- Email: [email protected]
The 108-kDa peptidylglycine α-amidating monooxygenase precursor contains two separable enzymatic activities involved in peptide amidation
Vol.
171, No.
September
3, 1990 28,
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
1990
926-932
THE 108~kDA PEPTIDYLGLYCINE a-AMIDATING MONOOXYGENASE PRECURSOR CONTAINS TWO SEPARABLE ENZYMATIC ACTIVITIES INVOLVED
IN PEPTIDE
AMIDATION
Susan N. Perkins, E. Jean Husten and Betty A. Eipper Department
of Neuroscience, Johns Hopkins University Baltimore,
Received
August
10,
School of Medicine,
MD 21205
1990
SUMMARY: A 43-kDa protein factor that increases the ability of purified bovine peptidylglycine a-amidating monooxygenase (PAM)-A and -B to produce a-amidated peptides at physiological pH was purified to homogeneity from bovine neurointermediate pituitary. At each step of the purification, the amount of activity correlated with the amount of protein detected on Western blots by antibody to bovine PAM(561-579). In the bovine neurointermediate pituitary the lO&kDa PAM precursor protein is cleaved to form a peptidylglycine a-hydroxylating monooxygenase and a peptidyl-cr-hydroxyglycine Qamidating lyase, which function sequentially in the 2-step formation of or-amidated peptides. @ 1990
Academic
Press,
Inc.
Peptidylglycine
a-amidating
monooxygenase (PAM; EC 1.14.17.3) is responsible for
the conversion of peptides with a COOH-terminal a posttranslational
modification
glycine into a-amidated product peptides,
often required for biological activity (1,2). In the course of
investigating a protein factor that increases the ability of purified bovine PAM-A and -B (Fig. 1) to produce a-amidated peptides from their glycine-extended precursors at the acidic pH values characteristic of secretory granules, it became apparent that levels of stimulatory activity were closely correlated with levels of PAM activity (3). Although extracts of AtT20 cells transfected with cDNAs encoding either the entire 108~kDa bovine PAM precursor or a 38-kDa protein corresponding to PAM-B (Fig. 1) exhibited elevated levels of aamidation activity in test tube assays, only extracts of cells expressing the 108~kDa PAM precursor protein exhibited increased levels of stimulatory
activity. These results suggested
The abbreviations used are: PAM, peptidylglycine c*-amidating monooxygenase; PHM, peptidylglycine a-hydroxylating monooqgenase; PAL, peptidyls-hydroxyglycine a-amidating lyase; FPLC, fast protein liquid chromatography; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis. 0006-291X/90 Copyright All rights
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
926
Vol.
171, ,No. 3, 1990
BIOCHEMICAL
that a part of the remaining approximately
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
70 kDa of the PAM
45kDa stimulatory
precursor might
constitute
the
activity. We used antisera to a synthetic peptide derived
from the region of the intragranular domain of the bovine PAM precursor not included in PAM-A or -B (Fig. 1) to demonstrate that a protein derived from this region of the PAM precursor is indeed responsible for this stimulatory
activity.
Moreover,
we purified
an
active form of this factor more than 20,000-fold to homogeneity. The production of a-amidated peptides from their glycine-extended precursors is a 2-step process proceeding through peptidyl-a-hydroxyglycine (4-S). The enzyme catalyzing the first step requires copper, molecular oxygen and ascorbate (1,2,4,6) and is a peptidylglycine a-hydroxylating monooxygenase (PHM). Although conversion of peptidylcr-hydroxyglycine to a-amidated product occurs spontaneously at alkaline pH (6,7), an enzyme activity catalyzing this step at acidic-to-neutral pH values has recently been identified (7,8). The stimulatory activity that we have studied (3) has all of the properties expected of the enzyme catalyzing the second step of this reaction; this enzyme is a peptidylhydroxyglycine
a-amidating
lyase (PAL).
