From the
Peptidylglycine
Biosynthetic maturation of bioactive peptides in the neural and
endocrine system involves a variety of post-translational processing
steps during transit of precursor proteins through the secretory
compartments(1, 2) . Endoproteolytic cleavage and
removal of COOH-terminal basic amino acids take place initially in the trans-Golgi network (TGN),
Peptide
Post-translational modifications are highly compartmentalized
events, particularly in the secretory pathway. The compartments in
which specific processing events occur are determined by the
maturation/activation of the enzymes, restricted localization of the
enzymes, availability of substrate, and the physicochemical milieu of
the compartments such as pH, divalent cations, and
cofactors(11, 12) .
In general, endo- and
exoproteolytic cleavages of peptide precursors must precede peptide
In the case of peptide
Although PAM proteins are subjected to glycosylation,
sialylation, sulfation, and endoproteolytic
processing(23, 25, 26, 27) , little is
known about where in the secretory compartment newly synthesized PAM
proteins become enzymatically active. To address this question, we
attached the ER retention/retrieval signal, KDEL, or an inactive
homologue, KDEV (28), to the COOH terminus of a bifunctional soluble
PAM protein (PAM-3) (Fig. 1). Both proteins were examined in
stably transfected non-neuroendocrine cells (hEK-293) and
neuroendocrine cells (AtT-20). We found that the PAM-3-KDEL protein was
efficiently retained in the ER in both cell types and that ER-retained
PAM-3-KDEL protein had enzyme activities similar to those of wild type
PAM-3 and PAM-3-KDEV. Thus, acquisition of enzyme activities by PAM
does not require transit beyond the cis-Golgi network.
PHM and PAL
activities were measured as described (5, 6) using 0.5
µM
hEK-293 cells and AtT-20 cells were stably transfected with
expression vectors encoding PAM-3-KDEL or PAM-3-KDEV. We chose KDEV as
a control sequence since it was inactive in ER retention and had no
effect on normal secretion and processing when attached to the COOH
terminus of proneuropeptide Y(28) . To examine the effect of the
COOH-terminal KDEL sequence on the intracellular distribution of PAM-3,
transfected cells were fixed, permeabilized, and visualized with a PAM
antibody. Both hEK-293 and AtT-20 cells expressing PAM-3-KDEL showed a
typical ER-staining pattern with a diffuse, reticular network-like
pattern extending throughout the cytoplasm (Fig. 2, A and E). In contrast, hEK-293 and AtT-20 cells expressing
PAM-3-KDEV protein exhibited staining patterns similar to those of wild
type PAM-3 in the same cell type (Fig. 2, B, C and F, G)(23, 29) . PAM-3-KDEV
expressed in hEK-293 cells exhibited concentrated staining in the
perinuclear region, overlapping but more diffuse than a Golgi marker,
wheat germ hemagglutinin. In transfected AtT-20 cells, PAM-3-KDEV
displayed punctate staining throughout the cell body with concentrated
staining at the tips of processes and some staining in the perinuclear
region.
In transfected AtT-20 cells, which
possess a regulated secretory pathway and secretory granules, PAM-3
protein (95 kDa) is targeted to the secretory granules and cleaved into
a 75-kDa bifunctional PAM protein lacking its COOH-terminal domain (Fig. 1)(23) . This endoproteolytic cleavage is blocked
upon incubation at 20 °C and is thought to occur after exit from
the TGN, presumably in immature secretory granules(18) .
Extracts of AtT-20 cells expressing PAM-3-KDEL or PAM-3-KDEV were
analyzed by SDS-PAGE and immunoblot with PHM antibody (Fig. 4).
Extracts from PAM-3-KDEL cells showed only a 95-kDa PAM-3-KDEL protein;
no 75-kDa cleavage product was detected, indicating that PAM-3-KDEL
protein failed to reach the site where the cleavage normally occurs.
