Proc. Natl. Acad. Sci. USAVol. 82, pp. 4745-4749, July 1985Cell Biology

Immunochemical characterization of carboxypeptidase J3-likepeptide-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 bo-vine pituitary carboxypeptidase processing enzyme (which hasalso been referred to as "enkephalin convertase") have beenprepared and characterized. The antisera recognized both thepurified soluble and the membrane-bound forms of the enzymewith equal affinity, suggesting that these two forms of theenzyme 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 maybe a structurally distinct form of'carboxypeptidase. Carboxy-peptidase inmunostaining, as seen by light microscopy, wasfound throughout the rat brain and in bovine adrenal medulla,reflecting the widespread distribution of neuropeptides. Elec-tron microscopic immunocytochemistry of rat paraventricularnucleus and other brain areas showed that the enzyme waspresent in some dendrites and nerve terminals, which containstorage vesicles. These findings support the hypothesis that thiscarboxypeptidase is involved in the processing of many peptidehormone precursors.

Adrenocorticotropin (1), opiate peptides (1-5), vasopressin(6), corticotropin-releasing factor (7), and other peptidehormones (8, 9) are initially synthesized at the roughendoplasmic reticulum as large precursors which must thenundergo proteolytic processing to yield the biologically activepeptides. Within the precursors, the hormones are typicallyflanked by pairs of basic amino acid residues (lysine and/orarginine), suggesting that trypsin-like endopeptidase(s) andcarboxypeptidase B-like exopeptidase(s) are required forprocessing. Such enzymes are thought to be present insecretory granules, where prohormones and their cleavageproducts have been found (9-12).

Carboxypeptidase B-like activities involved in processingseveral peptide precursors have been identified. Carboxy-peptidase activities that process enkephalin precursors havebeen detected in crude bovine adrenomedullary chromaffingranules (13, 14). The carboxypeptidase present in purifiedchromaffin granules, which has been referred to as "enkeph-alin convertase" (14) and is hereafter referred to ascarboxypeptidase processing enzyme, appears to be a thiol-metallocarboxypeptidase with an acidic pH optimum (15). Itis stimulated by Co2' and inhibited by the active site-directedinhibitors guanidinoethylmercaptosuccinic acid, guanidino-propylsuccinic acid, aminopropylmercaptosuccinic acid,and 2-mercaptomethyl-3-guanidinoethylthiopropionic acid(GEMSA, GPSA, APMSA, and MGTA) (14-19). Similarcarboxypeptidase B-like activity has been found in brains andpituitaries from cow and rat (14, 16); secretory granules fromanterior, intermediate, and posterior lobes of rat pituitary

(20); and secretory granules of rat pancreatic islet tumor cells(21). These activities may be involved in processing pro-opiomelanocortin, provasopressin, proinsulin, and otherprohormones. Purification and characterization (18, 19) hasrevealed that the bovine adrenal medulla, pituitary, and brainforms of this Co2"-stimulated carboxypeptidase are indistin-guishable and that many oftheir biochemical properties, suchas the acidic pH optimum, thiol dependence, potency ofGEMSA inhibition, and molecular weight, differ from thoseof other known carboxypeptidases (13-20), including pan-creatic carboxypeptidase B or plasma carboxypeptidase N(22, 23). However, the carboxypeptidase processing enzymeresembles carboxypeptidases B and N in its specificity forbasic amino acid residues and stimulation by Co2".We have prepared specific rabbit antisera against the

purified bovine pituitary carboxypeptidase. These antiserado not crossreact with other known carboxypeptidases,which suggests that this processing enzyme may be struc-turally unique. Use of the antibody in light- and electron-microscopic immunocytochemistry has shown that the pro-cessing carboxypeptidase is localized in many peptidergicneurons. Our immunohistochemical data are compatible withresults from [3H]GEMSA autoradiography (24). The pres-ence of this carboxypeptidase along with many neuropep-tides in neuroendocrine tissues suggests that it may beinvolved in the processing of a variety of peptide hormoneprecursors.

