Vol. 85, No. 4, 1978

8lOCHEMlCAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

December 29,1978

Pages

ACCELERATIVE

AUTOACTIVATION

PROSTAGLANUIN BIOSYNTBESIS Martin 1

E. Hemler',

Gustav

Graff',

Received

October

24,

OF

BY PGGz

and William L

Department of Biological Chemistry The University of Michigan Ann Arbor, Michigan 48109

1325-1331

E. M. Lands'

Department University Minneapolis,

of Pharmacology of Minnesota 55455 Minnesota

1978

Summary : Cyclooxygenase catalysis is stimulated by its product, PGG2, and by other lipid hydroperoxides. The endoperoxide, PGH , was not stimulatory. The results provide a direct demonstration of an essen & ial role for lipid hydroand show how the biosynthetic interperoxides in prostaglandin biosynthesis, mediate PGG2 has a positive accelerative effect.

INTRODUCTION A -bis-dioxygenation the

endoperoxide

thromboxanes logic vity

which

and pharmacologic (l-3).

product

and the

perhaps

feature

of peroxide This

peroxide

oxygenase

assays

ment was clearly glutathione

substrates,

(5) cyclooxygenase

have noted

in the

available

overproduction

requirement

is

and as a result,

(GSP) inhibited and interrupted

the

are extent

the

requirement

for

apparent

in the routine

oxyqenation

overlooked.

of peroxide by crude

(4,5)

a minimal

catalysis.

removal

of

Another

cyclooxygenase

prostaglandin

required

acid,

In addition,

can be easily

when the

fatty

acti-

of the cyclooxygenase

is

not readily it

of physio-

of the

of prostaglandins.

mechanism

and maintain

prostaglandins,

a variety

levels

and unesterified --in ~vitro.

produces

of cyclooxygenase

by a self-inactivation

reaction

demonstrated

subsequent

the regulation

formation

to initiate

peroxidase

for

by cyclooxygenase

of all

reviews

oxygen

aqainst

of the

acids

formation

reductians

is limited

protects

fatty

Recent

prostaqlandin

formation

important

to the

mechanisms

For example,

known to reduce

level

leads

and prostacyclin.

heme cofactor

which

of unsaturated

cyclo-

The requireby additions (4,6)

biosynthesis

Abbreviations: DDC, diethyldithiocarbamate; GSP, glutathione peroxidase; GSH, glutathione; HPET, hydroperoxyeicosatetraenoic PGG2, prostaglandin Gp; PGH2, prostaglandin Hz.

of

and purified

already

in

acid;

0006-291X/78/0854-1325$01.00/0 1325

Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved

Vol. 85, No. 4, 1978

BIOCHEMICAL

progress

studies,

(7).

up to 50-60 inhibited

seconds)

physiologically

levels

After

resulting

crude

lagging,

To clearly

gland

lipid

electrophoretically

requirement

with

systems

(I)),

accelerative

with

the

peroxide

pure

conditions

concePt

may

essential

of

ether,

the

back to

self-generated, was not

the

the

(GSH),

ðyl

factor(s),

isolated

reaction

the pure

but

cyanide,

when added

oxygenation

cyclooxygenase,

PGG2 and PGH2, and established peroxide

by removal

reaction

were

supporting

biosynthesis

was stimulatory

pure

with

glutathione

mixture

(lags

preparations

(a),

fraction

the autoactivating

peroxide

cosubstrate,

of a reaction

for

for

synergistically

peroxide

responsible identify

requirement

was inhibitory

prostaglandin

extraction

RESEARCH COMMUNICATIONS

cyclooxygenase

Acting

of GSP and its

accelerating

peroxide

(8,9).

vesicular

regulate

peroxide(s).

tory

cyanide

in bovine

the --in vivo

an enhanced

was seen when crude

by sodium

GSP present that

In later

AND BIOPHYSICAL

stimulacharacterized.

we secured reaction

suitable

for

products showing

the

enzyme.

