Purification of rat heart cyclic nucleotide phosphodiesterase on column of immobilized phenylbutenolide inhibitor

Vol.

95, No.

August

3, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages 1080-1089

14, 1980

PURIFICATION

OF

RAT HEART CYCLIC NUCLEOTIDE OF IMMOBILIZED PHENYLBUTENOLiDE Annie

Laboratoire des Sciences

Received

F.

Prigent,

G.

Nemoz

de

Chimie Biologique, Appliquees de Lyon, 69621 Vi I leurbanne

PHOSPHODIESTERASE INHIBITOR and

H.

ON COLUMN

Pacheco

B$t 406, lnstitut 20 Avenue Albert Cedex - France.

National Einstein,

June 19,198O

SUMMARY : Cyclic nucleotide phosphodiesterase was purified over 200-fold in a single step from the rat heart cytosolic fraction, using affinity chromatography on phenylbutenolide inhibitor immobilized to AH Sepharose; After elimination of the contaminating proteins by washing with the loading buffer and ther with 0.4 M KCI buffer, without any loss in enzymatic activity, the cyclic nucleotide phosphodiesterase was eluted in good /isIds with a linear KCI gradient from 0.4 M to 1.8 M. Enzymatic activity determination performed with both cyclic AMP and cyclic GMP as substrate, either at low (0.25 PM) or at high (25 pM) concentration, pointed out the presence of several phosphodiesterase forms with different substrate specificities, in the elution prof i les.

INTRODUCTION

: Since

chromatography macromolecules dy

employed

rases

from

several

to

a solid

linked tion

in

: dyes, proteins

cyclic

nucleotide

not

terases Abbreviations phenvlacetic

been

and

specially

the

purification

do,

used

and

been blue c-like

but

also

et

the

reported (2)

protein

doxantrazole

enzymes

which

bind

for

various

other

proteins

: phenylbutenolide acid

acetic

related

(41

or

O,~c,,

of

been

Blue

of

Iigands,

which

purifica-

dyes

to

bind

alrea-

phosphodieste-

types

(3),

calcium

(CDR) papaverine

(5,6,7), and

dextran-Sepharose

nucleotides,

:

various

has

calmodulin

related (9).

acid

affinity

phosphodiesterases

and

cyclic

the

nucleotide Three

inhibitors

or

(11,

technique

cyclic

origins.

F3GA

al

purification

affinity of

species

have

Cibacron

(8)

for

This

phosphodiesterases

retains

Cuatrecasas

procedure

troponin

xanthine only

of

enzymes.

matrix, as

work

extensively

tissular

as

methylisobutyl gel

early

has

such

binding

the

as linear

phosphodiesnucleotides

(Z-oxo,2,5-dihydro,4-furyl)4-

-COOH

A-H-Sepharose 48 : aminohexyl Sepharose 48 ; EDC, HCI : I-ethyl-3-(-3-dimethylamino-propyl)carbodiimide, HCI ; DMF : N-N dimethylformamide ; Tris Tr i s (hydroxymethy I )-ami nomethan ; DTE : (erythro-2,3-dihydroxy)-1,4-butan dithiol ; EGTA : ethylene glycol 2 (2-aminoethyl) tetracetic acid ; CAMP 3’5’cyclic monophosadenosine 3’5’ cyclic monophosphate ; cGMP : guanosine dependent regulator (calmodulin) phate ; CDR : calcium 0006-291X/80/151080-10$01.00/0 Copyrtghi 1~‘ I980 b.v Academic Press, Inc. All rights of reproduction in any form reserved.

1080

: :

Vol.

95, No.

such

BIOCHEMICAL

3, 1980

as

ATP,

Sepharose

NAD

not

several

other are

on

of

The

only

a minor

only

phosphodiesterases of

columns

of

step

but

and

heart.

