Purification and characterization of rat urinary esterase A, a plasminogen activator.

A plasminogen activator, previously designated as rat urinary esterase A (Nustad, K., and Pierce, J. V. (1974) Biochemistry 13, 2312-2319), was separated from kallikrein of rat urine and purified to homogeneity. In polyacrylamide slab gel electrophoresis, the purified enzyme showed three closely migrating protein bands which were labeled with [14C]diisopropylphosphorofluoridate and stained on a zymogram using the chromogenic substrate methionine-alpha-naphthyl ester. Two chains, heavy chain(s) (Mr approximately 15,800, 14,200) and light chain(s) (Mr approximately 8,850, 8,550), were separated in SDS-polyacrylamide gel under reducing conditions, while two bands (Mr approximately 24,500 and 23,000) were seen under nonreducing conditions. The active site of the enzyme was associated with the heavy chain. The purified enzyme was stained for carbohydrate by the periodic acid-Schiff reagent. Five bands were distinguished in slab gel electrofocusing with isoelectric points ranging from 5.05 to 5.45. The purified enzyme lysed fibrin clots containing plasminogen but not plasminogen-free fibrin. It hydrolyzed benzyloxylcarbonyl-Gly-Gly-Arg-amino-4-trifluoromethyl coumarin, and a Km of 53 microM and a Vmax of 63 mumol/min/mg of enzyme were obtained at pH 8.0 and 37 degrees C. The enzyme cleaved kininogen substrates to produce kinin which was measured by bioassay or radioimmunoassay. The enzyme was inhibited by soybean or lima bean trypsin inhibitor, aprotinin, alpha 1-antitrypsin, phenylmethanesulfonyl fluoride, D-Phe-Phe-ArgCH2Cl, antipain, leupeptin, benzamidine, and pentamidine. Its pH optimum was 8.5 to 9.0; it was unstable on dilution and on heating. On immunoelectrophoresis, an antiserum to the esterase formed precipitin arcs with rat plasma and this enzyme at identical positions, which in turn were different from those formed with kallikrein. This urinary enzyme belongs to the family of serine proteinases and is immunologically related to urinary kallikrein.

and showed it to be a urinary alkaline esterase and kininogenase with properties distinctly different from urinary or glandular kallikrein (2). In order to understand its bioregulatory actions, we developed a direct radioimmunoassay for esterase A and localized the enzyme in salivary gland, kidney, and other glandular tissues.' Recently, we reported that the level of arginine esterase A, measured by direct radioimmunoassay, is regulated by testosterone in rat submaxillary gland and kidney (3). The present work describes the purification and properties of rat urinary esterase A and indicates that it is also a plasminogen activator. Collection of Rat Urine-Thirty-two male or female Sprague-Dawley rats (200-350 g) were kept in 16 stainless steel metabolic cages (20 X 24 X 18 cm) and were given water but no food during the 22 h per day of urine collection. Urine was collected through a stainless steel funnel into plastic tubes. Solid debris was deflected at the curved end of the funnel. Each lot of urine (100-400 ml) was filtered through several layers of cheesecloth and stored at -20 "C until use.

