©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification and Characteristics of the Candidate Prohormone Processing Proteases PC2 and PC1/3 from Bovine Adrenal Medulla Chromaffin Granules (*)

(Received for publication, November 14, 1994; and in revised form, January 30, 1995)

Anahit V. Azaryan (2) Timothy J. Krieger (2)(§) Vivian Y. H. Hook (2) (1)(¶)

From the  (1)From theDepartment of Medicine, University of California, San Diego, California 92103-8227 and (2)Department of Biochemistry, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The prohormone-processing proteases PC1/3 and PC2 belong to the family of mammalian subtilisin-related proprotein convertases (PC) possessing homology to the yeast Kex2 protease. The presence of PC1/3 and PC2 in secretory vesicles of bovine adrenal medulla (chromaffin granules) implicates their role in the processing the precursors of enkephalin, neuropeptide Y, somatostatin, and other neuropeptides that are present in chromaffin granules. In this study, PC1/3 and PC2 were purified to apparent homogeneity from the soluble fraction of chromaffin granules by chromatography on concanavalin A-Sepharose, Sephacryl S-200, pepstatin A-agarose, and anti-PC1/3 or anti-PC2 immunoaffinity resins. PC1/3 and PC2 were monitored during purification by measuring proteolytic activities with S-enkephalin precursor and Boc-Arg-Val-Arg-Arg-methylcoumarin amide (MCA) substrates and by following PC1/3 and PC2 immunoreactivity with specific anti-PC1/3 and anti-PC2 sera generated in this study. Purified PC1/3 and PC2 on SDS-polyacrylamide gels each show a molecular mass of 66 kDa. PC2 in the soluble fraction of chromaffin granules was present at 5- and 10-fold higher enzyme protein and activity, respectively, compared with that of PC1/3. PC1/3 and PC2 cleaved paired basic and monobasic sites within peptide-MCA substrates, with Boc-Arg-Val-Arg-Arg-MCA and pGlu-Arg-Thr-Lys-Arg-MCA as the most effectively cleaved peptides tested. PC1/3 and PC2 showed pH optima of 6.5 and 7.0, respectively. Kinetic studies indicated apparent K values for hydrolysis of Boc-Arg-Val-Arg-Arg-MCA as 66 and 40 µM, with V(max) values of 255 and 353 nmol/h/mg for PC1/3 and PC2, respectively. Specificity of the PC enzymes for dibasic sites was confirmed by potent inhibition by the active site-directed peptide inhibitors (D-Tyr)-Glu-Phe-Lys-Arg-CH(2)Cl and Ac-Arg-Arg-CH(2)Cl. Inhibition by EGTA and activation by Ca indicated PC1/3 and PC2 as Ca-dependent proteases. In addition, PC enzymes were activated by dithiothreitol and inhibited by thiol-blocking reagents, p-hydroxymercuribenzoate and mercuric chloride. These results illustrate the properties of endogenous PC1/3 and PC2 as prohormone-processing enzymes.


INTRODUCTION

The posttranslational processing of prohormones and proneuropeptides requires proteolytic cleavage at paired basic residues and less frequently at monobasic residues, which flank active peptide sequences within the precursors (Docherty and Steiner, 1982; Hook et al., 1994). Recently, candidate mammalian subtilisin-related proprotein convertases have been cloned based on sequence homology to yeast Kex2, a processing protease for pro-alpha-mating factor and pro-killer toxin (Julius et al., 1984; Mizuno et al., 1988; Fuller et al., 1989). Seven members of this proprotein convertase family thus far identified are furin (Roebroek et al., 1986; Hatsuzawa et al., 1990; Van den Ouweland, 1990), PC1/3 (Seidah et al., 1991a; Smeekens et al., 1991; Nakayama et al., 1991), PC2 (Seidah et al., 1990; Smeekens and Steiner, 1990), PACE 4 (Barr et al., 1991; Kiefer et al., 1991), PC4 (Nakayama et al., 1992a; Seidah et al., 1992), PC5/6 (Lusson et al., 1993; Nakagawa et al., 1993), and PC7 (Tsuji et al., 1994). Among these members, PC1/3 and PC2 are most relevant to neuropeptide production since their expression is restricted to neuroendocrine cells, as demonstrated by Northern analysis and in situ hybridization studies (Smeekens et al., 1991; Seidah et al., 1990, 1991a, 1991b; Schafer et al., 1993).

Evidence supporting the role of these PC proteases in the maturation of proproteins and prohormones is based on numerous studies of coexpression of potential proprotein substrates and PC proteases in eukaryotic cell lines (Seidah et al., 1991b; Benjannet et al., 1991; Thomas et al., 1991; Smeekens et al., 1992) or by in vitro experiments using recombinant PC2 and PC1/3 (Shennan et al., 1991; Jean et al., 1993; Rufaut et al., 1993; Zhou and Lindberg, 1993). With respect to PC activities in vivo, PC2 activity has been identified in secretory vesicles of insulinoma cells (Bennett et al., 1992), in intermediate pituitary (Estivariz et al., 1992), and in pancreatic islets of anglerfish (Mackin et al., 1991).

