A cyclopeptidic suicide substrate preferentially inactivates urokinase-type plasminogen activator

Vol. 178, No. 1, 1991 July 15, 1991

A

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 352-359

CYCLOPEPTIDIC SUICIDE SUBSTRATE PREFERENTIALLY UROKINASE-TYPE PLASMINOGEN ACTIVATOR

Michele

INACTIVATES

Reboud-Ravauxa, Anne-CBcile Vilaina, Nicole Boggettoa, Jean Maillarda, Favreaua, Juan Xieb, Jean-Paul Mazaleyratb and Michel Wakselmanb

aLaboratoire bLaboratoire

d’Enzymologie

Mol&ulaire et Fonctionnelle, lnstitut Jacques Monod, VII, 2 place Jussieu, 75251 Paris Cedex 05, France

de Chimie

Organique

May

1991

Received

28,

Siologique,

CNRS-CERCOA,

2 rue Henry

Catherine

Universitb

Dunant,

94320

de Paris Thiais,

France

c[Arg-aB-(CH26CH3$)-Gly4] was designed and studied as a mechanism-based inactivator (suicide substrate) for plasminogen activators (U-PA and t-PA) and plasmin. This compound inhibited U-PA and fulfills criteria expected for the involvement of an enzyme-activated inhibitor : first-order and irreversible process, saturation kinetics, protection by substrate. The limiting first-order rate constant kinact and the apparent enzyme-inhibitor dissociation constant KI were 0.021 s-l and 9 PM, respectively at pH 7.5 and 25 “C. The activation of plasminogen by U-PA is compromised after this enzyme has been treated by the reagent. Plasmin and t-PA were inactivated 40- and 2330-fold less efficiently than U-PA, respectively. 0 1991 Academic Press, Inc.

(EC 3.4. 21.7), a serine protease

with a broad

enzymatically

Plasmin

inactive

by action

plasminogen

activators

(U-PA).

In addition

zymogen

(EC 3.4.21.31),

to fibrinolysis

physiological

and pathological

implantation

(4) and tumor

expression

of tumor

plasminogen

potential

(11,

This paper using

are known,

analysis

among

and

as

A selective

alkylation

of the active

a further

novel

approach

inhibitor,

0006-291x/91 $1.50 Copyright 0 1991 by Academic Press. inc. All rights of reproduction in any form reserved.

synthetic

the

inhibitors

for plasmin

Few

of

generation, roles and synthetic

of lysyl or arginyl

halomethylated

derivatives

inactivators

site histidine

was demonstrated

to mechanism-based

352

between

derivatives

mechanism-based

c[Arg-aB-(CH26

of

(3), embryonic

shown

proteolysis.

them peptidic

behaving

derivative

with a number

of their biological

of extracellular

12)

presents

Specific

responsible

by affinity labeling

by a dihydrocoumarin a

They include

has been

from the unrelated

and urokinase

(2), cell migration

(5-8).

protease

the control

(9, 10) acting

(13-I 5).

urokinase

activators

applications.

of these enzymes

3,4-dihydrocoumarins weight

the initial

uses through

chloromethylketones

substrates)

by inhibiting

(t-PA)

is associated

(5). A correlation invasion

is generated

immunologically

activator

activation

like angiogenesis

cell metastasis

specificity

of two

plasminogen

(l), plasminogen processes

in different

therapeutic

inactivators

tissue

cell U-PA and tumor

activators,

are of interest

plasminogen

of

(suicide

of high-molecular-

(14). inhibition

C H 3@)-GIy4]

of plasminogen (Fig.

1, aB

=

Vol.

178,

No.

1, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

HZ0 I Hydrolysis poduar Figure

1.

Postulated mechanism for the plasmin by c[Arg-aB-(CH2SCH3@)-Gly4].

3-aminobenzoic which

acid residue).

has been designed

into a cyclopeptide

Other

chymotrypsic

by including

the formation

function

19, 20).

