Purification of protease nexin II from human fibroblasts.

Normal human fibroblasts secrete a protein named protease nexin II (PN II) which previously was shown to form sodium dodecyl sulfate (SDS)-stable complexes with epidermal growth factor-binding protein (EGF-BP). These complexes then bind to the same cells and are rapidly internalized and degraded (Knauer, D.J., and Cunningham, D.D. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2310-2314). Here we describe a procedure for purifying PN II to apparent homogeneity from serum-free culture medium conditioned by human fibroblasts. The first step employed dextran sulfate-Sepharose affinity chromatography. Further purification was achieved by ion-exchange chromatography on DEAE-Sepharose followed by gel filtration on Sephacryl S-400. Sequence analysis of purified PN II identified 33 amino-terminal amino acids; a computer search of several protein sequence data banks failed to reveal homologies with other reported amino acid sequences. Purified PN II had an apparent Mr of 106,000 and an isoelectric point of approximately 7.2. It retained full activity after incubation in the presence of 0.05% SDS or at a pH of 1.5. PN II formed SDS-stable complexes with EGF-BP, the gamma subunit of 7 S nerve growth factor, and trypsin with estimated Mr of 120,000, 120,000, and 110,000, respectively. PN II was metabolically labeled with [35S]methionine and purified; the metabolically labeled protein formed complexes with EGF-BP. Complexes between purified PN II and EGF-BP bound to human fibroblasts. These results show that the purified protein possesses the properties previously attributed to PN II in cell culture medium.

There is evidence that the linkage between a PN and its protease involves an ester bond with the catalytic site serine of the protease. First, PN-protease complexes are stable to boiling in SDS suggesting that they are covalent (1, 3, 4).
Second, the complexes are disrupted by pH 12 or 1 M hydroxylamine (1,9). Third, derivatization of the protease active site serine with &isopropyl fluorophosphate blocks the formation of complexes (1,5,8). The acyl-linked PN-protease complex is probably a stable intermediate of proteolysis of the PN since a fragment is released from native PN during complex formation (9).
Since the linkage of these proteases to their respective PN involves the catalytic site serine, this inactivates these proteases and provides a mechanism whereby cells can regulate the activity of certain proteases at or near the cell surface. Studies with purified PN I showed that it can modulate thrombin-stimulated cell division (ll), levels of plasminogen activator at the cell surface (S), and prevent degradation of extracellular matrix by fibrosarcoma cells (12). The present purification of PN I1 was undertaken so that its biochemical properties and biological functions could also be examined. PN I has been purified from serum-free culture medium conditioned by HF cells grown on microcarrier beads (9) or in tissue culture roller bottles (13). Here we describe the purification of PN I1 from serum-free culture medium conditioned by HF cells maintained in microcarrier cultures.

EXPERIMENTAL PROCEDURES
Materials-Gelatin microcarrier beads were obtained from KC Biological. Microcarrier flasks and stir plates were from Wheaton Scientific, Millville, NJ. Fetal bovine serum and all cell culture media were from Gibco. All tissue culture plasticware was from Corning. Sepharose CL-GB, DEAE-Sepharose, Sephacryl S-400, and dextran sulfate (M. = 500,000) were from Pharmacia P-L Biochemicals. 1,4-Butanediol diglycidyl ether and N,N'-diallyltartardiamide were obtained from Aldrich. Aquacide 11-A was froh Behring Diagnostics. Ultrapure acrylamide and ampholytes were from BioRad. Isoelectric focusing marker proteins were from BDH Chemicals. Na'251 was from New England Nuclear and IODO-GEN was from Pierce Chemical Co.
[%S]Methionine was from Amersham Corp. Mono-Tris was synthesized and purified as previously described (14). All other chemicals were reagent-grade and obtained from Sigma.
Mouse EGF-BP was purified as previously described (15) and was iodinated by the chloroglycouril method using IODO-GEN and Na'*'I (16). The specific activities ranged between 30,000 and 70,000 cpm/ ng.
