomplex assembly is the ATP- and hsp40-dependent priming of the GR by hsp70 to form a GR·hsp70 complex that can then bind Hop and hsp90 and undergo a second ATP-dependent step in which the steroid binding cleft is opened (3Morishima Y. Murphy P.J.M. Li D.P. Sanchez E.R. Pratt W.B. J. Biol. Chem. 2000; 275: 18054-18060Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 9Murphy P.J.M. Morishima Y. Chen H. Galigniana M.D. Mansfield J.F. Simons S.S. Pratt W.B. J. Biol. Chem. 2003; 278: 34764-34773Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). It is the ATP-dependent conformation of hsp70 that initially binds to the GR LBD, rather than the ADP-dependent conformation that favors interaction of hsp70 with hydrophobic peptides (10Kanelakis K.C. Shewach D.S. Pratt W.B. J. Biol. Chem. 2002; 277: 33698-33703Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). A short segment of the GR that lies at the extreme amino terminus of the LBD is required for LBD·hsp90 heterocomplex assembly and steroid binding activity (11Xu M. Dittmar K.D. Giannoukos G. Pratt W.B. Simons S.S. J. Biol. Chem. 1998; 273: 13918-13924Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). This segment lies at the rim of the ligand binding cleft of the receptor (12Giannoukos G. Silverstein A.M. Pratt W.B. Simons S.S. J. Biol. Chem. 1999; 274: 36527-36536Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), and mutational analysis reveals that hsp90 binding requires the presence of the segment but no defined amino acid composition (13Kaul S. Murphy P.J.M. Chen J. Brown L. Pratt W.B. Simons S.S. J. Biol. Chem. 2002; 277: 36223-36232Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The work on the mechanism of GR·hsp90 heterocomplex assembly led to the notion that the chaperone machinery interacts with the region where the hydrophobic cleft merges with the surface of the receptor. Such regions are a general topologic feature of virtually all proteins in native conformation, and this cleft recognition hypothesis could account for the ability of the chaperone machinery to interact with a variety of proteins in native conformation regardless of sequence or structure (1Pratt W.B. Toft D.O. Exp. Biol. Med. 2003; 228: 111-133Crossref PubMed Scopus (1266) Google Scholar). The ability of the chaperone machinery to recognize hydrophobic clefts and to open and stabilize them in the open state, even transiently, could facilitate the entry of a number of hydrophobic ligands and prosthetic groups to catalytic centers in the interior of many enzymes. As a start to testing this general proposal, we have examined the role of hsp90 in facilitating the binding of heme by apo-neuronal nitric-oxide synthase (apo-nNOS). The NOS enzymes are important signaling proteins that function as cytochrome P450-type hemoproteins to catalyze the formation of nitric oxide (NO) and citrulline from l-arginine, O2, and NADPH (14Stuehr D.J. Biochim. Biophys. Acta. 1999; 1141: 217-230Crossref Scopus (811) Google Scholar). The NOS enzymes are active as homodimers, with each monomer binding tightly 1 eq each of FAD, FMN, tetrahydrobiopterin, and heme (14Stuehr D.J. Biochim. Biophys. Acta. 1999; 1141: 217-230Crossref Scopus (811) Google Scholar, 15Hevel J.M. White K.A. Marletta M.A. J. Biol. Chem. 1991; 266: 22789-22791Abstract Full Text PDF PubMed Google Scholar, 16Stuehr D.J. Cho H.J. Kwon N.S. Weise M.F. Nathan C.F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7773-7777Crossref PubMed Scopus (731) Google Scholar, 17White K.A. Marletta M.A. Biochemistry. 