Activation of Human Neutrophil Procollagenase by Nitrogen Dioxide and Peroxynitrite: A Novel Mechanism for Procollagenase Activation Involving Nitric Oxide

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 342, No. 2, June 15, pp. 261–274, 1997 Article No. BB970127

Activation of Human Neutrophil Procollagenase by Nitrogen Dioxide and Peroxynitrite: A Novel Mechanism for Procollagenase Activation Involving Nitric Oxide1 Tatsuya Okamoto,*,† Takaaki Akaike,* Tetsuo Nagano,‡ Seiya Miyajima,* Moritaka Suga,† Masayuki Ando,† Koji Ichimori,§ and Hiroshi Maeda*,2 *Department of Microbiology and †Department of Internal Medicine I, Kumamoto University School of Medicine, Kumamoto 860, Japan; ‡Faculty of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113, Japan; and §Department of Physiology, Tokai University School of Medicine, Kanagawa 259-11, Japan

Received December 30, 1996, and in revised form April 1, 1997

The involvement of nitric oxide (NO) and its reactive intermediates such as nitrogen dioxide (NO2) and peroxynitrite (ONOO0) in the activation of matrix metalloproteinase was investigated. The human neutrophil procollagenase (matrix metalloproteinase-8) (Mr, 85 kDa) was purified to homogeneity from human neutrophils by using column chromatography. After incubation of human neutrophil procollagenase with various nitrogen oxide-generating systems, collagenolytic activity in each reaction system was measured. In addition, neutrophil collagenase activity was determined by assessment of proteolysis of human a1-protease inhibitor. NO was formed by the propylamine NONOate, and NO2 was generated by oxidation of NO with 2-(4carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO). NO2 , formed by NONOate and carboxy-PTIO, and the synthetic ONOO0 exhibited strong activation of the procollagenase at 1–20 mM. Significant activation of the procollagenase was observed with use of authentic NO2 gas as well. Constant flux infusion of ONOO0 into the procollagenase solution resulted in stronger procollagenase activation than did a bolus addition of ONOO0 to the reaction mixture. However, NO showed only weak activating potential under the aerobic (ambient) condition; an NO concentration of more than 10 mM was needed for appreciable activation of the procollagenase. Of considerable importance was the fact that NO participates in activa1 This work was supported by grants-in-aid for Scientific Research from Ministry of Education, Science and Culture of Science to H.M. and T.A. 2 To whom correspondence should be addressed at the Department of Microbiology, Kumamoto University School of Medicine, 2-2-1 Honjo, Kumamoto 860, Japan. Fax: (81) 96-362-8362. E-mail: [email protected].

tion of the neutrophil collagenase through its conversion to NO2 or ONOO0 in human neutrophils. These results suggest that NO2 and ONOO0 may be potent activators of human neutrophil procollagenase. q 1997 Academic Press

Key Words: human neutrophil procollagenase; nitric oxide; peroxynitrite; nitrogen dioxide; reactive nitrogen oxides.

Much concern has been raised about hazards of nitrogen oxides such as nitric oxide (NO) and nitrogen dioxide (NO2), which are gaseous radical components in cigarette smoke and the polluted atmosphere (1–5). Evidence has accumulated showing potent tissue damage caused by nitrogen oxides (3, 6, 7). It was reported that NO2 inhaled by animals causes severe pulmonary edema and emphysema (1), characterized by destruction of the extracellular matrix (ECM).3 It has also been well documented that NO being generated endogenously by NO synthase (NOS) exerts multiple functions in biological systems (8–11). NO is transformed into other types of reactive nitrogen oxides by reaction with 3 Abbreviations used: carboxy-PTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide; carboxy-PTI, 2-(4-carboxyphenyl)4,4,5,5-tetramethyl-1-oxyl; MMP-8, matrix metalloproteinase-8; ONOO0, peroxynitrite; a1-PI, a1-protease inhibitor; DAN, 2,3-diaminonaphthalene; NOS, nitric oxide synthase; ECM, extracellular matrix; NEM, Nethylmaleimide; DTT, dithiothreitol; Suc-Ala-Ala-Ala-MCA, succinylalanyl-alanyl-alanyl-4-methylcoumaryl-7-amide; L-NMMA, Nv-monomethyl-L-arginine; PCMB, p-chloromercuribenzoate; BSA, bovine serum albumin; PBS, 10 mM phosphate-buffered 0.15 M NaCl (pH 7.4); PMA, phorbol myristate acetate; KRP, Krebs’ Ringer’s phosphate; SOD, superoxide dismutase; DTPA, diethylenetriaminepentaacetic acid; HSA, human serum albumin.