MATERIALS AND METHODSPreparation of Antisera. Male National Institutes of Health

rabbits were injected (s.c. and i.m.) three times, at 2-weekintervals, with 30 Ag of purified, soluble, bovine pituitarycarboxypeptidase processing enzyme in Freund's adjuvant;booster injections were given at monthly intervals for 7months. Rabbits were bled 5-7 days after the last injection.Antisera were screened by a solid-phase RIA (25). Microtiterwells (Dynatech, Alexandria, VA) were coated with purifiedbovine pituitary carboxypeptidase at 6 ,ug/ml in phosphate-buffered saline (P1/NaCl: 0.15 M NaCl/100 mM phosphate,pH 7.4; 40 A.l per well), blocked with 10% (wt/vol) bovineserum albumin (BSA) in P1/NaCl, and incubated with anti-serum diluted in 10% fetal calf serum/P1/NaCl. Carboxypep-tidase-antibody complexes were detected with 125I-labeledantibody to rabbit immunoglobulins (Amersham).Immunoblot Analysis. Rat pituitary anterior, intermediate,

and neural lobes and bovine adrenal medulla were dissectedand homogenized in 50 mM Tris-HCl (pH 7.4) by sonicationfor 15 sec. Samples were subjected to NaDodSO4/10%PAGE (26) in 1.5 mm thick slab gels (200 V, 4-6 hr) and thenwere transferred electrophoretically onto nitrocellulose

Abbreviations: GEMSA, guanidinoethylmercaptosuccinic acid;BSA, bovine serum albumin.

4745

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Proc. Natl. Acad. Sci. USA 82 (1985)

membranes (0.2 A, 16-18 hr) (27). Immunoblots were pre-pared by blocking membranes with 10% fetal calf serum inPi/NaCl for 30 min at room temperature and then incubatingthem with antiserum (rabbit A) at a final dilution of 1:1000.Anti-carboxypeptidase complexes were visualized with per-oxidase-conjugated goat anti-rabbit immunoglobulins(Boehringer Mannheim). Protein was determined by themethod of Lowry et al. (28) with BSA as standard.RIA of Carboxypeptidase Processing Enzyme. The RIA was

carried out as described (38). 125I-labeled carboxypeptidasewas prepared by a lactoperoxidase method using Enzymo-beads (Bio-Rad). Nonspecific binding of 125I-labeled carboxy-peptidase was 4-5% of the total cpm in each tube, andantibody at 1:15,000 dilution bound 19-22% of the total125I-labeled carboxypeptidase. Crossreactivity of the anti-body was tested with purified bovine carboxypeptidase B (agift from T. H. Plummer, Jr., New York Department ofPublic Health), porcine carboxypeptidase B (Sigma), bovinecarboxypeptidase N (a gift from T. H. Plummer, Jr.), bovinecarboxypeptidase A (Boehringer Mannheim), yeast carboxy-peptidase Y (Boehringer Mannheim), and Penicilliumjanthinellum carboxypeptidase P (Sigma).

For measurement of tissue content of carboxypeptidaseprocessing enzyme, the anterior, intermediate, and neurallobes of rat pituitary were dissected, placed in 0.75 ml ofice-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 adrenalmedulla homogenates were prepared in the same mannerexcept that a glass/glass homogenizer was used instead ofsonication. Samples were centrifuged in a BeckmanMicrofuge for 5 min, and the supernatants (2-12 ,ul) wereassayed for carboxypeptidase processing enzyme immuno-reactivity. Tissue concentrations were calculated as ng ofcarboxypeptidase processing enzyme/,g of supernatant pro-tein. Two to twelve microliters of homogenizing buffer didnot interfere with the carboxypeptidase standard curve.

