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• W2066431883 abstract "Immunostimulants trigger vascular smooth muscle cells (VSMC) to express both the inducible isoform of NO synthase (iNOS) and argininosuccinate synthetase (AS). With constitutively expressed argininosuccinate lyase (AL), AS confers cells with an Arg/Cit cycle that can sustain NO production via continuous regeneration of the NOS substrate, l-arginine (Arg), from the NOS coproduct, l-citrulline (Cit). To assess whether NO synthesis can be rate-limited by Arg recycling, we tested whether AS-overexpressing cells have an enhanced capacity for immununostimulant-induced NO synthesis. Rat VSMC were stably transfected with human AS cDNA in a eukaryotic cell expression vector, driven by a strong viral promoter. AS activity in transfected VSMC exceeded that induced in untransfected cells treated for 24 h with a combination of bacterial lipopolysaccharide and interferon-γ (LPS/IFN). AS activity was predominantly associated with membranes but was also found in cytosol. Recombinant AS was purified from cytosol and possessed a specific activity exceeding that reported for native AS. Western blotting verified the basal expression of AS antigen in membranes from untreated AS-transfected VSMC and from untransfected VSMC after 24 h exposure to LPS/IFN. Epifluorescence histochemistry revealed a punctate distribution of AS antigen in transfected cells, consistent with a predominant membrane localization. Remarkably, on a per cell basis, LPS/IFN-induced NO production was 3–4-fold greater in AS-transfected cells than untransfected VSMC. In untransfected VSMC, maximal NO production during 48 h required millimolar Arg; notably, Cit was needed at ≈3-fold higher concentrations than Arg for a comparable NO synthesis rate. In contrast, AS-transfected VSMC utilized Arg and Cit equi-effectively and at much lower concentrations; 100 μm of either precursor supported a maximal rate of NO synthesis for 48 h. The enhanced ability of AS-transfected cells to produce NO, compared with untransfected cells, could not be ascribed to differences in iNOS protein content or LPS/IFN potency for immunoactivation. We conclude that transfection with AS provides a continuous flux of Arg which drives NO synthesis in immunoactivated VSMC. Arg regeneration by AS is rate-limiting to NO synthesis and apparently provides iNOS with a preferred cellular source of Arg. In accord with the reported “channeling” of substrates by urea cycle enzymes, we hypothesize that the Arg/Cit cycle sequesters a discrete pool of recyclable substrate that sustains high-output NO synthesis. Immunostimulants trigger vascular smooth muscle cells (VSMC) to express both the inducible isoform of NO synthase (iNOS) and argininosuccinate synthetase (AS). With constitutively expressed argininosuccinate lyase (AL), AS confers cells with an Arg/Cit cycle that can sustain NO production via continuous regeneration of the NOS substrate, l-arginine (Arg), from the NOS coproduct, l-citrulline (Cit). To assess whether NO synthesis can be rate-limited by Arg recycling, we tested whether AS-overexpressing cells have an enhanced capacity for immununostimulant-induced NO synthesis. Rat VSMC were stably transfected with human AS cDNA in a eukaryotic cell expression vector, driven by a strong viral promoter. AS activity in transfected VSMC exceeded that induced in untransfected cells treated for 24 h with a combination of bacterial lipopolysaccharide and interferon-γ (LPS/IFN). AS activity was predominantly associated with membranes but was also found in cytosol. Recombinant AS was purified from cytosol and possessed a specific activity exceeding that reported for native AS. Western blotting verified the basal expression of AS antigen in membranes from untreated