Complete amino acid sequence of Scytalidium lignicolum acid protease B
Descrição do Produto
J. Biochem. 95, 465-475
Complete
Amino
Acid Protease
Acid Sequence
of Scytalidium
(1984)
lignicolum
B1
Tetsuo MAITA,* Shuichi NAGATA,* Genji MATSUDA,* Shinsaku MARUTA,** Kohei ODA,*** Sawao MURAO,*** and Daisuke TSURU**,2 *Department
of Biochemistry
Sakamoto-machi, Sciences, and
Nagasaki
The
protease and
of
acetic
acid.
tional
methods
were The
composed
and
carboxyl-termini,
established
to
Scytalidium tryptic
Cys47-126
of
SLB
and
acid
residues Locations and
hitherto
In 1974, Murao and Oda (1) reported that Scytali dium lignicolum, a strain of fungi imperfecti, pro duces three types of acid proteases. These en I This
work
from
was
supported
the Ministry
of Education,
Japan. 2 To whom
requests
Faculty
Pharmaceutical
of
versity,
SLB,
for
Bunkyo-machi
Abbreviations: c ysteic
DTNB, protease
S-car
pepsin
of
Vol.
95,
No.
should
Nagasaki
Nagasaki,
Nagasaki
boxy
Scytalidium
propane;
methylated;
.
Uni 852.
acid); DAN,
EPNP,
1,2
Streptomyces
(acetylpepstatin).
2, 1984
carboxymeth
were
isolated
peptides
disulfide
by
slight
of
enzymatic homology
by
SLB. valine
and
and
conven
The as
enzyme
its
bridges
dilute
amino
were
also
fragmentation was
found
of in
the
reported.
zymes, designated acid proteases A-1, A-2, and B, were highly purified and their properties were characterized (1-3). Interestingly, these three en zymes were exceptionally insensitive to diazoacetyl DL-norleucine methylester (DAN) and Strepto myces pepsin inhibitor (S-PI, acetylpepstatin), both of which were known to be specific inhibitors for acid proteases (carboxyl proteases) such as pepsin, rhymosin and those of micrnbial origin (4) There are comprehensive studies on reaction mechanism of proteolytic enzymes, especially of serine proteases (5) and metallopeptidases (6). However, the reaction mechanism of acid pro teases has remained somewhat unclear, though the three-dimensional structures of pepsin, penicil
CysO3,
lignicolum;
S-PI,
of
be addressed
Sciences,
methylester;
3-(p-nitrophenoxy) inhibitor
and Culture
5,5•Œ-dithiobis(2-nitrobenzoic B
diazoacetyl-DL-norleucine epoxy
by a Grant-in-Aid
Science
reprints
1-14,
Cm-,
acid; acid
in part
a
the
and
three
Cys192-201
Only
proteases
and sequence
threonine of
and
thermolysin,
acid
with
SLB. acid
reduced
fragments
fragments
amino
Cys140-163
other
these
complete
unmodified
was Five
with ƒ¿-chymotrypsin,
analysis
amino
lignicolum digestion.
respectively.
and of
29, 1983
the
204
852,
591
digested
sequence
be
denatured
sequences
further
of
Osaka
to
established
was
of Osaka
of
subjected
them
Nagasaki
University
August
B (SLB)
then
some
Nagasaki,
Chemistry,
Sakai,
for publication,
acid
ylated
Bunkyo-machi,
of Agricultural
Mozuume-cho,
Received
the
University,
***Department
Prefecture,
, Nagasaki University School of Medicine, Nagasaki 852, **Faculty of Pharmaceutical
Nagasaki,
465
466
T. MAITA
lopepsin,
and
Rhi=opus
(Rhizopus pepsin) Among the three lignicolum,
chinensis
acid
protease
have been established (4, 7). acid proteases of Scrtalidium
SLB
is the
lowest
protein (Mr: about 21,000) bohydrate (1-3), indicating target for determining the
molecular
weight
and contains no car that SLB is a good amino acid sequence
and comparing its active site structure with those of DAN-sensitive enzymes. Thus, we attempted to determine the amino acid sequence of SLB. The present paper describes the complete amino acid sequence of this enzyme and compares it with those of other acid proteases so far reported.