This inhibitor

is based

functionalized

specificity

of

is a member

activators

quinonimmonium

of the

series

(11, 12,

cyclopeptides

have

in the dihydrocoumarin been

inactivators

targeted

against

the conformational

peptidic

backbone

report

features

inhibitor

possessing

of urokinase

and plasmin

a latent thioanisole

the salient

proteases

flexibility

of the inactivation

by c[Arg-aB-(CH2&H30)-Glyd], leaving group;

mode

of

(18, 21). The NMR conformational

has demonstrated

activators

(16. 17)

unmasking

findings

of c[Arg-aB-(CH,)-Gly4]

plasminogen

methide

with the simultaneous

of an acyl-enzyme

We now

substrates

Its presumed

analysis

(22).

and

dictates specificity (18).

upon previous

and act as enzyme

plasminogen

of a new family of suicide

a latent electrophilic

ring which amino acid sequence

of action through electrophilic

inactivation

its influence

a potential on the fibrinolytic

of the of both suicide activity

is also analyzed. MATERIALS

AND METHODS

Human urinary urokinase (RFU 203) was obtained from Choay. Based on quantitative gels, this preparation was more than 95 % pure in the high-molecular-weight form. Human plasmin was purchased from Kabi Vitrum, France. Two-chain t-PA, obtained from cultured melanoma cells, was purchased from Biopool, Sweden. Its concentration in solution was determined by amino acid analysis. Active-site titrations were carried out with 4-methyl-umbelliferyl-p-guanidinobenzoata for urokinase (23) and p-nitro-p’-guanidinobenzoate for plasmin (24). The chromogenic substrates L-pyroglutamyl-L-glycyl-L-arginine-pNA (S-2444) of urokinase, D-isoleucyl-L-prolyl-L-argininepNA (S-2288) for t-PA and D-valyl-L-leu+cyl-L-lysine-pNA (S-2251) of plasmin were from Kabi Vitrum. The synthesis of c[Arg-m-aB-(CH2SCH3$)-Gly4] will be reported elsewhere. . . rtv assu The amidolytic activity of urokinase was determined towards S-2444 (100 FM) in 0.025 M phosphate, 0.1 M NaCI, 0.05 % (v/v) Tween 80 at pH 7.5 and 25 “C by measuring the production of nitroaniline at 405 nm using a Lambda 5 Perkin Elmer spectrophotometer. Plasmin was assayed similarly with 280 FM S-2251 in 0.1 M sodium phosphate, 25 % (WV) glycerol at pH 7.5 and 25 “C. t-PA was assayed with 280 pM S-2288 in 0.05 M Tris, 0.1 M NaCI, 0.01 % Tween 80 at pH 7.5. bvme inactivation (incubation method), Urokinase (0.55 FM) was incubated with various concentrations of inhibitor (2.75-41.2 PM). Aliquots (10 11) were withdrawn with time and their residual activity was determined in standard conditions after filtration-centrifugation at 4 “C on Centricon 30 microconcentrator to remove the inhibitor. The apparent pseudo-first-order constants

353

Vol.

178,

No.

for inactivation, remaining 1.9 PM, “C. The enzymes

BlOCHEfvllCAL

1, 1991

kobs,

were

obtained

from

AND

BlOPHYSlCAL

least-squares

analysis

RESEARCH

of semilog

COMMUNlCATlONS

plots

of percentage

of

activity against time. This method was also used to analyze plasmin inhibition with [E] = [I] = 34-112 FM and t-PA inhibition with [E] ~0.75 ~.LM, [I] = 0.37-l mM at pH 7.5 and 25 buffers were identical to that used in the activity assays. In some cases, the filtered were treated for 30 min by 0.75 M NH20H at pH 8.5, 25 “C and filtered again before

determination of activity. For the determination of the partition ratio, incubations with urokinase (0.55 FM) were carried out in duplicate as described above, using inhibitor concentrations ranging from 0.89 to 11 pM. Enzyme activity towards S-2444 was determined 18 h after the addition of the inhibitor relative to a blank that did not contain inhibitor. trate foroqress curve method). Urokinase (25) in the inhibition was assayed by progress curve analysis according to Hart and O’Brien following experimental conditions : [E] = 31 nM; [S-2444] = 75 PM; [I] = 5-21.9 PM; pH 7.5 and 25 “C ([I] = 0 in the reference cell). The kinetics were treated according to the scheme shown in eq. 1. Species E, I, E’I. E-l, ES and P represent, respectively, enzyme, inhibitor, enzyme-

+I

--

E’I

kinact -

+s

__

ES

-

K1

E-l

E

(eq. 1) KS

inhibitor

complex

E+P kC

formed

prior

to kinact,

inhibited

enzyme

with

the

inhibitor

covalently

attached,

enzyme-substrate (S-2444) complex, hydrolysis product of S-2444. The simplified inactivation scheme (line 1, eq. 1) is derived from a more complete scheme that includes the acylation step (eq. 2) where El is the Michaelis complex and E-l the acyf-enzyme. The expression in eq. 1 (line 1) is obtained ‘(1 __

E+I

k2 El

-

k3 E-l

-

E-l

Wt.

