©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of the Region within the Neuroendocrine Polypeptide 7B2 Responsible for the Inhibition of Prohormone Convertase PC2 (*)

(Received for publication, December 13, 1994; and in revised form, March 20, 1995)

A. Martin Van Horssen Wilhelmina H. Van den Hurk (1) Elaine M. Bailyes (2) John C. Hutton (2) Gerard J. M. Martens Iris Lindberg (1)(§)

From the  (1)Department of Animal Physiology, University of Nijmegen, Toernooiveld, 6525 ED Nijmegen, The Netherlands, the Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans, Louisiana 70112, and the (2)Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QR, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The highly conserved polypeptide 7B2 and the subtilisin-related prohormone convertases PC1/PC3 and PC2 are broadly distributed in neurons and endocrine cells and are localized to secretory granules. We recently showed that recombinant 7B2 is in vitro a potent inhibitor of PC2 activity, but not of PC1/PC3, and that newly synthesized 7B2 is transiently associated with proPC2 in vivo. In the present study, in vitro mutagenesis was used to identify the region within the 7B2 sequence responsible for the inhibition of PC2. Mutant proteins were produced in a prokaryotic expression system and their effects on PC1/PC3 and PC2 activities were studied by two different in vitro enzyme assays. None of the 7B2 mutant proteins inhibited PC1/PC3 activity. Truncation studies revealed that a short segment within the COOH-terminal portion of 7B2 is critical for its inhibitory effect on PC2. This segment contains a pair of basic amino acid residues which may represent a recognition motif for PC2. Single amino acid substitutions within this Lys-Lys site strongly diminished and a double mutation abolished the inhibitory potency of 7B2. Our results indicate that, although amino acid residues directly surrounding this dibasic pair also contribute to PC2 inhibition, the Lys-Lys site is particularly important for the ability of 7B2 to inhibit PC2.


INTRODUCTION

Many peptide hormones, neuropeptides, and other biologically active peptides and proteins are produced through intracellular proteolytic cleavage of larger precursor proteins at pairs of basic amino acid residues (Douglass et al., 1984). Recently, a mammalian family of processing enzymes related to bacterial subtilisins has been identified. This family specifically participates in the endoproteolytic cleavage of proproteins in the secretory pathway (Barr, 1991; Steiner et al., 1992; Seidah and Chrétien, 1992). The family includes the prohormone convertases PC1 (also known as PC3) and PC2, which are localized to the regulated secretory pathway of neuroendocrine cells, and the more broadly distributed transmembrane proteinase furin, involved in the proteolysis of proproteins in the constitutive pathway of secretion.

The neuroendocrine polypeptide 7B2 is exclusively present in prohormone-producing cells possessing a regulated secretory pathway. In vivo, proteolytic processing of the 27-kDa precursor form of 7B2 occurs at a site marked by basic amino acids in the COOH-terminal domain by an unidentified proteinase, resulting in the production of a 21-kDa 7B2 form (Ayoubi et al., 1990; Lazure et al., 1991; Paquet et al., 1994). The processed 21-kDa 7B2 form was initially purified from porcine and human pituitary glands (Hsi et al., 1982; Seidah et al., 1983) and was found to be released via the regulated secretory pathway (Ayoubi et al., 1990; Iguchi et al., 1987; Vieau et al., 1991). Immunohistochemical analyses have shown that 7B2, PC1/PC3, and PC2 are present in a wide range of endocrine glands and in the central nervous system (Waldbieser et al., 1991; Marcinkiewicz et al., 1987; Steel et al., 1988; Smeekens et al., 1991; Seidah et al., 1991; Cullinan et al., 1991).

The NH(2)-terminal half of 7B2 is distantly related to a subclass of molecular chaperones, the so-called chaperonins of the 60-kDa heat shock protein family (Braks and Martens, 1994). The COOH-terminal half of the 7B2 protein sequence, on the other hand, shares a low degree of homology with members of the potato inhibitor I family, which includes subtilisin inhibitors (Martens et al., 1994). Intact recombinant 27-kDa 7B2, but not processed 21-kDa 7B2, is in vitro a potent inhibitor of PC2 activity and prevents pro-PC2 cleavage. The activity of PC1/PC3, however, was not affected by either form of 7B2, indicating that 7B2 specifically inhibits PC2 (Martens et al., 1994). Furthermore, newly synthesized 7B2 transiently and selectively associates with pro-PC2 in vivo, consistent with its putative role as a chaperone (Braks and Martens, 1994). In the present study, 7B2 mutant proteins were produced and studied as possible inhibitors of PC2 and PC1/PC3 in order to define the region within 7B2 responsible for the potent and selective inhibition of PC2.


