N-terminal sequence of swine pepsinogen and pepsin. The site of pepsinogen activation

N-terminal sequence of swine pepsinogen and pepsin. The site of pepsinogen activation

Vol. 54, No. 3, 1973 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS N-TERAKINAL SEQUENCE OF SWINE PEPSINOGEN AND PEPSIN. THE: SITE OF PEPSINOG...

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Vol. 54, No. 3, 1973

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

N-TERAKINAL SEQUENCE OF SWINE PEPSINOGEN AND PEPSIN. THE: SITE OF PEPSINOGEN ACTIVATION. V.M.Stepanov,

L.A.Baratova, I.B.Pugacheva, L.P.Revina, E.A.Timokhina.

L.P.Belyanova,

Institute of Genetios and Selection of Industrial Mioroorganisms, Post Box 379, Moscow D-182, Laboratory of Bioorganic Chemistry, Moscow State University, Moscow, USSR. Received

August

21,

1973

SUMMARY. The sequence of 119 amino acids of swine pepsinogen comprising the fragment released during the 5ymogen activation as well as the N-terminal part of pepsin is established. The activation of swine pepsinogen is shown to be accompanied by specific cleavage of Leu-Ile bond in the sequence: 41 46 Ala Ala Ala Leu Ile Gly where Ile+ represents the N-terminal residue of pepsin.This sequence is attacked in the course of pepsinogen activation by external enzymes - neutral protelnases and elastase. INTRODUCTION. Although became recently

the mechanism of pepsinogen

an object

of vivid

discussion

of amino aoids of the zymogen preceding which appears Pepsinogen minal acids.

as the N-terminal

activation

fragment

isoleucine

Ong and Perlmann established

the N-terminal

and presumed that

bond which is cleaved

pepsinogen

of one-two

not excluded

(6).

re ( cf.7) rather

Cop.vright AN rights

but it

it

activation

acid preceds

Itzc. reserved.

is Glu-Ile although

was accepted pepsin

1134

was

in the literatu-

has been shown in our laboratory

0 19 73 by Academic Press, of reproductim in any /bun

sequence of

amino acids between these residues

This assumption

than glutamio

of N-ter-

paper 44 amino

41 amino acids of pepsinogen the presence

residue

by the cleavage

as shown in this

during

the sequence

(l-4)

of pepsin was not known.

is accompanied

containing

activation

N-terminal

that

leucine

residue

-

BIOCHEMICAL

Vol. 54, No. 3, 1973

isoleuoine

in pepainogen

ded N-terminal

sequence (4).

in definite

during

pepsinogen

allows

terms the change of primary

AND METHODS. The preparation has been described

acid sequences were determFned Sequenoer,

struoture

of swine pepsinogen elsewhere

40 per cent pyridine.

I-Chlorobutane

by treatment

contained

with

isolated lysates were

chromatography

Pepsinogen as follows.

N-terminal

kept for

To the solution

(Fig.?) (12)

pH 5.0,

20

mg of elastase

At this

was performed in 50 ml in 10 ml 20 h.

were added and the solution time the activity

ensyme. In the control

of elastase

hydro-

was inoubated

showed almost quantitative

active

were

structures

of 300 mg of pepsinogen

then IO mg of elastase

the addition

(PTH)

conversion

elastase

were added. The mixture

of the zymogen into

after

B-2. Their

pancreatic

with

measured by milk-clotting

out

fragment

activation

24 h. at 36'.

in

min. PTH were snalysed

or thermolytic

procedures.

of the same buffer at 36*,

(9-11)

buffer,

dissolved

the extraction

to the sequence 45-119

by aonvent.ional.

of 0.1 88 acetate

370

(8).

from cbymotrgptic of pepsin

with

phenylthiohydantoins

when necessary

derivatives

corresponding

studied

Protein-

5.10W5?d dithiotreitol.Thiaaolinones

HCl at 80' for110

IN

to trimethylsilyl Peptides

used for

to the corresponding

by gas-liquid

Amino

Model 890. The runs were performed snd 300 nmol of pepsinogen

were converted

(4).

using BecbmanSpinco

nmol of leucyl-pepsin of thi~solinones

us to de-

aotivation.

and of leucyl-pepsin Peptide

The study of an exten-

sequence of swine pepsinogen

scribe

MATEIUAI&

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

was

of the mixture conversion mixture

with-

only 2.5 per cent of pepsinogen

were activated. The mixture

was applied

on DEWS-cellulose 1165

column (2 x 40 cm)

Vol. 54, No. 3, 1973

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Fia.1. The N-terminal sequence of swine pepsinoaen (P.). The line C. shows the homologous sequences of calf proohymosln (13). 15

P. Leu Val Iga Vsl PRO Ll!XJVsl Arg Lys LYS SER LEU AEG Gln Asn C. Ile Thr Arg Ile PRO LEU !Qr rips Gly LYS SER LEU AZ I.ys Ala 30

