Purification of nitric oxide synthase from rat macrophages.

Nitric oxide (NO) synthase (EC 1.14.23) has been purified to apparent homogeneity from rat macrophages. The purification procedure involves affinity chromatography with adenosine 2',5'-diphosphate-agarose and gel filtration chromatography on a Superose 12 HR 10/30 column. The apparent molecular weight is 300,000 by gel filtration. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the enzyme migrates as a single protein band with Mr = 150,000. The purified enzyme is colorless, and an absorption maximum is observed at 280 nm. The half-life of the enzyme activity is 6 h at pH 7.4 and 4 degrees C. The enzyme activity required the presence of NADPH, (6R)-5,6,7,8-tetrahydro-L-biopterin, and dithiothreitol. Although the cerebellar and endothelial enzyme require Ca2+ and calmodulin, these are not required by the macrophage enzyme. The macrophage nitric oxide synthase (an inducible enzyme) seems to be different from the cerebellar and endothelial enzyme (a constitutive enzyme).

Gubrin vaccine (12). Macrophages were placed on 10-cm culture dishes containing Dulbecco's modified Eagle's medium (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan), 10% fetal calf serum (Gibco Laboratories), and 10 ng/ml lipopolysaccharide. Cells were incubated for 24 h in humidified 5% CO, and 95% air at 37 "C. After incubation, nonadherent peritoneal exudate cells were removed by washing three times with phosphate-buffered saline. The adherent cells were scraped with a rubber policeman and collected.
Cells were then disrupted by sonication for 10 s (Dial 6, UR-BOOP, Tomy, Tokyo, Japan), and the sonicate was centrifuged a t 100,000 X g for 60 min a t 4 "C. The supernatant was removed and used as the cytosol fraction.
All other chemicals were from Wako Pure Chemical Industries (Osaka, Japan), unless otherwise specified.

Methods
Enzyme Assay-The activity of nitric oxide synthase was determined by measuring the generation of nitrite and nitrate, breakdown products of nitric oxide. The complete reaction mixture (600 p1) contained purified enzyme, 50 mM Tris-HC1 (pH 7.4), 1 mM NADPH, 1 mM L-arginine, 1 mM DTT, 0.1 mM (GR)-BH,, and 1 p M FAD. When necessary, 5 mM EGTA was added in the absence of Ca'+. The mixture was incubated for 20 min at 37 "C, and the reaction was stopped by boiling a t 100 "C for 30 s. The tube was centrifuged a t 15,000 X g for 20 min, and the resulting supernatant was passed through a Millipore TGC filter (Millipore Corp., Bedford, MA). The filtrate was analyzed for nitrite and nitrate. The total recovery of nitrite and nitrate was 90%. Nitrite and nitrate were determined independently. Enzyme activity was expressed as nitrite + nitrate formation/min/mg of protein.
Nitrite (NO;) Determination-Nitrite in the enzyme reaction mixture was determined by the post-column method using the diazocoupling reaction (13). NADPH required as a substrate in the enzyme reaction was found to interfere with this reaction. Therefore, we separated nitrite from NADPH using ion-pair chromatography.
Nitrate (NO;) Determination-Nitrate was determined by ion chro-  were injected through an autosampler (SII,-GH) onto an anion-exchange column (a quaternary ammonium is incorporated in polymethacrylate resin)(Shim-pack IC-AI, 100 X 5.0 mm, Shimadzu Corp.). The column temperature was 40 "C. The carrier was 1.2 mM potassium hydrogen phthalate (pH 4.2) a t a constant flow rate of 1.5 ml/ min. The detector was CDD-6A (electrical conductivity detector). The detection limit and coefficient of variation ( n = 10) were 0.5 pmol and 0.4'76, respectively.
Arginine and Citrulline Determination-Arginine loss and citrulline formation were 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.
Purification of Nitric Oxide Synthase-The purification started from 15 ml of cytosol. The cytosol (7.5 ml) was applied to a column of 2',5'-ADP-agarose (1 X 2 cm, Sigma) equilibrated with 50 mM Tris-HCI buffer a t pH 7.4 containing 1 mM DTT. The column was washed with 10 ml of 50 mM Tris-HCI containing 1 mM DTT and 750 mM NaCl and with 10 ml of 50 mM Tris-HCI containing 1 mM DTT, successively. The enzyme was then eluted with 2 ml of 50 mM Tris-HCI containing 1 mM DTT and 1 mM NADPH and was concentrated to 50 p1 using a Centricon 10 (Amicon, Danvers, MA). This sample was injected onto Superose 12 HR l0/30, a gel filtration column (300 X 10 mm, Pharmacia LKR Biotechnology Inc., Uppsala, Sweden), which was equilibrated with 50 mM Tris-HCI containing 1 mM DTT. Superose resin is a cross-linked agarose-based medium with average particle size of 10 * 2 mm. This step was carried out by FPLC system (Pharmacia LKB Biotechnology Inc.) a t room temperature. Eluents were fractionated by 0.5 ml. Sodium Ilodecyl Sulfate (SI)S)-pol.vacrvlamidr Grl Elrctrophoresis-Electrophoresis was performed using the I'hast-System (Pharmacia LKR Biotechnology Inc.) on a 10-1596 gel according to the manufacturer's directions. Proteins were stained with Coomassie Blue. Molecular weight standards (Pharmacia LKR Riotechnology Inc.) were: thyroglobulin, 330,000; ferritin, 220,000; alhumin, 67,000; catalase, 60,000; and lactate dehydrogenase, 36,000.
Other Determinations-The ahsorption spectrum of nitric oxide synthase at pH 7.4 was determined using a Beckman DU-65 spect.rophotometer with a microcell. The protein concentration was determined by a dye-binding microassay (Rio-Itad). Bovine serum alhumin was used as the standard. Table I shows the the purification steps of macrophage nitric oxide synthase.