AS-transfected VSMC and from untransfected VSMC after 24 h exposure to LPS/IFN. Epifluorescence histochemistry revealed a punctate distribution of AS antigen in transfected cells, consistent with a predominant membrane localization. Remarkably, on a per cell basis, LPS/IFN-induced NO production was 3–4-fold greater in AS-transfected cells than untransfected VSMC. In untransfected VSMC, maximal NO production during 48 h required millimolar Arg; notably, Cit was needed at ≈3-fold higher concentrations than Arg for a comparable NO synthesis rate. In contrast, AS-transfected VSMC utilized Arg and Cit equi-effectively and at much lower concentrations; 100 μm of either precursor supported a maximal rate of NO synthesis for 48 h. The enhanced ability of AS-transfected cells to produce NO, compared with untransfected cells, could not be ascribed to differences in iNOS protein content or LPS/IFN potency for immunoactivation. We conclude that transfection with AS provides a continuous flux of Arg which drives NO synthesis in immunoactivated VSMC. Arg regeneration by AS is rate-limiting to NO synthesis and apparently provides iNOS with a preferred cellular source of Arg. In accord with the reported “channeling” of substrates by urea cycle enzymes, we hypothesize that the Arg/Cit cycle sequesters a discrete pool of recyclable substrate that sustains high-output NO synthesis. Nitric oxide is a cell signaling gas with diverse functions and global importance to mammalian cell physiology (1Sadrzadeh S.M. Nathan C. Xie Q.W. Cell. 1994; 78: 915-918Abstract Full Text PDF PubMed Scopus (2754) Google Scholar). NO synthases (NOS, 1The abbreviations used are: NOS, nitric-oxide synthase; AS, argininosuccinate synthetase; AL, argininosuccinate lyase; iNOS, the high-output inducible form of nitric-oxide synthase; LPS, lipopolysaccharide; IFN, interferon-γ PCR, polymerase chain reaction; VSMC, vascular smooth muscle cells; Arg,l-arginine; Cit, l-citrulline; MBP, maltose-binding protein; PAGE, polyacrylamide gel electrophoresis. EC 1.14.13.39) catalyze two sequential oxygenations of l-arginine (Arg), producing stoichiometric amounts of NO and l-citrulline (Cit) (2Griffith O.W. Stuehr D.J. Annu Rev Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar, 3Nathan C. Xie Q.W. J. Biol. Chem. 1994; 269: 13725-13728Abstract Full Text PDF PubMed Google Scholar). All nucleated mammalian cells have the capacity for high-output NO production upon transcription of the gene that encodes inducible NOS (iNOS). While the iNOS gene lies dormant in cells at rest, iNOS transcription can be initiated by various immunostimulants, cytokines, and growth factors (3Nathan C. Xie Q.W. J. Biol. Chem. 1994; 269: 13725-13728Abstract Full Text PDF PubMed Google Scholar). In macrophages, iNOS-derived NO appears to be a major weapon in the arsenal of molecules that mediate host defense (1Sadrzadeh S.M. Nathan C. Xie Q.W. Cell. 1994; 78: 915-918Abstract Full Text PDF PubMed Scopus (2754) Google Scholar). On the other hand, inappropriate expression of iNOS mediates a variety of pathophysiological conditions caused by NO excess (4Gross S.S. Wolin M.S. Annu. Rev. Physiol. 1995; 57: 737-769Crossref PubMed Scopus (821) Google Scholar),e.g. iNOS induction in vascular smooth muscle cells (VSMC) has been implicated in the genesis of lethal septic shock (5Kilbourn R.G. Griffith O.W. J. Natl. Cancer Inst. 1992; 84: 827-831Crossref PubMed Scopus (261) Google Scholar). Appreciation of the factors that control iNOS activity should provide a rationale for the design of therapeutics that effectively limit pathophysiological NO overproduction. Inasmuch as high-output NO synthesis requires a continuous supply of substrate, it is important to identify where and how this Arg originates. Possible sources of cellular Arg include