Extracts of PAM-3-KDEV cells contained similar amounts of 95-kDa
PAM-3-KDEV and 75-kDa PAM derived from cleavage of PAM-3-KDEV. Thus the
presence of the COOH-terminal KDEL signal specifically blocked
targeting of PAM-3 to the secretory granules.
hEK-293 cells expressing wild type PAM-3, PAM-3-KDEL, or PAM-3-KDEV
were assayed for PHM and PAL activities. To compare the specific
activities of wild type and mutant PAM-3 proteins, equal amounts of PHM
activity (1 nmol/h) were subjected to immunoblot analysis (Fig. 5A). The amounts of wild type and mutant PAM-3
protein detected by the COOH-terminal domain antibody were almost equal
(densitometric values differed by <10% of wild type PAM-3, n = 2), indicating that the PHM specific activities of all
three PAM-3 proteins were very similar. The ratios of PAL to PHM
activity for PAM-3-KDEL and PAM-3-KDEV (
When PAM proteins were expressed in neuroendocrine cells (i.e. AtT-20) or in non-neuroendocrine cells lacking a
regulated secretory pathway (i.e. hEK-293), active PHM and PAL
were produced(23, 29) . These findings suggest that the
regulated secretory pathway and secretory granules are not necessary
for the maturation of active PAM proteins. For example, the 10 amino
acid pro-region of PAM, which is well conserved in evolution, is
removed in a post-TGN compartment in AtT-20 cells and is not removed in
hEK-293 cells; PHM activity is unaffected by the presence of the
pro-region (34). We wanted to identify the compartment in which
maturation of PAM occurred. In this study, we created a mutant soluble
bifunctional PAM protein with the COOH-terminal KDEL ER
retention/retrieval signal (PAM-3-KDEL) to examine the effect of ER
retention on enzyme activities. Based on immunofluorescence staining,
pulse-chase experiment and Endo H sensitivity, the PAM-3-KDEL protein
was efficiently retained in the ER/cis-Golgi network. The ER
retention of PAM-3-KDEL was mediated by the genuine KDEL ER
retention/retrieval mechanism since attachment of an inactive
homologue, KDEV, to PAM-3 had little effect on trafficking of PAM-3.
COOH-terminal attachment of the KDEL sequence was sufficient to
cause extremely efficient retention/retrieval of PAM-3. In other
secretory proteins examined after addition of the KDEL sequence,
inefficient ER retention was often observed(35, 36) .
Proteins that are efficiently retained in the ER are often
distinguished by the presence of acidic amino acid residues upstream of
the KDEL sequence(37, 38) . Addition of an acidic amino
acid sequence found in an ER resident protein (i.e. protein
disulfide isomerase) upstream of a COOH-terminal KDEL signal enhanced
the ER retention efficiency of the secretory protein,
interleukin-6(36) . Thus certain features of upstream sequence,
such as acidic residues, may determine the efficiency of recognition by
the KDEL receptor, and the acidic residues in the COOH-terminal
During passage through the secretory
pathway, newly synthesized PAM proteins undergo extensive
post-translational processing. Signal sequence removal and N-linked glycosylation occur in the ER (25). As PAM proteins
travel through the Golgi stacks, O-linked glycosylation,
sialylation, and Tyr- or O-linked oligosaccharide sulfation
take place(26, 27) . In transfected AtT-20 cells, all of
the post-signal peptide cleavages of PAM occur in a post-TGN
compartment(18, 23) . These endoproteolytic cleavages
have a modulatory effect on the PHM activity of PAM-3 and PAM-1 (34, 39) but are not essential for the generation of
activity. Other peptide-processing enzymes such as
furin(13, 21) , PC1, and PC2 (17, 19), which are
synthesized as proenzymes, are inactive until activated by
post-translational events following intra- (i.e. furin) or
intermolecular autocatalytic cleavage in the ER. The 10-amino acid
NH
Newly synthesized
PAM proteins are catalytically competent in the ER. It seems unlikely
that the peptidylglycine substrate acted upon by PAM would be available
in the ER. Endo- and exoproteolytic cleavages of peptide precursors are
required to reveal COOH-terminal Gly residues in most prohormones, and
these cleavages occur first in the TGN and largely in secretory
granules(1, 2) . Accordingly, compartments in which
peptide amidation occur are restricted to the TGN and secretory
granules by the availability of substrates as seen for the
proopiomelanocortin-derived joining peptide amide immunoreactivity in
the TGN and secretory granules(10) . It is not clear whether
PAM-3-KDEL could
We thank Drs. Dick Mains, Luc Paquet, and Sharon
Milgram for critically reading this manuscript. The monoclonal antibody
to PAM COOH-terminal domain was prepared in collaboration with Dr.
