Bredt and Snyder (4), our enzyme differs in the following points: 1) calmodulin ... Acknowledgment-We express our appreciation to Daniel Mrozek for help with ...
THEJOURNALOF BIOLOGICAL CHEMISTRY Vol. 266, No. 6, Issue of February 25, pp. 3369-3371,1991
Communication
0 1991 hy The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S A .
Calmodulin-independentNitric Oxide Synthase from Rat Polymorphonuclear Neutrophils* (Received for publication, November 7, 1990) Yoshiki YuiQ, Ryuichi Hattori, Kunihiko Kosuga, Hiroshi Eizawa, Kazuaki Hiki, Shigenori OhkawaS, Kohji OhnishiS, Shinji TeraoS, and ChuichiKawai From the ThirdDiuision, Department of Internal Medicine, Faculty of Medicine, Kyoto University, Kyoto 606, Japan and the$Chemistry Research Laboratories, Takeda Chemical Industries Ltd., Osaka 532, Japan
Recently, the purification of nitric oxide synthase (EC 1.14.23) from rat cerebellum has been reported, and the enzyme is a calmodulin-requiring enzyme (Bredt, D. S., and Snyder, S . H. (1990) Proc. Nutl. Acad. Sci. U.S. A. 87, 682-685). In this paper, nitric oxide synthase has been purified to near homogeneity from the cytosol fraction of rat polymorphonuclear neutrophils. The purification procedure involves affinity chromatography with adenosine 2',5'-diphosphateagarose and an anion exchange column, DEAE-Bio-Gel A. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the enzyme migrated as a single protein band with M , = 150,000. The molecular weight was estimated to be 150,000 by gel filtration on a Superose 12 HR 10/30. The purified enzyme was unstable with a half-life of 3 h at pH 7.4 and 4 "C. The enzyme activity required the presence of Ca2+, NADPH, FAD, and (6R)-5,6,7,8-tetrahydro-l"iopterin. Calmodulin antagonists (W5, W7, W13, and trifluoperazine dihydrochloride) did not inhibit the enzyme activity, and the addition of calmodulin was also ineffective for the increase in the enzyme activity. The neutrophil enzyme appears to be a calmodulinindependent type of nitric oxide synthase.
sidered to generate nitric oxide as a cytotoxic agent (8). Recently, Bredt et al. (9) have reported that they purified nitric oxide synthase with M, = 150,000 from rat cerebella and that this enzyme requires calmodulin. We developed a new assay system for NO, determination coupled with specific separation by ion pair high performance liquid chromatography. Using this highly sensitive system, we purified nitric oxide synthase from rat PMN. Nitric oxide synthase from PMN is a monomeric protein of M , = 150,000 that is dependent on Ca", NADPH, and (6R)-BH, with no calmodulin requirement. EXPERIMENTALPROCEDURES
Materials Peritoneal neutrophils were obtained from male Kbl Wistar rats (200-230 g) given anintraperitonealinjection of oyster glycogen (Type 11) by the method of Rimele et al. (5). PMN cells were suspended in 50 mM Tris-HC1 buffer, pH 7.4, and 1 mM dithiothreitol (DTT) at a cell density of 1 X 10' cells/ml. The following protease inhibitors were added: phenylmethylsulfonyl fluoride (0.1 mg/ml), trypsin inhibitor(0.01 mg/ml), leupeptin (0.01 mg/ ml), antipain (0.01 mg/ml), and pepstatin (0.01 mg/ml) (Sigma). This preparation resulted in a cell population of >95% neutrophils. Cell viability as assessed by trypan blue exclusion consistently exceeded 95%. Cells were then disrupted by sonication for 30 s (Dial 6, UR-SOOP, Tomy, Tokyo, Japan) and centrifuged at 100,000 X g for 60 min a t 4 "C, and the enzyme was purified from the supernatant (cytosol). Calmodulinantagonists,W5 (N-(6-aminohexyl)-l-naphthalenesulfonamidehydrochloride),W7 (N-(6-aminohexyl)-5-chloro-lnaphthalenesulfonamide hydrochloride), and W-13 (N-(4-aminobutyl)-5-chloro-2-napthalenesulfonamide hydrochloride were obtained from Seikagaku Kogyo (Tokyo, Japan). Trifluoperazinedihydrochloridewas obtainedfromResearch BiochemicalsInc.Bovine brain calmodulin was obtained from Calbiochem and Amano Pharmaceutical Co. (Osaka, Japan). (6R)-5,6,7,8-tetrahydro-~-biopterin ((6R)BH,) was obtained from Research Biochemicals Inc. All other chemicals were from Wako Pure Chemical Industries (Osaka, Japan), unless otherwisespecified. Methods
