From the Departament de Bioquímica i Biologia Molecular, Unitat de Ciències and the Institut de Biologia Fonamental, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain
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ABSTRACT |
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The cDNA of human procarboxypeptidase
A2 has been overexpressed in the methylotrophic yeast
Pichia pastoris and secreted into the culture medium by
means of the -mating factor signal sequence, yielding a major
protein of identical size and N-terminal sequence as the wild-type
form. Two other forms containing the proenzyme have also been
overexpressed: one of them resulted from an incomplete processing of
the signal peptide, whereas the other was a glycosylated derivative.
Recombinant procarboxypeptidase A2 was purified to homogeneity, and it
was shown that its mature active form displays functional properties
similar to those of the enzyme directly isolated from human pancreas.
The overall yield was ~250 mg of proenzyme or 180 mg of mature
enzyme/liter of cell culture. The proteolysis-promoted activation
process of the recombinant proenzyme has been studied in detail. During
maturation by trypsin, the increase in activity of the enzyme is a
rapid and monotonic event, which reflects the rate of the proteolytic
release of the inhibitory pro-segment and the weaker nature of its
interactions with the enzyme moiety compared with procarboxypeptidases
of the A1 type. Three main forms of the pro-segment (96, 94, and 92 amino acids), with no inhibitory capability in the severed state, and a
single mature carboxypeptidase A2 are produced during this process. No further proteolysis of these pro-segments by the generated
carboxypeptidase A2 occurs, in contrast with observations made in other
procarboxypeptidases (A1 and B). This differential behavior is a result
of the extreme specificity of carboxypeptidase A2.
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INTRODUCTION |
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Pancreatic carboxypeptidases (CPs)1 are digestive metalloenzymes involved in the hydrolysis of alimentary proteins and peptides from their C-terminal end. Their participation as proenzymes in the digestive cascade (promoted by limited proteolysis) is a well documented process (1-3). Also, their specificity classification between the A forms (CPA, with preference for apolar C-terminal residues) and the B forms (CPB, with preference for basic C-terminal residues) and the tertiary structures of both forms are well known (4). In recent years, there has been an increasing interest in the study of the synthesis, storage, activation, and three-dimensional structure of procarboxypeptidases (pro-CPs), the precursors of such proenzymes (4-6).
The classification of metallocarboxypeptidases has been widened in the last few years with reports about new non-digestive pancreatic-like carboxypeptidases in different extra-pancreatic tissues and biological fluids, with the same evolutionary ancestors as pancreatic carboxypeptidases (4, 7-10). Additionally, the traditional classification of pancreatic carboxypeptidases and their zymogens into the A and B forms has been expanded with the identification of the A1 and A2 isoforms in rat and humans (11, 12). CPA1 and CPA2 differ in specificity for peptide substrates: the former (assignable to the traditional A form) shows a wider preference for aliphatic and aromatic residues, whereas the latter is more restrictive for aromatic residues; this reflects significant differences in the specificity pocket of the enzymes (13). Recently, we have reported the cloning and sequence analysis of the human pro-CPA2 cDNA as well as its three-dimensional modeling (14). CPA2 isoforms have also been reported in rat extra-pancreatic tissues such as brain, testis, and lung (15); these CPA2 isoforms are shorter and have a distinct role from the pancreatic isoform. The high sequence identity found between human pro-CPA2 and rat pro-CPA2 (89% homology) as compared with human pro-CPA1 (64% homology) corroborates the proposal that locates the appearance of the two isoforms by gene duplication before speciation of mammals (11).
Comparison of the prodomain structures in the family of pancreatic proenzymes shows close similarities in conformation between the A1 and A2 forms in regions assumed to be critical for their inhibition and proteolytic activation (6, 14) and significant differences from the corresponding regions in the B form (5). Accordingly, the A2 proenzyme could be expected to show a bimodal and slow proteolytic activation behavior, as previously reported for the A1 form (16), and to differ from the monotonic and quick activation behavior found for the B proenzyme (17). However, earlier proteolytic activation experiments carried out on natural pro-CPA2 isolated from human or rat pancreas (12, 18) do not fit with these expectations and assumptions. Therefore, this is an issue that requires clarification.
