carboxypeptidase B-like exopeptidase(s) are required for processing. Such enzymes ..... We thank Dr. Solomon H. Snyder (Johns Hopkins University. School of ...
Proc. Natl. Acad. Sci. USA Vol. 82, pp. 4745-4749, July 1985 Cell Biology
Immunochemical characterization of carboxypeptidase J3-like peptide-hormone-processing enzyme (prohormone processing/peptidase/neuropeptides/neuroendocrine)
VIVIAN Y. H. HOOK*, EVA MEZEY*, LLOYD D. FRICKERt, REBECCA M. PRUSS*, RUTH E. SIEGEL*, AND MICHAEL J. BROWNSTEIN* *Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, MD 20205; and tDepartment of Chemistry, University of Oregon, Eugene, OR 97403
Communicated by Julius Axelrod, March 1, 1985
ABSTRACT Specific rabbit antisera against purified bovine pituitary carboxypeptidase processing enzyme (which has also been referred to as "enkephalin convertase") have been prepared and characterized. The antisera recognized both the purified soluble and the membrane-bound forms of the enzyme with equal affinity, suggesting that these two forms of the enzyme may possess many regions of structural homology. Since the antisera did not crossreact with carboxypeptidases B, N, A, Y, and P, the carboxypeptidase processing enzyme may be a structurally distinct form of 'carboxypeptidase. Carboxypeptidase inmunostaining, as seen by light microscopy, was found throughout the rat brain and in bovine adrenal medulla, reflecting the widespread distribution of neuropeptides. Electron microscopic immunocytochemistry of rat paraventricular nucleus and other brain areas showed that the enzyme was present in some dendrites and nerve terminals, which contain storage vesicles. These findings support the hypothesis that this carboxypeptidase is involved in the processing of many peptide hormone precursors.
Adrenocorticotropin (1), opiate peptides (1-5), vasopressin (6), corticotropin-releasing factor (7), and other peptide hormones (8, 9) are initially synthesized at the rough endoplasmic reticulum as large precursors which must then undergo proteolytic processing to yield the biologically active peptides. Within the precursors, the hormones are typically flanked by pairs of basic amino acid residues (lysine and/or arginine), suggesting that trypsin-like endopeptidase(s) and carboxypeptidase B-like exopeptidase(s) are required for processing. Such enzymes are thought to be present in secretory granules, where prohormones and their cleavage products have been found (9-12). Carboxypeptidase B-like activities involved in processing several peptide precursors have been identified. Carboxypeptidase activities that process enkephalin precursors have been detected in crude bovine adrenomedullary chromaffin granules (13, 14). The carboxypeptidase present in purified chromaffin granules, which has been referred to as "enkephalin convertase" (14) and is hereafter referred to as carboxypeptidase processing enzyme, appears to be a thiolmetallocarboxypeptidase with an acidic pH optimum (15). It is stimulated by Co2' and inhibited by the active site-directed inhibitors guanidinoethylmercaptosuccinic acid, guanidinopropylsuccinic acid, aminopropylmercaptosuccinic acid, and 2-mercaptomethyl-3-guanidinoethylthiopropionic acid (GEMSA, GPSA, APMSA, and MGTA) (14-19). Similar carboxypeptidase B-like activity has been found in brains and pituitaries from cow and rat (14, 16); secretory granules from anterior, intermediate, and posterior lobes of rat pituitary The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
(20); and secretory granules of rat pancreatic islet tumor cells (21). These activities may be involved in processing proopiomelanocortin, provasopressin, proinsulin, and other prohormones. Purification and characterization (18, 19) has revealed that the bovine adrenal medulla, pituitary, and brain forms of this Co2"-stimulated carboxypeptidase are indistinguishable and that many oftheir biochemical properties, such as the acidic pH optimum, thiol dependence, potency of GEMSA inhibition, and molecular weight, differ from those of other known carboxypeptidases (13-20), including pancreatic carboxypeptidase B or plasma carboxypeptidase N (22, 23). However, the carboxypeptidase processing enzyme resembles carboxypeptidases B and N in its specificity for basic amino acid residues and stimulation by Co2". We have prepared specific rabbit antisera against the purified bovine pituitary carboxypeptidase. These antisera do not crossreact with other known carboxypeptidases, which suggests that this processing enzyme may be structurally unique. Use of the antibody in light- and electronmicroscopic immunocytochemistry has shown that the processing carboxypeptidase is localized in many peptidergic neurons. Our immunohistochemical data are compatible with results from [3H]GEMSA autoradiography (24). The presence of this carboxypeptidase along with many neuropeptides in neuroendocrine tissues suggests that it may be involved in the processing of a variety of peptide hormone precursors.
