A new serum component which specifically inhibits thiol proteinases

Vol. 76, No. 3, 1977

BIOCHEMICAL

AND BIOPHYSICAL

A NEW SERUM COMPONENT WHICH SPECIFICALLY Makoto

RESEARCH COMMUNICATIONS

INHIBITS

THIOL PROTEINASES

Sasaki, Kayoko Minakata, Hiroyasu Yamamoto, Masaaki Niwa, Taiji Kato and Nobuo Ito*

Department of Biochemistry and Department of Physiology*, School of Medicine, Nagoya City University, Mizuhoku, Nagoya 467, Japan Received

April

27,1977 SUMMARY

A serum proteinase inhibitor specific for thiol proteinases was prepared in a functionally pure state by Sephadex G-200 gel filtration, starch block electrophoresis and immunoaffinity chromatography. This component was distinct from the known serum proteinase inhibitors. It was demonstrated by immunoelectrophoresis that the incubated mixture of thiol proteinase and this inhibitor produced a soluble complex possessing both antigenicities. The molecular weight of the inhibitor was found to be 90,000 by gel filtration on Sephadex G-150 column, and the electrophoretic mobility was in the @2-region. A tentative term, Oc2-thiol proteinase inhibitor, was given because of its mobility and inhibition spectrum. Most with not

serum proteinase

serine only

ases.

proteinases

serine

to thiol

Sephadex

G-200

characteristics

(1) with

proteinases

In a previous

specific

inhibitors

also

investigation

of this

other

proteinases

between

The purpose component

including that

the

of this

as a proteinase

MATERIALS

interact

of Cc2-macroglobulin,

(2) we noticed appeared

fractionation.

have been characterized

the exception

but

proteinases

that

which thiol

an inhibitory

second study

and third

binds

proteinactivity peaks

was to elucidate

of the

inhibitor.

AND METHODS

Materials. Bovine trypsin[EC 3.4.21.41 and Q-chymotrypsin [EC 3.4.21.11 were purchased from Worthington Biochemical Corporation and nagarse (SubtilisinBPN') [EC 3.4.21.141 from Nagase Company Limited. Papain [EC 3.4.22.21 and ficin [EC 3.4.22.31 were purified in our laboratory by the methods of Kimmel and Smith (3) and of Englund et al (4), respectively, from crude enzyme preparations from Worthington Biochemical Corporation. Cathepsin Bl [EC 3.4.22.11 was purified from fresh porcine liver according to the procedure of Greenbaum Stem [EC 3.4.22.41 and fruit bromelains were prepared as and Fruton (5). described previously (6). Casein for enzyme assay was a product of Wako Company limited. Hemoglobin for the assay of cathepsin Bl, was purchased from Miles Laboratories Incorporation. Pure agar, Agarose, monospecific antisera and immunoplates (Partigen) were preparations of Behringwerke HOchst. ThelAbbreviations used: @2TPI, ti2-thiol proteinase inhibitor; globulin; &lAT, O(1-antitrypsin; @1X, @l-antichymotrypsin; thrombin III; IUI, inter Q-trypsin inhibitor; qs1, Cp, ceruloplasmin; and @2HS, @z-heat stable glycoprotein. Copyright 0 1977 by Academic Press, Inc. AN rights of reproduction in any form reserved.

Cl

U2M, d2-macroAth III, Antiesterase inhibitor:

917 ISSN

0006-291

X

Vol. 76, No. 3, 1977

globulin sulfate order to of these laboratory than ten

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

fractions of these specific antisera were fractionated by ammonium precipitation (7) (40% saturation once and 33% saturation twice) in exclude the coexistent proteinase inhibitors in the @l- and Dc;l-regions antisera. Antiwhole human serum and anti-OI2TPI were prepared in our by isnnunizing rabbits (8). Human serum was collected from more healthy persons.

