Studies on the Limulus coagulation system: Inhibition of activation of the proclotting enzyme by dimethyl sulfoxide

Vol. 105, No. 2, 1982 March

AND BIOPHYSICAL

BIOCHEMICAL

RESEARCH COMMUNICATIONS

30, 1982

STIIDIES

Pages

ON THE

INHIBITION OF ACTIVATION LIJIULUS COAGULATION SYSTEfl: THE PROCLOTTING ENZYME BY DTIJETHYI, SULFOYIDE Shu-1Iei

Division

of

Riceived

553-559

Liang

Biochemistry and Administration,

February

II,

and

Teh-Yung

Biophysics, Bethesda,

OP

Liu

Bureau of Ilaryland

Biologics, 10205

Food

and

Drug

1982

Dimethyl sulfoxide inhibits the endotoxin-induced clotting activity of The inhibition is dependent upon the concentration Limulus amoebocyte lysate. of dimethyl sulfoxide and is competitive with endotoxin. Further studies showed that dimethyl sulfoxide inhibited the clot formation by reversibly inhibiting the activation of the proclotting enzyme to the clotting enzyme. Dimethyl sulfoxide had no effect on the active form of the clotting enzyme. Based on these findings, a procedure has been developed for the isolation of the proclotting enzyme from J,imulus amoehocyte in the presence of dimethyl sulfoxide. In

1964,

Relation

of

Levin Limulus

extreme

sensitivity,

variety

of

mechanism

of

the

dependent

activation

Since

amounts

enzyme,

endotoxin-free

the

conditions

activation

of

procedure

(8).

resulted (9).

Abbreviations:

Limulus the

the

(6)

endotoxin

isolation

of

(2).

proclotting This

endotoxin-induced

in

very

poor

In

this

communication,

EDTA,

Ethylenediamine

the

occurs

even we

form

the

1~s

tetraacetic

553

that

found

and

endotoxin

enzyme

followed

the

that the

proclotting

isolating dimethyl

acid

The

necessitates

during

in

(5)

7).

of

the

(2)

amoebocyte.

(3,

enzyme

a

protein

clotting

activation

of

failure

enzyme

Ca i+

a clot

been

in

its

the

binding

frequently

report

and

investigating

the

activation or

endotoxin

involves

to to

it

detect

Limulus

proclotting

However, enzyme

yields

the

property

endotoxin

lysate

cause

induce

unique

been

from

coagulin

rapidly

a proclotting

an

enzyme

the

to

purified

amoebocyte

of of

used

and

proclotting

could

this

has

(4),

activity

cleavages

minute

proclotting

enzyme

of

been

Me have enzyme

in

of

laboratory

gelntion.

clotting

proteolytic

has

Our

a clotting

endotnxin

Because

lysate

fluids. lysate

that

(1).

calmodulin-like

of

has

amoehocyte

(3),

possesses

the

discovered

Limulus

mechanism

by

Bang

biological

a cnagrllogcn that

and

J)PJSD,

the sulfoxide Dimethyl

purification enzyme proclotting (DHSO) sulfoxide

0006-291X/82/060553-07$01.00/0 C’opyrghr 8 1982 t1.v Academic Press. Inc. .A II rr,yh/l of- reproduction rn on! .fonn rc~.~erved.

Vol. 105, No. 2, 1982 inhibits

this

adding

DPISO to

in

chromatographic

two

BIOCHEMICAL

endotoxin-induced the

AND BIOPHYSICAL

activation

buffer

solution,

of

the

the

RESEARCH COMMUNICATIONS

proclotting

proclotting

enzyme.

enzyme

has

By

been

purified

steps. MATERIALS

AND METHODS

Pyrogen-free water (Abbott labs); Escherichia coli 0113:HlO (Dr. Donald Hochstein, Food and Drug Administration); dimethyl (Pierce labs); frozen Limulus amoebocytes and pyrotell Limulus lysate (Association of Cape Cod); Sephadex G75 and DEAE Sephadex (Pharmacia Fine Chemicals); S-2222 (Kabi Diagnostica); glassware from pyrogens by heating at 180°C for at least 4 h.