uptake from plasma, intracellular protein degradation, and de novobiosynthesis from Cit. Although de novo biosynthesis of Arg in ureotelic organisms is accomplished principally by the kidney, and to a lesser extent the small intestine (6Morris Jr., S.M. Annu. Rev. Nutr. 1992; 12: 81-101Crossref PubMed Scopus (214) Google Scholar, 7Takiguchi M. Mori M. Biochem. J. 1995; 312: 649-659Crossref PubMed Scopus (97) Google Scholar), it has become clear that other tissues can also produce Arg from Cit. Biosynthesis of Arg requires two enzymes, argininosuccinate synthetase (AS, EC 6.3.4.5) and argininosuccinate lyase (AL, EC 4.3.2.1), that together catalyze the conversion of Cit, l-aspartate, and ATP to Arg, fumarate, AMP, and pyrophosphate. AS and AL are typically considered in the context of their contribution to the five-enzyme urea cycle of liver; however, in conjunction with NOS, these enzymes endow cells with an Arg/Cit cycle that can continually regenerate Arg from Cit for sustained NO production (8Hattori Y. Campbell E.B. Gross S.S. J. Biol. Chem. 1994; 269: 9405-9408Abstract Full Text PDF PubMed Google Scholar). Accordingly, Arg production from Cit has been observed in endothelial cells, which basally produce NO, and has been shown to increase as a function of NO synthesis rate (9Hecker M. Sessa W.C. Harris H.J. Anggard E.E. Vane J.R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8612-8616Crossref PubMed Scopus (408) Google Scholar). We reported earlier that along with iNOS, immunostimulants synergistically induce VSMC to express AS mRNA and activity (8Hattori Y. Campbell E.B. Gross S.S. J. Biol. Chem. 1994; 269: 9405-9408Abstract Full Text PDF PubMed Google Scholar), allowing for NO synthesis from Cit in the absence of extracellular arginine. Induction of AS is rate-limiting to Arg synthesis in VSMC, since AL mRNA and activity were found to be constitutively expressed. Notably, AS is also the rate-limiting enzyme of the urea cycle (6Morris Jr., S.M. Annu. Rev. Nutr. 1992; 12: 81-101Crossref PubMed Scopus (214) Google Scholar). Similar induction by immunostimulants of AS mRNA and activity, and constitutive expression of AL, were also found in murine macrophages (10Nussler A.K. Billiar T.R. Liu Z.-Z. Morris Jr., S.M. J. Biol. Chem. 1994; 269: 1257-1261Abstract Full Text PDF PubMed Google Scholar) and human pancreatic β-cells (11Flodstrom M. Niemann A. Bedoya F.J. Morris Jr., S.M. Eizirik D.L. Endocrinology. 1995; 136: 3200-3206Crossref PubMed Google Scholar) in culture. Coinduction of AS and iNOS has more recently been shown in vivo in spleen, heart, and lung of LPS-treated rats (12Hattori Y. Shimoda S. Gross S.S. Biochem. Biophys. Res. Commun. 1995; 215: 148-153Crossref PubMed Scopus (28) Google Scholar, 13Nagasaki A. Gotoh T. Takeya M. Yu Y. Takiguchi M. Matsuzaki H. Takatsuki K. Mori M. J. Biol. Chem. 1996; 271: 2658-2662Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). In contrast with in vitro findings, AL expression was also observed to be up-regulated by LPS in vivo. These findings suggest that together AS and AL play a special function in providing substrate for high-output NO synthesis. The present study was performed to investigate whether the availability of AS may limit NO synthesis in VSMC. Toward this end, we engineered VSMC to overexpress human AS and examined how this impacted on immunostimulant-induced NO synthase activity and substrate preference. While it is predictable that AS-overexpressing cells would more efficiently produce NO from Cit, it is remarkable that maximal NO production from Arg was also increased 3–4-fold on a per cell basis. This suggests that the Arg/Cit cycle provides the preferred source of Arg for NO synthesis by VSMC and can be rate-limiting to high-output NO production. Human AS cDNA, kindly