Enzyme assay. Caseinolytic activity was assayed by Kunitz's method (9) with an appropriate modification. To 1.0 ml of enzyme solution (enzyme, lo-20 pg) was added 3.0 ml of 1.2% casein in 0.05 M potassium phosphate buffer, pH 7.5 (In the case of thiolproteinases, substrate solution contained 6 mM cysteine for maximum activationofenzyme.). After 30 min-incubation at 30°C, 4.0 ml of 6% trichloroacetic acid in 0.05 M acetate buffer, pH 4.5 was poured into the reaction mixture. Coagulated precipitate was removed by filtration after standing for an additional 30 min. The absorption of the filtrate was then determined at 275 nm using a Hitachi Perkin-Elmer 139 spectrophotometer. Proteolytic activity of cathepsin Bl (10) was assayed by using hemoglobin as substrate in 0.05 M acetate buffer, pH 4.5, instead of the casein solution. Inhibition assay. Enzyme was first incubated at room temperature for 30 min with an appropriate amount of the serum fraction containing proteinase inhibitor. The subsequent procedures were the same as for the enzyme assay described above. Suppression. The inhibitor was incubated for 30 min with monospecific antibody (7-globulin fraction), followed by adding 15 ug of enzyme and adjusting the volume to 1.0 ml. After 30 minincubation, casein and then trichloroacetic acid were added, followed by the procedures described above. Inhibition effect of U TPI on factor XIII. The effect of 0(2TPI on factor XIII activity was measured z-y means of an assay system from Behringwerke Hbchst (a kit for determining coagulation factor XIII). Immobilization of protein. Antibody was attached to BrCN-activated Chromagel A-2 (Dojin Yakkagaku Institute) for immunoaffinity chromatography (11). Starch block electrophoresis. It was performed in 0.05 M veronal-acetate buffer, pH 8.6, with a voltage gradient of 5 V/cm for 60 hr at 4'C. Immunoelectrophoresis. It was carried out in 1.2% agar (1:l mixture of Pure agar and Agarose) dissolved in 0.025 M veronal-acetate buffer, pH 8.6, with a voltage gradient of 8 V/cm for 90 min at 4'C. RESULTS Partial filtration.

both

The former latter

using in the

and 02-heat

activity

casein first

inhibitory

depended

proteinase

ti2TPI

human serum was fractionated

The inhibitory

was estimated found

Fresh

Purification.

peak

stable

their

the

coincided

with

antigenicities

(which

toward inhibitory

second

and third

The elution

show very

on immunoplates

918

similar

analyses), containing

G-200

peaks

were (figure

activity positions

1).

and the of other

ceruloplasmin

behaviors were

gel

proteinases

activities

serum components,

and electrophoretic

thiol

Oc2-macroglobulin

inhibitor.

and of two additional glycoprotein

fraction

Dominant

and between

on a new proteinase

in chromatographic

measuring

as substrate.

activity

inhibitors

in every

by Sephadex

to those

determined

specific

by

antisera

of

Vol. 76, No. 3, 1977

BIOCHEMICAL

OL 30

35

40

AND BIOPHYSICAL

45

50

FRACTION

Figure

55

60

RESEARCH COMMUNICATIONS

65

NUMBER

1.

Distribution of the inhibitory activity against thiol proteinase and of several plasma proteins on Sephadex G-200 gel filtration. 25 ml of human serum was applied on Sephadex G-200 gel columr. (3 x 140 cm) equilibrated with 0.02 M borate buffered saline, pH 8.0. Gel filtration was performed with the same buffer at a flow rate of 10 ml/hr. Each fraction volume was 15 ml. Inhibitory activity was estimated as described in the Materials and Methods section. Open circles denote inhibitory activity against thiol proteinase and solid line W absorption at 280 nm. The locations of several plasma proteins estimated by radial immunodiffusion plates are shown with bar symbols.

(-)

5

0

5 FRACTION

Figure

10

15

20

25(+)

NUMBER

2.

