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Endocrinology Vol. 140, No. 8 3744-3754
Copyright © 1999 by The Endocrine Society

ARTICLES

Evidence for Functional Localization of the Proenkephalin-Processing Enzyme, Prohormone Thiol Protease, to Secretory Vesicles of Chromaffin Cells1

Vivian Y. H. Hook, Stephen Noctor, Catherine A. Sei, Thomas Toneff, Sukkid Yasothornsrikul and Yuan-Hsu Kang

Department of Medicine, University of California-San Diego, La Jolla, California 92093; the Department of Biochemistry, Uniformed Services University of the Health Sciences (S.N.), Bethesda, Maryland 20814; and the Naval Medical Research Institute (Y.-H.K.), Bethesda, Maryland 20814

Address all correspondence and requests for reprints to: Dr. V. Hook, Department of Medicine, University of California-San Diego, 9500 Gilman Drive, #0822, La Jolla, California 92093-0822. E-mail: vhook{at}ucsd.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The biosynthesis of enkephalin opioid neuropeptides as well as numerous peptide hormones and neurotransmitters requires proteolytic processing of the respective prohormone precursors. We previously identified a novel cysteine protease known as prohormone thiol protease (PTP) as the major proenkephalin-processing enzyme in chromaffin granules (secretory vesicles) of bovine adrenal medulla. In this study, colocalization of PTP with (Met)enkephalin in regulated secretory vesicles was assessed by immunochemical approaches. Western blots demonstrated the presence of PTP in chromaffin granules, with equivalent levels of PTP protein in the soluble and membrane components of the vesicle. The presence of PTP in pituitary was also demonstrated by immunoblots. Immunoelectron microscopy demonstrated immunogold-labeled PTP and (Met)enkephalin within isolated chromaffin granules. In primary cultures of chromaffin cells, the discrete pattern of PTP and (Met)enkephalin immunofluorescence staining in neuritic extensions and cytoplasmic (perinuclear) regions of chromaffin cells is consistent with localization to secretory vesicles. Moreover, cosecretion of PTP and (Met)enkephalin from chromaffin cells occurred upon KCl depolarization in a calcium-dependent manner, indicating the localization of PTP and (Met)enkephalin within regulated secretory vesicles. Calcium-dependent secretion is a well known property of regulated secretory vesicle exocytosis. Overall, these results are consistent with the localization of PTP to functional, regulated secretory vesicles that contain (Met)enkephalin.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ENKEPHALIN opioid peptides and numerous peptide hormones are synthesized as precursor proteins that require proteolytic processing to generate the smaller, active neuropeptides. A major site of prohormone processing occurs within secretory vesicles (1, 2, 3). Preprohormones are initially synthesized from their respective messenger RNAs at the rough endoplasmic reticulum (RER), where the NH2-terminal signal peptide is removed by signal peptidase. The resultant prohormone is routed to the Golgi apparatus and packaged into newly formed secretory vesicles. Proteolytic processing of prohormones at dibasic residues as well as at monobasic residues (Lys and Arg) generates the smaller active peptide hormones and neuropeptides that are stored within secretory vesicles for subsequent secretion. The presence of prohormone substrates and peptide products within secretory vesicles indicates that many of the corresponding processing enzymes are colocalized within the same organelle.

Secretory vesicles of bovine adrenal medulla, known as chromaffin granules, contain high levels of enkephalin peptides (4, 5), as well as proenkephalin and proenkephalin-derived intermediates (6, 7, 8, 9). Chromaffin granules also contain several other neuropeptides such as neuropeptide Y (10), vasoactive intestinal polypeptide (11), and somatostatin (5). These vesicles thus serve as an excellent model neurosecretory vesicle system for studies of proenkephalin- and prohormone-processing enzymes. Our search for proenkephalin-processing enzymes in chromaffin granules led to the finding that the major enkephalin precursor-cleaving activity is represented by a novel cysteine protease known as prohormone thiol protease (PTP) (12, 13, 14, 15, 16). PTP represents approximately 80% of the total enkephalin precursor-cleaving activity in the granules. In addition, lower levels of activity (15–20%) are contributed by the subtilisin-like PC1/3 and PC2 proteases (17) and a 70-kDa aspartyl protease that resembles the pituitary POMC-converting enzyme (18).

PTP is a 33-kDa glycoprotein that cleaves at paired basic and monobasic residue sites within proenkephalin (12, 13, 14). PTP converts proenkephalin into a series of intermediates that lack the COOH-terminal domains of proenkephalin (12, 14) and generates the final peptide product (Met)enkephalin (13). The in vitro products generated by PTP are similar to proenkephalin products in vivo in adrenal medulla, indicating that PTP produces relevant enkephalin peptide products. PTP is optimally active at an acidic pH of 5.5, which is compatible with the acidic intragranular environment of pH 5.5–6.0 (19). Furthermore, a potent cysteine protease inhibitor of PTP inhibits cAMP-mediated elevation of (Met)enkephalin levels in chromaffin cells (15). These findings provide evidence for PTP as a proenkephalin-processing enzyme.