2)

k- 1 by combining kg and kg. The rate v of change in absorbance at 405 nm due to the hydrolysis of S-2444 was obtained from computer-assisted spectrophotometer. A plot of In v versus time gives a straight line characterized by a slope of -x. The analysis by statistical treatment of Wilkinson (26) of the plot 11~ VefSUS l/[l](l-a), with a = [S]/KM+[S], (eq. 3), allows the determination of Kl and kinact, the apparent enzyme-inhibitor dissociation inhibitor concentration, respectively.

= KI /

1 Ir

kinact

constant

VI (1-a) +

and

’

’

the

inactivation

rate

constant

at infinite

kinact

.Flbrinolvsls assav, After treatment of urokinase (47 nM) by the inhibitor (0.86-7.95 FM) during 30 min at pH 7 (25 mM sodium phosphate, 0.1 M NaCI, 0.05 % Tween 80), and removal of the inhibitor (Centricon 30), the plasminogen-activating properties of the treated enzyme was evaluated by fibrin clot lysis experiments. 1251-labeled fibrin clots were prepared as previously described (15) and incubated at 37 ’ C in 200 ~1 of enzyme sample. The extent of fibrinolysis upon addition of urokinase previously treated by the inhibitor (final concentration 1.6 nM; no inhibitor for the blank) was calculated at 30 min intervals from the amounts of radioactivity released from the clot into supernatant after the digestion of the clot had been stopped by adding cold 0.15 M NaCI. Experiments were performed in duplicate.

RESULTS -iOn

t-PA

urokinase,

observed

c[Arg-aB-(CH2~CH3~)-Gly41 is

kinetics.

and plasmin

after elimination

by filtration

for 16 h. In the same conditions, 5%. Addition resulted kinetics

of buffered

in less than characterized

for the inactivation

(Figure

2). For all enzymes, (Centricon

the spontaneous

hydroxylamine

the rate constants

of plasmin

inactivator

no reactivation

30) of the reagent

and incubation

loss of activity of a control

The

activity

losses

followed

kobs. The second-order

and t-PA were 354

40 and 1 M-ls-1,

of

(< 1 %) was at 4 ’ C

sample was <

(0.75 M, pH 8.5, 25 ‘-‘C) to the inhibited

2 % reactivation. by

a time-dependent

enzymes

pseudo-first-order

rate constants respectively

kobs/[l] (Table

I).

Vol.

178, No. 1, 1991

BIOCHEMICAL

0

AND BiOPHYSICAL

40

RESEARCH COMMUNICATIONS

120

80

160

Time (s) Figure 2.

Kinetics of inactivation of urokinase ( l , +0.55 pM), t-PA ( = , 0.75 FM) and plasmin ( A , 1.9 pM) by c[Arg-aB-(CHpSCH 8@)-Gly4] (2.75, 1000 and 67 pM, respectively) at pH 7.5 and 25 OC. Corresponding blanks : ( 0, q , A , respectively).

Inactivation

rates for urokinase

conditions.

Therefore

substrate method

S-2444

the kinetic

by progress

(28), the partition

from the intercept [E]/[E],

were

too fast to be measured

parameters

curve analysis

ratio k,at/kinact

with the x axis minus

at infinite time versus

were

accurately

determined

(25) (Figure

first-order

in the presence

3, Table

for the inactivation

under

of the

I). Using the titration

of urokinase

was determined

1 of the linear plot of the fraction of enzyme

the molar excess

of inhibitor

over enzyme

[I], /[El,

activity and was

found equal to 4 (Figure 4). This ratio represents the average number of enzyme turnovers per inactivation event since kcat is the turnover rate constant for the hydrolysis of the inhibitor

catalyzed

by the enzyme.

From the partition

Increasing concentration

TABLE

(progress

1. Kinetic two-chain

curves)

constants t-PA and 25

~

“C)

protect

for plasmin and

ratio, kcat was evaluated

amounts

of substrate

urokinase

against

S-2444

inactivation.

inactivation of high-molecular-weight c[Arg-aB-(CH2&CH3@)-Gly4]

the by

3-benzyl-6-chloromethyl-3,4-dihydrocoumarin

at 0.084 s-l.

at fixed

inhibitor

This was confirmed

(I)

(pH

7.5

urokinase, and

(II)

~ t-PA

Urokinase

Inhibitor

kinact’ (M-l,-‘)

KI WV

-I

9.0x10-6

a

0.021b

IId ak

1.4~10-~

bi

(27) 7.3,

dData 4 “C).

from

0.002 references

c k.,nact/

Kf was

14 (urokinase,

obtained pH

2 330

-.lC

2 100

187

as k,bs/[l] 6.8,

355

4 “C)

and

al

Plasmin KI

kinact’ (M-l,-‘) 4oc 136

low concentrations

15 (t-PA,

KI

pH 6.8,

of 4 “C;

inhibitors

plasmin,

pH

Vol.