Immunohistochemistry. For immunostaining of rat pitu-itary and brain, animals were perfused with 4% (wt/vol)paraformaldehyde/0.2% picric acid/0.1 M sodium phosphatebuffer, pH 7.4. The pituitaries and brains were removed andincubated in the same fixative for 1 hr. After washingovernight at 4°C in P,/NaCl containing 5% (wt/vol) sucrose,4-10 ,um thick frozen sections were cut in a cryostat, thawedon gelatin-coated slides, and incubated with the primaryantiserum (from rabbit B) diluted 1:250 in Pi/NaCl containing0.6% Triton X-100 at 4°C for 24-48 hr. The sections then werewashed with Pi/NaCl, incubated with rhodamine-conjugatedanti-rabbit immunoglobulins (1:100 dilution, BoehringerMannheim) for 30 min at 37°C, washed again, mounted withglycerol/P,/NaCl, and examined by epifluorescence with aZeiss fluorescence microscope. For brain sections, the anti-gen was visualized in 40 ,um thick Vibratome sections usingthe avidin-biotin (ABC) method (29).For immunostaining of bovine adrenal gland, 0.5 cm thick

sections were fixed overnight at 4°C in 4% paraformalde-hyde/0.1 M phosphate buffer, pH 6.0, and then immersedovernight in 5% sucrose/0.1 M phosphate buffer, pH 7.0.Sections (14 ,um) were cut and processed as described abovefor the anterior and intermediate lobes, except that theprimary antiserum was from rabbit A and diluted 1:100 andthat fluorescein-conjugated goat anti-rabbit immunoglobulins(1:100 dilution, Cappel Laboratories, Cochranville, PA) wasused as the secondary antibody.

Cultured bovine adrenal medullary chromaffin cells wereprepared as described (30) and grown on glass coverslips.After 7 days in culture, cells were fixed in 4% formaldehydein 0.1 M phosphate buffer (pH 7.2) for 10 min, washed with0.1 M phosphate buffer (pH 7.4), incubated with antiserum

(1:100 dilution, rabbit B) in the same buffer at 370C for 45 min,washed, and incubated with rhodamine-conjugated goat anti-rabbit immunoglobulins at 370C for 45 min. Cells were viewedby use of filters appropriate for rhodamine fluorescence or byphase-contrast optics.

Staining was blocked when tissue sections or chromaffincells were incubated with antiserum plus excess purifiedsoluble carboxypeptidase processing enzyme.

Electron Microscopic Immunocytochemistry. Rats wereperfused through the ascending aorta with 4%paraformaldehyde/0.05% glutaraldehyde/0.2% picricacid/0.1 M sodium phosphate buffer, pH 7.4. The brains thenwere removed and fixed overnight at 40C in the same fixativebut with no glutaraldehyde. Vibratome sections (50 ttm) thenwere cut and washed with Pi/NaCl. Sections were incubatedwith the carboxypeptidase antiserum (1:400 dilution, rabbitB) for 24 hr at 40C, washed with Pi/NaCl, incubated withanti-rabbit gamma globulin (1:100 dilution, Cappel Labora-tories) for 1 hr at 240C, washed, and then incubated withrabbit peroxidase-antiperoxidase (30) for 1 hr at 240C. Theperoxidase was detected with 0.04% diaminobenzidinetetrahydrochloride (Polyscience, Warrington, PA)/0.0015%H202/0.05 M Tris HCl, pH 7.6, for 10 min. Sections weretreated with OSO4, dehydrated in ethanol, and embedded inPolybed 812 (Polyscience). Ultrathin sections were cut withan LKB ultramicrotome and examined with a Zeiss 109electron microscope.

RESULTSRabbit antisera (from two rabbits, A and B) against thesoluble form ofbovine pituitary carboxypeptidase processingenzyme were obtained. The antisera were positive in thesolid-phase RIA, even at dilutions of 1:200,000, when com-pared to preimmune serum (data not shown). To obtaininformation on the specificity of the carboxypeptidase pro-cessing enzyme antibody, we prepared immunoblots of ratpituitary and bovine adrenal medulla homogenates (Fig. 1).The antisera recognized purified soluble and membraneforms of bovine pituitary carboxypeptidase. One majorprotein band was stained by the antiserum in homogenatesfrom anterior, intermediate, and posterior rat pituitary andfrom bovine adrenal medulla; this band comigrated withstandard soluble and membrane forms of bovine pituitarycarboxypeptidase. Because the soluble and membrane formsof the enzyme differ in size by only 2500 Da (18), they maynot be resolved in the same sample by this electrophoresissystem. Therefore, the major anti-carboxypeptidase stainedprotein band seen in the pituitary and adrenal medullahomogenates may represent both soluble and membraneforms of the enzyme. The immunoblot data suggest that theantibody recognizes primarily the carboxypeptidase process-ing enzyme molecules contained in pituitary and adrenalmedulla.To determine whether the antisera recognized the soluble