EXPERIMENTAL PROCEDURES

Production of Mutant 7B2 Proteins

The prokaryotic expression constructs encoding the intact 27-kDa form and the processed 21-kDa form of human 7B2 have been described previously (Martens et al., 1994). The cDNAs for the expression of mutated and truncated forms of human 7B2 were generated by polymerase chain reactions (PCR)^1(^1)using specific primers. The human 27-kDa 7B2 cDNA cloned in the prokaryotic expression vector pQE30 (Qiagen, Chatsworth, CA) was amplified in a 50-µl sample mixture containing SuperTaq polymerase buffer, 50 pmol of the appropriate 5` and 3` primers, 200 µM dNTP, and 0.5 unit of SuperTaq DNA polymerase (HT Biotechnology, Cambridge, United Kingdom). PCR amplifications were carried out for 30 cycles in a thermal cycler (Perkin-Elmer) with 1 min of denaturation at 93 °C; 1.5 min of primer annealing at 60 °C; and 1 min of extension at 70 °C. A final 10-min incubation at 70 °C was performed to ensure full extension of all PCR products. To facilitate cloning, the specific PCR primers introduced internal restriction sites, as indicated in bold.

The cDNAs for NH(2)- and COOH-terminally truncated 7B2 proteins were generated by PCR using specific primers: the 5` primers Deg-5` (5`-GC AGG ATC CCC TGT/C CCT GTG/ATC GGG/ATC AAA/G AC-3` for pCP93), CD125-5` (5`-TT CGA GCT CTA GAA GGA TCC GAT CCG GAA CAT GAC TAT CC-3` for pCD125), and 7B2-5` (5`-GCA GGA TCC ATG GGA CAT ATG TAC AGC CCC CGG ACC CCT GAC CG-3`) and 3` primers NG162-3` (5`-T TCT AGA AGC TTA TCC TTG TAG ATA TGG ATT GAC-3` for pNG162), NN167-3` (5`-T TCT AGA AGC TTA ATT ATC CAG TCT CTG TCC TTG-3` for pNN167), NF177-3` (5`-T TCT AGA AGC TTA AAA ATG GGG GAC AGA CTT CTT-3` for pNF177) and 7B2-3` (5`-GTG TCT AGA AGC TTA CTC TGG GTC CTT ATC CTC ATC-3`). The resulting PCR products were cloned into the BamHI and HindIII sites of the appropriate prokaryotic expression vectors pQE30 and pQE32. The plasmid pNG86 was constructed by digesting the 27 kDa 7B2 construct with KpnI and HindIII followed by low melting point-agarose purification and blunt-end ligation. The plasmid pNK171 was constructed by digesting the PCR product also used for the construct pSTTP with BamHI and SalI followed by cloning into pQE30 (SalI site indicated in bold in primer STTP-5`).

Amplification of 7B2 cDNA representing amino acids Tyr^1-Pro of the human 7B2 protein (numbering according to that in Martens(1988)) was accomplished by PCR using the 5` primer 7B2-5` and 3` primer Subt-3` (5`-CG GAT CCC GGG ATA GTC ATG TTC CGG ATC-3`). The plasmid p7B2BS was constructed by cloning the resulting PCR product into the BamHI and SmaI sites of pQE30. Mutations in the potential PC2 recognition site Lys-Lys of human 7B2 were introduced by PCR using the following primers: the 5` primers Fur-5` (5`-AG GAT CCC GGG TTG GGC AAG AGG AAC AAG AGA CTC CTT TAC-3` for pFur), and Chymo-5` (5`-AG GAT CCC GGG TTG GGC AAG TGG AAC ATG GAA CTC CTT TAC GAG-3` for pChymo) and the 3` primer 7B2-3`. The resulting PCR products corresponding to amino acids Pro-Glu of the human 7B2 protein were cloned into the SmaI and HindIII sites of p7B2BS.