Peptides nm
AND
after
prussid
method
Analyses
tides
carried
acid
HCl
in
Procedures
of
ylated
digestion
(Cm-)
SLB
of
with
(13)
Worthington
Biochemical
fragmentation trypsin
of (twice
(Nakarai
in
10)
Co.),
the
and
conditions
unmodified
thermolysin
were
bridges.
cedures
are
also
of
matographic
(200-400
ditions and
by in
legends
matography pyridine min,
with
also
carried of "
each of on
the
figures. paper buffer, upper acid-water
tified
by
using
a
high
dena Co.)
locate
in
(Merck)
(20,
21).
and
was
carried
and
that
8.1,
LC-3A
peptides,
iden
a
column
equilibrated
buffer
system on
a
(19)
silica
solvent with
gel
system
carboxypep
Biochemical Tris-HCl
in
program. were
with
Cm-SLB
0.1M
sequenator
ODS
conventional
in
net
chromatography
(Worthington
out of
Jeol
liquid
of
B
assign the Edman
acids
acetate
Digestion A
the
chromatography
with
(17) papers
peptide
ZORBAX
layer
Edman
from
a
long
performance type
(14).
Automated
amino
of
acid
Sasaki
methods,
(18).
Shimadzu
color
amino
previous
deduced
a
22
procedure in
latter
6 N
for
subtractive
was
using
200A
Ehrlich
the and
performed
47K)
thin
tidase
shown
Co.)
buffer,
0.02M
pH
borate
8.0,
buffer,
pH
respectively.
the
experimental
pro
RESULTS
chro
Chromo
Beads
Technicon
are
to
JAS
by
(15),
phenylthiohydantoin
and
110"C
by
Se pep
RESULTS."
on
Bio-Rad
case
are
(Sigma
out
the
was
(Model
plate and
of
pepsin
peptides
degradation
detected
the
the
acetonitrile-sodium
Fragments-Column
Co.),
methods,
and
mentioned
in
pH
6.4,
(4
at
nal
1•~2
mated
were
car
to
the
con
and
performed
phase
Terminal
(cat
2,200
of
a
mixture
:1
: 5,
v/v/v),
V
chro
of 1-
of
Cm-SLB
up
The was
70
(mot
after
to
the
by 15-th
auto residue
typical
C-terminal to
be
digestion,
sequence
-Ser-Tyr-Val
which
by
released
Ser,
mol
protein);
Tyr,
0.4;
and
Val,
0.8
and
Ser,
Tyr,
0.9;
and
Val,
0.9
incubation Isolation
a
deduced A
per
10min
after 1h
respec
N-termi
determined
Thr-Val-Glu-Ser-Asn-Trp-Gly-Gly-Ala-Ile
Cm-SLB
0.1
Cm-SLB-The was
degradation
carboxypeptidase
10% for
of
Edman be
of
using
Sequence
sequence
Leu-Ile-Gly-Ser-Asp-.
"RESULTS"
Electrophoresis were
P AG
Laboratories)
conventional
acetate and
butanol-acetic tively.
Peptide
resin,
mesh, out
SLB
in
separation
exchange
ried
shown
case
of residues
by
papers
digestions
cases
and and
with
at
described
amide of
tubes
dansyl-Edman
as
and
Co.)
treatment
hydrolysis
Matsubara
the
with
previous
each
In of
6AH
method and
performed 10).
Jeol
determined
Edman (16),
ment
with
to
further
thermolysin
(Sigma in
of
Details
Isolation
ion
subtilisin described
Enzymatic
and
disulfide
Co.),
(9,
protein
was and
(4.6•~250mm)
of
5,5•Œ-dithiobis(2 alkali
of
sealed
ketone,
with ƒ¿-chymo
Sigma
were
"RESULTS."
tured
with L-(1-
and
peptides
crystallized,
acid
and
Co.]
larger
Chemical acetic
(9,
[treated chloromethyl
CN-nitro
Composition
after
according
were
The
S-earboxymeth
trypsin
tosylamido-2-phenyl)ethyl
dilute
the
with after
Acid
out
Tryptophan
phoresis at pH 2.3 and 4.3. The enzyme was S-carboxymethylated by the method of Crestfield et al. (8). Peptides
Cystine
by
analyses
analyzers
evacuated,
72h.
charge
and
Amino acid
were
amino
method
Proteins
reaction (DTNB)
of
quence-Amino
Manual
of
(11).
followed
230
and/or
(12).