MATERIALS

AND MRTHODS

Hemin chloride (Calbiochem), manganese protoporphyrin IX (Porphyrin Products, Logan, Utah), arachidonic acid (99% pure, Supelco), and sodium diethyldithiocarbamate (Sigma) were used as supplied. Cyclooxygenase apoenzyme was obtained as previously described (5), and mangano-heme cyclooxygenase was prepared by incubating apoensyme with a S-lo-fold molar excess of mangano-heme and then removing the unbound heme by chromatography on DEARcellulose equilibrated with 20 mM sodium phosphate (pH 7.0) containing 20% glycerol. PGG2 was isolated and purified from cyclooxygenase reaction mixtures (10) and PGH2 and 15-hydroperoxyeicosatetraenoic acid were prepared as previously described (11). Enzyme activity was determined polarigraphically (12) in reaction vessels with 50 uM arachidonate (20:4) in a total volume of 3 ml of Tris chloride 0.1 M (pH 8.5) at 30°C. Continuous analysis of oxygenation rates was facilitated by an electronic differentiator. RESULTS AND DISCUSSION Although

previous

preparations

were

requirement,

assays

because recently lag

rapid

more suitable

adequate for

that

(compare assay

for

using

inhibited

establishing

peroxide

acceleration

observed

phase

studies

mangano-heme

ferri-heme system

phase

1326

of a peroxide

somewhat

relatively

cyclooxygenase

screening

existence

were

and mangano-heme for

cyclooxygenase

the

stimulation

made the lag

ferri-heme

potential

inconvenient short.

We have

has a much more obvious in Fig.

2A) and provides

peroxide

activators.

a

BIOCHEMICAL

Vol. 85, No. 4, 1978

200

1:

RESEARCH COMMUNICATIONS

2

Figure

AND BIOPHYSICAL

The accelerative activity.

4

6 Time (mid

effect

6

IO

of peroxide

12

on mangano-heme

cyclooxygenase

Mangano-heme cyclooxygenase EO.062 PM subunit, based on 70,00072,000 daltons per subunit (17,25)) was added (first arrow) to standard reaction mixtures and after 20 seconds, reactions were initiated (second arrow) by the addition of 10 )~l of ethanol which contained: no 20:4 (curve a), SnCl -pretreated 20:4 (curve b), untreated 20:4 (curve c), and SnC12-pretrea z ed 20:4 plus 15-hydroperoxy eicosatetraenoic acid (15~IiPET) (curve d). Final concentrations were 20:4 (50 uM) and 15-HPET (1 PM). After 'L 9 minutes (arrows), fresh enzyme (0.062 uM) was added to reactions b and c. The dashed lines are tangents representing the velocities at 15 seconds. For pretreatment, 20:4 in toluene was made 4 mM with ethanolic SnC12 and mixed vigorously. After several hours, and acidification with aqueous citric acid, aliquots of the extracted organic layer were dried under N2, taken up in ethanol, and used immediately.

the mangano-enzyme

Also,

associated this

with

ferri-heme

free

The Mn-catalyzed

(curve

some

b). acid

ferences

of peroxide oxygenation

was accentuated to remove

stimulation

activity

reaction

is clearly

needed

of any added

when the substrate

arachidonate

of the contaminating

peroxides

On the other practically in early

at 15 seconds

(13)

Thus the kinetic

to cyclooxygenase

even in the absence

rate

activity

that

is

results

that

is

with

uncomplicated

mechanism.

The importance

optimum

of the peroxidase

cyclooxygenase.

enzyme can be attributed

by the peroxidase

noic

is

hand,

eliminated reaction

(represented

rates

addition

evident

than

more

a minute

inhibitors (20:4) which

lag

are

particularly

by the dashed

1327

(15-HPET,

line

1.

to reach

(curve

c).

was treated

curve obvious

tangents)

(1).

its

This with

had spontaneously

of 1 )J?I 15-hydroperoxy

the

in Fig.

lag SnC12

formed

eicosatetraeThese

dif-

when the velocities are

compared.