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molecular oxygen and reactive oxygen species (12–15). It is therefore critical to clarify the mechanism of tissue destruction induced by a series of nitrogen oxides. ECMs such as collagen, a major structural component of various tissues and organs, are known to be modulated by a number of matrix metalloproteinases (MMPs) under physiological and pathological conditions, e.g., wound healing (16), arthritis (17), periodontal disease (18), immune complex-induced alveolitis (19), and tumor invasion and metastasis (20). MMPs, a group of zinc neutral endopeptidases, are produced by a variety of cells and are released extracellularly as inactive precursors (proMMPs) (21, 22). Activation of proMMP can be achieved either by limited proteolysis of the zymogens (21–23) or by chemical means, including chaotropic agents, organomercurial compounds, or reactive oxygen species (21–25). Human neutrophils produce a procollagenase (human neutrophil procollagenase), now known as proMMP-8, which plays an important role in remodeling and destruction of the ECM (21, 23–28). In this experiment, we investigated a mechanism of tissue injury induced by nitrogen oxides by analyzing the effect of a series of nitrogen oxides on the activation of human neutrophil procollagenase. EXPERIMENTAL PROCEDURES Substances. Human buffy coats were kindly supplied by Kumamoto Red Cross Blood Center (Kumamoto, Japan). Bovine Achilles’ tendon type I collagen, human placenta type I collagen, trypsin from bovine pancreas, and p-chloromercuribenzoate (PCMB) were purchased from Sigma Chemical Co. (St. Louis, MO). Porcine pancreatic elastase was from Elastin Products Co., Inc. (Owensville, MO). NvMonomethyl-L-arginine (L-NMMA) monoacetate and crystalline bovine serum albumin (BSA) were from Calbiochem (La Jolla, CA). Succinyl-alanyl-alanyl-alanyl-4-methylcoumaryl-7-amide (Suc-AlaAla-Ala-MCA), a fluorescent substrate for elastase, was from the Peptide Institute, Inc. (Osaka, Japan). A polyamine-zwitterion derivative of NONOate (CH3N[N(O)NO]0(CH2)3NH/ 2 CH3 , 1-hydroxy-2-oxo3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene) that spontaneously releases NO in solution (29), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO), and 2,3-diaminonaphthalene (DAN) were supplied from Dojindo Laboratories (Kumamoto, Japan). N-Ethylmaleimide (NEM), dithiothreitol (DTT), and phorbol myristate acetate (PMA) were from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). o-Phenanthroline was from Nacalai Tesque, Inc. (Kyoto, Japan). Human recombinant Cu,Zn-superoxide dismutase (SOD) (4390 unit/mg protein) was provided by Nippon Kayaku Co., Ltd. (Tokyo, Japan). Catalase (65,000 unit/mg protein) was a product of Boehringer Mannheim GmbH (Mannheim, Germany). Human hemoglobin was purified from red blood cells of healthy volunteers as was reported previously (30). Human a1-protease inhibitor (a1-PI) was purified from human plasma according to our previous method (31). Peroxynitrite (ONOO0) was synthesized by use of a quenched flow reactor as described by Radi et al. (32), in which a mixture of 0.7 M H2O2/0.6 M HCl was reacted with 0.6 M NaNO2 in a flow reactor (flow 10 ml/min of each), immediately followed by neutralization with 1.5 M NaOH (20 ml/min). NO2 gas (5.22 ppm) was provided by Taiyo Toyo Sanso Co., Ltd. (Osaka, Japan). All other chemicals were of the highest analytical grade commercially available. Isolation and purification of human neutrophil procollagenase. Human neutrophil procollagenase was purified as reported pre-

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viously (27, 33, 34), with modification. Briefly, leukocytes isolated from fresh buffy coats were homogenized, and the supernatant of the homogenate obtained by ultracentrifugation was applied to a DEAE-cellulose column (DE 52; Whatman, Maidstone, England). Subsequently, isolation of the procollagenase was performed serially on Matrex Red A, QAE Sephadex A50 (Pharmacia Fine Chemicals, Uppsala, Sweden), and HiTrap Blue (Pharmacia) column chromatographies. The procollagenase fraction was concentrated on an Amicon Centriplus-30 concentrator (Amicon, Inc., Boston, MA) and was finally purified on a column (1.5 1 85 cm) of Bio-Gel P100 (Bio-Rad Laboratories, Richmond, CA). Activation of neutrophil procollagenase by NO and its oxidized intermediates. Purified neutrophil procollagenase (1 mg, 600 nM) was incubated in various concentrations of propylamine NONOate (1– 10,000 mM) with or without 100 mM carboxy-PTIO in 20 ml of 10 mM phosphate-buffered 0.15 M NaCl (PBS; pH 7.4) at 357C for 60 min. We also examined the effect of authentic NO2 on the procollagenase. The procollagenase was treated with NO2 by bubbling the procollagenase solution in PBS (pH 7.4) with 5.22 ppm of NO2 gas at a flow rate of 1.0 ml/min for various time periods. Similarly, ONOO0 at various concentrations (0.4–1200 mM) was added to the procollagenase solution followed by incubation at 357C. ONOO0 has a short half-life (1.4 s) in the PBS solution (pH 7.4) (32). Thus, ONOO0 was repeatedly added to the reaction mixture containing procollagenase (three times, every 300 s). To maintain the effective concentration of ONOO0 in the reaction mixture, a constant flux infusion system was employed as was reported by Castro et al. (35). Briefly, to 400 ml of 500 mM Tris–HCl buffer (pH 8.2) with 10 mM CaCl2 containing the procollagenase at 1 mM was added constantly 2 mM ONOO0 in 10 mM NaOH at a flow rate of 40 ml/min (200 mM/min at a final concentration of ONOO0). Although the halflife of ONOO0 in 500 mM Tris–HCl buffer (pH 8.2) is 4.2 s, a steady level of ONOO0 was sustained at approximately 20 mM by this constant flux infusion. The effective concentration of ONOO0 in the mixture was monitored by measuring absorbance at 302 nm, and the activity of the collagenase generated was quantified as follows. The procollagenase (1 mg), treated or untreated with various nitrogen oxides, was incubated with human placenta type I collagen (3 mg) at 357C for 180 min. Then, collagen hydrolysis was analyzed by sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS–PAGE) in 7.5% polyacrylamide gel under reducing conditions with DTT according to the method of Laemmli (36). After electrophoresis, the protein band was stained with Coomassie brilliant blue R250. Collagenase activity was assessed by measuring the amount of the collagen fragmented proteolytically by densitometric analysis of the SDS–PAGE gel on a Macintosh computer (Quadra 800) combined with an Image Scanner (GT6500 ART2; Epson Co., Ltd., Tokyo, Japan) using the public domain NIH Image program (37). Furthermore, change in molecular size of neutrophil procollagenase was investigated by SDS–PAGE after treatment with either NO/ carboxy-PTIO or ONOO0. Briefly, the purified procollagenase (5 mg) was incubated in the reaction mixture (25 ml in PBS) containing 100 mM NONOate with or without 100 mM carboxy-PTIO or 100 mM ONOO0 at 357C for 60 min. The procollagenase treated with or without nitrogen oxides was then electrophoresed on SDS–PAGE (10% polyacrylamide) under reducing condition. After staining the protein band, the mobility of the collagenase in the gel was analyzed by using NIH Image program as just described. Analysis for proteolysis and inactivation of a1-PI by neutrophil collagenase activated by nitrogen oxides. Purified human neutrophil procollagenase (1 mg) was incubated either with NONOate in the presence or absence of carboxy-PTIO or with ONOO0 in PBS (pH 7.4) as just described. After the procollagenase treated or untreated with various nitrogen oxides was incubated with a1-PI (1.4 mg) at 357C for 180 min, a1-PI hydrolysis was analyzed by SDS–PAGE on 10% polyacrylamide gel as described above. Simultaneously, the