In this work, pro-CPA2 has been overexpressed in Pichia pastoris to produce the protein in quantities amenable to the study of the structural and functional determinants of its behavior and activation. The methylotrophic yeast P. pastoris was chosen because of its high yield and capacity of secreting heterologous proteins when linked to the appropriate secretion signal (19). The development of the system reported here to obtain large quantities of fully activable human pro-CPA2 should facilitate not only the characterization of this form, but probably also that of other structurally related forms to which the same procedure could be applied. It could also facilitate its potential biotechnological use, such as the large-scale production of carboxypeptidases able to act as activators of antitumoral prodrugs (20, 21). The efficient expression of human pro-CPA2 in P. pastoris has allowed us to investigate the different events in the proteolytic activation and processing of this proenzyme in detail and to compare them with the processes described in other pancreatic procarboxypeptidases. An overall maturation scheme of such zymogens emerges from this study.
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EXPERIMENTAL PROCEDURES |
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Materials--
Restriction endonucleases, T4 DNA ligase,
Vent polymerase, deoxynucleotide stocks, and
N-glycosidase F were purchased from Boehringer Mannheim.
Salts and media for Escherichia coli and P. pastoris growth were purchased from Difco. The P. pastoris expression kit was purchased from Invitrogen. Trypsin
(treated with tosylphenylalanyl chloromethyl ketone) was from
Worthington. Trifluoroacetic acid,
N-p-tosyl-L-lysine
chloromethyl ketone (TLCK), and
N-(3-(2-furyl)acryloyl)-L-phenylalanyl-L-phenylalanine (FAPP) were from Sigma. Electrophoretic studies were carried out in a
Bio-Rad Mini-Protean system. HPLC studies were carried out in a Waters
chromatograph.
Plasmid Constructs--
DNA manipulations were carried out
essentially as described by Sambrook et al. (22) using
E. coli strains JM83 and MC1061 as hosts. The cDNA of
human pro-CPA2 was amplified by polymerase chain reaction
from a pUC9 vector to introduce an XhoI site at the 5-end
and an EcoRI site at the 3
-end of the cDNA using the following primers: sense primer,
5
-GTATCTCTCGAGAAAAGACTAGAAACATTTGTGGGAGA-3
; and antisense
primer, 5
-CTAGAATTCATGGCTCTTGTTTCTTCC-3
. After restriction enzyme digestion of the polymerase chain reaction product,
the cDNA was cloned and subcloned into M13 pBluescript to confirm
the entire sequence and the changes made in the polymerase chain
reaction. pBluescript-pro-CPA2 was digested by
XhoI and EcoRI, and the cDNA was ligated to
the P. pastoris shuttle vector pPIC9 between the 5
-promoter
and the 3
-terminator of the alcohol oxidase gene (AOX1).
pPIC9 provides the
-mating factor signal for secretion and the
HIS4 gene for selection of the recombinant yeast clones.
pHIL-D2 was also used for the expression and secretion of
pro-CPA2 using its own signal sequence. In the latter case, the original pUC9-pro-CPA2 clone was digested and ligated
to the P. pastoris pHIL-D2 shuttle vector using the
EcoRI site of the polylinker.
Transformation and Selection of the Productive Clones-- After linearization of the corresponding P. pastoris pPIC9-pro-CPA2 and pHIL-D2-prepro-CPA2 vectors with BglII and NotI, respectively, the P. pastoris GS115 (his4) strain was transformed either by electroporation or by the spheroplast method. After simultaneously plating the transformants in MM and MD agar (1.34% yeast nitrogen base, 0.00004% biotin, and 0.5% methanol or 1% dextrose, respectively), those clones that suffered homologous recombination with the AOX1 sequence (slow growing in MM agar) were selected. To test for the most productive clones, colonies were grown in 10 ml of buffered liquid BMGY medium (1% yeast extract, 2% peptone, 90 mM potassium phosphate, pH 6.0, 1.34% yeast nitrogen base, 0.00004% biotin, and 1% glycerol) at 30 °C for 4 days. Cells were collected by centrifugation and gently resuspended in 2 ml of buffered liquid BMMY medium (same as BMGY medium but containing 0.5% methanol instead of 1% glycerol) and cultured for another 2 days to induce the expression of the recombinant protein. The production of the clones was monitored after 6, 24, and 48 h by electrophoretic analysis of the supernatant on SDS-12% polyacrylamide gels. Western blotting was carried out as described previously using 1:500 anti-human pancreatic procarboxypeptidase antiserum (14). The functionality of the expressed protein was analyzed with the synthetic substrate FAPP after activation of the proenzyme with trypsin (at a 40:1 ratio by weight).