P. LEU Ile Iys Asp GLY Lys LEU Iys ASP PHE LEU LYS Thr his Lys C.IlEU~sGlyRisGLY~uLWJGlyASP~LEUUSS 45

P. His Aan Pro Ala Ser Iya Tyr Phe Pro Glu Ala Ala Ala Leu Ile Gly Glu Val C. P. Gly Asp Glu PROLHJ Glu ASN TYP LED ASP Thr Glu TYR PHE & C.AlaSerValPROLEUThrASNTYRLEUASPSerGlnTYRPHXGLY P. Thr ILE Gly Ile

GLY THR PROAla Gln Asp PEE Thr Val Ile %

C.~sILETyrLeuGLYTHRPROProGlyGluPHE 90

P. ASP THR GLY SEE SEEiAsn Leu TRP Val Pro Ser Vsl Tyr CYS Ser C.ASPTRBGLYSERSER AspPhe TRIP CYS Ip 105

P. SER Leu ALA CYS Ser Asp HIS Asn Gln PH3 Aan PROAsp Ser Asp C.SEBAanllWLCY8~sAsn~pSGlnArgPIIEAspPIK)hrg 119

P. Ser Thr Phe Glu Ala Thr Ser Glu Glu Leu Ser Ilr Fia.la.

The internal

homology in swix~ pepainogea.

16

IZJ ILELysASPGlyLysLJW IysAspPhe ~ILEGlyb8PGluProLIWGlyAan~~Asp~~ 44 equilibrated

with 0.1 M acetate buffer,

was achieved by linear (

300

ml ) to

containing

0.5

dex G-25 and lyophilized ensyme was inactivated

LEJ zips %R

pH 5.6.

gradient from the starting

M N&l

the active

Thr Tyr

in the same buffer

ensyme was collected, to give

146.7

The elution buffer

(Fig.2).

The perk

desalted on Sepha-

mg of the protein.

by phenol treatment

The

(4) snd used for

N-terminal assay and the automatic sequenoing. 14 mg of this protein gave after dinitrophenylation, acid hydrolysis and TIC 100 nmol of DNP-Als, 100 nmol of DNP-Leu and 70 nmol of

1166

BIOCHEMICAL

Vol. 54, No. 3, t 973

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I,5

’ 70

095

0 200

400

600

800

ml

Pig.2. DEAB-cellulose chromatography of the mixture obtsined by pepsinogen activation with elaetase. Conditions as specified in the text. The peak which shows milk-clotting autivitg is cross-hatched. The peak I contains elastase and the peptides cleaved off in the course of activation. The pesk 2 - pepsinogen . DNP-Ile,

the latter

hydrasinolysis

two derivatives

being identified

and emino acid analysis.

amino acids

of the enzyme obtainsd

equal to that

of pepsin

( Iys,,

that

gave the sequence I-50

of pepsin.

of basic

activation

of the sequenator

(Fig.1)

was

>.

Arg2

RESULTS AND DISCUSSION. Applioation pepsinogen

The content

by elastase

his,,

after

overlapping

The sequence 'l-39 confirmed

to with

the data obtained

by Ong and Perlmann but the sequenoe Gluaa

was found for

residues

by these authors

(6).

40-41 rather

The sequeme

ed in this

sequenaing agreement peptides

41-43

Identification

the struoture of I&u-pepsin with

for

by Ile-45

the first

time revesl-

- the N-terminal

of Leu as residue

of leuayl-pepsin

(4).

emino

44 once more

The automatic

gave the sequence 4W3g which was in

the struotures

of thermolytio

suggested

Ala-Ala-Leu

work was followed

aoid of pepsin. confixmed

than Ala&lu

the

of N-terminsl

hydrolyeate

1167

54-58,

nonapeptide 59-61,

62-70,

(7), 71-

Vol. 54, No. 3, 1973

BIOCHEMICAL

Elaatase

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

~-,

CPepein

Ala Ala Ala Le?i Ile

Gly Asp Glu

from Asp,orysae Fig.3. 82, 87-91

The activation

and of ohymotryptic

The sequence determined of residues

87-119 established

The N-terminal obviously

with

site

of swine

hydrolysate

pe~einogan.

60-71,

the sequenator earlier

overlaps

sin (13)

(Fig.1).

internal

homology that

that

of calf

There is rather

(g-12).

with

that

in t&is laboratory

sequenue of swine pepsinogen

homologous with

84-88

(12).

and pepsin

c-sin

is

and proohymo-

suggestive

indioation

on the

appears when sequences 16-28 and 44-56

are compared (Fig.la). The sequence 74-82 coincides aspartic

acid residue

propane

(14).