178,

No.

BIOCHEMICAL

1, 1991

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

i

0

10

5

6

Time (min) Figure

3.

A.Time absence Gly4] 21.9

using

: (b)

S-2444

5, (c) 6.25,

method.

in a significant

for the inactivation

of hydrolysis in the presence (d)

PM at pH 7.5 and 25°C.

the incubation

resulted

PM

course (a) and

I/ [I] (i-a) (xlP)

(e)

(75 pM) by concentrations

11.2,

B. Determination

(1) 12.5,

(g)

urokinase (31 of c[Arg-aB-(CH2

nM

13.7,

(i)

(h)

15.6,

in the A CH3$)17.5,

(j)

of Kt and kinact.

of substrate

in the inactivation

of urokinase

was decreased

9.37,

Addition

decrease

of S-2444 of various

(W)

to the urokinase rate constant.

(0.66 FM) by the inhibitor

by a factor of 1.6 compared

incubation

mixture

The rate constant

(4 uM) in the presence to that obtained

kous/[l] of

260

in the absence

of

S-2444.

1.0

0.8 100 ,o w v 3 -

0.6

0.4

3 k .E

80

‘s o .E

60

e

40

0.2 20

0 4

0

5

10

15

0 5

0

20

40

60

60

100

120

140

160

Time (min)

Pld[El,

Figure

4.

Determination of the c[Arg-aB-(CH24CH3+)-Gly4] activity versus [l]o/[E]o.

Figure

5.

Lysis of 1251-labeled before ( o ) Bnd c[Arg-aB-(CH2SCH3$)-Glyq]:

partition

ratio for the (pH 7.5 and 25 “C)

inactivation of urokinase by : plot of the fraction of remaining

fibrin clots by urokinase (final concentration after previous treatment of urokinase (47 0.88 (o), 2.65 (m) and 7.95 (A) PM.

356

1.6 nM)

nM) by

180

Vol.

178, No. 1, 1991

BIOCHEMICAL

. .

of fm of 1251-labelled when

fibrin clots induced

the enzyme

([IJo /[El,

. .

clot IVSIS wed

bv urow.

by urokinase

has been previously

=19-170).

Complete

AND BIOPHYSICAL

treated

RESEARCH COMMUNICATIONS

Figure is

5 shows

prevented

by various

that the dissolution

in a dose-dependent

amounts

of inhibitor

manner

over enzyme

lysis of clots gives the 100 % value. DISCUSSION

In this work, two-chain Gly,]

t-PA was

the differential and plasmin

examined

enzyme-mediated compared

and

process

interaction

can be discussed

as follows

(b)

inactivators.

substrate

selectivity

In the presence

c[Arg-aB-(CH&XH&)-

: (a) and

urokinase,

characteristics

efficiency

of urokinase,

t-PA and plasmin,

was observed

with no significant

of activity. The lack of reactivation

after treatment

by hydroxylamine

of a stable

pathway.

First-order

acyl-enzyme kinetics

for urokinase

substrate

protects

and indicates

are observed

fit to the minimal

urokinase

occurs at the active site.

inactivation

(28) are met supporting acyl-enzyme,

nucleophilic

the

enzymic

Alternatively,

thus leading

acyl-enzyme.

A good partition

ratio (equal

1.7 to 91 have

been

(chymotrypsin)

by

haloenol

lactones

the chemical

in Figure be

by a

inactivation.

deacylation

for urokinase.

inactivation

1. In the

attacked

and enzyme

after water-mediated

for the

results

for mechanism-based

may

modification

to 4) is observed

reported

that

described

derivative

activity may be restored

the

is the major

The kinetic

indicating

expected

mechanism

to covalent

excludes

above (eq. 1, line 1). A synthetic

criteria

p-aminobentyl

a

spontaneous

alkylation

process.

by the reagent

Consequently,

ranging

from

reported

the postulated

reactive

residue,

the enzyme

scheme

against inactivation

modification

transient

that the enzyme

for the inactivation

of the

of the inhibitor

inhibition

formation

obtained

high-molecular-weight

suicide

time- and concentration-dependent recovery

human

of a new potential

of inhibition;

to other known

with

of the

Partition

of a serine

ratios

protease

(29).