MATERIALS
AND METHODS
Assays. PAL activity was assayed by adding 0.2 ~1 purified bPAM-A+B (which catalyze the first step of the reaction but fail to produce a-amidated product at acidic pH) to samples and measuring production of a-amidated product (3); standard assay conditions included 0.5 PM a-N-acetyl-Tyr-Val-Gly, 20,000 cpm [““I]-a-N-acetyl-Tyr-Val-Gly, 175 pg/ml catalase, 0.1 or 1 PM CuSO, (optimal concentration determined at each step), 0.5 mM ascorbate, and loo-150 mM NaMES, pH 6.0. Protein was assayed with the BCA reagent (Pierce Chemical Co., Rockford, IL) or with Integrated Separation Systems Protein-Gold reagent (Hyde Park, MA). Purification of PAL. The resuspended 25-45% (NH,),SO, fraction prepared from 130 frozen bovine neurointermediate pituitaries (9) was thawed and applied to a 45-ml column of phenylSepharose (Pharmacia, Piscataway, NJ) equilibrated with 10 mM NaTES, pH 7.5, 1.0 M (NH&SO.,; proteins were eluted with 500 ml of the same buffer containing 0.7, 0.3 or 0 M (NH&SO, or with a linear gradient to 0 M (NH,),SO,. Fractions containing PAL activity were pooled, concentrated 20-fold with an Amicon (Danvers, MA) ultrafiltration cell using a PM-10 membrane and applied to a 2 x 55 cm column of Sephadex G75 equilibrated and eluted with 20 mM TrisHCl, pH 8.0,40 mM NaCl. Subsequent steps in the purification were carried out by FPLC (Pharmacia). Fractions containing stimulatory activity were pooled and applied directly to a MonoQ HR S/5 column equilibrated with the same buffer; proteins were eluted with a gradient to 1.0 M NaCl. Peaks of activity were pooled and applied to a MonoP HR 5/20 column equilibrated with either 25 mM BisTrisHCl, pH 6.0, or 25 mM 1-methylpiperazine (Fluka, Ronkonkoma, NY) HCI, pH 5.7, and eluted by chromatofocusing with Polybuffer 74 (Pharmacia) diluted lo-fold and titrated to pH 3.94 with HCl. Fractions were neutralized by adding 50 ~1 1 M NaTES, pH 7.0, per 1.0 ml fraction and stored in the presence of 0.02% Lubrol-PX (Pierce). Pooled fractions were concentrated with a Centricon 10 microconcentrator (Amicon) and applied to a Superose 6 HR lo/30 column equilibrated and eluted with 0.2 M NH,HCO, containing 0.02% Lubrol-PX. Pooled fractions were applied to the MonoQ column equilibrated with 25 mM BisTrisHCl, pH 6.0, containing 0.02% Lubrol-PX, and eluted with a gradient to 0.5 M NaCl in the same buffer. 927
Vol.
171, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Western blots Samples were fractionated on slab gels containing 10% polyacrylamide, 0.27% N,K-methylenebisacrylamide, transferred to Immobilon P membranes (Millipore, Bedford, MA) and visualized with affinity-purified antibody to bPAM(561-579) (Ab68 or 69) (10) or purified bPAM-A+B (Ab36) (11) and [‘*‘I]-Protein A as described (10). RESULTS To test the hypothesis that the intragranular domain of the PAM precursor encodes PAL activity, proteins cross-reactive with antibody to bPAM(561-579), a synthetic peptide from the highly conserved intragranular domain (Fig. l), were monitored by Western blot analysis during purification of PAL activity. In the soluble fraction prepared from frozen bovine neurointermediate pituitaries, two rabbit polyclonal antibodies to bPAM(561-579) detected major proteins of 50&l-kDa (sometimes resolved into a doublet of proteins differing by about l&Da material;
in size) and 30~1 kDa, with lesser amounts of 43- and 36-kDa
inclusion of bPAM(561-579)
appearance
of these signals.
(10 erg/ml) during incubation with antibody blocked
PAM-A
and -B were separated from PAL activity by
adsorption to a D-Tyr-Trp-Gly substrate affinity resin (3) or by chromatography on phenylSepharose (Table 1); the 30-kDa protein remained bound to the phenyl-Sepharose column and was not further studied. PAL activity was further purified by gel filtration and anion exchange chromatography on a MonoQ column (Fig. 2A). A single broad peak of PAL activity was eluted from the MonoQ column. Aliquots of the active fractions were analyzed by SDSPAGE. Upon visualization with antibody to bPAM(561-579), a protein of 50-kDa and a doublet of 43/44-kDa proteins were identified (Fig. 2A, inret), with the total intensity of the signal generated by this antibody roughly correlating with the amount of PAL activity in each fraction; fractions lacking PAL activity also failed to generate a signal with the antibody. The MonoQ column afforded partial resolution of the 50-kDa and 43/44kDa species, and in subsequent purifications, the eluate was divided into an early, higher molecular-weight amino
r 0
4
signal
200
300
400
paired basic residues I
possible
QlyCoSylatiln
700 SiteS
600
900
+CHO I
I
II
I
I,
I I imIll
+ T PAM-0
600
500
Cl-pI
II
pool (Pool 2).
acids 100
‘t
pool (Pool 1) and a late, lower molecular-weight
n
PAM-A bPAM
Introgranular Domain
memb-span
(56 l-579)
Cytoplatmic Domain
m Bovine PAM Precursor. The predicted signal and membrane-spanning domains of the bovine PAM precursor are labeled. Vertical lines denote potential paired basic amino acid cleavage sites; sites yielding NH,-terminal proteins thought to correspond to PAM-A and -B are identified. The synthetic peptideusedto generateantiserais indicated. 928
Vol.
171, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Table 1. Purification of PAL SamDle
(ml)
Protein (rnp)
P4S
12.0
272
kd
Phenyl-Sepharose 73
PAL. activitv nmol/h Recoverv
Fold Purification
3120
100%
1
80.3
6000
192
6
G7.5
12.3
2.3
2220
71
,84
pH 8, MonoQ-1 -2
;::
0.135 0.325
978 1330
31 43
630 360
MonoP-1 -2
1.5 2.0
0.0031 0.0043
389 319
12 10
11,000 6,500
Superose-1 -2
1.93 1.96
---
208 157
7 5
---
pH 6, MonoQ-1 -2
0.95 0.70
0.0011 0.00033
178 84
6 3
14,000 22,000
The 25-45%(NH,),SO, fraction (P4S)from 130frozen bovineneurointermediatepituitaries wasappliedto a phenylSepharosecolumnand elutedwith a gradient. The broad peak of PAL activity from the MonoQ column,pH 8.0, wasdivided into an earlier (Pool 1) and later (Pool 2) eluting fraction; subsequentpurification of the poolswasidentical. When fractions from a similar
chromatofocusing
on a MonoP
preparation
were pooled and further purified by
column (Fig. 2B), a single peak of stimulator-y activity
eluting at pH 4.1 was recovered. Upon Western blot analysis, the amount of material cross-reactive with antibody to bPAM(561-579) was again found to correlate with the amount of PAL activity (Fig. 2B, ilzret). Gel filtration
of this material
revealed a single broad peak of activity with an
apparent molecular weight of approximately
45 kDa.
Western blot analysis revealed that
the Superose column partially resolved the Xl-kDa and 43/44-lcDa proteins.
The overall
intensity of the signal generated by antibody to bPAM(561-579) again correlated with the level of PAL activity, suggesting that both the 50-lcDa and 43/44-kDa proteins were active. Subsequent chromatography PAL activity coincident
on the MonoQ
column at pH 6.0 yielded a peak of
with a peak of UV-absorbing
material
(Fig. 3).
SDS-PAGE
revealed a single 43-kDa protein coincident with the protein visualized by antibody to bPAM(561-579), together with a small amount of the 50-kDa protein (Fig. 3, inset.) The overall yield of PAL, activity was 3 to 6% following a 14,000- to 22,000-fold purification (Table 1). A similar fold purification was required for the isolation of PAM-A and -B (9). DISCUSSION The major proteins detected by antibody to bPAM(561-579) in bovine neurointermediate pituitary extracts have apparent molecular weights of 50 and 30 kDa
Vol. 171, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ti ;
0.5
! 0.0
0.0 0
10
20
30 fraction
40
50
60
6.5 p400 -. ,
c---d
2
6.0
E
5,300 h c 2 t IJ 200
5.5 , 5.0
;i’ LL t
! -0 I
4.5 100 4.0
I 0
3.5
0 10
20
30 fraction
40
50
60
Fie. Ion ExchaneeandCh o atofocus’g A. Fractionsfrom the G75 columncontaining PAL activity were pooled aid:pplied tra MonoQ column at pH 8.0. Flowrate was 1 ml/min and fraction size 1 ml. The solidline indicatesabsorbanceof the effluent at 280 nm (0.2 AU full scale). Assays of PAL activity included 0.1 PM CuSO,; the total activity/fraction is plotted. Inret showsWestern blot of 20 ~1 of fractions 34-38 with antibody to bPAM(561-579). B. Activity from a separatepreparationwasapplied to the MonoQ column, and early fractions containingPAL activity were pooled (Pool 1) and appliedto a MonoP column equilibratedwith the BisTris buffer systemand eluted with a descendingpH gradient, asindicated (0.02AU full scale).Columnparameters,assayand imet asin A.
