Aminopeptidase B (Ap-B) is a Zn2+-dependent exopeptidase which selectively removes Arg and/or Lys residues from the N terminus of several peptide substrates. Isolated and characterized from rat testes, this ubiquitous enzyme may participate in the final stages of precursor processing mechanisms. To test this hypothesis, we have investigated the secretion and subcellular localization of this enzyme in a rat cell line of pheochromocytoma (PC12 cells). By using a combination of biochemical and immunocytochemical methods, the following observations were made: (i) the level of aminopeptidase B detectable in the cell culture medium increased with time; (ii) 8-bromo-adenosine 3′-5′-cyclic monophosphate and the Ca2+ ionophore A23187 both stimulated enzyme liberation in the culture medium; (iii) brefeldin A, an inhibitor of vesicular transport from the endoplasmic reticulum to the Golgi apparatus, decreased enzyme secretion in a time-dependent manner; (iv) whereas nocodazole, a microtubule depolymerizing agent, inhibited enzyme secretion, cytochalasin D, a microfilament disruption agent, had no effect on released aminopeptidase B level; (v) immunofluorescence demonstrated the presence of aminopeptidase B in the Golgi apparatus; (vi) immunofluorescence, electron microscopy and tests of enzyme activity on intact cells showed an association of the peptidase with the external face of the plasma membrane. Together these data strongly argued in favour of the enzyme secretion by PC12 cells. It is concluded that aminopeptidase B may participate in processing events occurring either during its intracellular transport along the secretory pathway or at the plasma membrane level, or both.

REFERENCES

Azaryan
A. V.
,
Hook
V. Y. H.
(
1994
).
Unique cleavage specificity of ‘prohormone thiol protease’ related to proenkephalin processing.
FEBS Lett
341
,
197
202
Belhacene
N.
,
Mari
B.
,
Rossi
B.
,
Auberger
P.
(
1993
).
Characterization and purification of T lymphocyte aminopeptidase B: a putative marker of T cell activation.
Eur. J. Immunol
23
,
1948
1955
Benjannet
S.
,
Rondeau
N.
,
Day
R.
,
Chretien
M.
,
Seidah
N. G.
(
1991
).
PC1 and PC2 are proprotein convertases capable of cleaving proopiomelanocortin at distinct paired basic residues.
Proc. Nat. Acad. Sci. USA
88
,
3564
3568
Bradford
M. M.
(
1976
).
A rapid and sensitive method for quantitation of microgram quantities of proteins utilizing the principle of protein dye binding.
Anal. Biochem
72
,
248
254
Brinkley
B. R.
,
Murphy
P.
,
Richardson
L. C.
(
1967
).
Procedure for embedding in situ selected cells cultured in vitro.
J. Cell Biol
35
,
279
283
Cadel
S.
,
Pierotti
A. R.
,
Foulon
T.
,
Creminon
C.
,
Barre
N.
,
Segretain
D.
,
Cohen
P.
(
1995
).
Aminopeptidase-B in the rat testes: isolation, functional properties and cellular localization in the seminiferous tubules.
Mol. Cell. Endoc
110
,
149
160
Cadel
S.
,
Foulon
T.
,
Viron
A.
,
Balogh
A.
,
Midol-Monet
S.
,
Noël
N.
,
Cohen
P.
(
1997
).
Aminopeptidase B from the rat testis is a bifunctional enzyme structurally related to leukotriene-A4hydrolase.
Proc. Nat. Acad. Sci. USA
94
,
2963
2968
Carraway
R. E.
,
Bullock
B. P.
,
Dobner
P. R.
(
1993
).
Induction of the neurotensin (NT) gene in PC12 cells gives rise to NT precursor (88%), NT (3–13)-like peptide (10%), and NT (2%).
Peptides
14
,
991
999
Castro
M. G.
,
Birch
N. P.
,
Peng Loh
Y.
(
1989
).
Regulated secretion of pro-opiomelanocortin converting enzyme and an aminopeptidase B-like enzyme from dispersed bovine intermediate lobe pituitary cells.
J. Neurochem
52
,
1620
1628
Chesneau
V.
,
Pierotti
A. R.
,
Barre
N.
,
Creminon
C.
,
Tougard
C.
,
Cohen
P.
(
1994
).
Isolation and characterization of a dibasic selective metalloendopeptidase from rat testes that cleaves at the amino terminus of arginine residues.
J. Biol. Chem
269
,
2056
2061
Cohen
P.
(
1987
).
Proteolytic events in the post-translational processing of polypeptide hormone precursors.
Biochimie
69
,
87
89
Darby
N. J.
,
Smyth
D. G.
(
1990
).