L

c[Arg-aB-(CH2SCH&)-Gly4j same

range

is found to be a good inactivator

of efficiency

that

This also holds for the inhibition (Figure period

5). A molar

of the fibrinolytic

activity

over enzyme

of 120 min. The same effect was observed = 11. Interestingly,

observed

with the cyclopeptidic

higher

for urokinase

factor

of 76

specifically

a better

(cyclopeptide). with

with one dihydrocoumarin between

urokinase

1) : the inactivation

plasminogen

significant

activators,

inactivation

of

and

potency

is

ktnact/Kt

is

derivative) inactivator

t-PA.

Some

inhibits

Glu-Gly-Arg-CH2CI

inactivates

18-fold faster than t-PA but the efficiency

is not negligible

(402

M-l 357

s-l)

compared

of urokinase

to the

and a

tripeptide

as efficient

of t-PA

with

plasmin

has been described

inactivation

after a

derivative

chloromethylketones

urokinase

inactivators

the

1).

by the cyclopeptide

by a factor of 15 (dihydrocoumarin

Among no

(Table

of urokinase

(Table

of 56 leads to 50 % fibrinolysis

discrimination

inactivator

than for plasmin

urokinase

acting in the

3-benzyl-6-chloromethyl-3,4-dihydrocoumarin

excess of inhibitor

[I],/[E],

of urokinase

(9, 10, 30). for the

cyclopeptide

(1

Vol.

178,

No.

BIOCHEMICAL

1, 1991

M -’ s-l).

In

contrast

(CH2&.CH3$)-Gly4] occurs

leading

the

is essentially to production

renal toxicity elastase

to

has been

AND

chemically

inert

of urokinase urokinase

(compared

displayed

to t-PA)

for the activation

establish

labels,

c[Arg-aB-

until the enzyme-catalyzed within

chloromethylketone

activation

the active

able to inhibit

the differential

by c[Arg-aB-(CH2$CH3o)-Gly4],

and alteration

of plasrninogen,

an activator

in cell migration

circumstances

(32, 33).

mediated

activation

degrade

surrounding

determines

site. A

neutrophil

Many observations

and matrix degradation

of procollagenases structures

is probably

(34). The balance

of proteolytic

distinct

types of natural

been described

(35). It has been demonstrated

by PAI 2 prevents Antibodies

Consequently, interesting

a specific to consider

in pathological

under

also

synthetic

normal

cascade a major

used inhibitor

as a potential

aiming

and also pathological implicating

mechanism

the plasmin-

enabling

PA and their natural area. Three

that the selective

to inhibit

degradation invasion

of U-PA like

drug to overcome

to

like the movement

cells to inhibitors

genetically

(PAI 1, PAI 2 and protease

the plasminogen-dependent were

of the modified

suggest an in viva role for U-PA as

between

inhibitors

inactivation

in experiments processes

activity in the pericellular

immunogenically

specific

parameters

are of interest

An u-PA-plasmin-collagenase

the degree

(36).

of the kinetic

role of u-PA and t-PA in physiological

of cells and the biology of reproduction.

matrix

affinity

form of the inhibitor

for a peptide

COMMUNJCATIONS

(31).

The two main features

u-PA

RESEARCH

chloromethylketones

of the reactive

reported

BIOPHYSICAL

inhibition

nexin) have of cancer

of labeled

by metastatic

and cell

extracellular cells

(5, 37).

c[Arg-aB-(CH2$CH3o)-Gly4]

an u-PA-natural

inhibitor

is imbalance

situations.

Acknowledgments. This work was funded by the Agence Recherche and the Association de la Recherche sur le Cancer.

Nationale

de Valorisation

de la

REFERENCES 1. Rijken, D. C., Hoylaerts, M. and Collen, D. (1982) J. Biol. Chem. 257, 2920-2925. 2. Gross, J. L., Moscatelli, D. and Rifkin, D. B. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 2623-2627.

Ossowski, L., Quigley, J. and Reich, E. (1979) In Proteases and Biological Reich, D. B. Rifkin and E. Shaw, Ed.) PP. 901-913. Cold Spring Harbor, New York. 4. Strickland, S. E., Reich, E. and Sherman, M. I. (1976) Cell, 9, 231-240. 5. Ossowski, L. and Reich, E. (1983) Cell, 35, 61 l-619.