4-fold)

with

lung

a 40% (9)

in

cyclic

nucleotide

active

drugs.

bution

of

and

of

conjugate in

cyclic

to

highly

use

purify, nucleotide

via

amide

a single

phosphodiesterases

total

from

guinea

(ll-12), for

several

cardio-

eventual

contri-

of

various

all from

backbone

Among

described

acetic

the

moderate

(151, could

carboxylic be

easly

This

report

acid

- AH Sepharose

the rat

pig

heart

a phenylbutenolide

they

to

various

effect

linkage.

step,

Iigand,

the

the

since

a phenylbutenolide

using

13

as

target

enzyme.

Iigands

(8)

eventual

inotropic

cardiac

al

authors

previously

potential

in

the

with

on

on

independent)

of

several

the

the few

purification

investigate

to

of

nucleotide

other low

a possible

affinity

Only

et

analog

further

EGTA.

chromatography

cyclic

follow

by

inhibitors

of

of the

cardenolides,

Sepharose and

to the

out

effects

convenient

tocu-aminoalkyl

sent

to

selective

proved

preparation

is

forms.

purification

is

aims

related

their

phenylbutenolide

48

pointed

this forms

affinity

yielded

not

retained by

phosphodiesterase

Mohindru

dependent,

phospho-

released

that

independent

forms

CDRalso

specifically and

noteworthy

reported.

did

that

CDR-dependent

by

inhibition

derivatives

the

was

our

competitive

linked

it

showed

Cat’

a do xantrazole

phosphodiesterase One

the

only

but

Iarly, but

a 20-40-fold is

the

excellent

activity

compounds

(13-14),

CDR

With an

phosphodiesterase

synthetic

and

Simi

proteins

gives

two

technique

(10)

of

of purify

COMMUNICATIONS

effecters.

al

It

were

recovery.

As

ly

isolated

obtained

presence.

the

purification

this

phosphodiesterase

forms

not

Ca++

Wrigglesworth

of

(4,101.

MIX-agarose

However

et

presence

usual

inhibitors

phosphodiesterases(one

or

RESEARCH

phosphodiesterases

Klee

purification

does

immobilized

bovine

the

phosphodiesterases

STMP-agarose

substrates

component

recovery

the

BIOPHYSICAL

nucleotide

in

a 40%

allows

as

proteins.

affinity with

attempts

A, cyclic

CDR-Sepharose

CDR-Sepharose

purification technique

coenzyme retains

CDR-dependent

diesterases a column

or

only

AND

multiple heart

describes

forms

pre-

cytosolic

fraction.

MATERIALS A&D METHODS : A-H Sepharose 46 was obtained from Pharmacia Fine Chemicals. EDC-HCI, snake venom (Ophiophagus hannah), unlabelled cyclic nucleotides were purchased from Sigma Chemical Co. AG 1 x 2 resin i200-400 mesh) was from Bio-Rad Laboratories. [3H] CAMP (20-30 Ci/mmol), [3H] cGMP [3H] guanosine (5-15 Ci/mmol) (10-X) Ci/mmolI, [3~] adenosine (20-25 Ci/mmol<, were supplied by the Radiochemical Centre Amersham. N-N dimethylformamide acetic acid, EGTA were obtained from Carlo Erba-Milano. All other chemicals were reagent grade. Preparation of cyfosolic rat heart phosphodiesterases prior to chromatogram. Hearts from Sprague Dawley rats (250 g) were rapidly removed and perfused with 0.32 M saccharose in Tris-HCI buffer 1 mM (pH 7.4) to remove blood. The fresh muscle was homogenized with a glass-glass potter in 3 v/w of the

1081

Vol.

95. No.

3, 1980

above-mentioned buffer. min.. The supernatant supernatant fraction terases.

BIOCHEMICAL

AND

BIOPHYSICAL

The homogenate was centrifuged was then centrifuged at 105,000 was stored at -2O’C and used as

