Effect of α1-protenase inhibitor and sulphated polysaccharides on the activity of m β-acrosin

Vol. 117, No. 1, 1983 November 30, 1983

BIOCHEMICAL

EFFECT OF a,-PROTEINASE

INHIBITOR

AND SULPHATED

POLYSACCHARIDES ON THE ACTIVITY William Dept. Received

F. Long and Frank

of Biochemistry,

October

RESEARCH COMMUNICATIONS Pages 319-323

AND BIOPHYSICAL

University

OF m+-ACROSIN

B. Williamson

of Aberdeen,

Aberdeen,

AB9 lAS,

U.K.

18, 1983

Purified m -acrosin catalysed amidolysis in vitro of several p-nitroanilides with C-ter 4. lnal arginine residues. or-Proteinase inhibitor inhibited amidolysis catalysed by the enzyme. This effect of cl,-proteinase inhibitor was not prevented by pre-incubation of the enzyme with heparin or any other glycosaminoglycan. Pre-incubation of the enzyme with sulphated dextran or sulphated cellulose alleviated the effect of a,-proteinase inhibitor. These results are discussed in terms of possible in vivo modulation by a,-proteinase inhibitor of acrosin activity. The proteinase within

the

acrosin

acrosome,

of fertilization

of acrosin

penetration

of the

the acrosomal of acrosomal effectiveness possible

an intracellular

in vitro

importance

during zona

ability

Because

considerations inhibitors

the action

sulphated a,-proteinase

inhibitor

of this

polysaccharides inhibitor

inhibitor (3-6),

system

In this the

on some proteinases

also

MATERIALS

spermatazoon

to release

of these

in or exposure

interest

activity,

present

the

in participation

of acrosin

to affect

the effects is

suggests

have stimulated

(1,2).

is

Prevention

in enabling

leading

modulators

contraceptives

of a,-proteinase

inhibitors

on acrosin

form,

organelle.

ovum and/or

of events

as physiological

use as pharmacological the

These

in zymogen

possibly

of the

a sequence

of proteinase

by acrosin

fertilization,

pellucida

contents.

largely

spermatazoon

and in vivo

reaction,

function

(EC 3.4.21.10),

in

the

and in their activity

and possible

communication action

in vitro

we report of mp-acrosin.

may be affected compounds

on the

by a+- acrosin-

examined. AND METHODS

Materials: Human plasma ol- proteinase inhibitor (preparation A: lot no. 7OF-9340; preparation 8: lot no. lllF-9355; preparation C: lot no. 42F-9420; preparation lot no. 51F-9345; preparation E: lot no. 92F-9325), heparin from porcine intestinal mucosa (lot no. 46C-0035) and Polybrene (1,5-dimethyl 1,5-diazaundecamethylene polymethobromide) were from Sigma Chemical Co., Poole, U.K. Dermatan sulphate

319

0006-291X/83 $1.50 Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.

D:

Vol. 117, No. 1, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

from porcine skin (lot no. S9401) was from Seikagaku Fine Biochemicals, Tokyo, Japan. River sturgeon notochord chondroitin 4-sulphate and 6-sulphate, bovine lung heparan sulphate and human umbilical cord hyaluronic acid, all produced under contract no. NOl-AM-5-2205 for the U.S.A. National Institues of Health, were kindly given by Professors M.B. Mathews and J.A. Cifonelli, Dept. of Pediatrics, University of Chicago, lL, U.S.A. Sulphated cellulose (lot no. 185) was obtained fromKelco,,San Diego, CA, U.S.A. Sulphated xylan (preparation SP54) Sulphated dextran from dextran was obtained from Benechemie GmbH, Munich, F.D.R. of molecular weight 5 x 10 was obtained from Pharmacia, Uppsala, Sweden. All polysaccharides were in the form of sodium salts. Chromogenic substrates S-2222 (N-benzoyl-L-isoleucyl-L-glutamyl-glycyl L -arginine-p-nitroanilide hydrochloride and its methyl ester), S-2238 (D-phenylalanyl-L-pipecolyl-L-argininep-nitroanilide dihydrochloride), S-2266 (D-valyl-L-leucyl-L-arginine-p-nitroanilide dihydrochloride), S-2288 (D-isoleucyl-D-prolyl-L-arginine-p-nitroanilide dihydrochloride), S-2302 (D-prolyl-L-phenylalanyl-L-arginine-p-nitroanilide dihydrochloride) and S-2444 (L-pyroglutamyl-glycyl-L-arginine-p-nitroanilide hydrochloride) were from KabiVitrum, Ealing, U.K. m -Acrosin (approximate activity 130 units/mg protein) purified from ovine spe-B matazoa (7) was kindly given by Dr. R.A.P. Harrison, Agricultural Research Council Institute of Animal Physiology, Cambridge, U.K. This is the major and most stable form of active ovine acrosin; for a definition of nomenclature and of unit of activity of ovine acrosins see (7). Reageets were dissolved and diluted in buffer (0.11 M-NaCl/O.O5M-Tris/HCl, pH 7.7 at 37 C). Methods: A 30~1 portion of polysaccharide solution was incubated at 37'C for 5 min with 2Oul of m -acrosin solution (4.4 g/ml). Then 20~1 of a sol;ut&n of a,-proteinase inhibiE 8 r was added and the mixture incubated for 5 min. 20~1 of a mixture of chromogenic substraie (0.12 mg/ml) and Polybrene (0.12 mg/ml) was added. Initial reaction rates at 37 C were determined by measuring released p-nitroaniline spectrophotometrically. In control experiments, polysaccharides, enzyme and a,-proteinase inhibitor were, in various combinations, replaced by appropriate volumes of buffer. The polycation Polybrene was included in order to prevent possible electrostatic interaction between polysaccharides and substrates (8). Concentrations quoted are final concentrations in the reaction mixture after addition of substrate and Polybrene. Methodology of experiments involving thrombin (EC 3.4.21.5) has been described previously. RESULTS In the assay inhibitor

