Download Purification and Partial Characterization of a Latent Serine Protease

Mol. Cells, Vol. 3, pp. 153-156
Purification and Partial Characterization of a Latent Serine Protease
in Escherichia coli
Jung Kwon Yoo, Kyung Chan Park, Seung Kyoon Woo, Kee Min Woo, Doo Bong Ha
and Chin Ha Chung*
Department of Molecular Biology, College of Natural Sciences, Seoul National University, Seoul
151-742, Korea
(Received on Arpil 7, 1993)
A new serine protease ~as been purified from Escherichia coli by conventional chromatographic
procedures using [3H]-casein as a substrate. It h' s an apparent size of about 34 kDa
as determined by gel filtration on a Superose-12 column. The purified enzyme, however, consisted of two polypeptides with 16 and l2-kDa when analyzed by polyacrylamide gel electrophoresis
in the presence of sodium dodesyl sulfate. These results suggest that the protease is a heterodimeric complex. The casein-degrading activity of the protease was completely inhibited by 1
mM diisopropyl fluorophosphate. It was also sensitive to inhibition by metal chelating agents
including EDTA and o-phenanthroline, suggesting that the enzyme belongs to a family of serine
proteases. Interestingly, the enzyme activity could be enhanced 5- to 20-fold by incubation
at 4 °C for about 3 weeks. Therefore, this new protease was named as LSP (Latent Serine
Protease). LSP was maximally active at pH between 5.5 and 9.5. The activation mechanism
and physiological role of this protease are presently unl own.
Intracellular proteolytic enzymes catalyze continuous degradation of proteins to small pep tides and
amino acids and limited cleavage of proteins with regulatory functions (Bond and Butler, 1987). The former
process is for the turnover of normal proteins and
for the rapid elimination of abnormal proteins, that
may arised from mutation, biosynthetic error or exposure to chemical damaging agents (Goldberg and St.
John, 1976). Limited proteolysis includes the processing of secretory proteins and the cleavage of critical
regulatory proteins with short half-lives (Gottesman,
1989).
Soluble extract of E. coli contains nine endoproteolytic activities that appear to be distinct (Goldberg et
al., 1981; Swamy et al., 1983; Chung and Goldberg,
1981, 1983; Park et al., 1988; Hwang et al., 1987, 1988).
Seven of these, named Do, Re, Mi, Fa, So, La and
Ti, are serine proteases that can hydrolyze casein and
globin. Two other enzymes, proteases Ci and Pi, are
metalloproteases that degrade insulin but not larger
proteins. While proteases Mi and Pi are periplasmic
enzymes, the others are localized to cytoplasm
(Swamy and Goldberg, 1982) and therefore may play
a role in the degradation of intracellular proteins. Of
these, proteases La and Ti require ATP for proteolysis
and reveal protein-activated ATPase activities. In addition, protease La has been shown to play a critical
role in the hydrolysis of polypeptides with aberrant
structure and certain regulatory proteins (Chung and
Goldberg, 1981; Gottesman, 1989).
Proteases Re and So have been shown to preferen-
* To whom all correspondences should be addressed.
tially hydrolyze the oxidized glutamine synthetase and
therefore suggested to play an important role in the
~limination of oxidatively damaged polypeptides (Lee
et al., 1988; Park et al., 1988). In addition, protease
Re is sirniliar in a number of criteria to Tsp (tail-specific protease) which specially degrades N-terminal-do- '
main variants of lambda phage with nonpolar C-terminal residues (Silber et al., 1992). Protease Do, which
is a heat-shock protein, degrades Ada protein, and
hence may play a rQle in the adaptive response (Lee
et al., 1990). However, other proteases have not yet
been purified and neither of theil physiological functions is clarified.
The present studies initially aimed to purify proteases Mi 1md Fa, which are known to have different
localization in E. coli but have the same size of about
110 kDa (Goldberg et al., 1981; Swamy and Goldberg,
1982). During purification of these proteases, we accidentally found a new serine proteolytic enzyme that
has relatively smaller molecular weight than previously
isolated proteases, including proteases Mi and Fa. In
the present paper, we describe the purification and
properties of this new enzyme, named latent serine
protease (LSP).
