A simple and sensitive competitive protein-binding assay for cyclic GMP

Vol.

67,

No.

BIOCHEMICAL

2, 1975

A

SIMPLE

AND

AND

SENSITIVE

PROTEIN-BINDING Kobayashi

Received

of

September

FOR

and

Laboratories, Chicago,

RESEARCH

COMMUNICATIONS

COMPETITIVE

ASSAY

Ryoji Endocrinology University

BIOPHYSICAL

CYCLIC

Victor

S.

GMP

Fang

Department of Chicago, Illinois

Medicine, 60637

lo,1975

A simple and sensitive competitive protein-binding assay for cyclic ‘w;P) has been developed using a specific cGMP-binding protein isolated from rat lungs. The contamin:tion of CAMP-binding protein was partially reThe most active moved by fractionated precipitation with ammonium sulfate. binding was observed when cGMP-binding protein was extracted with phosphate Magnesium (40 mM) and zinc (4 and buffer (50 mM) containing Z-mercaptoethanol. 40 mM) ions enhanced the binding of cGMP to the protein. The amount of protein which bound to cGMP decreased when the protein was extracted with buffer but without 2-mercaptoethanol. The binding constant of this protein was 5.5-16.3 x lo-’ M. The sensitivity of the assay was 0.2 picomole of cGMP. Other nucleotides exerted little or no interference.

The

role

of

CAMP*

as

documented

(1).

serotonin,

bradykinin

(4),

mammalian

tissues

of

cGMP

in

Recently,

cyclase

which

catalyzes

malian

tissues

and

several

the of

cGMP

messenger

acetylcholine

(2))

and

the has

mammalian system

(5)

shown protein

tissues

and

of

of to

were

second

messenger

to

been

found

from

adenylate

has

also

It

has

system

(3)) cause

levels.

8).

(7,

actions

agents

CAMP

activity

arthropods

a new

has

different

kinase

hormone

reported of

cGMP

be

various

mitogenic

elevation

formation

been

represents

insulin

without

cGMP-dependent

Furthermore, in

a second

is

well

histamine, accumulation

A guanylate in

several

mam-

(6).

cyclase been

discovered

been

suggested

independent

that

from

that

CAMP. Although

expensive,

several or

relatively

f Abbreviations: 3',5'- monophosphate, inosine-3’,5’-monophosphate, 3’,5’-monophosphate,

Cop.vright All rights

methods

are

insensitive.

available In

to the

measure

present

Adenosine-3’,5’-monophosphate, cyclic CM'; guanosine-3’,5’-monophosphate, clMP; thymidine-3-,5#-monophosphate, cUMP; xanthosine-3’,5’-monophosphate,

@ 19 75 by Academic Press. Inc. of reproduction it1 ar2.v form reserved.

493

cGMP, paper,

they we

AMP or cyclic cXMP.