In this study, the localization of PTP within regulated secretory vesicles was examined with an effective antibody generated against PTP, using combined immunochemical and cellular approaches. PTP was present in isolated chromaffin granules, as demonstrated by immunoblots, immunoelectron microscopy, and immunofluorescence cytochemistry. Importantly, demonstration of the cosecretion of PTP with (Met)enkephalin from primary cultures of chromaffin cells indicates the localization of PTP within regulated secretory vesicles. These findings establish the secretory vesicle localization of the cysteine protease PTP that is involved in the proteolytic conversion of proenkephalin, studied as a model prohormone, into active neuropeptides.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Development of PTP antisera and enzyme-linked immunosorbent assays (ELISAs)
PTP was purified to homogeneity from bovine adrenal medulla chromaffin granules, as previously described (12). Peptide microsequencing of the purified 33-kDa PTP (performed by the peptide-sequencing facility at the Medical College of Wisconsin, Milwaukee, WI) determined 17 residues of the NH2-terminal peptide sequence of PTP. This 17-residue sequence represented a unique peptide sequence based on comparisons with amino acid sequence databanks (BLAST and Human Genome Center).

To produce antisera against PTP, a synthetic peptide corresponding to the 17-residue NH2-terminal sequence of PTP was conjugated to thyroglobulin (peptide was synthesized and conjugated to thyroglobulin by Peninsula Laboratories, Inc., Belmont, CA). Rabbits were immunized with the peptide conjugates (by Hazelton Laboratories, Herndon, VA), and antisera (collected after each injection of PTP peptide conjugate) were tested in ELISAs. For ELISAs, 96-well microtiter plates were coated with the 17-residue PTP peptide by incubation with 100 µl 40 µg/ml PTP peptide overnight at 4 C. Wells were blocked with 3% BSA in PBS, pH 7.4, washed with PBS, and incubated (for 2 h at room temperature) with anti-PTP serum (100 µl, diluted in 10% FCS and PBS). After washing with PBS, wells were incubated (2 h at room temperature) with goat antirabbit conjugated to acid phosphatase. Acid phosphatase was detected with p-nitrophenylphosphate substrate (assayed according to the manufacturer’s protocol by Bio-Rad Laboratories, Inc., Richmond, CA) at room temperature for 45 min, measuring absorbance at 405 nm.

Immunodepletion of purified PTP activity
Immunodepletion of PTP activity used anti-PTP IgGs purified by protein A-Sepharose affinity chromatography. IgGs from immune and preimmune sera (from the same rabbit) were purified by incubating PTP serum (0.5 ml antiserum was diluted 1:10 in 10% FCS and 100 mM sodium phosphate buffer, pH 7.4) with protein A-Sepharose resin at 4 C overnight, washing the resin in a column with 100 mM sodium phosphate buffer, and eluting anti-PTP IgG proteins with 100 mM citric acid, pH 4.0. Eluted fractions (1.5 ml) were neutralized by the addition of 0.4 ml 1.0 M Tris-HCl, pH 8.3. Fractions containing anti-PTP IgGs were identified by ELISAs, pooled, dialyzed against 100 mM Tris-HCl, pH 7.4, and concentrated to 1.0 ml by lyophilization.

For immunodepletion of PTP activity, purified PTP enzyme [purified from bovine adrenal medulla as described previously (12)] was incubated with anti-PTP IgGs (1:10 or 1:5 final dilution) in 50 mM citric acid (pH 6.0) and 1 mM 3-[(3-cholamidopropyl)diethylammonio]-1-propanesulfonate (50 µl total volume) at 4 C for 18 h. Protein A-Sepharose [50 µl slurry in 50 mM citric acid (pH 6.0), 1 mM CHAPS, and 50 mM NaCl] was added to the sample, followed by incubation for 2 h at 4 C. PTP-antibody complexes bound to protein A-Sepharose were pelleted by centrifugation at 15,000 x g for 15 min at 4 C. The resultant supernatant was collected for assay of PTP activity. PTP activity was assayed by measuring the conversion of [35S]enkephalin precursor to trichloroacetic acid-soluble radioactivity, representing small 35S-labeled peptide products, as described previously (12).