provided by Drs. William O'Brien and Gerald Petajunas (Baylor College of Medicine), served as template for PCR amplification. Primers used were as follows: AS forward 24-mer (created to contain an EcoRV site), 5′-ATCCCAGACGATATCTCCAGCAAAGGC-3′ AS reverse 23-mer (created to contain a SalI site), 5′-CTCATTGTCGACGGGTCTATTTGG-3′. Thirty cycles of PCR were performed according to the following schedule: denaturation for 1 min at 94 °C, annealing for 1 min at 55 °C, and elongation for 1 min at 72 °C. PCR products were electrophoresed on a 1% agarose gel containing ethidium bromide and visualized by UV-induced fluorescence. This resulted in the amplification of a single product of the predicted size for human AS (1266 base pairs) that was ligated into the TA3 pCR™ vector (Invitrogen) and transformed into competent DH5α Escherichia coli. Successful subcloning of the PCR product was confirmed by restriction analysis of purified plasmid DNA (Wizard Minipreps, Promega). AS cDNA was then subcloned from the TA3 pCR™ plasmid into pMAL-P2 (New England Biolabs) to enable high level bacterial expression of AS as a fusion with maltose-binding protein. The pMAL-P2 plasmid was cut with bothXmnI and SalI, and the AS DNA/TA3 pCR™ plasmid was cut with both EcoRV and SalI. Cut plasmids were subjected to electrophoresis on 1% agarose, purified, ligated overnight at 16 °C, and transformed into DH5α E. coli. pMAL-P2/AS plasmid DNA was purified (Wizard Minipreps, Promega), sequenced by the dideoxynucleotide chain termination method, and found to be >99% identical to that for human AS cDNA (22Su T.-S. Bock H.-G.O. O'Brien W.E. Beaudet A.L. J. Biol. Chem. 1981; 256: 11826-11831Abstract Full Text PDF PubMed Google Scholar). Observed nucleotide differences from the reported human cDNA were three, an A to G substitution at nucleotide 410 and substitution of CT for AC at nucleotides 1050–1051. A clone harboring the pMAL-P2/AS plasmid was inoculated into 1 liter of LB medium containing 100 μg/ml ampicillin and grown at 37 °C until A 600 reached 0.5. Isopropyl-1-thio-β-d-galactopyranoside was added to a final concentration of 0.5 mm, and cultures were incubated for an additional 3 h at 37 °C. Bacteria were harvested by centrifugation at 4,000 rpm for 10 min, and pellets were stored at −20 °C until use. For AS purification, the stored pellets were resuspended in 50 ml of TEND buffer (20 mm Tris-Cl (pH 7.4), 1 mm EDTA, 200 mm NaCl, and 1 mm dithiothreitol) which was supplemented with 1 mm citrulline, 1 mm aspartate, and a mixture of protease inhibitors (10 μg/ml pepstatin, 10 μg/ml leupeptin, and 100 μm phenylmethylsulfonyl fluoride) and then subjected to two cycles of freezing at −70 °C and thawing at 37 °C. Sodium chloride was added to final concentration 1 m, and samples were lysed using a Branson sonicator. Lysates were centrifuged at 15,000 × g for 30 min at 4 °C, and supernatants were applied to a 5-ml column of amylose resin (New England Biolabs) that had been previously equilibrated with TEND buffer. The column was washed with 25 ml of TEND buffer two times. Finally, the column was eluted with 50 ml of 10 mm maltose in TEND buffer containing 1 mm citrulline and 1 mm aspartate. SDS-PAGE with Coomassie Blue staining revealed a predominant protein band which was of predicted size for the AS-fusion protein; this identity was confirmed by factor Xa cleavage (4 °C for 3 h, using factor Xa at a 1% w/w ratio with fusion protein), yielding two fragments of the predicted sizes. The products of factor Xa cleavage were separated by ion exchange fast protein liquid chromatography using a Mono Q column (Pharmacia Biotech Inc.) and a NaCl gradient as follows: 100% buffer A (20 mm Tris (pH 7.4), 1 mm aspartate, 1 mm citrulline) for 0–5 min, followed by a linear gradient to 100% buffer B (buffer A containing 25 mm NaCl) from 5 to 35 min. Purified AS eluted as a discrete peak at 24 min and gave a single protein band of ≈46 kDa on Coomassie Blue-stained SDS-PAGE. Rat aortic smooth muscle cells were isolated from thoracic aorta explants of Fisher rats and grown as described previously (14Gross S.S. Levi R. J. Biol. Chem. 1992; 267: 25722-25729Abstract Full Text PDF PubMed Google Scholar). Cells in passage 8–12 were seeded and grown to confluence in 96-well plates for nitrite assay or in 75-cm2 culture flasks for preparation of extracts for AS activity measurements. For transfection, cells in passage 4–6 were seeded and grown to 50% confluence in 6-well plates. Human AS cDNA was used as a template for PCR amplification of a cDNA encoding AS preceded by six N-terminal histidine residues and containing convenient restriction sites for ligation into the cytomegalovirus promoter-driven mammalian expression vector, pcDNA3 (Invitrogen). Primers for PCR were as follows: AS forward 49-mer (containing a BamHI site and His-Tag sequence), 5-CGGGATCCACGATGCACCACCACCACCACCACATGTCCAGCAAAGGCTC-3′ AS reverse 25-mer (containing an EcoR V site), 5′-CCAGCCGGGGATATCAAGTCACAAT-3′. Both the PCR product and pcDNA3 plasmid were digested with BamHI and EcoRV, purified, and ligated at 16 °C overnight. The resulting AS/pcDNA3 was transformed into DH5α E. coli. Plasmid DNA was purified from a positive clone for nucleotide sequencing and transfection into rat aortic smooth muscle cells. Sequencing revealed identity of the insert with that in our AS-pMAL-P2 plasmid (described above), encoding a predicted protein having >99% amino acid identity to human AS (22Su T.-S. Bock H.-G.O. O'Brien W.E. Beaudet A.L. J. Biol. Chem. 1981; 256: 11826-11831Abstract Full Text PDF PubMed Google Scholar). Transfection of VSMC was performed using 15 μg of AS/pcDNA3, 52.5 μl of LipofectAMINE (Life Technologies, Inc.), and 500 μl of Opti-MEM medium (Life Technologies, Inc.). While mixing the plasmid/LipofectAMINE mixture by inversion for 30 min, a plate of cells was twice washed with Opti-MEM and incubated with an additional 5 ml of Opti-MEM. The plasmid/LipofectAMINE mixture was then added to the cells and incubated for 6 h, followed by replacement with unmodified cell culture medium. After 48 h, G418 was added at a concentration of 500 μg/ml to initiate selection of stably transfected/resistant cells; culture medium was replaced at 2–3-day intervals. Approximately 3 weeks later, G418-resistant clones were isolated using an 8 × 8-mm cloning cylinder and analyzed individually for expression of AS activity. His6-AS protein was purified from a cell pellet prepared from 60 confluent T-75 flasks of a VSMC clone that was found to be positive for AS activity. The purification was performed using His-bind resin, according to the manufacturer's (Novagen) protocol. The purified and concentrated AS/maltose-binding protein fusion was used to develop polyclonal antibodies in each of two rabbits. Fusion protein in Freund's complete adjuvant was injected intradermally into four dorsal sites (50 μg/injection) in each rabbit. After 28 days the animals were boosted by intradermal injection of an additional 100 μg of fusion protein in incomplete adjuvant and thereafter boosted by subcutaneous injection of 50 μg of protein at 14-day intervals. Commencing at week 8, animals were bled every 2 weeks from a marginal ear vein. Serum was separated from the clotted blood and stored at −20 °C until use. Antibody titer was determined by enzyme-linked immunosorbent assay, and specificity for AS was established by Western blot analysis. Cells were washed twice with 10 ml of ice-cold phosphate-buffered saline and harvested with a Teflon cell scraper into an additional 10 ml of iced phosphate-buffered saline. Cell