Endopeptidases and prohormone processing.
Biosci. Reports
10
,
1
13
Das
B.
,
Sabban
E. L.
,
Kilbourne
E. J.
,
Fricker
L. D.
(
1992
).
Regulation of carboxypeptidase E by membrane depolarization in PC12 pheochromocytoma cells: Comparison with mRNA encoding other peptide and catecholamine-biosynthetic enzymes.
J. Neurochemistry
59
,
2263
2270
Devi
L.
,
Goldstein
A.
(
1985
).
Neuropeptide processing by single step cleavage: conversion of leumorphin (dynorphin B-29) to dynorphin B.
Biochem. Biophys. Res. Commun
130
,
1168
1176
Dittie
A. S.
,
Tooze
S. A.
(
1995
).
Characterization of the endopeptidase PC2 activity towards secretogranin II in stably transfected PC12 cells.
Biochem. J
310
,
777
787
Draoui
M.
,
Bellincampi
L.
,
Hospital
V.
,
Cadel
S.
,
Foulon
T.
,
Prat
A.
,
Barre
N.
,
Reichert
U.
,
Melino
G.
,
Cohen
P.
(
1997
).
Expression and retinoic modulation of N-arginine dibasic convertase and of an aminopeptidase-B in human neuroblastoma cell line.
J. Neurooncol
31
,
99
106
Eberlein
G. A.
,
Eysselein
V. E.
,
Davis
M. T.
,
Lee
T. D.
,
Shively
J. E.
,
Grandt
D.
,
Niebel
W.
,
Williams
R.
,
Moessner
J.
,
Zeeh
J.
,
Meyer
H. E.
,
Goebell
H.
,
Reeve
J. R.
(
1992
).
Patterns of prohormone processing order revealed by a new procholecystokinin-derived peptide.
J. Biol. Chem
267
,
1517
1521
Flores
M.
,
Aristoy
M. C.
,
Toldra
F.
(
1993
).
HPLC purification and characterization of porcine muscle aminopeptidase B.
Biochimie
75
,
861
867
Foulon
T.
,
Cadel
S.
,
Chesneau
V.
,
Draoui
M.
,
Prat
A.
,
Cohen
P.
(
1996
).
Two novel metallopeptidases with specificity for basic residues. Functional properties, structure and cellular distribution.
Ann. NY Acad. Sci
780
,
106
120
Fricker
L. D.
(
1988
).
Carboxypeptidase E.
Annu. Rev. Physiol
50
,
309
321
Fricker
L. D.
,
Das
B.
,
Angeletti
R. H.
(
1990
).
Identification of the pH-dependent membrane anchor of carboxypeptidase E (EC 3.4.17.10).
J. Biol. Chem
265
,
2476
2482
Gainer
H.
,
Russel
J. T.
,
Peng Loh
Y.
(
1984
).
An aminopeptidase activity in bovine pituitary secretory vesicles that cleaves the N-terminal arginine from-lipotropin.
FEBS Lett
175
,
135
139
Galanopoulou
A. S.
,
Kent
G.
,
Rabbani
S. N.
,
Seidah
N. G.
,
Patel
Y. C.
(
1993
).
Heterologous processing of prosomatostatin in constitutive and regulated secretory pathways.
J. Biol. Chem
268
,
6041
6049
Gluschankof
P.
,
Gomez
S.
,
Morel
A.
,
Cohen
P.
(
1987
).
Enzymes that process somatostatin precursors. A novel endoprotease that cleaves before the arginine-lysine doublet is involved in somatostatin-28 convertase activity of rat brain cortex.
J. Biol. Chem
262
,
9615
9620
Gomez
S.
,
Glushankof
P.
,
Lepage
A.
,
Cohen
P.
(
1988
).
Relationship between endo-and exopeptidase in a processing enzyme system: activation of an endoprotease by the aminopeptidase B-like activity in somatostatin-28 convertase.
Proc. Nat. Acad. Sci. USA
5
,
5468
5472
Greene
L. A.
,
Tischler
A. S.
(
1976
).
Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor.
Proc. Nat. Acad. Sci. USA
73
,
2424
2428
Halegoua
S.
,
Patrick
J.
(
1980
).
Nerve growth factor mediates phosphorylation of specific proteins.
Cell
22
,
571
581
Hopsu
V. K.
,
Kantonen
U. M.
,
Glenner
G. G.
(
1964
).
A peptidase from rat tissues selectively hydrolysing N-terminal arginine and lysine residues.
Life Sci
3
,
1449
1453
Hui
K. S.
,
Lo
E. S.
,
Hui
M. P. P.
(
1994
).