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ACTIVATION OF HUMAN NEUTROPHIL PROCOLLAGENASE BY NITROGEN OXIDES inhibitory activity of collagenase-treated a1-PI against a porcine pancreatic elastase was assayed as reported earlier (31). Specifically, after incubation of a1-PI with procollagenase treated with various nitrogen oxides, a1-PI was reacted with pancreatic elastase in 50 mM Tris–HCl buffer (pH 7.8) for 30 min at 377C, and then residual activity of the elastase was assessed fluorometrically with Suc-Ala-AlaAla-MCA as a substrate. Effect of DAN and hemoglobin on activation of neutrophil procollagenase by nitrogen oxides. To confirm the involvement of NO and NO2 in the activation of procollagenase, the effect of DAN and hemoglobin on NO-dependent activation of the neutrophil procollagenase was examined. It was recently clarified that DAN, which was originally used for NO0 2 measurement by Misko et al. (38), reacts directly with NOx such as NO2 or N2O3 to form naphthalene triazole at neutral pH (39). Thus, DAN can be applied as an NO2 quencher. Procollagenase was treated with nitrogen oxides in the presence or absence of various concentrations of DAN and hemoglobin in the reaction mixture of procollagenase with NONOate/carboxy-PTIO as described above. The collagenase activity was measured by assessing collagenolysis of type I collagen via SDS–PAGE. Simultaneously, the amount of naphthotriazole produced in each reaction system with DAN was quantified fluorometrically by measuring with the excitation wavelength at 375 nm and the emission wavelength at 425 nm as described previously (38). Activation of neutrophil procollagenase by nitrogen oxides released from human neutrophils. Human neutrophils were isolated from healthy volunteers by using a solution to separate monocytes and polymorphonuclear cells (Mono-Poly Resolving Medium; Dainippon Pharmaceutical, Osaka, Japan). Specifically, human peripheral blood was overlaid on the solution, followed by centrifugation at 400g for 30 min at room temperature. After the layer composed of neutrophils was collected, the cells were washed twice with Krebs’ Ringer’s phosphate (KRP, pH 7.4) without calcium ion and were centrifuged at 400g for 5 min at room temperature. The trace red blood cells, if the sample was contaminated, were removed by a red blood cell lysing buffer (140 mM NH4Cl/35 mM Tris–HCl, pH 7.6). Usually, human neutrophils with more than 95% purity and viability were obtained, as determined by microscopic observation after Giemsa staining and the trypan blue exclusion test, respectively. NO production by neutrophils was triggered by adding PMA (final concentration 100 ng/ml) to the cell suspension (cell density 4 1 107 cells/ml) in KRP (pH 7.4) with or without carboxy-PTIO (100 mM), propylamine NONOate (10 mM), DAN (10 mM), oxyhemoglobin (10 mM), catalase (3.25, 32.5, and 325 U/ml), SOD (43.9, 439, and 4390 U/ml), or L-NMMA (2 mM). Because PMA stimulation induces extracellular release of the neutrophil procollagenase as well as NO in this system, the action of nitrogen oxides on procollagenase can be tested simply by analyzing activity of procollagenase in the supernatant of the reaction mixture containing the neutrophils. Briefly, the supernatant of the cell suspension was harvested 1 h after initiating PMA stimulation in the presence or absence of various reagents and was incubated with type I collagen for 3 h at 357C. Collagen hydrolysis was then measured by SDS–PAGE as described. In some experiments, catalase, which was inactivated by boiling for 10 min, was used as a control to confirm the H2O2-scavenging effect of catalase on the procollagenase activation in the neutrophils. Similarly, SOD was treated with 10 mM H2O2 at 377C for 1 h and further incubated with 1.0 mM diethylenetriaminepentaacetic acid (DTPA) followed by dialysis against PBS (pH 7.4). By this treatment of Cu,Zn-SOD, SOD was completely inactivated, and copper iron in Cu,Zn-SOD, of which ligand was destroyed, was removed from the protein preparation as we reported previously (31). NO0 2 measurement and electron spin resonance (ESR) study using carboxy-PTIO for NO. The amount of NO0 2 produced in the reaction mixture of the NO2 gas bubbling system was quantified by highperformance liquid chromatography combined with Griess reagentflow reactor system (Eicom Corp., Kyoto, Japan) (40). During the

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NO-releasing reaction from the propylamine NONOate, the amount of NO generated in the reaction mixture was monitored continuously by ESR spectroscopy based on the change in ESR spectra of carboxyPTIO as we reported recently (41, 42). Specifically, NO released from NONOate is readily oxidized to NO2 by carboxy-PTIO. The carboxyPTIO is converted to 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-1-oxyl (carboxy-PTI), and generation of carboxy-PTI reflects well the stoichiometric conversion of NO to NO2 (41). Because carboxy-PTIO and carboxy-PTI give quite different ESR signals, the amount of NO and NO2 produced from the NONOate in the presence of carboxy-PTIO can be quantified by measuring carboxy-PTI via ESR spectroscopy at room temperature as reported earlier (41, 42).

