Observations on the specificity of a thermostable bacterial protease “thermolysin”

Vol.

21,

No.

BIOCHEMICAL

3, 1965

AND BlOPHYSlCAL

RESEARCH COMMUNICATIONS

OBSERVATIONS ON THE SPECIFICITY OF A THERMOSTABLE BACTERIAL PROTEASE “THERWOLYS IN”

Hiroshi

Matsubara,

Space

Sciences

Received

October

(1962)

University

of

with

the

commercial

&.

thermonroteolvticus

stable

at

high

temperatures

reported et

Thomas

H.

Jukes

Berkeley,

California’

al,

that

the

l%S).

We propose

Rokko, (t:80°)

enzyme

had the

was isolated

8’Thermoase”

name

of

recently

and

California,

cultures

comparatively

(Morihara

Sasaki

4, 1965

enzyme

from

Richard

Singer,

Laboratory,

A proteolytic

was

Alan

in

a strong trivial

and the

was

found

to

presence

of

Ca*.

elastase-like name

by

Endo be It

activity

thermolvsln

for

this

enzyme. The

substrate

cytochrome

s

dimensional

specificity

as

a substrate.

paper

found

that

groups

of

used.

Three

alanine

other

isolated

according

deionized

water

x

as

and

also

Methods:

the

described

by

were

in

the

amino

separated

and

by

twoIt

was

involving

g under of

heart

analysed.

bonds

cytochrome sites

beef

the

the

conditions

phenylalanine

and

fi

a

ised. Endo

(1953)

Thermolysin (1962).

All

pure

beef

was

dialysed

was other

heart

cytochrome against

purified

by

reagents

used

s

distilled

and

crystallization were

analytical

grade.

‘Supported Aeronautics 2We are

by

indebted

grant NsG 479 to the and Space Admlnistrat’on. to

Dr.

-NH2

hydrolysed.

Hagihara lyophil

using

12 peptide

Chromatographically to

and

produced

residues

involving

studied

electrophoresis,

10 of

leucine

bonds, were

and

4 times

and

was

peptides

hydrolysed

isoleucine

j’aterials

thermolysin

The

chromatography

thermolysin

residues,

of

Endo

for

University

a supply

242

of

of

the

California

crude

by

enzyme.

the

National

Vol.

21,

No.

3,1965

About was

10 mg of

adjusted

O..Ol

to

% enzyme

buffer, for

4.5 0.1

was

added 0.1

and

paper.

the

made

and

one

location

far

the

was

elution

of

was

hydrolysed

in

a sealed

analysed

by

the

and

cvonents

showed

analysed.

The

fragments

with

(Yasunobu

et

complicated

composition

al,

1963)

phenylisothiocyanate method or tide

was

isolated

Results

by and

on Ehrlich

Discussions:

the

method

et the

sufficient

al, of

in

the

cases

in

sequence

1956)

was

the

peptide.

sheet;

1 shows

g& & sheets

acetone

to

it

was

were identify

solution drying,

each

24

hr

pep-

at

105’-

was

accelerated al,

system

(1958).

clear

Minor

spots

to

identify

in

cytochrome

were the

1965)

or

a carboxypeptidase

to

identify

either

The

a pH

1959)

a modified

applied

15:lO:

and

which

6 Greenberg,

(Smith, Fig.

Spackman

by =

Three

an

3MM

water

composition

with

was

a few

then

After

acid 1208

of

peptides

(Light

reagent

Model

positions

and

6 N HC? for amino

acid dissolved

No.

ninhydrin

acid.

the

in

a different

acetic

mainly

their

residue

0.02%

and

except

et

terminal

identified

to

in

compositions of

method

(Fraenkel-Conrat

carboxyl

on

with

The

analyser

based

V/cm.

twice-distilled

tube.

automatic

respect

of

30

solution

30%

was

Whatman

Katz

8.0

at

chromatography v/v,

at

others

with

evacuated

cuvettes

of

the

1:10:289,

ninhydrin the

ml

for

out

acetic

material

a sheet

of

carried

glacial

dried

pH

ml

maintained

acid:

1.5 hr

for

and

0.5

pH was of

the

0.5

was

chromatography

used =

0.1%

peptides

with

a Spinco

sensitive

was

peptides

on paper

acid:water

the

the

The

was

added

reaction

1 drop

spotted

1953)

and

methane)

(butanol:pyridine:acetic

Watson,

with

water

was

The

reaction

by

of

COMMUNICATIONS

(hydroxymethylamino

reaction,

first

2 ml

solution

lyophilised.

made

sprayed

of

the

ml

which

this

M Tris

the

of was

was

electrophoresis

tne

10G°C

end

(pyridine:acetic

and

tide

during

RESEARCH

in

chloride.