RESEARCH

COMMUNICATIONS

at 20,000 g for 1 h. a source of

g for 15 The 105,OOOg phosphodies-

Preparation of a phenylbutenolide acetic acid substituted gel. A-H Sepharose 46 (5 g) washed following the manufacturer’s instruction s, was suspended in 20 ml of deionized water ,500 mg (2 mM) of phenylbutenolide acetic acid (2-0~0, 2,5-dihydro,4-furylj-4 phenyl acetic acid) Ueredissolved in 100 ml N.N-dimethylformamide and added to the A-H Sepharose 48 suspension. EDC-HCI (3 g) in 30 ml of deionized water was added dropwise. The pH was brought to 4.5 with dilute hydrochlorid acid and the reaction allowed to proceed for 48h at room temperature with ContinuoUSgentle mecanical stirring. pH was frequently readjusted to 4.5 during the first 24 h. A further overnight incubation in presence of 2 mM acetic acid was performed in order to block any remaining activated groups. The resulting substituted gel was washed with a N.N-dimethylformamide-water (2:l) mixture (1.5 I) to remove unreacted Iigand, (the disappearance of unbound phenylbutenolide acetic acid in the wash was monitored by determination of absorbance at 280 nm.), with 500 ml 1 M K HP04, 500 ml of deionized water and finally with 500 ml of storage buffer 7 50 mM Tris, 5 mM MgCl 0,l mM DTE pH 7.4). It was suspended in Tris buffer as a 2 : 1 slurry an 5’ stored at 4’ until1 use. In order to perform several control experiments, acetic acid - substituted gel was alternatively prepared following an identical procedure. In this case the coupling reaction was allowed to proceed overnight. The concentration of phenylbutenolide acetic acid attached to the gel was estimated to be 19 pmoles/g dried resin when determined from the ultraviolet spectrum of the conjugate measured as a suspension in polyethylenglycol and 21 pmoles/g/dried resin when determined from the ultraviolet spectrum of the hydrolysate (6N HCI hydrolysis at 120’ for 2 h.).

Chromatography procedure. Chromatography was performed as indicated in legends of fiqures 1 and 2. Gradient elution was followed by extensive washes of the column with 2 M KCI in Tris buffer. This procedure allows a good regeneration of the affinity column which can be reused at least three times with reproducible cyclic nucleotide phosphodiesterase elution patterns and constant recovery.

3’5r-cyclic nucleotide phosphodiesterase assay. CAMP and cGMP phosphodiesterase activities were assayed according to the method of Thompson and Appleman (16) as modified by Boudreau and Drummond (17). Percentages of adenosine and guanosine fixed on the resin were evaluated with tritiated nucleosides, and the phosphodiesterase activities corrected for these yields. No more than 15% of Assays were performed on an al iquot of substrate was hydrolyzed in an assay. each eluted fraction. On account of sample dilution, KCI concentration did not KCI, up to 0.5 M was shown to norsignifiexceed 0.45 M in the assay mixture. catively affect CAMP phosphodiesterase activity of the original cytosolic preparation when measured with both 0.25 pM and 25 pM substrate concentration ; cGMP phosphodiesterase sensitivity to KCI dependedon assay conditions : whereas it was only slightly inhibited (20% with 0.5 M KCI) when assayed at 25 pM cGMP concentration or at 0.25 FM cGMP in presence of calcium + calmoduIin, basal activity measured at 0.25 FM cGMP in presence of EGTA was decreased about 20% with 0.25 M KCI and 50% with 0.5 M KCI. Percent inhibition were determined up to 0.5 M KCI and basa I cGMP phosphodiesterase recovery was A boiled 105,000 g supernatant of rat cerebral corrected for these values. cortex was used as calmodulin source (18).

1082

Vol.

95,

3, 1980

No.

: Cyclic

RESULTS

tion

BIOCHEMICAL

was

nucleotide

entirely

teins

acetic

(50-60%

activity

was

Cyclic

eluted

kCI

(Fig.1).

by

with

vered

activity 6.6

M.

inhibitor

fore

recovered

0.6

was Cyclic

0.45

the

the

wash

fold

active

nucleotide Sepharose

in

and

the

same

in

to

fractions

of The

a narrow

range

this

10% only assay

KCI was

no

enzyme

of

the

conditions

activity

in

85%)

of

more

not

0.45

tightly

the

bound

detected

reco0.3 to

in

were

shown).