PTP Western blots of bovine adrenal medulla chromaffin granules and pituitary
For PTP Western blots, chromaffin granules were purified from fresh bovine adrenal medulla by differential centrifugation and discontinuous sucrose gradient centrifugation, as described previously (12, 20). Granules were lysed by freeze-thawing twice in 15 mM KCl and were separated into soluble and membrane fractions (illustrated in Fig. 4Go). Briefly, lysed granules (5 ml, at 15 mg/ml) were brought to 50 mM sodium acetate (pH 6.0) and 150 mM NaCl (isotonic buffer) and incubated at 4 C for 30 min (this sample is the chromaffin granule lysate, 150 min). After ultracentrifugation at 100,000 x g (SW60 rotor), the supernatant was collected as the soluble fraction (CGs150), and the pellet was taken as the membrane fraction (CGm150). Membranes were washed twice in the isotonic buffer by centrifugation at 100,000 x g (soluble wash fractions were CGs150-w1 and CGs150-w2) and resuspended in 5 ml isotonic buffer to obtain washed membranes (CGm150). The washed membrane fraction was then incubated in a high salt buffer (50 mM sodium-acetate, pH 6.0, and 500 mM NaCl) at 4 C for 30 min to dissociate membrane-associated proteins. This sample was subjected to ultracentrifugation at 100,000 x g; the supernatant was collected as a solubilized membrane-associated fraction (CGs500), and the pellet consisted of a high salt membrane fraction (CGm500), respectively. This membrane fraction was washed twice in the high salt buffer (washes were CGs500-w1 and CGs500-w2). These soluble and membrane fractions of chromaffin granules were subjected to PTP Western blot analyses, as described previously (14, 15).



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  		Figure 4. Separation of chromaffin granules into soluble and membrane fractions. Chromaffin granules in 150 mM NaCl buffer (CG lysate 150) were separated into soluble (CGs150) and membrane (CGm150) fractions. After washing the membranes in 150 mM NaCl buffer (CGs150-w1 and CGs150-w2), membranes were resuspended in 500 mM NaCl buffer (CGm500) and centrifuged to obtain membrane-associated proteins (CGs500) and resultant membranes (CGm500). After washing (obtaining CGs500-w1 and CGs500-w2), membranes were resuspended in 500 mM NaCl buffer (CGm500). The 150 and 500 mM NaCl buffers both included 50 mM sodium acetate, pH 6.0. During the fractionation, all fractions obtained were 5 ml, except for the CGm500, which was resuspended in 2.5 ml buffer.

 
PTP Western blots of adrenal medulla, anterior pituitary, and intermediate/neural lobe of pituitary used freshly dissected bovine tissues that were homogenized in ice-cold 25 mM histidine-HCl, pH 6.5, containing a cocktail of protease inhibitors (10 µM pepstatin A, 10 µM chymostatin, and 10 µM phenylmethysulfonylfluoride). Protein content was determined by the Lowry method (21) with BSA as standard. Tissue samples were subjected to PTP Western blot analyses (~20–50 µg protein) as described previously (14, 15).

PTP colocalization with (Met)enkephalin-containing chromaffin granules obtained by multistep sucrose density gradient fractionation
An enriched fraction of chromaffin granules, obtained after differential centrifugation in the Sorvall (before ultracentrifugation on a discontinuous sucrose gradient), was subjected to density gradient centrifugation on a multistep sucrose gradient consisting of 1.2–2.2 M sucrose (2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, and 1.2 M sucrose steps, each consisting of 2.5 ml), as previously described (20). Fractions (1 ml/fraction, beginning with 2.2 M sucrose) were analyzed for the presence of PTP by Western blots, (Met)enkephalin by RIA (as described previously (20), protein content by the method of Lowry (21), and acid phosphatase as previously described (20).

Immunoelectron microscopy of PTP in chromaffin granules
For PTP immunoelectron microscopy, chromaffin granules were isolated from homogenates of bovine adrenal medulla by differential centrifugation in 0.32 M sucrose. The first centrifugation at 400 x g resulted in granules in the supernatant. The supernatant was subjected to centrifugation at 13,000 x g to pellet chromaffin granules, and granules were washed three times in an equal volume of 0.32 M sucrose.

For electron microscopy, chromaffin granules were fixed in 3% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, for 60 min, washed three times in the cacodylate buffer, and stored in the same buffer overnight. Granule samples were dehydrated in 70% ethanol solution and embedded in LR white embedding medium at 60 C. Ultrathin sections were prepared with a diamond knife and mounted on nickel grids. The ultrastructure of isolated granules was examined by subjecting samples to osmium tetroxide and electron microscopy, as previously described (22, 23, 24).

For detection of PTP and (Met)enkephalin immunogold labeling of chromaffin granules, ultrathin sections were incubated in 3% normal goat serum in PBS for 15 min. Sections were then incubated with antienkephalin or anti-PTP antibody (1:20 final dilution, rabbit IgGs purified by protein A-Sepharose) in PBS for 30 min, washed in PBS, and then incubated with goat antirabbit IgGs linked to 5 nm colloidal gold (Janssen Life Sciences, Westbury, NY). After washing in PBS with 1% BSA and distilled water, sections were stained with lead citrate and examined in a JEOL 100 CX II transmission electron microscope (JEOL USA, Inc., Peabody, MA), as described previously (24, 25).

PTP in primary cultures of chromaffin cells: immunofluorescence staining and secretion
Primary cultures of chromaffin cells were prepared from fresh bovine adrenal medulla by enzymatic dissociation with collagenase and DNase, as described previously (15, 26). Chromaffin cells were plated in fibronectin-coated plates (Falcon six-well plates) at 2.0 x 106 cells/well (one well equals 9.6 cm2 growth area) in DMEM (high glucose, Life Technologies, Inc., Gaithersburg, MD) containing penicillin and streptomycin (100 U/ml and 100 µg/ml, respectively), 1 mM sodium pyruvate, 10% heat-inactivated FBS (Irvine Scientific, Santa Ana, CA), and 10-5 M cytosine arabinofuranoside (Sigma Chemical Co.). Cells were maintained in a humidified air/CO2 incubator (95% air-5% CO2).