RESULTS

Latency of neutrophil procollagenase and its activation by nitrogen oxides. Procollagenase from human leukocytes showed a homogeneous band, a single polypeptide chain of an apparent molecular size of 85 kDa on SDS–PAGE analysis under reducing conditions (Fig. 1A), consistent with the Mr reported previously (28). The protein band appears somewhat broad, possibly due to the highly glycosylated nature of the enzyme. Purified neutrophil procollagenase showed little collagenolytic action against type I collagen before activation. This indicates that the collagenase is in a latent form, i.e., zymogen (proenzyme) (Fig. 1B). However, strong collagenolytic activity was produced by treatment of the procollagenase with thiol-modifying agents such as NEM and PCMB (Fig. 1B). Also, this collagenase activity was almost completely inhibited by metalchelating agents such as o-phenanthroline and ethylenediaminetetraacetic acid (EDTA), indicating that collagen hydrolysis was catalyzed by metalloproteinase, i.e., MMP. To examine whether NO and its oxidized intermediates can activate procollagenase, the enzyme was treated with the propylamine NONOate, which spontaneously releases NO in neutral solution in the presence or absence of carboxy-PTIO. As shown in Fig. 2, carboxy-PTIO accelerates formation of NO2 from NO (15, 41, 42). The amount of NO and NO2 released from the NONOate/carboxy-PTIO system was quantified by ESR spectroscopy as assessed by the generation of carboxy-PTI, a product of carboxy-PTIO reacted with NO. As demonstrated in Figs. 2A and 2B, NO was generated in the reaction system in a quantitative manner; the amount of NO released was almost twice the amount of the NONOate added to the system. In addition, simultaneous production of NO2 can be expected by generation of carboxy-PTI (NO2 is produced through oxidation of NO by carboxy-PTIO to form carboxy-PTI) (41, 42). The rate of NO release from the NONOate is 6.65 mM/min (T1/2 , 1.7 min), when 10 mM propylamine NONOate was incubated with 100 mM carboxy-PTIO in PBS (pH 7.4) at 357C. According to this NO-releasing kinetics, we understand that almost 100% of the NONOate is consumed in the reaction mixture of the NONOate

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FIG. 1. SDS–PAGE of purified human neutrophil procollagenase (ProHNC) (A), and latency of the enzyme assessed by its collagenolytic activity before and after treatment with thiol-modifying agents (B). (A) The purified neutrophil procollagenase (5 mg) was electrophoresed using SDS–PAGE (10% polyacrylamide gel) under reducing conditions. (B) The purified procollagenase (1 mg) treated or untreated with either NEM (1 mM) or PCMB (1 mM) was incubated with human type I collagen (3 mg), followed by SDS–PAGE (7.5% gel) under reducing conditions. The protein bands were stained with Coomassie brilliant blue R250. Note that the procollagenase is activated by both NEM and PCMB and is inhibited by metal-chelating agents such as o-phenanthroline and EDTA. See text for details.

and carboxy-PTIO within the incubation period (60 min), where carboxy-PTIO is added at a concentration exceeding that of NONOate used. Treatment of procollagenase in the reaction mixture containing the propylamine NONOate alone showed weak collagenolysis only at high concentrations of the NONOate (10,000 mM or higher) (Figs. 3A and 3B). Any significant activation of procollagenase was not observed with NO under anaerobic condition without carboxy-PTIO, where the reaction mixture had been degassed and bubbled with helium gas before addition of the procollagenase and the NONOate. Thus, the procollagenase activation by the higher concentration of NONOate (more than 1 mM) under aerobic condition was produced via the formation of NO2 .

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In contrast, incubation of procollagenase with the propylamine NONOate and carboxy-PTIO resulted in marked activation of the neutrophil procollagenase, as evidenced by generation of fragments a1A and a2A; activation was dependent on the concentration of the NONOate added to the reaction system (Figs. 3A and 3B). Formation of the cleavage products of collagen (a1A, a2A, and a1B) (Fig. 3A) indicates that the collagenolysis was mediated by a specific catalytic action of the neutrophil collagenase. Activation of the procollagenase appears to occur at the NONOate concentration of 1 mM in the presence of carboxy-PTIO, and it became maximum at 10 mM (Figs. 3A and 3B). Activation of the procollagenase with NO2 was further substantiated by the reaction of the procollagenase with NO2 gas in solution. Specifically, as shown in Figs. 3C and 3D, activation of the neutrophil procollagenase was obtained clearly in a time-dependent fashion. During the NO2 bubbling in the reaction mixture, NO2 gas was efficiently dissolved in the solution as evidenced by increase in the concentration of NO20 (Fig. 3D), where pH of the solution was not changed during NO2 bubbling. This indicates that NO2 directly activates the human neutrophil procollagenase, although it may be that the molecular species responsible for the activation is not only NO2 per se but also HNO2 as well as N2O4 derived from NO2 . Furthermore, the activation potential of another important nitrogen oxide, ONOO0, was investigated (Fig. 4). As reported, ONOO0 possesses a short half-life in neutral solution; thus, ONOO0 was added repeatedly to the reaction mixture (three times, every 300 s) to maintain its effective concentration for activation of the procollagenase. When we compared the activation efficacy by ONOO0 between a single bolus and the repeated additions of 100 mM ONOO0, the procollagenase activation was potentiated with increasing number of times of ONOO0 addition (Fig. 4A). Incubation of the procollagenase with ONOO0 at 357C resulted in significant procollagenase activation in a ONOO0 concentration-dependent fashion (Figs. 4A and 4B). Cleavage of the collagen became apparent at 1.2 mM ONOO0, and maximal activation of the collagenase occurred at the ONOO0 concentration range of 36 to 120 mM. It should be noted that ONOO0 could activate the procollagenase at physiological concentrations ranging from 1.2 to 36 mM. On the contrary, no apparent procollagenase activation was obtained when ONOO0 was added to the reaction mixture 10 min before addition of the procollagenase solution (Fig. 4A). Moreover, the procollagenase activation by ONOO0 was examined by using the constant flux infusion system. As a result, a constant flux maintaining about 20 mM concentration of ONOO0 (Fig. 4C) gave a rapid and effective activation of the procollagenase within 1 min (Fig. 4D). During the constant infusion, the pH of the