Synthesis of Dextran Sulfate-Sephrose-250 ml of Sepharose CL-6B were washed thoroughly with distilled water. The caked Sepharose 8508 was then activated by adding 125 ml of 1 M NaOH containing 2 mg/ ml NaBH, and 125 ml of the diepoxide l,4-butanediol diglycidyl ether; the mixture was subsequently mixed on a rocking platform for 8 h at room temperature. After activation, the 250 ml of epoxyactivated Sepharose was washed thoroughly with distilled water and added to 250 ml of 0.2 M sodium bicarbonate (pH 11) containing 10 g of dextran sulfate (M, = 500,000). The coupling was performed by mixing on a rocking platform at 37 "C for 16 h. Then, ethanolamine was added to a final concentration of 1 M and mixing was continued at 37 ' C overnight to block any remaining reactive epoxide groups. The coupled Sepharose was then washed with 2 liters of 0.2 M sodium bicarbonate (pH l l ) , 2 litera of 0.1 M sodium acetate (pH 4), and 2 liters of phosphate-buffered saline. The dextran sulfate-Sepharose was stored at 4 "C in phosphate-buffered saline containing 0.02% NaN,.
Cell Culture-HF cells were isolated from explants of neonatal foreskins and were maintained in 100-mm culture dishes as previously described (1). To set up 850-cm2 roller bottle cultures, HF cells were treated with trypsin from two confluent 100-mm culture dishes and placed in 100 ml of DMEM buffered with 20 mM Hepes (pH 7.4) containing 100 units/ml of penicillin, 100 pg/ml streptomycin, and 10% fetal bovine serum. The roller bottles were maintained at 37 "C in a roller bottle apparatus (0.7 rpm); the HF cells reached confluency in approximately 7-10 days.
To prepare a 3-liter microcarrier culture, 35 g of gelatin microcarrier beads were swollen in phosphate-buffered saline, autoclaved, and rinsed in DMEM buffered with 20 m M Hepes (pH 7.4) containing 100 units/ml of penicillin, 100 pg/ml of streptomycin, and 10% fetal bovine serum. The entire 750-ml slurry of microcarrier beads in medium was added to a sterilized 3-liter microcarrier flask. HF cells obtained by trypsin treatment of eight 850-cm2 roller bottles were then added to the culture flask. The culture flask (900-lo00 ml total volume) was then incubated at 37 "C on a magnetic stir plate. The culture was stirred for 60 s at 30 rpm and then allowed to settle for 2 h. This alternating procedure of stirring and settling was continued for 10 h and then the culture was stirred at 40 rpm overnight. The volume of medium was then increased to 3 liters and changed every 4 days. The cells reached confluency on the microcarrier beads in approximately 2-3 weeks.
Collection of Serum-free Conditioned Medium-To remove the serum from the confluent microcarrier cultures, the microcarrier beads were allowed to settle; the serum-containing medium was removed by aspiration and replaced with 2 liters of DMEM buffered with 20 mM Hepes (pH 7.4) containing the antibiotics. The microcarrier beads were again allowed to settle; this rinse medium was removed by aspiration and replaced with 2 liters of DMEM buffered with 20 mM Hepes (pH 7.4) containing the antibiotics and 0.1% BSA. The culture was maintained in this medium for 24 h at 37 "C with stirring at 40 rpm. This medium was then removed and the cultures were incubated with the latter serum-free medium for two subsequent 3-day periods, after each period HF cell "conditioned medium was collected. Using two 3-liter microcarrier cultures, 4 liters of conditioned medium were harvested on each collection day. The cells were then returned to the growth medium containing 5% bovine serum for 5-7 days after which they were cycled again for collection of serumfree conditioned medium. The serum-free conditioned medium was aspirated into a siliconized flask, filtered to remove particulates, and chilled to 4 "C. Phenylmethanesulfonyl fluoride, butylated hydroxytoluene, and sodium azide were added to final concentrations of 200 p~, 50 p~, and 0.04%, respectively. Conditioned medium was used within 24 h after collection and all subsequent chromatographic steps were conducted at 4 "C.