The

M

a 75%

from

activity

(data

and column

at

resulted

was

recovered

pro-

(Fig.1

concentration

activity

the

acid-Sepharose of

(about

of

to

contaminating

buffer

peak

majority

and

frac-

phosphodiesterase

acetic peak

phosphodiesterase since

of

loading

the

a single

purification.

eluted

M KCI

as

cytosolic gel

bulk

the

COMMUNICATIONS

heart

detectable

with

bound

gradient

rat

substituted The

without

first

RESEARCH

the

acid gel.

material)

KCI the

of

acetic

phosphodiesterase

a lo-15

M KCI

to

substituted

in

a linear

coupled

before

both acid

applied

Pooling

yield

te

the

nucleotide

was

to

of

BIOPHYSICAL

phosphodiesterase

bound

phenylbutenolide

AND

eluted

the

the

eluabe-

whole

,l

KCI --- (M) -0.5

-0

I

1

I

30

60

Column

effluent

I

90

(ml)

FIGURE 1 : Chromatography using the acetic acid-AH Sepharose 46 conjugate. Theacetic acid-substituted gel was packed into a 1.0 x 2.0 cm column, washed and equilibrated with 50 mM Tris buffer containing 5 mM MgCl 0.1 mM CITE, 0. 1 mM CaCl ,pH 7.5.Enzyme samples (0.5 ml) containing 5.7 il proteins were applied to ? he column. After sample application, the column was washed with 36 ml of Tris buffer. 6 ml fractions were collected. The column was then eluted with a linear O-l M KCI gradient in Tris buffer (30-30 ml) at a flow rate of 25-30 ml/hour. 3 ml fractions were collected. CAMP phosphodiesterase activity (-) was determined in each eluted fraction on 100 pl aliquot using 2.5 pM CAMP as substrate. Assays were performed in triplicate as described in MATERIAL5 AND METHODS. Absorbance c------j of effluent fractions was monitored at 280 nm. Proteins were also determined according to Lowry et al. (25) using bovine serum albumin as standard.

1083

2)

Vol.

95, No.

BIOCHEMICAL

3, 1980

co

---

,(W) 0

AND

ml 0

m

a _..._..._...------,.::...............,...,..

1084

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Vol.

95. No.

3. 1980

cyclic

BIOCHEMICAL

nucleotide

coupled

ved

phosphodiesterase

Sepharose

column

with

column

0.4

prior

amount

material)

devoid

absorbing

material

was

was

generally

it

techniques

(for

dependent

upon

tration

FM)

peaks

2A).

In

vity

rat of

most

concentration lapping at

peaks

about

1.5 45%

with

level

(25

FM)

elution 2C and peaks fractions

FM)

were

eluted

The

CAMP which Cyclic

with

the from

was

these

activity

and

a 44%

ZOO-fold

and

150-fold

the with

totai

bulk

recovery

cGMP

very

1.15

M KCI

and

peaks

recovery purification

in

At

from

at

about

gave

a 68%

cGMP

phosphodiesterase respectively

recovery

(table low

Two

a minor

high

showed

acti-

at

phosphodiesterase

0.25

peak

appeared

activity

was

(Table

the

same

pM profiles

and in

over-

substrate

emerged 0.9

cGMP

broad

practically

the

activity

AMP

M (Fig.

purification

and

importance

1.5

assayed (Fig.26).

different

well

cyclic

about

ZOO-fold

1).

two

concen-

phosphodiesterase

M KCI

(Table

CAMP

recovered

0.8

cGMP

low into

at

cAMP

than

hydrolyzing

relative two

of

phosphodiesferase

proved

the

peak

as 1.5

two M KCI.

(Fig. large Pooled

CAMP

phosphodiesterase

activity

with

more

1).