(based on SDS-PAGE.) Acidic proteins (predicted PI’S of 5.2 to 5.9) of 38 to 49 kDa can be generated by endoproteolytic cleavage of the bovine PAM precursor at R”‘R or K”?K and R”‘K or K”“K (Fig. 1) (11); the 43/44&Da proteins, while enzymatically active, are largely generated by endoproteolytic cleavage occurring during the purification. Upon purification to homogeneity, PAL remains cross-reactive with antibody to bPAhI(561-579) (Fig. 3), but does not cross-react with antibodies directed against purified bovine PAM-A + B (Fig. 1) (data not shown). Thus in the bovine neurointermediate pituitary the PAM precursor undergoes endoproteolytic cleavage to yield two enzymes involved in peptide Qamidation. PAL shares many properties with a 41-kDa protein partially purified from rat brain based upon its ability to increase a-amidation of D-Tyr-Val-Gly at neutral pH (8,12) and 930
Vol. 171, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
0.25
0
10
20
30 fraction
40
50
60
Fig. 3. Final Ion Exchange. Active fractions from a MonoP column run similar to that in Fig. 2 (Pool 1) were applied to a Superose 6 column. Active fractions were applied to the MonoQ column at pH 6.0; the solid line indicates absorbance of the effluent at 280 nm (0.01 AZJ full scale). Column parameters and assaysas in Fig. 2. Inset: Aliquots containing 10 mnol/h purified PAL activity (fractions 32-34) were transferred to Immobilon P and visualized with AuroDye Forte (Janssen Life Sciences Products, Flanders, NJ) (left) or antibody to bPAM(561-579) (right). Molecular weight markers in kDa are labeled. with the 45kDa
a-hydroxyglycine
bovine neurointermediate a-amidation
amidating
dealkylase (HGAD)
partially
purified from
pituitary (7). The discrepant reports of pH optima for peptide
likely reflect the peptide substrate used, the tissue selected and the degree of
purity achieved. It is now clear that the production
of a-amidated
peptides from their glycine
extended precursors is a 2-step process (4-8): --- > Peptidyl-a-amide Peptidylglycine --- > Peptidyl-a-hydroxyglycine Reaction I Reaction II (spontaneous at high pH) (copper, 0,, ascorbate) The overall reaction is that of a peptidylglycine
a-amidating
monooxygenase,
proposed that the intact precursor continue to be referred to as PAM. is a peptidylglycine
a-hydroxylating
a-hydroxyglycine a-amidating
monooxygenase (PHM)
lyase (PAL).
+ Glyoxylate
and it is
The first enzyme
and the second is a peptidyl-
The soluble 75kDa enzyme purified from MTC
cells appears to retain both activities (4). In contrast, in tissues like the anterior and neurointermediate pituitary and in AtT-20 cells transfected with cDNA encoding bovine PAM, endoproteolytic
cleavage separates the two activities, yielding PHM and PAL, both
enzymes are found in the secretory granules (3). Although PHM and PAL can clearly function sequentially in vitro (7,8), how the two enzymes function in vivo and the stability of the peptidyl-a-hydroxyglycine intermediates within the acidic environment
of the secretory granule are unknown.
AtT-20 corticotrope
tumor cells transfected with cDNAs encoding the full-length bovine PAM precursor or a 38-kDa PHM (PAM-B) exhibit increased a-amidation of endogenous peptide products’; this ‘Mains, R.E., and Eipper, B.A., unpublished
observation. 931
Vol. 171, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
suggests that endogenous PAL is present in amounts sufficient to catalyze the conversion of any additional peptidyla-hydroxyglycine products generated by the exogenous PHM. Among the known multifunctional enzymes, peroxisomal enoyl-CoA hydratase:3hydroxy-CoA dehydrogenase is similar to PAM in that it contains 2 enyzymatic activities that sequentially catalytically
catalyze a Zstep reaction; partial digestion with elastase generates two
active fragments (13). Whether the intact PAM molecule is likewise capable
of catalyzing both steps of the amidation
reaction is under investigation.
ACKNOWLEDGMENTS We would like to thank Dr. AS. Mildvan for help with enzyme nomenclature
and Dr. R.E.
Mains for helpful discussions. REFERENCES 1. Eipper, B.A., and Mains, R.E. (1988) Ann. Rev. Physiol. 50, 333-344. 2. Bradbury, A.F., and Smyth, D.G. (1987) Biosci. Rep. 7, 907-916. 3. Perkins, S.N., Husten, E.J., Mains, R.E., and Eipper, B.A. (1990) Endocrinology 127, in press. 4. Young, S.D., and Tamburini, P.P. (1989) J. Am. Chem. Sot. 111, 1933-1934. 5. Bradbury, AF., and Smyth, D.G. (1987) Eur. J. Biochem. 169, 579-584. 6. Tajima M., Iida, T., Yoshida, S., Komatsu, K., Namba, R., Yanagi, M., Noguchi, M., and Okamoto, H. (1990) J. Biol. Chem. 265, 9602-9605. 7. Katapodis, A.G., Ping, D., and May, S.W. (1990) Biochemistry 29, 6115-6120. 8. Takahashi, K., Okamoto, H., Seino, H., and Noguchi, M. (1990) Biochem. Biophys. Res. Comm. 169, 524-530. 9. Murthy, A.S.N, Mains, R.E., and Eipper, B.A. (1986) J. Biol. Chem. 261, 1815-1822. 10. May, V., Ouafik, L’H., Eipper, B.A., and Braas, KM. (1990) Endocrinology 127,358364. 11. Eipper, B.A., Park, L.P, Dickerson, I.M., Keutmarm, H.T., Thiele, E.A., Rodriguez, H., Schofield, P.R., and Mains, R.E. (1987) Mol. Endocrinol. 1, 777-790. 12. Noguchi, M., Takahashi, K., and Okamoto, H. (1989) Arch. Biochem. Biophys. 275,505513. 13. Minami-ishii, N., Taketani, S., Osumi, T., and Hashimoto, T. (1989) Eur. J. Biochem. 185, 73-78.