Immunofluorescence cytochemistry of PTP and (Met)enkephalin was examined in chromaffin cells. Cells were plated on two-chamber glass slides (Lab-Tek slides, Nalge, Naperville, IL) coated with poly-D-lysine (according to the manufacturer’s directions, Sigma Chemical Co., St. Louis, MO) at a density of 0.5 x 106 cells/chamber. After 7 days in culture, cells were fixed in 3.7% formaldehyde (in PBS, pH 7.4) for 10 min, washed twice with PBS, pH 7.4, and stored in 0.5% formaldehyde at 4 C. Cells were permeabilized by incubating cells in ice-cold acetone for 2–3 min, and slides were then air-dried. Cells were washed twice with PBS, washed three times for 5 min each time with PBS-0.5% BSA (BSA, fraction V, Sigma Chemical Co.), and incubated with either primary or preimmune antibodies (final dilutions were anti-PTP IgGs at 1:500, anti-(Met)enkephalin serum at 1:2000, and preimmune serum at 1:500) in PBS-0.5% BSA for 60 min at room temperature. The primary antibody was removed, cells were washed three times for 5 min each time with PBS-0.5% BSA, and incubated with secondary goat antirabbit conjugated to a sulfonated rhodamine derivative known as Alexa 488 (from Molecular Probes, Inc., Eugene, OR; final concentration of secondary antibody, 1 µg/ml in PBS-0.5% BSA). Cells were again washed three times for 5 min each time in PBS-0.5% BSA. Coverslips were mounted with 50% PBS-buffered glycerol, and immunofluorescence was visualized in a Nikon Diaphot TE300 microscope using a fluorescein isothiocyanate cube.

Secretion experiments evaluated calcium-dependent secretion of PTP and (Met)enkephalin during KCl depolarization of chromaffin cells. For secretion studies, cells were washed and preincubated for 15 min at 37 C with standard release medium (SRM) with or without calcium. SRM buffer consisted of 25 mM HEPES (pH 7.3), 118 mM NaCl, 4.6 mM KCl, 10 mM D-glucose, 2.2 mM CaCl2, 1.2 mM MgSO4, penicillin (100 U/ml), streptomycin (100 µg/ml), and 0.5 µg/ml BSA. SRM without calcium included 2.2 mM MgCl2 (to replace 2.2 mM CaCl2) and 2.0 mM EGTA. After removal of preincubation medium, cells were incubated for 15 min at 37 C with fresh buffer consisting of 1) SRM with calcium, 2) SRM without calcium, 3) 50 mM KCl in SRM with calcium, or 4) 50 mM KCl in SRM without calcium. Media were collected, and a cocktail of protease inhibitors was added (final concentrations were 0.1 mM phenylmethylsulfonylfluoride, 10 µM chymostatin, 10 µM leupeptin, 10 µM pepstatin A, and 1 µM E64c). After addition of an equal volume of pH 6.0 buffer (100 mM sodium-phosphate, pH 6.0; 50 mM NaCl; and 1 mM EDTA), media were concentrated (by 100-fold) using a Millipore Corp. Ultrafree membrane (Ultrafree-15 centrifugal filter device, Biomax-10K NMWL membrane, Millipore Corp., Bedford, MA). Secretion samples were subjected to PTP Western blots on 12% SDS-PAGE gels (Novex) and electroblotting onto nitrocellulose membranes (Hybond ECL membrane, Amersham, Arlington Heights, IL). Membranes were blocked with 10% FCS in TTBS buffer (20 mM Tris-HCl, pH 7.5; 0.5 M NaCl; and 0.05% Tween-20), incubated for 2 h with PTP antiserum (1:1000 final dilution) in TTBS buffer containing 1% FCS, washed in TTBS buffer, and incubated for 2 h with antirabbit IgG conjugated to horseradish peroxidase (1:2500 final dilution; Amersham). Positive bands were visualized by chemiluminescence with the ECL Plus kit according to the manufacturer’s protocol (Amersham). (Met)enkephalin in the secretion media was measured by RIA, as described previously (15, 26).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PTP antisera and immunodepletion of PTP activity
Antisera (rabbit) were developed against a synthetic 17-residue peptide that corresponds to the determined NH2-terminus of PTP. The determined NH2-terminal 17 residues represent a unique sequence compared with those in protein sequence databases, suggesting that PTP is a novel cysteine protease. ELISA tests of antisera obtained after each injection of antigen indicated optimal production of antisera after the fifth, sixth, or seventh injections compared with preimmune serum that showed no binding to peptide antigen (Table 1Go).


