(Received for publication, November 14, 1994; and in revised form, January 30, 1995)
From the
The prohormone-processing proteases PC1/3 and PC2 belong to the
family of mammalian subtilisin-related proprotein convertases (PC)
possessing homology to the yeast Kex2 protease. The presence of PC1/3
and PC2 in secretory vesicles of bovine adrenal medulla (chromaffin
granules) implicates their role in the processing the precursors of
enkephalin, neuropeptide Y, somatostatin, and other neuropeptides that
are present in chromaffin granules. In this study, PC1/3 and PC2 were
purified to apparent homogeneity from the soluble fraction of
chromaffin granules by chromatography on concanavalin A-Sepharose,
Sephacryl S-200, pepstatin A-agarose, and anti-PC1/3 or anti-PC2
immunoaffinity resins. PC1/3 and PC2 were monitored during purification
by measuring proteolytic activities with S-enkephalin
precursor and Boc-Arg-Val-Arg-Arg-methylcoumarin amide (MCA) substrates
and by following PC1/3 and PC2 immunoreactivity with specific
anti-PC1/3 and anti-PC2 sera generated in this study. Purified PC1/3
and PC2 on SDS-polyacrylamide gels each show a molecular mass of 66
kDa. PC2 in the soluble fraction of chromaffin granules was present at
5- and 10-fold higher enzyme protein and activity, respectively,
compared with that of PC1/3. PC1/3 and PC2 cleaved paired basic and
monobasic sites within peptide-MCA substrates, with
Boc-Arg-Val-Arg-Arg-MCA and pGlu-Arg-Thr-Lys-Arg-MCA as the most
effectively cleaved peptides tested. PC1/3 and PC2 showed pH optima of
6.5 and 7.0, respectively. Kinetic studies indicated apparent K
values for hydrolysis of
Boc-Arg-Val-Arg-Arg-MCA as 66 and 40 µM, with V
values of 255 and 353 nmol/h/mg for PC1/3 and
PC2, respectively. Specificity of the PC enzymes for dibasic sites was
confirmed by potent inhibition by the active site-directed peptide
inhibitors (D-Tyr)-Glu-Phe-Lys-Arg-CH
Cl and
Ac-Arg-Arg-CH
Cl. Inhibition by EGTA and activation by
Ca
indicated PC1/3 and PC2 as
Ca
-dependent proteases. In addition, PC enzymes were
activated by dithiothreitol and inhibited by thiol-blocking reagents, p-hydroxymercuribenzoate and mercuric chloride. These results
illustrate the properties of endogenous PC1/3 and PC2 as
prohormone-processing enzymes.
The posttranslational processing of prohormones and
proneuropeptides requires proteolytic cleavage at paired basic residues
and less frequently at monobasic residues, which flank active peptide
sequences within the precursors (Docherty and Steiner, 1982; Hook et al., 1994). Recently, candidate mammalian
subtilisin-related proprotein convertases have been cloned based on
sequence homology to yeast Kex2, a processing protease for
pro--mating factor and pro-killer toxin (Julius et al.,
1984; Mizuno et al., 1988; Fuller et al., 1989).
Seven members of this proprotein convertase family thus far identified
are furin (Roebroek et al., 1986; Hatsuzawa et al.,
1990; Van den Ouweland, 1990), PC1/3 (Seidah et al., 1991a;
Smeekens et al., 1991; Nakayama et al., 1991), PC2
(Seidah et al., 1990; Smeekens and Steiner, 1990), PACE 4
(Barr et al., 1991; Kiefer et al., 1991), PC4
(Nakayama et al., 1992a; Seidah et al., 1992), PC5/6
(Lusson et al., 1993; Nakagawa et al., 1993), and PC7
(Tsuji et al., 1994). Among these members, PC1/3 and PC2 are
most relevant to neuropeptide production since their expression is
restricted to neuroendocrine cells, as demonstrated by Northern
analysis and in situ hybridization studies (Smeekens et
al., 1991; Seidah et al., 1990, 1991a, 1991b; Schafer et al., 1993).