Secretory vesicles of adrenal medulla, known as chromaffin granules, contain several neuropeptides including the enkephalins (Udenfriend and Kilpatrick, 1983; Liston et al., 1984; Spruce et al., 1988), neuropeptide Y (Carmichael et al., 1990), somatostatin (Lundberg et al., 1979), and others, which are generated by proteolytic processing of respective precursors. The presence of PC1/3 and PC2 in chromaffin granules is consistent with prohormone processing occurring in these vesicles. PC1/3 and PC2 proteins have been detected in chromaffin granules by microsequencing (Christie et al., 1991) and immunological analysis (Kirchmair et al., 1992). PC1/3 and PC2 activities in chromaffin granules have been shown by immunoprecipitation studies (Azaryan and Hook, 1992a, 1992b; Hook et al., 1993a). The high yield of chromaffin granules from bovine adrenal medulla should provide large quantities of purified PC1/3 and PC2 for characterizing their activities and understanding their role in processing proenkephalin and other adrenal medullary neuropeptide precursors.

In this study, our results indicate the purification of PC2, as well as lower levels of PC1/3, from the soluble fraction of bovine chromaffin granules. Characterization of endogenous PC1/3 and PC2 demonstrate their properties as Ca-dependent proteases cleaving at typical prohormone paired basic residue processing sites. The relative contribution of PC enzyme activities toward total enkephalin precursor cleaving activity in chromaffin granules is discussed.


EXPERIMENTAL PROCEDURES

Enzyme Assays

PC1/3 and PC2 were assayed by measuring enkephalin precursor cleaving activity and hydrolysis of peptide-MCA (^1)substrates. Enkephalin precursor cleaving activity utilized recombinant enkephalin precursor in the form of [[S]Met]preproenkephalin ([[S]Met]PPE), synthesized from the rat PPE cDNA (Yoshikawa et al., 1984) by in vitro transcription and translation, as described previously (Hook et al., 1990; Krieger and Hook, 1991). [[S]Met]PPE cleaving activity was measured by following the production of trichloroacetic acid-soluble radioactivity as described previously (Krieger and Hook, 1991).

Assay of PC1/3 or PC2 with peptide-MCA substrates was performed by incubating enzymes with 100 µM peptide-MCA in 0.1 M Tris-HCl, pH 6.5, 1 mM dithiothreitol, (160 µl) at 37 °C for 2 h. In some experiments, aminopeptidase M (2 µg, Sigma) was then added (with adjustment of pH to 8.8), and incubation continued at 37 °C for another hour. The rate of formation of free 7-amino-4-methylcoumarin was quantitated as described previously (Azaryan and Hook, 1992a, 1994a, 1994b).

Purification of PC1/3 and PC2

Chromatography of enkephalin precursor cleaving activity from the soluble extract of bovine chromaffin granules (from 650 bovine adrenal glands) on concanavalin A-Sepharose and Sephacryl S-200 columns was performed as described previously (Krieger and Hook, 1991). The S-200 column resulted in two peaks of [[S]Met]PPE cleaving activity. The second peak of activity eluting at 70 kDa apparent molecular mass was positive for PC1/3 and PC2 immunoreactivity.

Affinity chromatography on pepstatin A-agarose was performed by adjusting the pH of the 70-kDa fraction (25 ml) (from the S-200 column) to 4.5 with final buffer concentration of 50 mM sodium citrate and incubating this fraction with pepstatin A-agarose (5 ml, Pierce) at 4 °C for 4 h. This mixture, placed in a column, was washed with 50 mM sodium citrate, pH 4.5, and bound proteins were eluted with 0.1 M Tris-HCl, pH 8.5, 0.2 M NaCl buffer. The pH of the eluted fractions was adjusted to 6.0.

To produce affinity resins for immunoaffinity chromatography of PC1/3 and PC2, IgG immunoglobulins from anti-PC1/3 and anti-PC2 sera were linked to ImmunoPure IgG resin according to the manufacturer's procedure (Pierce). The unbound pool (25 ml) from the pepstatin A column, which contained PC1/3 and PC2, was dialyzed against 0.1 M Tris-HCl, pH 6.0, and concentrated by ultrafiltration to 2 ml. To 1 ml of pepstatin A unbound fraction, 1 ml of 10 mM Tris-HCl, pH 7.5, was added, followed by rocking with anti-PC1/3 or anti-PC2 immunoaffinity resin (2 ml bed volume) for 2 h at 4 °C. Unbound fractions were collected, and their pH was adjusted to 6.0 (by the addition of 0.4 M sodium citrate, pH 5.0). After application of 15 ml of washing buffer, pH 8.2 (from Pierce), bound fractions were eluted with 0.1 M glycine, pH 2.8, and their pH was adjusted to 6.0. Protein content was determined by the method of Lowry (Lowry et al., 1951) with bovine serum albumin as standard. Purified enzymes were assessed by SDS-polyacrylamide gel electrophoresis (as described previously, Krieger and Hook(1991)) and by Western blotting with anti-PC sera (immunoblots performed as described previously (Hook et al., 1993b)).