1992; 31: 6627-6631Crossref PubMed Scopus (578) Google Scholar, 18Stuehr D.J. Ikeda-Saito M. J. Biol. Chem. 1992; 267: 20547-20550Abstract Full Text PDF PubMed Google Scholar, 19Crane 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). The prosthetic heme is the site of oxygen activation, which is required for the metabolism of l-arginine. The heme-deficient monomer of NOS can be partially reconstituted in vitro in the presence of heme, tetrahydrobiopterin, and arginine to form the functional homodimer (20Baek K.J. Thiel B.A. Lucas S. Stuehr D.J. J. Biol. Chem. 1993; 268: 21120-21129Abstract Full Text PDF PubMed Google Scholar, 21Hemmens B. Gorren A.C.F. Schmidt K. Werner E.R. Mayer B. Biochem. J. 1998; 332: 337-342Crossref PubMed Scopus (31) Google Scholar, 22Bender A.T. Nakatsuka M. Osawa Y. J. Biol. Chem. 2000; 275: 26018-26023Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). We have shown previously that a portion of neuronal NOS (nNOS) is bound to hsp90 in vivo and that treatment of mammalian cells with an hsp90 inhibitor to block nNOS·hsp90 assembly leads to nNOS degradation via the ubiquitin-proteasome pathway (23Bender A.T. Silverstein A.M. Demady D.R. Kanelakis K.C. Noguchi S. Pratt W.B. Osawa Y. J. Biol. Chem. 1999; 274: 1472-1478Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). We have also shown that treatment of nNOS-expressing Sf9 cells with the hsp90 inhibitors geldanamycin and radicicol inhibits activation of apo-nNOS activity by exogenous heme (23Bender A.T. Silverstein A.M. Demady D.R. Kanelakis K.C. Noguchi S. Pratt W.B. Osawa Y. J. Biol. Chem. 1999; 274: 1472-1478Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 24Billecke S.S. Bender A.T. Kanelakis K.C. Murphy P.J.M. Lowe E.R. Kamada Y. Pratt W.B. Osawa Y. J. Biol. Chem. 2002; 277: 20504-20509Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Sf9 cells have very low levels of endogenous heme, and addition of exogenous heme to the culture medium increases nNOS activity in transfected cells. Inhibition of hsp90 is accompanied by a marked inhibition in heme binding and by an inability to form the dimeric state of nNOS, implying a role for hsp90 in heme-mediated assembly of the holo-enzyme (24Billecke S.S. Bender A.T. Kanelakis K.C. Murphy P.J.M. Lowe E.R. Kamada Y. Pratt W.B. Osawa Y. J. Biol. Chem. 2002; 277: 20504-20509Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The ferrous·carbonyl complex of nNOS is not formed when hsp90 is inhibited, indicating that functional heme insertion is prevented (24Billecke S.S. Bender A.T. Kanelakis K.C. Murphy P.J.M. Lowe E.R. Kamada Y. Pratt W.B. Osawa Y. J. Biol. Chem. 2002; 277: 20504-20509Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). To date, we have not been able to demonstrate a role for hsp90 in heme binding and nNOS activation in a cell-free system. Here, we show that cytosol prepared from Sf9 cells facilitates cell-free heme insertion into apo-nNOS and conversion to the active holo-nNOS enzyme. Cytosol from radicicoltreated Sf9 cells is not active in permitting heme activation of apo-nNOS or in converting the GR to the steroid binding state, and in both cases the inhibition is partially reversed by the addition of purified hsp90. However, the hsp90 machinery that dynamically facilitates heme access to its binding cleft in apo-nNOS is different from the hsp90 machinery that assembles stable hsp90 complexes with the GR to facilitate steroid binding access to its hydrophobic ligand binding cleft. Untreated rabbit reticulocyte lysate was purchased from Green Hectares (Oregon, WI). [6,7-3H]Dexamethasone (40 Ci/mmol) and 125I-conjugated goat anti-mouse and anti-rabbit IgGs were obtained from PerkinElmer Life Sciences. Dulbecco's modified Eagle's medium was from Bio-Whittaker (Walkersville, MD). The