Inhibition of cyclic nucleotide phosphodiesterase by adenosine 5′-triphosphate and inorganic pyrophosphate

Vol. 23, No. 2, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

INHIBITION OF CYCLICNUCLEOTIDE PHOSPHODIESTERASE BY ADENOSINE 5'-TRIPHOSPHATE ANDINORGANICPYROPHOSPHATE Wai Yiu Cheung Department of Biophysics and Pbsical Biochemistry Johnson Research Foundation, University of Pennsylvania Philadelphia, Pennsylvania

ReceivedMarch 23, 1966

Sutherland and Rall (1958) discovered a cyclic ase which hydrolyzes adenosine 3',5'-phosphate phosphate.

nucleotide phosphodiester-

(cyclic

Later Rall and Sutherland (1962) reported an adenyl cyclase cataly-

zing the formation of cyclic

AMP from adenosine 5'-triphosphate

comittant release of inorganic pyrophosphate (PP).

clase (Butcher and Sutherland, Kakiuchi (1965) after

1962; Sutherland -et al.,

have shown that cyclic decapitation

(ATP) with con-

Brain cortex is rich in both

enzymes, phosphodiesterase being present in much greater

tex shortly

AMP) to adenosine 5’-

excess than adenyl cyRall and

1962).

AMP increased markedly in the brain cor-

and in brain slices incubated in the presence of

neurohormones. The fact that cyclic

AMP accumulated with phosphodiesterase and

adenyl cyclase present in marked disparity

suggests that either

its level

is

subject to control mechanismsor the enzymes involved in its metabolism are localized in subcellular

structures

formed would not be freely

distinct

from each other.

accessible to phosphodiesterase.

here show that both ATP and PP are effective

inhibitors

Cyclic AMP thus Results reported

of phosphodiesterase.

The fact that ATP and PP are substrate and product of adenyl cyclase, respectively,

suggests that their

mechanismof plqsiological

inhibition

on phosphodiesterase might represent a

relevance. MATERIALSANDMETHODS

Brains from young male rats, of glass-distilled

water.

Wistar strain,

were homogenized in 5 volumes

The homogenate, with pH 6.8, was dialyzed

214

overnight

Vol. 23, No. 2, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

against 200-400 volumes of 20 mMTris-HCl, centrifuged

pH 7.5.

The dialyzed extract

for 30 min at 30,000 x g, and the supernatant fluid

immediately or stored at -20'.

Unless further

diluted,

was

was used

the extract

could be

kept at -20' for several weeks with no appreciable loss of enzymic activity. The reaction mixture contained in wales, HCl, pH 7.5; 1.8 MgS04; 2 cyclic temperature was 25'. after

AMP; protein

and ATP or PP as indicated.

The

At the end of 30 min, 0.1 ml of 3 g HC104was

added to terminate the reaction.

The pH was brought back to neutrality

and kept at 0' for 30 min.

speed centrifugation

and an aliquot

AMP using the coupled reaction hydrogenase (Adams, 1963). at the concentrations

with

Insoluble KC104was removed by low

of the supernatant fluid

assayed for 5’-

of myokinase, pyruvate kinase, and lactate

de-

Separate experiments established that ATP and PP

used did not appreciably

ployed in the 5'-AMP assay.

that hydrolyzes

interfere

with the enzymes em-

Protein was determined by the phenol reagent as

described by Lowry --et al. (1951). of protein

volume of 1 ml: 40 Tris-

The reaction was started by the addition of substrate

a 10 min pre-incubation.

KHCO:,crystals

in a final

One unit of enzyme is defined as the amount

1 wale cyclic

AMP in 30 min at 25'.

RESULTSANDDISCUSSION IOO-

0 0

I I

I 2

I I 3 4 Concentration

I 5

I I I, 6 7 8 Of ATP or PP mM

9

I IO

Inhibition of phosphodiesterase by ATP and PP. Standard assay conas described under Materials and Methods: protein concentration, 0.2 mg with specific activity 1.4 uni.ts/mg.