endotoxin sulfoxide amoebocyte A-25 was freed

The amidase activity of the Limulus proclotting enzyme was monitored by using a synthetic chromogenic substrate, benzoyl-Ile-Glu-(y-0CH3)-Gly-Arg-pnitroanilide (S-2222) (10). One hundred microliters of reconstituted lysate or an unknown amount of the proclotting enzyme was added to a buffer mixture containing 0.045M Tris-HCl pH 7.5, 3 mM Ca*, 0.1 m!l S-2222, and 0.3 ng of endotoxin in a total volume of 1 ml. After 30 min of incubation at 37"C, the reaction was stopped by the addition of 0.1 ml of glacial acetic acid and the released p-nitroaniline was estimated by its absorbance at 405 nm. All steps involved in the purification of the proclotting enzyme were performed at 4°C. Packed frozen amoebocytes (10 ml) were thawed and washed with 3% sterile saline. The cells were then lysed by vortexing them in 30 ml of pyrogen-free water containing 5% DFlSO and 1 m?l EDTA. The mixture was blended in a Waring blender for 30 set at high speed. After centrifugation at 1000 Xg for 20 min, the supernatant was applied to a G75 column (2.6 X 90 cm) and eluted with a buffer containing 0.05M NaCl, 5% DMSO, and 1 mP1 EDTA in pyrogen-free water. Eluent fractions containing the amidase activity were pooled, applied to a DEAE Sephadex A25 column (2 x 40 cm), previously equilibrated with 0.0511 Tris-HCl pH 6.8, 1 m!l EDTA, and 5% DMSO. The column was washed with the same buffer and eluted with a linear gradient of 0 to 1.0 The active fractions were analyzed by gel El NaCl in the same buffer. electrophoresis. Sodium performed

dodecyl according

sulfate to the

(SDS) method

polyacrylamide of Laemmli

(11)

gel electrophoresis using a 7.5%

was gel.

RESULTS The the

initial

objective

proclotting

amounts the

of

enzyme endotoxin

isolation the

purification this

inhibiting

the

Limulus

the

is

problem

endotoxin-induced that activity

DMSO and

lysate

was the

and

prone

a reagent

activation.

After

most

amidase

554

isolation with

to

clot

conditions

for

the

the

suitable. activity

thus

failure.

capable

of

screening D?ISO (Fig.

is the

entire

We,

therefore,

reversibly many

inhibited 1).

trace rendered

endotoxin during

to

of

even

and

Since

endotoxin-free

searching

improve

difficult.

time-consuming by

to

Contamination

amoebocyte enzyme

of

was

amoebocyte.

the

maintenance

we found clotting

the

study

proclotting

procedure

approached

reagents,

from

this

induced

of

ubiquitous,

of

The

potential both inhibition

the

vol. 105, No. 2. 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

lco90-

80 70 605040 30 20 10 01

Fig.

was

1

Effect of lysate. concentration assay was endotoxin respectively.

dependent

upon In

endotoxin. amidase

activity;

achieve

the

the

the

proclotting

the

clotting

to

30%)

the

the

being

activity

of

of

adding

gel

the

It

of

reduced

permeation

inhibition.

preincubated

with

1% DPISO inhibited DPISO was

inhibition

and the

with

clotting

57%

required

to

was

reversed

for

I hr

by

high of

1 & 2)

proclotting

it

enzyme

concluded,

therefore,

after

the

amidase

apparent

but

had

that

of endotoxin

is

no DNSO

convert

activity

concentrations DPISO with

to

of DNSO blocked

that

DPISO

effect

once

may

(up

the

faciliate

enzyme. to

proclotting

Sephadex

even

(Fig.

1 mPI EDTA

only

then

addition

lysate the

endotoxin

enzyme,

inhibited

was

to on

competitive

6.5% The

proclotting

5% DMSO

and

of

was

endotoxin,

endotoxin,

simultaneous

of

intact

TPlSO and

rig/ml

rig/ml

the

not

activation active.