and membrane-bound forms of bovine pituitary carboxypep-tidase with equal or different affinities, a RIA method wasdeveloped. Standard curves for the soluble and membraneforms of the enzyme (Fig. 2) appear identical, indicating thatthe antibody recognized these forms with equivalent affinity.The RIA could detect as little as 2 ng of carboxypeptidase andthe 50% displacement value for both soluble and membraneforms of the enzyme was 54 ng. The crossreactivity of theantisera with other carboxypeptidases was tested. 125i-labeled carboxypeptidase bound to the antibodies was notdisplaced by as much as 10 ,g of bovine or porcine carboxy-peptidase B, bovine carboxypeptidase N, bovine carboxy-peptidase A, yeast carboxypeptidase Y, or P. janthinellumcarboxypeptidase P (Table 1). Thus, the antibody against the

4746 Cell Biology: Hook et al.

Proc. Natl. Acad. Sci. USA 82 (1985) 4747

kDa

-94-68

43

30

-21

-14

1 2 3 4 5 6

FIG. 1. Immunoblot analysis of carboxypeptidase processingenzyme in homogenates of rat pituitary anterior, intermediate, andneural lobes and of bovine adrenal medulla. Lanes: 1, rat anteriorpituitary (200 ug of protein); 2, rat intermediate pituitary (75 ,ug ofprotein); 3, rat posterior pituitary (150 ,ug of protein); 4, bovineadrenal medulla (75 ,ug of protein); 5, standard soluble bovinepituitary carboxypeptidase (0.3 Ag); 6, standard membrane bovinepituitary carboxypeptidase (0.4 ,ug). Immunoblots with antisera fromrabbits A and B showed the same results. Positions of markerproteins (not shown) are indicated at right.

processing carboxypeptidase does not appear to crossreactwith other known carboxypeptidases.

Tissue levels ofcarboxypeptidase processing enzyme weremeasured in rat pituitary and bovine adrenal medulla. Unlikeantibodies against pancreatic carboxypeptidase B, whichshow species specificity (31, 32), the anti-carboxypeptidase-processing-enzyme antibody appears to recognize rat as wellas bovine forms of the enzyme. Increasing amounts of ratanterior pituitary homogenate displaced '25I-labeled carboxy-peptidase bound to antibody in a manner parallel to thestandard curve (Fig. 2 Inset). Anterior, intermediate, andneural lobes of rat pituitary homogenates contained, respec-tively, 0.17 + 0.10, 0.17 + 0.01, and 0.26 ± 0.09 ng ofcarboxypeptidase/Azg of protein (mean ± SD, n = 3), where-as 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. Neurallobe consistently contained higher concentrations than ante-rior lobe. Bovine adrenal medulla had 1.2 ± 0.35 ng/,ug ofprotein.The rabbit serum was useful for immunocytochemical

studies. In anterior and intermediate lobes of rat pituitary(Fig. 3 A and B), carboxypeptidase processing enzymeappeared to be discretely localized in the cytoplasm of allcells and was absent from the nuclei. Similar discrete local-ization ofthe enzyme was also found in the magnocellular cell

1008

>x Protein mg

50_ 50

30.T.

1020 50 100Carboxypeptidase, ng

FIG. 2. RIA of carboxypeptidase processing enzyme. Standardcurves for the purified soluble (o) and membrane-bound (o) forms ofbovine pituitary carboxypeptidase are shown. (Inset) Measurement

of processing carboxypeptidase in various amounts of rat anteriorpituitary homogenate. B/Bo, fraction of labeled carboxypeptidasenot displaced.