suspensions were centrifuged at 800 × g for 10 min, and pelleted cells from five 75-cm2 culture flasks were resuspended in 1 ml of ice-cold distilled H2O containing a mixture of protease inhibitors (10 μg/ml pepstatin, 10 μg/ml leupeptin, and 100 μm phenylmethylsulfonyl fluoride) and lysed by three cycles of freezing in liquid nitrogen and thawing in a 37 °C water bath. Lysates were centrifuged at 500 × g for 5 min, and the pellets were discarded. The resulting supernatants were recentrifuged at 15,000 × g for 5 min; this pellet provided the membrane fraction, and the supernatant, after a further centrifugation at 100,000 × g for 1 h, provided the cytosolic fraction. Both fractions were immediately assayed for AS activity. Protein was measured by the Bio-Rad dye binding assay (Bio-Rad), using bovine serum albumin as standard. After the protein concentrations of both cell cytosol and membrane samples were determined, they were added 1:1 with 2 × SDS-PAGE sample loading buffer and incubated for 10 min at 95 °C (Novex). Samples were then normalized for protein content and applied to lanes for SDS-PAGE on 8–16% gradient gels (Novex). Proteins were transferred by electroelution onto polyvinylidene difluoride membrane (0.2 μm, Trans-Blot medium, Bio-Rad). Western blot analysis of AS expression was performed using polyclonal AS antiserum from rabbit, at a dilution of 1: 1,000. Immunoreactive bands were detected by sequential incubation in biotinylated goat anti-rabbit IgG (1:3,000), streptavidin-alkaline phosphatase (1:3,000, Life Technologies, Inc.), and the chemiluminescent 1,2-dioxytene substrate, CSPD® (Tropix). Western blotting to demonstrate iNOS protein in VSMC cytosol was similar to the above, except that rabbit antibody was to iNOS holoprotein (Upstate Biotechnology) and used at 1:3,000 dilution. Binding of alkaline phosphatase-coupled second antibody was visualized using a chromogenic substrate (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium; Life Technologies, Inc.). AS activity was assayed using each of two techniques. The first was based on the conversion of [3H]aspartate to [3H]argininosuccinate, as described by O'Brien (15O'Brien W.E. Biochemistry. 1979; 18: 5353-5356Crossref PubMed Scopus (32) Google Scholar) with the exception that [3H]aspartate was present at a concentration of 400 μm. Each reaction mixture contained (final concentration) citrulline (5 mm), Tris-HCl (10 mm, pH 7.5), ATP (0.1 mm), MgCl2 (6 mm), KCl (20 mm), phosphoenolpyruvate (1.5 mm), pyruvate kinase (4.5 units), myokinase (4 units), and pyrophosphatase (0.2 units) in a final volume of 150 μl. Reactions were incubated in glass test tubes and allowed to proceed for 60 min at 37 °C, before enzyme activity was terminated with addition of 50 μl of 1 macetic acid and heating for 30 min at 90 °C. Following this procedure, 800 μl of distilled H2O was added to incubates, and the contents were applied to 0.5 × 4-cm columns of Dowex 1-X8 (200–400 mesh, Bio-Rad) in 0.05 m acetic acid. An additional 2 × 1 ml of 0.05 m acetic acid was then applied to the columns. Radioactivity in the column flow-through, reflecting [3H]argininosuccinate, was added to In-Flow BD liquid scintillant (IN/US Systems Inc.) and quantified by liquid scintillation spectrometry. Additionally, purified AS activity was assessed by measuring the formation of inorganic phosphate by the method of Fiske and Subbarow (36Fiske C.H. Subbarow Y. J. Biol. Chem. 1925; 66: 375-400Abstract Full Text PDF Google Scholar), as previously adapted to the 96-well microtiter plate format (37Sadrzadeh S.M.H. Vincenzi F.F. Hinds T.R. J. Pharmacol. Toxicol. Methods. 