Evidence supporting the role of these PC proteases in the maturation of proproteins and prohormones is based on numerous studies of coexpression of potential proprotein substrates and PC proteases in eukaryotic cell lines (Seidah et al., 1991b; Benjannet et al., 1991; Thomas et al., 1991; Smeekens et al., 1992) or by in vitro experiments using recombinant PC2 and PC1/3 (Shennan et al., 1991; Jean et al., 1993; Rufaut et al., 1993; Zhou and Lindberg, 1993). With respect to PC activities in vivo, PC2 activity has been identified in secretory vesicles of insulinoma cells (Bennett et al., 1992), in intermediate pituitary (Estivariz et al., 1992), and in pancreatic islets of anglerfish (Mackin et al., 1991).
Secretory vesicles of adrenal medulla, known as chromaffin granules, contain several neuropeptides including the enkephalins (Udenfriend and Kilpatrick, 1983; Liston et al., 1984; Spruce et al., 1988), neuropeptide Y (Carmichael et al., 1990), somatostatin (Lundberg et al., 1979), and others, which are generated by proteolytic processing of respective precursors. The presence of PC1/3 and PC2 in chromaffin granules is consistent with prohormone processing occurring in these vesicles. PC1/3 and PC2 proteins have been detected in chromaffin granules by microsequencing (Christie et al., 1991) and immunological analysis (Kirchmair et al., 1992). PC1/3 and PC2 activities in chromaffin granules have been shown by immunoprecipitation studies (Azaryan and Hook, 1992a, 1992b; Hook et al., 1993a). The high yield of chromaffin granules from bovine adrenal medulla should provide large quantities of purified PC1/3 and PC2 for characterizing their activities and understanding their role in processing proenkephalin and other adrenal medullary neuropeptide precursors.
In this study, our
results indicate the purification of PC2, as well as lower levels of
PC1/3, from the soluble fraction of bovine chromaffin granules.
Characterization of endogenous PC1/3 and PC2 demonstrate their
properties as Ca-dependent proteases cleaving at
typical prohormone paired basic residue processing sites. The relative
contribution of PC enzyme activities toward total enkephalin precursor
cleaving activity in chromaffin granules is discussed.
Assay of PC1/3 or PC2 with peptide-MCA substrates was performed by incubating enzymes with 100 µM peptide-MCA in 0.1 M Tris-HCl, pH 6.5, 1 mM dithiothreitol, (160 µl) at 37 °C for 2 h. In some experiments, aminopeptidase M (2 µg, Sigma) was then added (with adjustment of pH to 8.8), and incubation continued at 37 °C for another hour. The rate of formation of free 7-amino-4-methylcoumarin was quantitated as described previously (Azaryan and Hook, 1992a, 1994a, 1994b).
Affinity chromatography on pepstatin A-agarose was performed by adjusting the pH of the 70-kDa fraction (25 ml) (from the S-200 column) to 4.5 with final buffer concentration of 50 mM sodium citrate and incubating this fraction with pepstatin A-agarose (5 ml, Pierce) at 4 °C for 4 h. This mixture, placed in a column, was washed with 50 mM sodium citrate, pH 4.5, and bound proteins were eluted with 0.1 M Tris-HCl, pH 8.5, 0.2 M NaCl buffer. The pH of the eluted fractions was adjusted to 6.0.
To produce affinity resins for immunoaffinity chromatography of PC1/3 and PC2, IgG immunoglobulins from anti-PC1/3 and anti-PC2 sera were linked to ImmunoPure IgG resin according to the manufacturer's procedure (Pierce). The unbound pool (25 ml) from the pepstatin A column, which contained PC1/3 and PC2, was dialyzed against 0.1 M Tris-HCl, pH 6.0, and concentrated by ultrafiltration to 2 ml. To 1 ml of pepstatin A unbound fraction, 1 ml of 10 mM Tris-HCl, pH 7.5, was added, followed by rocking with anti-PC1/3 or anti-PC2 immunoaffinity resin (2 ml bed volume) for 2 h at 4 °C. Unbound fractions were collected, and their pH was adjusted to 6.0 (by the addition of 0.4 M sodium citrate, pH 5.0). After application of 15 ml of washing buffer, pH 8.2 (from Pierce), bound fractions were eluted with 0.1 M glycine, pH 2.8, and their pH was adjusted to 6.0. Protein content was determined by the method of Lowry (Lowry et al., 1951) with bovine serum albumin as standard. Purified enzymes were assessed by SDS-polyacrylamide gel electrophoresis (as described previously, Krieger and Hook(1991)) and by Western blotting with anti-PC sera (immunoblots performed as described previously (Hook et al., 1993b)).