Evi-

vol. 85, No. 4, 1978

dence

for

BIOCHEMICAL

a self-generated

response

to second

concept

that

lags

for

also

(after

reaction

characteristic

effects

(PGG2),

[diethyldithiocarbamate the mangano-heme

240-300

seconds

lagging

system

early

period ties

(Figure it

rates (Fig.

were

continuously

addition

changing,

was graphed

ty at 60 seconds

in Figure

compared

2B.

of 1 ~.IM. The lipoxygenase

enoic

(15~HPET),

hydroperoxide

group

rather

promote

cyclooxygenase

earlier

report

catalyze

that

reduction

the hydroperoxide oxygenase

was free

was also than

GSP (which of the

to the

acceleration

60 seconds effect

lag the veloci-

after

conclusion

can remove

the activator moeity

of PGG2 (15).

To confirm

of peroxidase,

a reaction

1328

it is

well

is

the

needed

to

with

peroxide)

of PGH2, but that

eicosatetra-

which

agrees

it

the does

not

does reduce

the mangano-heme

was run

veloci-

at a con-

Apparently

group

peroxide

on reaction

15-hydroperoxy

endoperoxide

of

diminished in which

at 1 W.

these

of at least

gave maximum stimulation

This

endoperoxide

reaction

directly

a greatly

.

di-

Using

by a lag

rapid

product,

catalysis.

in the

added

the velocity

the

stimulatory

(14)l.

of reactions

stimulatory

are

by including

lags,

PGI12 had little

the

enzyme provided

have caused the

peroxide

and gave

to PGG2, which

reactions

cyclooxygenase

testing

was preceded

only

first

the

self-inactivating

be obtained

with

comparison

centration acid

for

When PGG2 was then

To simplify

support

of

might

more pronounced

consumption

b and

(PGH2) or an endoperox-

a concentration-dependent

of oxygen

curves

cyclooxygenase.

system

reaction

2A).

caused

2A).

recognized

of which

reacts

for

the

(6) ferri-heme

or both

in the rapid

addition

an endoperoxide

caused

seen

9 minutes

a second

PGG2 and PGH2 could which

also

during

mangano-heme

either

RESEARCH COMMUNICATIONS

enzyme additions)

the

A good

of the products

conditions,

the

pure

autoactivation.

mixtures

I),

can produce

ethyldithiocarbamate,

(at

(5) and purified

The cyclooxygenase

accelerative

enzyme

Since

(Fig.

of the

ide-hydroperoxide

is

accumulated

minutes.

of the crude

factor

the second

peroxide

several

uninhibited

properties

of fresh

a stimulatory

and remained further

stimulating

additions

The diminished

cl -

AND BIOPHYSICAL

in the absence

cycloof

BIOCHEMICAL

Vol. 85, No. 4, 1978

AND BIOPHYSKAL

RESEARCH COMMUNlCATlONS

A “cr, 0

0.3

I

0.75

\

\

\

\\

1.5

'1

'A_

--

0.”

120

480

240 360 TIME bc)

6OC

B

0

0.4

11

1

0.8 I.2 ENDOPEROXIDE

t

I .6 (,M

4

2.0

Figure

2A: The accelerative effect of PGG2 on cyclooxygenase action. Oxygenation of 20:4 (50 uM, standard reaction conditions), which had been preincubated with DDC (4 mM) for at least 5 minutes, was initiated at zero time by the addition of mangano-heme cyclooxygenase (0.031 PM), or cyclooxygenase (0.041 uM) in the presence of 0.9 pM ferri-heme (dashed line). various concentrations of PGG2 were added, and reaction At 30 seconds (arrow), velocities were determined. Figure

2B: Stimulation products,

of cyclooxygenase; comparison of the endoperoxide PGG~ and PGH2 . Reactions were carried out as in Fig. 2A using 0.041 )JM manganoheme cyclooxygenase. Various concentrations of PGG2 or PGH2 were added as shown and reaction velocities at 90 seconds (60 seconds after the addition of endoperoxide) were determined tangentially from the polarograph tracings.

diethyldithiocarbamate cosubstrate). that

and with The ratio

120 nmoles

migrated

of

without

a tank

(20%).