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FIG. 2. (A) A scheme for the NO-releasing reaction of the propylamine NONOate and for subsequent generation of NO2 via oxidation with carboxy-PTIO. (B) Time profile of NO release from NONOate (10 mM) in PBS (pH 7.4) at 357C; the amount of NO was quantified by ESR spectroscopy with use of carboxy-PTIO (100 mM). The change in the ESR spectrum of carboxy-PTIO during NO release is shown (B). Data are shown as means { SE of three different experiments. See text for details.

solution (pH 8.2) did not vary significantly. This is consistent with the procollagenase activation observed by the repeated addition of ONOO0 (Figs. 4A and 4B). Activation of neutrophil procollagenase determined by proteolytic inactivation of a1-PI. We tested activation of neutrophil procollagenase induced by nitrogen oxides by using purified human plasma a1-PI as a substrate for the collagenase (25). a1-PI was treated with the neutrophil collagenase activated in the reaction of NONOate/carboxy-PTIO as well as ONOO0. As shown in Fig. 5A, native a1-PI showed a 54-kDa homogeneous band by SDS–PAGE, and the specific cleavage product of 50 kDa was generated by incubating a1-PI with the procollagenase treated with either NONOate/carboxyPTIO or ONOO0 (Figs. 5A and 5B). The amount of the 50-kDa fragment of a1-PI was increased in a concentration-dependent manner with NONOate and ONOO0. These results indicate that a1-PI was proteolytically cleaved by the active form of the neutrophil collagenase produced in the reaction of the procollagenase with either NO2 or ONOO0, similar to the type I collagen as just described. In contrast, no appreciable proteolysis was observed for procollagenase treated with NO without carboxy-PTIO below the concentration of 10,000 mM NONOate (Fig. 5A). Also, the elastase inhibitory activity of a1-PI was significantly abrogated by treatment of neutrophil procollagenase with NONOate/carboxy-PTIO or ONOO0 (Fig. 5C), which was consistent with the proteolytic degradation of a1-PI by the neutro-

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phil collagenase activated by these nitrogen oxides (Figs. 5A and 5B). Change in molecular size of neutrophil procollagenase during activation with nitrogen oxides. To examine whether proteolytic modification of neutrophil procollagenase occurred during activation with nitrogen oxides, similar to the activation with various proteases (21–23, 34), the neutrophil procollagenase treated with NONOate/carboxy-PTIO or ONOO0 was subjected to SDS–PAGE analysis. After electrophoresis of the procollagenases, the mobility of each protein band of the collagenase was analyzed by use of the NIH Image program as described earlier. The results shown in Fig. 6A indicate that the molecular mass of the collagenase was not affected even when strong activation was obtained by the treatment with these nitrogen oxides. This is in clear contrast to the proteolytic activation with trypsin demonstrated in Fig. 6A (34). Reversibility of activity of neutrophil collagenase activated by nitrogen oxides. To characterize the chemical modification of the collagenase activated with nitrogen oxides, reversibility of the enzyme activity was tested. After activation of the procollagenase with either 100 mM ONOO0 or 10 mM NONOate and 100 mM carboxyPTIO in the same manner as described earlier, the procollagenase treated with these nitrogen oxides was incubated in the presence or absence of 100 mM DTT at 357C for 30 min, followed by treatment with or without 0.5 mM PCMB at 357C for 30 min. The procollagenase

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FIG. 3. Activation of human neutrophil procollagenase with an NO-releasing system with or without carboxy-PTIO. (A) SDS–PAGE showing activation of neutrophil procollagenase after treatment with the NONOate/carboxy-PTIO system (NO/NO2 releasing system) as was determined by collagenolytic activity against type I collagen in the same manner as shown in Fig. 1. (B) Collagenolytic activity generated by treatment with the NO/NO2-releasing system was quantified by measuring the amount of the collagen degraded (cleavage of a1 and a2 of native collagen to form a1A and a2A). In some experiments, the procollagenase was treated with the NONOate under anaerobic condition. (C) Collagenolysis by the procollagenase treated with NO2 gas. (D) Collagenolytic activity was measured by densitometry, and the amount of NO0 2 generated during NO2 bubbling in the solution is shown. Data are shown as means { SE of three different experiments. See text for details.

was further incubated with type I collagen and then subjected to SDS–PAGE as described for Fig. 4. The result demonstrated in Fig. 6B showed that the activation of procollagenase by NO2 and ONOO0 was reversible. Specifically, after the activation of the procollagenase by ONOO0, the enzyme could revert to its inactive form in the presence of excessive amounts of DTT. Further, the collagenase activity is generated again by treatment of the enzyme with PCMB. A similar result was obtained with the neutrophil procollagenase activated with the NO/carboxy-PTIO system (data not shown). Effect of various nitrogen oxide scavengers on activation of neutrophil procollagenase by NONOate/carboxy-PTIO or ONOO0. To examine further the chemical reactivity of NO, NO2 , and ONOO0 with procollagenase, the effect of various compounds on activation

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of procollagenase was investigated. We used oxyhemoglobin and DAN as scavengers for NO and NO2 , respectively. As demonstrated in Fig. 7, DAN markedly inhibited activation of neutrophil procollagenase in the NONOate/carboxy-PTIO system. Procollagenase activation induced by ONOO0, however, was not affected by DAN. A naphthotriazole derivative of DAN, which is a product in the reaction of NO2 or N2O3 and DAN (39), was produced in a concentration-dependent manner with DAN in the reaction system of NONOate/carboxy-PTIO (Fig. 7). This indicates that DAN indeed directly scavenged NO2 and thus prevented activation of the procollagenase. In fact, there was no appreciable generation of naphthotriazole in the reaction of DAN with ONOO0. Furthermore, oxyhemoglobin, but not methemoglobin, markedly reduced activation of procollagenase with NONOate/carboxy-PTIO. Oxyhemoglo-