Preparation of the Acid Protease B and the fllodification-The crystalline enzyme was prepared by the method described previously (1, 2), and its homogeneity was confirmed by disc gel electro
Fragmentations
or
at
ninhydrin
hydrolysis
were
acid)
absorption
with
alkali
peptides
- nitrobenzoic
by
reaction
fluorescamine containing
analysis
METHODS
monitored
280nm,
reaction
MATERIALS
were
and
et al.
of experiment,
of Tryptic
0.5;
Cm-SLB
at
Peptides 4ƒÊmol
30•Ž. of
of
Cm-SLB-In
Cm-SLB
was
J.
di
Biochem.
AMINO
ACID
gested
with
5h,
and
SEQUENCE
trypsin
the
(2mg)
resulting
chromatography in
Fig.
the paper
peaks
with
The
that
of
numbered
were
ments
the
in
T-3
to
the
and
T-4
in
The to
T-3.
IS
4
their
was
the
pH 3.1, were DE 52 column
are
peptides (Chy-1 to Chy-12) and 6 overlapping peptides (Chy-1 . 2, Chy-3 . 4, Chy-5 . 6, Chy-9. 10, Chy-9. 10. 11, and Chy-11 . 12) were obtained.
together
peptides final
a linked
align peptide
Their amino IIS.
T-4.
Sequence Studies of T-1 Peptide (Res. 1-113) -T-1 was a large peptide consisting of 113 resi
are Chymotryptic
trypsin
stirred
The
peptides
buffer
pH
45•Ž in
as
shown
fraction on
pH
system collidine
and
250ml
in
Fig.
1•~2
of
0.1M
acetic
Fig.
a
1.
The
8.5
Elution
tides were buffer:
Vol.
95,
No.
2, 1984
Beads
2%
of the tryptic
NH,HCO3,
45•Ž
digest gradient
pH 8.1.
were
the
conditions
so
as
peptides
of Cm-SLB
(80mg)
was dissolved to a column
of the first buffer
those Thm-5
that
the
on
DE
(700ml)
by Beads in
8.5
Pep
to the second
and Thm
successive P
column 2S)
Thm-6 of
of the first
to
Fig.
and
(1.8 was
pH
Thm-1
52 column
20cm).
. 4
Chy-4)
at
sequence
in 3ml (1.8
and
digest
Chromo
electrophoresis.
sequenced,
Chy-3
peptides,
a
was
manual
Chy-3
(0.5mg)
from
the
identification.
sequence,
major
on
Chy-4 by
ambiguous
of
se
method
method.
thermolysin Six
were
Edman
residue
its
peptide
isolated
same
an
Chy-5
manual
11-th
with
peptides
non-overlapping
and
the
complete
8h.
the
a
in Table
chymotryptic All
Chy-4
the
linked
are shown
Edman
to
with for
2.
by
chromatography
pH 8.5, and applied
a linear
a
paper 250
to
digested
(the
pyridine,
by
mol,
- 6,
chamber),
A lyophilized
NH,HCO3,
the
with
followed
with
P
chromatog
(mixing
pattern
eluted
1.0M
of
ƒÊ
for
method
order
the
Fig.
subtractive up
Edman
peptides)
in
the
sequenced
In
of
in
completely
and/or
suspension
peptides
400ml
acid
0.05M
The
studies
except
quenced
centrifuged.
by
acid compositions
summarized
peptides
0.05M
(0.9•~20cm)
chromatography. buffer:
of
Chromo
column
pH
4h.
and
purified
acetate,
for 3ml The
pep
chymo
(soluble
IS.
further
composed
of
3.1.
on
AG
in
30min
T-1
with
37•Ž
supernatant
were an
1%
and
for
the
The
suspended
buffer,
at
T-1: digested
8.5
chromatographed
column
of
further
was
acetate
was
raphy
at digest
pyridine
first
was
(1mg)
lyophilized
were
peptides
(3.6 ƒÊmol)
isolated by chromatography on (Fig. 2S). Twelve non-overlapping
Sequence
dues. tide
467
paper electrophoresis (Chy-1 and Chy-1 . 2). The insoluble peptides in 0.05M pyridine acetate buffer,
by
of
Table
according
and
electrophoresis,
protein.
sequence.
shown
purified
B
ml of 1M acetic acid (reservior), and by paper electrophoresis. The peptides in other fractions were purified by paper chromatography (Chy-3, Chy-4, Chy-6, Chy-9, Chy-12, and Chy-11 . 12) or
for
(T-2)
compositions
shown
PROTEASE
by
as
first
further
acid
original
37•Ž
separated
the
paper
ACID
and
column
in
amino are
T-1
52
and
peptides
8.5 were
DE
chromatography
isolated
pH
involved
(T-3)
respectively.
of
a
Peptides
second
at
peptides
on
1.