FIGURE 2 : Chromatography using the phenylbutenolide acetic acid-AH-Sepharose 48 conjugate. The phenylbutenolide acetic acid-substituted gel was packed into a 1.0 x 3.0 cm column, washed and equi Iibrated with the same Tris buffer as described in the legend to Figure 1. The 105,000 g supernatant of the rat heart phosphodiesterase preparation (1.5 ml , 7 - 16 mg proteins) was applied to the column. The column was washed with 42 ml of Tris buffer followed by 48 ml of 0.4 M KC1 in Tris buffer (as indicated by arrows). 6 ml fractions were collected. After th,ese two washes, the column was eluted with a linear KCI gradient from 0.4 to 1.8 M (84-84 ml) at a flow rate of 25-30 ml/ hour. 3 ml fractions were collected. Cyclic nucleotide phosphodiesterase activity (++) was measured at the following substrate concentrations : panel A, 0.25 FM CAMP ; panel 5, 0.25 pM cGMP ; panel C, 25 FM CAMP ; panel D, 25 pM cGMP. In panel B, (-) activity was determined in presence of 0.1 mM CaCl plus calmodulin in a saturating amount (40 pg protein/assay) ; (O-*..o) acti? vity was determined in presence of 1 mM EGTA. All assays were performed in triplicate on 100 ~1 of each eluted fraction. Absorbance c------l was measured at 280 nm. In order to avoid large losses in enzyme activity which occurred upon storage at -20°C, the purified fractions were stored at +4OC in presence of bovine serum albumin (1 mq/ml). Before BSA addition, aliquots of each fraction were taken up for protein determination.

1085

nm

M KCI.

strikingly

at

from

in

280 1.8

separation

resolved

of

remo-

applied

to

were

activity

purification

the

0.4

measured

(65%

more

0.925

nucleotide same

profiles

a minor

affinity

still

detectable

from

was peak

the

of

no

inhibitor

phosphodiesterases

When

M and

eluted at

and

Then, profiles

phosphodiesterase

a 150-fold

pattern 20).

0.7

yield

cGMP

(0.25

M.

about

67%

of

(lo-20%

elution

: a major at conditions,

in

part

elution,

phosphodiesterase

activity

recovered

The

heart

substrate

Washing

other

enzyme

the

(Fig.2).

conditions.

activity)

these

was

1).

191,

substrate

from

gradient

elution with

COMMUNICATIONS

KCI proteins

the

observed see

eluted

activity.

in

RESEARCH

M KCI

linear

contaminating

found

review

phosphodiesterase

the

phosphodiesterase

assay

(0.25

separated

of

was

0.5-1.8

to

of

BIOPHYSICAL

activity

between

M KCI,

a substantial

As

AND

than

Vol.

95, No.

3, 1980

BIOCHEMICAL

AND

1086

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Vol.

95, No.

The to

original

phosphodiesterase

exogenous

yed. At

In 25

ca lmodul

contrast,

were

by

the

eluted

0.25

45%

as

reported

purification

of

enzyme

The cGMP

no

to

whole

major

45%

form

decreases

activity. ratio

only

profi

les

determined

some

similitude

from

rat

from

human

heart

and

platelet,

cedure.

More

purified

to characterization

fraction

is

previously KCI

concentration

low

substrate

during

the

the

eluted

cGMP

well

from

4))

25

PM substrate D

with

III

the

DEAE

a high

a parent

affinity

form by higher

concentration.

will of

the

others than

obtained

be

needed

high

affinity

(20,21,24). 0.8 The

at

M hydrolyzed first

part

1087

et

AMP GMP cyclic

substrate

This

form and

presents Terasaki

and as

Asano

(20) pro-

if

cyclic

AMP

The

active

fractions

our

cyclic from

was

Further

specify

separation 0.7

M KCI

phosphodiesterase eluted nucleotides

0.8

(24)

separation

phosphodiesterase (21).

AMPlevel

phosphodiesterase

to

both

proteo-

cyclic

low

Hidaka

al

eluted

that

cGMP

Appleman

AMP

Thompson

that Es-

M KCl),more

and

by

presumed

Cyclic

before

chromatography

cyclic

by

be

substantial

(0.7

by

with

phosphodies-

can

occurred.

