Proc. Natl. Acad. Sci. USA Vol. 88, pp. 5984-5988, July 1991 Immunology
Identification of aminopeptidase activity in the secretory granules of mouse mast cells (protease/proteoglycan/exopeptidase)
WILLIAM E. SERAFIN*, URSULA A. GUIDRY, ELAHE T. DAYTON, MIKA M. KAMADA, RICHARD L. STEVENS, AND K. FRANK AUSTEN Department of Medicine, Harvard Medical School, and Department of Rheumatology and Immunology, Brigham and Women's Hospital, Boston, MA 02115
Contributed by K. Frank Austen, April 8, 1991
ABSTRACT Sonicates of mouse bone marrow-derived mast cells (BMMC) differentiated in vitro and of mouse serosal mast cells differentiated in vivo contained small but approximately equal amounts of aminopeptidase activity, as determined by cleavage of leucine-fl-naphthylamide and resolution of the reaction products by reverse-phase high-performance liquid chromatography. Aminopeptidase activity was exocytosed from antigen-activated, IgE-sensitized BMMC in proportion to the secretory granule enzyme (3-hexosaminidase, thereby localizing -60% of the total cell-associated aminopeptidase activity to the secretory granules of the mast cells. A prominent secretory granule location for aminopeptidase was confirmed by activity measurement in subcellular fractions of disrupted BMMC. The secretory granule aminopeptidase had a pH optimum of 6.0-8.0 and a Km of 0.36 ± 0.06 mM (mean + SD; n = 3) for leucine-I3-naphthylamide. When various amino acid j3-naphthylamides were used as substrates, the preference of the secretory granule enzyme was Ala > Leu > Phe >> Arg >> Asp = Tyr. Most of the aminopeptidase activity that was exocytosed from calcium ionophore-activated BMMC was bound to 35S-labeled proteoglycans in complexes of >1 x 107 kDa as dermed by exclusion during Sepharose CL-2B gel-filtration chromatography. We postulate that the aminopeptidase in the mast cell protease/proteoglycan complexes allows the removal of N-terminal amino acids from peptides that are generated by the action of mast cell endopeptidases.
BALB/c mice (The Jackson Laboratory) by peritoneal lavage, purified to >90%, resuspended in Tyrode's buffer, sonicated, and stored frozen at -70'C until used. BMMC were obtained by culturing bone marrow cells from BALB/c mice for 3-6 wk in 50% enriched medium (RPMI 1640 medium containing 10% fetal bovine serum, penicillin at 100 units/ml, streptomycin at 100,ug/ml, 2 mM L-glutamine, 0.1 mM nonessential amino acids, and 5 mM Hepes) and 50%6 WEHI-3 conditioned medium (15). In some experiments, BMMC were differentiated further by fibroblast coculture (16). After 30 days, the mast cell/fibroblast cocultures were washed twice with calcium- and magnesium-free Hanks' balanced salt solution (HBSS), dispersed with trypsin, enriched to a purity of >94% mast cells by discontinuous metrizamide centrifugation (12), resuspended at 1 x 106 cells per ml in HBSS, and sonicated. Activation of Mouse BMMC. BMMC were sensitized with mouse monoclonal anti-dinitrophenyl IgE (17) in Tyrode's buffer with 0.05% gelatin and activated with 0-40 ng of dinitrophenyl conjugated to bovine serum albumin as described (18). In some experiments, BMMC were activated with 0.5 ,M calcium ionophore A23187 (19). After immunologic or calcium ionophore activation, the mast cells were pelleted by centrifugation, the supernatants were removed, and the cell pellets were resuspended in Tyrode's buffer and sonicated. The cytosolic enzyme lactate dehydrogenase (20) and the secretory granule enzymes fB-hexosaminidase (21) and mast cell carboxypeptidase A (6) were routinely quantitated in samples of supernatants and sonicated cell pellets. The net percentage release (4) of each constituent was calculated. Identification and Quantitation of Aminopeptidase Activity. Samples (50 p1L) of supernatants or cell sonicates from 1 x 105 mast cells were incubated at 370C for 15 min with 200 /ul of 0.15 M NaCl/0.02 M Tris'HCl, pH 8.0, digestion buffer containing 1 mM leucine-j3-naphthylamide (22). The reactions were stopped with the addition of 250 p1L of 0.2% trifluoroacetic acid, and 20-plI samples were analyzed by reverse-phase high-performance liquid chromatography (RPHPLC). A 218TPS column (Vydac, Hesperia, CA) (150 x 4.6 mm) containing octadecylsilyl packing with 5-Axm porous particles was used in conjunction with a 25 x 4.6 mm column containing octadecylsilyl packing with 10-pAm porous particles. Solvent A consisted of water/trifluoroacetic acid (100:0.1; vol/vol), and solvent B consisted of water/ acetonitrile/trifluoroacetic acid (60:40:0.1; vol/vol). The solvent system was isocratic with 30% solvent A/70% solvent B; the flow rate was 1.5 ml/min. The aminopeptidase digestion product, /3-naphthylamine, was quantitated by measur-