BuGR2 monoclonal IgG used to immunoblot the GR was from Affinity Bioreagents (Golden, CO), and the FiGR monoclonal IgG used to immunoadsorb the GR was generously provided by Dr. Jack Bodwell (Dartmouth Medical School, Lebanon, NH). Rabbit antiserum against the C terminus of hsp90 used to immunoblot insect hsp90 and hsp70 was kindly provided by Dr. Christine Radanyi (UMR8612, CNR5, Paris, France), and the AC88 monoclonal IgG used to immunoblot rabbit hsp90 was from StressGen Biotechnologies (Victoria, BC, Canada). The affinity-purified rabbit IgG against brain NOS used to immunoblot nNOS was from BD Transduction Laboratories (Lexington, KY). (6R)-5,6,7,8-Tetrahydro-l-biopterin was purchased from Dr. Schirck's laboratory (Jona, Switzerland). Protein A-Sepharose, iron protoporphyrin IX, l-arginine, glucose 6-phosphate, glucose-6-phosphate dehydrogenase, calmodulin, radicicol, NADPH, and NADP+ were purchased from Sigma. 2′,5′-ADP-Sepharose 4B was purchased from Amersham Biosciences. The cDNA for rat nNOS was kindly provided by Dr. Solomon Snyder (The Johns Hopkins Medical School, Baltimore, MD). The baculovirus for expression of the mouse GR was generously provided by Dr. Edwin Sanchez (Medical College of Ohio, Toledo, OH). Expression and Purification of Apo-nNOS and Holo-nNOS—Recombinant baculovirus containing nNOS cDNA was produced as described previously (23Bender A.T. Silverstein A.M. Demady D.R. Kanelakis K.C. Noguchi S. Pratt W.B. Osawa Y. J. Biol. Chem. 1999; 274: 1472-1478Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Sf9 cells were grown in SFM 900 II serum-free medium (Invitrogen) supplemented with 1% Cytomax (Kemp Biotechnology, Rockville, MD) in suspension cultures maintained at 27 °C with continuous shaking (150 rpm). To express holo-nNOS, oxyhemoglobin (25 μm) was added as a source of heme during the last 24 h of expression. Cultures (2 × 106 cells/ml) were infected in log phase of growth with recombinant baculovirus at a multiplicity of infection of 1.0. After 48 h, cells were harvested, washed once with ice-cold phosphate-buffered saline (pH 7.4), and ruptured by Dounce homogenization in 1 pellet volume of a homogenization buffer containing 10 mm Hepes, pH 7.4, 10 μg/ml trypsin inhibitor, 10 μg/ml leupeptin, 2 μg/ml aprotinin, 6 mm phenylmethanesulfonyl fluoride, and 10 μm tetrahydrobiopterin. The lysates were spun at 100,000 × g for 60 min at 4 °C. The supernatants were collected, aliquoted, flash-frozen, and stored at –70 °C. nNOS was purified from 70 ml of cytosol prepared from infected Sf9 cells by adsorption to a column of 2′,5′-ADP-Sepharose and elution with 10 mm 2′-AMP, followed by gel filtration chromatography on Sephacryl S-300 as described previously (23Bender A.T. Silverstein A.M. Demady D.R. Kanelakis K.C. Noguchi S. Pratt W.B. Osawa Y. J. Biol. Chem. 1999; 274: 1472-1478Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Fractions containing nNOS were pooled and concentrated to 2 ml. The concentrated enzyme was divided into aliquots and stored at –70 °C. Activation of Apo-nNOS with Heme in Sf9 Cells—The activation of apo-nNOS in Sf9 cells by the addition of heme-albumin to the medium was performed as described previously (24Billecke S.S. Bender A.T. Kanelakis K.C. Murphy P.J.M. Lowe E.R. Kamada Y. Pratt W.B. Osawa Y. J. Biol. Chem. 2002; 277: 20504-20509Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). In the experiment of Fig. 4A, Sf9 cells (50 ml, 2 × 106 cells/ml) were infected with recombinant nNOS baculovirus for 36 h and then treated with vehicle (Me2SO) or 20 μm radicicol for 20 min. A 5-ml aliquot was treated with 25 μm heme, added as an albumin conjugate (25Asseffa A. Smith S.J. Nagata K. Gillette J. Gelboin H.V. Gonzalez F.J. Arch. Biochem. Biophys. 