Table 1. Antiserum against the carboxypeptidase processingenzyme does not crossreact with other carboxypeptidases

Competing protein Amount, ,ug (B/BO) x 100

None 100Bovine pituitary carboxypeptidase 0.045 50Bovine carboxypeptidase B 1 100

10 101Porcine carboxypeptidase B 1 97

10 94Bovine carboxypeptidase N 1 103

10 101Yeast carboxypeptidase Y 1 100

10 100Bovine carboxypeptidase A 1 100

10 100P. janthinellum carboxypeptidase P 1 100

10 100

B/Bo is the fraction of "M51-labeled bovine pituitary carboxy-peptidase not displaced.

bodies of the hypothalamic supraoptic and paraventricularnuclei (Fig. 3 C and D), which project to the posteriorpituitary where vasopressin and oxytocin are stored andreleased. In addition to the hypothalamic area, carboxypep-tidase immunostaining was present in cell bodies of all brainareas where neuropeptides are known to be present. Noimmunostaining of nerve fibers or terminals was seen withparaformaldehyde fixation, but staining in these structureswas seen by electron microscopy when glutaraldehyde wasincluded with paraformaldehyde in the perfusion solution.Carboxypeptidase processing enzyme immunostaining

(Fig. 3E) was present in bovine adrenal medulla, where largeamounts of enkephalin peptides are present (33, 34), but wasabsent in adrenal cortex, which does not contain enkephalins.Because the cellular location of the enzyme could not beresolved in whole adrenal gland sections, immunostaining inadrenomedullary chromaffin cells in culture was investigated(Fig. 3F). Carboxypeptidase and [Leu]enkephalin (data notshown) immunoreactivity were both found in chromaffincells and were not present in fibroblasts. Carboxypeptidaseimmunoreactivity, like the [Leu]enkephalin staining, wasfound in discrete cytoplasmic areas compatible with itspresence in secretory granules and Golgi apparatus fromwhich granules are formed. Carboxypeptidase and[Leu]enkephalin immunostaining were not seen in the nuclei.

Electron microscopic immunocytochemistry of the car-boxypeptidase processing enzyme in several areas of ratbrain was examined, and the immunostaining of the hypo-thalamic paraventricular nucleus is shown in Fig. 4. Theenzyme's subcellular localization was similar in all areas.Carboxypeptidase immunostaining was found in some nerveterminals (Fig. 4 A, B, and D) and dendrites (Fig. 4C), whichcontain storage vesicles. Some sections (Fig. 4 B and D)showed carboxypeptidase-labeled nerve terminals forming asynaptic contact with a nonlabeled cell body. Several nerveterminals (Fig. 4 A and D) did not show carboxypeptidaseimmunostaining.

DISCUSSIONSpecific rabbit antisera against the soluble form of thecarboxypeptidase processing enzyme of bovine pituitaryhave been prepared and characterized. The antisera pos-sessed equivalent binding affinities for both the soluble andmembrane-bound forms of the enzyme, suggesting that thesetwo enzyme forms may possess many regions of structuralhomology. Although the purified membrane carboxypepti-dase is slightly larger (52,500 Da) than the purified soluble

Cell Biology: Hook et al.

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) Bovineadrenal gland. (x300.) (F) Cultured bovine adrenomedullary chromaffin cells. (x420.)

form (50,000 Da) (18), they show identical substrate andinhibitor 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 knowncarboxypeptidases suggests that the peptide hormone pro-cessing enzyme is a structurally distinct carboxypeptidase.Immunohistochemical studies showed that the enzyme

was present in areas where neuropeptides have been foundpreviously. In anterior and intermediate lobes of rat pituitary,where proopiomelanocortin-derived peptides are synthe-sized, carboxypeptidase immunostaining was discretely lo-calized in the cytoplasm and not in the nuclei. The enzymewas also found in the cell bodies of the hypothalamicsupraoptic and paraventricular nuclei. Cells in the supraopticnucleus contain vasopressin and oxytocin (35). Cells in theparaventricular nucleus contain corticotropin-releasing fac-tor, somatostatin, enkephalins, dynorphins, oxytocin, thy-rotropin-releasing hormone, vasopressin, cholecystokinin,and bombesin (36). Discrete carboxypeptidase immunostain-ing in cell bodies was found throughout the brain, reflectingthe widespread distribution of brain neuropeptides. In theperiphery, immunostaining was present in adrenal medullarychromaffin cells, which contain enkephalin peptides.The localization of the carboxypeptidase processing en-