Dextran Sulfate-Sephrose Affinity Chromatography-4 liters of serum-free conditioned medium were applied to a column (2.5 X 40 cm) of dextran sulfate-Sepharose equilibrated with phosphate-buffered saline at a flow rate of 100 ml/h. After loading, the column was washed with phosphate-buffered saline until the Am returned to base-line. The adsorbed protein was eluted from the column with a 1.5-liter linear gradient from 0.15 to 1.2 M NaCl in phosphate-buffered saline and fractions of 19 ml were collected. Fractions were assayed for PN I1 activity with lz5I-EGF-BP as described below. PN IIcontaining fractions were pooled and protein content was measured employing the method of Bradford (17), as modified by Spector (18), using bovine y-globulin and ovalbumin as standards. Phenylmethanesulfonyl fluoride and butylated hydroxytoluene were added to the pooled fractions to final concentrations of 200 and 50 p~, respectively.
Anion Exchange Chromatography-PH 11-containing fractions from dextran sulfate-Sepharose were diluted with 20 mM potassium phosphate (pH 7.4) to reduce the conductivity to that of the starting buffer of the next column, 20 mM potassium phosphate, 0.2 M NaCl (pH 7.4). The resulting solution was applied to a column (0.5 X 12 cm) of DEAE-Sepharose equilibrated in the above starting buffer at a flow rate of 20 ml/h. The column was then washed with 4 column volumes of starting buffer and PN I1 was eluted with 0.75 M NaCl buffered with 20 mM potassium phosphate (pH 7.4). Fractions of 2 ml were collected and each was analyzed for PN I1 activity as described above.
Gel Exclusion Chromatography-The PN 11-containing fractions from DEAE-Sepharose were pooled and placed in M, = 12,OOO-14,000 cutoff dialysis tubing (Spectrapore) and concentrated to approximately 1.0 ml using Aquacide 11-A. Due to the high concentration of salt in the starting pooled DEAE-Sepharose fractions (0.75 M NaCl), loss of PN I1 activity was negligible. The entire 1.0 ml was applied to a column (1.1 X 110 cm) of Sephacryl S-400 equilibrated with 20 mM potassium phosphate, 0.5 M NaCl (pH 7.4). Fractions of 2 ml were collected at a flow rate of 5 ml/h. Aliquots of each fraction were analyzed by SDS-PAGE fractions containing apparently homogeneous PN I1 were pooled, aliquoted, and stored at -70 "C.
Isoelectric focusing was performed employing a modification of the procedure described by O'Farrell (21). 5-pg aliquots of PN I1 were focused in 5% total acrylamide gels cast as tubes (0.6 X 10 cm). Gels contained 1% of the cross-linker N,N"diallyltartardiamide, 2% total ampholytes (1.4% pH range 3-10, 0.6% pH range 6 4 , 0.0003% riboflavin, 0.12% K2S20s, and 1 p1 of Temed/ml of gel. The gel solution was degassed for 5 min; the glass gel tubes were filled, overlaid with distilled water, and photopolymerized under fluorescent light. The upper reservoir buffer consisted of 20 mM NaOH and the lower reservoir buffer consisted of 10 mM H3POI. Samples were focused at 250 V for 18 h at 4 "C. When focusing was complete, the pH gradient of the gels was determined with the use of colored isoelectric focusing marker proteins. The focused gels containing PN I1 were frozen and sliced into 2-mm sections. Each section was extracted into 0.2 ml of phosphate-buffered saline for 24 h at 4 "C. Each gel slice extract was then tested and quantitated for PN I1 activity by the formation of complexes with '"I-EGF-BP on slab gels as described above. Once the PN 11-containing extracts were identified, the isoelectric point was determined from the standard pH curve generated by the isoelectric focusing marker proteins. Alternatively, isoelectric focusing gels containing PN I1 were frozen, sliced into 2mm sections, and extracted into 0.2 ml of distilled water for 24 h at 4 "C. The pH of each gel slice extract was measured. 0.1 ml of phosphate-buffered saline was then added to each extract and aliquots of each extract were tested for PN I1 activity as described above.