1989; 274: 481-490Crossref PubMed Scopus (82) Google Scholar, 26Richards M.K. Marletta M.A. Biochemistry. 1994; 33: 14723-14732Crossref PubMed Scopus (100) Google Scholar), for 1 h at 27 °C. Cells were ruptured, and nNOS activity was measured as described below. The remainder of the cell suspension (45 ml) was washed twice with 25 ml of SFM 900 II medium, the cells were resuspended in 45 ml of medium, and the incubation was continued without heme. At various times, 5-ml aliquots were treated with heme for 1 h, ruptured, and assayed for nNOS activity. Cell-free Activation of Apo-nNOS with Heme—Aliquots (25 μl) of cytosol from nNOS-expressing Sf9 cells that were grown in heme-free medium were preincubated at 27 °C with 3 μl of an ATP-regenerating system (50 mm ATP, 100 units/ml creatine phosphokinase, 20 mm magnesium acetate, and 250 mm creatine phosphate in 10 mm Hepes, pH 7.4) in a total volume of 30 μl. After 5 min, 1.5 μl of heme·bovine serum albumin complex (25Asseffa A. Smith S.J. Nagata K. Gillette J. Gelboin H.V. Gonzalez F.J. Arch. Biochem. Biophys. 1989; 274: 481-490Crossref PubMed Scopus (82) Google Scholar, 26Richards M.K. Marletta M.A. Biochemistry. 1994; 33: 14723-14732Crossref PubMed Scopus (100) Google Scholar) was added to yield a final heme concentration of 25 μm. Aliquots of 3.5 μl were removed at various times for assay of nNOS activity. In some experiments, purified apo-nNOS (10 μg) was substituted for cytosol from nNOS-expressing Sf9 cells in the reconstitution mixture. Where indicated, 20 μl of cytosol prepared from uninfected Sf9 cells pretreated with radicicol or vehicle was added to the reconstitution mixture containing purified apo-nNOS. DE52 Chromatography of Sf9 Cytosol—Non-infected Sf9 cytosol (12 ml) was adsorbed to a 1.5 × 20-cm column of DE52 equilibrated with HE buffer (10 mm Hepes, 0.1 mm EDTA, pH 7.4); the column was washed with 100 ml of HE buffer, producing a “drop-through” fraction, followed by elution of bound proteins with 100 ml of HEK buffer (HE buffer containing 500 mm KCl) to produce a “retained” fraction. Each fraction was concentrated to 2 ml, and then a portion (6 μl) was used in place of cytosol for heme-mediated reconstitution of purified apo-nNOS. For the preparation of DE52-retained fractions A, B, and C, 30 ml of Sf9 cytosol was adsorbed to a 2.5 × 18-cm column of DE52 equilibrated with HE buffer; the column was washed with 250 ml of HE buffer, and proteins were eluted with a 400-ml gradient of 0–0.5 m KCl. hsp90 and hsp70 were assayed by Western blotting an aliquot of every other fraction using rabbit antiserum directed against the carboxyl terminus of hsp90 that also detects hsp70. Fractions were combined in three pools designated A–C as described by Dittmar et al. (27Dittmar K.D. Hutchison K.A. Owens-Grillo J.K. Pratt W.B. J. Biol. Chem. 1996; 271: 12833-12839Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Fractions A, B, and C were concentrated to 1.5 ml, and then a portion (2 μl) was used in place of cytosol for heme-mediated reconstitution of purified apo-nNOS. Assay of nNOS Activity—NO formation was assayed by the NO-mediated conversion of oxyhemoglobin to methemoglobin. nNOS-containing samples were added to an assay solution containing 100 μm CaCl2, 100 μml-arginine, 100 μm tetrahydrobiopterin, 100 units/ml catalase, 10 μg/ml calmodulin, 25 μm oxyhemoglobin, and an NADPH-regenerating system consisting of 400 μm NADP+, 10 mm glucose 6-phosphate, and 1 unit/ml glucose-6-phosphate dehydrogenase, expressed as final concentrations, in a total volume of 180 μl of 50 mm potassium