Evidence for the occurrence of one-step activation in porcine pepsinogen

Vol. 107, NIO. 3, 1982 August 16, 1982

AND BIOPHYSICAL

BlOCHEMlCAL

RESEARCH COMMUNICATIONS Pages 1117-1122

EVIDENCE FOR THE OCCURRENCE OF ONE-STEP ACTIVATION Takashi Department

July

6,

and

of Biochemistry,

Kyoto Received

Kageyama

University,

Kenji

Primate Inuyama,

IN PORCINE PEPSINOGEN

Takahashi Research

Aichi

Institute,

484,

Japan

1982

SUMVARY: Upon activation at pH 2.0 and 14'C, a significant portion of porcine pepsinogen was found to be converted directly to pepsin, releasing the 44-residue intact activation segment. The released segment was further cleaved to smaller peptides at pH 2.0, but at pH 5.5 it formed a tight complex with pepsin, and the complex was chromatographically indistinguishable from pepsinogen. This intact segment could be isolated for the first time. Thus one-step activation occurs in porcine pepsinogen along with the already known sequential activation.

Pep:inogenl

is known

conditions,

releasing

to be autocatalytically the so-called

part. of thle pepsinogen one is

the direct

peps.inogenl

to pepsin.

pep:inogen

in the

NH2-terminlal Christensen occurred

44-residue the:e

results In this

44-residue intermediate that.

presence

also

one-step

reported

that 16 17 bond Leu-Ile,

are

forming

in the absence

in support

of the sequential we report

protein activation

segment,

species

formed

occurs

inhibitor

cleavage

together

first with

activation.

in porcine

pepsinogen

pepsinogen

form(pseudopepsin) Moreover,

the

Therefore pathway.

the

isolation

some other

during

the

of porcine

isolated.

time

of

of porcine

intermediate

been

activation:

first(l).

conversion

the

the

of pepsin,

was released

the

acidic

conversion

of pepstatin(2).

had never

for

for

on activation

initial

segment

activation

that

under

the NH2-terminal

the sequential

l-16

activation

communication, intact

is

the

from

are possible

a potent

residues

was activated

intact

other

of pepstatin,

comprising

at the peptide

wherl pepsinogen

Two pathways

and the

to pepsin

peptides

Dykes and Kay reported

peptide et al.

activation

molecule.

conversion

activated

peptides

The results along

with

of the and

demonstrate the sequential

process. 0006-291X/82/151117-06$01.00/0 1117

All

Copyright 0 I982 rights oJ reproduction

by Academic Press, Inc. in any form reserved.

BIOCHEMICAL

Vol. 107. No. 3, 1982

AND BIOPHYSICAL

MATERIALS

AND

RESEARCH COMMUNICATIONS

METHODS

Activation of Pepsinogen and Isolation of Released Peptides and Resulting Protein Species.... .Ten mg of pepsinogen(grade I, from Sigma) was dissolved in 50 ml of 0.01 M sodium phosphate buffer, pH 7.0, and the solution was acidified to pH 2.0 by the addition of 12.5 ml of 0.1 N HCl. The reaction mixture was prepared and incubated with gentle stirring at 14'C. At desired intervals, aliquots were withdrawn to examine the extent of activation by SDSdisc gel electrophoresis according to Weber and Osborn(3). To isolate the released peptides, the reaction was terminated by the addition of 1 M NH40H to a final concentration of 0.2 M. The mixture was immediately frozen, lyophilized, and subjected to gel filtration on a column (1.6 x 150 cm) of Sephadex G-50 in 0.1 M sodium acetate buffer, pH 5.5, containing 8 M urea. Peptide was determined by the fluorometric method according to de Bernard0 Purity of each peptide fraction after --et a1.(4). gel filtration was examined by NH -terminal amino acid analysis by dansylation(fi) To isolate the resulting pro ? ein species, the activation reaction was stopped by raising the pH to near 5.5 by adding 2.5 ml of 5 M sodium acetate buffer, pH 5.5, containing about 3-fold molar excess of pepstatin over the initial amount of pepsinogen used. This preparation was subjected to chromatography on a column(l.15 x 25 cm160f DEAE-Toyoperal in 0.1 M sodium acetate buffer, pH 5.5, containing 7x10 M pepstatin. The adsorbed protein was eluted by a linear gradient of NaCl from 0 to 0.5 M using two 300-ml chambers in the same buffer. Amino Acid Analysis and Amino Acid Sequence Determination.....Amino acid analysis was performed according to Spackman et al.@) with a Hitachi model 835 amino acid analyzer. Amino acid sequence was determined by a modification (I) of the manual method of the sequential Edman degradation(8). Released PTH-amino acids were identified by thin layer chromatography@) and/or high performance liquid chromatography(llJ). The COOH-terminal amino acid sequence of the activation segment was analyzed by using carboxypephidase Y as follows: two n moles of each activation segment was incubated at 37 C with 20 pg of carboxypeptidase Y in 300 pl of 0.1 M sodium phosphate buffer, pH 6.5, containing 10 % methanol. Aliquots were withdrawn at desired times and released amino acids were determined by the amino acid analyzer. RESULTS Analysis