MATERIALS AND METHODS Preparation of Antisera. Male National Institutes of Health rabbits were injected (s.c. and i.m.) three times, at 2-week intervals, with 30 Ag of purified, soluble, bovine pituitary carboxypeptidase processing enzyme in Freund's adjuvant; booster injections were given at monthly intervals for 7 months. Rabbits were bled 5-7 days after the last injection. Antisera were screened by a solid-phase RIA (25). Microtiter wells (Dynatech, Alexandria, VA) were coated with purified bovine pituitary carboxypeptidase at 6 ,ug/ml in phosphatebuffered saline (P1/NaCl: 0.15 M NaCl/100 mM phosphate, pH 7.4; 40 A.l per well), blocked with 10% (wt/vol) bovine serum albumin (BSA) in P1/NaCl, and incubated with antiserum diluted in 10% fetal calf serum/P1/NaCl. Carboxypeptidase-antibody complexes were detected with 125I-labeled antibody to rabbit immunoglobulins (Amersham). Immunoblot Analysis. Rat pituitary anterior, intermediate, and neural lobes and bovine adrenal medulla were dissected and homogenized in 50 mM Tris-HCl (pH 7.4) by sonication for 15 sec. Samples were subjected to NaDodSO4/10% PAGE (26) in 1.5 mm thick slab gels (200 V, 4-6 hr) and then were transferred electrophoretically onto nitrocellulose Abbreviations: GEMSA, guanidinoethylmercaptosuccinic acid; BSA, bovine serum albumin.
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membranes (0.2 A, 16-18 hr) (27). Immunoblots were prepared by blocking membranes with 10% fetal calf serum in Pi/NaCl for 30 min at room temperature and then incubating them with antiserum (rabbit A) at a final dilution of 1:1000. Anti-carboxypeptidase complexes were visualized with peroxidase-conjugated goat anti-rabbit immunoglobulins (Boehringer Mannheim). Protein was determined by the method of Lowry et al. (28) with BSA as standard. RIA of Carboxypeptidase Processing Enzyme. The RIA was carried out as described (38). 125I-labeled carboxypeptidase was prepared by a lactoperoxidase method using Enzymobeads (Bio-Rad). Nonspecific binding of 125I-labeled carboxypeptidase was 4-5% of the total cpm in each tube, and antibody at 1:15,000 dilution bound 19-22% of the total 125I-labeled carboxypeptidase. Crossreactivity of the antibody was tested with purified bovine carboxypeptidase B (a gift from T. H. Plummer, Jr., New York Department of Public Health), porcine carboxypeptidase B (Sigma), bovine carboxypeptidase N (a gift from T. H. Plummer, Jr.), bovine carboxypeptidase A (Boehringer Mannheim), yeast carboxypeptidase Y (Boehringer Mannheim), and Penicillium janthinellum carboxypeptidase P (Sigma). For measurement of tissue content of carboxypeptidase processing enzyme, the anterior, intermediate, and neural lobes of rat pituitary were dissected, placed in 0.75 ml of ice-cold homogenizing buffer [100 mM sodium phosphate, pH 7.0/aprotinin (Sigma; 0.5 trypsin inhibitor units/ml)/O. 1% BSA/0.01% merthiolate/0.1% Triton X-100/10 mM EDTA], and sonicated for 15 sec. Bovine pituitary and adrenal medulla homogenates were prepared in the same manner except that a glass/glass homogenizer was used instead of sonication. Samples were centrifuged in a Beckman Microfuge for 5 min, and the supernatants (2-12 ,ul) were assayed for carboxypeptidase processing enzyme immunoreactivity. Tissue concentrations were calculated as ng of carboxypeptidase processing enzyme/,g of supernatant protein. Two to twelve microliters of homogenizing buffer did not interfere with the carboxypeptidase standard curve. Immunohistochemistry. For immunostaining of rat pituitary and brain, animals were perfused with 4% (wt/vol) paraformaldehyde/0. 