For immunoprecipitation of PC1/3 and PC2, the unbound
fraction from the pepstatin A-agarose column was preincubated with
preimmune, PC1/3, or PC2 antiserum (final dilution, 1:100) for 1 h at
room temperature in 50 µl of 0.1 M Tris-HCl buffer, pH
6.5. After further incubation at 4 °C for 16 h, the mixture was
rocked with 50 µl of Protein A-Sepharose CL-4B at 4 °C for 45
min. The sample was centrifuged at 13,000 g for 5 min,
and the supernatant was assayed for Boc-Arg-Val-Arg-Arg-MCA cleaving
activity. Removal of Boc-Arg-Val-Arg-Arg-MCA cleaving activity from the
supernatant by anti-PC enzyme sera indicated immunoprecipitation of
PC1/3 and PC2.
Further purification of the concanavalin A-bound pool by Sephacryl S-200 gel filtration revealed two peaks of enkephalin precursor cleaving activity (Fig. 1). The first peak has been purified and characterized as the novel cysteine protease, ``prohormone thiol protease,'' and has been demonstrated as a candidate proenkephalin processing enzyme (Krieger and Hook, 1991; Krieger et al., 1992; Azaryan and Hook, 1994a, 1994b). The second peak eluting at approximately 70 kDa was analyzed in this study for the PC1/3 and PC2 enzymes.
Figure 1:
Sephacryl S-200
chromatography. The concanavalin A-bound enkephalin precursor cleaving
activity was chromatographed by gel filtration on Sephacryl S-200.
[[S]Met]PPE-cleaving activity (
)
is expressed as total trichloroacetic acid soluble radioactivity
generated by a 5-µl aliquot from each column fraction. Relative
protein levels were measured by absorbance at 280 nm (
) in
column fractions. The arrow indicates the 70 kDa
peak.
Analysis of the 70-kDa fraction with protease inhibitors revealed
the presence of serine and aspartic proteases (Table 1).
Enkephalin precursor cleaving activity was partially inhibited by
soybean trypsin inhibitor, -antitrypsin, benzamidine,
and N-tosyl-L-lysine chloromethyl ketone, indicating
serine protease activity. The serine protease inhibitor N-tosyl-L-phenylalanine chloromethyl ketone was an
effective inhibitor. However, phenylmethylsulfonyl fluoride, another
serine protease inhibitor, had no effect. Inhibition by pepstatin A
indicated aspartic protease activity. No inhibition was detected by the
cysteine protease inhibitor cystatin C. Activity directed toward paired
basic residues was indicated by inhibition with the peptide inhibitor (D-Tyr)-Glu-Phe-Lys-Arg-CH
Cl that possesses a
Lys-Arg site. The effectiveness of protease inhibitors on enkephalin
precursor cleaving activity in the 70-kDa fraction indicates inhibition
of the production of small peptides (less than 5-8 kDa) that are
trichloroacetic acid-soluble (Krieger and Hook, 1991); intermediate
sized products greater than 10 kDa are trichloroacetic acid-insoluble
and, therefore, are not detected as trichloroacetic acid-soluble
S-labeled peptides in this assay (Krieger and Hook, 1991).
To examine the serine proteolytic activity in more detail, pepstatin
A-agarose was used to remove the aspartic proteolytic activity from the
70-kDa fraction (Fig. 2). The pepstatin A unbound and bound
pools contained 65 and 35%, respectively, of the activity recovered
from the column. This step recovered 50% of the total activity applied
to the column. The loss of activity may be due to instability of the
enzyme(s) at basic pH (the pepstatin A column was eluted with pH 8.5
buffer), as it is known that some secretory granule proteases are
unstable at neutral or basic pHs (Krieger and Hook, 1991). ()Pepstatin A inhibited enkephalin precursor cleaving
activity in the bound pool, but not in the unbound pool (data not
shown), indicating removal of aspartic protease activity by pepstatin
A-agarose. The tetrapeptide Boc-Arg-Val-Arg-Arg-MCA, a good substrate
for the PC enzymes (Jean et al., 1993; Shennan et
al., 1991; Rufaut et al., 1993; Zhou and Lindberg, 1993),
was readily cleaved by activity in the pepstatin A unbound pool but not
by activity in the bound pool (Fig. 2). The
Boc-Arg-Val-Arg-Arg-MCA cleaving activity in the pepstatin A unbound
pool demonstrates cleavage at an Arg-Arg paired basic residue
processing site.