OF Scytalidium
Chy-4
and were was
468
T. MAITA
et al.
J. Bioche,n.
AMINO
ACID
established
(Fig.
drolyzed
with
for
in
15h
peptides,
2).
5ml an
From
the
Ach-2,
sealed
tube
with
was
of
110•Ž .
Two
by
the
Chy-5
results
of
Chy-5
as
.
shown
in
shown
in on
of
order
of
the
N-terminal
should
be
it
a
had
to
8h,
3S.
Overlapping
(AC
18/42)
were
obtained by
consisted Ile, Val,
Asx,
and
was
chromato
shown and
and
Chy-9
(AC
fractions The
Ser5,
Gty4
Ser,
no
and
with 55•Ž,
overlapping
peptide
alignment
of
Chy-6
to
of the chymotryptic
total
residues
of the
agreement
with
the
12 peptides
was
composition
of
of T-2 (Res.
114-116)
and
T-3
up
of
(1.0mg)
of
the
at
peptides
chymotryptic
95, No.
. 4 pH
as
(Res.
12-th
and
those
2, 1984
128-204)-The 77
residue in
37•Ž by
used
for
of
T-1.
with for
the
3.
the An
isolation elution
same of
pattern
the
the
of
of
of
peptides
them
Chy-5
were was
of
manual
Chy-4.
residue
5
some
it (1.6 ƒÊmol) at
pH
was
8.5
and
isolated
Chromo
by
Beads
sequenced,
established
and
Chy-5 with
were on
were
peptides
Chy-4.
Then
of
(Thm-1
them
results
(0.75mg) 8
by
column
Thm-3.4)
and
18-th
and
so as
P that
shown
3.
T-3
. 4 was
the
sequence
of T-4 and
result the
the
subtilisin
Some
P
thermolysin
Chy-4
chromatography
sequence Fig.
to
7.0
isolated
major
some
the
from on
and
pH
Beads
digestion
2 h,
at
out
digested
were
and of
deduced
Alignment 3.8
The
almost
in
8 h
Seven
obtained
up
paper.
deduced
carried
(Thm-2•Œand
identifications.
for
on
was
further
Chromo
minor
sequenced
successive
manual
About
chymotrypsin
4 h.
using
was
for
a
degradations
37•Ž
the
residues, by
Fig.
digested
isolated
peptides
of
shown
was 8.5
were
T-4
consists
as
T-3
procedures
Vol.
to
method
mol
ing
Studies which
two
with
14h,
isolated
chromatog
were
peptides
Alignment
digested
for
were
Chy-3
was
(0.5mg)
was
hy
degradations
Chy-4
resulting
were
ambiguous
and
for
carboxypeptidase
was
of
to
was
110•Ž
and
electrophoresis.
Chy-4
at Ach-4,
(1.8ƒÊmol)
on
sequenced.
order
3.
the
and
Edman
acid
to
method In
Edman
studies
paper -7)
up
Edman
(1.2 ƒÊmol)
to
sequence
Chy-4
and
and
acetic
of
N-terminal
sequenced
electrophoresis
thermolysin
to
(Res. 117-127)-The sequences of T-2 and T-3 were determined to be Val-Ser-Lys and Ser-Phe - Ser-Asn-Glu-Ser-Ser-Gly-Leu-Cys-Arg, respec tively, by the subtractive Edman method and the manual Edman method.