DEAE
Conductiuity Determination-The conductivity of fractions from DEAE-cellulose chromatography was measured at 25 "C with a Yel- Electrophoresis in 12.5% Polyacrylamide Slab Gels-The method used for vertical slab gel electrophoresis was that described by Studier (5) with slab gels 1 mm thick, and solutions and buffers described by Laemmli (6), according to the procedures described previously (7). For active site labeling, esterase A or kallikrein (-4 pg) was incubated with ['4C]diisopropylphosphorofluoridate (0.2 pCi, specific activity > 100 mCi/mmol) in a total volume of 30 pl for I h at 37 "C followed by 16 h at 4 "C. The mixture was then added to 30 pl of buffer containing 0.0625 M Tris-HC1 (pH 6.8), 20% glycerol. Electrophoresis was performed in a 12.5% polyacrylamide gel in a Tris-glycine buffer, pH 8.8. The gels were stained for 30 min in 50% trichloroacetic acid containing 0.2% Coomassie blue and destained in 7% acetic acid. The gels were then dried on Whatman No. 3MM filter paper and treated with enhancer. Autoradiography was carried out by placing the gels in direct contact with Kodak NS2T x-ray film. Film was developed after 4 days of exposure.
Molecular Weight Determination-The molecular weight of urinary esterase A was estimated by gel filtration at room temperature on a Sephacryl S-200 column. The column was previously calibrated using blue dextran, bovine serum albumin, ovalbumin, and chymotrypsinogen A. The molecular weight of esterase A was also determined by electrophoresis on a 7.5 to 17.5% continuous SDS-polyacrylamide gel. Serum albumin, ovalbumin, aldolase, chymotrypsinogen, and ribonuclease were used as marker proteins, and the electrophoretic mobilities were measured relative to the marker proteins. The active site was labeled with [14C]diisopropylphosphorofluoridate as described previously. The mixture was then added to 30 pl of the extraction buffer containing 0.0625 M Tris-HC1 (pH 6.8), 20% glycerol, 5% mercaptoethanol, and 2.3% sodium dodecyl sulfate and heated to 100 "C for 5 mi n. Electrophoresis was performed in 0.1% sodium dodecyl sulfate. The same procedures were carried out for SDSpolyacrylamide electrophoresis in nonreducing conditions with the omission of mercaptoethanol in the extraction buffer.
Isoelectric Focusing on Polyacrylamide S l a b Gels-Isoelectric focusing was carried out in an LKB Multiphor electrophoresis system with a pH gradient of 3 to 7 as described previously (7). Separation was carried out for 1 h with increasing power settings: 20 watts for 20 min, 25 watts for 30 min, and 30 watts for 10 min. Following completion of focusing, the filter paper tabs and an edge portion of the gel were removed. The strip (1 cm width) was cut transversely at 0.5-cm intervals and each section was placed in a tube containing 1 ml of distilled water. After standing about 18 h, the pH values of the sections were determined. The pH gradient was obtained by plotting pH uersus gel distance. The remainder of the slab was fwed in 12.5% trichloroacetic acid for 10 min, washed 10 min each three times with 50% ethanol, 10% acetic acid, and finally with water. The protein bands on the gel were visualized with the sensitive silver staining technique (8).
Esterase Activity Stacning-Following the completion of electrophoresis, the slab gels were stained according to the procedures described by Schaller and von Deimling (9) using N-acetyl-L-methionine a-naphthyl ester or N-acetyl-L-alanine a-naphthyl ester.
Carbohydrate Staining-Following electrophoresis, non-SDS or SDS-polyacrylamide gels were stained for carbohydrate using the periodic acid-Schiff procedures of Fairbanks et al. (10). SDS was removed before periodic acid-Schiff staining. The gels were then transferred to Schiff reagent and left overnight. The gels were incubated in 0.1% sodium metabisulfite (Na2S20,), 0.01 M HCI, for several hours with gentle shaking and soaked repeatedly with the same solution until the rinse solution failed to turn pink upon addition of formaldehyde. Rose-pink bands appeared after 1 h in the Schiff reagent and slowly intensified.
Fibrin Plate Assay-The fibrin plate assay for the assessment of plasminogen activator by microradial diffusion was used according to the procedures of Schumacher and Schill (11). Bovine fibrinogen Preparations containing plasminogen or plasminogen free were used in this test. Ten pl each of standard urokinase (1 unit/ml to 100 units/ ml) or purified rat urinary esterase A was placed into each well in 1% agar gel Containing 0.5% fibrinogen and 0.1 unit/ml of thrombin. The diameter of the lysis zone was measured after 16 h of diffusion at 25 "C.
Fluorometric Assay-Fluorometric assay of rat urinary esterase A with the fluorogenic substrate, Cbz-Gly-Gly-Arg-AFC, was conducted at excitation and emission wavelengths of 400 and 505 nm, respectively, according to the procedures of Smith et al. (12) Fifty pl of substrate at concentrations ranging from 0.5 to 7.5 mM in dimethyl sulfoxide were added to 2.93 ml of 0.2 M Tris-HC1, pH 8.0, followed by 20 pl of enzyme solution (4 pg/rnl). Fluorescence was recorded for at least 5 min a. t 37 "C at a chart speed of 2 cm/min using an Aminco-Bowman spectrofluorometer.
Tos-ArgOMe Esterase Assay-The modified assay of Beaven et al. (13) was used routinely to determine Tos-Arg-OMe esterase activity, a measure of kallikrein-like activity. One Tos-Arg-OMe esterase unit is defined as that amount of enzyme which hydrolyzes 1.0 pmol of Tos-Arg-OMe per min at pH 8.0 and 30 "C (13).
Kininogenase Radioimmunoassay-The assay of Shimamoto et al. (14) was used to determine the kinin-generating activity of esterase A. The amount of kinin produced was measured by a kinin radioimmunoassay (15). Purified bovine low molecular weight kininogen (7.5 pg/ml) or heated dog, human, or rat plasma (1 -+ 50 dilution) were used as substrates. The reaction was carried out in 0.1 M phosphate buffer (pH 8.5) containing the kininase inhibitors, 3.0 m~ phenanthroline and 30 mM Na, EDTA in plastic tubes. After adding 0.10 ml of esterase A (17 ng) to 0.20 ml of buffer for kinin generation, the contents were mixed and incubated at 37 "C for 5 min followed by addition of the kininogen substrate in 0.2 ml of buffer. The rest of the procedure was carried out as described previously (15).
Kallikrein Biological Assay-The kinin-releasing activity of esterase A was determined by incubating 0.3 ml of 0.2 M Tris-HC1 buffer, pH 8.0, 0.1 ml of enzyme solution (200 ng/ml), and 0.1 ml of previously heated dog, human, or rat plasma (60 "C, 60 min) or a bovine low molecular weight kininogen substrate for 30 min at 37 "C. The kinin released was measured on the isolated guinea pig ileum or rat uterus preparation (16), using bradykinin as a standard. Blood pressure measurements were conducted essentially by the procedure described by Kettner et al. (17). Kallikrein or esterase A was dissolved in phosphate-buffered saline, pH 7.4 (40 pg/ml), and aliquots were injected in the right carotid artery of male Sprague-Dawley rats (300 g) anesthetized with pentobarbital ( 5 0 mg/kg). Blood pressure was monitored by a Grass polygraph and a Statham pressure transducer connected to a cannula in the left femoral artery.
Immunization Procedure-Purified rat urinary esterase A in Dulbecco's phosphate-buffered saline, pH 7.3 (ea2' and Mg2+ free), was emulsified with an equal volume of Freund's complete adjuvant, and 0.1-ml aliquots were injected subcutaneously at 10 dorsal sites in each of three 6 to 8-pound male New Zealand rabbits. One hundred pg of esterase A was administered to each rabbit. The first booster was given 1 month later, followed by additional boosters at 2-week intervals. Precipitating antibody was found after the second booster injection using Ouchterlony double immunodiffusion analysis (18).
Immunoelectrophoresis Analysis-Ten-microliter samples were placed in each well. Immunoelectrophoresis was carried out using an LKB 2117 Multiphor immunoelectrophoresis system with 1% agarose slides in 0.05 M barbiturate buffer, pH 8.6. Plates were electrophoresed for 2 h at 1 0 0 V (25-30 mA). Antiserum to esterase A (50 pl) was then placed in each trough, and diffusion was allowed to occur over a period of 16 h at room temperature.