1993; 30: 103-110Crossref PubMed Scopus (27) Google Scholar). Incubation mixtures were identical to above, except they were scaled to 100 μl total volume, the ATP concentration was increased to 300 μm, and the ATP regenerating system was omitted (i.e. phosphoenolpyruvate, pyruvate kinase, and myokinase). Nitrite was used as an indicator of cellular NO synthesis, as described previously (16Gross S.S. Stuehr D.J. Aisaka K. Jaffe E.A. Levi R. Griffith O.W. Biochem. Biophys. Res. Commun. 1990; 170: 96-103Crossref PubMed Scopus (260) Google Scholar). For experiments that assessed the dependence of NO production on citrulline or arginine concentration, cells were washed twice with serum-free and Arg-free RPMI 1640 cell culture medium and then incubated for 24 h with the same medium containing the desired concentration of test agents. In all other experiments, RPMI containing 10% newborn calf serum and standard levels of Arg (200 mg/liter) was used. The accumulation of nitrite in the cell culture medium was quantified colorimetrically after adding 100 μl of Griess reagent (1% sulfanilamide and 0.1% naphthalenediamine in 5% o-phosphoric acid) to an equal volume of sample. For immunofluorescence studies, rat aortic smooth muscle cells, stably-transfected with human AS, were grown on glass microscope slide chambers (Costar). Cells were fixed for 30 min at room temperature with neutral buffered 4% paraformaldehyde and washed 3 times with Ca2+/Mg2+-containing HEPES-buffered saline (pH 7.5; HBS). Cells were then permeabilized, and nonspecific binding was blocked by treatment for 2 h with 0.075% saponin in HBS containing 0.2% bovine serum albumin. After an additional wash in HBS, cells were incubated for 1 h at room temperature in 1:500 AS antiserum, diluted in HBS containing 0.2% bovine serum albumin and 0.075% saponin. Cells were washed 3 times in HBS and then incubated for 30 min at room temperature with fluorescein isothiocyanate-conjugated bovine anti-rabbit IgG (1:200, Vector Labs) in HBS/bovine serum albumin. After a final 3 washes in HBS, cells were washed in distilled H2O and coverslipped with glycerol-based mountant (Vectorshield, Vector Labs). Antibody binding was visualized by epifluorescence using an Olympus BX-60 microscope and a U-MWB fluorescence filter cube. Rat recombinant IFN-γ, RPMI culture medium, and cell culture reagents were from Life Technologies, Inc. [3H]Aspartic acid was purchased from Amersham Life Sciences, Inc. (specific activity = 40 Ci/mmol). Factor Xa was from New England Biolabs. Enzymes, LPS (E. coli serotype 0111:B4), and all other chemicals were obtained from Sigma or Calbiochem. AS protein was previously isolated from human (15O'Brien W.E. Biochemistry. 1979; 18: 5353-5356Crossref PubMed Scopus (32) Google Scholar, 17Kimball M.E. Jacoby L.B. Biochemistry. 1980; 19: 705-709Crossref PubMed Scopus (13) Google Scholar), bovine (18Rochovansky O. Kodowaki H. Ratner S. J. Biol. Chem. 1977; 252: 5287-5294Abstract Full Text PDF PubMed Google Scholar), and rat (19Saheki T. Kusumi T. Takada S. Katsunuma T. J. Biochem. ( Tokyo ). 1977; 81: 687-696Crossref PubMed Scopus (32) Google Scholar) and found to be homotetrameric. The human gene that encodes AS comprises 16 exons which span 63 kilobases (20Freytag S.O. Beaudet A.L. Bock H.G. O'Brien W.E. Mol. Cell. Biol. 1984; 4: 1978-1984Crossref PubMed Scopus (44) Google Scholar) and map to chromosome 9q34 (21Northrup H. Lathrop M. Lu S.Y. Daiger S.P. Beaudet A.L. O'Brien W.E. Genomics. 1989; 5: 442-444Crossref PubMed Scopus (26) Google Scholar). Based on cDNA cloning, the predicted polypeptide sequence is 412 amino acids and 46.4 kDa (22Su T.-S. Bock H.-G.O. O'Brien W.E. Beaudet A.L. J. Biol. Chem. 1981; 256: 11826-11831Abstract Full Text PDF PubMed Google Scholar). To obtain large quantities of human AS for antibody development and to confirm functionality of the recombinant protein, we sought to express AS in bacteria. Although AS has previously been purified after overexpression in bacteria, it was not recovered in a catalytically active state (23Yu Y. Terada K. Nagasaki A. Takiguchi M. Mori M. J. Biochem. ( Tokyo ). 