Rat and mouse mast cells contain in their secretory granules serine endopeptidases and the exopeptidase carboxypeptidase A (1-11). These proteases are active at neutral pH and are ionically linked in macromolecular complexes with acidically charged proteoglycans (1, 5, 6). Mast cells can be characterized in part by their content of these proteases. For example, mouse serosal mast cells contain '150-fold more carboxypeptidase A in their secretory granules than interleukin 3-dependent, mouse bone marrow-derived progenitor mast cells (BMMC). We have shown that when BMMC are cocultured with fibroblasts, the BMMC differentiate to become more like serosal mast cells in histamine content, carboxypeptidase A content, and [355]heparin proteoglycan synthesis (6, 12). We now identify and partially characterize a mouse mast cell exopeptidase that is located in the secretory granules of BMMC and that has aminopeptidase activity. This aminopeptidase activity is present in approximately equal amounts in BMMC, BMMC cocultured with fibroblasts, and serosal mast cells.
MATERIALS AND METHODS Isolation of Mouse Serosal Mast Cells and Culture of Mouse BMMC. Serosal mast cells (13, 14) were obtained from
Abbreviations: BMMC, bone marrow-derived mast cells; RP-HPLC, reverse-phase high-performance liquid chromatography. *To whom reprint requests should be sent at the present address: Allergy Research, 847 Light Hall, Vanderbilt Medical School, Nashville, TN 37232-0111.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 5984
Immunology: Serafin et al. ing the area under the peak that eluted at 1.6 min. Conversion of area units to gmol was accomplished by comparison to a standard curve that was generated by chromatography of 0-640 nmol of authentic f3-naphthylamine. One unit of aminopeptidase activity was defined as that amount of enzyme that cleaved leucine-f3-naphthylamide to produce 1 Amol of /3-naphthylamine per min. Because other types of cells have extracellularly oriented plasma membrane-localized aminopeptidases, we measured the aminopeptidase activity of intact cells compared to sonicated cells. Inasmuch as leucine-pB-naphthylamide lysed the BMMC, a second assay was developed with Ile-His-Pro-Phe (Peninsula Laboratories) (23) as the substrate to measure the aminopeptidase activity of intact cells. Portions (100 pl) of intact or sonicated BMMC were mixed with 100 Al of Tyrode's buffer containing 1.0 mM Ile-His-Pro-Phe. After a 2-min incubation at 220C, the reactions were stopped by the addition of 250 Al of 0.2% trifluoroacetic acid. Loss of substrate and generation of the degradation product were monitored by RP-HPLC as described above except that the solvent system was isocratic with 55% solvent A/45% solvent B. Amino acid analysis, performed according to the method of Hirs (24), confirmed that the digestion product eluting at 2.0 min was His-Pro-Phe. The aminopeptidase activity was quantitated based on the area under the HisPro-Phe peak and was expressed as a percentage of the activity of the whole cell sonicates. Characterization of the Aminopeptidase Present in the Secretory Granules of BMMC. The aminopeptidase present in the supernatants of calcium ionophore-activated BMMC was characterized by its susceptibility to inhibition by the aminopeptidase-specific inhibitor bestatin (25), the serine protease inhibitor diisopropylfluorophosphate (4), the carboxypeptidase A inhibitor derived from potatoes (6, 26), and the divalent cation chelating agent bathophenanthroline (Sigma). Samples (250 pl) of supernatants from 2.5 x 106 activated BMMC were incubated at 370C for 1 hr with an equal volume of 0.15 M NaCI/0.2 M Tris HCI, pH 8.0, buffer alone or containing 2 mM bestatin, 20 mM diisopropylfluorophosphate, 300 ,uM potato inhibitor, or 20 mM bathophenanthroline. The inhibitor-treated and untreated supernatants were incubated with leucine-,f-naphthylamide as described above, and the fraction of residual aminopeptidase activity was calculated. The pH optimum for the BMMC