zyme, or "enkephalin convertase", in rat brain has recentlybeen demonstrated by use of [3H]GEMSA autoradiography(24). GEMSA has been found to be a potent inhibitor of thisenzyme (19). Although [3H]GEMSA binding sites correspond

closely to the distribution of enkephalinergic neurons, theareas found to bind [3H]GEMSA also contain many otherneuropeptides. In addition, [3H]GEMSA binding sites werealso abundant in areas that do not contain enkephalins, suchas the anterior pituitary which contains adrenocorticotropin.No discrepancies were found between our immunocyto-chemical localization of the enzyme and the [3H]GEMSAautoradiography done by Lynch et al. (24). Both im-munohistochemistry and [3H]GEMSA autoradiography ofthe carboxypeptidase processing enzyme show that thisenzyme is localized with a number of neuropeptides.The colocalization of processing carboxypeptidase with

many neuropeptides suggests that this enzyme may be usefulas a marker for peptidergic cells. Carboxypeptidase im-munostaining was present in a few cell bodies in globuspallidus (data not shown), but no neuropeptide has yet beenfound in these cell bodies. Neuropeptide immunostaining inglobus pallidus has been found only in nerve fibers (35).Perhaps cell bodies of globus pallidus contain an undiscov-ered neuropeptide.Although the discrete localization of carboxypeptidase

immunostaining is compatible with its presence in storagevesicles, its subcellular localization can only be determinedby electron microscopy. Electron microscopy revealed thatthe carboxypeptidase was present in dendrites and nerveterminals that contain storage vesicles. Some nerve terminalsand dendrites showed no immunostaining. It is possible thatthese may represent nonpeptidergic cells.

4748 Cell Biology: Hook et A

Proc. NatL. Acad. Sci. USA 82 (1985) 4749

FIG. 4. Electron microscopic immunocytochemistry of carboxy-peptidase processing enzyme. Stars in A, B, and D indicate carboxy-peptidase immunostaining in nerve terminals. Two anti-carboxypep-tidase 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 letterD (in C) designates dendritic areas. Bar at top right of each panelrepresents 1 jm.

Visualization of enzyme immunostaining in cell body andnerve terminal areas each required different fixation condi-tions. Light microscopy (paraformaldehyde fixation) re-vealed carboxypeptidase immunostaining in cell bodies butnot in nerve fibers or terminals. On the other hand, im-munostaining in nerve fibers and terminals as well as cellbodies was seen when glutaraldehyde was included withparaformaldehyde in the perfusion solution. The differentfixation conditions required to visualize the enzyme in thesesubcellular areas could be explained by two hypotheses.First, the accessibility of the antibody to the enzyme mayrequire specific conditions at the different subcellular struc-tures in the cell body (rough endoplasmic reticulum, Golgiapparatus, and newly formed secretory vesicles) and in thenerve fibers and terminals (mature secretory vesicles). Alter-natively, different forms of the enzyme could be present incell body and nerve fiber and terminal regions. It is possiblethat the soluble and membrane forms of the enzyme may bepresent in different proportions in immature compared tomature secretory vesicles. In situ, the antibody may prefer-entially stain one of these forms. In addition, recent studieshave shown that the carboxypeptidase in mature chromaffingranules is more catalytically active than the enzyme in aheterogeneous population (immature and mature) of chro-maffin granules (37). The factor(s) responsible for regulationof the enzyme's activity may possibly influence the ability ofthe antibody to recognize the enzyme.

In summary, use of specific rabbit antisera against theprocessing carboxypeptidase have shown that (i) the solubleand membrane forms of this enzyme may possess manyregions of structural homology, (ii) this enzyme may bestructurally different from other carboxypeptidases, and (iii)the enzyme is colocalized with many neuropeptides, whichsuggests that it may be involved in the processing of manypeptide hormone precursors.

We thank Dr. Solomon H. Snyder (Johns Hopkins UniversitySchool of Medicine) for generously providing purified bovine pitu-itary carboxypeptidase. We also thank Dr. Lee Eiden (Laboratory ofCell Biology, National Institute of Mental Health) for helpfuldiscussions. V.Y.H.H. is a recipient of a Wellcome Senior ResearchFellowship.

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