Figure 2: Pepstatin A-agarose chromatography. The 70-kDa fraction from the Sephacryl S-200 column was subjected to affinity chromatography on pepstatin A-agarose. a, total enkephalin precursor cleaving activity was determined in the 70-kDa fraction from the S-200 column (S-200: 70 kDa) and in the unbound (U), wash (W), and bound (B) pools from the pepstatin A-agarose column. b, total Boc-Arg-Val-Arg-Arg-MCA-cleaving activity was determined in the 70-kDa fraction from the S-200 column, and in the unbound (U), wash (W), and bound (B) pools from the pepstatin A-agarose column.
Anti-PC1/3 and anti-PC2 immunoblots indicated that chromaffin granules contained PC1/3 and PC2 as 66-kDa bands (Fig. 3, a and b), which is consistent with the molecular size of PC enzymes detected in neuroendocrine tissues (Bennet et al., 1992; Kirchmair et al., 1992; Mackin et al., 1991), and in cells expressing recombinant PCs (Rufaut et al., 1993; Zhou and Lindberg, 1993). Immunoblots showed that the pepstatin A-agarose unbound, but not the bound, pool contains both PC1/3 and PC2 (Fig. 3, c and d); results suggest higher levels of PC2 than PC1/3 in the unbound pool. Protein staining by Amido Black of the unbound pool showed a single 66-kDa band (Fig. 3e); this band presumably contains both PC1/3 and PC2 based on anti-PC immunoblots (Fig. 3, c and d). Additionally, immunodepletion of 31 and 40% of the Boc-Arg-Val-Arg-Arg-MCA cleaving activity (data not shown) from the pepstatin A unbound pool by anti-PC1/3 and PC2 sera, respectively, but not by preimmune serum, indicated the presence of relevant PC enzyme activities. These results confirmed the presence of PC1/3 and PC2 in the pepstatin A-agarose unbound pool.
Figure 3: PC1/3 and PC2 immunoreactivity in CG and unbound fraction of pepstatin A-agarose. a, anti-PC1/3 immunoblot (anti-PC1/3 antiserum at 1:200) of CG lysate (18 µg). b, anti-PC2 immunoblot (anti-PC2 serum at 1:200) of CG lysate (18 µg). c, anti-PC1/3 immunoblot (anti-PC1/3 serum at 1:200) of pepstatin A unbound (U) and bound (B) fractions. d, anti-PC2 immunoblot (anti-PC2 serum at 1:200) of pepstatin A unbound (U) and bound (B) fractions (2.5 µg). e, Amido Black staining of pepstatin A unbound fraction (1 µg).
Chromatofocusing of the pepstatin A unbound pool was performed to determine whether the the Boc-Arg-Val-Arg-Arg-MCA-cleaving activity in the pepstatin A-agarose unbound pool possesses a pI typical for PC enzymes. A single peak of Boc-Arg-Val-Arg-Arg-MCA cleaving activity was detected at pH 5.2-4.8, with maximal activity at pH 5.0 (data not shown). These results indicate Boc-Arg-Val-Arg-Arg-MCA cleaving activity with pI value of approximately 5.0, which is consistent with the detection of PC1/3 and PC2 as glycoprotein H with pI of approximately 4.9-5.0 (Christie et al., 1991).
Immunoaffinity chromatography was used to separate PC1/3 and PC2 in the pepstatin A unbound pool. PC2 was purified using the anti-PC1/3 immunoaffinity column, with PC2 eluting as the unbound material. Analogously, PC1/3 was purified using the anti-PC2 immunoaffinity column, with PC1/3 eluting as the unbound material. Total PC1/3 and PC2 activities obtained after the immunoaffinity step, yielded 10 times greater levels of PC2 activity compared with PC1/3 activity (Fig. 4). There was no proteolytic activity in the bound pools eluted from the immunoaffinity columns at pH 2.8. Evidently, PC enzymes are not stable to the large drop in pH during elution from the immunoaffinity column. Collection of the unbound pool results in isolation of active PC enzymes.
Figure 4: Immunoaffinity chromatography of PC1/3 and PC2. Total PC1/3 activity obtained as the unbound pool from the anti-PC2 affinity column, and total PC2 activity obtained as the unbound pool from the anti-PC1/3 affinity column are shown.