Sharon Milgram. We also thank Zina Garrett for secretarial assistance,
Carla Berard for help with tissue culture, and Marie Bell for general
laboratory assistance.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-amidating monooxygenase (PAM) catalyzes
the COOH-terminal
-amidation of neural and endocrine peptides via
a two-step reaction carried out in sequence by the monooxygenase and
lyase domains contained in this bifunctional protein. Peptide
-amidation is thought to take place primarily in the secretory
granules in which mature bioactive peptides are stored, and it is not
known where in the secretory compartment newly synthesized PAM protein
becomes enzymatically active. To address this question, PAM-3, a
soluble bifunctional protein, was modified by addition of the KDEL
endoplasmic reticulum (ER) retention/retrieval signal to its COOH
terminus. PAM-3-KDEL protein stably expressed in hEK-293 cells or in
AtT-20 cells was efficiently retained in the ER based on
immunocytochemistry, pulse-chase experiments, and maintained
endoglycosidase H sensitivity. The effect of the KDEL sequence was
specific since PAM-3 with an inactive ER retention/retrieval signal
(PAM-3-KDEV) moved through the secretory pathway like wild type PAM-3.
In AtT-20 cells, PAM-3-KDEL was not subjected to the COOH-terminal
endoproteolytic cleavage that generates a 75-kDa PAM protein from PAM-3
and PAM-3-KDEV. PAM-3-KDEL protein exhibited both monooxygenase and
lyase activities with specific activities similar to those of the wild
type PAM-3 and PAM-3-KDEV proteins. Thus, although PAM catalyzes a
reaction that occurs primarily in the secretory granules, newly
synthesized PAM protein becomes enzymatically competent in the ER.
(
)continue in
secretory granules and often result in the production of COOH-terminal
Gly-extended peptides that are subsequently
-amidated to produce
active peptides. Peptide
-amidation is often rate-limiting and
essential for generating bioactive peptides(3, 4) .
-amidation is a two-step reaction catalyzed in sequence
by monooxygenase and lyase domains contained in a bifunctional enzyme,
peptidylglycine
-amidating monooxygenase (PAM; EC 1.14.17.3). The
first step of the reaction is catalyzed by peptidylglycine
-hydroxylating monooxygenase (PHM) and requires copper, ascorbate,
and molecular oxygen; the second step is catalyzed by
peptidyl-
-hydroxyglycine
-amidating lyase (PAL) and generates
-amidated peptide and
glyoxylate(5, 6, 7, 8, 9) . This
modification is a late step in the maturation of bioactive peptides
beginning in the TGN after endo- and exoproteolytic cleavages but
occurring mostly in secretory granules(3, 10) .
-amidation. The subtilisin-like endoproteases are involved in
processing of prohormones. Furin, PC1, and PC2 undergo an autocatalytic
cleavage that removes the pro-region and is required for production of
fully active enzyme(13, 14, 15, 16) .
Pro-region cleavage of furin and PC1 takes place in the endoplasmic
reticulum
(ER)(13, 17, 18, 19, 20) .
Nevertheless, generation of catalytically active furin requires a
post-Golgi event(21, 22) .
-amidation, there is evidence suggesting the importance of both
the targeting of PAM and compartment physicochemical milieu in
establishing the restricted location of peptide
-amidation. First,
PAM protein is targeted to the secretory granules when expressed in
AtT-20 corticotrope tumor cells(23) , and PAM proteins are
enriched in dense core vesicles in the central nervous
system(24) . Second, proopiomelanocortin-derived
-amidated
joining peptide immunoreactivity is detected in the TGN and secretory
granules (10). Third, the TGN and secretory granules are the
compartments in which Gly-extended peptide substrates and optimum pH
are available.
Figure 1:
Mutant PAM-3
proteins. A schematic diagram of mutant PAM-3 proteins with
COOH-terminal ER retention signal is shown. PAM-3 is a splicing variant
of the longest form of PAM (PAM-1) lacking both the non-catalytic
region between PHM and PAL and the transmembrane domain between PAL and
the COOH-terminal domain (CD). Irregular closed
curve, N-linked oligosaccharide on Asn (25); arrowhead with vertical tic mark, paired
basic amino acid endoproteolytic cleavage sites that may be used in
AtT-20 cells (23). Tyr
of PAM-3 is sulfated (26). All
sites are numbered as in PAM-1, which is 976 residues in
length.