Mutations in the potential furin recognition site Arg-Arg-Lys-Arg-Arg and in the potential PC2 recognition site Lys-Lys of 7B2 were introduced by a PCR strategy involving the production of a double-stranded DNA megaprimer (Barik, 1993). The appropriate megaprimer was generated using the 5` primers RLKLR-5` (5`-GGA GAG AGA CTA AAG CTG AGG AGT GTC-3` for pRLKLR), KS-5` (5`-GTT GTT GCA AAG TCG TCT GTC CCC CAT-3` for pKS), KDEG-5` (5`-GTT GTT GCA AAG (C/G) (A/T/C)G TCT GTC CCC CAT-3` for pKV), KR-5` (5`GTT GTT GCA AAG AGG TCT GTC CCC CAT-3` for pKR), SS-5` (5`-AAT GTT GTT GCA TCG TCG TCT GTC CCC CAT-3` for pSS), RSK-5` (5`-AAT GTT GTT GCA AG(A/T) AAG TCT GTC CCC-3` for pRK and pSK), STTP-5` (5`-GTT GTT GCA AAG TCG ACT ACC CCC CAT TTT TCA-3` for pSTTP), and RAKR-5` (5`-CTG GAT AAT GTT CGT GCA AAG AGG TCT GTC CCC CAT-3` for pRAKR) and the 3` primer 7B2-3` in the first round of PCR amplification. The PCR product for the pRAKR mutant was generated by using pRLKLR cDNA as template. After low melting point-agarose purification and phenol-chloroform extraction, the resulting megaprimers served in the second round of PCR amplification as the 3` primer introducing the appropriate mutation in 7B2 cDNA in combination with primer 7B2-5`. Purification of the PCR products was performed with low melting point-agarose, and reamplification was carried out with primers 7B2-5` and 7B2-3`. The PCR products were cloned into the BamHI and HindIII sites of pQE30.

The sequences of all 7B2 constructs were confirmed by double-stranded DNA sequence analysis using T7 DNA polymerase.

Recombinant human 7B2 proteins were produced in Escherichia coli as fusion proteins with an NH(2)-terminal hexahistidine tag by induction of the cells with 1 mM isopropyl-beta-D-thiogalactopyranoside. Recombinant proteins were purified by nickel nitriloacetate-agarose affinity chromatography according to the manufacturer's instructions (Qiagen).

Enzyme Assays

PC1/PC3 and PC2 activities were studied by two different in vitro assays. For the first assay, PC1/PC3 and PC2 proteinases were purified from the culture medium of overproducing CHO cells and the conditioned medium of betaTC3 cells, respectively, essentially as described previously (Zhou and Lindberg, 1993; Shen et al., 1993; Lindberg et al., 1995). PC1/PC3 and PC2 activities were measured in the presence or absence of recombinant 7B2 by cleavage of a fluorogenic substrate as described previously (Martens et al., 1994). Recombinant 7B2 proteins were reconstituted to about 1 mg/ml. Duplicate reactions were performed in 0.1 M sodium acetate, pH 5.5; 5 mM calcium chloride; 0.2 mM fluorogenic substrate (Z-Arg-Ser-Lys-Arg-AMC, where AMC is aminomethylcoumarin); 0.1% Brij; 2 µg of bovine serum albumin; 1-10 µl of diluted recombinant 7B2 or 0.1% Brij; 1 µM pepstatin A, 1 µM E-64, 140 µM TLCK, 280 µM TPCK; and 10 µl of purified PC1/PC3 or immunopurified PC2 in a final volume of 50 µl. At least five different concentrations of each mutant were used. Enzyme was preincubated with mutant 7B2 protein for 30 min at room temperature prior to the addition of substrate. Incubations were carried out at 37 °C in polypropylene microtiter plates, and the amount of the fluorescent product AMC was estimated in a microtiter plate fluorometer every hour for 5-7 h. Production of AMC was linear after the first hour. The rate of AMC production in control reactions (no 7B2 protein) was about 100 pmol/h. Data are calculated from 6-7 h time points; nonlinear regression was used to derive the point of 50% inhibition for each protein.