are

describe

laborious, a simple

CAMP; cytidineGMP or cGMP; cTMP; uridine-

Vol. 67, No. 2, 1975

and

sensitive

BIOCHEMICAL

protein-binding

tion

of

cGMP-binding

tion

of

the

protein

assay

protein are

also

from

AND BIOPHYSICAL

for rat

cGMP. lung

and

RESEARCH COMMUNICATIONS

The

procedure

the

preliminary

for

the

prepara-

characteriza-

included. MATERIALS

AND

METHODS

Nucleotides and resin: 3H-cGMP (21 Ci/mmole) was purchased from AmershamSearle Corp., _ Arlinqton Heiqhts. III.. and 3H-cAMP (37.7 Ci/mrnole) was purchased from New England Nuclear; Boston, Mass. Al 1 -unlabeled nucleotidks were purchased from Sigma Chemical Co., St. Louis, MO. Ion exchange resin, AGI-X2 (chloride form) was purchased from Bio-Rad Laboratories, Richmond, Calif. Preparation of binding protein: The lungs were quickly removed from SpragueDawley rats (80-120 g) under ether anesthesia and rinsed in cold phosphate buffer (56 mM), pH 7.0, containing 2-mercaptoethanol (6 mM). Lungs from IO to 20 animals in a batch were homogenized in 5 volumes of the same buffer with UltraTurrax (Janke & Kunkel KG), and the homogenate was filtered through 4 layers of gauze and centrifuged at 50,000x g for 60 min. Ammonium sulfate was added to a concentration of 22 g per 100 ml of the supernatant. The precipitate was discarded after centrifugation. An additional amount of ammonium sulfate (24 g per 100 ml of the original volume) was added to the supernatant. The precipitate collected after centrifugation was redissolved in the same buffer and contained cGMP-binding protein. The protein preparation was dialyzed against the same phosphate buffer (50 mM) containing 2-mercaptoethanol (6 mM) and glycerol (10%). The whole procedure was carried out at O-4” C. The final preparation was analyzed for protein content by the method described by Lowry et al. (9) and stored in a freezer. The cGMP-binding protein preparation was diProtein-binding assay for cGMP: luted in sodium acetate buffer (50 mM). DH 4.0. Any acid precipitable protein was removed by centrifugation. ‘The bind’ing assay for cGMP’ was carried out using the same acetate buffer (50 mM), pH 4.0, containing 1 picomole of 3H-cGMP, 200-220 pg of binding protein, and either standard, other nucleotide or unknown sample in a final volume of 150 1-11. When tested, divalent cations were dissolved in acetate buffer directly. After 60 min incubation at 0” C, I ml of Tris-HCI buffer (10 mM), pH 8.0, containing 40 mM MgClz was added to stop the reaction (IO). The contents of each assay tube were transferred to a cellulose filter (Millipore HAWP O25OO), and the filter was washed with 5 ml of Tris-HCl The radioactivity retained by the filter was determined by liquid scinbuffer. tillation spectrometer (Packard Model 3380) in 10 ml of Bray’s solution. Kinetic study of cGMP-protein binding: Mixtures of tein and varied amounts of ‘H-cGMP (l-50 picomoles) of acetate buffer were incubated at 0” C for 60 min. was separated according to the same assay procedure Cyclic Determination of CAMP: binding assay method described from rat liver as described by tion, a dextran-charcoal mixture Preparation extracted Guidotti,

pro200 ~1

AMP was determined by the competitive proteinby Gilman (11), using a binding protein extracted Instead of Millipore filtraKumon et al. (12). was used to separate the free from the bound

Cyclic GMP samples: from each other by column prior to an

of tissue and separated using alumina

200 pg of cGMP-binding in a final volume of The bound radioactivity employed for cGMP.

and the ion

CAMP contents method described exchange resin

in

tissues were by Mao and column (13).

RESULTS The

specificity

of

cGMP-binding

protein

494

isolated

from

rat

lung

is

demon-

CAMP

Vol. 67, No. 2, 1975

05

BIOCHEMICAL

I

5

IO

AND BIOPHYSKAL

I. 1000 0 of nucleotide

50 100 Amount

0.5 1 [picomolesltube]

RESEARCH COMMUNICATIONS

5

10

50 lb0

1000

lOdO

Fig. 1. A. Dose-binding curves of cyclic nucleotides, including cGMP clMP (AA) , cXMP (A--d) , cUMP (I--s) , cCMP (.--.), cAMP (-1, (a-), and cTMP (*--;’ ). 6. Dose-binding curves of guanosine (A+), ;i-GMP (M , GDP (:c-jk) , GTP (@--@I), adenosine (A---A) , 5’-AMP ---a), ADP (x---x), and ATP (o---o). As compared to that of cGMP (o--e), all nucleotides are inactive.

strated to

in

the

Fig.

protein

competition

of

various

cross-reaction,

cyclic

nucleotides

than

the

binding

protein

the effect

40

mM.

by The

influence. prepare

could

was

be

Zn2+

at

other

protein,

50

detected

and

(Fig. l/26

lower

that

and totally

carried

out

in

both

the

divalent

on

1.

binding

buffer the

cations,

cGMP,

containing

50

mM of

binding

increased

495

Ca 2+,

the

showed

an

appre-

The cTMP

other did

not other

1-B). preparation

2-mercaptoethanol

of

cGMP-

(6

mM),

of cGMP was tested,

cGMP

to

protein

40 mM, Mn*+

without

was

and

and

Co

2-mercaptoethanol even

Of

derivatives

When of

The

cGMP.

and

(Fig.