Expression and Purification of Recombinant Human Pro-CPA2-- 1-Liter shake-flask cultures were grown at 30 °C for 4 days in buffered BMGY medium (20-30 A units at 600 nm; 20 g of cells (dry weight)/liter). Cells were collected by centrifugation at 1500 g, gently resuspended in 200 ml of BMMY medium, and cultured for another 2 days to induce the production of pro-CPA2. The culture medium was separated from the cells by centrifugation, and after equilibrating its ionic strength, it was processed through a two-step chromatographic purification: hydrophobic interaction chromatography on a butyl-Toyopearl 650M column eluted with a decreasing gradient of ammonium sulfate and fast protein liquid chromatography on a preparative anion-exchange column (TSK-DEAE 5PW) eluted with a gradient of ammonium acetate in 30 mM MES, pH 5.7. The elution of recombinant pro-CPA2 was observed at 9% ammonium sulfate and 6 mM ammonium acetate, respectively. The identity of recombinant pro-CPA2 was confirmed after automated Edman degradation analysis of its N-terminal sequence and MALDI-TOF spectrometry analysis.
Activation Studies of Recombinant Human
Pro-CPA2--
Recombinant human pro-CPA2 at 1 mg/ml in 50 mM Tris-HCl and 1 µM
ZnCl2, pH 8.0, was treated with trypsin at 40:1 and 400:1 (w/w) ratios at 0 °C. At given times after trypsin addition,
aliquots were removed for activity measurements, for electrophoretic
reverse-phase HPLC and mass spectrometry analyses, and for
quantification of the released amino acids. For activity measurements,
10 µl of the activation mixture were added to 190 µl of aprotinin
(bovine pancreas trypsin inhibitor) at 0.1 mg/ml in 20 mM
Tris and 0.1 M NaCl, pH 7.5, and 10 µl of this new
mixture were used to carry out spectrophotometric activity measurements
with the FAPP substrate at 330 nm. For electrophoretic analysis, 20 µl of the activation mixture were mixed with 2 µl of 22 mM TLCK in water to reach a final trypsin inhibitor
concentration of 2 mM. Each sample was immediately mixed
with electrophoretic loading buffer containing 1% SDS and 3%
-mercaptoethanol, heated at 90 °C for 1 min, and stored at
20 °C until analysis. Electrophoresis was carried out on
Tricine-polyacrylamide gels (23). For HPLC and mass spectrometry analyses, 90-µl samples were removed from the activation mixture, made 0.5% in trifluoroacetic acid to inhibit proteolysis, and immediately chromatographed or kept at
20 °C for subsequent
analysis. For quantitation of the amino acids released into the
activation mixture, 90-µl samples were taken (2 nmol of initial
pro-CPA2) and mixed with trifluoroacetic acid up to a final
concentration of 0.5%. 1.5 nmol of norleucine were added as a
quantitative reference before the addition of 3 volumes of ethanol to
precipitate proteins and large peptides. The supernatant was
lyophilized and analyzed for amino acid composition.
Chromatographic Analysis by Reverse-phase HPLC-- Samples removed from the activation mixtures were analyzed by reverse-phase HPLC on Vydac C4 supports. A 214TP54 column (250 × 4.6 mm, 5-µm particle size, 0.3 µm-pore) was used, and elution was followed at 214 nm. Chromatographies were performed in 0.1% trifluoroacetic acid with an eluting linear gradient between water (solvent A) and acetonitrile (solvent B) according to the following steps: 10% solvent B from 0 to 10 min, 10-32% solvent B from 10 to 30 min, and 32-52% solvent B from 30 to 130 min.
Mass Spectrometry-- The activation mixtures were analyzed by mass spectrometry with a MALDI-TOF spectrometer (Biflex with Reflectron, Bruker). 50 pmol of each sample in 1 µl were mixed with 1 µl of 50% synapinic acid as a matrix and loaded.