Edman
Fig.
follows:
Ala,
Chy-3
Ach-1
the
in
the
was
and
peptide
and
manual
Subtractive
peptides,
Val3,
Chy-3 the
0.25M
Sequence as
T-3
identifications.
paper
nona
Chy-2
overlapping
of
Fig. Fig.
below.
sequences,
an
com
isolated
N-terminal
their
by
From
given the
From
T-4.
peptides,
shown
a
sequenced
with
the
composition
be
pep
acid
for
acid
sequence,
successive
as
T 1.
peptide,
to
amino
amino
ambiguous
4
raphy.
in
Sequence
with
the
the
the sum
Sequences
studies
identical
of
some
peptides
in
sequence
dipeptide
is
insoluble
Table HIS and should consult
the
in
acetate
P column
the
studies
was
residue
complete
peptides was assumed to be Chy-1 to Chy-12 from the above results and suggested by the fact that of the
The
chromatography
obtained,
reasonably
5S.
sequence.
with
Fig.
Pro,
in Fig.
T-3
16-th
66/75)
Ala6,
Glx2,
the
these
peptide
of
considered
Chy-5
in
former
Pro, of
Fig.
that
sequence
C-terminal
by
in
and
pyridine
Beads
summarized in The readers
of
drolyzed at
Trp2.
Although Chy-8
was as
de
4S,
52,
pentapeptide
and
acid
Chy-4
latter
the
partially
of
the
was
were
was
peptides
Thr3,
Chy-12
the
acetic
indicated
terminal
Chy-12
hydrolysate
the
and
to
0.25M
Chy-8
from
peptides
column
of
peptide
a Chromo
and
3 to follow
because
establishing
electrophoresis.
Trp,
Tyr2,
T-4
DE
in 0.05M
on
Fig.
peptides are 3, respectively.
T-1:
Chy-9
P-4
from
of
and
linked
(2.0 ƒÊmol) of
the
Biogel
and
paper
in
T-1
T-1,
Chy-6,
Chy-11
20ml and
a
of The
aided
Then
on
Cm-SLB.
to
and
with for
of
in
deduced
C-terminus
Chy-5
they
order.
hydrolyzed
was
lysine.
Chy-4,
and
peptides
Chy-3
the
Chy-11,
obtained,
3S
at
C-terminal
Chy-10
110•Ž
to
sequence
to
graphed
chymotryptic
Chy-1
placed
Chy-3
scribed
the
peptides
3.1,
Chy-1
The
of
469
soluble pH
2. Alignment
ƒÊ
the
positions
and
deduced
B
buffer,
tides
suc
chromatography.
on
Chy-5
PROTEASE
hy
at
isolated
and together
of
was
acid
were
degradation
sequence
Fig.
M acetic and
sequences
Edman
ACID
(1.2 ƒÊmol)
0.25
electrophoresis
their
manual
Chy-5 of
and
paper
OF Scytalidium
evacuated
Ach-1
cessive
6,
SEQUENCE
of
easily
the deduced
of T-3 Chy-4.
and
chymotryptic as shown the
N-terminal
peptides in Fig.
in
3 from
sequences
5.
Alignment of the Tryptic Peptides in SLB Alignment of the tryptic peptides in the protein was easily deduced to be T-1 to T-4 from following
470
T. MAITA
J.
et al.
Biochem.
AMINO
ACID
SEQUENCE
OF Scytalidium
ACID
PROTEASE
events: The N-terminal sequence of T-1 was iden tical with that of the protein, the C-terminal se quence of T-4 was identical with that of the pro tein, the linked peptide of T-3 and T-4 (T-3 .4) was obtained and the sum of the total residues of the 4 peptides (T-1 to T-4) was reasonably in agreement with the composition of the protein (Table IS). Locations SLB
of
was
of
0.01
denatured N
pepsin
was
freeze-dried,
pyridine
at
and
for
pH
of
were
Pep-1,
3.1,
in
tained
as
major acid
on Pep-3,
peptides Pep-1
Gly, Gly
was Asx, as
consisting Ser
as
Pep-2a
composed
Thr,
the
and
N-terminus; of
Ser,,
N-terminus.
and
Pep-2b,
Fig. 4.
Vol.
95,
No.