Nitrite (NO;) Determination-Samples were injected through an Endothelium-derived relaxing factor has been shown to be autosampler (SIL-GB) onto a C-18 reverse phase column (YMC A5 mM tetra-n-butyl ammonium identical to nitric oxide (NO) (1,2).In additionto endothelial 301,100 X 4.6 mm, Kyoto, Japan) with phosphate as the carrier solution at a constant flow of 1.0 ml/min. cells, nitric oxide has been found to be generated in macro- T o column effluents, thefollowing reagents for diazo formation (10) phages (3), cerebellum (4), and neutrophils (5). With L-argi- were added sequentially by the reaction pump (FIU-300N, Nihon nine serving as a substrate, guanidino a nitrogen of L-arginine Bunko Co., Tokyo, Japan): 1% (w/v) sulfanilic acid in 2 N HCl (0.3 is oxidized to form nitric oxide and L-citrulline. Small mole- ml/min) and 1% (w/v) N-(1-naphthy1)ethylenediamine dihydrochlocule cofactors, NADPH and (GR)-BH,,'were identified as ride (0.3 ml/min). The wavelength of the detector (SPD-6AV) was 548 nm. This postcolumn systemwas operated a t room temperature. being necessary for the enzyme reaction (6, 7). equipment, unless otherwise specified, was from Shimadzu Corp. Nitric oxide leads to an increase in the second messenger, All (Kyoto, Japan). The detection limit and CV were 5 pg and 0.5% ( n = cyclic GMP, by directly activating the enzyme guanylate lo), respectively. cyclase. In macrophages and neutrophils, these cells are conNitrate (NO:) Determination-Nitrate was determined by ion chromatography (Shimadzu Corp., Kyoto, Japan). * This research was supported by a research grant for cardiovasEnzyme Assay-The complete reaction mixture (600 pl final volcular diseases from the Ministry of Health and Welfare and by grants ume) contained the purified enzyme, 50 mM Tris-HC1 (pH7.4), 1 mM from the Ministry of Education, Science, and Culture, Japan. The NADPH, 1 mM L-arginine, 1 mMCa", 10 p~ FAD, 0.5 mM DTT, costs of publication of thisarticle were defrayed inpart by the and 0.1 mM (6R)-BH4. The mixture was incubated for 20 min at payment of pagecharges. Thisarticlemustthereforebe hereby 37 "C, and the reaction stopped by boiling at 100 "C for 30 s. The marked "aduertisement" in accordance with 18 U.S.C. Section 1734 tube wascentrifuged a t 15,000 X g for 20 min, and the resulting solely to indicate thisfact. supernatant was passed through a Millipore TGC filter (Millipore § To whom correspondence shouldbe addressed. Corp.). The filtratewas injected onto theabove nitrite determination ' The abbreviationsused are: (6R)-BH,, (6R)-5,6,7,8-tetrahydro-~-system. The total recovery rate of nitrate was 90%. Enzyme activity biopterin; PMN, polymorphonuclear neutrophils; DTT, dithiothreiwas expressed as (NC; + NO,) generation (nmol)/min/mg protein. tol; SDS, sodium dodecyl sulfate. Arginine and Citrulline Determination-Arginine loss and citrul-
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Calmodulin-independent Nitric
3370