171, No.
September
3, 1990 28,
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
1990
926-932
THE 108~kDA PEPTIDYLGLYCINE a-AMIDATING MONOOXYGENASE PRECURSOR CONTAINS TWO SEPARABLE ENZYMATIC ACTIVITIES INVOLVED
IN PEPTIDE
AMIDATION
Susan N. Perkins, E. Jean Husten and Betty A. Eipper Department
of Neuroscience, Johns Hopkins University Baltimore,
Received
August
10,
School of Medicine,
MD 21205
1990
SUMMARY: A 43-kDa protein factor that increases the ability of purified bovine peptidylglycine a-amidating monooxygenase (PAM)-A and -B to produce a-amidated peptides at physiological pH was purified to homogeneity from bovine neurointermediate pituitary. At each step of the purification, the amount of activity correlated with the amount of protein detected on Western blots by antibody to bovine PAM(561-579). In the bovine neurointermediate pituitary the lO&kDa PAM precursor protein is cleaved to form a peptidylglycine a-hydroxylating monooxygenase and a peptidyl-cr-hydroxyglycine Qamidating lyase, which function sequentially in the 2-step formation of or-amidated peptides. @ 1990
Academic
Press,
Inc.
Peptidylglycine
a-amidating
monooxygenase (PAM; EC 1.14.17.3) is responsible for
the conversion of peptides with a COOH-terminal a posttranslational
modification
glycine into a-amidated product peptides,
often required for biological activity (1,2). In the course of
investigating a protein factor that increases the ability of purified bovine PAM-A and -B (Fig. 1) to produce a-amidated peptides from their glycine-extended precursors at the acidic pH values characteristic of secretory granules, it became apparent that levels of stimulatory activity were closely correlated with levels of PAM activity (3). Although extracts of AtT20 cells transfected with cDNAs encoding either the entire 108~kDa bovine PAM precursor or a 38-kDa protein corresponding to PAM-B (Fig. 1) exhibited elevated levels of aamidation activity in test tube assays, only extracts of cells expressing the 108~kDa PAM precursor protein exhibited increased levels of stimulatory
activity. These results suggested
The abbreviations used are: PAM, peptidylglycine c*-amidating monooxygenase; PHM, peptidylglycine a-hydroxylating monooqgenase; PAL, peptidyls-hydroxyglycine a-amidating lyase; FPLC, fast protein liquid chromatography; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis. 0006-291X/90 Copyright All rights
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
926
Vol.
171, ,No. 3, 1990
BIOCHEMICAL
that a part of the remaining approximately
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
70 kDa of the PAM
45kDa stimulatory
precursor might
constitute
the
activity. We used antisera to a synthetic peptide derived
from the region of the intragranular domain of the bovine PAM precursor not included in PAM-A or -B (Fig. 1) to demonstrate that a protein derived from this region of the PAM precursor is indeed responsible for this stimulatory
activity.
Moreover,
we purified
an
active form of this factor more than 20,000-fold to homogeneity. The production of a-amidated peptides from their glycine-extended precursors is a 2-step process proceeding through peptidyl-a-hydroxyglycine (4-S). The enzyme catalyzing the first step requires copper, molecular oxygen and ascorbate (1,2,4,6) and is a peptidylglycine a-hydroxylating monooxygenase (PHM). Although conversion of peptidylcr-hydroxyglycine to a-amidated product occurs spontaneously at alkaline pH (6,7), an enzyme activity catalyzing this step at acidic-to-neutral pH values has recently been identified (7,8). The stimulatory activity that we have studied (3) has all of the properties expected of the enzyme catalyzing the second step of this reaction; this enzyme is a peptidylhydroxyglycine
a-amidating
lyase (PAL).