2, 1984
latter
Leu,
the
acid
Complete
bridge
by into former
residues, Glx,
two acid
covalent
Arg,
is between confirm
The of
with
1 mg
at of
pended
with
peptides, oxidation.
structure
in
trophoresis peptides, major
of Scytalidium
carried
65•Ž
out
for
of
pH
0.1M
6.3
of
and
cystine-containing
lignicolurm
Biogel
on
protease
for were After
P-4.
purified
were
components.
acid
45•Ž
with 5h. sus cen
chromatographed
further
Thm-2,
20 by
digested
acid.
chromatography
Thm-1
in
denatured
and
was
were
denatured
peptides
acetic
(1.6•~150cm)
the
then
the
thermolysin
dissolved
and
and
supernatant
peptides and
5.8,
20min at
for was
pH
freeze-dried, 2ml
the
2.5ƒÊmol,
CaCl2,
the
a column
containing
conclusion,
thermolysin
being
trifugation, on
also
enzyme,
10mM
After
Cysl40 and Cys163 this
was
SLB:
heating
a hexapeptide and
gave
being composed of 11 amino acid residues, Thr2, Asx2, CysO3, Sera, Val, Gly, and Glx, with Thr as its N-terminus, suggesting that another disulfide
ml
resulting
separated
and
was
residues, Glx, Asx2 Val3, Thr3, (CysO3)2, Ser4, and Gly, indicating that Cys192 and Cys201 form a disulfide bond (see Fig. 4). Pep-4 was separated into two peptides, Pep-4a and Pep-4b, after oxi dation. The former peptide was a decapeptide consisting of Phe, Glx2, CysO3, Asx3, Ser, and Gly, with a N-terminal phenylalanine, the latter
digestion
ob
subjected
The
CysO3,
performic
pep
separated
amino
Pep-2 after
and were
in Pep-lb. These results suggest that one of the disulfide bridges is between Cys47 and Cys136, as shown in Fig. 4. Pep-3 gave a single spot after oxidation and was composed of 16 amino acid
To
were
were
(CysO3)
the Gly,
its
5
acid
with
Four
Pep-lb.
of
cysteic
under
cystine
Pep-4,
was
a
471
Pep-2a showed the same amino acid composition as that of Pep-la, and Pep-2b contained Thr and Asx in addition to the amino acid residues found
electropho
paper.
(22),
acid-containing
P
The reaction
They
electrophoresis.
peptide
centrifu
by
and
oxidation
Pep-la
0.05M
on
1S. by
paper
peptides,
digest
of after
purified
cysteic
two
20ml
The
Beads
Fig.
components.
performic
2 h.
and
detected further
Pep-2,
in
incubation
chromatographed
chromatography
tides,
of
5ml
Chromo
as
peptides (12)
resis
by
in
was
conditions
DTNB
5min
37•Ž
buffer,
(0.9•~20cm)
containing
for
digested
dissolved
supernatant
same
90•Ž
then
(2.5mg)
the
column
to
and
acetate
gation
Bonds-Five ƒÊmol
at
HCl
with
the
Disulfide
B
B.
Cystine by
paper.
elec Two
obtained Each
as of
472
T. MAITA
them were oxidized with performic acid, and the resulting cysteic acid-containing peptides were sep arated by paper electrophoresis. From Thm-1, three peptides, Thm-la, Thm-lb, and Thm-1c, were obtained. Thm-la was a pentapeptide con sisting of Val, Thr, Asx, CysO3, and Ser, and Thm-lb contained 4 amino acid residues, Val, Sera, and CysO3. Thm-lc was a tripeptide lacking one Ser in comparison with Thm-lb. All of these peptides gave Val as their N-termini. These data indicate that Thm-1 was a mixture of two peptides and both were derived from the Cys192-Cys201 region. Thm-2 was separated, after oxidation, into two peptides, Thm-2a and Thm-2b, by paper electrophoresis. The former was a heptapeptide composed of Ile, Asx2, Gly, Thr, CysO3, and Glx, with Ile as the N-terminus. Thm-2b was a tetra peptide consisting of Gly, Leu, CysO3, and Arg, with Gly as its N-terminus. These data suggest that Thm-2 is the peptide linked at Cys47 and Cys126. Although we failed to isolate another cystine peptide containing Cys140 and Cys163 from thermolysin-digest of SLB, the results shown above lead us to conclude that the three disulfide bonds in SLB are Cys47-126, Cys140-163, and Cys192-201 (see Fig. 4).
Scy.lignicolum Porcine Cow
DISCUSSION
The complete amino acid sequence of SLB was established as shown in Fig. 4 from the results described above. The enzyme is a single poly peptide composed of 204 amino a molecular weight of 21,969.
able, because sequence analysis showed that car boxyl group of the methionyl residue of SLB links to amino group of the Beryl residue, and peptide bonds of Met-Ser and Met-Thr have been reported to be resistant to cyanogen bromide cleavage (23).