line formationwere determined with an amino acid analyzing system by postcolumn derivatization with o-phthaldehyde (Shimadzu Corp.). In the stoichiometry experiment, 50 p~ arginine was used as the substrate concentration. Enzyme Purification-The cytosol (20 ml) was applied to a column of 2’,5’-ADP-agarose (1 X 1 cm, Sigma) equilibrated with 50 mM Tris-HCI buffer at pH 7.4 containing 1 mM DTT. The column was washed with 5 ml of 50 mM Tris-HCI, 1 mM DTT, and 500 mM NaCl and 10 ml of 50 mM Tris-HCI with 1 mM DTT, successively. The enzyme was eluted with 2 ml of 50 mM Tris-HCI, 1 mM DTT, and 1 mM NADPH and applied to an anion exchange column, DEAE-BioGel A (0.5 X 1 cm, Bio-Rad). After washing the column with 50 mM Tris-HCI, 1 mM DTT, and 80mM NaC1, the enzyme was eluted with a step gradient of 50 mM Tris-HCI, 1 mM DTT, and 120 mM NaC1. Sodium Dodecyl Sulfate (SDS)-Polyacrylamide Gel Electrophoresis-Electrophoresis was performed by the Phast-System (Pharmacia LKB Biotechnology, Uppsala, Sweden) ona 10-15% gel according to the manufacturer’s directions (Fig. 1). Proteins were stained with Coomassie Blue. Molecular weight standards (Pharmacia) were: thyroglobulin 330,000, ferritin 220,000, albumin 67,000, catalase 60,000, lactate dehydrogenase 36,000, and ferritin 18,500. Estimation of Molecular Weight byGel Filtration-Analytical gel filtration chromatography was carried out ona Superose 12 HR 10/ 30 (Pharmacia) equilibrated with 50 mM Tris-HCI, pH 7.4, 1 mM DTT, and 100 mM NaCl at a flow rate of 0.5 ml/min. The eluent was monitored a t 280 nm, and protein concentration was assayed. The standard proteins (Bio-Rad) and their molecular weight were: bovine thyroglobulin 670,000, bovine y-globulin 158,000, chicken ovalbumin 44,000, and horse myoglobin 17,000. Determination of Isoelectric Point-The isoelectric point of the purified enzyme was determined by Phast Gel isoelectric focusing (Pharmacia). Other Determinations-Ca2+ concentration was determined using stability constants by Martell et al. (11).The protein concentration was determined by a dye-binding microassay (Bio-Rad). Bovine serum albumin was used as the standard. RESULTS AND DISCUSSION
The result of a representative purification of nitric oxide synthase startingfrom cytosol (20 ml) of PMN is summarized in TableI. On SDS-polyacrylamidegel electrophoresis, DEAE eluate exhibited a single band on SDS-polyacrylamide gel electrophoresis with M , = 150,000. The molecular weight of the enzyme was estimated to be 150,000 on superose 12 HR 10/30 gel filtration chromatography. Thus, this protein appears to be a monomeric protein. The purified enzyme was very unstable with a half-life of 3 h at 4 “C, whereas the cytosol preparation was stable after 24 h. A poor yield in the purification step is considered to be because of the instability of the enzyme due to the loss of the stabilizing factor, which we have recently found (12), at the step of affinity chromatography. The loss of cofactors during the purification steps may also induce the instabilityof the enzyme, which may not be completely ameliorated by the additionof the cofactors at the enzyme reaction. The PIvalue of the enzyme estimated by Phast Gel isoelectric focusing was 5.6, and this enzyme functioned optimally at pH 7-8. Compared with the enzyme from cerebellum reported by Bredt and Snyder (4), our enzyme differs in the following points: 1)calmodulin independence and 2) necessities of FAD and (GR)-BH,. TABLE I Purification of nitric oxide synthase Step
Total protein pg
fraction Cytosol 24,800 Z’,B’-ADP-agarose eluate 136 DEAE eluate 1.8
Total activity
Specific activity
prnolfrnin nrnolfrninlrng
23,114 4,136 220
0.93 30 122
Yield %
100 18
1.0
Oxide Synthase oriQin
C
-
Enzyme
c
330,000 220,000 6 7 000 60:OOO
36,000 18.500
0.5
>
9
NO
synthase thyroglobulin
I
0 10,000
gamma globulin I
100,000
1.000.000
Log Molecular Weight
FIG.1. SDS-polyacrylamide gel electrophoresis of nitric and the estimation of the molecoxide synthase (upper panel) ular weight of the native enzyme (lower panel). The Phast systemwith a 10-15%gel wasusedforSDS-polyacrylamide gel electrophoresis, and gels were stained with Coomassie Blue. Left lane, 4 pg of the purified enzyme; right lane, molecular markers. Molecular weightwas estimated by a Superose 12 HR 10/30 withmolecular standards.