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  		Table 1. ELISA assay of antisera against PTP

 
To test whether the antipeptide antibody recognizes native, purified PTP enzyme, the ability of the antibody to immunodeplete purified PTP activity from solution was evaluated. Because PTP activity is stable at pH 6.0, but not at a neutral pH of 7.4 (the pH at which most antibody-antigen interactions occur), it was first necessary to assess antibody binding at pH 6.0 for immunoprecipitation experiments. ELISAs showed that antibody binding was nearly 2-fold greater at pH 6.0 than at pH 7.4 (Table 2Go). Moreover, antibody binding in PTP buffer (50 mM citric acid, pH 6.0; 1 mM CHAPS, and 100 mM NaCl), was more than 2 times greater than binding at pH 7.4. These results indicated antibody binding at pH 6.0 under buffer conditions that stabilize PTP activity. Therefore, immunoprecipitation of the enzyme could be performed in PTP buffer.


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  		Table 2. Conditions for effective PTP antibody binding

 
Binding of the anti-PTP peptide IgG fraction to purified, native PTP was assessed by immunodepletion of PTP activity from solution after immunoprecipitation. Immunoprecipitation was conducted by incubating purified PTP with immune or preimmune IgGs, antibody-PTP complexes were bound to protein A-Sepharose, and these complexes were removed by centrifugation. PTP activity remaining in the supernatant was assayed for [35S]enkephalin precursor-cleaving activity. Results showed that PTP activity was specifically removed from solution by immune, but not by preimmune, antibodies (Fig. 1Go). Immunodepletion of PTP was dependent on the amount of antibody used, as the addition of 4 and 10 µl of anti-PTP IgGs removed 43% and 95% of PTP activity, respectively. Results also showed that preimmune IgGs did not affect control PTP activity (no IgGs added). These findings indicate that the antibody specifically recognizes PTP.



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  		Figure 1. Immunodepletion of purified PTP activity. Purified PTP was incubated overnight at 4 C without addition of antibodies (control), with addition of preimmune IgGs (10 µl), or with addition of anti-PTP IgGs (4 or 10 µl). All IgGs were purified from antisera by protein A-Sepharose affinity chromatography, as described in Materials and Methods. Antibody-PTP complexes were bound to protein A-Sepharose and pelleted by centrifugation. PTP activity remaining in the supernatant was assayed by measuring the conversion of [35S]enkephalin precursor into trichloroacetic-acid soluble 35S-labeled peptides, as described previously (12 ). PTP activity is expressed as the percentage of activity remaining in the supernatant of preimmune (control) immunoprecipitation (100%). Immunoprecipitations and PTP assays were performed in duplicate.

 
PTP in chromaffin granules and neuroendocrine tissues detected by Western blots
The PTP antibody allowed detection of PTP by Western blots, which showed a PTP band of 33 kDa that corresponds to purified 33-kDa PTP (12). This band was not detected by preimmune serum (Fig. 2Go). The 33-kDa PTP band was also detected in homogenates of bovine adrenal medulla as well as in anterior and intermediate/neural lobes of pituitary (Fig. 2Go). In addition, PTP was detected by Western blots in rat neuroendocrine tissues, including pituitary, adrenal, and brain homogenates (data not shown). These data indicate the presence of PTP in adrenal medulla and pituitary neuroendocrine tissues.



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  		Figure 2. Western blots detect PTP in chromaffin granules and neuroendocrine tissues. a, PTP Western blots of chromaffin granules. The PTP antiserum (final dilution of 1:500) detected PTP of 33 kDa in isolated chromatin granules (CG; lane 2; ~100 µg). The preimmune serum (1:500 final dilution) did not detect PTP. b, PTP Western blots of neuroendocrine tissues. Tissue of homogenates of bovine adrenal medulla (AM), anterior lobe of pituitary (AL), and intermediate/neural lobe of pituitary (IL/NL) were subjected to Western blots with the PTP antiserum (final dilution of 1:500), shown in lanes 1–3, respectively.

 
The presence of PTP with (Met)enkephalin in secretory vesicle fractions was further indicated by PTP’s colocalization with (Met)enkephalin-containing chromaffin granule fractions obtained on a multistep density gradient from 1.2–2.2 M sucrose (Fig. 3Go). Western blots of gradient fractions indicated the presence of PTP in (Met)enkephalin-containing granule fractions (fractions 12–26). These results indicate the colocalization of PTP and (Met)enkephalin to secretory vesicles (chromaffin granules) of similar density. In addition, the lack of a peak of acid phosphatase activity (a lysosomal enzyme marker) in less dense fractions 54–55, where lysosomes would be located (20), indicated the absence of lysosomes in this preparation of highly purified chromaffin granules. Overall, these results provide evidence for the presence of PTP in (Met)enkephalin-containing secretory vesicles (chromaffin granules).