2% picric acid/0.1 M sodium phosphate buffer, pH 7.4. The pituitaries and brains were removed and incubated in the same fixative for 1 hr. After washing overnight at 4°C in P,/NaCl containing 5% (wt/vol) sucrose, 4-10 ,um thick frozen sections were cut in a cryostat, thawed on gelatin-coated slides, and incubated with the primary antiserum (from rabbit B) diluted 1:250 in Pi/NaCl containing 0.6% Triton X-100 at 4°C for 24-48 hr. The sections then were washed with Pi/NaCl, incubated with rhodamine-conjugated anti-rabbit immunoglobulins (1:100 dilution, Boehringer Mannheim) for 30 min at 37°C, washed again, mounted with glycerol/P,/NaCl, and examined by epifluorescence with a Zeiss fluorescence microscope. For brain sections, the antigen was visualized in 40 ,um thick Vibratome sections using the avidin-biotin (ABC) method (29). For immunostaining of bovine adrenal gland, 0.5 cm thick sections were fixed overnight at 4°C in 4% paraformaldehyde/0.1 M phosphate buffer, pH 6.0, and then immersed overnight in 5% sucrose/0.1 M phosphate buffer, pH 7.0. Sections (14 ,um) were cut and processed as described above for the anterior and intermediate lobes, except that the primary antiserum was from rabbit A and diluted 1:100 and that fluorescein-conjugated goat anti-rabbit immunoglobulins (1:100 dilution, Cappel Laboratories, Cochranville, PA) was used as the secondary antibody. Cultured bovine adrenal medullary chromaffin cells were prepared as described (30) and grown on glass coverslips. After 7 days in culture, cells were fixed in 4% formaldehyde in 0.1 M phosphate buffer (pH 7.2) for 10 min, washed with 0.1 M phosphate buffer (pH 7.4), incubated with antiserum
Proc. Natl. Acad. Sci. USA 82 (1985)
(1:100 dilution, rabbit B) in the same buffer at 370C for 45 min, washed, and incubated with rhodamine-conjugated goat antirabbit immunoglobulins at 370C for 45 min. Cells were viewed by use of filters appropriate for rhodamine fluorescence or by phase-contrast optics. Staining was blocked when tissue sections or chromaffin cells were incubated with antiserum plus excess purified soluble carboxypeptidase processing enzyme. Electron Microscopic Immunocytochemistry. Rats were perfused through the ascending aorta with 4% paraformaldehyde/0.05% glutaraldehyde/0.2% picric acid/0.1 M sodium phosphate buffer, pH 7.4. The brains then were removed and fixed overnight at 40C in the same fixative but with no glutaraldehyde. Vibratome sections (50 ttm) then were cut and washed with Pi/NaCl. Sections were incubated with the carboxypeptidase antiserum (1:400 dilution, rabbit B) for 24 hr at 40C, washed with Pi/NaCl, incubated with anti-rabbit gamma globulin (1:100 dilution, Cappel Laboratories) for 1 hr at 240C, washed, and then incubated with rabbit peroxidase-antiperoxidase (30) for 1 hr at 240C. The peroxidase was detected with 0.04% diaminobenzidine tetrahydrochloride (Polyscience, Warrington, PA)/0.0015% H202/0.05 M Tris HCl, pH 7.6, for 10 min. Sections were treated with OSO4, dehydrated in ethanol, and embedded in Polybed 812 (Polyscience). Ultrathin sections were cut with an LKB ultramicrotome and examined with a Zeiss 109 electron microscope.