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FIG. 4. Activation of neutrophil procollagenase by ONOO0. After purified neutrophil procollagenase was treated with various concentrations of ONOO0, collagenolysis was assessed by SDS–PAGE as described in Fig. 1, and collagenase activity produced was quantified. (A) SDS–PAGE showing collagenolysis by the procollagenase treated either with three times addition of ONOO0 (A-a) or with different times of addition (A-b). In the lane 120* (A-a), 120 mM ONOO0 was given three times 10 min before addition of the procollagenase to the reaction mixture. (B) The collagenolysis observed was quantified by densitometric analysis. (C) Time profile of the concentration of ONOO0 in a constant flux infusion system. (D) Collagenolysis induced by the constant flux infusion of ONOO0; constant infusion was performed in a same manner as in (C) except that ONOO0 was given continuously to the reaction mixture without pause of syringe pump. Data are shown as means { SE of three different experiments. See text for details.

bin only marginally inhibited the ONOO0-induced procollagenase activation, but methemoglobin did not suppress the activation with ONOO0 (Fig. 8). A weak inhibition of the ONOO0-dependent procollagenase activation by oxyhemoglobin may be due to a slow reaction rate of ONOO0 with oxyhemoglobin as was reported recently by Denicola et al. (43). In a separate experiment, a product analysis was performed with the reaction mixture of oxyhemoglobin and NONOate/carboxy-PTIO. The result showed a quantitative and stoichiometric conversion of oxyhemoglobin to methemoglobin that depended on NO generated from NONOate (data not shown). All these results indirectly substantiate the direct interaction of NO2 and ONOO0 with neutrophil procollagenase resulting in activation of the zymogen.

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Evidence for involvement of nitrogen oxides in procollagenase activation in human neutrophils. After human neutrophils were stimulated with PMA in the presence or absence of carboxy-PTIO or NONOate, the activity of collagenase released in the supernatant of the neutrophil sample was quantified by measuring collagen hydrolysis by SDS–PAGE as described above. Collagenase was released from the neutrophils on stimulation with PMA (Fig. 9). The following experiments were constructed to see involvement of NO, peroxynitrite (via O20 and NO), NO2 , and myeloperoxidase (H2O2), as was assessed by the effect of oxyhemoglobin, L-NMMA, NONOate, SOD, carboxy-PTIO, DAN, and catalase (Figs. 9 and 10). Collagenase activity was enhanced by addition of carboxy-PTIO to the reaction mixture of the neutrophils

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FIG. 5. Activation of neutrophil procollagenase by NO2 (A) and ONOO0 (B) determined by proteolytic cleavage of a1-PI with the collagenase. (A) Neutrophil procollagenase treated with propylamine NONOate in the presence or absence of carboxy-PTIO (100 mM) was reacted with a1-PI, and cleavage of a1-PI was examined by SDS–PAGE. (B) Activation of procollagenase with ONOO0 was analyzed in the same manner as in (A). (C) Inactivation of a1-PI by neutrophil collagenase activated with NO2 and ONOO0. Residual activity of a1-PI after incubation with the collagenase was assayed by measuring its elastase inhibitory activity fluorometrically by using a synthetic peptidyl substrate for a pancreatic elastase. Data are means { SE (n Å 3). See text for details.

with PMA and was significantly inhibited by both LNMMA and oxyhemoglobin (Figs. 9 and 10). In addition, procollagenase activation was further potentiated by NO given exogenously with the propylamine NONOate and was inhibited by oxyhemoglobin similar to other systems. SOD had little effect on activation of the procollagenase in PMA-stimulated neutrophils without addition of carboxy-PTIO and NONOate (Fig. 10A), and it weakly inhibited its activation in the neutrophils with carboxyPTIO (Fig. 10B). Interestingly, the activation of the procollagenase in the neutrophils with NONOate was strongly suppressed by SOD (Fig. 10C). On the contrary, catalase attenuated the procollagenase activation in all three PMA-stimulated neutrophil systems (Fig. 10). Although catalase showed inhibition of the procollagenase activation in neutrophils with or without carboxy-PTIO (Figs. 10A and 10B), reduction of the activation by catalase was more prominent in the neutrophils with NONOate (Fig. 10C) than in other neutrophil systems. In each experiment with catalase and SOD, heat-inactivated catalase and H2O2-inactivated/DTPA-treated SOD did not show significant suppressive effect on the procollagenase activation, except that the highest concentration of the inactivated SOD

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(4390 U/ml) reduced marginally the procollagenase activation in all systems (data now shown). Although DAN strongly abrogated the generation of collagenase activity in PMA-stimulated neutrophils in the presence of carboxy-PTIO (Fig. 10B), it did not appreciably inhibit collagenase activation in a reaction mixture without carboxy-PTIO (Fig. 10A). It is intriguing that the procollagenase activation enhanced by NONOate was also significantly suppressed by DAN (Fig. 10C). DISCUSSION

It has been shown that bioactive molecules can be chemically modified by various nitrogen oxide species such as NO2 , HNO2 , N2O4 , N2O3 , and ONOO0; almost all proteins and enzymes are inactivated or degraded (44, 45), and lipids, especially those containing unsaturated bonds, are oxidized, which triggers lipid peroxidation reaction. It has been reported that NO activated soluble guanylate cyclase via formation of nitrosyl heme of the prosthetic group of the enzyme (46–48). A similar activation by NO has been described with cyclooxygenase, although its molecular mechanism is not fully understood (49). Of interest is a recent finding