For the second assay, the proinsulin endoproteinases PC1/PC3 and PC2 were solubilized by detergent extraction of insulin secretory granules prepared from rat insulinoma cells (Bailyes and Hutton, 1992). PC1/PC3 and PC2 proteinase activities were measured in the presence or absence of recombinant 7B2 through analysis of human I-proinsulin cleavage essentially as described previously for PC1/PC3 (Bailyes and Hutton, 1992). The assay mixture contained 50 mM sodium acetate, pH 5.5; 1 mM calcium chloride; 0.1% Triton X-100; 10 µM E-64; 10 µM pepstatin A; 100 µM TPCK and 1 mM phenylmethylsulfonyl fluoride. In control incubations, calcium chloride was replaced by 2 mM EDTA. Enzyme and recombinant 7B2 proteins were preincubated for 30 min before the enzyme assay was started by the addition of substrate. Incubations (total volume: 55 µl) were conducted at 30 °C for 2 h and terminated by the addition of Tris, pH 8.0, to a final concentration of 0.1 M. Proinsulin and its cleavage products were separated through immunoprecipitation using the cellulose-coupled monoclonal antibodies A6 (directed against the B-chain/C-peptide junction of proinsulin) or ANT-1 (directed against the C-peptide/A-chain junction of proinsulin; Crowther et al., 1994) in order to determine PC1/PC3 or PC2 enzyme activity, respectively. Radioactivity remaining in the supernatant after immunoprecipitation was determined and the activity was expressed as the percentage conversion of the initial radioactivity.

Cleavage Studies

Cleavage of recombinant 7B2 proteins by purified recombinant PC1/PC3 and immunopurified PC2 was analyzed by incubating 1 µg of 7B2 for 16 h at 37 °C in 0.1 M sodium acetate, pH 5.5; 5 mM calcium chloride; 0.1% Brij; 2 µg of bovine serum albumin; 10 µl of diluted recombinant 7B2 or its diluent, 0.1% Brij; 1 µM pepstatin A, 1 µM E-64; 140 µM TLCK; and 280 µM TPCK. Control reactions contained 5 mM EDTA instead of 5 mM calcium chloride. One-quarter of each reaction was subjected to SDS-polyacrylamide gel electrophoresis and Western blotting performed as described previously (Martens et al., 1994) using a combination of two anti-7B2 monoclonal antibodies (MON-102 and MON-144; Van Duijnhoven et al., 1991).


RESULTS AND DISCUSSION

The effect of recombinant 7B2 on PC2 activity was studied by two different in vitro assays. One assay was based on PC2-mediated cleavage of a fluorogenic substrate (Z-Arg-Ser-Lys-Arg-AMC) (Martens et al., 1994) using PC2 obtained by immunopurification (Shen et al., 1993; Lindberg et al., 1995) from the conditioned medium of the mouse pancreatic cell line betaTC3. The second assay used I-proinsulin as a substrate and was based on the separation of cleavage products by immunoprecipitation (with a monoclonal antibody directed against the PC2 cleavage site in the C-peptide/A-chain junction of proinsulin), followed by determination of the radioactivity that remained in the supernatant. As a source for the enzyme in this assay, solubilized secretory granules of rat insulinoma cells were used (Bailyes and Hutton, 1992). In both assays, recombinant 27-kDa 7B2 produced half-maximal inhibition of PC2 at nanomolar concentrations (Fig. 1A and Table I). In contrast, at submicromolar concentrations, the COOH-terminally truncated recombinant 21-kDa form of 7B2 did not affect PC2 activity (Fig. 1A and Table I). Granular PC1/PC3 was not inhibited by either form of 7B2 (Fig. 1B).


Figure 1: Recombinant 27-kDa 7B2 inhibits granular PC2 enzyme activity. Solubilized insulin secretory granules were incubated with recombinant 7B2 proteins. A, recombinant 27-kDa 7B2 (bullet) but not COOH-terminally truncated 21-kDa 7B2 (circle) inhibited PC2-mediated cleavage of proinsulin at nanomolar concentrations. B, recombinant 27-kDa 7B2 (bullet) and recombinant 21-kDa 7B2 (circle) did not inhibit PC1/PC3-mediated cleavage of proinsulin. PC2 and PC1/PC3 activities are expressed as a percentage of the activity obtained without added 7B2 protein.