Materials and Methods
Materials
E. coli 3302 (ptr) strain (Cheng and Zipser, 1979)
The abbreviations used are: DFP, diisopropyl fluorophosphate; LSP, latent serine protease; PMSF, phenylmethylsulfonyl fluoride.
© 1993 The Korean Society of Molecular Biology
154
A Latent Serine Protease in E. coli
was obtained from Dr. Cheng (Cold Spring Harbor
Laboratory). DEAE-cellulose (DE-52) was purchased
from Whatman; DEAE-Sepharose and Superose-12
from Pharmacia; [ 3HJformaldehyde and Na l25I from
New England Nuclear. All other chemicals were obtained from Sigma. [ 3HlGlobin and [ 3H}casein were
prepared by reductive methylation as described by Jentoft and Dearborn (1979). Insulin B-chain was radioiodinated using chloramin T (Greenwood et al., 1963).
60
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20
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Determination of proteolytic activity
Proteolysis was assayed by measuring the conversion of protein substrates to acid-soluble products.
Reaction mixtures (100 ~) contained 50 mM Tris-HCl
(PH 7.8), 10 llg of radiolabeled proteins, and 0.01 to
5 llg of protease preparations depending on purification stage. Incubations were carried out at 37 °C for
2 h. The pH-optimum of the purified enzyme was
determined as described previously (Chung and Goldberg, 1983). Protein concentration was determine~ as
described by Bradford (1976) using bovine serum albumin as a standard.
Electrophoresis
Polyacrylamide gel electrophoresis in the presence
of sodium dodecyl sulfate (SDS) was performed in
15% (w/v) slab gels as described by Laemmli (1970).
Protein bands were stained by following the ammonical silver staining method (Oakley et al., 1980; Merril
et al., 1981).
Results
The crude extract (8 g) obtained from 100 g of frozen E. coli cells (3302) was loaded on a DEAE-cellulose (5 X 40 cm) equilibrated with 20 mM Tris-HCl
(PH 7.8) containing 5 mM MgCh. Mter washing the
column with the same buffer, proteins were eluted
with a linear gradient of 0 to 80 mM NaCl (total
2 liters). Fractions of 25 ml were collected at a flow
rate of 250 ml/h. Unlike the previous report (Swamy
and Goldberg, 1981), the fractions containing proteases
Mi and Fa showed relatively insignificant activity
against casein (Fig. 1). However, after incubation for
about 3 weeks at 4 °c , they became to reveal much
higher casein-degrading activity. Fractions with high
activity were pooled, concentrated by ultraflltration
using a YM 10 membrane (Amicon), and dialyzed
against 20 mM KH 2POJK2HP04 buffer (PH 6.5) containing 5 mM MgCh.
The resulting samples were loaded on a hydroxyla-
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Preparation of crude extracts
E. coli crude extract was prepared as described previously (Chung and Goldberg, 1983). Frozen E. coli
(3302) cells were suspended in 200 ml of 20 mM TrisHCl buffer (PH 7.8) containing 5 mM MgCh. The
cells were disrupted in a French press at 14,000 psi
and centrifuged at 100,000 X g for 3 h. The resulting
supernatant was dialyzed overnight against the same
buffer and referred to as crude extract.
Mol. Cells
0
, 0
20
30
40
50
o
20 z
o
FRACTION NUMBER
Elution proflle of the latent casein-degrading actia DEAE-cellulose column. E. coli crude extract
was chromatographed on a DEAE-cellulose column as described in the text Fractions of 25 ml were collected at a
flow rate of 250 ml/h and assaysed for casein hydrolysis
inunediately after the chromatography CO) or after incubation at 4 t for 3 weeks ce). The fractions under the bar
were pooled and used in subsequent purification steps.
Figure 1.
vity from
patite column (1 X 4 cm) equilibrated with the same
buffer. The flow-through fractions showing high activity were pooled and dialyzed overnight against 20
mM Tris-HCl (PH 7.8) containing 5 mM MgCb. The
dlaiyzed p'roteins were adsorbed to a DEAE-Sepharose
column (1 X 4 cm) equilibrated with the same buffer.