0.25

25

was

to

Since

of

of

dialysis.

lysate

process

simplified

by

2).

isolation

purification

DHSO

was

became

By

magnitude

enzyme

the

of at

enzyme

amidase

enzyme

presence

Limulus

(Fig.

inhibited

concentration

while

of

the

the

the

same

removal If

DNSO on amidase activity of the Limulus amoebocyte The amidase activity of the lysate was plotted versus the The amidase activity of DMSO on a logarithmic scale. performed as described under Methods. The concentrations 2.5 rig/ml (0) and 25 rig/ml (a) were 0.25 rig/ml (o), The points plotted were the means of duplicate assay.

two

all enzyme

successive G75

and

555

buffer from

solutions,

the

Limulus

amoehocyte

chromatographic the

second

being

steps, ion-exchange

was the

first

Vol. 105, No. 2, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

100 90 I30 70 60 50

!

40 30 20 10 0

0i

I

I

I

I

I

1

2

5

10

30

% DMSO Fig.

2

Effect of DMSO on the endotoxin-preincubated lysate. The amidase activity of the concentration of DMSO on a logarithmic was added to the lysate 1 hr before the addition of DMSO.

chromatography

on

amoehocyte

lysate

on

0.8 0.7 E

0.6

i G

0.5

8

0.4

: E 0 07 z

II&Xl? a

Sephadex

A25.

Sephndex

(;75

The

Limulus amoebocyie lysate was plotted versus scale. Endotoxin (2.5 (0) or simultaneously with

elution

column

is

profile shown

in

of Fig.

the

crude

the rig/ml) (0)

Limulus

3.

Sephadex G 75 (2.5 x 92 cm)

0.3 0.2 0.1

FRACTION Fig.

3

NUMBER

Gel filtration of the crude amoebocyte column. The column was eluted with Fractions of 5 ml were collected at portions which contained the amidase bar.

556

15 ml!tube)

lysate on a Sephadex G75 0.05 !I NaCl, 5% DMSO and 1mM EDTA. a flow rate of 50 ml/hr. The activity are indicated by the

Vol. 105, No. 2, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

0.4 DEAE

Sephadex

A 25

12 x 40 cm) 0.3

0.2

0.1

J

FRACTION

Fig.

4

llpon first

to

proclotting

The

first

activity

(Fig.

composition

contained 4).

is

the

Using were

unable

from

the

same

to

of

,activity

immediately

12.S

times

reported mnrc

found

after

the

a small

amidnse

void

amid&se Ohki.

et

activity

the

Following

a peak

volume,

having

the

‘RIP the

557

al.

two

the

shoun

ahsorhe
prnclotting

ahsorhed

Tai

and

lwocedure

ac,iii

purified

Liu that

enzyme

(2). they

activity hut

with

the

enzyme

l:ig.

5.

Tn ,?ddition,

traction enzyme

frarti,)n.

ax~i.no this

proclotting in

peaks. enzyme

and

reported

same

major

that

proclotting

the the

proclnttin~

by (9)

of

containing

weight,

the as

in

(5)). than

yielded

the

containinK

activity al.

column

of

conrained

fraction,

isolated et

containing

peak

indicated

enzyme

lysate.

this

fraction

Ohki

shoulder

this

moleclllar

column,

amoebocyte

hy

protein

proclotting

fractions

we

A25

homogeneous

Sephnrose

5% DIISO,

detected

Sephadex

trailing

that of

electrophoresis,

this the

isolate

addition

as

as

a Heparin

Limulus

WC also

of

the

chromatography

a DEAE

SDS gel

in

indicating

a homogeneous

analysis

protein

on

eluates

activity

Subsequent

enzyme, peak

the

amidasc

enzyme.

proclotting

27-70)

endotoxin,

exlribited

tire

(6 ml/tube)

DEAF. Sephadex A25 chromatography of proteins from Sephadex C75 15 ml sample was dialyzed against the elating buffer, fractions. containing 0.05 I1 Tris-HCl pH 6.8, 1mM EDTA and 5% DPlSO before application to the column. The column was first washed with ?O!: ml eluting buffer at a flow rate of 15 ml/hr and then elated with a linear gradient from 0 to 1.0 )I NaCl. The fractions which contained the amidase activity are indicated by the bar.

exposure

peak

NUMBER

(Fractions fracti.on In

contrdst

i:ad tc,

Vol. 105, No. 2, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

HEPARIN-SEPHAROSE 1.4x2.3 cm

0.7

5

0.6

gI)

0.5

$

0.4

?J m

0.3

3

1

45 FRACTION

Fig.