CAMP

described form

2-5-fold

attests

no

importance both

ce I I u lose

homogeneity

that-

by

procedure.

concentration.

form

it

CAMP

given

nucleotide ,

peak

major of

F III

us i ng

(20-211,

column

eluted

of

a shoulder

s-tudies

described

and

as

respec-

phosphodiesterase

and

yields

for

that

chromatographic

affinity

first

good

150-fold

than

cyclic

(22-23)) the

and

condition

recovered

step

phosphodiesterases,

cellulose

lost

acid-

one with

better

in

substrate

The

the

recently, apparent

DEAE

far

importan-

1).

a rapid

ZOO-fold

yields

was

about

at with

with

eluted

appears

cGMP

with of

acetic

for

than

calcium-dependence

phosphodiesterase (A/G

and

each

are

Table

phenylbutenolide

is

considers

of

was

FM),

CAMP

inhibiof

range

phosphodiesterase,

(more

CAMP

was

sensitivity

same

lB,

PM,

investigated

the

(Fig.

in

inhibition

phosphodiesterase

specific

one

in

acti-

0.25

the

emplo-

conditions.

hydrolysis

only

be

assay

; at

cGMP

procedure

for

lo-40-fold

If

forms

(which

to

respectively) :

calcium-dependent

and

and

obtained

EGTA

10.25

nucleotide

phosphodiesterase

therefore

on

cyclic

cGMP

1mM EGTA

insensible

condition on

results,

chromatography

obtained

enzymatic

was

nearly

dependent

by

above

shown

a convenient

(68%

(20).

pecially,

was

is

purification

activity

the

be

COMMUNlCATiONS

substrate

while

chromatography

procedures

lysis

to

20%)

to

every

and

shown)

RESEARCH

found

was

CAMP

EGTA

and

here

filtration

terase

of

phosphodiesterases

classical gel

account

affinity

recovery

tively). and

not

prior

: The

Sepharose

EGTA

(about (data

substrate,

observed

DISCUSSION

in

On

in

to

phosphodiesterase

that

adjunction

inhibited

1).

BIOPHYSICAL

was

both

unmodified

(Table

FM cGMP as

+ Ca

concentration

was

ted

2+

sensitivity

slightly

hydrolysis

AND

preparation

in

its

FM substrate

vities

ce

BIOCHEMICAL

3, 1980

to

1.05

at at M hydro-

Vol.

95, No.

lyzed

3, 1980

as

much

ferentially

CAMP

might

form

cGMP

last

represent

trate

level

activity)

the

P II

(50

and

60%

form

the

D II

reported the

by

affinity

gel”

from

its

assume

arms

of

acid

on

non

bring

REFERENCES

2. 3. 4. 5. 6. 7. 8. 9. 10.

Terasaki

be

subs-

compared

(24)

and

achieved Iity

by

to

the

that

accounted

al I,

part

caused

by

a smal

either

stronger

the

the

mock very

different being

less So,

hydrophobic I part

in

cyclic

of of

the the

we

spacer our

chroma-

nucleotides

phenylbutenolide

nature

a

chromato-

results).

with

than

separation

of

phosphodiesterase

proved

in

for

bioelution,

from

use

(phosphodiesterase

related

on

activity

acid-Sepharose,“a at

gel

a better

may

However,

acetic

I igand

informations allow

high

acetic

enzyme-support multiple

forms

with

purification.

ACKNOWLEDGEMENTS. This Sante et de la Recherche and by the Centre National

1.

on

inhibitors

might

was

possibi

adsorption

of

specific

both.

phosphodiesterase

nonbiospecific.

gel,as

only

Attemps

or

phosphodiesterase

the

recognizable

I Sepharose

further and

out

biospecific

a cGMP

at

fraction and

gel

been

or

(20).

inhibitor-coupled

phenylbutenolide

will

have

AcOH-Sepharose

that

interactions higher

may

to

procedure.

with

Asano

phosphodiesterase no

ami nohexy

tographic

Appleman

pre-

phosphodiesterase

peak,

recovered This

nucleotide

rule

carrying

bound

could

gel of

behaviour

strongly

cGMP

by

and

enzyme

M zone

important

nucleotides.

cyclic

we can’t

the

behavior

and

of

specific

particularly

COMMUNICATIONS

1.05-1.4

bulk

of

CAMP

acetic-acid-Sepharose

to

graphic

CAMP

the

This form

eluted

RESEARCH

while

= 0.5).