It has been reported
that DAN-sensitive
penicillopepsin, shows about 40% homology to animal pepsin in the primary structure (7). Most of these DAN-sensitive acid proteases have almost the same molecular weight of 32,000-36,000, and two aspartic acids, one of which is reactive to
Present
B
(7) (7) (7)
chymosin
(7)
Penicillopepsin Rhizopus
(24)
pepsin
Scy.lignicolum
Calf
Present
B
(7)
chymosin
* ** 5.
Homology
acid proteases.
result
(7)
pepsin
(7)
Penicillopepsin
Fig.
result
(7)
pepsin
Porcine
acid
proteases show high homology in their three dimensional structures as well as in their amino acid sequences, regardless of their origin (4, 7). An acid protease from Penieillium janthinellum,
pepsin
Calf
acid residues with The three intra
molecular disulfide bridges are deduced to be Cys47-126, Cys140-163 and Cys192-201. 1he enzyme contains one methionine reidu. We attempted to cleave the protein at methionine residue by cyanogen bromide but failed to obtain two fragments. This failure seems to be reason
pepsin
Human
et al
EPNP-reactive DAN-reactive in the Identical
amino residues
acid
aspartic
acid
aspartic
acid
sequences
are shown
of Scytalidium
acid
protease
B and
some
DAN-sensitive
in boxes.
J. Biochem.
AMINO
ACID
SEQUENCE
OF Scytalidium
ACID
PROTEASE
DAN, have been concluded to participate in ac tivity of these acid proteases (7) . On the other hand, DAN-insensitive SLB has a molecular weight of about 22,000, and the present results indicated that the amino acid sequence is quite different from those of DAN-sensitive enzymes such as
6.
(Fig. 5). Interestingly, Asp32 of pepsin is reactive to a specific pepsin inhibitor, 1,2-epoxy-3-(p-nitro phenoxy)propane (EPNP) and is considered to be one of the active site amino acids in DAN-sensitive acid proteases (7), and SLB is also inactivated by EPNP (1). Furthermore, the above sequence in SLB is also somewhat homologous to those around DAN-reactive Asp215 of pepsin-type acid proteases (Fig. 5). It in
remains
SLB
unclear
is one
SLB
and
lems
are
of
the
reactive now
to
under
at
present
active EPNP
site or
not.
whether amino These
Asp" acids
of
prob
7.
8.
9.
11.
2, 1984
(1976)
Acid
T.,
Maita,
T., Eur.
Moore, 238,
Odani,
S.
Smith, Matsubara,
I.
16.
&
Matsuda,
(1963)
G.
360,
Kato,
Y.,
(1979)
1483-1495 &
Matsuda,
G.
45-49
Chemistry
of
Vol.
&
II,
Proteins pp.
Wetlaufer,
Nature
H. Res.
Blomback,
W.H.
(Narita,
277-332,
K. Tokyo
D.B.
(1975)
Anal.
493-502
(1953)
Biophys.
London
Tokyo
67,
14.
Function and
Stein,
Chem.
114,
eds.)
W.L.
13.
B.
in
Dozin,
Anderson,
&
K., Phvsiol. T.,
T.,
York
S.,
Biochem.
(1976)
Murachi,
284-305,
622-627
Umegane, J.
pp.
Structure, New
Morokuma,
Biochem.
15.
Press,
Z.
(1980)
Metalloproteins
eds.)
Proteases:
Plenum
Chem.
Maita,
of K.,
Tokyo
A.M.,
Biol.
Kagaku 12.
&
171,
Common. B.,
R.M. 35,
Blomback,
(1966)
Biochim.
Konigsberg,
W.
43-44
Sasaki,
M.,
Biophys. &
(1969)
Edman, Acta
Hill,
Biochem.
175-181
R.J.
P.,
115,
&
Hassel,
371-396
(1962)
J.
Biol.
Client.
237,2547-2561 17.
Hartley,
18.
Offord,
19.
Zimmerman,
B.S.
(1970)
R.E.
(1977)
Biochem
(1966) C.L.,
Anal.
Jeppson,
&
211,
Appella,
Biochem.
J.O.
J.
Nature
77,
119,
805-822
591-593 E.,
&
Pisano,
J.J.
569-573
Sjoquest,
J.
(1967)
Anal.
&
Robinowitz,
Biochem.
