Isolation of a human urinary trypsin inhibitor

Vol. 109, No. 4, 1982 December

BIOCHEMICAL

BALDUYCK M.*, Lab.

RESEARCH COMMUNlCATlONS Pages 1247-1255

31, 1982

ISOLATION

*

AND BIOPHYSICAL

KERCKAERT J.P.+,

HAYEM A.**,

de Biochimie,

Av. de Pharmacie, FRANCE

Fat.

**Unit@

16 INSERM, Pl.

'Unite Received

OF A HUMAN URINARY TRYPSIN INHIBITOR

du Pr.

de Verdun

59045

124 INSERM, BP 311 59020

November

22,

MIZON C.*

and MIZON J.*

Laguesse LILLE

LILLE

59045

LILLE

Cedex

Cedex FRANCE

Cedex FRANCE

1982

of antitrypsin activity of human urine was SUMMARY - The stabilization obtained by storage at neutral pH in the presence of EDTA. Taking the results into account, a urinary trypsin inhibitor was isolated in a pure state by a new preparation procedure, and partially characterized.

Inter

atrypsin

accounts

for

However,

inter

smaller

only

1 to

and are

in

These

inhibitors

modify

them

affinity

in

without

conditions,

been

characteristics which

obtained

depending

Using

methods

urine

two

having

trypsin

ship

with

was

a degradation

fractions

intera

on the

could

structural studying

the

fractions the

be obtained

in

conditions

this

paper, which

for

other

permit

:

acid

which

we

Under

physico-chemical the

M, estimations

isolated

from

(A).

(7).

However, quantity

present

the

stabilization

to

results

relation-

these

inhibitory

allow

a thorough

obtained of

human

One of them (B)

while

native

Abbreviations : EDTA : ethylenediaminetetraacetic, disodium salt sodium dodecyl sulfate ; PAS : periodic acid-Schiff reagent polyacrylamide gel electrophoresis ; L BAPNA : N benzoyl L-arginin anilid hydrochlorid.

1247

to

treatment, (2-6).

an immunological

established

one

the

able

and 68,000.

a sufficient we

denaturation.

by methods

Therefore,

of

a characteris-

variable

effect,

has been

of

In

used.

14,000

serum.

biological

or acid

activity showed

denaturing

inhibitor

product not

study.

least

possess urine

the

precursor

chymotrypsin-Sepharose

methods

between

inhibitory trypsin

or

glycoproteins

vary the

from

of the

different

to thermic

biological

trypsin

be

into

components

isolated

their

to

diffuse

These

previously

on

have been reported

to

in human serum,

capacity

considered

activity

changing the

able

(1,2).

inhibitors

antitrypsin

is

antitrypsin

chromatography

these

total

are

urine

their have

the

inhibitor which

found

resistance

one of the trypsin

5% of

a trypsin

antiproteases

fluids tic

inhibitor,

urinary

;

;

SDS : PAGE : 4-nitro-

0006-291X/82/241247-09$01.00/0 Copyright 0 I982 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 109, No. 4, 1982 trypsin

BlOCHEMlCAL

inhibitor

(UTI)

and partially

; it

AND BIOPHYSKAL

was further

isolated

RESEARCH COMMUNICATIONS from

urine

in a pure

state

characterized.