The protease activity, which was eluted in the flowthrough fractions unlike the first ion-exchange chromatography, were pooled and concentrated by ultraflltration using a centricon (Amicon). Mter concentration, the samples were loaded on a Superose-12 column (1 X 25 em) equilibrated with the same buffer
containing 200 mM NaCl. Fractions of 0.5 ml were
collected at a flow rate of 30 ml/h and assayed for
casein hydrolysis. As shown in Figure 2A, two peaks
of casein-degrading activities were eluted from the column.
We then examined the purity of the activity peaks
by polyacrylamide gel electrophoresis in the presence
of SDS followed by staining with silver nitrate. Figure
2B shows that the first peak consists of two polypeptides with different sizes. However, no protein band
could be detected in the fractions corresponding to
the second peak even by the silver staining. Therefore,
we concentrated our further studies only on the first
proteolytic peak, which was named as LSP. The purified LSP was stable at least for several months when
stored at - 20 °C in 20 mM Tris-HCl (PH 7.8) containing 5 mM MgCh. However, repeated freezing and
thawing inactivated the enzyme.
The native and subunit sizes of LSP were estimated
using the data obtained in Figure 2. LSP behaved
as a 34-kDa protein upon the gel flltration under nondenaturing condition, but could be separated as two
bands with sizes of 16- and l2-kDa upon the gel electrophoresis with SDS (Fig. 3). These results suggest
that LSP is a heterodimeric complex. However, it is
also possible that LSP consists of two identical subunits of 16~kDa and the smaller polypeptide is derived
Vol. 3 (1993)
lung Kwon Yoo et al.
26
27
28
29
30
31
90
40
~ 16 kDa
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40
60
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FRA CTION NUMBER
Figure 2. Gel fIltration chromatography of the casein-degrading actiVity on a Sup erose-12 column. The protein preparations from the DEAE-Sepharose step were subjected to chromatography on a Superose-12 column as described in the
text. Fractions of 30 III were assayed for casein hydrolysis
(A) and subjected to gel electrophoresis in the presence of
SDS (B). Proteins in the gel were then visualized by silverstaining.
,
/
•
8
6
10
pH
Figure 4. pH-dependence of the casein-degrading activity of
LSP. Effect of pH on the activity of LSP was determined
using the following buffers: glycine-HCl for pH 3.0-3.5, sodium acetate for 4.0-5.5, MES for 6.0-6.5, MOPS for 7.0-7.5,
Tris-HCl for 8.0-8.5, sodium bicarbonate for 9.0-10.5.
Table 1. Effects of various protease inhibitors on LSP.
Inhibitor
.
-
DFP
•
~
I
0
2 .0
~
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::
•
1.5
::lE
% Inhibition
0.5
1
53
100
30
94
0
1.5
"
0
./
h
1.0
1.~
1.6
1.6
Ve/Vo
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2.0
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<
PM SF
)(
::lE
./
~
"0
Concentration (ruM)
2 .0
2.5
...J
1. 0
0.9
Rf
Figure 3. Estimation of the molecular weight of LSP. Both
it, native and subunit sizes of LSP were estimated using
the data from Figure 2. Arrows indicate where LSP eluted
from the Superose-12 column and the subunits of LSP migrated in 15% slab gel containing SDS (B). The size markers
used for the gel fIltration are (a) alcohol dehydrogenase (150
kDa), (b) bovine serum albumin (66 kDa), (c) carbonic anhydrase (29 kDa), and (d) cytochrome c (12.4 kDa), and those
for the electrophoresis are (e) bovine serum albumin (66
kDa), (f) ovalbumin (45 kDa), (g) trypsin inhibitor (24 kDa),
and (h) RNase A (14 kDa).
from the 16-kDa subunit, such as by an autolytic process. Clarification of the relationship between these
subunits requires immunochemical studies and their
amino acid sequence analysis.