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  		Figure 3. PTP and (Met)enkephalin in chromaffin granule fractions obtained from a multistep sucrose gradient. a, PTP Western blots of sucrose gradient fractions. Purified chromaffin granules were subjected to a multistep gradient (1.2–2.2 M sucrose) ultracentrifugation, as described in Materials and Methods. Fractions (0.5 ml/fraction) were collected (fractions 1–50 represent 2.2 to 1.2 M sucrose) and were analyzed for PTP by Western blots. b, (Met)enkephalin in sucrose gradient fractions. (Met)enkephalin content (•) in sucrose gradient fractions was determined by RIA, to identify enkephalin-containing chromaffin granule fractions. Protein content in fractions was also measured ({circ}). In addition, acid phosphatase activity in fractions ({triangleup}) was determined to confirm the lack of a peak of lysosomes.

 
It is noted that two peaks of (Met)enkephalin-containing granule fractions were observed (Fig. 3Go, fractions 11–18 and 18–27) that differ in density. It is known that secretory granule formation involves the biogenesis of immature secretory granules that develop into mature, denser granules, which involves numerous budding and fusion events that alter the protein composition of granules during their development (27). Thus, the two populations of (Met)enkephalin-containing secretory granules that differ in density may reflect granules at different stages of maturation. It is also known that the varied protein composition occurs during secretory granule biogenesis (28, 29). This knowledge provides an explanation for the slight differences observed in the localization of PTP to fractions of somewhat lighter density compared with those that contain (Met)enkephalin. Overall, however, the majority of PTP is present in (Met)enkephalin-containing fractions.

The localization of PTP to soluble or membrane components of chromaffin granules was examined. Soluble and membrane components of chromaffin granules were prepared (Fig. 4Go), as described in Materials and Methods. Western blots showed that PTP was present in both soluble and membrane fractions at similar levels (Fig. 5Go, lanes 2 and 5, respectively, as SOL and MEM fractions). PTP was not detected in the solubilized membrane-associated fraction (Fig. 5Go, lane 6), indicating that PTP is not a membrane-associated protein. PTP was present in high salt-treated membranes (Fig. 5Go, lane 9), suggesting that PTP also exists as a membrane protein. The presence of PTP immunoreactivity at equivalent levels in soluble and membrane components of chromaffin granules contrasts with detection of the majority (~85%) of enkephalin precursor-cleaving activity in the soluble fraction of chromaffin granules (12). These findings suggest that PTP in the soluble component of granules may be more active than that in the membrane component of chromaffin granules.



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  		Figure 5. PTP in soluble and membrane components of chromaffin granules. The chromaffin granule soluble and membrane fractions obtained from the fractionation procedure (Fig. 4Go) were subjected to PTP Western blots. Lanes 1–9 contained the granule fractions (from Fig. 4Go) CG lysate 150 (CG), CGs150 (SOL), CGs150-w1 (W1), CGs150-w2 (W2), CGm150 (MEM.), CGs500 (MEM. ASSOC.), CGs500-w1 (W1), CGs500-w2 (W2), and CGm500 (MEM/NaCl), respectively. Identical volumes of each fraction (25 µl) were subjected to 12% polyacrylamide SDS-PAGE gels followed by PTP Western blots.

 
Immunoelectron microscopy of chromaffin granules
The integrity and homogeneity of isolated chromaffin granules that contain (Met)enkephalin was assessed by electron microscopy (EM). EM demonstrated isolation of highly enriched chromaffin granules that possess a diameter of approximately 0.1 µm (Fig. 6aGo). Some differences in granule diameters may represent sections through different planes of the vesicles. The electron-dense nature and morphology of isolated chromaffin granules is similar to the appearance of these granules in situ, that is, granules observed by EM within chromaffin cells (30). These results indicate that the integrity of the chromaffin granules has been retained during purification from chromaffin cells.



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  		Figure 6. EM of chromaffin granules. a, Electron microscopy of chromaffin granules. Isolated chromaffin granules were prepared for EM as described in Materials and Methods. EM shows intact granules with an average diameter of approximately 0.1 µm for these granules. b, Immunoelectron microscopy of (Met)enkephalin in chromaffin granules. Isolated chromaffin granules were prepared for (Met)enkephalin immunolabeling with 5-nm gold particles linked to the secondary antirabbit IgGs, as described in Materials and Methods. Immunoelectron microscopy shows the presence of (Met)enkephalin (indicated by gold particles) within isolated chromaffin granules. Controls indicated that immunogold labeling was not detected when the primary antibody [anti-(Met)enkephalin IgGs] was omitted from the immunostaining procedure (not shown).

 
Immunoelectron microscopy demonstrated the localization of (Met)enkephalin in chromaffin granules (Fig. 6bGo), indicated by the localization of 5-nm gold particles to these granules. These results are consistent with the high levels of (Met)enkephalin detected by RIA of chromaffin granules extracts (15). The localization of (Met)enkephalin to chromaffin granules demonstrates effective isolation of intact (Met)enkephalin-containing secretory vesicles.

Importantly, immunoelectron microscopy of PTP demonstrated localization of immunogold-labeled PTP within chromaffin granules (Fig. 7Go). Quantitation of PTP-labeled immunogold particles in chromaffin granules by immunoelectron microscopy indicated significant numbers of immunogold-labeled PTP particles within these granules (Table 3Go), compared with control sections that demonstrated lack of background immunostaining when the anti-PTP primary antibody was omitted from the staining procedure. Thus, these immunoelectron microscopy results demonstrate localization of PTP within chromaffin granules that contain (Met)enkephalin.