aminopeptidase was examined over the range 5.0-11.0 using a 0.15 M NaCl/0.2 M 2-(N-morpholino)ethanesulfonic acid buffer adjusted to pH 5.0, 6.0, or 7.0; a 0.15 M NaCI/0.2 M Tris-HCI buffer adjusted to pH 7.0, 8.0, or 9.0; and a 0.15 M NaCI/0.2 M glycine buffer adjusted to pH 9.0, 10.0, or 11.0. For each assay, 10-pl samples of supernatant from calcium ionophoreactivated BMMC were mixed with 20 pl of 2 mM leucine-/3naphthylamide dissolved in the various buffers. After a 15-min incubation at 37°C, the reactions were stopped by the addition of 5 vol of 0.2% trifluoroacetic acid, and the digests were assessed by RP-HPLC. For kinetic studies of the secretory granule aminopeptidase of BMMC, 20-pA samples of the supernatant from calcium ionophore-activated BMMC were each incubated with 80 ,ul of leucine-f3-naphthylamide dissolved in the pH 8.0 digestion buffer to final concentrations ranging from 0.09 to 0.64 mM. After the reactions were allowed to proceed for 2 min at 22°C, they were stopped with the addition of 100 pl of 0.2% trifluoroacetic acid, and the digestion products were resolved and quantitated by RPHPLC. The Km for the hydrolysis of leucine-,8-naphthylamide was calculated by the use of double reciprocal plots (27). To calculate the Ki (27) for the aminopeptidase inhibitor bestatin, 20-1.d samples of the supernatant from calcium ionophore-activated BMMC were incubated with 0.13-0.76 mM leucine-f-naphthylamide as described above but in the presence of bestatin. For each concentration of the substrate,
Proc. Natl. Acad. Sci. USA 88 (1991)
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bestatin was added to the reaction mixture to give a final concentration of 0-69 AM. To characterize the substrate preference of the BMMC secretory granule aminopeptidase, various amino acid ,3-naphthylamides (Leu, Ala, Phe, Arg, Tyr, and Asp) (Sigma) were incubated individually with supernatants from calcium ionophore-activated BMMC, and the rate of cleavage ofeach substrate was determined. Each ofthe amino acid p8-naphthylamides (0.1 mM in 200 pl of the pH 8.0 buffer) was mixed with 50 ,ul of the supernatants from calcium ionophoreactivated BMMC and incubated for 15 min at 370C. This concentration of substrate was chosen to completely dissolve each ofthe amino acid f-naphthylamides. The reactions were stopped by the addition of 250 pA of 0.2% trifluoroacetic acid, and 20-A.l samples were injected onto the RP-HPLC column. RP-HPLC was performed as described above except that a 20-min linear gradient program starting with 60% solvent A/40o solvent B and ending with 30o solvent A/70%o solvent B was used. The rate of cleavage for each substrate was expressed relative to that of leucine-f3-naphthylamide. Gel-Filtration Chromatography. BMMC (3-7 x 107 cells per experiment) were incubated for 17 hr in 60 ml of culture medium containing 1 mCi of [35S]sulfate (4000 Ci/mmol; 1 Ci = 37 GBq; New England Nuclear). Radiolabeled BMMC were processed and activated with calcium ionophore as described above for unlabeled cells. Samples (3 ml) of supernatant from 3 x 107 activated BMMC were applied at 4°C to a 0.8 x 80 cm column of Sepharose CL-2B (Pharmacia) that had been equilibrated with 0.15 M NaCI/0.01 M Tris HCl, pH 7.2 (5). The 35S-labeled macromolecules in the collected 1-ml fractions were quantitated by liquid scintillation counting of 50-pl samples of the eluate. Aminopeptidase and carboxypeptidase A activities were assessed in separate 50-plA samples of the column fractions by monitoring the cleavage of leucine-f3-naphthylamide and hippuryl-L-phenylalanine, respectively. The void and total volumes of the Sepharose CL-2B column were determined with blue dextran 2000 (Pharmacia) and [35S]sulfate, respectively. To determine the hydrodynamic size of the exocytosed aminopeptidase after removal of the ionically linked proteoglycans, the supernatant from 1 x 108 activated BMMC was made 5 M in NaCI and then applied to a column (0.8 x 2 cm) of phenyl-Sepharose CL-4B (Pharmacia) that had been previously equilibrated in 5 M NaCI/0.01 M Tris HCI, pH 7.2. BMMC proteoglycans do not bind to the phenyl-Sepharose CL-4B column under these high salt conditions (5). After the column was washed with 5 column vol of the high salt buffer, bound proteins were eluted with a low salt buffer of 0.15 M NaCI/0.01 M Tris-HCI, pH 7.2. A 1-ml sample ofthis