Chromatofocusing

The pH of the pepstatin A-agarose unbound fraction was brought to 6.0 with chromatofocusing elution buffer prepared as described previously (Krieger and Hook, 1991). After concentration by ultrafiltration, 4 ml of chromatofocusing elution buffer, pH 5.0, was added, and the sample was loaded on a Polybuffer Exchanger 94 column (0.9 times 10 cm, Pharmacia Biotech Inc.) equilibrated with 25 mM Tris acetic acid, pH 8.3. Fractions (2 ml) were eluted with chromatofocusing elution buffer and assayed for hydrolysis of Boc-Arg-Val-Arg-Arg-MCA.

Production of Anti-PC1/3 and Anti-PC2 Sera and Immunoprecipitation of Boc-Arg-Val-Arg-Arg-MCA-cleaving Activity

Anti-PC1/3 and anti-PC2 sera, generated against COOH-terminal peptide sequences, detected PC1/3 and PC2 in the pepstatin A-agarose unbound fraction (anti-PC sera were a gift from Dr. Y. Peng Loh, National Institutes of Health). To obtain quantities of antisera needed for immunoaffinity purification of these enzymes, polyclonal antibodies were raised in rabbits (ImmunoDynamics, La Jolla, CA.) to synthetic peptides corresponding to the COOH-terminal sequence 625-636 of mouse PC1/3, RLLQALMDILNE (Seidah et al., 1991a), and to the COOH-terminal sequence 740-751 of human PC2, EAVERSLKSILN (Smeekens and Steiner, 1990). Peptide synthesis included Cys at the NH(2) terminus for conjugation to KLH protein. Antisera were produced in rabbits and tested in enzyme-linked immunosorbent assays, as described previously (Hook et al., 1985).

For immunoprecipitation of PC1/3 and PC2, the unbound fraction from the pepstatin A-agarose column was preincubated with preimmune, PC1/3, or PC2 antiserum (final dilution, 1:100) for 1 h at room temperature in 50 µl of 0.1 M Tris-HCl buffer, pH 6.5. After further incubation at 4 °C for 16 h, the mixture was rocked with 50 µl of Protein A-Sepharose CL-4B at 4 °C for 45 min. The sample was centrifuged at 13,000 times g for 5 min, and the supernatant was assayed for Boc-Arg-Val-Arg-Arg-MCA cleaving activity. Removal of Boc-Arg-Val-Arg-Arg-MCA cleaving activity from the supernatant by anti-PC enzyme sera indicated immunoprecipitation of PC1/3 and PC2.

Determination of Kinetic Constants, K(m)and V(max)

Affinity, K(m), and maximal velocity, V(max), of PC1/3 and PC2 were determined by assaying enzymes with 2.5-25 µM Boc-Arg-Val-Arg-Arg-MCA in 0.1 M Tris-HCl, pH 6.5, 1 mM dithiothreitol (1-h incubation at 37 °C). Kinetic constants were determined from a reciprocal plot of 1/[S] versus 1/v (Lineweaver and Burke, 1934), where [S] represents substrate concentration and v is enzyme velocity.

Determination of pH Optimum and Effect of Protease Inhibitors

Purified PC1/3 or PC2 were assayed at pH values between 3.0 and 8.5 using 0.1 M sodium citrate, pH 3.0-6.0, and 0.1 M Tris-HCl, pH 6.5-8.5, with Boc-Arg-Val-Arg-Arg-MCA as substrate. For inhibitors, enzymes and inhibitors were preincubated for 30 min at room temperature in 0.1 M Tris-HCl, pH 6.5, and reactions were initiated by adding Boc-Arg-Val-Arg-Arg-MCA at 100 µM.

Rate of PC Enzyme Inactivation by the Peptide Inhibitor Ac-Arg-Arg-CH(2)Cl

Inactivation of PC enzymes by Ac-Arg-Arg-CH(2)Cl was assessed by determining the second-order rate constant k(2), computed as described previously (Azaryan and Hook, 1994a, 1994b).


RESULTS

Purification of PC1/3 and PC2

Identification of potential enkephalin precursor cleaving enzymes in chromaffin granules initially began with [[S]Met]PPE as a model enkephalin precursor substrate (Hook et al., 1990; Krieger and Hook, 1991). The majority (about 90%) of enkephalin precursor cleaving activity in chromaffin granules is present in the soluble rather than in the membrane component (Krieger and Hook, 1991). Chromatography of the soluble fraction on concanavalin A-Sepharose, provided a 25-fold purification with nearly full recovery of enkephalin precursor cleaving activity (Krieger and Hook, 1991).