Plasmid Construction
pBluescript plasmids
encoding PAM-3 with COOH-terminal KDEL or KDEV sequences were created
using the polymerase chain reaction; pBS.KrPAM-3 was amplified using a
sense primer in the PAL domain (nucleotides 2517-2533) and an
antisense primer with an XbaI site (boldface) and codons for
KDEL or KDEV following the COOH-terminal end of PAM-3 (underlined):
5`-CCTCTA-GACTACAGCTCGTCCTTGGAGGAAGGTGCAGGCTT-3` for KDEL and
5`-CCTCTAGACTACACCTCGTCCTTGGAGGAAGGTGCAGGCTT-3` for KDEV. The
amplified fragment obtained was digested with XbaI and AatII (nucleotide 2620) and inserted into pBS.KrPAM-3 (23) from which the XbaI/AatII fragment had
been removed, creating pBS.KrPAM-3-KDEL and pBS.KrPAM-3-KDEV. The cDNA
region derived from polymerase chain reaction was verified by
sequencing. The KrPAM-3-KDEL and KrPAM-3-KDEV cDNA was inserted into
the pCIS.2CXXNH expression vector by ligation of the Bsp106I/XbaI fragment of pBS.KrPAM-3-KDEL or
pBS.KrPAM-3-KDEV and the Bsp106I/XbaI vector fragment
of pCIS.KrPAM-3.
Cell Culture and Transfection
hEK-293 cell lines
were maintained in DMEM/F-12 (Life Technologies, Inc.) containing 10%
fetal clone serum (Hyclone, Logan, UT) and antibiotics at 37 °C in
5% CO as described(23) . AtT-20 cells were
maintained in the same medium supplemented with 10% NuSerum
(Collaborative Research, Bedford, MA). Cells were passaged weekly.
pCIS.KrPAM-3-KDEL and pCIS.KrPAM-3-KDEV plasmids were transfected into
hEK-293 cells and AtT-20 cells using Lipofectin (Life Technologies,
Inc.) as described (23). Transfected cells were grown in DMEM/F-12
containing antibiotics and 0.5 mg/ml G418 supplemented with 10% fetal
clone serum (for hEK-293 cells) or 10% fetal clone serum and 10%
NuSerum (for AtT-20 cells). G418-resistant cells were screened for
expression of PAM proteins by immunocytochemical staining using PHM
antibody or PAL antibody as described(29) .
-N-acetyl-Tyr-Val-Gly and 0.5
µM
-N-acetyl-Tyr-Val-
-hydroxyglycine.
Western blot analyses were carried out as described(30) . PAM
proteins were detected using ECL (Amersham Corp.) with rabbit
polyclonal antisera against the PHM domain (antibody 475; Ref. 25) and
mouse monoclonal antibody against the COOH-terminal domain of PAM
(6E6).
(
)
Biosynthetic Labeling and
Immunoprecipitation
Stably transfected cells were plated on
12-mm culture dishes coated with 25 µg/ml fibronectin (for hEK-293
cells) or 0.1 mg/ml poly-L-lysine (for AtT-20 cells) and grown
a minimum of 36 h and were 50-75% confluent before experiments
were begun. Biosynthetic labeling with
[S]cysteine/methionine (Amersham Corp.) (0.3
mCi/well, 1-2 µM methionine) was performed as
described (31). Cells were labeled for 15 min (pulse) and rinsed once
with complete serum-free medium. Cells were then either extracted with
20 mM TES (pH 7.4), 10 mM mannitol, 1% Triton X-100
containing protease inhibitors as described (23) or incubated for
varying periods of time in 300 µl of complete serum-free medium
(chase). Immunoprecipitation of cell extracts and media was carried out
as described using rabbit antibody to recombinant COOH-terminal domain
of PAM (antibody 571)(25, 31) . Immunoprecipitated
proteins were fractionated by SDS-PAGE on 10% acrylamide (0.27% N,N`-methylenebisacrylamide) gels and visualized by
fluorography. The apparent molecular masses of the immunoprecipitated
proteins were determined by comparison with prestained molecular weight
standards (Rainbow standards; Amersham Corp.). Densitometric analyses
were performed using an Abaton Scan 300/GS linked to an Apple Macintosh
IIci and NIH Image 1.35 software (National Institute of Mental Health). N-glycanase and endoglycosidase H treatment of
immunoprecipitated PAM proteins were performed as
described(27, 32) .