material was applied at 4°C to a 0.8 x 80 cm column of Sephadex G150 (Pharmacia) that had been equilibrated in the low salt Tris*HCI buffer. Fractions (0.5 ml) were collected and assessed for aminopeptidase activity using leucine-,3-naphthylamide as the substrate. Catalase (232 kDa), aldolase (158 kDa), bovine serum albumin (67 kDa), ovalbumin (45 kDa), chymotrypsinogen A (25 kDa), and ribonuclease A (13.7 kDa) were used to standardize the Sephadex G-150 column. The void and total volumes of the column were determined with blue dextran 2000 and [35S]sulfate, respectively. Density-Gradient Subcellular Fractionation of Aminopeptidase Activity. BMMC (108 cells) were suspended in 2 ml of disruption buffer (0.25 M sucrose/0.01 M Hepes, pH 7.2) and gently sonicated (setting 1; 20%o duty cycle; five pulses) at 4°C. The sonicate was filtered sequentially through prewetted 5-pAm and 3-,um filters (Nuclepore) to remove nuclei and nonlysed cells. The filtrate was adjusted to 5 ml with disruption buffer, layered onto 20 ml of 48% (vol/vol) Percoll (Pharmacia) in the disruption buffer, and centrifuged at 60,000 x g for 55 min (28). Fractions of 0.8 ml were collected from the top downward with the aid of an Autodensiflow II
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Immunology: Serafin et al.
(Haacke Buchler, Saddlebrook, NJ). Samples (50 Al) of each fraction were assessed for aminopeptidase activity, carboxypeptidase A activity, histamine content (29), and lactate dehydrogenase activity.
Proc. Natl. Acad. Sci. USA 88 (1991) 16
14 '
x
RESULTS Identification of Aminopeptidase Activity in Supernatants of Calcium Ionophore-Activated Mouse BMMC. When supernatants of calcium ionophore-activated BMMC were incubated for 0-60 min with leucine-13-naphthylamide, progressive degradation of the substrate occurred with the concurrent formation of a digestion product that had a retention time on RP-HPLC of 1.80 min, identical to that of the fB-naphthylamine standard. No measurable product was formed when leucine-f3-naphthylamide was incubated in the absence of the BMMC supernatants. In three determinations with separate cultures of BMMC and separate preparations of mouse serosal mast cells, 0.003 ± 0.002 (mean ± SD) and 0.002 ± 0.001 unit, respectively, of aminopeptidase activity was found per 106 sonicated cells. Cleavage of Ile-His-Pro-Phe by the proteases present in the supernatant of calcium ionophore-activated BMMC also occurred in a linear manner with time, resulting in the appearance of a digestion product that was His-Pro-Phe by amino acid analysis (data not shown). Characterization of the Aminopeptidase Activity Exocytosed from Calcium Ionophore-Activated BMMC. Digestion of leucine-f3-naphthylamide was not inhibited by a pretreatment of the supernatant for 1 hr with 10 mM diisopropylfluorophosphate or 150 ,M potato carboxypeptidase A inhibitor. In contrast, when samples of the same supernatants were preincubated with 10 mM bathophenanthroline or 1 mM bestatin, the degradation of this substrate was inhibited 41% ± 3% or 91% ± 2% (mean ± SD; n = 3), respectively. This aminopeptidase possessed a broad pH optimum of 6.0-8.0, and the Km for the degradation of leucine-p-naphthylamide was 0.36 ± 0.06 mM (mean + SD; n = 3) in 0.02 M Tris buffer (pH 8.0) (data not shown). The presence of varied amounts of the inhibitor bestatin did not alter the Vmax of the reaction, indicating a competitive-type inhibition with a Ki of 1.0 ,uM (data not shown). To characterize the substrate preference of the exocytosed aminopeptidase, supernatants from calcium ionophore-activated BMMC were incubated with various amino acid P-naphthylamides. The preferred substrate was alanine-,B-naphthylamide, with the rate of cleavage of the leucine and phenylalanine derivatives being within the same order of magnitude (Table 1). To determine whether the aminopeptidase activity was released in association with proteoglycans, 35S-labeled BMMC were activated with calcium ionophore, and the resulting supernatant was filtered on a Sepharose CL-2B column. As shown in Fig. 1, 69% of the exocytosed aminopeptidase activity, 78% of the carboxypeptidase A activTable 1. Substrate preference of the aminopeptidase that is exocytosed from calcium ionophore-activated BMMC
,8-Naphthylamine Relative rate Amino acid produced, pmol 1.0 137 ± 21 Leu 1.9 254 + 12 Ala 0.6 87 + 11 Phe 0.1 23 1 Arg