The effects of varying pH on the casein degrading '
activity of LSP were examined by using various buff-
TPCK
EDTA
o-phenanthroline
NEM
5
1
10
1
10
46
52
57
0
The purified LSP was incubated with the inhibitors for 10
min at 37 °C prior to the addition of [3HJ-casein. DFP
(diisopropyl fluorophosphate); PMSF (phenylmethylsulfonyl
fluoride); TPCK (Tosyl-L-phenylalanine chloromethyl ketone); NEM (N-ethylmaleimide).
ers. LSP was maximally active over broad pH range
of 5.5-9.5, but without a sharp pH optimum (Fig. 4).
However, it became inactive at pH below 5 or above
I O. We also examined the effects of various inhibitors
and site-specific reagents on the enzyme activity by
incubating it with the agents for lO min at 37 °C prior
to the addition of casein. As shown in Table I, diisopropyl fluorophosphate (DFP) at I mM completely
blocked the casein-degrading activity of LSP. Phenylmethylsulfonyl fluoride (PMSF) also inhibited the activity. In addition, LSP was sensitive to inhibition by
metal chelating agents, such as EDTA and o-phenanthroline, but not by sulfhydryl blocking agents, including N-ethylmaleirnide. These results clearly demonstrate that LSP belongs to a group of serine proteases.
156
A Latent Serine Protease in E. coli
Discussion
During purification of proteases Mi and Fa, we fortuitously isolated a new, additional serine protease
(LSP) in E. coli. From 8 g of the crude extract, approximately 1 flg of the purified LSP was obtained. However, the final yield as well as -·the specific activity
could not be calculated until the last step of purification, because the crude extract contained many other
proteases which are active against casein and because
the final Superose-12 column step eventually separates
LSP from the unidentified, low molecular weight proteolytic activity.
In a number of criteria, LSP appears distinct from
other known E. coli proteases, including proteases Do,
Re, Mi, Fa, So, La, Ti, Ci and Pi (Goldberg et al.,
1981; Swamy et al., 1983; Chung and Goldberg, 1981 ,
1983; Park et al., 1988; Hwang et al., 1987, 1988). LSP
has a relatively small molecular mass (i.e., 34-kDa),
while the sizes of the previously isolated serine proteases are in range of 100-500 kDa. Its activity is latent
(see below), unlike the others. It is the most sensitive
enzyme to inhibition by DFP or PMSF among the
serine endoproteases so far been isolated. Of particular
interest is the finding that LSP is a latent enzyme,
whose activity can be observed only after the incubation at 4 °c for about 3 weeks. Incubation for shorter
periods can also result in the enzyme activation but
to a lesser extent. No other treaments so far been tested can overcome the latency under condition without
the incubation at 4 t . Therefore, the mechanism and
physiological signiflCance' of the latency of LSP are
presently unknown.
Noteworthy is the unusual chromatographic behavior of LSP during cation-exchange column chromatography. This enzyme was adsorbed to a DEAE-cellulose column, which was used as the initial step for
purification, although its affinity was relatively weak
(i.e., eluted from the column at a salt concentration
of about 20 mM) (Fig. 1). On the contrary, LSP was
recovered in the flow-through fractions when it was
again chromatographed on a DEAE-Sepharose column as the later step for purification. Therefore, it
appears possible that a certain, unidentified molecule
(s) is bound to LSP, and the removal of the molecule
by incubation at 4 °c for 3 weeks or during subsequent purification steps may result in the loss of affinity to the ion-exchange resin as well as the loss of
its latency. However, the latter possibility is less likely
because LSP was recovered in the flow-through fractions even when the LSP fractions from the DEAEcellulose column were ·directly applied to the second
DEAE-Sepharose column (data not shown). Perhaps
the regulatory molecule is slowly hydrolyzed by LSP
Mol. Cells
in the complex during the incubation at low temperature and this proteolysis may be responsible for activation of the enzyme and the changes in chromatographic behavior.
Acknowledgment
This was supported by grants from Korea Science
and Engineering Foundation through SRC for Cell
Differentiation and The Ministry of Education.
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