MATERIALS AND METHODS Urines were collected from patients suffering from disseminated cancers and inflammatory syndromes (Oscar Lambret, Lille Cancer Center and Calmette Hospital, Lille). Their trypsin inhibitory capacity was measured according to FRITZ et al. (8) with L-BAPNA as substrate. Sephacel, Sephacryl S200 and ConA-Sepharose were purchased from Pharmacia Fine Chemicals. DEAE-Trisacryl and Agarose were from IBF. Acrylamide, N-N'-methylene bisacrylamide and N-N-N'-N" -tetramethylene diamine were from BDH Chemicals Ltd. L BAPNA and trypsin were obtained from MERCK as well as other chemicals of analytical grade. Immunoelectrophoretic studies : Crossed immunoelectrophoresis was carried out, according to CLARKE and FREEMAN (9), using a specific antiserum which has been previously prepared (7), human plasma protein antiserum (Behring lot NO2987 A) and rabbit anti-human urine serum (Dako lot 100 A). Analytical electrophoresis was carried out on polyacrylamide gel slabs, using the technique described by KERCKAERT (10). Electrophoresis was performed on 7.5 or lo-25% gel gradient, with or without prior reduction, using LAEMMLI's buffer system (11). The Pharmacia low M electrophoresis calibration kit was used for M estimation. After migratron, proteins were stained overnight by Coomassie Brilliant Blue 6250 (10 ml 0.25% aqueous solution per 100 ml 12.5% trichloracetic acid). Characterization of trypsin inhibitors in polyacrylamide gels was done according to URIEL and BERGES (12). Agarose electrophoresis gel was carried out on 0.9% Agarose slabs (7 x 2.5 cm) using a 0.1 M pH 8.2 barbital buffer system. Electrophoresis was performed for 1 hr at 2.66V per cm and was further stained either with Toluidine Blue or PAS or Amidoschwartz (13). Chromatographic separations were performed at room temperature and monitored by absorbance at 280 nm. All concentrations were carried out by ultrafiltration in a Diaflo apparatus with a PM 10 membrane (Amicon). Amino acid analysis were performed with a Multichrom Beckman analyser after hydrolysis of the sample in 5.6 N HCl for 24 hours at 110°C in a vacuum sealed tube. The cysteine content was measured as cysteic acid after performic oxidation and hydrolysis. Quantitative gas-liquid chromatography of carbohydrate was performed by the slightly modified method of REINHOLD according (14) with columns containing 3% OV17 on Chromosorb to ROUSSEL et al. The sialic acid content measured with the W.AW.OMCS 80-lOO-Tiiesh (Supelco). thiobartituric assay of AMINOFF (15) after hydrolysis with 0.05 N H2S04 for 30 min. at 80°C.

RESULTS of UT1 stability

Study

A time-course out

on

temperature agents.

study

concentrated at Samples

analyzed

variable were

forms

by their antitrypsin

inhibitory activity

presence

of

urinary

pH withdrawn

by alkaline

degradated

conservation

of

urine.

7.5% (UTI-B)

trypsin

It

was

and

in

inhibitors

stored the

on the have

lst,

3rd,

of EDTA (fig.

was

clearly

a lesser

improved

lc). 1248

of

days 7th

(fig.

9th

(fig. lb),

days

and its

can both

degradation

laboratory stabilizing

and

UTI-A

mobility, day

was carried at

different

5th,

inhibitor,

At acid pH (4,5) capacity. disappeared on the 7th UTI-A

stability nine

presence

PAGE. The native which

for

be revealed

was rapid la).

At

specially

and

partially and the

pH 7,

the

in

the

Vol. 109, No. 4, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

UTI

UTI

la

lb

Id

: Study of UT1 stability on alkaline 7.5% PAGE, with visualization by technique ; la : concentrated urine stored at pH 4.5, withdrawn on the 1 st, 3rd, 5th, 7th and 9th days. lb and lc : id. on concentrated urine stored at pH 7 ; lb : without EDTA ; lc : with EDTA. Id : fresh urine withdrawn on the 1 st, 3rd, 5th, 7th and 9th days after collection on conservation mixture.

'&s

These collected

results directly

led

us to

sodium

azide

native

fresh

urine,

on the

9 th

day of

storage

pH of

UTI-A

at

(0.5

alkaline

modify

on Na2COS (1 g/l) g/l).

Fig.

the

Id

was

preparation

avoid

shows

that

degradation at

our to

products

laboratory

acid

procedure. ptl,

under only

these

appear

temperature.

slightly

more

was

g/l)

and

conditions, in

than

in

a small

However,

rapid

Urine

on EDTA (1.5

that

a

quantity

the migration of

the

native

inhibitor. UT1 isolation Urines ted

to

having

pH 7 with

dialyzed

for

against

a

15 hrs Tris-HCl

DEAE-Sephacel concentrated gel

sedimented

M pH Tris-HCl

7.4

antitrypsin

activity

M NaOH and

concentrated.

against 0.01

gel

equilibrated

urine.

After

water M

containing

pH

Then,

M, NaCl 0.45

7.4

in

were

was

with

mechanical with

M pH 7.4

eluted buffer.

1249

g/l)

from

stirring

Tris-HCl the

gel

The treatment

for

EDTA. added 0.01 by

adjusurine

then,

containing

same buffer

filtered,

concentrated

EDTA (1.5

was washed

UT1 was

pooled,

The

buffer,

the

adsorption

by centrifugation,

buffer. 0.01

a high 0.1

100

was

24 hrs, ml

of

ml

of

to

800

for

1 hr,

the

M, NaCl 0.05

stirring was repeated

with once.

a

I

10

30

\\ : v

I

0/ 50 Fraction

/ Number

to

NaCl.