On SDS-polyacrylamide gel electrophoresis, the purified PC2 and PC1/3 appeared as single bands of 66 kDa, indicating purification to apparent homogeneity (Fig. 5a). Immunoblots showed that purified PC2 was recognized by anti-PC2 serum but not by anti-PC1/3 serum (Fig. 5b); PC1/3 was recognized by anti-PC1/3 serum but not by anti-PC2 serum (Fig. 5c). These results indicate the effective separation of PC1/3 from PC2 by the immunoaffinity step.
Figure 5: Purified PC2 and PC1/3 on SDS-polyacrylamide gel electrophoresis and immunoblots. a, purified PC2 and PC1/3 proteases (4 µg of each, lanes1 and 2, respectively), were subjected to gel electrophoresis on a 12% SDS-polyacrylamide gel that was stained with Coomassie Blue. b, anti-PC2 immunoblot of purified PC2 and PC1/3 (lanes1 and 2, respectively). Lane1 illustrates positive immunochemical identification of purified PC2, and lane2 shows the lack of PC2 in the purified PC1/3. c, anti-PC1/3 immunoblot of purified PC2 and PC21/3 (lanes1 and 2, respectively). Lane2 shows PC1/3 immunoreactivity in the purified sample of PC1/3, and lane1 shows the lack of PC2 in the sample of purified PC1/3. Immunoblots utilized anti-PC1/3 and anti-PC2 sera at 1:200 dilution.
Based on Boc-Arg-Val-Arg-Arg-MCA cleaving activity in chromaffin granules, purification at the pepstatin A step represents approximately a 363-fold purification of PC1/3 and PC2 from chromaffin granules. With some loss of activity at the pepstatin A-agarose and immunoaffinity steps, the purification results in a 46- and 95-fold purification of PC1/3 and PC2, respectively, from chromaffin granules (Table 2). An excellent yield (from 650 adrenal medullae) is illustrated by the isolation of 100 and 550 µg of PC1/3 and PC2, respectively (Table 2).
Figure 6: pH dependence of PC1/3 and PC2. The purified PC1/3 (panela) and PC2 (panelb) were assayed with Boc-Arg-Val-Arg-Arg-MCA as substrate at different pH values.
The kinetic constants apparent K and V
were assessed with
Boc-Arg-Val-Arg-Arg-MCA, the best peptide-MCA substrate, by
Lineweaver-Burk plots (Fig. 7, a(i) and a(ii)). PC1/3 and PC2 showed apparent K
values of 66 and 40 µM and V
values of 255 and 353 nmol of 7-amino-4-methylcoumarin
released/h/mg, respectively. Further characterization of PC enzymes
utilized 100 µM Boc-Arg-Val-Arg-Arg-MCA.
Figure 7:
Kinetic parameters of PC1/3 and PC2. a, kinetic constants, apparent K and V
, for PC1/3 and PC2.
Lineweaver-Burke plot of PC1/3 (i) and PC2 (ii)
hydrolysis of Boc-Arg-Val-Arg-Arg-MCA (8.8 and 5.0 µg of PC1/3 or
PC2, respectively, per assay). b, rate of PC1/3 (i)
and PC2 (ii) inactivation by Ac-Arg-Arg-CH
Cl.
Inactivation of PC1/3 and PC2 (8.8 and 5.0 µg of PC1/3 or PC2,
respectively, per assay) by Ac-Arg-Arg-CH
Cl (2
10
M) was examined as a function of
preincubation time of inhibitor with
enzyme.
Protease
inhibitor studies showed that the PC enzymes were potently inhibited by
the active-site directed peptide inhibitors (D-Tyr)-Glu-Phe-Lys-Arg-CHCl and
Ac-Arg-Arg-CH
Cl (Table 4), providing further evidence
for paired basic residue cleavage specificity. The second order rate
constants for Ac-Arg-Arg-CH
Cl inactivation of PC1/3 and PC2
were 32,000 and 31,200 M
s
, respectively (Fig. 7, b(i) and b(ii)). Both PC enzymes were inhibited by the metal
chelators EDTA and EGTA; the addition of calcium ions (5 mM)
reversed the inhibitory effect of EGTA. The thiol blocking agents, p-hydroxymercuribenzoate and mercuric chloride, inhibited
activity, while dithiothreitol stimulated activity of PC1/3 and PC2 by
10-fold. These results suggest the presence of functional cysteine
residue(s) in the vicinity of the enzyme active site. E-64c, a cysteine
protease inhibitor, had no effect.