of

pepsinogen

Time

species

weight

with

between

were

those as

a long

period

of

peptide

be maximum

at

1 or

progress Isolation After

weight

of

a few

incubation

of

Activation

activation

for

Activation.....As

minutes

two

bands;

pepsin

and

the

other

and

pepsin.

pepsinogen,

incubation.

of

molecular

Pepsinogen

as

with

amount

DISCUSSION

detected

pepsinogen

compared of

of

within

authentic

stable

low

Course

disappeared

protein

The

the

AND

in

high

and

to

2 min peptide

band

had

but

of

them

were weight

rather

appeared

to

form

converted

peptides

decrease

the

to

also peptide

rapidly, increase

while gradually

molecular

intermediate was

relatively

pepsin

detected band

la,

resulting

same

weight

intermediate

Fig.

The

had

a molecular

gradually

molecular

in

acidification.

one

The

Released the

after

shown

during as

two

appeared that

in

with

bands. to

the the

time. Peptides 2 min,

the

and reaction

Evidence products

1118

for

One-Step were

Activation..... fractionated

by

Sephadex

BIOCHEMICAL

Vol. 107, No. 3, 1982

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

b

“‘-qp-*-..-0

I

---

_.-

2

3

5

TIME

17

7

---HPd CLPd

30

(min)

-HPd

SDS-disc gel electrophoresis. a) Activation mixtures of porcine FigA pepsinogen at different times of incubation. b) Pepsinogen fraction after Pg,pepsinogen; 1,intenediate form; DEAE-Toyopearl chromatography. P,pepsin; HPd,high molecular weight peptide; LPd,low molecular weight peptide. Arrows indicate the position of bromphenol blue.

G-5(1

gel

volume.

F'eptides

Eactl

fraction

composed and

The

filtration(Fig.2).

had of

III

were

intact

using

Fraction

I)

segment,

were

as

Ala(1.77);

and

trace

amounts

the

known

COOH-terminal

24

and

follows:

h,

Leu(l.lZ),

of

Lys,

amino

sequence

and

was

confirmed

by

Y.

The

released

amino

Leu(0.56)

and

and

These

each

of

I,

to

be

COOH-terminal

60

were

Leu(0.81)

Pro(0.85), consistent

(-Pro-Glu-Ala-Ala-Ala-Leu-OH)

the

of

min,

Glu(O.861,

of

the

one-step

of

the

44-residue

activation.

activation However,

segment as

1119

shown

in

gave Fig.la

the and

direct as

II,

sequence

acids(moleslmole

results

was

the

deduced

Ala(0.47);

Phe(0.881,

Ser.

the

III).

Fractions

1-16

I was

Fraction

void

that of

17-44,

the and

indicating

this

Ala(2.85),

II,

compositions

l-44,

I).

near

I,

acid,

acid

residues

2 min,

Tyr,

eluted

with the

segment(ll-13). -Isolation

for

of

carboxypeptidase

and

activation

those

amino

respectively(Table

activation

ana‘ysis

The

was

3 peaks(Fractions

NH2-terminal

to

segment,

mixture

into

peptide.

corresponded

activation

separated

a single

a single

protein

evidence reported

Vol.

107,

No.