PN ZZ Assay-Known quantities of '"I-EGF-BP were incubated with aliquots of samples containing PN I1 for 20 min at 37 "C. An equal volume of SDS sample buffer was then added; the mixtures were subjected to SDS-PAGE as described above. After autoradiography, PN I1 activity was monitored by the formation of a 120-kDa complex with the '"I-EGF-BP. To quantitate PN I1 activity, the autoradiograms were aligned with the dried gels, the '"I-labeled complexes were located, excised, and measured in a y-counter.
Protein Sequencing-Amino-terminal sequencing was performed on purified PN I1 as previously described (22).
Stability of PN ZZ-To investigate the stability of PN I1 under denaturing conditions, the purified protein (30 pg/ml) was incubated in phosphate-buffered saline containing various concentrations of SDS for 60 min at 37 "C. Five volumes of phosphate-buffered saline and 20 ng of '"I-EGF-BP were then added to each sample which was incubated for an additional 20 min at 37 "C. An equal volume of SDS-PAGE sample buffer was then added to each sample and 50 pl of each reaction were subjected to SDS-PAGE. The completed gels were dried and autoradiography was performed. As described above, the gels were aligned, the lZ5I-EGF-BP.PN I1 complexes were located, excised, and measured in a y-counter. PN I1 (30 pg/ml) was also incubated in 30 pl of 0.2 M glycine-HC1 (pH 1.5) for 60 min at 37 "C. After that 10 pl of 3 M Tris-HC1 (pH 8.3) and 20 ng of lZ5I-EGF-BP were added and incubated for an additional 20 min at 37 "C. The samples were subjected to SDS-PAGE and the '%I-EGF-BP.PN I1 complexes were quantitated as described above. In each study, samples incubated with phosphate-buffered saline instead of SDS or glycine-HC1 served as controls.
Metabolic Lubeling of PN I1 by HF Cells-PN I1 was also purified from conditioned medium collected from HF cells that were cultured in the presence of [35S]methionine. Three confluent 100-mm cultures of HF cells were each incubated for 24 h in 10 ml of a minimal essential medium buffered with 20 mM Hepes (pH 7.4) containing 0.1% BSA and only 15% of the normal methionine concentration. This medium was then replaced with 10 ml/dish of the same medium containing 10 pCi/ml of [35S]methionine (1450 Ci/mmol). After a 4day incubation the medium was collected as described above, combined with 90 ml of unlabeled conditioned medium, and loaded onto a 5-ml dextran sulfate-Sepharose column equilibrated with phosphate-buffered saline. The column was washed with 30 ml of phosphate-buffered saline and the protein was eluted with a 40-ml linear gradient of 0.15-1.2 M NaCl in phosphate-buffered saline. Fractions of 1 ml were collected at a flow rate of 3 ml/h. The fractions were assayed for PN I1 activity by incubation with I2'I-EGF-BP as described above; the appropriate fractions were then subjected to preparative SDS-polyacrylamide tube gel electrophoresis employing the mono-Tris/bis-Tris/Bicine/SDS gel system as described above. Since PN I1 retained full activity in the presence of 0.05% SDS the preparative gel electrophoresis was performed at this SDS concentration. After electrophoresis, the gels were frozen, sliced into 2-mm sections, and extracted with 0.3 ml of phosphate-buffered saline for 24 h at 4 "C. Each gel slice extract was then tested for PN I1 activity with '*SI-EGF-BP as described above. The resulting gel slice extracts that contained PN I1 were then stored at -20 "C.

Cellular Binding of ' Y -E G F -B P . PN I1
Comple~es-'*~1-EGF-BP (150 ng) was incubated alone or with an equimolar or 4-fold molar excess of purified PN I1 for 30 min at 37 "C in 200 pl of 20 mM potassium phosphate, 0.2 M NaCl (pH 7.4). Each of these incubation mixtures was then added to 2.8 ml of DMEM buffered with 20 mM Hepes (pH 7.4) containing 0.1% BSA (binding medium). The serumcontaining growth medium on 35-mm culture dishes of confluent HF cells was replaced with binding medium for 24 h at 37 "C. This binding medium was removed and the cells were rinsed once with fresh binding medium (2 ml/dish). This rinse medium was removed and replaced with one of the binding media containing the 9 -E G F -BP. PN I1 complexes (1 ml/dish) and maintained at 37 'C for 20 min. The culture dishes were then rapidly cooled to 0 "C and rinsed five times with ice-cold phosphate-buffered saline containing 0.1% BSA (2 ml/dish/rinse). The cells in each dish were then solubilized in 2 ml of 1 M NaOH for 60 min at 37 "C. The cell-associated radioactivity was then measured in a y-counter. In addition, one dish of cells from each incubation mixture was solubilized in SDS-PAGE sample buffer and an aliquot of each was analyzed by SDS-PAGE and subsequent autoradiography.