Further purification of the concanavalin A-bound pool by Sephacryl S-200 gel filtration revealed two peaks of enkephalin precursor cleaving activity (Fig. 1). The first peak has been purified and characterized as the novel cysteine protease, ``prohormone thiol protease,'' and has been demonstrated as a candidate proenkephalin processing enzyme (Krieger and Hook, 1991; Krieger et al., 1992; Azaryan and Hook, 1994a, 1994b). The second peak eluting at approximately 70 kDa was analyzed in this study for the PC1/3 and PC2 enzymes.


Figure 1: Sephacryl S-200 chromatography. The concanavalin A-bound enkephalin precursor cleaving activity was chromatographed by gel filtration on Sephacryl S-200. [[S]Met]PPE-cleaving activity () is expressed as total trichloroacetic acid soluble radioactivity generated by a 5-µl aliquot from each column fraction. Relative protein levels were measured by absorbance at 280 nm (box) in column fractions. The arrow indicates the 70 kDa peak.



Analysis of the 70-kDa fraction with protease inhibitors revealed the presence of serine and aspartic proteases (Table 1). Enkephalin precursor cleaving activity was partially inhibited by soybean trypsin inhibitor, alpha(1)-antitrypsin, benzamidine, and N-tosyl-L-lysine chloromethyl ketone, indicating serine protease activity. The serine protease inhibitor N-tosyl-L-phenylalanine chloromethyl ketone was an effective inhibitor. However, phenylmethylsulfonyl fluoride, another serine protease inhibitor, had no effect. Inhibition by pepstatin A indicated aspartic protease activity. No inhibition was detected by the cysteine protease inhibitor cystatin C. Activity directed toward paired basic residues was indicated by inhibition with the peptide inhibitor (D-Tyr)-Glu-Phe-Lys-Arg-CH(2)Cl that possesses a Lys-Arg site. The effectiveness of protease inhibitors on enkephalin precursor cleaving activity in the 70-kDa fraction indicates inhibition of the production of small peptides (less than 5-8 kDa) that are trichloroacetic acid-soluble (Krieger and Hook, 1991); intermediate sized products greater than 10 kDa are trichloroacetic acid-insoluble and, therefore, are not detected as trichloroacetic acid-soluble S-labeled peptides in this assay (Krieger and Hook, 1991).



To examine the serine proteolytic activity in more detail, pepstatin A-agarose was used to remove the aspartic proteolytic activity from the 70-kDa fraction (Fig. 2). The pepstatin A unbound and bound pools contained 65 and 35%, respectively, of the activity recovered from the column. This step recovered 50% of the total activity applied to the column. The loss of activity may be due to instability of the enzyme(s) at basic pH (the pepstatin A column was eluted with pH 8.5 buffer), as it is known that some secretory granule proteases are unstable at neutral or basic pHs (Krieger and Hook, 1991). (^2)Pepstatin A inhibited enkephalin precursor cleaving activity in the bound pool, but not in the unbound pool (data not shown), indicating removal of aspartic protease activity by pepstatin A-agarose. The tetrapeptide Boc-Arg-Val-Arg-Arg-MCA, a good substrate for the PC enzymes (Jean et al., 1993; Shennan et al., 1991; Rufaut et al., 1993; Zhou and Lindberg, 1993), was readily cleaved by activity in the pepstatin A unbound pool but not by activity in the bound pool (Fig. 2). The Boc-Arg-Val-Arg-Arg-MCA cleaving activity in the pepstatin A unbound pool demonstrates cleavage at an Arg-Arg paired basic residue processing site.


Figure 2: Pepstatin A-agarose chromatography. The 70-kDa fraction from the Sephacryl S-200 column was subjected to affinity chromatography on pepstatin A-agarose. a, total enkephalin precursor cleaving activity was determined in the 70-kDa fraction from the S-200 column (S-200: 70 kDa) and in the unbound (U), wash (W), and bound (B) pools from the pepstatin A-agarose column. b, total Boc-Arg-Val-Arg-Arg-MCA-cleaving activity was determined in the 70-kDa fraction from the S-200 column, and in the unbound (U), wash (W), and bound (B) pools from the pepstatin A-agarose column.