Identification of aminopeptidase activity in the secretory granules of mouse mast cells (protease/proteoglycan/exopeptidase)
WILLIAM E. SERAFIN*, URSULA A. GUIDRY, ELAHE T. DAYTON, MIKA M. KAMADA, RICHARD L. STEVENS, AND K. FRANK AUSTEN Department of Medicine, Harvard Medical School, and Department of Rheumatology and Immunology, Brigham and Women's Hospital, Boston, MA 02115
Contributed by K. Frank Austen, April 8, 1991
ABSTRACT Sonicates of mouse bone marrow-derived mast cells (BMMC) differentiated in vitro and of mouse serosal mast cells differentiated in vivo contained small but approximately equal amounts of aminopeptidase activity, as determined by cleavage of leucine-fl-naphthylamide and resolution of the reaction products by reverse-phase high-performance liquid chromatography. Aminopeptidase activity was exocytosed from antigen-activated, IgE-sensitized BMMC in proportion to the secretory granule enzyme (3-hexosaminidase, thereby localizing -60% of the total cell-associated aminopeptidase activity to the secretory granules of the mast cells. A prominent secretory granule location for aminopeptidase was confirmed by activity measurement in subcellular fractions of disrupted BMMC. The secretory granule aminopeptidase had a pH optimum of 6.0-8.0 and a Km of 0.36 ± 0.06 mM (mean + SD; n = 3) for leucine-I3-naphthylamide. When various amino acid j3-naphthylamides were used as substrates, the preference of the secretory granule enzyme was Ala > Leu > Phe >> Arg >> Asp = Tyr. Most of the aminopeptidase activity that was exocytosed from calcium ionophore-activated BMMC was bound to 35S-labeled proteoglycans in complexes of >1 x 107 kDa as dermed by exclusion during Sepharose CL-2B gel-filtration chromatography. We postulate that the aminopeptidase in the mast cell protease/proteoglycan complexes allows the removal of N-terminal amino acids from peptides that are generated by the action of mast cell endopeptidases.
BALB/c mice (The Jackson Laboratory) by peritoneal lavage, purified to >90%, resuspended in Tyrode's buffer, sonicated, and stored frozen at -70'C until used. BMMC were obtained by culturing bone marrow cells from BALB/c mice for 3-6 wk in 50% enriched medium (RPMI 1640 medium containing 10% fetal bovine serum, penicillin at 100 units/ml, streptomycin at 100,ug/ml, 2 mM L-glutamine, 0.1 mM nonessential amino acids, and 5 mM Hepes) and 50%6 WEHI-3 conditioned medium (15). In some experiments, BMMC were differentiated further by fibroblast coculture (16). After 30 days, the mast cell/fibroblast cocultures were washed twice with calcium- and magnesium-free Hanks' balanced salt solution (HBSS), dispersed with trypsin, enriched to a purity of >94% mast cells by discontinuous metrizamide centrifugation (12), resuspended at 1 x 106 cells per ml in HBSS, and sonicated. Activation of Mouse BMMC. BMMC were sensitized with mouse monoclonal anti-dinitrophenyl IgE (17) in Tyrode's buffer with 0.05% gelatin and activated with 0-40 ng of dinitrophenyl conjugated to bovine serum albumin as described (18). In some experiments, BMMC were activated with 0.5 ,M calcium ionophore A23187 (19). After immunologic or calcium ionophore activation, the mast cells were pelleted by centrifugation, the supernatants were removed, and the cell pellets were resuspended in Tyrode's buffer and sonicated. The cytosolic enzyme lactate dehydrogenase (20) and the secretory granule enzymes fB-hexosaminidase (21) and mast cell carboxypeptidase A (6) were routinely quantitated in samples of supernatants and sonicated cell pellets. The net percentage release (4) of each constituent was calculated. Identification and Quantitation of Aminopeptidase Activity. Samples (50 p1L) of supernatants or cell sonicates from 1 x 105 mast cells were incubated at 370C for 15 min with 200 /ul of 0.15 M NaCl/0.02 M Tris'HCl, pH 8.0, digestion buffer containing 1 mM leucine-j3-naphthylamide (22). The reactions were stopped with the addition of 250 p1L of 0.2% trifluoroacetic acid, and 20-plI samples were analyzed by reverse-phase high-performance liquid chromatography (RPHPLC). A 218TPS column (Vydac, Hesperia, CA) (150 x 4.6 mm) containing octadecylsilyl packing with 5-Axm porous particles was used in conjunction with a 25 x 4.6 mm column containing octadecylsilyl packing with 10-pAm porous particles. Solvent A consisted of water/trifluoroacetic acid (100:0.1; vol/vol), and solvent B consisted of water/ acetonitrile/trifluoroacetic acid (60:40:0.1; vol/vol). The solvent system was isocratic with 30% solvent A/70% solvent B; the flow rate was 1.5 ml/min. The aminopeptidase digestion product, /3-naphthylamine, was quantitated by measur-