RESULTS
Purification of PN ZZ-We purified P N I1 from serum-free culture medium conditioned by HF cells maintained in microcarrier cultures utilizing the procedures outlined under "Experimental Procedures." Table I summarizes the purification. An early observation t h a t P N I1 adsorbed to highly sulfated molecules such as heparin and dextran sulfate provided a useful initial step in the purification. Earlier we synthesized sulfated-dextran beads according to the procedure of Miletich et al. (23) which utilizes Sephadex G-50. The major drawback of this affinity gel is that the column bed volume decreases drastically upon exposure to high ionic strength, thus preventing the use of a salt gradient to elute the adsorbed proteins. T o circumvent this problem, we coupled dextran sulfate t o epoxy-activated Sepharose CL-GB to produce a gel which possesses a high affinity for PN I1 and is unaffected by changes in ionic strength. This affinity matrix not only permitted enrichment for PN 11, but also provided a means for concentrating the large volume of starting material. Four liters of HF cell serum-free conditioned medium were applied directly to this dextran sulfate-Sepharose column followed by elution of the adsorbed protein with a linear salt gradient. PN I1 eluted from the column between 0.5 and 0.6 M NaCl (Fig. 1) with a yield of 95% ( Table I) (Table I). It should be stressed that earlier attempts at using low salt buffers in these steps resulted in severe losses of P N I1 (data not shown). This gel filtration step effectively removed the high M, contaminants (Fig. 2) and resulted in apparently homogeneous protein as assessed by SDS-PAGE and silver stain analysis (Fig. 3, lane I ) . In addition, amino-terminal sequence analysis of purified PN I1 yielded a single sequence of 33 amino acids, further demonstrating the homogeneity of this protein (Table 11). Preliminary Characterization of P N ZZ-PN I1 is a singlechain polypeptide with an estimated M , of 106,000 (Fig. 3, lanes I and 2). When PN I1 was electrophoresed under reducing conditions it migrated slower on the SDS gel with an estimated M , of 112,000 (Fig. 3, lane 3 ) , suggesting t h a t P N  I1 possesses intrachain disulfide bonds. PN I also shares this type of behavior on SDS gels after reduction (10). The isoelectric point of PN I1 was determined as described under "Experimental Procedures" and found to be 7.2 (data not shown). The amino-terminal amino acid sequence of PN I1 is shown in Table 11. A computer search of several protein sequence data banks failed to reveal any homology with other reported amino acid sequences. When purified PN I1 was incubated with EGF-BP and then analyzed by SDS-PAGE, a 1:l stoichiometric complex was observed between the protease and inhibitor (Fig. 4) with an estimated M, of 120,000. A molar excess of EGF-BP was used in this experiment to show that almost all of the PN I1 was reactive.
The stability of the complexing activity of PN I1 under denaturing conditions was examined. Incubation of purified PN I1 in 0.2 M glycine-HC1 (pH 1.5) for 60 min at 37 "C had no effect on its ability to complex lZ5I-EGF-BP (102 f 3.0% of control value; data not shown). The ability of PN I1 to form complexes with lZ5I-EGF-BP was not significantly af- fected upon incubation with 0.05% SDS for 60 min at 37 "C (data not shown). Incubation with higher concentrations of SDS did begin to affect the inhibitor activity of PN 11, but even at 0.1% SDS, greater than 80% of the control activity remained. Together, these data suggest that PN I1 is a stable protein. In contrast, similar treatments readily destroy the inhibitor activity of PN I (24).