3.

6.

Ossowski,

L. (1968)

Cell,

52,

Control

(E.

321-326.

7. Hearing, V. L., Law, L. W., Corti, A., Appella, E. and Blasi, F. (1988) Cancer Res. 1270-l 278. 8. Brunner, G., Pohl, J., Erkell, L. J., Radler-Pohl, A. and Schirrmacher, V. (1989) J. Cancer Res. Clin. Oncol. 115, 139-144. 9. Kettner, C. and Shaw, E. (1979) Biochim. Biophys. Acta, 569, 31-40. 10. Lijnen, Ii. R., Van Hoef, B. and Collen, D. (1987) Eur. J. Biochem. 162, 351-356. 11. BBchet, J.-J., Dupaix, A., Yon, J., Wakselman, M., Robert, J.-C. and Vilkas, M. (197’3) Eur. J. Biochem., 35, 527-539. 12. Nicolle, J. P., Hamon, J. F. and Wakselman, M. (1977) Bull. Sot. Chim. Fr., 83-88. 358

Vol.

13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37.

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RESEARCH

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Reboud-Ravaux, M., Desvages, G. and Chapeville, F. (1982) FEBS Let!., 140, 58-62. Reboud-Ravaux, M. and Desvages, G. (1984) Biochim. Biophys. Acta, 791, 333-341. Biochim. Biophys. Mor, A., Maillard, J., Favreau, C. and Reboud-Ravaux, M. (1990) Acta, 119-l 27. Wakselman, M. (1973) Nouv. J. Chimie, 7, 439-447. Decodts, G. and Wakselman, M. (1983) Eur. J. Med. Chem., 18, 107-l 11. Wakselman, M., Mazaleyrat, J.-P., Xie, J., Montagne, J.-J., Vilain, A.-C. and Reboud-Ravaux, M. (1991) Eur. J. Med. Chem., in press. BBchet, J.-J., Dupaix, A., Raucous, C. and Bonamy, A. M. (1977) Biochimie, 59, 241-246. Vilain, A. C., Okochi, V., Vergely, I., Reboud-Ravaux, M., Mazaleyrat, J.-P. and Wakselman. M. (1991) Biochim. Biophys. Acta, 1076, 401-405. Mazaleyrat, J.-P., Reboud-Ravaux, M. and Wakselman. M. (1987) Int. J. Pept. Prot. Res., 30, 622-633. Convert, O., Mazaleyrat, J.-P., Wakselman, M., Morize, 1. and Reboud-Ravaux, M. (1990) Biopolymers, 30, 583591. Jameson, G. W., Roberts, D. V., Adams, R. W., Kyle, W. S. A. and Elmore, D.T. (1973) Biochem. J. 131, 107-117. Chase, T. and Shaw, E. (1970) Methods Enzymol. 19, 20-27. Hart, G. .J. and O’Brien R. D. (1973) Biochemistry, 12, 2940-2945. Wilkinson, G. N. (1961) Biochem. J., 80, 324-332. Kitz, R. and Wilson, I. B. (1962) J. Biol. Chem. 237, 3245-3249. Silverman, R. B. (1988) In Mechanism-Based Enzyme Inactivation : Chemistry and Enzymology, Vol. 1, pp. 3-30, CRC, Boca Raton. Daniel% S. B., Cooney, E., Sofia, M. J., Chakravarty, P. K. and Katzenellenbogen, J. A. (1983) J. Biol. Chem. 258, 15046-15053. Lijnen, H R., Uytterhoeven, B. and Collen, D. (1984) Thromb. Res., 34, 431-437. Ranga, V., Kleinerman, J., Ip, M. P. C., Sorensen, J. and Powers, J. C. (1981) Am. Rev. Respir. Dis. 124, 613-618. Saksela, 0. and Rifin, D. B. (1968) Ann. Rev. Cell Biol. 4, 93-126. Reboud-Ravaux, M. (1985) Biochimie, 67, 11097-l 2012. Moscatelli, D. and Rifkin, D. B. (1988) Biochim. Biophys. Acta, 948- 67-85. Kruithof, E. K. 0. (1988) Enzyme 40, 113-121. Baker, M. S., Bleakley, P., Woodrow, G. C. and Doe, W. F. (1990) Cancer Res. 50, 4676-4684. Reich, R., Thompson, E. W., Iwamoto, Y., Martin, G. R.. Deason. J. R.. Fuller, G. C. and Miskin, R. (1988) Cancer Res., 48, 3307-3312.

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