phosphate, pH 7.4. The mixture was incubated at 37 °C, and the rate of oxidation of oxyhemoglobin was monitored with a microtiter plate reader as described previously (28Feelisch M. Kubitzek D. Werringloer J. Feelish M. Stamler J.S. Methods in Nitric-Oxide Research. John Wiley & Sons, Inc., New York1996: 455-478Google Scholar). P450 Assay—The heme present in nNOS was assessed by measuring the ferrous·CO complex. Because the cell cytosols contained compounds that absorbed in the 420-nm region, we partially purified nNOS from the cytosols. Cytosols (8-mg total) were loaded onto a 2′,5′-ADP-Sepharose column (2.0 cm × 0.8 cm) and eluted with 2′-AMP as described above, except that the gel filtration step was omitted. The ferrous carbonyl complex was measured as a difference spectrum and quantified with an extinction coefficient of 91 mm–1, as previously described (29McMillan K. Bredt D.S. Hirsch D.J. Snyder S.H. Clark J.E. Masters B.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11141-11145Crossref PubMed Scopus (357) Google Scholar). Expression of Glucocorticoid Receptor in Sf9 Cells—Sf9 cultures infected in log phase of growth with recombinant baculovirus containing the mouse GR cDNA at a multiplicity of infection of 3.0 were supplemented with 0.1% glucose at infection and 24 h post-infection as described by Srinivasan et al. (30Srinivasan G. Post J.F. Thompson E.B. J. Steroid. Biochem. Mol. Biol. 1997; 60: 1-9Crossref PubMed Scopus (28) Google Scholar). In the experiment of Fig. 4, at 48 h post-infection, cells were treated with Me2SO or 20 μm radicicol (or geldanamycin) for 20 min followed by washing and resuspension as described for nNOS-infected cells. At various times after washing, cells were harvested, resuspended in 1.5 volume of HEM buffer (10 mm Hepes, pH 7.4, 1 mm EDTA, 20 mm molybdate) with 1 mm phenylmethylsulfonyl fluoride and 1 tablet of Complete-Mini protease inhibitor mixture per 3 ml of buffer (Roche Applied Science), and ruptured by Dounce homogenization. The lysate was then centrifuged at 100,000 × g for 30 min, and the supernatant was collected, aliquoted, flash-frozen, and stored at –70 °C. Immunoadsorption of GR—Receptors were immunoadsorbed from 50-μl aliquots of Sf9 cytosol by rotation for 2 h at 4 °C with 18 μl of protein A-Sepharose precoupled to 9 μl of FiGR ascites suspended in 200 μl of TEG buffer (10 mm TES, pH 7.6, 50 mm NaCl, 4 mm EDTA, 10% glycerol). Prior to incubation with Sf9 lysate, or the DE52-retained fractions of Sf9 lysate, or the purified proteins of the chaperone machinery, immunoadsorbed receptors were stripped of associated hsp90 by incubating the immunopellet for an additional 2 h at 4 °C with 350 μlof0.5 m NaCl in TEG buffer. The pellets were then washed once with 1 ml of TEG buffer followed by a second wash with 1 ml of Hepes buffer (10 mm Hepes, pH 7.4). GR·hsp90 Heterocomplex Reconstitution—FiGR immunopellets containing GR stripped of chaperones were incubated with unfractionated rabbit reticulocyte lysate or with the five-protein assembly system (20 μg of purified hsp90, 15 μg of purified hsp70, 0.6 μg of purified human Hop, 6 μg of purified p23, 0.125 μg of purified YDJ-1 (see Ref. 31Kanelakis K.C. Pratt W.B. Methods Enzymol. 