1995; 117: 952-957Crossref PubMed Scopus (64) Google Scholar). AS was directionally cloned into pMAL-P2 in an orientation that would result in the N terminus of AS fused to the C terminus of maltose-binding protein. Relative to the reported human AS cDNA, the plasmid we constructed was found to contain an A to G substitution at nucleotide 410 and a substitution of CT for AC at nucleotides 1050–1051. These nucleotide changes are non-conservative, resulting in predicted differences in amino acid sequence from that reported for human AS: Lys112 to Glu and Leu325-Arg326 to Phe-Trp. Although Lys112 is conserved in human, rat, murine, and bovine AS cDNA, it is notable that Saccharomyces cerevisiae, Streptomyces lavendulae, and E. coli each have a different amino acid at the corresponding position (Phe, Gln, and Asn, respectively). Thus, although conserved in mammals, a basic residue in this position is not a requirement for AS activity. Substitution in human AS of Phe-Trp for Leu325-Arg326 is also consistent with enzymatic activity. Indeed, sequence alignment reveals that rat, mouse, and bovine AS sequences each have Phe-Trp at the corresponding sites. Therefore, the reported Leu325-Arg326 sequence in human AS cDNA is the exception, rather than the rule. This Phe-Trp is partially conserved in AS cDNAs from S. cerevisiae, S. lavendulae, and E. coli AS cDNAs, which encode Phe-Leu, Arg-Trp, and Arg-Trp, respectively. Thus, the sequence we observed at this site makes our predicted enzyme identical with that from three other mammalian isoforms and more closely in agreement with more evolutionarily divergent species. Since all other nucleotides in our human AS-containing plasmids were common to human rather than rodent AS cDNA, we are confident that we have not inadvertently amplified from rodent template cDNA. This substitution could conceivably represent a correction to the previously reported sequence or perhaps results from human gene pleiotropy. E. coli harboring the AS/pMAL-P2 plasmid expressed the predicted 89.1-kDa fusion protein to an extent of 10–20% total cellular protein (estimated from Coomassie staining of SDS-PAGE). A single step purification on amylose resin gave near-homogeneous AS/MBP (>90% purity; see Fig. 1 A, lane 1), the most prominent contaminant being free MBP. Yields of 10–15 mg of purified fusion protein were obtained per liter of culture broth. The purified AS/MBP fusion protein was found to be catalytically active, as indicated by Fig. 1 B. While the specific activity of the preparation described in Fig. 1 B was 5.4 μmol/mg/h, other preparations ranged in activity to 11.9 μmol/mg/h (based on the initial rate of AS formation). Considering that the fusion protein is ≈50% AS by weight, the specific activity of AS/MBP fusion protein was 20–40% that observed with AS purified from a human lymphoblast cell line (17Kimball M.E. Jacoby L.B. Biochemistry. 1980; 19: 705-709Crossref PubMed Scopus (13) Google Scholar). As shown in lane 2 of Fig. 1 A, cleavage of AS/MBP by treatment with factor Xa yielded a mixture of free AS (46.4 kDa) and MBP (42.7 kDa). Subsequent purification of free AS on Mono Q resin was performed using f" @default.
• W2066431883 created "2016-06-24" @default.
• W2066431883 creator A5000612299 @default.
• W2066431883 creator A5034085605 @default.
• W2066431883 date "1997-06-01" @default.
• W2066431883 modified "2023-09-26" @default.
• W2066431883 title "Argininosuccinate Synthetase Overexpression in Vascular Smooth Muscle Cells Potentiates Immunostimulant-induced NO Production" @default.
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