215

Vol. 23, No. 2, 1966

Figure

1 depicts

hyperbolic half

BIOCHEMICAL

that

and reaches

maximum around

tends

Table hibition

half

obtained

while

900/o

either

effects

through

Additions (in mM)

inhibition

by PP is

less

inhibition

of both

is

agrees

additions.

It

represents

reached

should

maximum,

The extent with

greater

out that

inhibition

91 58 (54) 80 (78) 81 (84)

10 PP 1 ATP + 1 PP

1 PP

11

3 PP

35

2 ATP + 3 PP 10 ATP + 10 PP

1 ATP + 3 PP 2 ATP + 1 PP

90 100

(100) (100)

TABLE I. Additive inhibition of phosphodiesterase by ATP and PP. parenthesis are calculated from individual addition of ATP or PP. assay conditions with 0.3 mg protein, sp. act. 1.2 units/mg.

spectively.

In Figure

fall

inhibition Figure similar

varies

a mixed

3 illustrates

to those

no PP to 0.50

competitive

on the ordinate

but

type

(Dixon

that

mM in the presence of inhibitor

inhibition

that

The fact

this

is

Figures Standard

to 0.33

increases that

not a simple

the

intercept

competitive

and Webb, 1964).

the

inhibition

2.

Again

by PP exhibits the Km increases

of 5 mM PP. concentrations.

216

In both

in

by ATP or PP re-

no inhibitor

inhibition.

suggests

of ATP in Fig.

as a function

of the

2 a Km of 0.‘20 mM with

mM at 3 mM ATP, indicating does not

the nature

by

O/O Inhibition

43 73 96

2 and 3 indicate

sum of

I.

2 ATP 10 ATP

Figures

of in-

the

be pointed

Additions (in mM)

0

None

and the curve

virtually

very well

as shown in Table

O/O Inhibition

1 ATP

severe,

4 ti.

inhibitors

be obtained

by ATP is

at 3 mM with

of ATP and PP are additive.

usually

PP could

of phosphodiesterase

of PO/o

of 900/o

individual

inhibition

ATP or

The inhibition

maximum at about

in the presence

curve level

An asymptote

I shows the

that

inhibition

a saturation

1 ml&

to be sigmoid.

at 10 mM with

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Figs

characteristics from 0.28 mM with 2 and 3, Vmax

Vol. 23, No. 2, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

3mm 1.5 -1

/ /

1.0 i/

/

-5

-4

-3

-2 [Cyclil

/

f

Ir/’

-I

0

AMP]

2mM

J

I “OS

2

3

4

’

Standard assay conditions with 0.57 mg Fig. 2. Nature of ATP inhibition. protein, specific activity 1.2 units/mg protein; ATP concentrations as indicated; v = A O.D./tube/jO min at 25'.

5mM 4mM

No PP

Conditions identical to those of Figure 2; Fig. 3. Nature of PP inhibition. PP concentrations as indicated, v = A O.D./tube/30 min at 25'.

Corden (1950) has noted an extremely active the rat brain, but PP itself

if detectable

in tissues examined for it

(Kornberg, 1962).

217

inorganic pyrophosphatase in

is present in very small amounts In view of the possible prefer-

BIOCHEMICAL

Vol. 23, No. 2, 1966

ential

distribution

by Whittaker

of ATP in the

Pertinent

to be published) is

to our that

associated

synaptosome

a localized

(1.965),

unlikely.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

with

and transient

discussion

considerable

a fraction

fraction

in the brain

accumulation

is the

finding

comparable

of PP might

(Cheung

phosphodiesterase

as claimed not be

and Salganicoff,

activity

of the rat

to the synaptosome

fraction

brain

described

by Whittaker. Studies those

along

used in our

different

lines

experiment

by Chance --et al.

showed

that

(1966)

the level

on rats

similar

to

of ATP in the brain

is 1.8

L

1 0.2

Cunoles/g

reach

4 mM, estimated

?O'/o

and the

sideration,

would

inhibited

The fact synthesis

its

destruction.

lase

appear

both

-b kinase

(Krebs

action

longer

necessary.

been

hibits caffeine

AMP is

capability

delay

a cyclic

and not

and Sutherland nucleotide

at 20 mM gave 500/o

favor

is

into

--in vivo

of the becomes

and its stored

suggests

activities

conin a

would

possible.