Anti-PC1/3 and anti-PC2 immunoblots indicated that chromaffin granules contained PC1/3 and PC2 as 66-kDa bands (Fig. 3, a and b), which is consistent with the molecular size of PC enzymes detected in neuroendocrine tissues (Bennet et al., 1992; Kirchmair et al., 1992; Mackin et al., 1991), and in cells expressing recombinant PCs (Rufaut et al., 1993; Zhou and Lindberg, 1993). Immunoblots showed that the pepstatin A-agarose unbound, but not the bound, pool contains both PC1/3 and PC2 (Fig. 3, c and d); results suggest higher levels of PC2 than PC1/3 in the unbound pool. Protein staining by Amido Black of the unbound pool showed a single 66-kDa band (Fig. 3e); this band presumably contains both PC1/3 and PC2 based on anti-PC immunoblots (Fig. 3, c and d). Additionally, immunodepletion of 31 and 40% of the Boc-Arg-Val-Arg-Arg-MCA cleaving activity (data not shown) from the pepstatin A unbound pool by anti-PC1/3 and PC2 sera, respectively, but not by preimmune serum, indicated the presence of relevant PC enzyme activities. These results confirmed the presence of PC1/3 and PC2 in the pepstatin A-agarose unbound pool.


Figure 3: PC1/3 and PC2 immunoreactivity in CG and unbound fraction of pepstatin A-agarose. a, anti-PC1/3 immunoblot (anti-PC1/3 antiserum at 1:200) of CG lysate (18 µg). b, anti-PC2 immunoblot (anti-PC2 serum at 1:200) of CG lysate (18 µg). c, anti-PC1/3 immunoblot (anti-PC1/3 serum at 1:200) of pepstatin A unbound (U) and bound (B) fractions. d, anti-PC2 immunoblot (anti-PC2 serum at 1:200) of pepstatin A unbound (U) and bound (B) fractions (2.5 µg). e, Amido Black staining of pepstatin A unbound fraction (1 µg).



Chromatofocusing of the pepstatin A unbound pool was performed to determine whether the the Boc-Arg-Val-Arg-Arg-MCA-cleaving activity in the pepstatin A-agarose unbound pool possesses a pI typical for PC enzymes. A single peak of Boc-Arg-Val-Arg-Arg-MCA cleaving activity was detected at pH 5.2-4.8, with maximal activity at pH 5.0 (data not shown). These results indicate Boc-Arg-Val-Arg-Arg-MCA cleaving activity with pI value of approximately 5.0, which is consistent with the detection of PC1/3 and PC2 as glycoprotein H with pI of approximately 4.9-5.0 (Christie et al., 1991).

Immunoaffinity chromatography was used to separate PC1/3 and PC2 in the pepstatin A unbound pool. PC2 was purified using the anti-PC1/3 immunoaffinity column, with PC2 eluting as the unbound material. Analogously, PC1/3 was purified using the anti-PC2 immunoaffinity column, with PC1/3 eluting as the unbound material. Total PC1/3 and PC2 activities obtained after the immunoaffinity step, yielded 10 times greater levels of PC2 activity compared with PC1/3 activity (Fig. 4). There was no proteolytic activity in the bound pools eluted from the immunoaffinity columns at pH 2.8. Evidently, PC enzymes are not stable to the large drop in pH during elution from the immunoaffinity column. Collection of the unbound pool results in isolation of active PC enzymes.


Figure 4: Immunoaffinity chromatography of PC1/3 and PC2. Total PC1/3 activity obtained as the unbound pool from the anti-PC2 affinity column, and total PC2 activity obtained as the unbound pool from the anti-PC1/3 affinity column are shown.



On SDS-polyacrylamide gel electrophoresis, the purified PC2 and PC1/3 appeared as single bands of 66 kDa, indicating purification to apparent homogeneity (Fig. 5a). Immunoblots showed that purified PC2 was recognized by anti-PC2 serum but not by anti-PC1/3 serum (Fig. 5b); PC1/3 was recognized by anti-PC1/3 serum but not by anti-PC2 serum (Fig. 5c). These results indicate the effective separation of PC1/3 from PC2 by the immunoaffinity step.


Figure 5: Purified PC2 and PC1/3 on SDS-polyacrylamide gel electrophoresis and immunoblots. a, purified PC2 and PC1/3 proteases (4 µg of each, lanes1 and 2, respectively), were subjected to gel electrophoresis on a 12% SDS-polyacrylamide gel that was stained with Coomassie Blue. b, anti-PC2 immunoblot of purified PC2 and PC1/3 (lanes1 and 2, respectively). Lane1 illustrates positive immunochemical identification of purified PC2, and lane2 shows the lack of PC2 in the purified PC1/3. c, anti-PC1/3 immunoblot of purified PC2 and PC21/3 (lanes1 and 2, respectively). Lane2 shows PC1/3 immunoreactivity in the purified sample of PC1/3, and lane1 shows the lack of PC2 in the sample of purified PC1/3. Immunoblots utilized anti-PC1/3 and anti-PC2 sera at 1:200 dilution.



Based on Boc-Arg-Val-Arg-Arg-MCA cleaving activity in chromaffin granules, purification at the pepstatin A step represents approximately a 363-fold purification of PC1/3 and PC2 from chromaffin granules. With some loss of activity at the pepstatin A-agarose and immunoaffinity steps, the purification results in a 46- and 95-fold purification of PC1/3 and PC2, respectively, from chromaffin granules (Table 2). An excellent yield (from 650 adrenal medullae) is illustrated by the isolation of 100 and 550 µg of PC1/3 and PC2, respectively (Table 2).