90

column

: fractions

110

-.--. --‘--i.\~ k. ..-.-.N.\&

: Ion-exchange chromatography on a DEAE-Trisacryl 5 ml/15 min were eluted by a O-O.3 M gradient of : trypsin inhibition in percentage a -0-e : absorbance at 280 nm -.-.-

w

i

I !‘\ y! !\ ’ \‘\.-J ./

Vol. 109, No. 4, 1982 The

two

8lOCHEMlCAL

combinated

in

two

parts,

in

a Tris-HCl

eluates

loaded

antitrypsin

the a flow

rate

of 15 ml/hr

volume

: 600 ml).

zone

(fig.

2).

After

dialysis

mM, MgC12 1 mM, NaN3 0.2 fractions cm).

(about

After

linear

washing

fractions

were

equilibrated

in the

allowed

under

applied

to

the

and

followed

chromatographied

on

0.01

elimination

of

of collected

M, NaCl

fractions.

the

last

The

last

equilibration

by by

form

on

gradient

of

a wide

0.05

M, NaCl 0.2 M, CaC12 1 the

was performed 12% PAGE (fig.

Sephacryl M,

S200

EDTA 1.5

(2.5 3).

(2.5 g/l

UT1 fractions

x 15

by a O-O.1 x

M

The

UT1

95

cm)

pH 7 buffer.

of contaminant

eluted

inhibitory

column

by 0.1

(total

and heterogeneous

elution

traces

UT1 was

of NaCl

and concentration,

a

a DEAE-

same buffer,

a ConA-Sepharose

same buffer,

a phosphate

the

the

concentration

chromatographied

a Tris-HCl buffer

and

M linear

the

containing

After

with

by a O-O.3

pH 7.6

D-glucose

then

This

SDS-PAGE

with of

were

divided

x 97 cm) equilibrated

buffer

washing

against

g/l

70 mg) were

gradient

serumalbumin.

After

UT1 was eluted

(2.5

and,

The fractions

M pH 7.5

fractions

x 25 cm).

at

with

inhibitory

(1.5

eluted

S200 column

0.05

column

RESEARCH COMMUNICATIONS

by ultrafiltration

M pH 7 buffer.

eluted

a Tris-HCl

ultrafiltration, Trisacryl

0.2

were

against

concentrated

a Sephacryl

M, NaCl

activity

dialysis

were

onto

0.05

AND BIOPHYSICAL

as shown have

in

a faster

migration.

UT1 characterization When relatively (12).

subjected diffuse

After

modified.

(fig.

specific

4).

tly

The higher The

values 1).

of UTI-A

After peak

serum

without

Coomassie

acid

human plasma

amino more were

In

protein

was

prepared

acids

than

both

determined

and very

serum (7).

(data

and

low

be stained

and with

not

of

the

No precipitation shown). significan-

characterized

content

20% carbohydrate.

not

42,000

immunoelectrophoresis,

urine

antiserum

was

between

UT1 could

a

method

on SDS gel

crossed

in

URIEL's

be estimated

anti-human already

by

of UT1 was 1.6U/mg and was therefore obtained values for UTI-A.

composition

dicarboxylic contained

Blue. been

mobility

could

UT1 migrated

as

electrophoresis,

a rabbit had

SDS, Blue

the

gel

Toluidine with

which

with

N-acetylglucosamine

or

conditions

agarose

specific activity than the previous amino

by

these

PAS and

was obtained

with

by mercaptoethanol,

a single anti-B

PAGE,

revealed

UT1 Mr under

by Amidoschwartz, UT1 gave

7.5%

zone,

reduction The

and 46,000

line

to

histidine

by

high (table

N-acetylgalactosamine

and

present.

DISCUSSION The which

is

UT1 also

an acid medium active at neutral

lability stabilized

is

very

close

by EDTA (16).