2003; 364: 159-173Crossref PubMed Scopus (20) Google Scholar for details)) adjusted to 55 μl with HKD buffer (10 mm Hepes, pH 7.4, 100 mm KCl, 5 mm dithiothreitol) containing 20 mm sodium molybdate and 5 μl of an ATP-regenerating system. The assay mixtures were incubated for 20 min at 30 °C with suspension of the pellets by shaking the tubes every 2 min. At the end of the incubation, the pellets were washed twice with 1 ml of ice-cold TEGM buffer (TEG with 20 mm sodium molybdate) and assayed for steroid binding capacity and for receptor-associated proteins. Assay of Steroid Binding Capacity—Immune pellets to be assayed for steroid binding were incubated overnight at 4 °Cin50 μl of HEM buffer plus 100 nm [3H]dexamethasone. Samples were then washed three times with 1 ml of TEGM buffer and counted by liquid scintillation spectrometry. The steroid binding is expressed as counts/min of [3H]dexamethasone bound/FiGR immunopellet prepared from 50 μl of cell cytosol. For supernatants to be assayed for steroid binding, a 50-μl aliquot of supernatant was incubated overnight at 4 °Cin50 μl HEM buffer with 100 nm [3H]dexamethasone in the absence or presence of a 1000-fold excess of unlabeled dexamethasone. Samples were mixed with dextrancoated charcoal and, after centrifugation, counted by liquid scintillation spectrometry. The steroid binding is expressed as counts/min of [3H]dexamethasone bound/50 μl of cell cytosol. Gel Electrophoresis and Western Blotting—Samples (immune pellets, aliquots of cytosol, and aliquots of DE52 fractions) were resolved on 12% SDS-polyacrylamide gels and transferred to Immobilon-P membranes. The membranes were probed with 0.25 μg/ml BuGR for GR or 0.01% rabbit antiserum to the carboxyl terminus of hsp90 for insect hsp90 (this antibody also weakly detects insect hsp70), or 1 μg/ml AC88 for rabbit hsp90. The immunoblots were then incubated a second time with the appropriate 125I-conjugated counterantibody to visualize immunoreactive bands. Immunoblots for nNOS were run on 6% SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and probed with 0.1% anti-nNOS polyclonal antibody as described (24Billecke S.S. Bender A.T. Kanelakis K.C. Murphy P.J.M. Lowe E.R. Kamada Y. Pratt W.B. Osawa Y. J. Biol. Chem. 2002; 277: 20504-20509Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). In studies where the SDS-resistant dimer was measured, the reaction mixture was quenched with sample buffer supplemented with 100 μm BH4 and 100 μml-arginine. The samples were kept on ice prior to loading for analysis by SDS-PAGE as described above. This method has previously been described by Klatt et al. (32Klatt P. Schmidt K. Lehner D. Glatter O. Bachinger H.P. Mayer B. EMBO J. 1995; 14: 3687-3695Crossref PubMed Scopus (266) Google Scholar) to prevent the dissociation of nNOS dimers prior to and during electrophoresis. Radicicol Treatment of Sf9 Cells Inhibits Subsequent Cell-free nNOS Activation—The major cell-free system used to study hsp90 action on signaling proteins is rabbit reticulocyte lysate (1Pratt W.B. Toft D.O. Exp. Biol. Med. 2003; 228: 111-133Crossref PubMed Scopus (1266) Google Scholar), but reticulocyte lysate contains very high levels of heme in addition to the necessary machinery for hsp90 heterocomplex assembly. Thus, although we have been able to activate apo-nNOS with reticulocyte lysate, we have not been able to show that the activation is both heme-dependent and hsp90-dependent (23Bender A.T. Silverstein A.M. Demady D.R. Kanelakis K.C. Noguchi S. Pratt W.B. Osawa Y. J. Biol. Chem. 1999; 274: 1472-1478Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Because Sf9 cells grown in heme-free medium have low levels of endogenous heme, we asked whether concentrated lysate from cells grown in the absence of exogenous heme would support heme-dependent activation of apo-nNOS in vitro. In the experiment of Fig. 1," @default.
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• W2049032587 title "The Role of hsp90 in Heme-dependent Activation of Apo-neuronal Nitric-oxide Synthase" @default.
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