-antitrypsin and
-antichymotrypsin, serpin protease inhibitors, reduced
PC enzyme activities. However, the serine protease inhibitors
diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, and
benzamidine had no effect. The aspartic protease inhibitor pepstatin A,
also had no effect. These protease inhibitor studies illustrate PC1/3
and PC2 as subtilisin-like proteases that are sensitive to
Ca
and reducing conditions.
In this study, we report the purification and characteristics
of two endogenous subtilisin-related proprotein convertases, PC1/3 and
PC2, from the soluble extract of bovine adrenal medulla chromaffin
granules. Using
[[S]Met]preproenkephalin and
Boc-Arg-Val-Arg-Arg-MCA as substrates, in conjunction with anti-PC1/3
and anti-PC2 sera to identify PC enzyme immunoreactivity, PC1/3 and PC2
of 66 kDa were purified by concanavalin A-Sepharose, Sephacryl S-200,
pepstatin A-agarose, and immunoaffinity chromatography. Greater levels
of PC2 than PC1/3 were present in the soluble fraction, since the
purification yielded 550 µg of PC2 and only 100 µg of PC1/3.
This is consistent with previous immunoblot studies showing PC2 as the
major soluble PC enzyme in chromaffin granules (Kirchmair et
al., 1992). The purified PC1/3 and PC2 show localization to
isolated secretory vesicles (chromaffin granules), appropriate
specificity for cleavage at paired basic residue sites, pH dependence
consistent with the intragranular pH, and apparent affinity (K
) and maximal velocity (V
) consistent with in vivo levels of
adrenal medullary neuropeptide precursors. Furthermore, studies with
protease inhibitors and activators indicate PC1/3 and PC2 as
Ca
-dependent proteases with sensitivity to reducing
conditions. These results provide supportive evidence for PC1/3 and PC2
as processing proteases with appropriate cleavage specificity for
paired basic residue sites present within prohormones.
Studies of
cleavage specificity of chromaffin granule (CG) PC1/3 and PC2 indicated
that the most effective peptide-MCA substrates for both PC enzymes were
Boc-Arg-Val-Arg-Arg-MCA and pGlu-Arg-Thr-Lys-Arg-MCA (Table 3),
which contain Arg-Arg and Lys-Arg paired basic cleavage sites.
Recombinant PC1/3 (produced in a mouse L cell line,
GHC
cells, or Chinese hamster ovary cells) also
shows excellent activity with Boc-Arg-Val-Arg-Arg-MCA and
pGlu-Arg-Thr-Lys-Arg-MCA (Rufaut et al., 1993; Jean et
al., 1993; Zhou and Lindberg, 1993). Thus, native and recombinant
PC1/3 and PC2 resemble one another in preference for tetrapeptide
substrates. These results suggest that Arg in the P
position of the Arg-X-Lys/Arg-Arg motif is important for
cleavage specificity requirements of PC1/3 and PC2. However,
Arg-Gln-Arg-Arg-MCA was a poor substrate for CG PC1/3 and PC2, even
though this peptide contains the same Arg-X-Lys/Arg-Arg motif
as Boc-Arg-Val-Arg-Arg-MCA and pGlu-Arg-Thr-Lys-Arg-MCA (Table 3). The free amino-terminal Arg residue of
Arg-Gln-Arg-Arg-MCA may be less desirable than the NH
terminus blocked by t-butoxycarbonyl. Indeed, an
8.4-fold reduction in activity of recombinant PC1/3 upon removal of t-butoxycarbonyl from Boc-Arg-Val-Arg-Arg-MCA has been
demonstrated (Jean et al., 1993).
CG PC1/3 and PC2 cleavage of paired basic residues occurs preferentially at the COOH-terminal side of paired basic residues. The yeast Kex2 protease shows a similar cleavage specificity (Brenner and Fuller, 1992). The CG PC enzymes also resemble Kex2 with respect to equivalent effectiveness in cleaving at paired basic and single basic residues (Lys or Arg) within tripeptide substrates. The ability of CG PC1/3 and PC2 to hydrolyze single basic residue sites (Arg or Lys) is in accord with mono-arginyl cleavages of prorenin by recombinant PC1 (Nakayama et al., 1992b) and with in vitro cleavage at a single arginine site of prodynorphin by recombinant PC1 (Dupuy et al., 1994).