Purification of Rat Urinary Esterase A
The following steps in the purification of rat urinary esterase A were carried out at room temperature and the activity was measured by the Tos-Arg-OMe radiochemical method and are summarized in Table I. Step 1. Ammonium Sulfate Fractionation-Three liters of rat urine were centrifuged at 5000 X g for 30 min to remove precipitates. Solid ammonium sulfate (209 gm/liter) was added to the supernatant until the salt concentration reached 35% saturation. The mixture was stirred for an additional 60 min and centrifuged at 5000 X g for 30 min. The resulting supernatant was brought to 65% saturation of ammonium sulfate (200 gm/liter). 0.2 ml of 1 M NaOH was used per 10 g of solid ammonium sulfate added to adjust the pH to 7.0. The precipitate collected by centrifugation was dissolved in distilled water. The crude protein solution was dialyzed overnight against distilled water a t 4 "C to remove a considerable amount of amber-colored material into the dialysate and then dialyzed overnight again a t 4 "C against 0.1 M NaC1-0.01 M sodium phosphate, pH 7.0.
Step 2. DEAE-cellulose Chromatography-The solution from ammonium sulfate fractionation in 0.1 M NaCl-0.01 M sodium phosphate, pH 7.0, was passed through a DEAEcellulose column (2.5 X 30 cm), previously equilibrated with the same buffer and eluted until absorbance of the effluent at 280 nm dropped below 0.05 unit. The flow rate was 40 ml per h. The Tos-Arg-OMe esterase A activity which flowed through the DEAE-cellulose column was combined and passed through a second DEAE-cellulose column. Rat urinary kallikrein binds to the column under the specified conditions and can be eluted from the column with a sodium chloride concentration of 0.2-0.3 M. The flow through fractions containing Tos-Arg-OMe esterase activity were precipitated by 65% ammonium sulfate, dissolved in 100 ml of distilled water, and dialyzed against 0.2 M NaCl, 0.02 M sodium phosphate, pH 7.0.
Step 3. Aprotinin-Agarose Affinity Chromatography-The dialyzed enzyme solution was applied to an aprotinin-agarose affinity column (Affl-Gel 10) and equilibrated with 0.2 M NaCl-0.02 M Na phosphate, pH 7.0. The column was washed with the dialysis buffer until protein was not detected in the effluent. Adsorbed Tos-Arg-OMe esterase was then eluted with 0.1 M acetate buffer, pH 3.5. Five-ml fractions were collected into tubes containing 1 ml of 0.5 M Tris-HC1, pH 8.5. Most of the contaminant nonesterase proteins flowed through the column at pH 7.0 and esterase A was eluted a t 0.1 M acetate buffer, pH 3.5 (Fig. 1). About 180-fold purification was achieved with this step.
Step 4. Sephacryl S-200 Gel Filtration-Esterase A fractions eluted from the aprotinin-affinity column at pH 7.0 were combined and concentrated with Immersible-CX (Millipore). The enzyme solution was dialyzed against 0.1 M NaCl-0.01 M sodium phosphate, pH 7.0, and gel filtered through a Sephacryl S-200 column (2.6 X 95 cm) equilibrated with the same buffer. The Tos-Arg-OMe esterase activity eluted as a single peak between ovalbumin (43,000) and chymotrypsinogen A (23,000). The molecular weight was estimated to be 33,000 on the calibrated column of Sephacryl S-200.