system

catalysed

containing

C-terminal

initial

reaction

rates

S-2266

3.9;S-2238

3.5;

addition

S-2288

S-2302

substrate

the absence

Incubation

of heparin

did with

of heparin not affect thrombin

2.7;

inhibited

when al-proteinase

in

the

in the absence

release

arginine

S-2444

2.3.

residues

S-2288

In subsequent

inhibitor

(Table

preparations

from

at the following

of al-proteinase

enzyme activity

of a,-proteinase

of p-nitroaniline

substrates

Incubation

was used.

of substrate

acrosin

(A@~l/minx103):

was observed

inhibitor

"p-

or polysaccharide

p-nitroanilides

substrate

described,

5.8;

S-2222

4.0

experiments,

with

enzyme

before

1).

No amidolysis

were

incubated

with

of enzyme. with the

!p -acrosin

action

before

before

of the

addition

addition

inhibitor;

of al- proteinase 320

of al-

in contrast, inhibitor

proteinase incubation prevented

;

Vol. 117, No. 1, 1983

BIOCHEMICAL

AND BIOPHYSICAL Table

Concentration of inhibitor (mg/ml x lo*)

Initial

Human preparations B C D

1.3 3.4 5.4 5.8 N.D. N.D.

Amidolytic rate 5.8 (Ak~~ul/min

the action ation

of the

inhibitor

of m - acrosin -P before addition

xylan inhibitor

(Fig.

or with

a sulphated

prevented

out

catalvsed x 103)

the

other

cellulose of the

inhibitor,

absence

not affect

E

1.1 1.6 3.3 5.7 N.D. N.D.

co.2 co.2 co.2 1.4 4.8 5.4

addition

(Fig.

or with

a sulphated

with

action

a sulphated

of al-proteinase

dextran

lc).

incubated

polysaccharides,

Incub-

inhibitor

when any of the polysaccharides, were

la).

of a,-proteinase

of al-proteinase

(Fig.

was

amidolysis

the

of trip-acrosin

inhibitor

a,-proteinase

in the

did

before

was observed

None of the

Bovine

1.2 3.5 5.8 5.9 N.D. N.D.

glycosaminoglycans

Incubation

No amidolysis

of enzyme.

1.0 3.0 5.5 5.7 N.D. N.D.

by .T: acrosin in lbitor

by mgacrosin in absence of inhibitor N.D. = not done

of inhibitor

action

0.8 1.4 3.0 5.6 N.D. N.D.

on thrombin-catalysed

with

lb,c).

1

amidolytic rate catalysed in presence of a,-proteinase (A~~~ll/rninxlO~)

A

200 100 50 10 5 1

RESEARCH COMMUNICATIONS

with

substrate

when incubated

inhibitor,

with

altered

or with-

in the absence

with

enzyme

the amidolvtic

rate.