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FIG. 6. (A) Effect of treatment with nitrogen oxides on the molecular size of human neutrophil procollagenase (ProHNC). Procollagenase was incubated with NONOate (100 mM)/carboxy-PTIO (100 mM), ONOO0 (100 mM), or trypsin (4.4 mg/ml) at 357C for 60 min and was electrophoresed using SDS–PAGE (10% polyacrylamide gel) under reducing condition as shown in Fig. 1. Mobility of the protein band of collagenase measured by densitometric analysis is shown. (B) Reversibility of activation of the neutrophil procollagenase with ONOO0. After activation of the procollagenase with 100 mM ONOO0, the procollagenase treated with these nitrogen oxides was incubated with 100 mM DTT at 357C for 30 min, followed by treatment with or without 0.5 mM PCMB at 357C for 30 min. Data are means { SE (n Å 3). See text for details.

that the NOS inhibitor L-NMMA inhibited collagenolytic activity in bovine and human articular cartilage that expressed the inducible isoform of NOS on stimulation with interleukin 1b (50). In the present experiment, it became apparent that nitrogen oxides, e.g., NO2 and ONOO0, activate a latent form of human neutrophil procollagenase. Activation of the neutrophil procollagenase was brought about by reacting the purified procollagenase with micromolar concentrations of NO2 and ONOO0. In our current study, the NO-releasing compound propylamine NONOate (Fig. 2) was used as an NOgenerating system. A series of NO-amine complexes such as propylamine NONOate, which were originally

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described by Keefer’s group (29), released NO according to the reaction scheme in Fig. 2A. One molecule of the NONOate contains two molecules of NO, both of which can be released spontaneously in neutral solution, as was verified by our recent investigation using liposomeencapsulated PTIO (42). Carboxy-PTIO, which we found to oxidize and scavenge NO effectively in solution (41), reacts with NO to form NO2 in a completely stoichiometric manner (Fig. 2) (42). Propylamine NONOate with carboxy-PTIO showed a strong activating potential for human neutrophil procollagenase at micromolar concentrations of the NONOate (Fig. 3). This indicates that NO2 can effectively activate the latent collagenase. However, NO is 1000

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FIG. 7. Effect of DAN on activation of neutrophil procollagenase in the NONOate/carboxy-PTIO system or with ONOO0. Neutrophil procollagenase was treated either with 10 mM NONOate plus 100 mM carboxy-PTIO or with 10 mM ONOO0 (three additions, once every 300 s) in the presence of various concentrations of DAN, and collagenase activity was measured by SDS–PAGE as shown in Fig. 3. Simultaneously, the amount of naphthotriazole produced in each reaction system is demonstrated. Data are means { SE of three different experiments. See text for details.

times less potent in procollagenase activation; in the absence of carboxy-PTIO, more than 1 mM NONOate was needed to obtain significant activation of the procollagenase, although this activation was apparent only under aerobic (ambient) condition. It is therefore highly plausible that nitrogen oxides such as NO2 or N2O3 , rather than NO itself, are implicated in enzyme activation. Furthermore, possible involvement of NO2 , which depends on the L-arginine-dependent NO biosynthesis pathway in human neutrophils (51), was suggested in activation of procollagenase released from the neutrophils on stimulation with PMA. Peroxynitrite anion (ONOO0) also showed a strong activating potential for neutrophil procollagenase (Fig. 4). It is well documented that ONOO0 is formed via a diffusion-limited rapid reaction between O20 and NO (rate constant 6.7 1 109 M01 s01) and has oxidizing and toxic actions under various pathological conditions (52–57). Once ONOO0 is protonated, peroxynitrous acid (ONOOH) rapidly undergoes intramolecular rearrangement to form NO30 , a highly oxidizing intermediate species, which has not yet been identified, is supposed to be generated and to exert a potent oxidizing action (54).

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It was recently reported that DAN, which has been used for quantification of NO20 under acidic conditions (38), can react directly with NO2 and N2O3 to produce naphthotriazole in neutral solution (39). In the present experiment, we applied DAN as an NO2 scavenger, and DAN strongly suppressed activation of the procollagenase induced by both the NONOate/ carboxy-PTIO system and human neutrophils in culture stimulated with PMA. In contrast, DAN did not significantly affect activation of the procollagenase mediated by ONOO0, indicating that NO2 is not involved in activation of the procollagenase by ONOO0 in the cell-free reaction system. The mechanism of the procollagenase activation in the PMA-stimulated neutrophils appears complex comparing with the procollagenase activation in cell-free systems. However, based on the effect of various substances on the activation of procollagenase in three different reaction systems of PMA-stimulated neutrophils in culture, it is interpreted that somewhat different molecular mechanisms might be operative in each procollagenase activation systems in the neutrophils. Namely, at first, in PMA-stimulated neutrophils without addition of carboxy-PTIO or NONOate, the H2O2-

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FIG. 8. Effect of oxyhemoglobin (OxyHb) and methemoglobin (MetHb) on activation of neutrophil procollagenase in the NONOate/carboxyPTIO system or with ONOO0. Procollagenase treated with NONOate/carboxy-PTIO or ONOO0 with various concentrations of either OxyHb or MetHb was assayed for collagenase activity in the same manner as shown in Fig. 3. Data are shown as means { SE of three different experiments. See text for details.

myeloperoxidase system is a main pathway for the procollagenase activation as evidenced by a strong inhibition of the activation by catalase (Fig. 10A). In the presence of carboxy-PTIO, NO2 which possibly originated from NO produced by the neutrophils seems to exhibit activating potential for the procollagenase (Fig. 10B). However, the H2O2-myeloperoxidase system is partly involved in the procollagenase activation in this neutrophil system with carboxy-PTIO, because moderate inhibition of the activation was also brought about by catalase. In contrast, in the neutrophil system with NONOate, both ONOO0 and the H2O2-myeloperoxidase system seem to be involved in the procollagenase activation in the neutrophils incubated with NONOate, because both SOD and catalase profoundly attenuated the procollagenase activation in the PMA-stimulated neutrophils with NONOate (Fig. 10C). Intriguingly, DAN, which did not inhibit the procollagenase activation by