RESULTS Rabbit antisera (from two rabbits, A and B) against the soluble form of bovine pituitary carboxypeptidase processing enzyme were obtained. The antisera were positive in the solid-phase RIA, even at dilutions of 1:200,000, when compared to preimmune serum (data not shown). To obtain information on the specificity of the carboxypeptidase processing enzyme antibody, we prepared immunoblots of rat pituitary and bovine adrenal medulla homogenates (Fig. 1). The antisera recognized purified soluble and membrane forms of bovine pituitary carboxypeptidase. One major protein band was stained by the antiserum in homogenates from anterior, intermediate, and posterior rat pituitary and from bovine adrenal medulla; this band comigrated with standard soluble and membrane forms of bovine pituitary carboxypeptidase. Because the soluble and membrane forms of the enzyme differ in size by only 2500 Da (18), they may not be resolved in the same sample by this electrophoresis system. Therefore, the major anti-carboxypeptidase stained protein band seen in the pituitary and adrenal medulla homogenates may represent both soluble and membrane forms of the enzyme. The immunoblot data suggest that the antibody recognizes primarily the carboxypeptidase processing enzyme molecules contained in pituitary and adrenal medulla. To determine whether the antisera recognized the soluble and membrane-bound forms of bovine pituitary carboxypeptidase with equal or different affinities, a RIA method was developed. Standard curves for the soluble and membrane forms of the enzyme (Fig. 2) appear identical, indicating that the antibody recognized these forms with equivalent affinity. The RIA could detect as little as 2 ng of carboxypeptidase and the 50% displacement value for both soluble and membrane forms of the enzyme was 54 ng. The crossreactivity of the antisera with other carboxypeptidases was tested. 125ilabeled carboxypeptidase bound to the antibodies was not displaced by as much as 10 ,g of bovine or porcine carboxypeptidase B, bovine carboxypeptidase N, bovine carboxypeptidase A, yeast carboxypeptidase Y, or P. janthinellum carboxypeptidase P (Table 1). Thus, the antibody against the
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Proc. Natl. Acad. Sci. USA 82 (1985)
Table 1. Antiserum against the carboxypeptidase processing enzyme does not crossreact with other carboxypeptidases Amount, ,ug (B/BO) x 100 Competing protein 100 None Bovine pituitary carboxypeptidase 0.045 50 Bovine carboxypeptidase B 1 100 10 101 1 97 Porcine carboxypeptidase B 94 10 Bovine carboxypeptidase N 1 103 10 101 1 100 Yeast carboxypeptidase Y 10 100 1 100 Bovine carboxypeptidase A 10 100
kDa
-94 -68 43
30
-21
-14 1
2
3
5
4
6
FIG. 1. Immunoblot analysis of carboxypeptidase processing enzyme in homogenates of rat pituitary anterior, intermediate, and neural lobes and of bovine adrenal medulla. Lanes: 1, rat anterior pituitary (200 ug of protein); 2, rat intermediate pituitary (75 ,ug of protein); 3, rat posterior pituitary (150 ,ug of protein); 4, bovine
adrenal medulla (75 ,ug of protein); 5, standard soluble bovine pituitary carboxypeptidase (0.3 Ag); 6, standard membrane bovine pituitary carboxypeptidase (0.4 ,ug). Immunoblots with antisera from rabbits A and B showed the same results. Positions of marker proteins (not shown) are indicated at right.
processing carboxypeptidase does not appear to crossreact with other known carboxypeptidases. Tissue levels ofcarboxypeptidase processing enzyme were measured in rat pituitary and bovine adrenal medulla. Unlike antibodies against pancreatic carboxypeptidase B, which show species specificity (31, 32), the anti-carboxypeptidaseprocessing-enzyme antibody appears to recognize rat as well as bovine forms of the enzyme. Increasing amounts of rat anterior pituitary homogenate displaced '25I-labeled carboxypeptidase bound to antibody in a manner parallel to the standard curve (Fig. 2 Inset). Anterior, intermediate, and neural lobes of rat pituitary homogenates contained, respectively, 0.17 + 0.10, 0.17 + 0.01, and 0.26 0.09 ng of carboxypeptidase/Azg of protein (mean SD, n = 3), whereas the three lobes of bovine pituitary had 1.62 0.07, 3.85 0.20, and 3.87 0.25 ng/,ug of protein, respectively. Neural lobe consistently contained higher concentrations than anterior lobe. Bovine adrenal medulla had 1.2 0.35 ng/,ug of protein. The rabbit serum was useful for immunocytochemical studies. In anterior and intermediate lobes of rat pituitary (Fig. 3 A and B), carboxypeptidase processing enzyme appeared to be discretely localized in the cytoplasm of all cells and was absent from the nuclei. Similar discrete localization of the enzyme was also found in the magnocellular cell ±
±
±
±
±
±
100
8
> x
Protein
mg
50 _ 50
P. janthinellum carboxypeptidase P
1
100
10 100 B/Bo is the fraction of "M51-labeled bovine pituitary carboxypeptidase not displaced.
