Complete amino acid sequence of Scytalidium lignicolum acid protease B

May 27, 2017 | Autor: Kohei Oda | Categoria: Biochemistry, Amino Acid Sequence, Hydrolysis, Biochemistry and cell biology, Chymotrypsin
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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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