A BPAW system

Walley

3.7 buffer

0.1

0.01-0.02

map

is .

v/v,

and

and

To

M NaOH.

and the

BlOPHYSlCAL

dissolved

M calcium

solution

water

rophores

3:12,

for

the

of

0.002

At

A peptide

e I ect

0.1

35’-37’C

N NaOH.

Ewas

containing

and

at

ml

with

solution

hr.

AND

cytochroma

8.0

pH 8.0,

with

in

BIOCHEMICAL

g

Edmanps

the

amino

tryptophanrcontaining

separated

only

pepby

BPAW and

1953). the

243

peptide

map

of

the

digest

of

beef

Vol.

21,

No. 3, 1965

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

: ‘I ‘\ : -. I \

, 4

-.

I-\ . -’ ’

0 14

tit---

P”

3.7,

30 “/cm

1.5 hr. ___t

3 2 m

w

Fig. 1. Peptide nrap of thermlysin digest of beef cytochrotne A. Ten mg of cytochrome E was digested with 0.05 mg of thermoiysin for 3.5 hr. at 35O-37’C. The peptide map was made by two dimensional chromatography (first, BPAW), and eiectrophoresis (second, pH 3.7, 30 v/cm, 1.5 hr.). Spots were developed by spraying with 0.1% ninhydrin solution.

cytochroma

E with

obtained

by

showed and

the

was

rately

tris.

Table in

Peptides as (Fig.

two

2,

good

chromatography

of

Peptide

cytochrome 14 was

and

4.

the

separation

the

peptides

electrophoresis.

Peptide

tryptophan

of

Peptide

13 showed peptide

was

yellow

which

3

color

was

located

sepa-

above). the

sequence 3,

corresponding 2).

color

I shows the

Fairly

dimensional

heme

(see

tion

thermolysin.

of

5,

10, to

The

amino

yield

each I2

peptide

composition,

peptide

separated

14 were

identified

and

regions of

acid

in

the 14,

known

amino-acid

containing

244

relative

yield,

as

and

shown

solely

by

numbered their

sequence tryptophan

and

posiin

Fig.

I.

composition of

and

the

cytochrome

obtained

E sepa-

Vol.

21,

No.

3,1965

rately, or

BIOCHEMICAL

was

not

carboxyl

calculated.

terminal

68 with

67

or

65

C4 or

82

to

85.

Since

one

must

be

81

to

84

4,

6, 7, 8 and

residues

of

tively.

Peptide

could

not

separate

to

since

met-i

Ten

out

,G were

I1

found

were

so

that

to

95

and of

component

67

released

of

the

Edman

examined between amount

peptide to

97

peptide

its to

of

peptide

found.

bonds

tyr-leu(2),

component

sequence

45

or

hydrolysed:

aspNH2-leu,

are

its

orlgin in

corresponding

in

Fig.

a to

No peptide

32

to

fragment

2.

gly-ileu(2), gly-leu,

respec-

However,

36 to 45.

results

Peptides

and

46.

be

amino-terminal

resldue to

spot, must

lsoleucine

fragment to

The

were

37

have

and

81 to

a main

85 to 94.

to

terminal

9 corresponds was

minor

the

residues

residues

as

procedure

36

to

the

amino

to

tyrosine

leucine for

their

mainly

and

leucine,leucine, not

for

corresponding

sequence,

by

COMMUNICATIONS

examined

I corresponded

consisted

a small

leu-ileu,

leu-phe,

to

eleven

following

leu,

64

distinguished

r:orresponding The

the

RESEARCH

were

carboxy-peptidase

9 was

found,

peptides

a minor

phenylalanine,

be

BlOPHYSlCAL

Peptide

peptide

experiment

115 was

Other

residues.

Cl4 to

peptide

AND

lys-ileu,

thr-leu,

gly-phe,

lys-ala. 12

(83%)

hydrolysed

of at

the the

isoleucine amino

site

and of

leucine

their

I 4cNH-Gly-Asp-Vai-Glu-Lys-Gly-Lys-Lys-~-Phe-Val-GluN-Lys-C~s-Ala-GluNH2-CysT 20 His-Thr-Val-Glu-Lys-Gly-Gly-Lys-His-Lys-Thr-Gly-Pro-AspNH2-~-His-Gly-~-Phe-

residues

peptide

in

linkages.

