Effect of N6, 2′-O-dibutyryl cyclic AMP upon the interconvertible forms of cyclic AMP phosphodiesterase from human platelets

Vol. 64, No. 1,1975

BIOCHEMICAL

AND BIOPHYSICAL

EFFECT OF N6, 2'-0-DIBUTYRYL THE INTERCONVERTIBLE

RESEARCH COMMUNICATIONS

CYCLIC AMP UPON

FORMS OF CYCLIC AMP

BHOSPHODIESTERASE FROM HUMAN PLATELETS Anne-Lise

Pichard

and Jean-Claude


Faculte de Medecine Cochin Port-Royal Institut de Pathologie Moleculaire Universite Paris V, 75014 Paris, France Received

March

21,1975

SUMMARY - The influence of dibutyryl cyclic AMP upon the equilibrium existing between three interconvertible forms - 2.5 S, 4.8 S and 7 S - of cyclic AMP phosphodiesterase from human platelets was investigated. It shifted the equilibrium towards the lighter form. It also exerted a protective effect against the thermo-inactivation of the enzyme. It is suggested that the analogue-induced equilibrium shift towards the dissociated high Km form of the phosphodiesterase might reflect a regulatory mechanism occuting also with natural cyclic nucleotides.

INTRODUCTION - Human

platelets

phosphodiesterase already that

activity,

been after

defined

to

1o-4

M)

and

species

depending

on

the

whose

"affinity"

specific

dissociated activity

observed It

equilibrium of

form,

was

the

which

a lower specific CAMP

is

lower.

forming

in In

an

(Km

this

latter

that

such

They

with

g

7 S correspond

enzyme

at

low

(3). enzyme

(Km

enzyme

: 3-5.

shown

association-disso-

CAMP

high

have

a 33,000

of

for At

we

S and

predominant

equilibrium

greater

4.8

concentration

"affinity"

is is

S, obtained.

(CAMP) have

(3), of

2.5

is

AMP

characteristics report

activity.

form for

its

were

molecular

a higher

of

3':5'-cyclic

sedimentation

activity

possesses

tion,

nism

- respectively

dissociated

concentration,

are

peaks

phosphodiesterase

the

some

a high

a previous

gradient

equilibrium

vitro

In

density

interconvertible

ciation In

three

CAMP

and

(1,2).

sucrose

supernatant, with

possess

: 3-5.

concentra-

associated 10m5M)

condition

forms

and both

whose Km's

(3). tempting also

regulation.

to takes

assume place

Any

Cop.vrigh t o 19 75 by Academic Press. Im. AN rights of reproduction in ary form reserved.

1 igand

in

vivo modify

342

a transconformational

and might ing the

represent equilibrium

a mechawould

BIOCHEhdd

Vol. 64, No. 1,1975

induce

modifications

other

hand

sion,

or

fully

associated

single

it

of

would

even

to

be

AMP

as well

as

of

outdated

platelet

National

de

and

ending

with

As

previously

vol.

of

S04,

and

dies.

in

enzyme

(3),

CAMP which

is

to

the

added

the

curve

600 ce

ug of

When

some

shifted

protein

per

This sedimentation. (3),

three

values peaks

the was

compatible

with

in

sucrose in

of

the

sucrose

3

stu-

by

level was

of

due by

the

of

me-

2mM

carried

out

pg

wt

against previously

a concentration

dibutyryl

CAMP,

(5),

concentration

protein

per

dibutyryl

of

is

an lmM,

prolonged ml

in

CAMP

to

the

absen

prevents

to

enzyme. by

sucrose (fig.

2),

density gradient as already reported

were

ratios

of

1 :

obtained,

When

density

gradient, shifted

343

with

2 : 4 . Any

reconcentration (3).

was

as

plateau

phosphodiesterase after

plotted

phosphodiesterase

the

that

the

to

a final

260

suggests

was

If

and

gradient

phosphodiesterase

in

2 mM Mg

we obtained, is

at

right,

mol.

-20°C.

subsequent

measured

activity

which

of

others

at

prepared

all

gradient

assay,

demonstrated conditions

the

steps,

containing was

degraded

instead

CAMP

(Centre erythrocy-

stored

a saturating

In standard of

several

donors

were for

(fig.1).

not

This

further

generated

incorporated

at

enzyme

the

aggregation

peaks

dimentation pattern

ml

CAMP,

fig.2.

the

the

of

combining

was

used

density

extract to

analogue.

extent

is

one

2'-0-dibutyryl

N6,

blood

pH 7.4, was

curve

platelet

is

the

which

equilibrium

of

with

centrifugation

extracts

(4)

in

analogue

only

Contaminating

platelets

to

a triphasic

from

activity

legend

aggregation

by

sequential

buffer,

concentration

the

analogue

platelet

sucrose

RESULTS AND DISCUSSIONreported

by

Appleman

the

deal of

Paris).

g supernatant

in

to

prepared

CAMP phosphodiesterase

as described

dependent

of (2),

and

interconver-

towards

strength.

units

removed

pellet

33,000

thod of Thompson CAMP. Sedimentation

the

ionic

were

50 mM Tris-Hcl

the

The

and

plasma

described

as

the

metabolised

Sanguine, were

a pure

cold

heat

enriched

leukocytes

block

influence

a non

platelets

Transfusion

so

the

the

Human

equilibrium

On the

studies.