Rat and mouse mast cells contain in their secretory granules serine endopeptidases and the exopeptidase carboxypeptidase A (1-11). These proteases are active at neutral pH and are ionically linked in macromolecular complexes with acidically charged proteoglycans (1, 5, 6). Mast cells can be characterized in part by their content of these proteases. For example, mouse serosal mast cells contain '150-fold more carboxypeptidase A in their secretory granules than interleukin 3-dependent, mouse bone marrow-derived progenitor mast cells (BMMC). We have shown that when BMMC are cocultured with fibroblasts, the BMMC differentiate to become more like serosal mast cells in histamine content, carboxypeptidase A content, and [355]heparin proteoglycan synthesis (6, 12). We now identify and partially characterize a mouse mast cell exopeptidase that is located in the secretory granules of BMMC and that has aminopeptidase activity. This aminopeptidase activity is present in approximately equal amounts in BMMC, BMMC cocultured with fibroblasts, and serosal mast cells.
MATERIALS AND METHODS Isolation of Mouse Serosal Mast Cells and Culture of Mouse BMMC. Serosal mast cells (13, 14) were obtained from
Abbreviations: BMMC, bone marrow-derived mast cells; RP-HPLC, reverse-phase high-performance liquid chromatography. *To whom reprint requests should be sent at the present address: Allergy Research, 847 Light Hall, Vanderbilt Medical School, Nashville, TN 37232-0111.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 5984
Immunology: Serafin et al. ing the area under the peak that eluted at 1.6 min. Conversion of area units to gmol was accomplished by comparison to a standard curve that was generated by chromatography of 0-640 nmol of authentic f3-naphthylamine. One unit of aminopeptidase activity was defined as that amount of enzyme that cleaved leucine-f3-naphthylamide to produce 1 Amol of /3-naphthylamine per min. Because other types of cells have extracellularly oriented plasma membrane-localized aminopeptidases, we measured the aminopeptidase activity of intact cells compared to sonicated cells. Inasmuch as leucine-pB-naphthylamide lysed the BMMC, a second assay was developed with Ile-His-Pro-Phe (Peninsula Laboratories) (23) as the substrate to measure the aminopeptidase activity of intact cells. Portions (100 pl) of intact or sonicated BMMC were mixed with 100 Al of Tyrode's buffer containing 1.0 mM Ile-His-Pro-Phe. After a 2-min incubation at 220C, the reactions were stopped by the addition of 250 Al of 0.2% trifluoroacetic acid. Loss of substrate and generation of the degradation product were monitored by RP-HPLC as described above except that the solvent system was isocratic with 55% solvent A/45% solvent B. Amino acid analysis, performed according to the method of Hirs (24), confirmed that the digestion product eluting at 2.0 min was His-Pro-Phe. The aminopeptidase activity was quantitated based on the area under the HisPro-Phe peak and was expressed as a percentage of the activity of the whole cell sonicates. Characterization of the Aminopeptidase Present in the Secretory Granules of BMMC. The aminopeptidase present in the supernatants of calcium ionophore-activated BMMC was characterized by its susceptibility to inhibition by the aminopeptidase-specific inhibitor bestatin (25), the serine protease inhibitor diisopropylfluorophosphate (4), the carboxypeptidase A inhibitor derived from potatoes (6, 26), and the divalent cation chelating agent bathophenanthroline (Sigma). Samples (250 pl) of supernatants from 2.5 x 106 activated BMMC were incubated at 370C for 1 hr with an equal volume of 0.15 M NaCI/0.2 M Tris HCI, pH 8.0, buffer alone or containing 2 mM bestatin, 20 mM diisopropylfluorophosphate, 300 ,uM potato inhibitor, or 20 mM bathophenanthroline. The inhibitor-treated and untreated supernatants were incubated with leucine-,f-naphthylamide as described above, and the fraction of residual aminopeptidase activity was calculated. The pH optimum for the BMMC aminopeptidase was examined over the range 