Amino Acid Analysis--
Amino acid analyses were carried out by
the 4-dimethylaminoazobenzene-4-sulfonyl derivatization method (24)
using materials and protocols from Beckman. A reverse-phase HPLC
NovaPak C18 column was used to separate the amino acids and
oligopeptides produced during the activation.
N-terminal Sequence Analysis-- After analysis by SDS-PAGE or analysis and purification by HPLC, N-terminal sequence analysis of pro-CPA2 and the activation products was performed by blotting the samples on polyvinylidene difluoride membranes, followed by direct analysis on a Beckman LF3000 Protein Sequencer.
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RESULTS |
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Construction of the Expression Vector and Transformation into P. pastoris--
A 1254-base pair cDNA encoding human
preprocarboxypeptidase A2 has been cloned from a pancreatic library by
immunological and radioactive approaches (14). This cDNA codes for
a short leader signal peptide of 16 amino acids, a pro-segment of 96 amino acids, and an active enzyme of 305 amino acids (see below). In this work, this cDNA was modified by polymerase chain reaction to
add an XhoI site at the 5-end and an EcoRI site
at the 3
-end to amplify only the proenzyme. The complete human
prepro-CPA2 cDNA was also cloned into the
EcoRI site of the P. pastoris pHIL-D2 expression
vector to test whether the native signal sequence of pro-CPA2 could release the proenzyme into the extracellular
medium.
Overexpression and Purification of Recombinant Human
Pro-CPA2--
The clones transformed with the
-MF-pro-CPA2 fusion product secreted a dominant 45-kDa
protein in the P. pastoris supernatant upon induction by
methanol. Those transformed with pHIL-D2-prepro-CPA2 did
not secrete any protein into the medium. The 45-kDa protein was found
to correspond to human pro-CPA2 by Western blot analysis. Upon induction with 0.5% methanol, the Mut
phenotypes
expressed more protein than the Mut+ phenotypes. However, a
3-fold increase in protein secretion was observed in the
Mut+ phenotypes when induction was assayed with 5%
methanol (data not shown). One of these high productivity clones was
selected for large-scale production of human pro-CPA2.
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Activation of Recombinant Human Pro-CPA2 with Different Proteases-- Analysis of the action of trypsin, chymotrypsin A, and elastase (the major active endoproteolytic counterparts in pancreatic secretion) upon correctly processed recombinant pro-CPA2 indicates that trypsin is, by far, the most efficient activator (data not shown). At the pro-CPA2/endoprotease ratio used, 90% activation of the former was achieved by trypsin in ~10 min, whereas the same level of activation was only achieved after 125 min of treatment with elastase; in the latter time span, chymotrypsin was able to generate only 10% of active CPA2. This is in agreement with previous studies that reported trypsin as the main enzyme responsible for the activation of pancreatic procarboxypeptidases (3).
Trypsin Activation of Recombinant Human Pro-CPA2-- The action of trypsin at 37 °C on recombinant pro-CPA2 at a 40:1 (w/w) pro-CPA2/trypsin ratio is excessively quick for detailed mechanistic analysis, in agreement with previous studies on natural human proenzymes (12). The study of the activation process was therefore carried out at 0 °C and at a 400:1 ratio. Under these conditions, the appearance of carboxypeptidase activity was measured with the synthetic substrate FAPP; it followed a quick and monotonic activation curve, which can be fitted to a pseudo first-order kinetics (Fig. 4A). Using Tricine/SDS-PAGE, the rapid proteolytic severing of the pro-segment, the concomitant appearance of CPA2, and a straight correlation of this proteolysis with the monotonic activity curve were observed (Fig. 4B). Nearly all pro-CPA2 was converted into its active form in 20 min, and a maximum value in the activity curve was achieved. The generated pro-segment appeared as a single band on Tricine/SDS-PAGE (Fig. 4B), although mass spectrometry analysis indicated heterogeneity for this species (see below).