described

of

buffer”,

adsorption

the

Hidaka

elution

1)

a CAMP-cGMP

cyclic

form

phenylbutenolide

“deforming

ratio

peak

of

BIOPHYSICAL

about

previously

both

CAMP-cGMP

Since

(A/G

phosphodiesterase

hydrolyzed

the

AND

ratio

either

by

eluted

(A/G

cGMP

contaminated

The

or

as

hydrolyzed

activity

to

BIOCHEMICAL

work was Medicale de la

supported (INSERM, Recherche

by the lnstitut National de la FRA no5 and grant 77.4.096.3) Scientifique (CNRS, ERA no 560).

:

Cuatrecasas, P., Wilchek, M. and Anfinsen, C.B. (1968) Proc.Natl.Acad.Sci. USA, 61, 636-643 . Morrill, M.E., Thompson, S.T. and Stellwagen, E. (1979) J.Biol.Chem., 254, 437 l-4374 . Ash-ton, A.R. and Polya, G.M. (1978) Biochem.J., 175, 501-506. Watterson, D.M. and Vanaman, T.C. (1976) Biochem.Biophys.Res.Commun., 73, 40-46 Miyake, M., Daly, J.W. and Creveling, C.R. (1977) Arch.Biochem.Biophys., 181, 39-45 Klee, C.B., Crouch, T.H. and Krinks, M.H. (1979) Biochemistry, 18, 722-729 Kincaid, R.L. and Vaughan, M. (1979) Proc.Natl .Acad.Sci .USA, 76, 4903-4907 Mohindru, A., Chenet, A. and Rhoads, A.R. (1978) Biochemistry, 17, 32973304 ’ Wrigglesworth, R. (1977) FEBS Letters, 80, 141-144 Klee, C.B. and Krinks, M.H. (1978) Biochemistry, 17, 120-126 .

1088

. .

Vol.

95, No.

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

17. 18. 19. 20. 21. 22. 23. 24.

25.

3, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Amer, M.S. and Kreighbaum, W.E. (1975) J.Pharm.Sci., 64, l-37 . Weiss, B. and Hait, W.N. (1977) Ann.Rev.Pharmacol.Toxicol., 17, 441-477 . Nemoz, G., Prigent, A.F. and Pacheco, H. (1977) Biochem.Pharmacol., 27, 2769-2774 . Prigent, A.F., Nemoz, G., Roche, M. and Pacheco, H. (1979) Arch.lnt.Pharmacodyn., 241, 131-152 . Duperray, B., Nemoz, G., Prigent, A.F. and Pacheco, H. (1980) Biochem.Phar29, 115-118. macol., Thompson, Y.J., Brooker, G. and Appleman, M.M. (1974) in Methods in Enzymology (Hardman, J.G. and O’Malley, B.W.Eds) pp 205-212, Academic Press, New York . Boudreau, R.J. and Drummond, G.J. (1975) Analyt.Biochem., 63, 388-399 . Egrie, J.C., Campbell, J.A., Flangas, A.L. and Siegel, F.L. (1977) J.Neu28, 1207-1213 . rochem., Thompson, W.J., Terasaki, W.L., Epstein, P.M. and Strada, S.J . ( 1979) Adv. Cyclic Nucleotide Res., 10, 69-92 . Hidaka, H. and Asano, T. (1976) Biochem.Biophys.Acta, 429, 485-497. Thompson, W.J., Epstein, P.M. and Strada, S.J. (1979) Biochemistry, 18, 5228-5237. Sakai, T., Yamanaka, H., Tanaka, R., Makino, H. and Kasai, H. (1977) Biochem.Biophys.Acta, 483, 121-134 . Epstein, P.M., Pledger, W.J., Gardner, E.A., Stancel, G.M., Thompson, W.J. ( 1978) Biochem. Bi ophys.Acta, 527, 442-455 e and Strada, S.J. W.L. (1975) Adv.Cyclic Nucleotide Res., 5, Appleman, M.M. and Terasaki, 153-162 . Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J.Biol. Chem., 249, 4943 .

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