Identification as a Serine Proteinase and a Glycoprotein
Electrophoresis on polyacrylamide slab gel showed the different mobilities of rat urinary esterase A and kallikrein (Fig.  2). Purified rat urinary esterase A gave three closely migrating protein bands which were all labeled with ['4C]diisopropylphosphorofluoridate (Fig. 2 A ) and also stained on a zymogram using the chromogenic substrates, methione-a-naphthyl ester or alanine a-naphthyl ester (data not shown). In addition, the Coomassie blue staining bands of esterase A were also stained for carbohydrate with periodic acid-Schiff reagent. The bands corresponded to the protein bands in either non-SDS or SDSpolyacrylamide gel. Each 1-mm section of non-SDS gel slice from duplicate gels was eluted with 0.5 ml of H20, and the elutes from the three protein bands coincided with three peaks of Tos-Arg-OMe esterase and kinin-releasing activity with purified bovine low molecular weight kininogen substrate.

Electrofocusing in Polyacrylamide Gel
Analytical electrophoresis of purified rat urinary esterase A was performed on polyacrylamide slab gels in a pH gradient of 3 to 7 formed with Ampholine. Fig. 3 shows that five bands were separated with isoelectric points ranging from 5.05 to 5.45.

Fibrinolytic Activity
Rat urinary esterase A converts plasminogen to plasmin as determined by the radial diffusion in plasminogen-fibrinogencontaining agar gel. A linear relationship was found between the diameter of the lysis zone and the logarithm of the urokinase concentration within a range of 1 to 100 units/ml. Control experiment using a fibrin plate containing plasminogen-free fibrinogen showed that neither urokinase nor rat urinary esterase A lysed the fibrin clot under the same conditions. Furthermore, Tos-Arg-OMe esterase activity of esterase A is not inhibited by €-amino-n-caproic acid in a concentration range of 1O-'-1O" M.