Selectivity of CG
PC1/3 and PC2 for paired basic residues was further demonstrated by
potent inhibition by the active site-directed peptide inhibitor (D-Tyr)-Glu-Phe-Lys-Arg-CHCl. This inhibitor
corresponds to the Glu-Phe-Lys-Arg sequence at the junction of ACTH and
-lipotropin within proopiomelanocortin that is cleaved by Kex2,
and PC1/3 or PC2 in DNA cotransfection experiments (Thomas et
al., 1988, 1991; Benjannet et al., 1991). The CG PC
enzymes were also effectively blocked by another dibasic
site-containing inhibitor, Ac-Arg-Arg-CH
Cl, with the
second-order rate constants of inactivation being 32,000 and 31,200 M
s
for PC1/3 and PC2,
respectively.
The kinetic parameters of Boc-Arg-Val-Arg-Arg-MCA
hydrolysis by CG PC1/3 and PC2 indicated apparent K values of 66 and 40 µM, and V
values of 255 and 353 nmol/h/mg, respectively. The affinity of
PC1/3 and PC2 is compatible with the estimated in vivo levels
of proenkephalin in chromaffin granules at 10-100 µM (Ungar and Phillips, 1983). The apparent K
values of CG PC1/3 and PC2 are also of similar range with the K
of 19 µM reported for Kex2
catalyzed hydrolysis of Boc-Arg-Val-Arg-Arg-MCA (Bennet and Fuller,
1992).
CG PC1/3 was most active at pH 6.5, while the pH optimum for CG PC2 was 6.5-7.0. Importantly, PC1/3 and PC2 show 50% of maximum activity at the intragranular pH of 5.5-6.0 (Pollard et al., 1979), indicating that these enzymes would be active in vivo. The endogenous PC enzymes in CG and recombinant PC enzymes show similar pH optima (Rufaut et al., 1993; Shennan et al., 1991).
Studies with protease inhibitors indicated
that CG PC1/3 and PC2 were sensitive to metal chelators and
thiol-reactive inhibitors, similar to yeast Kex2 (Julius et
al., 1984; Brenner and Fuller, 1992). CG PC1/3 and PC2 are
calcium-dependent proteases, illustrated by EGTA inhibition of PC
enzyme activities, with reversal by Ca ions to
generate fully reactivated proteases. The CG PC1/3 and PC2 were also
sensitive to thiol-reactive inhibitors p-hydroxymercuribenzoate and mercuric chloride. The effect of
sulfhydryl reagents has been also reported for recombinant PC1/3
(Rufaut et al., 1993; Jean et al., 1993; Zhou and
Lindberg, 1993) and PC2 (Shennan et al., 1991). Activation of
PC1/3 and PC2 by dithiothreitol indicates the presence of functional
cysteine near the active site catalytic triad of these proteases. Of
interest is the inhibition of the PC enzymes by
-antichymotrypsin that is present within chromaffin
granules (Hook et al., 1993b), suggesting
-antichymotrypsin as a possible regulator of PC
enzymes in vivo. The protease inhibitor profile of chromaffin
granule PC1/3 and PC2 is in accord with that of recombinant furin,
PC1/3, and PC2 (Hatsuzawa et al., 1992; Brennan and Nakayama,
1994; Rufaut et al., 1993; Jean et al., 1993; Zhou
and Lindberg, 1993; Shennan et al., 1991). This investigation
of endogenous PC1/3 and PC2 from adrenal medullary chromaffin granules
indicates active PC enzymes as single chain glycoproteins with
molecular mass of 66 kDa and pI 5.0, that are sensitive to
Ca
and sulfhydryl reagents.
It is important to consider that this study finds soluble PC1/3 and PC2 in CG as secondary enkephalin precursor cleaving activities, accounting for approximately 20% of total CG enkephalin precursor cleaving activity. PC enzymes in the membrane fraction of CG (Kirchmair et al., 1992) may contribute, in part, to the granule membrane-bound enkephalin precursor cleaving activity that represents about 10% of total enkephalin precursor cleaving activity in CG (Krieger and Hook, 1991). In addition, the present study indicates that proteolytic activity of the aspartic protease class represents 10% of total granule enkephalin precursor cleaving activity. In contrast to the PC enzymes and the aspartic protease, the cysteine protease `prohormone thiol protease' (PTP) has been shown as the major contributor of 60% of total enkephalin precursor cleaving activity in CG (Krieger and Hook, 1991). It will be necessary in future studies to understand the coordinate regulation of multiple prohormone processing enzymes in peptide hormone and neurotransmitter biosynthesis.