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DISCUSSION |
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One of the aims of this study was the development of a highly efficient recombinant expression system for a native and activable form of human procarboxypeptidase A2 in the P. pastoris heterologous system to study its proteolytic activation and maturation pathway in vitro. It is worth mentioning that pancreatic carboxypeptidases and their precursors are difficult to express in native and soluble forms in E. coli (28) and that reports about the expression of their precursor forms in eukaryotic cells (i.e. Saccharomyces cerevisiae) have indicated a moderate yield (<10 mg/liter) until now (21, 29). In fact, the primary production of the proenzymes by recombinant approaches followed by proteolytic activation is probably the best strategy to obtain active carboxypeptidases given that it takes advantage of the high folding capability of the pro-segment in heterologous systems (29, 30). The recent biotechnological interest in metallocarboxypeptidases as prodrug activators for cancer therapy (21, 31, 32) makes the availability of large-scale procedures to obtain pro- and carboxypeptidases, particularly the human forms, very useful. The efficient heterologous production of extra-pancreatic metalloprocarboxypeptidases, with many of them involved in important biological functions and some with a tertiary structure or domains structurally related to the pancreatic ones (4), could also facilitate their detailed characterization. Hence, the development of efficient heterologous expression systems for enzymes and proenzymes such as the one reported here is highly desirable.
The experiments designed to establish an easy and robust expression system capable of providing large amounts of soluble human pro-CPA2 were restricted to shake-flask cultures, which rendered a production of ~250 mg of recombinant proenzyme/liter or 180 mg of active enzyme/liter. This production is more than enough for studies such as those presented here and for subsequent structural determinations as well as for other analytical and semipreparative purposes. However, P. pastoris can increase this production 10-fold by scaling up from shake-flask to high density fermentation (19).
The original target for the removal of the -MF propeptide is the
sequence KREAEAEA, which is cleaved by the yeast endopeptidase KEX2
after the dibasic peptide and undergoes subsequent elimination of
Glu-Ala dipeptides by dipeptidyl aminopeptidase A (33). Thus, according
to the design of the pPIC9-pro-CPA2 expression plasmid, the
pro-segment of the
-mating factor was expected to be cleaved after
the dibasic residues Lys-Arg by a single KEX2 endopeptidase action.
However, the N-terminal sequence of expressed recombinant pro-CPA2 displayed microheterogeneity at the amino
terminus, indicating that cleavage by the KEX2 endopeptidase is only
partial and that other endoproteinases might be responsible for the
unspecific cleavages (see Fig. 3). The microheterogeneity observed did
not become an important problem since the principal product found in
all of the cultures was native pro-CPA2. Part of the
secreted fusion product appeared glycosylated; however, this product
was converted to normal pro-CPA2 after deglycosylation. In
any case, all of these products can generate fully active
CPA2 by limited proteolysis.
A correctly processed -MF propeptide has been reported for
recombinant proteins expressed in P. pastoris without the
C-terminal Glu-Ala extension (34, 35); other reports indicated
heterogeneity at the N terminus of the secreted proteins
(i.e. aprotinin and coffee bean galactosidase), also
resulting from different cleavage points in the processing of the
-MF pro-segment (36, 37). Thus, the microheterogeneity observed in
some cases could be dependent on the nature of the recombinant protein
expressed (33).
The trypsin-promoted maturation mechanism of human
pro-CPA2, as derived from our studies, is shown in Fig. 6.
Due to the presence of several arginines at the boundary region between
the pro-segment and the enzyme moiety, a rapid tryptic cleavage at the
most exposed arginine of this region and subsequent cleavages at the
remaining trypsin targets should be expected. Alternatively,
simultaneous cleavages could occur, but our results indicate that it is
the first mechanism that takes place. In contrast to other
procarboxypeptidases studied, the Arg96-Ser97
peptide bond is the first target for trypsin action observed in
pro-CPA2, releasing a pro-segment of 96 residues into the
medium (-fragment in Figs. 5 and 6). From sequence alignments and
structure comparisons (14), this peptide bond is considered to belong to the enzyme moiety in porcine pro-CPA1 and pro-CPB, where
the cleavage occurs two residues farther along N-terminally (16, 17).
The released primary activation fragment and mature enzyme are
therefore two residues larger and shorter, respectively, in pro-CPA2.
The second step in the proteolytic processing is the rapid elimination
of the C-terminal Glu-Arg dipeptide end from the -fragment. The
product of this cleavage (
-fragment in Figs. 5 and 6) is due to a
tryptic action on arginine 94 of the
-fragment, as shown by mass
spectrometry analysis and by the lack of release of free amino acids.