MATERIALS
AND METHODS
Assays. PAL activity was assayed by adding 0.2 ~1 purified bPAM-A+B (which catalyze the first step of the reaction but fail to produce a-amidated product at acidic pH) to samples and measuring production of a-amidated product (3); standard assay conditions included 0.5 PM a-N-acetyl-Tyr-Val-Gly, 20,000 cpm [““I]-a-N-acetyl-Tyr-Val-Gly, 175 pg/ml catalase, 0.1 or 1 PM CuSO, (optimal concentration determined at each step), 0.5 mM ascorbate, and loo-150 mM NaMES, pH 6.0. Protein was assayed with the BCA reagent (Pierce Chemical Co., Rockford, IL) or with Integrated Separation Systems Protein-Gold reagent (Hyde Park, MA). Purification of PAL. The resuspended 25-45% (NH,),SO, fraction prepared from 130 frozen bovine neurointermediate pituitaries (9) was thawed and applied to a 45-ml column of phenylSepharose (Pharmacia, Piscataway, NJ) equilibrated with 10 mM NaTES, pH 7.5, 1.0 M (NH&SO.,; proteins were eluted with 500 ml of the same buffer containing 0.7, 0.3 or 0 M (NH&SO, or with a linear gradient to 0 M (NH,),SO,. Fractions containing PAL activity were pooled, concentrated 20-fold with an Amicon (Danvers, MA) ultrafiltration cell using a PM-10 membrane and applied to a 2 x 55 cm column of Sephadex G75 equilibrated and eluted with 20 mM TrisHCl, pH 8.0,40 mM NaCl. Subsequent steps in the purification were carried out by FPLC (Pharmacia). Fractions containing stimulatory activity were pooled and applied directly to a MonoQ HR S/5 column equilibrated with the same buffer; proteins were eluted with a gradient to 1.0 M NaCl. Peaks of activity were pooled and applied to a MonoP HR 5/20 column equilibrated with either 25 mM BisTrisHCl, pH 6.0, or 25 mM 1-methylpiperazine (Fluka, Ronkonkoma, NY) HCI, pH 5.7, and eluted by chromatofocusing with Polybuffer 74 (Pharmacia) diluted lo-fold and titrated to pH 3.94 with HCl. Fractions were neutralized by adding 50 ~1 1 M NaTES, pH 7.0, per 1.0 ml fraction and stored in the presence of 0.02% Lubrol-PX (Pierce). Pooled fractions were concentrated with a Centricon 10 microconcentrator (Amicon) and applied to a Superose 6 HR lo/30 column equilibrated and eluted with 0.2 M NH,HCO, containing 0.02% Lubrol-PX. Pooled fractions were applied to the MonoQ column equilibrated with 25 mM BisTrisHCl, pH 6.0, containing 0.02% Lubrol-PX, and eluted with a gradient to 0.5 M NaCl in the same buffer. 927
Vol.
171, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Western blots Samples were fractionated on slab gels containing 10% polyacrylamide, 0.27% N,K-methylenebisacrylamide, transferred to Immobilon P membranes (Millipore, Bedford, MA) and visualized with affinity-purified antibody to bPAM(561-579) (Ab68 or 69) (10) or purified bPAM-A+B (Ab36) (11) and [‘*‘I]-Protein A as described (10). RESULTS To test the hypothesis that the intragranular domain of the PAM precursor encodes PAL activity, proteins cross-reactive with antibody to bPAM(561-579), a synthetic peptide from the highly conserved intragranular domain (Fig. l), were monitored by Western blot analysis during purification of PAL activity. In the soluble fraction prepared from frozen bovine neurointermediate pituitaries, two rabbit polyclonal antibodies to bPAM(561-579) detected major proteins of 50&l-kDa (sometimes resolved into a doublet of proteins differing by about l&Da material;
in size) and 30~1 kDa, with lesser amounts of 43- and 36-kDa
inclusion of bPAM(561-579)
appearance
of these signals.
(10 erg/ml) during incubation with antibody blocked
PAM-A
and -B were separated from PAL activity by
adsorption to a D-Tyr-Trp-Gly substrate affinity resin (3) or by chromatography on phenylSepharose (Table 1); the 30-kDa protein remained bound to the phenyl-Sepharose column and was not further studied. PAL activity was further purified by gel filtration and anion exchange chromatography on a MonoQ column (Fig. 2A). A single broad peak of PAL activity was eluted from the MonoQ column. Aliquots of the active fractions were analyzed by SDSPAGE. Upon visualization with antibody to bPAM(561-579), a protein of 50-kDa and a doublet of 43/44-kDa proteins were identified (Fig. 2A, inret), with the total intensity of the signal generated by this antibody roughly correlating with the amount of PAL activity in each fraction; fractions lacking PAL activity also failed to generate a signal with the antibody. The MonoQ column afforded partial resolution of the 50-kDa and 43/44kDa species, and in subsequent purifications, the eluate was divided into an early, higher molecular-weight amino
r 0
4
signal
200
300
400
paired basic residues I
possible
QlyCoSylatiln
700 SiteS
600
900
+CHO I
I
II
I
I,
I I imIll
+ T PAM-0
600
500
Cl-pI
II
pool (Pool 2).
acids 100
‘t
pool (Pool 1) and a late, lower molecular-weight
n
PAM-A bPAM
Introgranular Domain
memb-span
(56 l-579)
Cytoplatmic Domain
m Bovine PAM Precursor. The predicted signal and membrane-spanning domains of the bovine PAM precursor are labeled. Vertical lines denote potential paired basic amino acid cleavage sites; sites yielding NH,-terminal proteins thought to correspond to PAM-A and -B are identified. The synthetic peptideusedto generateantiserais indicated. 928
Vol.
171, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Table 1. Purification of PAL SamDle
(ml)
Protein (rnp)
P4S
12.0
272
kd
Phenyl-Sepharose 73
PAL. activitv nmol/h Recoverv
Fold Purification
3120
100%
1
80.3
6000
192
6
G7.5
12.3
2.3
2220
71
,84
pH 8, MonoQ-1 -2
;::
0.135 0.325
978 1330
31 43
630 360
MonoP-1 -2
1.5 2.0
0.0031 0.0043
389 319
12 10
11,000 6,500
Superose-1 -2
1.93 1.96
---
208 157
7 5
---
pH 6, MonoQ-1 -2
0.95 0.70
0.0011 0.00033
178 84
6 3
14,000 22,000
The 25-45%(NH,),SO, fraction (P4S)from 130frozen bovineneurointermediatepituitaries wasappliedto a phenylSepharosecolumnand elutedwith a gradient. The broad peak of PAL activity from the MonoQ column,pH 8.0, wasdivided into an earlier (Pool 1) and later (Pool 2) eluting fraction; subsequentpurification of the poolswasidentical. When fractions from a similar
chromatofocusing
on a MonoP
preparation
were pooled and further purified by
column (Fig. 2B), a single peak of stimulator-y activity
eluting at pH 4.1 was recovered. Upon Western blot analysis, the amount of material cross-reactive with antibody to bPAM(561-579) was again found to correlate with the amount of PAL activity (Fig. 2B, ilzret). Gel filtration
of this material
revealed a single broad peak of activity with an
apparent molecular weight of approximately
45 kDa.
Western blot analysis revealed that
the Superose column partially resolved the Xl-kDa and 43/44-lcDa proteins.
The overall
intensity of the signal generated by antibody to bPAM(561-579) again correlated with the level of PAL activity, suggesting that both the 50-lcDa and 43/44-kDa proteins were active. Subsequent chromatography PAL activity coincident
on the MonoQ
column at pH 6.0 yielded a peak of
with a peak of UV-absorbing
material
(Fig. 3).
SDS-PAGE
revealed a single 43-kDa protein coincident with the protein visualized by antibody to bPAM(561-579), together with a small amount of the 50-kDa protein (Fig. 3, inset.) The overall yield of PAL, activity was 3 to 6% following a 14,000- to 22,000-fold purification (Table 1). A similar fold purification was required for the isolation of PAM-A and -B (9). DISCUSSION The major proteins detected by antibody to bPAM(561-579) in bovine neurointermediate pituitary extracts have apparent molecular weights of 50 and 30 kDa
Vol. 171, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ti ;
0.5
! 0.0
0.0 0
10
20
30 fraction
40
50
60
6.5 p400 -. ,
c---d
2
6.0
E
5,300 h c 2 t IJ 200
5.5 , 5.0
;i’ LL t
! -0 I
4.5 100 4.0
I 0
3.5
0 10
20
30 fraction
40
50
60
Fie. Ion ExchaneeandCh o atofocus’g A. Fractionsfrom the G75 columncontaining PAL activity were pooled aid:pplied tra MonoQ column at pH 8.0. Flowrate was 1 ml/min and fraction size 1 ml. The solidline indicatesabsorbanceof the effluent at 280 nm (0.2 AU full scale). Assays of PAL activity included 0.1 PM CuSO,; the total activity/fraction is plotted. Inret showsWestern blot of 20 ~1 of fractions 34-38 with antibody to bPAM(561-579). B. Activity from a separatepreparationwasapplied to the MonoQ column, and early fractions containingPAL activity were pooled (Pool 1) and appliedto a MonoP column equilibratedwith the BisTris buffer systemand eluted with a descendingpH gradient, asindicated (0.02AU full scale).Columnparameters,assayand imet asin A.