BIOCHEMICAL

3, 1982

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

0 40 Fig.2

BO and salts,

by

Sephadex salt indicate respectively.

others&x),

form

the

between

step

process

yield

in

Table

This

occurred

to

be

in

the

form

conversion

to

Protein

Species

was pepsin

a 2-min activation of blue dextran

of

through

mixture. and inorganic

the

intermediate

about

early

based to

be

by

releasing

of

the

the

of

stable

the

each

onepeptide

the the

results

rather

by

activation

cleavage

on

the

activated 40 % from

period by

shown

Under

pepsinogen

maximally

was

Ix)

simultaneously.

exclusively

assumption

IO0

pathway

proportion

that formed

intermediate

further

the

estimated

was

released. the

pepsin

assuming

peptide

segment

its

and

be

I

activation

used,

could

28-residue

which

sequential

conditions

60 NUMBER

G-50 gel filtration of the elution positions Fraction size, 3ml.

pepsinogen

activation

60 FRACTION

44-residue

in

Fig.la,

in

when

formed

and

28-residue

peptide

proceeded

gradually. Isolation

of

. . . . . After

activation

to

pepsinogen,

on

DEAE-Toyopearl

graphic reported

for

pepsin, in

pattern

was

porcine

pepsinogen

mediate

form

were

the

contained

although

this

fraction

2 min,

the

the

intermediate

presence

were

23,

identified

and

from sequences(data

the

NH2-terminal had

the

protein

same

species

were

with

%,

their

that of

amino 1120

of these

shown). human

acid

shown). of acid

chromatography

protein

chromatospecies

species

Pepsin

and

from the

compositions(Table However, both

composition

Complex

separated

The

protein

respectively.

amino not

by

not

yields 36

Segment

form(pseudopepsin)

sequences same

a Pepsin-Activation

pepstatin(data

relative 41,

of

resulting

of the

The

-

5 residue

fraction

Presence

essentially

previously(14).

NH2-terminal

and

and

the

interI)

pepsinogen

pepsinogen

and

as

authentic

pepsin,

and

BIOCHEMICAL

Vol. 107, No. 3, 1982 TABLE

I.

Amino

acid

Number Amino acid

residues

of

per

and

of

peptide

molecule

Protein

II

9.3(9)

5.9(6)

1.8(2)

1.8(Z)

RESEARCH COMMUNICATIONS

peptides

fraction

I LYS His

compositions

of

Peptide

AND BIOPHYSICAL

III

Pepsinogen

2.9(3)

9.5(10) 2.2(

2)

1.8(2)

3.7(

4)

1.0(l)

46.0(46) 44.3(44)

proteins. or

protein

fraction Intermediate

Pepsin

6.7(

7)

1.3(

1)

2.5(

3)

0.9(

1)

2.0(

2)

1.9(

2)

Arg

2.2(2)

Asp

4.0(4)

3.0(3)

Thr

0.9(l)

0.9(l)

Ser

1.8(2)

1.0(l)

0.9(l)

Glu

2.0(2)

1.1(l)

0.9(l)

28.2(28)

Pro

2.5(3)

1.6(2)

0.7(l)

18.2(18)

17.0(17)

15.7(16)

GUY Ala

1.5(l)

1.3(l)

35.6(36)

35.0(35)

33.9(34)

3.5(4)

3.9(4)

19.4(19)

20.7(21)

18.8(19)

Val

3.1(3)

25.0(25)

23.6(24)

23.7(24)

28.8(

2.5(3)

Met

29)

2.3(

Ile

1.1(l)

0.9(l)

Leu

7.4(7)

3.1(3)

Tyr

0.2(l)

0.8(l)

Phe

1.8(2)

1.9(2)

Total

44

N-Terminus

Leu

Yield(%)

10

3.9(4)

28

2)

45.0(45)

42.0(42)

27.9(28)

26.6(27)

44.7(45)

43.7(44)

27.7(28)

27.5(28)

2.6(

3)

2.6(

3)

27.0(27)

26.2(26)

25.3(25)

32.6(33)

29.4(29)

27.3(27)

15.1(15)

15.5(16)

15.5(16)

17.1(17)

16.3(16)

15.8(16)

16

Ile

Leu Ile

Leu

24

78

Ile

12.0(23)

Ile

21.9(41)

19.0(36)