bodies of the hypothalamic supraoptic and paraventricular nuclei (Fig. 3 C and D), which project to the posterior pituitary where vasopressin and oxytocin are stored and released. In addition to the hypothalamic area, carboxypeptidase immunostaining was present in cell bodies of all brain areas where neuropeptides are known to be present. No immunostaining of nerve fibers or terminals was seen with paraformaldehyde fixation, but staining in these structures was seen by electron microscopy when glutaraldehyde was included with paraformaldehyde in the perfusion solution. Carboxypeptidase processing enzyme immunostaining (Fig. 3E) was present in bovine adrenal medulla, where large amounts of enkephalin peptides are present (33, 34), but was absent in adrenal cortex, which does not contain enkephalins. Because the cellular location of the enzyme could not be resolved in whole adrenal gland sections, immunostaining in adrenomedullary chromaffin cells in culture was investigated (Fig. 3F). Carboxypeptidase and [Leu]enkephalin (data not shown) immunoreactivity were both found in chromaffin cells and were not present in fibroblasts. Carboxypeptidase immunoreactivity, like the [Leu]enkephalin staining, was found in discrete cytoplasmic areas compatible with its presence in secretory granules and Golgi apparatus from which granules are formed. Carboxypeptidase and [Leu]enkephalin immunostaining were not seen in the nuclei. Electron microscopic immunocytochemistry of the carboxypeptidase processing enzyme in several areas of rat brain was examined, and the immunostaining of the hypothalamic paraventricular nucleus is shown in Fig. 4. The enzyme's subcellular localization was similar in all areas. Carboxypeptidase immunostaining was found in some nerve terminals (Fig. 4 A, B, and D) and dendrites (Fig. 4C), which contain storage vesicles. Some sections (Fig. 4 B and D) showed carboxypeptidase-labeled nerve terminals forming a synaptic contact with a nonlabeled cell body. Several nerve terminals (Fig. 4 A and D) did not show carboxypeptidase
immunostaining.
30.T.
50 100 Carboxypeptidase, ng
1020
FIG. 2. RIA of carboxypeptidase processing enzyme. Standard for the purified soluble (o) and membrane-bound (o) forms of
curves
bovine pituitary carboxypeptidase are shown. (Inset) Measurement
of processing carboxypeptidase in various amounts of rat anterior
pituitary homogenate. B/Bo, fraction of labeled carboxypeptidase not displaced.
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DISCUSSION Specific rabbit antisera against the soluble form of the carboxypeptidase processing enzyme of bovine pituitary have been prepared and characterized. The antisera possessed equivalent binding affinities for both the soluble and membrane-bound forms of the enzyme, suggesting that these two enzyme forms may possess many regions of structural homology. Although the purified membrane carboxypeptidase is slightly larger (52,500 Da) than the purified soluble
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Proc. Natl. Acad. Sci. USA 82 (1985)
D
a
4
FIG. 3. Light-microscopic immunohistochemistry of the carboxypeptidase processing enzyme. (A) Rat pituitary anterior lobe. (x420.) (B) Rat pituitary intermediate lobe. (x300.) (C) Rat supraoptic nucleus. (x87.) (D) Rat hypothalamic paraventricular nucleus. (x87.) (E) Bovine adrenal gland. (x300.) (F) Cultured bovine adrenomedullary chromaffin cells. (x420.)