TABLE I1 ECso values for cofactors and Ca2+in the purified enzyme FAD 100 nM (6R)-BH4 100 nM cay+ 150 nM
Calmodulin antagonists (100 p~ W5, W7, W13, and trifluoperazine dihydrochloride) had no effect on the enzyme activity of the cytosol preparation, the sample after 2‘,5’ADP-agarose affinity chromatography,and a purified enzyme after DEAE chromatography. Moreover, the addition of calmodulin (1 p ~ to) each above sample also did not increase the activity. Thus, ourenzyme does not seem to require calmodulin for activation,but Ca’+ was necessary for the enzyme reaction. In theenzyme reaction using cytosol, the additionof (6R)BH, was not necessary for enzyme activation. However, in the absence of (GR)-BH,, enzyme activities after 2’,5’-ADPagarose and DEAE chromatographies were almost lost. ECSO values for cofactors and Ca2+were presented in Table11. Bredt and Snyder (9) have reported that the molecular weight of monomeric nitric oxide synthase from rat cerebella is M, = 150,000, and the molecular weight of nitric oxide synthase from PMN is also 150,000. The K, for arginine and the V,, at 37 “C and pH 7.4 are 22 p M and 485 nmol/min/mg protein, respectively. In the stoichiometry experiment using 50 p~ arginine as thesubstrate, 12.0 p~ arginine consumptionleads to the formation of 11.8 pM citrulline and 12.3 pM NO; + NOT. Thus, the stoichiometry of arginine loss to the formation of citrulline andNO; + NO, appears to be 1:l:l. Fromourdataanddata by Bredtand Snyder (91, the formation of L-citrulline andnitric oxide appeared to be mediated by one single enzyme, and 7,8-dihydro-~-biopterin (BH,)formed from (6R)-BH, is considered to be reconverted
Calmodulin-independentNitric Oxide Synthase to (6R)-BH4by a coupling reaction with NADPH and dihydrofolate reductase (7). Nitric oxide synthase may differ in the cerebellum and PMN. As Moncada et al. (13) have suggested, a seriesof nitric oxide synthase enzymes may exist. There may be two kinds of nitric oxide synthase; one is the constitutive enzyme in the cerebellum and in the endothelial cell. These enzymes require Ca"-calmodulin (4, 14, 15). The other is inducible enzyme in inflammatory neutrophiland cytotoxic activated macrophage.' These are calmodulin-independent. Nitric oxide synthase in cerebellum and the endothelial cells may respond quickly only when signals come. Nitric oxide synthase in PMN and macrophage may be synthesizing nitric oxide continuously. The molecular cloning of these enzymes and their comparisons will contribute to the clarification of the regulatory mechanism of NO synthase. Acknowledgment-We express our appreciation to Daniel Mrozek for help with preparation of the manuscript. REFERENCES 1. Palmer, R. M. J., Ferridge, A. G., and Moncada, S. (1987) Nature 327,524-526
* Y. Yui, R. Hattori, K. Kosuga, H. Eizawa, K. Hiki, and C. Kawai, manuscript in preparation.
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2. Ignarro, L. J., Buga, G. M., Wood,K. S., Byrns, R.E., and Chaudhuri, G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,92659269 3. Hibbs, J. B., Taintor, R. R., Vavrin, Z., and Rachilin, E. M. (1988) Biochem. Biophys. Res. Commun. 157,87-94 4. Bredt, D. S., and Snyder, S. H. (1989) Proc. Natl. Acad. Sci. U. S. A. 86,9030-9033 5. Rimele, T. J., Sturm, R. J., Adams, L. M., Henry, D. E., Heaslip, R. J., Weichman, B. M., and Grimes, D. J. (1988) J. Pharmacol. Exp. Ther. 245,102-111 6. Tayeh, M. A., and Marletta, M. A. (1989) J. Biol. Chern. 264, 19654-19658 7. Kwon, N. S., Nathan, C. F., and Stuehr, D. J. (1989) J . Biol. Chem. 264,20496-20501 8. Ignarro, L. J. (1990) Annu. Reu. Pharmacol. Toxicol. 3 0 , 535-560 9. Bredt, B. S., and Snyder, S. H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87,682-685 10. Yui, Y., Ohkawa, S., Ohnishi, K., Hattori, R., Aoyama, T., Takahashi, M., Morishita, H., Terao, Y., and Kawai, C. (1989) Biochem. Biophys. Res. Commun. 164,544-549 11. Martell, A. E., and Smith, R. (1977) Critical Stability Constants: Other Organic Ligands, Ed. 1, p. 269, Plenum Press, New York 12. Kosuga, K., Yui, Y., Hattori, R., Eizawa, H., Hiki, K., and Kawai, C. (1990) Biochem. Biophys. Res. Commun. 172,705-708 13. Moncada, S.. Palmer. R. M. J.. and Hiees,. E. A. (1989) . . Biochem. Pharmkol. 38,1709-1715 14. Gross. S. S.. Stuehr. D. J.. Aisaka. K.. Jaffe, E. A.. Levi. R.. and Griffith, 0.W. (1990) Biochem.' Biophys. Res. Comrnun. 1 7 0 , 96-103 15. Busse, R., and Miilsch, A. (1990) FEBS Lett. 2 6 5 , 133-136 "
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