Fluorometric Assays
The initial rate of Cbz-Gly-Gly-Arg-AFC hydrolysis was proportional to esterase A concentration with saturating amounts of substrate (0.5 mM). The rate of hydrolysis varied as a function of substrate concentration according to the Michaelis-Menten model. Double reciprocal plot analyses 7 I\ 6 I".,

TABLE I1
Inhibition of rat urinary plasminogen activator IC:,,, is the inhibitor concentration giving 50% inhibition. Twenty pl of rat urinary esterase A (2 X 10.' esterase unit/ml in 0.1% bovine serum albumin), 10 pI of inhibitor (final concentrations as indicated) incubated at 37 "C for 30 min, and 30 pI of 0.2 M Tris-HC1 buffer, pH 8.0, were mixed and allowed to stand 10 min. Tos-Arg-OMe (3.0 X 10' cpm. 10 pl) was added, mixed, and allowed to incubate for 30 min at room temperature. The [,"H]methanol released was measured in a Beckman "355 liquid scintillation spectrometer, and the enzyme activity is expressed as a per cent of control esterase activity in the absence of inhibitor. Each value represents the average of three experiments in duplicate. showed that K,, and Vn,ax of esterase A were 53 p~ and 63
Esterolytic a n d Biological Activity Pure rat urinary esterase A had Tos-Arg-OMe specific activity of 556 esterase units/AtHo a t pH 8.0. The enzyme also released kinin from either purified bovine low molecular weight kininogen substrate or from dog, human, or rat heated plasma, and the generated kinins were measured by radioimmunoassay or bioassay. The kininogenase activity of this enzyme using purified bovine low molecular weight kininogen or heated dog plasma kininogen was 9.6 pg of kinin generated/ min/A2Ro and 194 pg of kinin generated/min/AZM, respectively. Preincubation of esterase A with kininogen substrates produced a kinin-like contractile response in isolated guinea pig or rat uterus tissue, and the response was abolished or prevented by carboxypeptidase B (data not shown). Rat urinary esterase A did not induce contraction of rat uterus in the absence of exogenous kininogen, a response known to occur with glandular kallikrein (1, 19). Furthermore, rat urinary kallikrein (-4 pg) when injected into the rat carotid artery induced a transient decrease in blood pressure by 12-15 mm Hg while rat urinary esterase A at the same or 5-fold higher amounts had no hypotensive effect.

Inhibition a n d Activation Studies
The effects of serine proteinase inhibitors on rat urinary esterase A Tos-Arg-OMe esterase activity are shown in Table  11. These inhibitors included SBTI, LBTI, aprotinin, wantitrypsin, PMSF, D-Phe-Phe-ArgCHZCl, antipain, leupeptin, benzamidine, and pentamidine. SBTI was a potent inhibitor of esterase A, whereas it stimulated kallikrein activity a t low concentrations and inhibited at higher concentrations (7). LBTI stimulated esterase A a t low concentrations but produced inhibition at high concentrations, while it only stimulated kallikrein activity over the concentration range used (7). OMTI stimulated both esterase A and kallikrein activity (data not shown).

Heat and Dilutional Stability
The pH profile of Tos-Arg-OMe esterase activity of esterase A followed a Gaussian distribution with an optimum of 8.5- Immunoelectrophoresis of urinary esterase A, kallikrein, and rat plasma against an esterase A antiserum from rabbit. Ten pl of rat urinary kallikrein (4 pg) (wells 1 and 4), rat plasma (well 2), rat urinary esterase A (2 pg) (well 3), and concentrated rat urine (20 esterase units/ml) (well 5) were placed in the wells. Immunoelectrophoresis was carried out using a LKB 2117 Multiphor immunoelectrophoresis system with prepared 1% agarose slides in 0.05 M barbiturate buffer, pH 8.6. Plates were electrophoresed for 2 h at 100 V (25-30 mA). Undiluted rabbit antiserum (50 1. 11) was then added to each trough and diffusion allowed to occur over a period of 16 h at room temperature.
buffer (pH 8.5-10.0) as described previously (20). The enzyme lost 72 to 82% of its activity after 30-60 min of incubation at 55 "C. The enzyme solution (0.2 mg/ml) was stable for several months when stored at -20 "C in 0.01 M sodium phosphate buffer, pH 7.0. However, esterase A lost more than 90% of its activity upon 1:lOO or further dilution in Tris or phosphate buffer, pH 7-8, or in water. When diluted similarly in the presence of 1 mg/ml of bovine serum albumin, activity was maintained.