As an initial step toward the identification of the region within the 7B2 sequence responsible for the inhibition of PC2, we have produced the NH(2)- and COOH-terminal halves of 7B2 in E. coli and purified the recombinant proteins to near homogeneity by affinity chromatography. The NH(2)-terminal domain of 7B2 did not inhibit PC2 activity (mutant pNG86, representing amino acid residues Tyr^1 to Gly; Table Iand Fig. 2). This portion of the protein is chaperonin-related and may function in vivo as a molecular chaperone to act in the proper folding or prevent aggregation of proPC2 (Braks and Martens, 1994). The COOH-terminal half of 7B2 (mutants pCP93 and pCD125 corresponding to Pro-Glu and Asp-Glu, respectively) which carries regions structurally related to members of the potato inhibitor I family (Martens et al., 1994), inhibited PC2 activity with comparable potency to intact 7B2 ( Table Iand Fig. 2). Note that with these 7B2 fragments, similar effects on PC2 activity were observed in the two different assays ( Table Iand Fig. 2). This was in fact found to hold for all truncated and mutated 7B2 proteins examined in the present study (see below). Next, COOH-terminally truncated forms of 7B2 were produced to further localize the inhibitory domain within the COOH-terminal half of 7B2. The inhibitory potencies of the truncated proteins encompassing Tyr^1 to Gly, Tyr^1 to Asn, and Tyr^1 to Lys (mutants pNG162, pNN167, and pNK171, respectively) were dramatically diminished, as compared with the potency of intact 7B2 ( Table Iand Fig. 2). In contrast, the truncated 7B2 protein representing amino acid residues Tyr^1 to Phe (mutant pNF177) was a relatively potent inhibitor of PC2 ( Table Iand Fig. 2). These results indicate that a region of six amino acid residues within 7B2 (Lys to Phe) is crucial for PC2 inhibition. Interestingly, the COOH-terminal portion of 7B2 encompassing amino acid residues Val to His is identical among all mammalian, fish, and amphibian 7B2 sequences identified thus far, whereas the region surrounding this portion is much less conserved (Waldbieser et al., 1991; Brayton et al., 1988; Mbikay et al., 1989; Martens et al., 1989).


Figure 2: Inhibitory effect of 7B2 mutant proteins on granular PC2. Solubilized insulin secretory granules were incubated with 100 nM recombinant 7B2 proteins. PC2 convertase activities are expressed as a percentage of the activity obtained without added 7B2. The results shown are the mean ± S.E. of triplicates. Recombinant 7B2 proteins added are: -, no 7B2 protein added; 1, pFur; 2, 21-kDa 7B2; 3, 27-kDa 7B2; 4, pNG86; 5, pCP93; 6, pNG162; 7, pNN167; 8, pNK171; 9, pNF177; 10, pCD125; 11, pSTTP; 12, pKS; 13, pKV; 14, pRAKR; 15, pChymo; 16, pRLKLR; 17, pKR; 18, pSS; 19, pSK; and 20, pRK.



In prohormones, pairs of basic amino acid residues are thought to be potential recognition sites for prohormone convertases (Barr, 1991; Steiner et al., 1992; Seidah and Chretien, 1992). The potential PC2 binding site Lys-Lys is present within the portion of 7B2 which is highly conserved and now suspected to be responsible for the in vitro inhibition of PC2 activity. These observations prompted us to mutate this site. Substitution of Lys-Lys by Ser-Ser (mutant pSS) indeed abolished the inhibitory potency of 7B2 ( Table IIand Fig. 2). The potency of 7B2 was also impaired through the substitution of Lys with Val (pKV), but to a lesser extent than with the Lys to Ser mutant ( Table IIand Fig. 2). This indicates that the effects of the Lys mutations apparently depend on the nature of the substituting amino acid residue. Remarkably, substitution of Lys by Arg (mutant pKR) attenuated the inhibitory effect of 7B2, whereas the Lys to Arg mutant protein (mutant pRK) was as potent as nonmutated 7B2 ( Table IIand Fig. 2). Having demonstrated the critical role of Lys-Lys, we then decided to investigate whether other amino acid residues directly surrounding this dibasic pair are important for PC2 inhibition. Indeed, additional substitution of Val or Ser-Val (mutants pRAKR and pSTTP, respectively) further diminished the inhibitory potency of 7B2 ( Table IIand Fig. 2). The Lys-Lys site is probably a site of proteolytic cleavage in vivo, since the COOH-terminal 7B2 peptide corresponding to Ser to Glu has been recently identified in bovine adrenal medulla chromaffin vesicles (Sigafoos et al., 1993). The enzyme responsible for this cleavage remains to be identified. In contrast to the substitutions within Lys-Lys, mutations within the two other potential PC2 recognition sites in 7B2, namely Lys-Lys (mutants pChymo and pFur) and Arg-Arg-Lys-Arg-Arg (mutant pRLKLR), did not impair the inhibitory capacity of 7B2 ( Table IIand Fig. 2). This indicates that paired basic amino acids other than Lys-Lys do not contribute to the ability of 7B2 to inhibit PC2. Again, in the two different enzyme assays for both PC2 and PC1/PC3, similar results were obtained with these 7B2 mutants. None of the 7B2 mutant proteins described here was able to inhibit PC1/PC3 (data not shown).