CAMP),

-

to

the form

subsequent

kinetics.

useful

shift

(dibutyryl

EXPERIMENTAL

tes

extremely

investigated effect

RESEARCH COMMUNKATIONS

phosphodiesterase

dissociated

for

We therefore cyclic

the

completely or

species

AND BIOPHYSICAL

of

followed

by

1 mM dibutyryl the towards

S

these rese-

CAMP was

distribution the

lighter

peak

BIOCHEMICAL

Vol. 64, No. 1,1975

AND BIOPHYSICAL

untreated

200

400

RESEARCH COMMUNICATIONS

extract

1200

800

1600

pg Fig.

1

proteins/ ml

Influence of dibutyryl CAMP upon the enzyme concentration dance of CAMP phosphodiesterase activity (assayed with CAMP, in a final volume of 0.5 ml). -

: no dibutyryl

o-O-0

: 1 n$l dibutyryl

depen2 m M

CAMP added CAMP in the

extract

and in the

assay.

The inhibitory effect of dibutyryl CAMP was more pronounced at high enzyme concentration (50 %) than at low enzyme concentration (25 X)

(fig.

2).

tyryl ring

This

finding

CAMP did

confirms

influence

the

dissociated

The

dibutyryl

no min,

was loss

of of

be more

at in

absence

of

had

a protective

(fig.

favo-

of

suggests

for there

This the

finding, this

the

a platelet

exCAMP,

the

first

was

a 50

combined

equilibrium

that

on

1 mM dibutyryl

analogue 3).

shift

2),

of occurred

the

effect

When

presence

activity

induced

(fig.

by

dibu-

towards latter

form

20 % with the might

thermostable.

CONCLUSION

- The

ce

this

because

the

that

equilibrium

phosphodiesterase. in

activity

CAMP form

conclusion

enzyme.

also

the

51'C

the

enzyme

dibutyryl

dissociated

the

phosphodiesterase

although

decrease the

of

heated

foregoing

interconversion

of

derivative

thermo-inactivation tract

the form

our

effects

of

dibutyryl

compound

had

been

344

found

CAMP

are

to

be

of

great

significan-

a non-competitive

BIOCHEMICAL

vol. 64, No. 1,1975

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

NO dibutyrYlec/AMP

o.+

18 Dibutyryl-cAMP

10-i

I

I 5

Fig.

2

II Fractions

18

Effect of dibutyryl CAMP upon the sedimentation pattern of platelet CAMP phosphodiesterase in a sucrose density gradient. 0.2 ml of platelet extract containing 1 mg protein was layered onto 5 ml tubes containing a linear (5 to 20 %) sucrose density gradient made in the extraction medium. After centrifugation at 38,000 rpm in a Spinco S W 39 rotor, the bottom of the tubes was punctured, and 0.2 ml fractions were collected and assayed for cAyP phosphodiesterase (4) with 2 mM unlabelled CAMP and 20 nM (8 - H) CAMP (ca. 200,000 cpm in 0.5 ml). S values were calculated according to the method of Martin and Ames (6). When present,dibutyryl CAMP was 1 mmolar in the sucrose gradient.

Heating

:

ot 51° C

Without

dibutyryl.cAMP

15

50

2_

\ 4 x -1 103, 1

45

60

Minutes

Fig.

3

Effect of heating 12 mg prot/ml. e

:

l

O-04 No dibutyryl

at 51" C a platelet

: in the absence : in the presence cAMP was added

345

extract

of dibutyryl

CAMP

of 1 mM dibutyryl in the

test

containing

tube

CAMP during

the

assay.

BIOCHEMICAL

Vol. 64, No. 1,1975

inhibitor and

of

to

be

phosphodiesterase

unpublished tic

data). and

at This

substrate

substrate

suggests

possibly

concentration,

concentration

the

regulatory

We demonstrate induces

leading

the

That

enzyme this

cular

here

drastic

equilibrium

of

low

high

existence site

(personal

on

distinct

the

enzyme

from

the

of

cataly-

site.

logue of

at

uncompetitive

a binding,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

S)

analogue the

gands,

such

predominance

of

with

lower equilibrium

by

for

the

substrate.

its

represents

the

mole-

kinetics

possibility.

effecters,

anaform

cooperativity"

a likely

modifying

substrate

a dissociated

shift

"negative is

this

transconformational

"affinity"

pharmacological

effect

convertible

the

(7)

as

regulatory

of

enzyme

apparent

phosphodiesterase

binding the

induced

for

the in

to

(2.5

basis

that

changes

Artificial

might

also

equilibrium

li-

exert

between

a

the

inter-

forms. ACKNOWLEDGMENTS

We thank stimulating Recherche

Prof.

K. Goldberg

discussions. Scientifique

This

J. Hanoune for

and Dr. work

was supported

Institut

and the

much fruitful

by the Centre

National

de la Sante

and

National

de la

et de la Recherche

Medicale. REFERENCES

1.

Song, S.Y. 593 (1971).

2.

Pichard, Acta,

315,

and

Cheung,

W.Y.,

A.L., 370

Hanoune, (1973).

J.

A.L., (1975).

and

Kaplan

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4.

Thompson, (1971).

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Miller, J.P., Stewart, C.M., Biochemistry,

6.

Martin,

7.

Russell, T.R., Thompson, M.M., Proc. Nat. Acad.

N.J.

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