Purification of PN 1 1 from HF Cells Cultured in the Presence of r5S/Methionine-Since cultured cells have been shown to internalize serum protease inhibitors and subsequently release them back into the culture medium (26-29), we determined whether HF cells actually synthesize PN 11. To do this we incubated 100-mm confluent culture dishes with [35S]methionine. Since PN I1 was stable in 0.05% SDS, we purified 35S-PN I1 employing preparative SDS-PAGE as described under "Experimental Procedures." In Fig. 6, lane 1 is an autoradiogram of purified PN I1 demonstrating that it was metabolically labeled with [35S]methionine. Lane 2 of the same figure is an autoradiogram of the purified 35S-PN I1 incubated with unlabeled EGF-BP, clearly showing the formation of a 120-kDa 35S-PN 11. EGF-BP complex. The metabolic labeling of the protein clearly shows that it is indeed a biosynthetic product of the HF cells. The formation of an SDS-stable 120-kDa complex with EGF-BP correctly identified this metabolically labeled protein as PN 11. Cellular Binding of lZ5I-EGF-BP.PN 1 1 Complexes-We investigated whether complexes formed between EGF-BP and purified PN I1 bound to HF cells. lZ5I-EGF-BP was incubated with an equimolar or 4-fold molar excess of PN I1 and the resulting lZ5I-EGF-BP. PN I1 complexes were incubated with duplicate confluent cultures of HF cells for 20 min at 37 "C.
After rinsing the cells, the cell-associated radioactivity was measured as described under "Experimental Procedures.'' As shown in Fig. 7 , when free lZ5I-EGF-BP was incubated with the cultures some of the protease was bound to cells via PN I1 as evidenced by the small amount of 120-kDa complex (lanes 1 and 4). This binding probably occurred from PN I1  released during the length of the binding experiment or free P N I1 associated with the cells. However, when the binding was conducted with lz5I-EGF-BP incubated with purified P N I1 the amount of lZ5I-EGF-BP. P N I1 complex associated with the cells increased accordingly (Fig. 7, lanes 2, 3, 5, and 6). These results demonstrate that purified P N I1 can mediate the cellular binding of EGF-BP.

DISCUSSION
This report shows that PN I1 has been purified to apparent homogeneity from serum-free medium conditioned by HF cells maintained in microcarrier cultures. Initial attempts to purify PN I1 from HF cell monolayer cultures maintained in 1 2 FIG. 6. Metabolic labeling of PN 11. Confluent HF cells in three 100-mm culture dishes were incubated for 4 days with [35S]methionine (10 pCi/ml) in serum-free medium containing 15% of the normal methionine concentration. The labeled conditioned medium was harvested, combined with unlabeled conditioned medium, and the PN I1 was purified as described under "Experimental Procedures." Aliquots of "S-labeled PN I1 were analyzed under nonreducing conditions on a 7.5% polyacrylamide gel followed by autoradiography. The arrow denotes PN 11. EGF-BP complexes. Lune 1, 35S-labeled PN 11; lane 2, %-labeled PN I1 + unlabeled EGF-BP. roller bottles resulted in low yields due to the low concentrations of P N I1 in the starting conditioned medium. To increase the concentration of P N I in conditioned medium, Scott and Baker (9) maintained HF cells in microcarrier cultures using Cytodex-2 microcarrier beads. Therefore, we attempted to purify P N I1 from serum-free culture medium conditioned by HF cells cultured on the same microcarrier beads. However, we found very low levels of PN I1 activity in the conditioned medium collected from these cultures. The Cytodex-2 microcarrier beads are composed of a cross-linked dextran matrix which is coated with positively charged N,N,N-trimethyl-2hydroxyaminopropyl groups (30). These positively charged groups apparently enhance the adherence of cells to the beads. In the present studies we showed that PN I1 binds very tightly to the positively charged groups of DEAE-Sepharose. Therefore, a likely explanation for the very low levels of P N I1 we found in Cytodex-2 microcarrier cultures is that PN I1 released by the HF cells was adsorbed to the microcarrier beads.