Figure 2:
Immunofluorescence staining. hEK-293 cells (A-D) and AtT-20 cells (E-H)
expressing PAM-3-KDEL (A and E), PAM-3-KDEV (B and F), PAM-3 (C and G), or no
exogenous PAM (D and H) were fixed, permeabilized,
and stained with rabbit polyclonal antibody to PAL (antibody 471;
1:1500; A, B, E, and F) or to PHM
(antibody 475; 1:1500; C, D, G, and H) and fluorescein isothiocyanate-conjugated goat anti-rabbit
IgG (1:500; CalTag, San Francisco, CA). Scalebar, 20
µm.
To further investigate the effect of addition of the KDEL
sequence on the trafficking of PAM-3, hEK-293 cells expressing
PAM-3-KDEL or PAM-3-KDEV were incubated with
[S]cysteine/methionine for 15 min (pulse) and
then incubated with medium containing non-radioactive cysteine (200
µM) and methionine (114 µM) for 2 or 18 h
(chase). Cell extracts and chase media collected from each time point
were analyzed by immunoprecipitation and SDS-PAGE (Fig. 3A). After the 15-min pulse, 0.5-1% of the
protein synthesized (trichloroacetic acid-precipitable radioactivity)
could be identified as a 95-kDa PAM-3 protein using antiserum to the
PAM COOH-terminal domain. After 2 or 18 h of chase, the newly
synthesized PAM-3-KDEL protein was still retained in the cells without
significant loss of protein. Very little PAM-3-KDEL protein (<2% of
the newly synthesized PAM-3-KDEL) appeared in the chase medium even
after an 18-h chase. In contrast, about 50% of the newly synthesized
PAM-3-KDEV protein was secreted during the initial 2-h chase. This rate
of secretion is similar to the observed rate of constitutive secretion
of wild type PAM-3 from transfected hEK-293 cells (30-50% of
PAM-3 in the cell/h)(26, 29) . After 18 h of chase, all
of the PAM-3-KDEV found in the cell extract after the pulse was
recovered in the chase medium with little loss of protein. Thus, the
effect of the KDEL sequence on retention of PAM-3 in the cells is
specific and efficient.
Figure 3:
Pulse-chase experiment and endoglycosidase
H treatment. A, three identical wells of hEK-293 cells
expressing PAM-3-KDEL or PAM-3-KDEV were incubated with
[S]cysteine/methionine for 15 min and harvested (0 h cell) or chased for the indicated times in complete
serum-free medium. Equal amounts of cell extracts and chase media were
immunoprecipitated with an antibody to the COOH-terminal domain of PAM
(antibody 571) and analyzed by SDS-PAGE and fluorography. B,
selected samples from this and another pulse-chase experiment were
immunoprecipitated and subjected to Endo H or N-glycanase (N-gly) treatment; control samples (Con) were from
the same incubations without enzymes. Samples were analyzed by SDS-PAGE
and fluorography. Similar results were obtained in two independent
studies.
PAM-3 contains a single N-linked
oligosaccharide at Asn in the PAL domain (25) (Fig. 1). To test how far the cell-associated
PAM-3-KDEL protein progressed through the cell, PAM-3 proteins
immunoprecipitated from selected samples of cell extract or medium (Fig. 3A) were digested with endoglycosidase H (Endo H)
or N-glycanase (Fig. 3B); acquisition of
resistance to digestion with Endo H is diagnostic of transit from the
ER to the medial-Golgi compartment(33) . Immediately after the
pulse (0 h cell), both newly synthesized PAM-3-KDEL and KDEV proteins
were Endo H-sensitive. PAM-3-KDEL remained Endo H-sensitive up to 18 h
after its biosynthesis. Thus PAM-3-KDEL protein expressed in hEK-293
cells is efficiently retained in the ER without significant degradation
for up to 18 h. In contrast, as expected from its similarity to wild
type PAM-3,
50% of the newly synthesized PAM-3-KDEV protein was
Endo H-resistant after a 1.5-h chase. The behavior of PAM-3-KDEV is
consistent with its expected similarity to PAM-3, which has a t for ER to Golgi transit of about 1 h in AtT-20 cells(32) .
PAM-3-KDEV protein that had been secreted into the medium was
completely Endo H-resistant. These results together with
immunocytochemistry data clearly demonstrate that PAM-3-KDEL protein is
efficiently retained in the ER.