Distribution of the inhibitory activity against thiol proteinase and of several plasma proteins on starch block electrophoresis. The shaded portion of figure 1 was subjected to starch block electrophoresis. The size of the block was 4 x 1.5 x 45 cm. An arrow indicates the position the sample was applied. Electrophoresis was carried out as described in the Materials and Methods section. After electrophoresis the starch was cut into 1 cm-long small blocks, and the protein was extracted with chilled borate buffered saline, pH 8.0. Open circles denote inhibitory activity against thiol proteinase, solid circles, the amount of plasma proteins estimated by immunoplates, and solid line, W absorption.

919

Vol. 76, No. 3, 1977

BIOCHEMICAL

AND BIOPHYSICAL

Table Suppression Experimental

of the

inhibitory

Fit Fit Fit Fit Fit Fit Fit

+ + + + + +

Fit

+ (W2TPI + normal

activity

+ + + + +

of

anti+TPI) anti-Cp) anti-Ath [email protected]) anti-u2HS)

Inhibition (%)

antibody

Suppression (%)

0 78.6(100.0) 34.7( 44.1) 74.5( 94.8) 81.5c103.7) 80.8(102.8) 78.2( 99.5)

0 55.9 5.2 -3.7 -0.3 0.5

0.105

80.6c102.5)

(-2.5)

III)

IgG)

@2TPI by corresponding

a.542 0.116 0.354 0.138 0.100 0.104 0.118

(v2TPI) (@2TPI (v2TPI (@..TPI (W2TPI (o(2TPI

1

Absorbance (275nm)

systems

RESEARCH COMMUNICATIONS

Suppression corrected(%) 0 58.4 7.8 -1.1 -0.3 3.0

The extent of inhibition and suppression was estimated as described in the Materials and Methods section. Assay System: Enzyme; ficin, 15 pg/15 ~1. Buffer: 0.02 M borate buffered saline, pH 8.0, 0.2 ml (In the case without added antibody or without antibody and inhibitor, 0.7 or 1.0 ml of buffer solution was used in place of 0.2 ml of buffer solution). Inhibitor; C$TPI fraction purified by immunoadsorbent column, 0.3 ml. Antibody; Y-globulin fraction and rabbit normal JgG, 0.5 mg/O.S ml. Substrate; 1.2% casein in 0.05 M phosphate buffer, pH 7.0, 3.0 ml. Reagent for stopping the reaction; 6% trichloroacetic acid in 0.05 M acetate buffer, pH 4.5, 4.0 ml. Fit; ficin.

(shown

as bar

eliminate

three

(shaded

area),

concentration. @*-region trypsin III,

using listed

of six which

and other

was then

were

still

procedure inhibitors

subjected

in figure

distinguished

serum

filtration

known proteinase

the

made it

block

inhibitory

e ectrophoretic

inseparable

such as @*-heat by these

possible s2TPI

fraction after

was found

positions

antithrombin

glycoprotein

two successive

steps

in the

of q-anti-

inhibitors,

stable

to

electrophoresis

activity

One of the proteinase

components

the

from

o starch

2 the

from

and al-antichymotrypsin.

and cerulo-

of fractionations

C?2TPI activity.

Identification not

The gel

As shown being

plasmin, from

symbols).

separated specific in table

of inhibitor

by suppression

by the above procedure antibodies 1.

In this

were

corresponding assay

effect. tested

The remaining by the

to each component.

system

920

15 pg of ficin

effects

components of

suppression

The result digested

casein

is to an

Vol. 76, No. 3, 1977

absorption

BIOCHEMICAL

value

of 0.542

fractions

No.8,

digestion

was reduced

value,

at 275 nm, but

9 and 10 in

0.116).

the

the

Preincubation

inhibitory

Anti-ceruloplasmin

proteinase

inhibitor

molecular

the known

studying

the

inhibition

isolated

from

commercially

tation

prepared through nate

this

concentrated cathepsin sufficient fruit (about

and stem bromelains one-third).