The action of trypsin in this conversion is not as fast as in the first
cleavage, probably due to the less efficient tryptic endoproteinase
activity near the C-terminal end of proteins, and takes place while
some proenzyme molecules are still intact. The third and last sequence
of 92 residues (
-fragment in Figs. 5 and 6), resistant to further
proteolysis under these conditions, is generated by trypsin action in
the long run, releasing an Arg-Arg dipeptide into the activation
medium. It is also worth commenting upon that no release of free amino
acids into the medium is observed in the course of activation, in
contrast to previous observations in the corresponding activation
processes of pro-CPA1 and pro-CPB (16, 17, 38). This
indicates the high specificity shown by human CPA2, which
is unable to trim the C-terminal arginines from its activation
peptides, even at the high concentration of these species in the
activation medium.
According to this study, it seems clear that the pro-segments of
pro-CPA2 released into the medium do not inhibit the
activity of the active enzyme, even in its longer (primary) form (96 amino acids), since CPA2 reaches total activity following a
rapid hyperbolic activation curve at activation times when a
substantial concentration of the -fragment sequence is still present
in solution. In contrast to this behavior, porcine
pro-CPA1, which shows a slower and biphasic activation
process, needs subsequent trimmings at the C-terminal end and a second
tryptic cleavage inside the globular domain of the pro-sequence to
achieve full carboxypeptidase activity. In this sense,
pro-CPA2 behaves in a way that is more similar to pro-CPB
(17).
X-ray crystallography and modeling studies have shown the structural
similarities between pro-CPA1 and pro-CPA2,
both sharing a long C-terminal -helix at the region connecting the
globular activation domain with the active enzyme, and the differences of these two proenzymes from pro-CPB, whose corresponding connecting region has a much shorter
-helix and is less structured overall (5,
6, 14). These differences were proposed to be the primary determinant
responsible for the diversity observed in the rates of activation of
pro-CPA1 and pro-CPB, arguing that the larger regular
structure of the connecting region in pro-CPA1 would render
the interactions with the enzyme more stable and make the structural
relaxation needed for full release slower (4, 39). Modeling studies
with pro-CPA2 (14) have shown that such a connecting region
is also structured in a long
-helix, at least the same size as in
porcine pro-CPA1. Taking into account the former evidence
and the observation of a rapid and monotonic activation curve for
pro-CPA2, it can be concluded that the folding of the
connecting region of procarboxypeptidases in a long
-helix does not
give rise to inhibitory activation fragments and to slow activation
processes by itself. Other structural determinants, such as
surface complementarity and electrostatic and Van der Waals
interactions, should be considered to evaluate the stability of the
bimolecular complex between the pro-segment and the enzyme moiety once
the first proteolytic cleavage has taken place.
Preliminary x-ray diffraction studies2 on human pro-CPA2 show that the connecting region is highly structured and that extensive interactions take place between the enzyme moiety and both the globular domain region and the connecting region of the pro-segment. These observations confirm that the latter two regions are responsible for the activation behavior of pro-CPA2 and for its functional differences from other procarboxypeptidases.
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ACKNOWLEDGEMENTS |
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We thank Dr. S. Bartolomé (Laboratory for Image Analysis) for help in the elaboration of the figures and Dr. F. Canals (Servei de Seqüenciació i Biocomputació) for performing the N-terminal sequence analysis of the proteins.
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FOOTNOTES |
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* This work was supported by Grant BIO95-0848 from the Comisión Interministerial de Ciencia y Tecnología (Ministerio de Educación y Ciencia, Spain) and by the Center de Referencia de Biotecnologia (Generalitat de Catalunya).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 34-3-5811315;
Fax: 34-3-5812011; E-mail: FX.Aviles{at}blues.uab.es.
1
The abbreviations used are: CPs,
carboxypeptidases; pro-CPs, procarboxypeptidases; TLCK,
N-p-tosyl-L-lysine
chloromethyl ketone; FAPP,
N-(3-(2-furyl)acryloyl)-L-phenylalanyl-L-phenylalanine; HPLC, high pressure liquid chromatography; MES,
4-morpholineethanesulfonic acid; MALDI-TOF, matrix-assisted laser
desorption ionization time-of-flight; Tricine,
N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
-MF, prepro-
-mating factor; PAGE, polyacrylamide gel
electrophoresis.
2 I. García-Sáez, personal communication.
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REFERENCES |
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