The values were calculated by fixing the number of aspartic acid to the indicated values. Nearest integers are shown in parentheses. The values of Gly and Tyr of Peptide Peptide: hydrolysis time was 24 h. were assumed as 1 residue, respectively, allowing for contamination or Yields were calculated based on the amount loss during acid hydrolysis. of peptide determined by the amino acid analyzer. hydrolysis time was 24 and 72 h. Each value is an average of Proteins: these two values except for those of serine and threonine, which are Half-cystine and tryptoextrapolated values to zero time of hydrolysis. phan were not determined. Yields were calculated based on the amount Yields in parentheses of protein determined by the absorption at 280 nm. indicate relative values.

pepsinogen. bands to

gel

corresponding

the

this The

SDS-disc

high

molecular

fraction amino

identical together

to

by

adsorption

analysis

with

the its

pepsinogen weight

acid

with

electrophoresis

44-residue elution

and

this

pepsin,

position

in analysis

activation

segment. on

one

The

SP-Sephadex

NH2-terminal

fraction

and

peptide(Fig.lb). with

and

of

DEAE-Toyopearl

1121

the

gave peptide

peptide

band was

presence showed Judging

two

that

chromatography

corresponding from

8 M urea. the

from

protein

isolated

of

I

peptide these

was results

and

the

Vol.

107,

amino of

No.

acid

composition,

pepsinogen

and

From

the

estimated

complex

was

stable

decrease

of

while

The

44-residue

other

shorter

(16)

*

the

The

species(pepsin,

to

occupy

pH

5.5

complex

was

in

and

intermediate intermediate

to form

% of

absence

form

occupied form,

and

the

a mixture

residues,

pepsinogen

the

fraction. since in

affinity

the

essential absence

to to

NH*-terminal

The

no

converted

higher

value

be

NH2-terminal

pepstatin,

the

about

to

segment.

gradually

a much

a Ki

thought

chromatography

including have

RESEARCH COMMUNICATIONS

activation

the of

was

have

the the

during

peptides reported

50

the

to

was

of

observed

seems

activation was

pepsin

about

intermediate

segment

which

of

fraction

determination

at

the

AND BIOPHYSICAL

pepsinogen

complex

quantitative

was

pepstatin

the a 1:l

complex

peptides

BIOCHEMICAL

3, 1982

of

pepsin.

pepsin

than

16-residue

of

5.7~lO-~M(&)

or

46 % of

the

resulting

total

2.5x10e8M protein

complex).

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Dykes,C.W., and Kay, J.(1976) Bi0chem.J. 153, 141-144. Christensen,K.A., Pedersen, V.B., and Foltmann, B.(1977) FEBS Lett.76,214-218 Weber, K., and Osborn,M.(1969) J.Biol.Chem. 244, 4406-4412. de Benardo,S., Weigele, M., Toome,V., Manhart, K., Leimgruber,W., Bbhlen,P., Stein,S., and Udenfriend,S.(1974) Arch.Biochem.Biophys. 163, 390-399. Gray,W.R., and Hartley,B.S.(1963) Bi0chem.J. 89, 379-380. Spackman,D.H., Stein,W.H., and Moore,S.(1958) Anal.Chem. 30, 1190-1206. van Eerd,J-P., and Takahashi,K.(1976) Biochemistry 15, 1171-1180. Edman,P.(1970) in Protein Sequence Determination (Needleman,S.B., Ed) pp.211-255, Springer, New York. Kulbe,K.D.(1974) Anal.Biochem. 59, 564-573. Omichi,K., Nagura,N., and Ikenaka,T.(1980) J.Biochem. 87, 483-489. Ong,E.B., and Perlman,G.E.(1968) J.Biol.Chem. 243, 6104-6109. Pedersen,V.B., and Foltmann,B.(1973) FEBS Lett. 35, 255-256. Stepanov,V.M., Bartova,L.A., Pugacheva,L.B., Belyanova,L.P., Revina,L.P., and Timokhina,E.A.(1973) Biochem.Biophys.Res.Commun. 54, 1164-1170. Kageyama,T., and Takahashi,K.(1980) J.Biochem. 85, 571-582. Kumar,P.M.H., and Kassell,B.(1977) Biochemistry 16, 3846-3849. Dunn,B.M., Deyrup,C., Moesching,W.G., Gilbert,W.A.. Nolan,R.J., and Trach,M.L.(1978) J.Biol.Chem. 253, 7269-7275.

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