Unlike

coagulation

assay

thiol

system)

system

(1.2

purpose

III)

fractions

I-globulins

lower the

proteinases,

column.

(see Materials

extent

than

that

enzyme

passed elimiwas

ficin

and

preincubated of inhibition of papain

toward

serine 0 but

(factor

when estimated

and Methods).

921

were

was approximately

unaffected

The sample

fraction

papain,

the

inhibition

a thiol

was attached

to thoroughly

Although

shown),

were

precipi-

was repeatedly

when they

for

(anti-VI-antitrypsin,

The nonadsorbed

(not

sample

by ammonium sulfate

of these

completely

totally

D(2TPI

in their

V-globulin

x 4 cm) in order

and nagarse

was

of

that

pure

antisera

assay.

hand,

demonstrated

an immunoadsorbent

inhibition

other

chymotrypsin

coagulation

this

was considerably

On the

suppression

either

by electrophoresis

fraction

inhibitory

a functionally

A mixture

almost

of this

data

not

or antigenicity.

inhibitors.

the

inhibited

amounts

the highest. blood

for

of the

inhibitors,

monospecific

column

did against

insignificant

these

A-2 providing

proteinase

and used

such as trypsin,

but

III,

antibody

preparation

suppression

For

followed

anti-antithrombin

antibody

to obtain

and Methods).

three

Bl were

Only

and anti-antithrombin

filtration

with

of

(absorption

glycoprotein

a slight

available

immunoadsorbent

the above

stable

necessary

activity

fraction

mobility

is

Chromagel

by gel

2) the

serum proteinase

spectrum.

(see Materials

to BrCN-activated

in figure

Accordingly,

six

anti-@l-antichymotrypsin

electrophoresis

gTPI

showed

It

(mixture

OL2TPI.

electrophoretic

spectrum.

of s2TPI

a significant

activity.

weight,

Inhibition

the

of

displayed

in the presence

as 21% of the original

of

activity

activity.

from

block

or anti '-?-heat

0(2TPI fraction

differed

starch

to as low

anti-CJl-antichymotrypsin affect

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

XIII

with of

and ficin proteinases

up to 10% at in the

by the use of

Vol. 76, No. 3, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Anti-WHS Anti-ol*TPIfr: Anti-FUN

Figure 3. Immunoelectrophoretic demonstration of ficin-U2TPI complex. 3 ,ul of each antigen was applied in the wells as indicated. Only for the mixed solution a total of 6 pl was used after 15 min-incubation at room temperature. Ficin was activated with 5 mM cysteine before incubation. Ci2TPI fr.; mixture of fractions No.8, 9 and 10 in starch block electrophoresis. WHS; whole human serum. Electrophoretic condition, see Materials and Methods section.

Molecular

weight.

filtration with

The molecular

through

a Sephadex

some reference

and human IgG).

proteins

weight

Proteinase-inhibitor

plate patterns taneouly.

Ficin

the 02TPI ficin

and the

intermediate both.

This

indicates

(0.9

was measured

x 100

cm)

egg albumin, emerged

For

Q2TPI

in

(12)

by gel

in comparison

bovine

serum albumin

the position

corresponding

fraction toward toward

3 shows the

ficin

was applied

the

anode,

while

the cathode.

and ti2TPI

w2TPI formed

922

all

the

However, appeared

were

simul-

in the mixture

which

complex

run

serum components

migrated

and had antigenic

a soluble

on a agar

immunoelectrophoretic

and the above mixture

a new component

between

of a proteinase-inhibitor

and o(zTPI fraction

Figure

migrated

that

the elucidation

of ficin

mixture

0(2TPI fraction position

inhibitor

of 90,000.

migrated

fraction

column

activity

immunoelectrophoresis. when ficin,

of this

(U-chymotrypsin,

complex.

an incubated for

G-150

The inhibition

to a molecular

complex,

weight

with

of

in an

properties ficin.