18,264-269 21.
No.
J. Biology
Crestfield,
&
Chemistry
Yamanaka,
Hoppe-Seyler's 10.
in
&
Scientific,
Tang, and
REFERENCES
95.
(1982) S.
Kodansha
20.
Vol.
D.
(Otsuka,
investigation.
1. Oda, K. & Murao. S. (1974) Agric. Biol. Client. 38, 2435-2444 2. Oda, K., Murao, S., Oka, T., & Morihara, K. (1975) Agric. Biol. Chem.39, 477-484 3. Oda, K., Murao, S.. Oka, T., & Morihara, K. (1976) Agric. Biol. Chem. 40, 859-866 4. Tang, J. (1976) Trends Biochem. Sci. 1, 205-208 5. Sakiyama, F. (1983) Seikagaku (in Japanese) 55, 166-176
473
Tsuru,
J.
pepsin and penicillopepsin. However, only a slight sequence homology was observed between SLB and other acid pro teases. The sequence Asp98 to Leu105 in SLB showed some homology to those around Asp32 of pepsin and other DAN-sensitive acid proteases
B
Cherbulez,
E.,
Helv. 22.
23.
Chins. C.H.W.
(1967)
(Hirs,
C.H.W.,
ed.)
Press,
Inc.,
Koide, &
K.
New
Kagaku Nakamura, 81,805-807
Dozin,
in Vol.
in T.,
T.
(1964)
1350-1353
York
(1976)
Murachi,
B.,
47,
Hirs,
K.
24,
Baehler,
Acta
Methods 11,
&
pp.
Enzymology Academic
London
Chemistry
eds.)
in 197-199,
in
Vol. ‡U,
Proteins
pp.
(Narita,
234-254,
Tokyo
Tokyo S.
&
Takahashi,
K.
(1977)
J.
Biochem.
474
T.
MAITA
et al.
J. Biochem.
AMINO
ACID
SEQUENCE
OF Scytalidium
ACID
PROTEASE
Legends
B
of
supplementary materlais
Fig. 1S
Elution
Chrome the
Beads
was
the
pH
Peptides
were
peptide
analyzer
3.1;
dissolved 30cm).
reaps
1.0ml
of
of
Elation
of
peptides
of
ninhydrin
4,
and
120ml
2.0M
of
pyridine
reaction
using
on of
(see
were 1
T-1
suspension
pH 3.1,
Chambers and
of
the
the
eluted 2,
at
120ml
of
0.2M
pyridine
acetate,
pH
an
5.0.
automatic
Co.).
pattern
2ml
3
of
buffer,
The system:
chambers
of
the
chromatography.
in
acetate
buffer
peptides
supernatant
(0.9•~17cm).
5 and 6,
by
chymotryptic
The
pH 3.1;
(Technicon
column
soluble
pyridine
elution
chambers
Elution
the
0.05M
column
detected
2S 52
a
acetate,
acetate,
DE
in
following
pyridine
Fig.
to
of
chromatography.
digest
applied
55•Ž with 0.05M
pattern
P column
chymotryptic
text)
on
475
insoluble The
0.050 NH4HC03, peptides
pH was
chyotryptic
insoluble 8.1,
peptides
peptides and
carried out
e
(see
applied
to
described
of T-1
the a
in
text)
column
were (1.8•~
Fig.
1
in
the
text.
Fig.
3S
acetic text)
as
95,
No.
2, 1984
Beads
5S
T-3.4
on
other
conditions
Chy-4.5.
0.1M
Elution
DE
1.
column
peptide(s)
of The
T-1
with
0.25M
hydrolysate of
(see
peptides
the
was
acid.
of
the
soluble
chromatography.
chymotryptic The
peptides
of
chromatography
T-3.4
carried
1S. of
the
insoluble chymotryptic
chromotography. the
peptides
(1.0•~150cm). Elution
acetic
Fig,
hydrolytic
chromatography.
column
pattern
were of
a
the
column
pattern
Elution 52
of
F-4
P column
described
Fig.
composition
to
with
4S
Chrome
out
pattern
Biogel
applied out
Fig.
Vol.
on
was
carried
on
Elution
acid
same in
as fraction ‡W
those
Size
of
in
Fig. as
peptides column I
almost
vas in
the identical
of
0.9•~50cm. text.
The Amino
with
acid that
of
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