(uropepsin) (17) pH, and partially

to

that

urine might responsible 1251

of

In addition also for

inter

a trypsin

inhibitor

to the

proteases

active

contain metalloproteases, UT1 degradation.

in

Vol. 109, No. 4, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

too w =s

75

I E ,c

i\ ‘I 1I I I

50

E : c

1; I I ! i

25

-I

10

20

30

40

50

60

Fig. 3 : Affinity chromatography on a Concanavalin-A of 4 ml a O-O.1 M gradient of glucose ; fractions : trypsin inhibition in percentage 0 -0-o : transmittance at 280 nm - .... -

The which

purification

is

rapid

concentration

been

used

by

Sepharose

also

The the

a

degraded

(5)

of

Mr

could the

reported The

amino

not

give

character

exchanger.

operation,

However,

the

was also

on

of

UT1 (7) allows

a

has already

adsorption

on Arginine-

results.

by WACHTER (18) noticed

with

This which

us reproducible

A used

Chromatography

the

different

measurement

generally

of

acid

: elution

for

isolation

by SALIER et fi. ConA-Sepharose

of

(19)

for

represents

an

procedure.

activity

UT1

did

Concanavalin

glycopeptides

yield

character

by a batch

recommended for

on the

Number

Sepharose collected.

were

on an anion

ROUTH (17).

in our

forms

based

inhibitor

inhibitor.

step The

the

is retained

PROKSCH and

trypsin

antitrypsin

keep the not

be

steps is

not

accurately

acid

composition

be

for

UTI,

(5)

in

evaluated

because

SOS-PAGE.

Our

the

obtained

of

the

the

the

results

and by BROMKE : 44,000 of

since

furthermore

UT1

activity.

determined

zone

: 43,000

not

same biological

electrophoretic

by TOKI

could specific

diffuse

agree

with

(6).

preparation

is

similar

by SUM1 et -al. (20) and by MURAMATU et fi. (21). Surprisingly, close to the composition which can be calculated for the sequenced by WACHTER et _.al (18) which is smaller than UTI.

to

given

is also inhibitor

Furthermore, isolated

of

affinity

essential

those

strongly

HI 30 inhibitor

inter

those

procedure

therefore

Fraction

UT1

by BROMKE et -al.

appears

(6)

to

specially

be

clearly

by its 1252

distinct

methionine

from

content.

the

it HI

inhibitor

30

Vol. 109, No. 4, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

100 rc) 0 Y

50

i 30

20

I

I

095

1 relative

Fig. 4 : M determination ;b: soyb&an trypsin d : ovalbumin (43,000) (94,000).

The reported

carbohydrate

in our the

divided

study,

results

in

; 6

composition

that

The presence in

The

presence

difficult

to

affinity

of

; the

been

SDS-PAGE

of

molecular

the

of

of

of the

(5,

20,

in

the carbohydrate (23).

It

carbohydraMr.

However,

which

approach

21).

described

reported

presence

half

from

the

20 to 25% of UTI,

reported

the

different

side was

by UACHTER for by

risk

of

sulfate

the

chain

found

our

HI 30 (181.

same author,

contamination

is

more

after

may explain

of

in

the

ConA

coloration

Blue.

pointed of

that

also

because

On polyacrylamide heterogeneity

only

glycoprotein been

than

glucose,

chromatography

already

for

very

in which

represents

this

already

be

HI 30 inhibitor

chains,

for

quantity

interprete

of UT1 by Toluidine

to

N-acetylgalactosamine has

a lower

the

account

inhibitor

preparation

for

obtained of

seems

two glycan

carbohydrates generally

interatrypsin

lower

by SDS-PAGE : M, markers ; a : lactalbumin (14,000) inhibitor (20,100) anhydrase (30,000) ; c : carbonic ; e : bovin albumin (67,000) ; f : phosphorylase

by HOCHSTRASSER (22)

te moiety,

mobility

gel, out

the

other

carbohydrate

trailing size

for

UT1 migrates

zone of

the

as a diffuse

glycoproteins

and was

moiety

(24).

The

on Sephacryl

5200

might

chain,

due

carbohydrate 1253

band.

fact

has

to

the

attributed

highest therefore to

This

action

mobility indicate of

urinary

in a

Vol. 109, No. 4, 1982

BIOCHEMICAL

AND BlOf’HYSlCAL TABLE

al

Amino acid residues)

our

ASP THR SER GLU PRO GLY ALA VAL CYS 2 MET ILE LEU TYR PHE LYS HIS ARG bl

composition

of

results

Sumi

10.82 5.71 5.13 13.96 4.52 14.88 6.18 5.43 6.91 1.31 1.18 5.96 4.66 4.78 5.14 0.28 3.70

Carbohydrate weight)

UT1

1

(results

et al (20)-

RESEARCH COMMUNICATIONS

expressed

Wachter

et (181

as

al -

2.: 15:2 5.2 14.9 ::2" 6.5 0.6 ::: ;:: 4.6

UT1

(results

expressed

as

Fucose Mannose Galactose Glucose N-acetylgalactosamine N-acetylglucosamine Sialic acid Sulfate

osidases.