To circumvent this problem we maintained HF cells on gelatin microcarrier beads and indeed found much more PN I1 in the conditioned medium. Employing a three-step procedure, an approximate 6000-fold purification was achieved with a recovery of 83%. Using dextran sulfate-Sepharose affinity chromatography for the initial step not only concentrated and enriched for P N 11, but also produced a very high yield of 95%. Further purification was achieved by taking advantage of the very tight binding that PN I1 exhibits toward DEAE-Sepharose. Apparent homogeneity was achieved by gel filtra- tion. It is noteworthy that the high recoveries of PN I1 were dependent on the use of high salt buffers throughout the purification. Examination of purified PN I1 by SDS-PAGE revealed that it is a single-chain polypeptide with an estimated M, of 106,000 (Fig. 3). It appears to be glycosylated as evidenced by its binding to lectin-Sepharose colums (data not shown). Purified PN I1 forms SDS-stable complexes with EGF-BP with an estimated M, of 120,000 (Fig. 4) as previously shown for PN I1 in conditioned medium (3). PN 11, unlike many serine protease inhibitors, is an extremely stable protein.
Purified PN I1 retained full activity upon incubation at a pH of 1.5 or in the presence of 0.05% SDS.
At a pH of 7.4, PN I1 did not elute from DEAE-Sepharose at salt concentrations less than 0.45-0.55 M (data not shown), suggesting that it is a highly negatively charged protein. In light of this behavior, it was unexpected to find a somewhat basic isoelectric point of 7.2 for PN 11. Thus, PN I1 may possess an extremely anionic region which is responsible for the high affinity binding to DEAE-Sepharose. Furthermore, it is interesting that PN I also possesses a somewhat basic isoelectric point of 7.5 (9). In contrast, several plasma serine protease inhibitors including antithrombin I11 (31), heparin cofactor I1 (32), and C1-inhibitor (33) are acidic in nature.
Since the HF cells were cultured in the presence of bovine serum we considered the possibility that PN I1 is a bovine protein.
In earlier studies we observed that bovine serum contains a protein which forms SDS-stable complexes with lZ5I-EGF-BP similar in M, to EGF-BP-PN I1 complexes.
However, we purified this bovine protein and demonstrated that it possesses structural and functional properties distinct from PN I1 (34). Importantly, this was the only bovine serum protein that formed complexes with EGF-BP that were similar in M, to EGF-BPePN I1 complexes. In order to definitively demonstrate that PN I1 is synthesized by HF cells, we

Purification 8513
purified the inhibitor from medium conditioned by these cells cultured in the presence of [35S]methionine. A radiolabeled protein of 106 kDa was purified which co-migrated with PN 11. More importantly, when this metabolically labeled protein was incubated with unlabeled EGF-BP, labeled SDS-stable EGF-BP PN I1 complexes were formed (Fig. 6), demonstrating that PN I1 is a biosynthetic product of the HF cells. Next, we determined if complexes between purified PN I1 and EGF-BP bound to cells as previously described (3, 5). lZ5I-EGF-BP PN I1 complexes exhibited a dose-dependent increase in binding over lZ5I-EGF-BP alone (Fig. 7). It is noteworthy that, unlike lZ51-EGF-BP -PN I1 complexes, complexes between lZ5I-EGF-BP and other plasma protease inhibitors failed to bind to HF cells (data not shown).
Although PN I1 was originally identified using 'T-EGF-BP as a probe (3), the present studies showed that purified PN I1 also formed complexes with NGF-7 and bovine trypsin (Fig. 7). We also investigated whether other serine proteases which possess trypsin-like activities are complexed by PN 11. Screening of numerous proteases from the coagulation, fibrinolytic, and complement pathways, as well as several chymotrypsin-like and elastase-like proteases failed to reveal any which formed SDS-stable complexes with PN 11. Although no complex formation was observed, noncovalent inhibition of these proteases may occcur. In contrast, PN I appears to have a broader specificity towards serine proteases (9). The reasons for the more limited specificity of PN I1 are as yet unclear.
However, now that PN I1 can be purified in relatively large quantities, it will be possible to conduct further biochemical and structural studies on it, including its protease specificity, so that physiological roles for this protein may be determined.