beef

cytochrome

Calculated

IO

Heme 1

30 t

40 Gly-Arg-Lys-Thr-Gly-GluNH2-AIa-Pro-Gly-Phe-Ser-Tyr-Thr-Asp-Ala-AspNH2-Lys-AspNH2Ip

60

+

70

Lys-Gly-m-Thr-Try-Gly-Glu-Glu-Thr-L T

I+

u-Met-Glu-Tyr-A-Glu-AspNH2-Pro-Lys-LysT

80

90

Tyr-~-Pro-Gly-Thr-Lys-Met-~-Phe-Ala-Gly-~-Lys-Lys-Lys-Gly-Glu-Arg-GlurF 100 Asp-&N-J&&-Ala-Tyr;~-Lys-Lys;Ala-Thr-AspNH2-GIuCOOH e Amino Fig. 2. by thermolysin are under1 ined.

t

50

T

104

acid sequence of cytochrome The isoleucine (arrows).

245

G , showing and leucine

peptide residues

bonds hydrolysed in cytochrome

c

Vol. 21, No. 3, 1965

BIOCHEMICAL

AND BlOPHYSlCAL

ml

WI.

-I

r; . -

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0. . -

RESEARCH COMMUNICATIONS

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.

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246

I . -. 4-N

-I

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a; d

a; d

Vol.

on

21, No.

the

3, 1965

basis

of

hydrolysis

in

and

An

peptide

ant

phenylalanine

The

fact

suggests enzyme insulin

the

and

isolated

from

bonds

involving

glucagon

of

residues

tha.

primary

the

-NH2

group

used

is

as

Further protein.

further

a rapid

test

seem

to

lead

to

in

the

time

the

in

that

the

number

of

progress,

using

of

hydrolysis

hydrolysis

of

(Morihara

et

tyrosine, al,

1965).

&,a,L,

reported

amino

1965)

to

sites

ic

hydrolyse

of

leucine

leucine

and

1965).

usefulness

that

hydrolysis

it

enables

the

residues

so

insulin or

other

to

A proteolyt

attacking

fact

between

found

valine,

was

potential

interest for

are

the

The

recently

enzyme.

atrox

of

varied

(Morihara

Pseudomonas

preferentially is

the

leucine,

insulin

E Krauss,

linkage

was of

involving

77%

contrast,

hydrolysed

activity

Crotalus

about

neutral.

sites

the

in

In

bonds and

of

COMMUNICATIONS

enzyme,

aeruqinosa

amino

bonds

the

residues.

basic

it

and

RESEARCH

by

the

elastase

proteins.

of

Prolonging

of

(Pfleiderer

of

studies

sites

snake

peptide

structure

leucine

B chain

thermolysin

in

and

a high

peptide

residues

split

between

the

the

at

bonds

the

in

of

BlOPHYSlCAL

Pseudomonas

has

venom

property

leucine

and

between

phenylalanine The

carboxyl

thermolysin

from

ratio

the

a similarity

of

isoleucine

residues

that

AND

numbe,r

hydrophobic,

elastase

hydrolyse

be

total at

involved

hydrophilic

to

the

occurred

residues

ant

BIOCHEMICAL

and

in

studies

takes

place

DNP

reaction

isoof at

liberated. tobacco

increasing

mosaic

the

virus

enzyme:

substrate

bonds.

Rei’erences : Endo, S., J. Fermentation Tech., g, 346 (1962). Fraenkel-Conrat, H., Harris, J. I. and Levy, A. L., in D. Glick (ed). Methods of Biochemical Analvsis, Vol. II, Interscience Publishers, Inc., New York, (1956) p. 397. Hagihara, B., Tagawa, K., Horikawa, I., Shin, M., and Okunuki, K., J. Biochem., Tokyo, 4fi, 725 (1958). W. J. and Anfinsen, C. B., J. Biol. Chem., 234, 2897 Katz, A. H., Dreyer, (1959). Light, Morihara,

A.

(1965) Pf leiderer, Smith, I., Spa&man, Walley, S. Yasunobu, Biophvs.

and K., P.

Greenberg, Tsuzuki,

J., H.,

3. Biol. and Oka,

T.,

Chem.. The

240, 17th

258 (1965). Svmoosium on

Enzvme

Chemistry,

293.

G., and Krauss, A., B’ochem. Nature, 171, Y. (1953 ). D. H., Stein, W. H. and Moore, G. and Watson, J., Biochem. K., Nakashima, T., Higa, H., Acta, a, 791 (1963).

Z.,

&&

85

(1965).

S., An a 1 . Fhem. J., 55, 328 1953) Matsubara, H. and

247

2,

1190

Benson,

(1958). A.,

Biochim.