Immunological Characterization
Immunoelectrophoresis of rat plasma, esterase A, urinary kallikrein, and rat urine against an esterase A antiserum is shown in Fig. 4. The antiserum formed immunoprecipitin arcs with rat plasma (well 2) and rat urinary esterase A (well 3) at identical positions which differed from those formed with urinary kallikrein (wells 1 and 4). The antiserum also formed a continuous immunoprecipitin arc with concentrated rat urine (well 5).

DISCUSSION
The present study reports on the purification and properties of a plasminogen activator from rat urine. This enzyme was first identified as "esterase A" by Nustad and Pierce (1) and later designated as "esterase A; ' by McParkland et al. (21,  22). Esterase A is an acidic glycoprotein. It cleaves kininogen substrate to produce kinin, lyses fibrin clots, and hydrolyzes the fluorogenic substrate Cbz-Gly-Gly-ArgAFC for urokinase or plasminogen activator. This enzyme belongs to the family of serine proteinases and is a kininogenase and a plasminogen activator.
This urinary plasminogen activator as well as urinary kallikrein possess kinin-releasing activities using kininogen substrates from bovine, dog, or rat sources. However, in contrast to rat glandular kallikrein (19), neither rat urinary plasminogen activator nor glandular kallikreins from other species induce rat uterine contraction in the absence of kininogen substrate. These results imply the presence of a specific receptor for rat glandular kallikrein in the rat uterus but not for other serine proteinases. Furthermore, the urinary plasminogen activator has no blood pressure-lowering effects when injected into an anesthetized rat. As the plasminogen activator (Table 11) but not glandular kallikrein (data not shown) is inhibited by cul-antitrypsin in the in vitro assay, the former could bind rapidly to a,-antitrypsin which is present in large quantity in rat plasma and thus be inactivated as soon as it reaches the blood stream. Therefore, despite its kinin-releasing activity against rat plasma kininogen (1, 2) the enzyme exhibits no hypotensive response.
A much greater lability of esterase A than urinary kallikrein was observed by Nustad and Pierce (1) and confirmed in this laboratory. The enzymatic activities were lost when diluted in buffer alone. However, addition of camer protein such as bovine albumin (1 mg/ml) protects it.
Immunodiffusion analysis showed that antibody to urinary plasminogen activator formed an immunoprecipitin arc with rat plasma at a corresponding position and with purified rat urinary kallikrein at a different position to that of esterase A. The results indicated that an esterase A-like immunoreactive substance is present in rat serum. Whether this immunoreactive material is present as the free active form or bound to an inhibitor or substrate in the serum remains to be seen. Undiluted antiserum to rat plasminogen activator also formed an immunoprecipitating arc with rat urinary kallikrein (Fig. 4). Furthermore, antiserum to purified rat urinary kallikrein cross-reacts with purified urinary plasminogen activator:' and many other nonkaliikrein esterases in rat submaxillary gland (23). Recently, a direct radioimmunoassay for rat plasminogen activator was developed in our laboratory for measuring this immunoreactive enzyme level in urine and various tissues (3). In the radioimmunoassay, antiserum to plasminogen activator was diluted to 160,000-fold, and about 2-556 of cross-reactivity of the antiserum to rat urinary kallikrein was observed. Therefore, the urinary plasminogen activator appears to be immunologically related but different from rat urinary (glandular) kallikrein.
Rat urinary plasminogen activator forms a covalent linkage with [14C]diisopropylphosphorofluoridate and is also inhibited by phenylmethanesulfonyl fluoride. Furthermore, D-Phe-Phe-Arg-CH2Cl inactivates the enzyme. SBTI appears to be an extremely potent inhibitor for the enzyme but not for glandular kallikrein. In addition, leupeptin and antipain also are strong inhibitors with ICm of -4 X lo-' M. As SBTI, leupeptin, and antipain have been demonstrated to be effective in retardation of tumor growth or cell transformation (24, 25), the role of plasminogen activator in situ is of importance for further investigation.
The cellular localization of plasminogen activator is being pursued in this laboratory. With a direct radioimmunoassay and immunohistochemical techniques, we found rat plasminogen activator to be present in various glandular tissues, such as salivary gland, kidney, pancreas, colon, intestine, and others. The enzyme level in the salivary gland appears to be androgen dependent (3).

activator.
Purification and characterization of rat urinary esterase A, a plasminogen