This

tic

mobility

for

the

observed

large In

elution

all

establish : inter

mechanism

the atrypsin

mode of formation

cases,

could

zone only

relationship

the

conservation

increase (fig.

of UT1 in DEAE-Trisacryl a more

extensive

between It

of other

related

ACKNOWLEDGMENT We are grateful to Amino Acid and Carbohydrate

explain

urine

inhibitor.

percentage

of

dry

/ 2.2 3.8 4.2 9.2 3.0 2.1 1.5

also

during

100

10.2

;:: 1.85 5.91 5.44 6.07 4.92 0 4.43 of

per

Muramutu et al (211 '-

8.74 4.9 3.96 12.23 4.37 15.11 6.42 5.07

composition

residues

this

could

inhibitor therefore

of UT1 electrophorelel

and could

chromatography structural and its be possible

study

account

(fig. of

supposed

UT1

2). will

precursor

to understand

the

inhibitors.

G. LAMBLIN and determinations.

C. RICHET

for

their

assistance

in

REFERENCES 1. 2. 3.

Laskowski M. and Kato I. (1980) Ann. Rev. Biochim. 49, 593-626. Hochstrasser K., Bretzel G., Feuth H., Hilla W. and Lempart K. (1976) Hoppe Seyler's Z. Physiol. Chem. 357, 153-162. M. and Toccacx N. (1975) Italian J. Biochem. Casati P., Grandi -'24 188-198. 1254

Vol. 109, No. 4, 1982 4. 5. 6. 7. 8. 9. 10. 11. 1':: 14. 15. C: 18. 19. 20. 21. 22. 23. 24.

8lOCHEMlCAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Kessner A., Hodgins L.T. and Troll W. (1979) Anal. Biochem. 92, 383-389 S., Hayashi M., Ukai R. and Sumi H. T1980) Inv. Toki N., Maehara Urology 17, 465-469. Bromke BTJ. and Kueppers F. (1982) Biochem. Med. 27, 56-67. Barthelemy-Clavey V., Yapo E.A., Vanhoutte G., -Aayem A. and Mizon J. (1979) Biochim. Biophys. Acta 580, 154-165. Fritz H., Trautschold I. anaWerle E. (1974) Methods of Enzymatic Analysis 2, 1064-1074 Academic Press New York. Clarke HTG.M. and Freeman T. (1967) Protides Biol. Fluids, Proc. Colloq. 503-509 Elsevier Amsterdam. Kerckaert J.P. (1978) Anal. Biochem. 84, 354-360. Laemmli U.K. (19701 Nature 227, 680-685. Uriel J. and Berges J. (196rNature 218, 578-580. G. anmavez R. (1973) Biochim. Biophys. Degand P., Roussel P., Lamblin Acta 320, 318-330. Rouss?l-P., Lamblin G., Degand P., Walker-Nassir E. and Jeanloz R.W. (1975) J. Biol. Chem. 250, 2114-2122. Aminoff D. (19611 BiocG. J. 81, 384-392. Steinbuch M. (1976) Methods in%zymology 45, 760-772. Proksch G.J. and Routh J.J. (1972) J. Lab.Tlin. Med. 79, 491-499. Wachter E. and Hochstrasser K. (1981) Hoppe Seyler'sT Physiol. Chem. 362, 1351-1355. mier J.P., Faye L., Vercaigne D. and Martin J.P. (1980) Electrophoresis 1, 193-197. Sumi-H., Toki N., Takasugi S., Maehara S., Maruyama M., Akazawa K., Matsuo 0. and Mihara H. (19821 Thromb. Haemost. 47, 14-18. Muramatu M., Mori S., Matsuzawa Y., HoriguchTY., Nakanishi Y. and Tanaka H. (1980) J. Biochem. 88, 1317-1329. Hochstrasser K., Schbnbergera.L., Rossmanith I. and Wachter E. (1981) Hoppe Seyler's Z. Physiol. Chem. 362, 1357-1362. Uhlenbruck G., Reese I., Vaith P.and Haupt H. (19791 J. Clin. Chem. Clin. Biochem. 17, 29-34. Tsuji T., IrimuE T. and Osawa T. (1981) Carb. Res. -92, 328-332.

1255