RESULTS AND DISCUSSION
The molecular weight of the enzyme is 300,000 on Superose 12 HR 10/30 gel filtration chromatography (shaded area in Fig. 1). On SDS-polyacrylamide gel electrophoresis, the active fractions exhibit a single band with M, = 150,000 (Fig. 2).
Thus, this protein appears to be a dimeric protein.
The purified enzyme exhibited a half-life of 6 h at pH 7.4 and 4 "C, whereas the cytosol preparation was stable after 24 h (Fig. 3). This might be due to the loss of a stabilizing factor, as reported for a neutrophil nitric oxide synthase (14) or might simply be due to the low protein concentration. The addition of NADPH, (6R)-BH,, and FAD did not increase stability. A poor yield during purification is considered to reflect instability of the enzyme due to loss of stabilizing factor. The loss of cofactors during purification may also induce instability of the enzyme. The half-life of the nitric oxide synthase purified from neutrophils was 3 h at pH 7.4 and 4 "C, and the specific activity was 122 nmol/min/mg protein. 2 The specific activity of the macrophage nitric oxide synthase was 944 (Table I). This increased activity may derive from the longer half-life of the macrophage enzyme.
The enzyme was colorless, and an absorption maximum was observed a t 280 nm (Fig. 4).  values for cofactors of the purified enzyme are shown in Table 11. Table I11 shows the percent decrease in activity by omitting each of the cofactors. NADPH, (6R)-BH4, and DTT were necessary for enzyme activity; however, the contribution of FAD was not great.
Calmodulin antagonists (100 p~ of W5, W7, W13, and trifluoperazine dihydrochloride) did not inhibit purified enzyme activity. Addition of calmodulin (1 PM) to the purified enzyme did not increase activity. The addition of EGTA to the reaction mixture without Ca2+ did not affect enzyme activity. Therefore, the macrophage enzyme was considered not to require Ca2+ and calmodulin for activation.
The apparent K,,, values for arginine and the V,,, at 37 "C and pH 7.4 are 32.3 mM and 1052 nmol/min/mg protein, respectively (Fig. 5). This enzyme functions optimally at pH 7-8.   mixture on percent inhibition of enzyme activity Enzyme activity was determined in the absence of NADPH, (6R)-BH,, or FAD using the purified enzyme containing 50 mM Tris-HC1 and 1 mM DTT and is expressed as the percent decrease of the activity obtained in the presence of all agents. DTT was added from the preparation of cytosol. In the DTT experiment, the purified enzyme was again injected into a Superose 12 HR 10/30 column (Pharmacia LKB Biotechnology Inc.), which was equilibrated with 50 mM Tris-HC1 (pH 7.4) buffer without DTT. The active fraction was then incubated with or without 1 mM DTT and each enzyme activity was compared. The other conditions of the enzyme reaction were the same as those stated under "Experimental Procedures." -NADPH

10%
In the stoichiometry studies, 10.5 p M arginine consumption led to the formation of 9.7 p~ citrulline and 10.8 pM (nitrite + nitrate) formation. Thus, the stoichiometry of arginine loss to the formation of citrulline and nitrite + nitrate formation seems to be 1/1/1.
In rat, nitric oxide synthase consists of 0.017% of the protein in the 15,000 x g supernatant of cerebellum (4). The content of macrophage nitric oxide synthase is 0.22% of the 100,000 X g supernatant. This may represent the difference in the nature of these enzymes. Nitric oxide synthase of the cerebellum and endothelial cell are constitutive enzymes, but enzyme is independent of Ca2+ and calmodulin. Thus, these two enzymes seem to be differently regulated. The molecular cloning and comparison of these enzymes may clarify the differences.