Obviously,

Asp-76 as well

as with

ence Ile-Val-Asp Strong

this

I-p-nitrophenolcy-2,3-epoxy

inhibitor

might interaot aoid residue

also with

homology of the corresponding the suggestion

It

is shown that

the hydrolysis

of Leu-Ile

by two hydrophobic main features sider

of pepsin

trait

the cluster

promote oonteining

activation

bond (Fig.3).

with inhibitors.

of abymosin sup-

of Asp-76. is accompanied

specificity.

of alanine of pepsinogen

It

residues sequence.

of amino aoid residues

the binding

of the activating

the %ypersensitive'*

Leu-Ile

1168

by

This bond is flanked

amino acids whinh is in agreement with

the cluster

important

role

the

in the sequ-

diasoacetyl

fragment

on the essential pepsinogen

which contain

with

the aspartic

which reacts

ports

that

reacting

with peptides

is tempting

the

to con-

41-43 as funotionally It might be suggested with

short

side Chains

enzyme to the sequence bond. Peptide

Ala-Ala-

BIOCHEMICAL

Vol. 54, No. 3,1973

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Thr-Leufoundbg

Kas~ellet-- al. in the aotiVa#ngmixture of pepsinogen (15) is homologous with the sequence 4%

bovine

44 of swine pepsinogen. lysis

aocompanies

(la),

that

Apparently,

the point

that

this

part

activation external

of pepsinogen we applied

of this

traits

amino acid of pep-

evokes the suggestion the specificity

with

neutral

proteinases

residues

for

and isoleucine

the activation

1st step Ala+Leu,

resulted

product and Ala-Ala

sin is also present

the mixture

site, at

alanine,

The applica-

of following

amino

3d step Ile+Gly, data ahow that

the

of leucyl-pepsin

as a result

and

of hydrolysis

bonds of the zymogen. "Conventional" although

ding amino acids were oleaved The bond Leu-Ile

as the

far

activation

amino acids.

in cleavage

arise

So

contained

2nd step Leu+Ile,

contains

only trace off

of pep-

amounts of correspon-

during

might be bydrolysed 1169

by

with high yield

product

as N-terminal

attack

at the activation

pepsinogen

The active

alanyl-leucyl-pepsinwhich

cing.

of

by the activation

(Fig.3).

was found

elastase

slowly.

earlier

4th step Gly+Asp, 5th step Asp+Glu.These

Ala-I&u

pepsin

The fact

the proteolytic

had been proved

pancreatic rather

activation

(17)

cannot de-

attack.

the N-terminal

sequence for

of the sequenator

acids:

vari-

- in calf

residue

proteinase

This enzyme performed

leucine

glycine

of the sequence governs

of alanine

although

(16) and chicken

B (lg),

characteristic

prcteinases

pH 5.0.

are remarkably

process.

Availabi1i.Q

cluster

pepsinogen.

the N-terminal

the sequence preceding

bond &ydro-

in human (16) aad bovine

of activating

sin beers rather

tion

is N-terminal

- in swine pepsin

chymosin (20). termine

of bovine

- in human gastricsin

serine

alanine

that Leu-Val

amino acids of pepsins

Thus, valine

pepsins,

appears

the activation

The N-terminal able.

It

the automatic by elastase

sequen-

or

Vol. 54, No. 3, 1973

eventuslly

BIOCHEMICAL

by pepsin

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

derivatives

appearing

in the course

of activation. REFERENCES. I. 2. 3. 4.

5.

6. 7. 8.

9. IO. II. 12.

13. 14.

15. 16.

17.

18. lg. 20.

Bustin AL, Jacobs B.C., J.Biol.Chem.,B, 615 (1971). Funatsu M., Harada P., Hsyasbi K., Kaheda T.,Agriculturel and Biologioal Chemistry (Japan), 36, 305 (1972). Al-Janabi J., Hartsuck J.A., Tsng J., J.Biol.Chem,, x, 4628, (1972). Stepanov V.M., Timokbina E,A., Baratova LA., Belyanova L.P., Korzhenko V.P., Zhukova I.G., Bioohem.Biopbys.Res. comlnun., 43w43.2 (1971). Mc Phie P., J.Biol,Chem., 247, 4277 (1972). 6104 (19Qe). Ong E.Bo, Perlmann G.E., J.Biol.Chem.,m, g, 697 (1970) Tang J., Bioohem.Biopbys.Res~Commu.n., F'isano J., Bromert T.J., J.Biol.Chem.,~, 5597 (1969). Vakhitova LA., Amirkhsxyan M,M,,Stepanov V,M,, Biok.bimiya, Bi, 1164 (1970). Velulis B.A., Stepanov V.M., Biokbimiya, $3, 358 (1971). Pugacheva I,B., Stepanov V.M., Amirkhanyen !&AL, Biokhimiss, z, 993 (1971). Revina L.Po, Vakhitova E.A., Pugacheva I.B., Lapuk Y.I., Stepanov V.M., Biokbimiya, 22, 1074 (1972). Foltmann B., in “Atlas of Protein Sequence and Structure',' v.5, (M.o.Dayhoff, ed.) Silver Spring, Md, 1972, p.D-123. Chen K.C.S., Tang J., J,Biol.Chem.,x, 2566 (1972). Kassell B., Kay J., Urciniszan J.P.,jr., Chemistry and Biology of Peptides, Ann Arbor Science Publ. ,1972,p.713. Tang K.Jo, Tang J., Ped.Proc.,~, 239 (1961). Meitner P,A., Kassell B., Biochem.J.,121, 249 (1971). Bohak L., J.Biol.Chem.,a, 4638 (196z Ryle A.P., Porter R.R., Biochem,J.,jU, 7S (1959). Jirgensons B., Ikenaka T., Gorgyrski V., Macromol.Chem., 28, 96 ('1958).

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