form (50,000 Da) (18), they show identical substrate and inhibitor specificities (14, 17, 18). The antibody against the carboxypeptidase processing enzyme did not crossreact with carboxypeptidases B, N, A, Y, and P. This lack of crossreactivity with other known carboxypeptidases suggests that the peptide hormone processing enzyme is a structurally distinct carboxypeptidase. Immunohistochemical studies showed that the enzyme was present in areas where neuropeptides have been found previously. In anterior and intermediate lobes of rat pituitary, where proopiomelanocortin-derived peptides are synthesized, carboxypeptidase immunostaining was discretely localized in the cytoplasm and not in the nuclei. The enzyme was also found in the cell bodies of the hypothalamic supraoptic and paraventricular nuclei. Cells in the supraoptic nucleus contain vasopressin and oxytocin (35). Cells in the paraventricular nucleus contain corticotropin-releasing factor, somatostatin, enkephalins, dynorphins, oxytocin, thyrotropin-releasing hormone, vasopressin, cholecystokinin, and bombesin (36). Discrete carboxypeptidase immunostaining in cell bodies was found throughout the brain, reflecting the widespread distribution of brain neuropeptides. In the periphery, immunostaining was present in adrenal medullary chromaffin cells, which contain enkephalin peptides. The localization of the carboxypeptidase processing enzyme, or "enkephalin convertase", in rat brain has recently been demonstrated by use of [3H]GEMSA autoradiography (24). GEMSA has been found to be a potent inhibitor of this enzyme (19). Although [3H]GEMSA binding sites correspond
closely to the distribution of enkephalinergic neurons, the areas found to bind [3H]GEMSA also contain many other neuropeptides. In addition, [3H]GEMSA binding sites were also abundant in areas that do not contain enkephalins, such as the anterior pituitary which contains adrenocorticotropin. No discrepancies were found between our immunocytochemical localization of the enzyme and the [3H]GEMSA autoradiography done by Lynch et al. (24). Both immunohistochemistry and [3H]GEMSA autoradiography of the carboxypeptidase processing enzyme show that this enzyme is localized with a number of neuropeptides. The colocalization of processing carboxypeptidase with many neuropeptides suggests that this enzyme may be useful as a marker for peptidergic cells. Carboxypeptidase immunostaining was present in a few cell bodies in globus pallidus (data not shown), but no neuropeptide has yet been found in these cell bodies. Neuropeptide immunostaining in globus pallidus has been found only in nerve fibers (35). Perhaps cell bodies of globus pallidus contain an undiscovered neuropeptide. Although the discrete localization of carboxypeptidase immunostaining is compatible with its presence in storage vesicles, its subcellular localization can only be determined by electron microscopy. Electron microscopy revealed that the carboxypeptidase was present in dendrites and nerve terminals that contain storage vesicles. Some nerve terminals and dendrites showed no immunostaining. It is possible that these may represent nonpeptidergic cells.
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Proc. NatL. Acad. Sci. USA 82 (1985)
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We thank Dr. Solomon H. Snyder (Johns Hopkins University School of Medicine) for generously providing purified bovine pituitary carboxypeptidase. We also thank Dr. Lee Eiden (Laboratory of Cell Biology, National Institute of Mental Health) for helpful discussions. V.Y.H.H. is a recipient of a Wellcome Senior Research
Fellowship.
FIG. 4. Electron microscopic immunocytochemistry of carboxypeptidase processing enzyme. Stars in A, B, and D indicate carboxypeptidase immunostaining in nerve terminals. Two anti-carboxypeptidase stained nerve terminals in B and D form synaptic contacts (indicated by arrows) with cell bodies showing no immunostaining. T designates nerve terminals showing no immunostaining. The letter D (in C) designates dendritic areas. Bar at top right of each panel represents 1 jm.
Visualization of enzyme immunostaining in cell body and terminal areas each required different fixation conditions. Light microscopy (paraformaldehyde fixation) revealed carboxypeptidase immunostaining in cell bodies but not in nerve fibers or terminals. On the other hand, immunostaining in nerve fibers and terminals as well as cell bodies was seen when glutaraldehyde was included with paraformaldehyde in the perfusion solution. The different fixation conditions required to visualize the enzyme in these subcellular areas could be explained by two hypotheses. First, the accessibility of the antibody to the enzyme may require specific conditions at the different subcellular structures in the cell body (rough endoplasmic reticulum, Golgi apparatus, and newly formed secretory vesicles) and in the nerve fibers and terminals (mature secretory vesicles). Alternatively, different forms of the enzyme could be present in cell body and nerve fiber and terminal regions. It is possible that the soluble and membrane forms of the enzyme may be present in different proportions in immature compared to mature secretory vesicles. In situ, the antibody may preferentially stain one of these forms. In addition, recent studies have shown that the carboxypeptidase in mature chromaffin granules is more catalytically active than the enzyme in a heterogeneous population (immature and mature) of chromaffin granules (37). The factor(s) responsible for regulation of the enzyme's activity may possibly influence the ability of the antibody to recognize the enzyme. In summary, use of specific rabbit antisera against the processing carboxypeptidase have shown that (i) the soluble and membrane forms of this enzyme may possess many regions of structural homology, (ii) this enzyme may be structurally different from other carboxypeptidases, and (iii) the enzyme is colocalized with many neuropeptides, which suggests that it may be involved in the processing of many peptide hormone precursors. nerve
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