5

the

previous

the

proclotting

finding

indicating the

NUMBER

Heparin-Sepharose column chromatogrphy of Limulus amoebocyte lysate. 10 ml of protein from the selected Sephadex G75 fractions were applied to the column, previously equilibrated with 0.05 M Tris-HCl pH 7.2, 1 mtl CaC12, 0.15 11 NaCl and 5% DWSO. The elution was performed at 4” C, first with the equilibration buffer and then with a linear gradient buffer from 0.154 M NaCl to 0.54 M NaCl in the same buffer. Arrow indicates the beginning of the linear gradient. Fractions of 3 ml were collected at a flow rate of 10 ml/hr. Solid line represents the absorbance at 280 nm and dotted line indicates the amidase activity.

of

enzyme that

absorbed

the

Ohki

et

fraction

activity

of

al.

(9),

addition

did

not

the

proclotting

of

increase

the

absorbed

the

amidase

enzyme

is

fraction

to

activity, not

dependent

upon

fraction. DISCUSSION

The

endotoxin-induced

activation

several

subsequent

enzymatic

Limulus

amoebocyte

lysate

endotoxin-induced enzyme

reported

that

enzyme.

However,

irreversibly inhibited conversion

reactions

4tl

of

not

sodium

this

proclotting

in

thus

Limulus enzyme

enzyme

to

capable

the of

the

amoebocyte to

558

the

inhibiting isolation

the

proclotting

lysate enzyme

found by

without

of et

of

we

of

the

this

activation

study,

initiates formation

Schleef

condition, present

clot

the

previoulsy.

salt the

lead

facilitating

found

high

proclotting

that

inhibited

In

(8).

formation the

been

chloride

under

denatured clot

and

have

the

Reagents

(2-4).

activation

proclotting

of

al. the

the (8)

proclotting enzyme

that preventing any

DMSO the concomitant

was

Vol. 105, No. 2, 1982 denaturation catalytic

BIOCHEMICAL It

of the protein. role

in

the conversion

the Limulus

lysate

active

of the enzyme is

that

form

DEISO rendered

the proclotting proclotting inhibitory

system

(4).

endotoxin

AND BIOPHYSICAL has been suggested

that

of the proclotting

enzyme to the enzyme in

The finding in agreement ineffective

that with

reaction

Studies

this

hypothesis.

as a catalyst

are underway

endotoxin

plays

DlISO had no effect

enzyme to the enzyme by preventing enzyme.

RESEARCH COMMUNICATIONS

to elucidate

its

for

the

a

on the

We suggest conversion

association the mechanism

with

of its

of this

by DIlSO. REFERENCES

Levin, J., and Bang, F.B. (1964) Bull. Johns Hopkins Hosp. 115, 265-274. Tai , J.Y., and Liu, T.-Y. (1977) J. Biol. Chem. 252, 2178-2181. Seid, R.C. Jr., Huhn, R.D., and Liu, T.-Y. (1977) J. Biol. Tai, J.Y., Chem. 252, 4773-4776. Seid, R.C., Jr., Tai, J.P., Liang, S.-PI., Sakmar, T.P., and 4. Liu, T.-Y., J.B. (1979) Biomedical Applications of the Horseshoe Crab Robbins, (Limulidae), pp. 147-158, Alan R. Liss, Inc., New York. S.+l., Sakmar, T.P., and Liu, T.-Y. (1980) J. Hiol. Chem. 255, 5. Liang, 5586-5590. S.-M., Liang, C.-t!., and Liu, T.-Y. (1981) J. Biol. Chem. 256, 6. Liang, 4968-4972. 7. Young, X-S., Levin, J., and Prendergast, R.A. (1972) J. Clin. Invest. 51, 1790-1797. 8. Schleef, R.R., Kenney, D.El., and Shepro, D. (1979) Thrombos. Haemostas (Stutty.), 41, 329-336. T., and Iwanaga, S. (1980) FEBS Letters 9. Ohki, Ft., Nakamura, T., tlorita, 120, 217-220. 10. Sakamura, S., plorita, T., Iwanaga, S., Niwa, )I., and Takahashi, K. (1977) J. Biochcm. 81, 1567-1569. 11. Laemmli, U.K. (1970) Nature 227, 680-685. 1. 2. 3.

559