We recently reported that intact recombinant 7B2 was cleaved in vitro by PC1/PC3 but not by PC2 (Martens et al., 1994). In order to examine whether our 7B2 mutants were cleaved, we incubated the recombinant proteins in the presence of PC2 or PC1/PC3 and analyzed the cleavage products by Western blotting. No PC2-mediated cleavage of recombinant 27 kDa 7B2 nor of the pFur, pChymo, and pRLKLR mutant proteins was observed under the present conditions (data not shown). These results are in line with the finding that these recombinant proteins potently inhibit PC2 activity. In vitro, PC1/PC3 has been shown to cleave intact recombinant 7B2 at Arg-Arg but not at Lys-Lys (Martens et al., 1994). Western blot analysis revealed that the pFur and pRLKLR mutant proteins were effectively cleaved by PC1/PC3 (Fig. 3). The size of the fragment (18 kDa) generated from the pFur mutant protein indicated that the introduction of a potential furin recognition site through replacement of Trp-Asn-Lys-Lys by Arg-Asn-Lys-Arg resulted in cleavage at this site by PC1/PC3. Interestingly, alteration of the natural furin site in 7B2, Arg-Arg-Lys-Arg-Arg to Arg-Leu-Lys-Leu-Arg (mutant pRLKLR), still resulted in the production of a fragment of 21 kDa by PC1/PC3 (Fig. 3), consistent with other studies which indicate that PC1/PC3 is able to cleave at single basic amino acid residues (Nakayama et al., 1992; Dupuy et al., 1994). The findings of these cleavage studies are in accordance with the observation that none of the 7B2 mutant proteins examined in the present study affected PC1/PC3 activity (data not shown).


Figure 3: 7B2 proteins are cleaved in vitro by PC1/PC3. After incubation of recombinant 7B2 proteins with PC1/PC3, the samples were subjected to Western blot analysis using anti-7B2 monoclonal antibodies. From left to right: lanes 1 and 2, pRLKLR cleavage; lanes 3 and 4, pFur cleavage; and lanes 5 and 6, recombinant 27-kDa 7B2 cleavage. Either calcium or EDTA was included in the reaction mixture, as indicated.



Taken together, our data indicate that a short segment located near the COOH terminus of 7B2 is responsible for the potent and selective inhibition of PC2 activity. The potential PC2 binding site Lys-Lys present within this segment appears to be essential, but not fully responsible, for the inhibitory potency of 7B2 and probably acts in concert with other amino acid residues in the direct vicinity of this site.

  Table I:


 


  Table II: Inhibitory potency of mutated 7B2 proteins




FOOTNOTES

*
This work was supported by a PIONIER grant from the Netherlands Organization for Scientific Research (to G. J. M. M.); the British Diabetic Association, Wellcome Trust (to J. C. H.); European Community Grant BIOT-CT91-0302 (to G. J. M. M. and J. C. H.); and National Institutes of Health Grant DA05084 (to I. L., who was supported by a Research Scientist Development Award from NIDA). 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.

§
To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Louisiana State University Medical Center, 1901 Perdido St., New Orleans, LA 70112. Tel.: 504-568-4799; Fax: 504-568-3370; E-mail: ilindb@lsumc.edu.

(^1)
The abbreviations used are: PCR, polymerase chain reaction; AMC, aminomethylcoumarin; E-64, trans-epoxysuccinic acid; PC, prohormone convertase; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone; TLCK, N-p-tosyl-L-lysine chloromethyl ketone.


ACKNOWLEDGEMENTS

We thank J. A. M. Braks, C. A. M. Broers, C. A. M. De Haan, and J. J. Finley for their contributions.


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