Confirmation

migration acid

with

(50:40:1)

liner)

with that

with

a tank

normally results

Since

show that

very

in the presence

with the

2.0.

the

Also, little

ferri-heme

stimulatory

activity

action

1329

that

co-

PGD, PGE, PGF

was obtained

by co-

(isopentane:butanone:acetic was reducible

PGH2 was formed

to have no peroxidase

showed

(50:50:0.5)

and combined

the material

of the peroxidase

Analysis

acid

product

system

a peroxidase

to products

PGH2 (4%),

in another liner).

enzyme appears

associated

was near

acetate:isooctane:acetic

PGG2 was the major

2 standard

(potentially

in 3 minutes

PGG2 (76%),

PGG

heme enzyme even

consumed

converted

ethyl

by triphenylphosphine.

mangano-heme

of 02/20:4

20:4 were

on TLC (using

0.67 mM phenol

by the mangano-

cosubstrate activity (16,17).

of hydroperoxide

to PGF2a

phenol,

the

of the type Thus,

our

is clearly

dis-

BIOCHEMICAL

Vol. 85, No. 4, 1978

tinct

from

its

ferri-heme that

action

enzyme.

occurs

catalyzed

both

breakdown

Our present and 20:4,

--in vitro

role

as proinflammatory

been

found

for

tions

facilitate

which

prostaglandin hypothesis

our

of the

product

of self-catalyzed and not

removal

have clearly

the

(PGG2) contri-

may have an important activity

(19-23). also

has

The

means that

condi-

inhibitory

for

in terms

of the

potentially

may be significant (24).

early

can be due to accumulation

of small

amounts

PGG 2, which

aspect

are

shown that

may trigger

accelerative

feedback

associated

are

available.

the

endoperoxide,

inactivation

product

may be antiinflanunatory

action

hydroperoxy

insight

also

lipoxygenase

peroxide

formation

action

prostaglandins

removal

This

are

hydroperoxides

and significantly,

of 02

prostaglandin

as its

of an activator

results

catalyze

by lipoxygenase

to produce

a peroxidase-

(18).

of a hydroperoxide

tissue

known

levels

positive

may not

require

even in the presence

accelerates

Thus,

also

peroxide

of cyclooxygenase

reaction

then

hydroperoxide

phases

This

also

of the

self-inactivation

earlier

that

of HPET produced

biosynthesis. that

suggest

amounts

agents,

trace

In summary,

activity.

does not

which

in tissues

activity

of cyclooxygenase

catalysis.

requirement

the peroxidase characteristic

findings

amounts

RESEARCH COMMUNICATIONS

the

--in vivo

activity

to further

we find

sufficient

trace

cyclooxygenase

for

of PGG2 to PGH2 as suggested

unless

example,

butes

types

cyclooxygenase

efficiently For

as a substrate In addition

with

AND BIOPHYSICAL

and the negative

characteristic

peroxidase

enhances

cyclooxygenase

feedback

property

of the cyclooxygenase

reaction.

ACKNOWLEDGMENTS This work was supported in part by an Upjohn Graduate Research Fellowship (M.E.H.) and a grant from the National Science Foundation (MS-7513157). We thank Ms. Mary O'Donnell for technical assistance, and Dr. Harold Cook for helpful discussions. REFERENCES 1. 2.

Samuelsson, B., Granstrijm, E., S. (1975) Ann. Rev. Biochem. Lands, W. E. M., and Rome, L. Biochemical Aspects &rim, S. Ltd., Lancaster, England.