DISCUSSION Inhibitors

immunologically

inhibitor

have been

Fallopian

tube

detected

fluids

(9).

to penetrate that

endogenous

by their effectively proteinase

in seminal Although

of such naturally-occurring may be limited

indistinguishable

high wide

from

plasma,

the

cervical

to sites inhibitors

of acrosin modulate 321

contraceptive

proteinase

and by their activity acrosin

qproteinase

mucous and

pharmacological

molecular-weight

specificity

plasma

inhibitors

probable (9),

inability

it

is possible

activity

in vivo.

potential

BIOCHEMICAL

Vol. 117, No. 1, 1983

a

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

b

C

f

0

-

O

1

0

0

2

1

3

PQ

1 b Y x

B: 2

1

3

2

3

4

Effect of polysaccharides on inhibition of I+- acrosin and thrombin $k$iroteinase inhibitor. (a) kffect of heparin on inhibition of (0) F- acrosin; (0) thrcmbin; (b) Effect of (0) heparan sulphate, (0) den tan sulphate, (A) chondroitin 4-sulphate, (A) chondroitin 6-sulphate on inhibition of - acrosin. (c) Effect of (0) cellulose sulphate, (0) dextran sulphate (i"4 xylan sulphate on inhibition of Initial amidolytic rate is expres Y- ed acrosi as a percentage n* of that initial rate occurring when enzyme was incubated with substrate and Polybrene and in the absence of inhibitor and polysaccharide; these control rates (Amul/min x 103) were 5.8 for m+-acrosin YT 0.26 for thrombin. Concentrations of al-proteinase inhibitor were 0.1 g/ml for m -acrosin experiments, and 0.05 mg/ml for thrombin experiments. Reaction rates ( s percentage control) in the presence of enzyme and these concentrations of inhibitor, and in the absence of polysaccharide were: m -acrosin 24%; thrombin 58%. 4

Such modulation during high

may include

anovulatory

periods.

concentrations

reported

(2 .

an apparently purified excess activity.

A partial

and complete

a,-proteinase

the enzyme, Inhibition

system

was incubated

of enzyme with

upon polysaccharide-proteinase

significant

(3).

In contrast,

(EC 3.4.21.

') by al-proteinase

in this

present with

in 9-10

ovine

a+-

(Fig.

inhibitor la;

interaction, inhibitor

of factor

inhibitor

is 322

by very

3-6);

has been

occurred

fold

acrosin

and

communication,

of enzyme activity

by a,-proteinase heparin

acrosin

preparations

described

inhibition

at fertilization

of ovine

inhibitor

inhihitor,

of thrombin

activity

inhibition

of human al-proteinase

rapid

by pre-incubation depends

on acrosin

In the experimental

bovine over

effects

when

molar of high

specific

may be prevented this

effect

and may be clinically Xa (EC 3.4.21.6)

not prevented

by heparin

and plasmin (4,5)

Vol. 117, No. 1, 1983

presumably the

because

BIOCHEMICAL

of the

glycosaminoglycan

in this

respect

inhibitor of acrosin

the

avid

interaction

Results

reported

factor

results

here

Xa and plasmin

of this

study

It

seems likely

that

RESEARCH COMMUNICATIONS

between

rather

using

endogenous

these

suggests

enzymes that

than

purified

and m - acrosin in vitro suggest possible 4 activity in vivo by endogenous inhibitors

inhibitor. affect

(10).

resembles

In sununary,

less

AND BIOPHYSICAL

"p-

and acrosin

thrombin.

a,-proteinase

physiological

modulation

resembling

o1 -proteinase

glycosaminoglycans

would

not

such modulation.

ACKNOWLEDGEMENTS: We thank the Council and Nuffield Foundation laboratory. We thank Dr. R.A.P.

Cancer Research Campaign,Medical Research for grants supporting work in our Harrison for the gift of the rnp- acrosin. REFERENCES

1.

Hartree,

2.

Morton, D.B. (1977) A.J. Barrett, (Ed.) Amsterdam, New York

3.

Danishefsky 91, 862-870.

4.

Long,

W.F.

and Williamson,

F.B.

(1980)

Brit.J.Pharmacol.

5.

Long,

W.F.

and Williamson,

F.B.

(1981)

Biochem.Soc.Trans.

6.

Takahara,

H. and Sinohara,

7.

Harrison,

R.A.P.

8.

Teien, A.N., 859-867.

9.

Zaneveld, L.J.D., Polakoski, K.L. and Schumacher, G.F.B. (1975) In Proteases and Biological Control. E.Reich, D.B. Rifkin and E. Shaw (Eds.) Cold Spring Harbor Conferences on Cell Proliferation Volume 2. pp. 683-706.

10.

E.F.

(1977)

I.

Biochem.Soc.Trans.

5, 375-394.

In Proteinases in Mammalian Cells Elsevier/North Holland Biomedical and Oxford, pp. 445-500.

and Pixley,

(1982)

Abildgaard,

R. (1979)

Biochem.Biophys.Res.Commun.

H. (1982)

Thromb.Res.

J.Reprod.Immunol. U. and Htll)k,

Jordan, R.E., Oosta, G.M., Gardner, J.Biol.Chem. 255, 10073-10080.

and Tissues. Press,

9, 408.

27, 45-50.

4, 231-249. M. (1976)

W.T.

323

73, 505-509.

Thromb.Res.

and Rosenberg,

R.D.

8,

(1980)