(based on SDS-PAGE.) Acidic proteins (predicted PI’S of 5.2 to 5.9) of 38 to 49 kDa can be generated by endoproteolytic cleavage of the bovine PAM precursor at R”‘R or K”?K and R”‘K or K”“K (Fig. 1) (11); the 43/44&Da proteins, while enzymatically active, are largely generated by endoproteolytic cleavage occurring during the purification. Upon purification to homogeneity, PAL remains cross-reactive with antibody to bPAhI(561-579) (Fig. 3), but does not cross-react with antibodies directed against purified bovine PAM-A + B (Fig. 1) (data not shown). Thus in the bovine neurointermediate pituitary the PAM precursor undergoes endoproteolytic cleavage to yield two enzymes involved in peptide Qamidation. PAL shares many properties with a 41-kDa protein partially purified from rat brain based upon its ability to increase a-amidation of D-Tyr-Val-Gly at neutral pH (8,12) and 930
Vol. 171, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
0.25
0
10
20
30 fraction
40
50
60
Fig. 3. Final Ion Exchange. Active fractions from a MonoP column run similar to that in Fig. 2 (Pool 1) were applied to a Superose 6 column. Active fractions were applied to the MonoQ column at pH 6.0; the solid line indicates absorbance of the effluent at 280 nm (0.01 AZJ full scale). Column parameters and assaysas in Fig. 2. Inset: Aliquots containing 10 mnol/h purified PAL activity (fractions 32-34) were transferred to Immobilon P and visualized with AuroDye Forte (Janssen Life Sciences Products, Flanders, NJ) (left) or antibody to bPAM(561-579) (right). Molecular weight markers in kDa are labeled. with the 45kDa
a-hydroxyglycine
bovine neurointermediate a-amidation
amidating
dealkylase (HGAD)
partially
purified from
pituitary (7). The discrepant reports of pH optima for peptide
likely reflect the peptide substrate used, the tissue selected and the degree of
purity achieved. It is now clear that the production
of a-amidated
peptides from their glycine
extended precursors is a 2-step process (4-8): --- > Peptidyl-a-amide Peptidylglycine --- > Peptidyl-a-hydroxyglycine Reaction I Reaction II (spontaneous at high pH) (copper, 0,, ascorbate) The overall reaction is that of a peptidylglycine
a-amidating
monooxygenase,
proposed that the intact precursor continue to be referred to as PAM. is a peptidylglycine
a-hydroxylating
a-hydroxyglycine a-amidating
monooxygenase (PHM)
lyase (PAL).
+ Glyoxylate
and it is
The first enzyme
and the second is a peptidyl-
The soluble 75kDa enzyme purified from MTC
cells appears to retain both activities (4). In contrast, in tissues like the anterior and neurointermediate pituitary and in AtT-20 cells transfected with cDNA encoding bovine PAM, endoproteolytic
cleavage separates the two activities, yielding PHM and PAL, both
enzymes are found in the secretory granules (3). Although PHM and PAL can clearly function sequentially in vitro (7,8), how the two enzymes function in vivo and the stability of the peptidyl-a-hydroxyglycine intermediates within the acidic environment
of the secretory granule are unknown.
AtT-20 corticotrope
tumor cells transfected with cDNAs encoding the full-length bovine PAM precursor or a 38-kDa PHM (PAM-B) exhibit increased a-amidation of endogenous peptide products’; this ‘Mains, R.E., and Eipper, B.A., unpublished
observation. 931
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BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
suggests that endogenous PAL is present in amounts sufficient to catalyze the conversion of any additional peptidyla-hydroxyglycine products generated by the exogenous PHM. Among the known multifunctional enzymes, peroxisomal enoyl-CoA hydratase:3hydroxy-CoA dehydrogenase is similar to PAM in that it contains 2 enyzymatic activities that sequentially catalytically
catalyze a Zstep reaction; partial digestion with elastase generates two
active fragments (13). Whether the intact PAM molecule is likewise capable
of catalyzing both steps of the amidation
reaction is under investigation.
ACKNOWLEDGMENTS We would like to thank Dr. AS. Mildvan for help with enzyme nomenclature
and Dr. R.E.
Mains for helpful discussions. REFERENCES 1. Eipper, B.A., and Mains, R.E. (1988) Ann. Rev. Physiol. 50, 333-344. 2. Bradbury, A.F., and Smyth, D.G. (1987) Biosci. Rep. 7, 907-916. 3. Perkins, S.N., Husten, E.J., Mains, R.E., and Eipper, B.A. (1990) Endocrinology 127, in press. 4. Young, S.D., and Tamburini, P.P. (1989) J. Am. Chem. Sot. 111, 1933-1934. 5. Bradbury, AF., and Smyth, D.G. (1987) Eur. J. Biochem. 169, 579-584. 6. Tajima M., Iida, T., Yoshida, S., Komatsu, K., Namba, R., Yanagi, M., Noguchi, M., and Okamoto, H. (1990) J. Biol. Chem. 265, 9602-9605. 7. Katapodis, A.G., Ping, D., and May, S.W. (1990) Biochemistry 29, 6115-6120. 8. Takahashi, K., Okamoto, H., Seino, H., and Noguchi, M. (1990) Biochem. Biophys. Res. Comm. 169, 524-530. 9. Murthy, A.S.N, Mains, R.E., and Eipper, B.A. (1986) J. Biol. Chem. 261, 1815-1822. 10. May, V., Ouafik, L’H., Eipper, B.A., and Braas, KM. (1990) Endocrinology 127,358364. 11. Eipper, B.A., Park, L.P, Dickerson, I.M., Keutmarm, H.T., Thiele, E.A., Rodriguez, H., Schofield, P.R., and Mains, R.E. (1987) Mol. Endocrinol. 1, 777-790. 12. Noguchi, M., Takahashi, K., and Okamoto, H. (1989) Arch. Biochem. Biophys. 275,505513. 13. Minami-ishii, N., Taketani, S., Osumi, T., and Hashimoto, T. (1989) Eur. J. Biochem. 185, 73-78.