5.0-11.0 using a 0.15 M NaCl/0.2 M 2-(N-morpholino)ethanesulfonic acid buffer adjusted to pH 5.0, 6.0, or 7.0; a 0.15 M NaCI/0.2 M Tris-HCI buffer adjusted to pH 7.0, 8.0, or 9.0; and a 0.15 M NaCI/0.2 M glycine buffer adjusted to pH 9.0, 10.0, or 11.0. For each assay, 10-pl samples of supernatant from calcium ionophoreactivated BMMC were mixed with 20 pl of 2 mM leucine-/3naphthylamide dissolved in the various buffers. After a 15-min incubation at 37°C, the reactions were stopped by the addition of 5 vol of 0.2% trifluoroacetic acid, and the digests were assessed by RP-HPLC. For kinetic studies of the secretory granule aminopeptidase of BMMC, 20-pA samples of the supernatant from calcium ionophore-activated BMMC were each incubated with 80 ,ul of leucine-f3-naphthylamide dissolved in the pH 8.0 digestion buffer to final concentrations ranging from 0.09 to 0.64 mM. After the reactions were allowed to proceed for 2 min at 22°C, they were stopped with the addition of 100 pl of 0.2% trifluoroacetic acid, and the digestion products were resolved and quantitated by RPHPLC. The Km for the hydrolysis of leucine-,8-naphthylamide was calculated by the use of double reciprocal plots (27). To calculate the Ki (27) for the aminopeptidase inhibitor bestatin, 20-1.d samples of the supernatant from calcium ionophore-activated BMMC were incubated with 0.13-0.76 mM leucine-f-naphthylamide as described above but in the presence of bestatin. For each concentration of the substrate,
Proc. Natl. Acad. Sci. USA 88 (1991)
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bestatin was added to the reaction mixture to give a final concentration of 0-69 AM. To characterize the substrate preference of the BMMC secretory granule aminopeptidase, various amino acid ,3-naphthylamides (Leu, Ala, Phe, Arg, Tyr, and Asp) (Sigma) were incubated individually with supernatants from calcium ionophore-activated BMMC, and the rate of cleavage ofeach substrate was determined. Each ofthe amino acid p8-naphthylamides (0.1 mM in 200 pl of the pH 8.0 buffer) was mixed with 50 ,ul of the supernatants from calcium ionophoreactivated BMMC and incubated for 15 min at 370C. This concentration of substrate was chosen to completely dissolve each ofthe amino acid f-naphthylamides. The reactions were stopped by the addition of 250 pA of 0.2% trifluoroacetic acid, and 20-A.l samples were injected onto the RP-HPLC column. RP-HPLC was performed as described above except that a 20-min linear gradient program starting with 60% solvent A/40o solvent B and ending with 30o solvent A/70%o solvent B was used. The rate of cleavage for each substrate was expressed relative to that of leucine-f3-naphthylamide. Gel-Filtration Chromatography. BMMC (3-7 x 107 cells per experiment) were incubated for 17 hr in 60 ml of culture medium containing 1 mCi of [35S]sulfate (4000 Ci/mmol; 1 Ci = 37 GBq; New England Nuclear). Radiolabeled BMMC were processed and activated with calcium ionophore as described above for unlabeled cells. Samples (3 ml) of supernatant from 3 x 107 activated BMMC were applied at 4°C to a 0.8 x 80 cm column of Sepharose CL-2B (Pharmacia) that had been equilibrated with 0.15 M NaCI/0.01 M Tris HCl, pH 7.2 (5). The 35S-labeled macromolecules in the collected 1-ml fractions were quantitated by liquid scintillation counting of 50-pl samples of the eluate. Aminopeptidase and carboxypeptidase A activities were assessed in separate 50-plA samples of the column fractions by monitoring the cleavage of leucine-f3-naphthylamide and hippuryl-L-phenylalanine, respectively. The void and total volumes of the Sepharose CL-2B column were determined with blue dextran 2000 (Pharmacia) and [35S]sulfate, respectively. To determine the hydrodynamic size of the exocytosed aminopeptidase after removal of the ionically linked proteoglycans, the supernatant from 1 x 108 activated BMMC was made 5 M in NaCI and then applied to a column (0.8 x 2 cm) of phenyl-Sepharose CL-4B (Pharmacia) that had been previously equilibrated in 5 M NaCI/0.01 M Tris HCI, pH 7.2. BMMC proteoglycans do not bind to the phenyl-Sepharose CL-4B column under these high salt conditions (5). After the column was washed with 5 column vol of the high salt buffer, bound proteins were eluted with a low salt buffer of 0.15 M NaCI/0.01 M Tris-HCI, pH 7.2. A 1-ml sample ofthis material was applied at 4°C to a 0.8 x 80 