Cleavage Specificity and Substrate Preference of PC1/3 and PC2 with Peptide-MCA Substrates

PC1/3 and PC2 both showed similar orders of preference for the peptide-MCA substrates tested. PC1/3 and PC2 readily cleaved peptide-MCA substrates containing the paired basic residues Arg-Arg, Lys-Arg, and also cleaved Lys-Lys within such peptide substrates (Table 3). Cleavage on the COOH-terminal side of Arg-Arg within Boc-Arg-Val-Arg-Arg-MCA showed the highest level of activity for both PC enzymes. The Lys-Arg-containing peptide pGlu-Arg-Thr-Lys-Arg-MCA was also an excellent substrate for the PC enzymes. Lower activity was observed with peptide-MCA substrates containing monobasic Arg or Lys cleavage sites. To assess cleavage of basic residues on their NH(2) terminal sides, aminopeptidase M was added to reactions after incubation with PC1/3 or PC2. Cleavage of basic residues on their NH(2) terminal sides would not be detected without aminopeptidase M, since only free 7-amino-4-methylcoumarin and not peptide-MCA are fluorimetrically detected. For most of the paired basic residue-containing peptides, there was no significant increase in the PC activity with aminopeptidase M, indicating preference of the PC enzymes for cleavage on the COOH-terminal side of the dibasic site. In the case of the monobasic containing peptides Boc-Val-Leu-Lys-MCA and Boc-Gln-Gly-Arg-MCA, treatment with aminopeptidase M indicated the ability of PC enzymes to cleave at the NH(2)-terminal side of monobasic Arg and Lys. These results indicate preference of the PC1/3 and PC2 for cleaving paired basic residues at their COOH-terminal side; however, cleavage of monobasic residues at both COOH- and NH(2)-terminal sides of the basic residue occurs.



pH Dependence, Kinetics, and Protease Inhibitors

PC1/3 and PC2 showed pH optima of pH 6.5 and pH 7.0, respectively (Fig. 6). Both enzymes showed approximately 50% of maximum activity near the intragranular pH of 5.5-6.0 (Pollard et al., 1979), indicating that they would be active in vivo. The possibility of varied stability of the enzyme at different pHs may be considered.


Figure 6: pH dependence of PC1/3 and PC2. The purified PC1/3 (panela) and PC2 (panelb) were assayed with Boc-Arg-Val-Arg-Arg-MCA as substrate at different pH values.



The kinetic constants apparent K(m) and V(max) were assessed with Boc-Arg-Val-Arg-Arg-MCA, the best peptide-MCA substrate, by Lineweaver-Burk plots (Fig. 7, a(i) and a(ii)). PC1/3 and PC2 showed apparent K(m) values of 66 and 40 µM and V(max) values of 255 and 353 nmol of 7-amino-4-methylcoumarin released/h/mg, respectively. Further characterization of PC enzymes utilized 100 µM Boc-Arg-Val-Arg-Arg-MCA.


Figure 7: Kinetic parameters of PC1/3 and PC2. a, kinetic constants, apparent K and V(max), for PC1/3 and PC2. Lineweaver-Burke plot of PC1/3 (i) and PC2 (ii) hydrolysis of Boc-Arg-Val-Arg-Arg-MCA (8.8 and 5.0 µg of PC1/3 or PC2, respectively, per assay). b, rate of PC1/3 (i) and PC2 (ii) inactivation by Ac-Arg-Arg-CH(2)Cl. Inactivation of PC1/3 and PC2 (8.8 and 5.0 µg of PC1/3 or PC2, respectively, per assay) by Ac-Arg-Arg-CH(2)Cl (2 times 10M) was examined as a function of preincubation time of inhibitor with enzyme.



Protease inhibitor studies showed that the PC enzymes were potently inhibited by the active-site directed peptide inhibitors (D-Tyr)-Glu-Phe-Lys-Arg-CH(2)Cl and Ac-Arg-Arg-CH(2)Cl (Table 4), providing further evidence for paired basic residue cleavage specificity. The second order rate constants for Ac-Arg-Arg-CH(2)Cl inactivation of PC1/3 and PC2 were 32,000 and 31,200 M s, respectively (Fig. 7, b(i) and b(ii)). Both PC enzymes were inhibited by the metal chelators EDTA and EGTA; the addition of calcium ions (5 mM) reversed the inhibitory effect of EGTA. The thiol blocking agents, p-hydroxymercuribenzoate and mercuric chloride, inhibited activity, while dithiothreitol stimulated activity of PC1/3 and PC2 by 10-fold. These results suggest the presence of functional cysteine residue(s) in the vicinity of the enzyme active site. E-64c, a cysteine protease inhibitor, had no effect. alpha(1)-antitrypsin and alpha(1)-antichymotrypsin, serpin protease inhibitors, reduced PC enzyme activities. However, the serine protease inhibitors diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, and benzamidine had no effect. The aspartic protease inhibitor pepstatin A, also had no effect. These protease inhibitor studies illustrate PC1/3 and PC2 as subtilisin-like proteases that are sensitive to Ca and reducing conditions.