Several

influence

activity,

result

it

felt,

will

of glyco-

it

not

is important

be called

would

in a waste

for

the

as the main

observed

phosphodiesterase inhibition

of phosphory-

regulation

upon

be a warranty

of energy.

AMP on phosphorylase

the advantage

(1962)

effects

By

ATP and PP keep the potentiality

of cyclic

to imply

to

in the presence

of the phosphodiesterase

which

dur-

leading

the activation

energy

that

nucleotide

a key enzyme in the

so that

An excess

to illustrate

do not

formed

The stimulation

merely

Butcher

exist

phosphodiesterase

thus

1939),

phosphodiesterase

control.

reserved

observations

might

AMP, among them is

terminated

any unnecessary

cited

these

conceivably

of the brain

content

phosphodiesterase

accumulation

--et al.,

to have its

proper

Taking

conditions

to cyclic

Once cyclic

suppressing

55’/0.

phosphodiesterase

genolysis.

against

ATP,

A transient

attributed

than

that

the water

ATP and PP inhibit

of cyclic

of an overwhelming have been

water

that

of ATP could

state.

that

ring

concentration

on the basis

intracellular

it

greatly

Intracellular

tissue.

that

caffeine

and is therefore

2 kinase

has

to have a large

function

from beef

218

cell

under

in the brain. competitively

heart.

in-

In our hands,

much less

potent

Vol. 23, No. 2, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

than either PP or ATP. Of the compoundsreported to inhibit

phosphodiester-

ase, the most potent is ATP, followed by PP. The inhibition

of phosphodies-

terase b;y both ATP and PP might well be a regulatory the intact

mechanismoperative

in

cell. ACKNOWLEDGEMENTS

The author is grateful

to Professor B. Chance for his interest

couragement throughout the course of this study.

and en-

This work was supported by

U.S. Public Health Service Grant 12202-02. REFERENCES Adams, H:., in Bermeyer, H., (Ed.), Methods of enzymatic analysis, Academic Press, NewYork, P. 573 (1963). Butcher, R. W., and Sutherland, E.W., 2. Biol. Chem., 237, 1244 (1962). Chance, B., Jamieson, D., and Williamson,J.R., SymposG on High Pressure Oxygen, Duke University, Durham, North Carolina, Nov., 1965. Dixon, Hi., and Webb, E.C., Enzymes, Longmans,p. 324 (1964). Garden, J.J., Biochem. J., g 96 (1950). Kornberg, A., in Kasha, M. and Pullman, B., (Eds.), Horizons in biochemistry, Academic Press, NewYork, p. 251 (1962). Krebs, EI.G., Graves, D.J., and Fischer, E.H., J. Biol. Chem., 234, 2867 (1959). Biol. Chem., Lowry, O.H., Rosebrough, N.J., Farr, A.L., and-Ra?i?il, R.J., 2. -Q3, 265 (1951). Rail, T.W., and Sutherland, E.W., J. Biol. e., -,237 1228 (19962). Rail, T.W., and Kakiuchi, S., Abst?acF 150th Am. Chem. Sot. Meeting, 68~

(1965).

Sutherland, Sutherland, Whittaker, physics

E.W., and Rail, T.W., 2. -Biol. Chem., m 1077 (1958). E.W., Rail, T.W., and Menon, T., J. Biol. Chem., 237, 1220 (1962). V.P., in Butler, J.A.V. and Huxley: Hr (Eds.), Progress in bioand molecular biology, PergamonPress, p. 39 (1965).

219