cm column of Sephadex G150 (Pharmacia) that had been equilibrated in the low salt Tris*HCI buffer. Fractions (0.5 ml) were collected and assessed for aminopeptidase activity using leucine-,3-naphthylamide as the substrate. Catalase (232 kDa), aldolase (158 kDa), bovine serum albumin (67 kDa), ovalbumin (45 kDa), chymotrypsinogen A (25 kDa), and ribonuclease A (13.7 kDa) were used to standardize the Sephadex G-150 column. The void and total volumes of the column were determined with blue dextran 2000 and [35S]sulfate, respectively. Density-Gradient Subcellular Fractionation of Aminopeptidase Activity. BMMC (108 cells) were suspended in 2 ml of disruption buffer (0.25 M sucrose/0.01 M Hepes, pH 7.2) and gently sonicated (setting 1; 20%o duty cycle; five pulses) at 4°C. The sonicate was filtered sequentially through prewetted 5-pAm and 3-,um filters (Nuclepore) to remove nuclei and nonlysed cells. The filtrate was adjusted to 5 ml with disruption buffer, layered onto 20 ml of 48% (vol/vol) Percoll (Pharmacia) in the disruption buffer, and centrifuged at 60,000 x g for 55 min (28). Fractions of 0.8 ml were collected from the top downward with the aid of an Autodensiflow II
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(Haacke Buchler, Saddlebrook, NJ). Samples (50 Al) of each fraction were assessed for aminopeptidase activity, carboxypeptidase A activity, histamine content (29), and lactate dehydrogenase activity.
Proc. Natl. Acad. Sci. USA 88 (1991) 16
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RESULTS Identification of Aminopeptidase Activity in Supernatants of Calcium Ionophore-Activated Mouse BMMC. When supernatants of calcium ionophore-activated BMMC were incubated for 0-60 min with leucine-13-naphthylamide, progressive degradation of the substrate occurred with the concurrent formation of a digestion product that had a retention time on RP-HPLC of 1.80 min, identical to that of the fB-naphthylamine standard. No measurable product was formed when leucine-f3-naphthylamide was incubated in the absence of the BMMC supernatants. In three determinations with separate cultures of BMMC and separate preparations of mouse serosal mast cells, 0.003 ± 0.002 (mean ± SD) and 0.002 ± 0.001 unit, respectively, of aminopeptidase activity was found per 106 sonicated cells. Cleavage of Ile-His-Pro-Phe by the proteases present in the supernatant of calcium ionophore-activated BMMC also occurred in a linear manner with time, resulting in the appearance of a digestion product that was His-Pro-Phe by amino acid analysis (data not shown). Characterization of the Aminopeptidase Activity Exocytosed from Calcium Ionophore-Activated BMMC. Digestion of leucine-f3-naphthylamide was not inhibited by a pretreatment of the supernatant for 1 hr with 10 mM diisopropylfluorophosphate or 150 ,M potato carboxypeptidase A inhibitor. In contrast, when samples of the same supernatants were preincubated with 10 mM bathophenanthroline or 1 mM bestatin, the degradation of this substrate was inhibited 41% ± 3% or 91% ± 2% (mean ± SD; n = 3), respectively. This aminopeptidase possessed a broad pH optimum of 6.0-8.0, and the Km for the degradation of leucine-p-naphthylamide was 0.36 ± 0.06 mM (mean + SD; n = 3) in 0.02 M Tris buffer (pH 8.0) (data not shown). The presence of varied amounts of the inhibitor bestatin did not alter the Vmax of the reaction, indicating a competitive-type inhibition with a Ki of 1.0 ,uM (data not shown). To characterize the substrate preference of the exocytosed aminopeptidase, supernatants from calcium ionophore-activated BMMC were incubated with various amino acid P-naphthylamides. The preferred substrate was alanine-,B-naphthylamide, with the rate of cleavage of the leucine and phenylalanine derivatives being within the same order of magnitude (Table 1). To determine whether the aminopeptidase activity was released in association with proteoglycans, 35S-labeled BMMC were activated with calcium ionophore, and the resulting supernatant was filtered on a Sepharose CL-2B column. As shown in Fig. 1, 69% of the exocytosed aminopeptidase activity, 78% of the carboxypeptidase A activTable 1. Substrate preference of the aminopeptidase that is exocytosed from calcium ionophore-activated BMMC
,8-Naphthylamine Relative rate Amino acid produced, pmol 1.0 137 ± 21 Leu 1.9 254 + 12 Ala 0.6 87 + 11 Phe 0.1 23 1 Arg