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ONOO0 in the cell-free system, also significantly attenuated the procollagenase activation in this NONOate system. This result may suggest an indirect involvement of ONOO0 in the procollagenase activation, possibly through the formation of another unidentified reactive intermediate species, which is derived from ONOO0 and the H2O2-myeloperoxidase system. In this context, generation of a potent oxidizing species such as NOCl has been proposed by Koppenol in the reaction of ONOO0 with HOCl (58). A previous study showed that reactive oxygen species, such as O20 , hydroxyl radical, and OCl0 (produced by neutrophil myeloperoxidase), can activate procollagenase (23). The Zn atom in the active center of the collagenase is bound with three ligands of His in the mature domain of the activated form of collagenase. At another coordinate binding site for the Zn atom in the procollagenase, the Zn atom is coordinated with a thiolate anion of a free Cys 71 which is located in the pro-

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FIG. 9. SDS–PAGE analysis of activation of procollagenase in PMA-stimulated human neutrophils. The activation of procollagenase in neutrophils stimulated with PMA with or without carboxyPTIO was tested by collagenolytic activity against type I collagen as described for Fig. 3, and the effect of various compounds on the activation was examined. All compounds were added to the reaction mixture of the neutrophils during stimulation with PMA in the presence or absence of carboxy-PTIO, except that PCMB (1 mM) was added to the supernatant of the reaction mixture of the neutrophils, followed by assay with SDS–PAGE. See text for details.

peptide domain, also called the autoinhibitory domain. The oxidative modification is considered to occur on the thiolate moiety of the Cys residue of the propeptide domain, not in the ligands of the mature enzyme. In the cysteine switch hypothesis proposed by Van Wart and Birkedal-Hansen (28), a zinc atom in the active site of the enzyme is complexed with a cysteine residue at the PRCG(V/N)PD region of the propeptide domain that is well conserved among a series of MMPs, and dissociation of this cysteine–Zn complex results in substrate binding to the enzyme active site (59). Nitrogen oxides may cause oxidation, nitration, or nitrosation of the thiol moiety (32, 60) of Cys 71 and thus cause dissociation of coordinate binding between Cys and the Zn atom, which would confer activation of the procollagenase. Several lines of evidence show that NO2 directly degrades various proteins and peptides. It was proposed by Hood et al. that NO2 would react with Lys and Arg residues of a1-PI, which are exposed outside its peptide

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backbone, and would bring about peptide bond cleavage (44). In our experiments, however, no apparent change in molecular size of the collagenase was observed during activation of procollagenase by both NONOate/carboxy-PTIO and ONOO0 (Fig. 6A), suggesting that the proteolytic processing of the propeptide of procollagenase does not occur during its activation by these nitrogen oxides. As demonstrated in Fig. 6B, the neutrophil collagenase activated by nitrogen oxides (NO2 and ONOO0) reverted to the inactive form of the enzyme by the treatment with DTT, which can be activated again by PCMB. This indicates that the Zn atom in the collagenase remains intact even after ONOO0/NO2 treatment. It is well documented that nitrogen oxides (NOx) such as HNO2 , N2O3 , and alkylnitrites serve as potential sources of NO/ (60), which appears to be the most responsible molecular species for formation of nitrosothiols (thionitrite). NO itself does not possess a nitrosating potential at neutral pH. In fact, a recent study by DeMaster et al. revealed that NOx produced from NO under aerobic condition reacts with a single thiol moiety (Cys) in human serum albumin (HSA), producing thionitrite in the Cys residue (60). Although formation of sulfenic acid of the Cys moiety in HSA has been suggested (60), possible formation of other types of RSNOx products, e.g., thionitrate (RSNO2), as a transient intermediate (61) is also proposed by Radi et al. in BSA treated with ONOO0 (32). Thus, based on the fact that the neutrophil collagenase activated by nitrogen oxides is reversibly converted to the inactive proenzyme by DTT, it is likely that thionitrite or thionitrate formation of Cys 71 in the propeptide domain of the neutrophil procollagenase resulted in dissociation of coordinate binding of the Cys–Zn complex in the active center of the enzyme. In addition, it is reported that Trp and Tyr residues in the protein were considerably decreased by the treatment with NO2 ; nitroindole derivatives and 3-nitrotyrosine as well as dityrosine were formed, and bovine serum albumin and g-globulin became crosslinked by nondisulfide bonds (62). Therefore, it is plausible that the target for NO2 is not only the thiol moiety of Cys 71 in procollagenase but also other amino acid residues such as Trp, Tyr, Lys, and Arg. More specifically, oxidation or nitration of these amino acids could confer a conformational change of procollagenase, and instability of the thiol moiety in the cysteine switch of the zymogen may facilitate dissociation of the Cys–Zn complex. Based on the present findings, we suggest that oxidized intermediates of NO, i.e., NO2 and ONOO0, could play important roles in tissue injury in inflammation and various diseases via activation of the neutrophil procollagenase. In addition, these reactive nitrogen oxides may have physiological functions in tissue remod-

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FIG. 10. Effect of various substances on activation of procollagenase in PMA-stimulated human neutrophils. Activation of procollagenase in neutrophils stimulated with PMA with or without carboxy-PTIO was examined by SDS–PAGE in a same manner as shown in Fig. 3. The effect of various substances on the activation was assessed by densitometric analysis as shown in Fig. 3. Data obtained from four different SDS–PAGE experiments (Fig. 9) are shown as means { SE. See text for details.

eling of the ECM which is typically observed in tissue injury and wound healing. ACKNOWLEDGMENT We thank Judith B. Gandy for editorial work and Rie Yoshimoto for preparing the manuscript.

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