An endogenous aminoenkephalinase inhibitor: purification and characterization of Arg0-Met5-enkephalin from bovine striatum.
J. Neurochem
63
,
1748
1756
Jasmin
B. J.
,
Cartaud
J.
,
Bornens
M.
,
Changeux
P.
(
1989
).
Golgi apparatus in chick skeletal muscle: changes in its distribution during end plate development and after denervation.
Proc. Nat. Acad. Sci. USA
86
,
7218
7222
Kawata
S.
,
Takayama
S.
,
Ninomiya
K.
,
Makisumi
S.
(
1980
).
Porcine liver aminopeptidase B.
J. Biochem
88
,
1601
1605
Mantle
D.
,
Lauffart
B.
,
Mc Dermott
J. R.
,
Kidd
A. M.
,
Pennington
R. J. T.
(
1985
).
Purification and characterization of two Cl-activated aminopeptidases hydrolysing basic termini from human skeletal muscle.
Eur. J. Biochem
147
,
307
312
McDermott
J. R.
,
Mantle
D.
,
Lauffart
B.
,
Gibson
A. M.
,
Biggins
J. A.
(
1988
).
Purification and characterization of two soluble Cl-activated arginyl aminopeptidases from human brain and their endopeptidase action on neuropeptides.
J. Neurochem
50
,
177
182
Misono
K. S.
,
Fukumi
H.
,
Grammer
R. T.
,
Inagami
T.
(
1984
).
Rat atrial natriuretic factor: complete aminoacid sequence and disulfide linkage essential for biological activity.
Biochem. Biophys. Res. Commun
119
,
524
529
Parish
D. C.
,
Tujeta
R.
,
Alstein
M.
,
Gainer
H.
,
Peng Loh
Y.
(
1986
).
Purification and characterization of a paired basic residue-specific prohormone converting enzyme from bovine pituitary neural lobe secretory vesicles.
J. Biol. Chem
261
,
14392
14397
Pedersen
R. C.
,
Brownie
A. C.
(
1983
).
Lys-3-melanortopin binds with high affinity to the rat adenal cortex.
Endocrinology
112
,
1279
1287
Picart
R.
,
Tixier-Vidal
A.
(
1974
).
Description d'une methode permettant la selection et l'etude ultrastructurale de plages cellulaires dans des monocouches heterogenes cultivees en flacons de plastique.
J. Microsc
20
,
80
–.
Radeke
M. J.
,
Misko
T. P.
,
Hsu
C.
,
Herzenberg
L. A.
,
Shooter
E. M.
(
1987
).
Gene transfer and molecular cloning of the rat nerve growth factor receptor.
Nature
325
,
593
597
Regoli
D.
,
Barabe
J.
(
1980
).
Pharmacology of bradikynin and related kinins.
Pharmacol. Rev.
32
,
1
46
Saraste
J.
,
Palade
G. E.
,
Farquhar
M. G.
(
1986
).
Temperature-sensitive steps in the transport of secretory proteins through the Golgi complex in exocrine pancreatic cells.
Proc. Nat. Acad. Sci. USA
83
,
6425
6429
Seidah
N. G.
,
Chretien
M.
(
1992
).
The family of subtilisin/kexin like pro-protein and pro-hormone convertases: divergent or shared functions.
Biochimie
76
,
197
209
Söderling
E.
,
Mäkinen
K. K.
(
1983
).
Modification of the Cl-activated arginine aminopeptidase from rat liver and human erythrocytes: a comparative study.
Arch. Biochem. Biophys
220
,
11
21
Schweitzer
E. S.
(
1993
).
Regulated and constitutive secretion of distinct molecular forms of acetylcholinesterase from PC12 cells.
J. Cell Sci
106
,
731
740
Tischler
A. S.
,
Greene
L. A.
(
1975
).
Nerve growth factor-induced process formation by cultured rat pheochromocytoma cells.
Nature
258
,
341
342
Tougard
C.
,
Picart
R.
,
Tixier-Vidal
A.
(
1980
).
Electron microscopic cytochemical studies on the secretory process in rat prolactin cells in primary culture.
Am. J. Anat
158
,
471
490
Tougard
C.
,
Picart
R.
(
1986
).
Use of pre-embedding ultrastructural immunocytochemistry in the localization of a secretory product and membrane proteins in cultured prolactin cells.
Am. J. Anat
175
,
161
177
Tooze
S. A.
,
Huttner
W. B.
(
1990
).
Cell-free protein sorting to the regulated and constitutive secretory pathways.
Cell
60
,
837
847
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