DISCUSSION

In this study, we report the purification and characteristics of two endogenous subtilisin-related proprotein convertases, PC1/3 and PC2, from the soluble extract of bovine adrenal medulla chromaffin granules. Using [[S]Met]preproenkephalin and Boc-Arg-Val-Arg-Arg-MCA as substrates, in conjunction with anti-PC1/3 and anti-PC2 sera to identify PC enzyme immunoreactivity, PC1/3 and PC2 of 66 kDa were purified by concanavalin A-Sepharose, Sephacryl S-200, pepstatin A-agarose, and immunoaffinity chromatography. Greater levels of PC2 than PC1/3 were present in the soluble fraction, since the purification yielded 550 µg of PC2 and only 100 µg of PC1/3. This is consistent with previous immunoblot studies showing PC2 as the major soluble PC enzyme in chromaffin granules (Kirchmair et al., 1992). The purified PC1/3 and PC2 show localization to isolated secretory vesicles (chromaffin granules), appropriate specificity for cleavage at paired basic residue sites, pH dependence consistent with the intragranular pH, and apparent affinity (K(m)) and maximal velocity (V(max)) consistent with in vivo levels of adrenal medullary neuropeptide precursors. Furthermore, studies with protease inhibitors and activators indicate PC1/3 and PC2 as Ca-dependent proteases with sensitivity to reducing conditions. These results provide supportive evidence for PC1/3 and PC2 as processing proteases with appropriate cleavage specificity for paired basic residue sites present within prohormones.

Studies of cleavage specificity of chromaffin granule (CG) PC1/3 and PC2 indicated that the most effective peptide-MCA substrates for both PC enzymes were Boc-Arg-Val-Arg-Arg-MCA and pGlu-Arg-Thr-Lys-Arg-MCA (Table 3), which contain Arg-Arg and Lys-Arg paired basic cleavage sites. Recombinant PC1/3 (produced in a mouse L cell line, GH(4)C(1) cells, or Chinese hamster ovary cells) also shows excellent activity with Boc-Arg-Val-Arg-Arg-MCA and pGlu-Arg-Thr-Lys-Arg-MCA (Rufaut et al., 1993; Jean et al., 1993; Zhou and Lindberg, 1993). Thus, native and recombinant PC1/3 and PC2 resemble one another in preference for tetrapeptide substrates. These results suggest that Arg in the P(4) position of the Arg-X-Lys/Arg-Arg motif is important for cleavage specificity requirements of PC1/3 and PC2. However, Arg-Gln-Arg-Arg-MCA was a poor substrate for CG PC1/3 and PC2, even though this peptide contains the same Arg-X-Lys/Arg-Arg motif as Boc-Arg-Val-Arg-Arg-MCA and pGlu-Arg-Thr-Lys-Arg-MCA (Table 3). The free amino-terminal Arg residue of Arg-Gln-Arg-Arg-MCA may be less desirable than the NH(2) terminus blocked by t-butoxycarbonyl. Indeed, an 8.4-fold reduction in activity of recombinant PC1/3 upon removal of t-butoxycarbonyl from Boc-Arg-Val-Arg-Arg-MCA has been demonstrated (Jean et al., 1993).

CG PC1/3 and PC2 cleavage of paired basic residues occurs preferentially at the COOH-terminal side of paired basic residues. The yeast Kex2 protease shows a similar cleavage specificity (Brenner and Fuller, 1992). The CG PC enzymes also resemble Kex2 with respect to equivalent effectiveness in cleaving at paired basic and single basic residues (Lys or Arg) within tripeptide substrates. The ability of CG PC1/3 and PC2 to hydrolyze single basic residue sites (Arg or Lys) is in accord with mono-arginyl cleavages of prorenin by recombinant PC1 (Nakayama et al., 1992b) and with in vitro cleavage at a single arginine site of prodynorphin by recombinant PC1 (Dupuy et al., 1994).

Selectivity of CG PC1/3 and PC2 for paired basic residues was further demonstrated by potent inhibition by the active site-directed peptide inhibitor (D-Tyr)-Glu-Phe-Lys-Arg-CH(2)Cl. This inhibitor corresponds to the Glu-Phe-Lys-Arg sequence at the junction of ACTH and beta-lipotropin within proopiomelanocortin that is cleaved by Kex2, and PC1/3 or PC2 in DNA cotransfection experiments (Thomas et al., 1988, 1991; Benjannet et al., 1991). The CG PC enzymes were also effectively blocked by another dibasic site-containing inhibitor, Ac-Arg-Arg-CH(2)Cl, with the second-order rate constants of inactivation being 32,000 and 31,200 M s for PC1/3 and PC2, respectively.