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  		Figure 7. Immunoelectron microscopy of PTP in chromaffin granules. Isolated chromaffin granules were prepared for PTP immunoelectron microscopy as described in Materials and Methods. PTP was detected as 5-nm gold particles linked to the secondary antirabbit IgG molecules. Immunoelectron microscopy illustrated the presence of PTP (indicated by electron-dense gold particles) within chromaffin granules. Controls showed a lack of immunogold labeling when the primary antibody was omitted (data not shown).

 

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  		Table 3. PTP and (Met)enkephalin immunogold labeling of chromaffin granules by immunoelectron microscopy

 
Quantitation of immunogold-labeled PTP and (Met)enkephalin in chromaffin granules indicated differences in their relative abundance within these secretory vesicles (Table 3Go). The lower density of immunogold labeling of PTP compared with (Met)enkephalin is consistent with lower levels of PTP in chromaffin granules, calculated as approximately 3 ng PTP/mg chromaffin granule protein (12), compared with the higher (Met)enkephalin content in chromaffin granules of 200 ng (Met)enkephalin/mg chromaffin granule protein. The different levels of PTP enzyme and (Met)enkephalin product are consistent with the fact that enzymes are normally present in vivo at levels that are several orders of magnitude lower than their substrates and products. Thus, these immunoelectron microscopy results demonstrate appropriate levels of PTP consistent with its role as a processing enzyme that generates (Met)enkephalin within chromaffin granules.

PTP in primary cultures of chromaffin cells: immunofluorescence staining and secretion
Immunofluorescence cytochemistry demonstrated the discrete pattern of cellular PTP immunostaining in primary cultures of chromaffin cells (Fig. 8Go). The majority of cells possess PTP (Fig. 8AGo), which is consistent with the presence of (Met)enkephalin in these chromaffin cells (Fig. 9AGo). The discrete pattern of PTP immunofluorescence staining (Fig. 8Go) in the perinuclear, cell body areas and in neuritic extensions are compatible with PTP’s localization to chromaffin granules of the secretory vesicle pathway, as (Met)enkephalin also shows a discrete pattern of immunostaining in chromaffin cells (Fig. 9Go). This pattern of (Met)enkephalin immunofluorescence staining represents enkephalins in the regulated secretory vesicle pathway involving chromaffin granules. It is also of interest to note that the lower intensity of PTP immunofluorescence staining (Fig. 8Go, C and D), compared with that of (Met)enkephalin (Fig. 9Go, C and D), is consistent with results from immunoelectron microscopy (Table 1Go) and biochemical studies (12), indicating lower levels of PTP compared with its (Met)enkephalin product in chromaffin granules. PTP and (Met)enkephalin were absent in the nucleus. Overall, the punctate, discrete localization of PTP and (Met)enkephalin in chromaffin cells is consistent with their presence in secretory vesicles (chromaffin granules).



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  		Figure 8. PTP immunofluorescence cytochemistry in primary cultures of chromaffin cells. Immunofluorescence cytochemistry of PTP was performed as described in Materials and Methods. PTP immunoreactivity in chromaffin cells was observed with x10X (a), x40 (b), and x100 objectives (c and d). The bars in each panel represent 10 µm. Controls showed no immunostaining when preimmune antiserum was used as the primary antibody (data not shown).

 


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  		Figure 9. (Met)enkephalin immunofluorescence cytochemistry in primary cultures of chromaffin cells. Immunofluorescence cytochemistry of (Met)enkephalin was performed as described in Materials and Methods. (Met)enkephalin immunoreactivity in chromaffin cells was observed with x10 (a), x40 (b), and x100 objectives (c and d). The bars in each panel represent 10 µm. Controls showed no immunostaining when the primary antibody [anti-(Met)enkephalin serum] was omitted from the procedure (data not shown).

 
The cosecretion of PTP and (Met)enkephalin from chromaffin cells was studied to assess the presence of PTP and (Met)enkephalin in functional secretory vesicles. These experiments examined regulated secretion induced by KCl depolarization in the presence or absence of calcium in the cell culture medium. Results show that KCl depolarization induces the cosecretion of PTP and (Met)enkephalin in a calcium-dependent manner (Fig. 10Go). Secretion of PTP, induced by KCl depolarization, occurred only in the presence of calcium. Similarly, the secretion of (Met)enkephalin induced by KCl depolarization occurs in a calcium-dependent manner. It was noted that secreted PTP (28–29 kDa) was slightly smaller in apparent molecular mass compared with 32- to 33-kDa PTP detected in chromaffin granules. It is known that some secreted proteins undergo limited proteolysis during secretion (31), which may be occurring with PTP. Overall, these results demonstrate the cellular colocalization of PTP and (Met)enkephalin to regulated secretory vesicles that require Ca2+ for secretion.