Green, K., Hamberg, M., and Hammarstrsm, Vol. 44, pp. 669-695. H. (1975) Prostaglandins: Chemical and M. M., ea.), Vol. 3, pp. 87-126, MTP Press,

1336

Vol. 85, No. 4, 1978

3.

4. 5. 6. 7.

8. 9.

11. 12. 13. 14. 15. 16. 17.

18. 19. 20.

21. 22. 23.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Lands, W. E. M., Hemler, M. E., and Crawford, C. G. (1977) Polyunsaturated Fatty Acids (Kanau, W. II., and Holman, R. T., eds.), pp. 193-228, American Oil Chemists' Society, Champaign, Ill. Smith, W. L., and Lands, W. E. M. (1972) Biochemistry 11, 3276-3285. hemler, M. E., Crawford, C. G., and Lands, W. E. M. (1978) Biochemistry 17, 1772-1779. Lands, W., Lee, R., and Smith, W. (1971) Annals of the New York Academy of Sciences 180, 107-122. Lands, W. E. M., Cook, H. W., and Rome, L. H. (1976) Advances in Prostaglandin and Thromboxane Research (Samuelsson, B., and Paoletti, R., eds.), PP. 7-17, Raven Press, New York. Cook, H. W., and Lands, W. E. M. (1976) Nature 260, 630-632. Cook, H. W., and Lands, W. E. M. (1975) Biochem. Biophys. Res. Commun. 65,

10.

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464-471.

Graff, G., Stephenson, J. H., Glass, D. B., Haddox, ?1. K., and Goldberg, N. D. (1978) J. Biol. Chem. 252 (in press). Crawford, C. G., Van Alphen, G. W., Cook, Ii. W., and Lands, W. E. M. (1978) Life Sciences 23, 1255-1262. Rome, L. H., and Lands, W. E. iul. (1975) Prostaglandins 10, 813-824. Ogino, N., Ohki, S., Yamamoto, S., and Hayaishi, 0. (1978) J. Biol. Chem. 252, 890-895. Ishimura, Y., and Hayaishi, 0. (1973) J. Biol. Chem 248, 8610-8612. Christ-Hazelhof, E., Nugteren, D. H., and Van Dorp, D. A. (1976) Biochim. Biophys. Acta 450, 450-461. Miyamoto, T., Ogino, N., Yamamoto, S., and Hayaishi, 0. (1976) J. Biol. Chem. 251, 2629-2636. Van der Ouderaa, F. J., Buytenhek, M., Nugteren, D. H., and Van Dorp, D. A. (1977) Biochim. Biophys. Acta 487, 315-331. Egan, R. W., Paxton, J., Kuehl, F. A., Jr. (1976) J. Biol. Chem. 251, 7329-7335. Borgeat, P., Hamberg, M., Samuelsson, B. (1976) J. Biol. Chem. 251, 7816-7820. Hanunarstr8m, S., Hamberg, Cl., Samuelsson, C., Duell, E. A., Stawiski, M. , and Voorhees, J. J. (1975) Proc. Nat. Acad. Sci. U.S.A. 72, 5130-5134. Ho, P. P. K., Walters, P., and Sullivan, Ii. R. (1977) Biochem. Biophys. Res. Cosnnun 76, 398-405. Nugteren, D. H. (1975) Biochim. Biophys. Acta 380, 299-307. Hamberg, M., and Samuelsson, B. (1974) Proc. Nat. Acad. Sci. U.S.A. 71, 3400-3404.

24.

Kuehl, G. C.,

25.

Hemler,

E'. A., Jr., Humes, J. L., Egan, R. W., Ham, E. A., Beveridge, and Van Arman, C. G. (1977) Nature 65, 170-173. Lipids 12, 591-595. M. E., and Lands, W. E. M. (1977)

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