The kinetic parameters of Boc-Arg-Val-Arg-Arg-MCA hydrolysis by CG PC1/3 and PC2 indicated apparent K(m) values of 66 and 40 µM, and V(max) values of 255 and 353 nmol/h/mg, respectively. The affinity of PC1/3 and PC2 is compatible with the estimated in vivo levels of proenkephalin in chromaffin granules at 10-100 µM (Ungar and Phillips, 1983). The apparent K(m) values of CG PC1/3 and PC2 are also of similar range with the K(m) of 19 µM reported for Kex2 catalyzed hydrolysis of Boc-Arg-Val-Arg-Arg-MCA (Bennet and Fuller, 1992).

CG PC1/3 was most active at pH 6.5, while the pH optimum for CG PC2 was 6.5-7.0. Importantly, PC1/3 and PC2 show 50% of maximum activity at the intragranular pH of 5.5-6.0 (Pollard et al., 1979), indicating that these enzymes would be active in vivo. The endogenous PC enzymes in CG and recombinant PC enzymes show similar pH optima (Rufaut et al., 1993; Shennan et al., 1991).

Studies with protease inhibitors indicated that CG PC1/3 and PC2 were sensitive to metal chelators and thiol-reactive inhibitors, similar to yeast Kex2 (Julius et al., 1984; Brenner and Fuller, 1992). CG PC1/3 and PC2 are calcium-dependent proteases, illustrated by EGTA inhibition of PC enzyme activities, with reversal by Ca ions to generate fully reactivated proteases. The CG PC1/3 and PC2 were also sensitive to thiol-reactive inhibitors p-hydroxymercuribenzoate and mercuric chloride. The effect of sulfhydryl reagents has been also reported for recombinant PC1/3 (Rufaut et al., 1993; Jean et al., 1993; Zhou and Lindberg, 1993) and PC2 (Shennan et al., 1991). Activation of PC1/3 and PC2 by dithiothreitol indicates the presence of functional cysteine near the active site catalytic triad of these proteases. Of interest is the inhibition of the PC enzymes by alpha(1)-antichymotrypsin that is present within chromaffin granules (Hook et al., 1993b), suggesting alpha(1)-antichymotrypsin as a possible regulator of PC enzymes in vivo. The protease inhibitor profile of chromaffin granule PC1/3 and PC2 is in accord with that of recombinant furin, PC1/3, and PC2 (Hatsuzawa et al., 1992; Brennan and Nakayama, 1994; Rufaut et al., 1993; Jean et al., 1993; Zhou and Lindberg, 1993; Shennan et al., 1991). This investigation of endogenous PC1/3 and PC2 from adrenal medullary chromaffin granules indicates active PC enzymes as single chain glycoproteins with molecular mass of 66 kDa and pI 5.0, that are sensitive to Ca and sulfhydryl reagents.

It is important to consider that this study finds soluble PC1/3 and PC2 in CG as secondary enkephalin precursor cleaving activities, accounting for approximately 20% of total CG enkephalin precursor cleaving activity. PC enzymes in the membrane fraction of CG (Kirchmair et al., 1992) may contribute, in part, to the granule membrane-bound enkephalin precursor cleaving activity that represents about 10% of total enkephalin precursor cleaving activity in CG (Krieger and Hook, 1991). In addition, the present study indicates that proteolytic activity of the aspartic protease class represents 10% of total granule enkephalin precursor cleaving activity. In contrast to the PC enzymes and the aspartic protease, the cysteine protease `prohormone thiol protease' (PTP) has been shown as the major contributor of 60% of total enkephalin precursor cleaving activity in CG (Krieger and Hook, 1991). It will be necessary in future studies to understand the coordinate regulation of multiple prohormone processing enzymes in peptide hormone and neurotransmitter biosynthesis.


FOOTNOTES

*
This work was supported by grants from the National Institute of Drug Abuse, the National Institute of Neurological Disorders and Stroke, National Institutes of Health, and the National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Cangene Corporation, Mississauga, Canada.

To whom correspondence should be addressed: Dept. of Medicine, University of California, UCSD Medical Center, 200 West Arbor Dr. #8227, San Diego, CA 92103-8227. Tel.: 619-543-7161; Fax: 619-543-7717.

(^1)
The abbreviations used are: MCA, methylcoumarin amide; PC, proprotein convertase; PPE, preproenkephalin; CG, chromaffin granule.

(^2)
Y. H. Hook and T. J. Krieger, unpublished observations.


ACKNOWLEDGEMENTS

We thank Kim Moran, Gail Hubbard, Rosayln Purviance, Uk Kwon, and Cathy Lee for excellent technical assistance. We also thank Dr. Y. Peng Loh (National Institutes of Health) for the gift of anti-PC1/3 and anti-PC2 sera.


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