Figure 4:
PAM-3-KDEL protein expressed in AtT-20
cells. Extracts of AtT-20 cells expressing PAM-3-KDEL or PAM-3-KDEV
were assayed for PHM activity, and equal amounts of PHM activity (10,
5, and 2.5 nmol/h) from each extract were analyzed by immunoblot with
PHM antibody (antibody 474) and ECL. The PHM specific activities of
PAM-3-KDEL and PAM-3-KDEV cell extracts were 1100 to 1300 and 50
to
100 pmol/µg/h, respectively. Similar data were observed on
two separate set of extracts.
Extracts of PAM-3-KDEL
and PAM-3-KDEV AtT-20 cells were assayed for PHM activity. Western blot
analysis indicated that samples with approximately equal amounts of PHM
activity contained similar amounts of PAM protein (Fig. 4;
densitometric values differed by <50% for PAM-3-KDEL and PAM-3-KDEV, n = 8). Since the endoproteolytic cleavage that
separates the COOH-terminal domain from PAL affects PHM
activity(34) , a more detailed analysis of the effect of the
KDEL/KDEV sequence on enzymatic activity was carried out in hEK-293
cells, where endoproteolytic cleavages of this type do not occur.
1.5) were
indistinguishable from each other (p < 0.05, n = 4) but were significantly less than the PAL:PHM ratio of
wild type PAM-3 (PAL:PHM
2; p < 0.05, n = 4). Addition of the COOH-terminal KDEL/KDEV tetrapeptide
does not cause more than a slight decrease of PAL activity. Thus we
conclude that the ER/cis-Golgi network provides an environment
sufficient for the maturation of fully active PAM-3 protein.
Figure 5:
PAM-3-KDEL protein expressed in hEK-293
cells. Extracts of hEK-293 cells expressing wild type PAM-3 (29),
PAM-3-KDEL, or PAM-3-KDEV were assayed for PHM and PAL activities. A, samples of each extract containing equal amounts of PHM
activity (1 nmol/h) were analyzed by immunoblot with monoclonal
antibody to the COOH-terminal domain of PAM (antibody 6E6) and ECL. B, PAL activity/1 nmol/h of PHM activity for each cell
extract; errorbars show standard errors obtained
from quadruplicate assays. Similar data were observed on two separate
sets of extracts. The specific activities of PHM and PAL in extracts of
wild type hEK-293 cells were less than 1% of those activities in the
transfected cells (29). WT, wild
type.
30
amino acid region of PAM-3 (11 of 30 residues are Asp or Glu) (Fig. 1) may contribute to the efficient ER retention observed
for the PAM-3-KDEL protein.
-terminal pro-region of PAM is not required during the
biosynthesis of PAM, and its removal has little or no effect on PHM
activity(34) . A truncated, soluble form of furin retained in
the ER by addition of a COOH-terminal KDEL sequence underwent
propeptide cleavage but was not active on exogenous substrates
coexpressed in vivo(16, 21) . Thus in the case
of furin, pro-region cleavage in the ER seems to be a prerequisite to
proceed to further post-ER events for complete maturation(21) .
Results of this study showing enzymatically active PAM-3-KDEL protein
in the ER ( Fig. 4and Fig. 5) suggest that
post-translational events occurring in the Golgi and later compartments
are not required for maturation of active PAM-3.
-amidate a peptidylglycine substrate presented to
it in the ER. Our assays were always carried out after the addition of
exogenous copper and ascorbate and do not address the issue of when
copper is loaded onto PHM or when ascorbate becomes available to the
enzyme. In addition, the lumenal pH in the ER is not as close to the
optimal pH (5.5-6) of PAM as the lumenal pH in the TGN and
immature secretory granules. Thus, this study strongly supports the
hypothesis that restricted localization of peptide amidation is
determined by the restricted activation of endo- and exoproteases along
with the lumenal milieu rather than by post-translational activation of
PAM.
-amidating monooxygenase; PHM, peptidylglycine
-hydroxylating
monooxygenase; PAL, peptidyl-
-hydroxyglycine
-amidating
lyase; DMEM/F-12, Dulbecco's modified Eagle's medium and
nutrient mixture F-12 mixed in equal volumes; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; PAGE,
polyacrylamide gel electrophoresis; ECL, enhanced chemiluminiscence;
ER, endoplasmic reticulum; Endo H, endoglycosidase H.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.