in

of

Vol. 76, No. 3, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

DISCUSSION ol2TPI

was separated

Cl esterase

inhibitor

@l-antitrypsin

inhibitor,

stable

Thus,

inhibited

very

The extent

of the

proteinases was less

it

selectively

inhibitory

sensitive,

Ficin

and fruit

the inhibition

well

known

is

that

other

proteinases

u2TPI

inhibits

inhibition The biological

inhibitor

at inflammatory the prolonged influenced

determine matory

by blood whether

loci

phase

ti2TPI

and by what

this

is

and papain

different

were

most

(2,

proteinases

o(2TPI is not

yet

from known.

be in the control

In fact,

Beloff

and tissue

and Tokaji

which

inhibitors.

thiol

It

is

interacts

with

the proteinases

mechanism

this

occurs.

is

but

(14).

In contrast,

that

the mechanism

of

Oc2-macroglobulin.

also

of

one of the probable proteihases

(16)

depends

It

proteinases

However,

of the

(15)

of inflammation,

that

sensi-

may suggest

combination.

proteinases

different

Bl

less

proteinases

serine

thiol

cathepsin

considerably

indicating

origins.

among several

stoichiometric

13) and acidic

proper-

and plant

sensitive,

were

not only

(ceruloplasmin

a new serum component.

among thiol

inhibits

from another

of animal

however,

and from

physicochemical

inhibitor

was,

and

mobilities.

gTP1

similar

proteinases

substantially

may

sites.

that

due to a simple

thiol

functionof

of this

that

thiol

selectively by @2TPI is

had very

weights,

serum components

and stem bromelains

a*-macroglobulin like

that

inhibitor

electrophoretic

distinguished

of susceptibility not

d-trypsin

molecular

two other

thiol effect

investigated.

that

role

and from

appears

Such a difference

tive.

of their

further

glycoprotein)

to qTP1.

0(2TPI

III,

inter

by their

of antigenicities

antithrombin

and @'*-heat

is

by the difference

and $-antichymotrypsin

The examination

ties

from C$macroglobulin,

have

on thiol

released indicated

proteinases,

of further

interest

to

released

in the

inflam-

REFERENCES 1. Heimburger, N., Haupt, H. and Schwick, H.G. (1971) Proceedings of the International Research Conference on Proteinase Inhibitors, ~~-1-22, Walter de Gruyter, Berlin-New York. 2. Sasaki,M.,Yamamoto, H., Yamamoto. H. and Iida S. (1974) J. Biochem. -'75 171-177. 3. Kimmel, J.R. and Smith, E.L. (1954) J. Biol. Chem. 207, 515-531. 4. Englund, T.R., King, T.P. and Craig, L.C. (1968) Biochemistry 1, 163-175.

923

Vol. 76, No. 3, 1977

5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Greenbaum, L.M. and Fruton, J.S. (1957) J. Biol. Chem. 226, 173-180. Iida, S., Sasaki, M. and Ota, S. (1973) J. Biochem. 13, 377-386. Arnon, R. and Shapira, E. (1967) Biochemistry 5, 3942-3950. Sasaki, M., Iida, S. and Murachi, T. (1973) J. Biochem. 73, 367-375. Kunitz, M. (1947) J. Gen. Physiol. 30, 291-310. Mycek, M.J. (1970) Cathepsins in "Methods in Enzymology" 19, 285-315. M. and Anfinsen, C.B. (1968) Proc. Nat. Acad. Cuatrecasas, P., Wilchek, Sci. U.S. 9, 636-643. Andrews, P. (1964) Biochem. J. 91, 222-233. Starkey, P.M. and Barrett, A.J. (1973) Biochem. J. 131, 823-831. Landurean, J.C. and Steinbuck, M. (1970) 2. Naturforsch. =, 231-232. Beloff, A. (1946) Biochem. J. 40, 108-115. Tokaji, G. (1971) Kumamoto Med. J. 2, 68-86.

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