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  		Figure 10. Cosecretion of PTP and (Met)enkephalin from chromaffin cells. a, Secretion of PTP from chromaffin cells. Western blots assessed PTP in the secretion medium from cells incubated without (lanes 1 and 2) or with 50 mM KCl (lanes 3 and 4) in the absence (lanes 2 and 4) or presence (lanes 1 and 3) of calcium (Ca2+). b, Secretion of (Met)enkephalin from chromaffin cells. (Met)enkephalin, measured by RIA, in the secretion medium from cells incubated without or with KCl (50 mM) in the absence or presence of Ca2+ was measured by RIA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrates that the major proenkephalin-processing enzyme, a novel cysteine protease known as PTP, is localized to regulated secretory vesicles of chromaffin cells (also known as chromaffin granules). In this study, antibodies against PTP were produced that recognize the unique NH2-terminal peptide sequence of PTP. These antibodies recognize native PTP, as shown by immunodepletion of PTP activity. Immunoblots indicated the presence of PTP in the soluble and membrane components of chromaffin granules. Immunoelectron microscopy indicated that intact, homogeneous chromaffin granules contain (Met)enkephalin and PTP, as demonstrated by immunogold labeling. Secretory vesicle localization of PTP was further illustrated by immunofluorescence cytochemistry showing the discrete, punctate localization of PTP in cytoplasmic, perinuclear regions and in neuritic extensions. Importantly, PTP cosecretion with (Met)enkephalin occurs upon KCl depolarization of chromaffin cells in a Ca2+-dependent manner, indicating regulated secretion. These results establish the localization of PTP to regulated secretory vesicles, a major site of proenkephalin and prohormone processing.

In the PTP immunodepletion experiments, the ability of the antibody to completely immunoprecipitate PTP activity indicates that the determined NH2-terminal sequence represents the enzyme. In addition, these results demonstrate that the NH2-terminus of the native enzyme is accessible to the antibody, suggesting that the NH2-terminal region may be located near the surface of the enzyme.

Within the chromaffin granule, PTP immunoreactivity is present at approximately equivalent levels in the soluble and membrane components of these vesicles. Previous studies, however, indicate that most of the enkephalin precursor-cleaving activity (~85%) is present in the soluble fraction compared with membranes. These observations suggest a higher specific activity for PTP in the soluble component, compared with PTP in the membrane component of these granules. PTP may reside in the membranes in a latent form. Similar to PTP, previous studies of the carboxypeptidase E/H-processing enzyme show that a more active form of carboxypeptidase E/H is present in the soluble compared with the membrane component of chromaffin granules (32).

The presence of PTP in the soluble component of chromaffin granules predicts that PTP should be cosecreted with (Met)enkephalin. Cosecretion of PTP and (Met)enkephalin was observed when primary cultures of chromaffin cells were subjected to KCl depolarization. Importantly, the secretion of both PTP and (Met)enkephalin was dependent on Ca2+. Exocytosis of regulated secretory vesicles to release peptide hormones or neurotransmitters is a calcium-dependent process (33, 34). These results, therefore, illustrate the presence of PTP with (Met)enkephalin in functional, regulated secretory vesicles.

Findings from this study illustrate that chromaffin granules are an excellent model neurosecretory vesicle system for identification of prohormone-processing enzymes. EM demonstrates that intact, homogeneous chromaffin granules are readily isolated from bovine adrenal medulla. These EM studies show that the integrity and morphology of isolated chromaffin granules in vitro are nearly identical to those of the same granules observed by EM in chromaffin cells in situ (24). Demonstration of PTP within chromaffin granules by immunoelectron microscopy and calcium-dependent secretion of PTP establish the localization of PTP to secretory vesicles. Chromaffin granules also contain several other prohormone-processing enzymes, including carboxypeptidase E/H (16, 32), subtilisin-like PC1/3 and PC2 enzymes (17, 36, 37, 38), a 70-kDa aspartyl protease (18) related to the POMC-converting enzyme (39), and a basic-residue cleaving aminopeptidase(s) (20). These proteases may be involved in processing several prohormones, as these granules contain, in addition to enkephalin, vasoactive intestinal polypeptide, neuropeptide Y, galanin, somatostatin, and other neuropeptides (10, 11, 40). It is apparent that chromaffin granules contain proteases required for processing several prohormones into active peptide hormones or neurotransmitters.

In summary, this study demonstrates that the primary proenkephalin-processing enzyme known as PTP is localized to regulated secretory vesicles of chromaffin cells. The secretory vesicle is an important subcellular site for proenkephalin and prohormone processing. It will be important in future studies to define the cellular role of PTP in the biosynthesis of peptide hormones and neuropeptides.


   Acknowledgments
 
The authors acknowledge the services of the peptide-sequencing facility at the Medical College of Wisconsin, directed by Liane Mende-Mueller. The assistance of Michael Byrne and Michelle Lyons is appreciated.


   Footnotes
 
1 This work was supported by grants from the NINDS and NIDA of the NIH. Back

Received November 11, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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