4 mM and

namely

clMP

phosphate

the binding

of

and

binding

cGMP.

respectively.

their

of

3H-cGMP

of

competition,

presence

cations

concentrations

picomole

inactive

2-mercaptoethanol:

The

unlabeled

CAMP

of

and

Table

of

0.2

of

cations

in

of

I-A),

degree

were

inhibition

picomoles with

adenosine,

some divalent listed

with

a much

Guanosine,

divalent

complete

examined

had

When the

achieved

nucleotides,

of are

A nearly

l/l5

cyclic of

hanced

be

al 1.

Effects

results

could

-B.

nucleotides

ciable

at

and

binding

cyclic

compete

1-A

by 2+

greatly

Mg*+

en-

only

at

, exerted was

the

and the

cations.

used

little to Under

Vol.

67,

No.

2‘1975

BIOCHEMICAL

AND

BIOPHYSICAL

TABLE Effects

of

divalent

3H-cGMP

and

to

Z-mercaptoethanol

cGMP-binding

Binding’

4

Cations’

control)

mM

40 mM

105.7

T

Ca2+

104.5

+_ 0.2

Mn2+

103.9

t

co2+

105.1

Zn2+

135.7

0.3

139.9

2 0.5

102.0

-f 0.2

0.2

95.1

+ 0.2

*

0.2

95.1

f

0.2

+

I.3

158.6

+

1.8

6

0

No cation

on

protein

(% of

Mg2+

2-Mercaptoethano13

COMMUNICATIONS

1

cations

binding

RESEARCH

61.5

+ 0.5

100

mM

50

mM

144.1

k

2.1

Mg2+

t 40

mM

41.5

? 0.3

123.7

+ 0.8

112.8

+ 1.6

Zn2+,

40

mM

45.9

2 0.2

138.5

+ 1 .o

177.8

+ 2.9

experiment expressed

as

1 The results

binding are

was performed Mean + 1 S.D.

in

triplicate,

and

the

2 The experiment was carried out using cGMP-binding protein extracted with phosphate buffer containing 6 mM of 2-mercaptoethanol. The control system did not contain any cation, and 100% binding represented 0.112 picomole of 3H-cGMP bound to 200 ug of protein. 3 The cGMP-binding protein was er containing no 2-mercaptoethanol, Three aliquots of the preparation containing glycerol and specified toethanol. The control system against buffer containing 6 mM Control not contain any cation. 0.12 picomole of 3H-cGMP bound

these tained tein neither

conditions,

40

a stimulatory extracted Mg

effect without

2+

nor

mM of

Zn

Mg2+ to

extracted

with phosphate buffup to the step of dialysis. were dialyzed against buffer concentrations of 2-mercapused the protein dialyzed of 2-mercaptoethanol and did of 100% binding represented to 200 pg of protein.

inhibited further

the increase

2-mercaptoethanol 2+

exerted

any

binding,

had enhancing

496

cGMP much effect.

lower

but

40 mM of

binding binding

to

Zn2+

protein. to

cGMP,

mainProand

Vol.

67, No.

BIOCHEMICAL

2, 1975

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

0.25 1

Fig. 2. Scatchard graphs of 3H-cGMP binding. tontrol (*--;‘:) has KD1 = 8.8 x IO-’ M and KD2 = 0.9 x lo-’ M. Mg2 (e) does not affect the binding kinetics, as KDI = 6.8 x IO-’ M and KD2 = 0.9 x lo-’ M. Zn2+ (o----o) affects the binding kinetics, exhibiting only a single KD = 7.4 x lo-’ M. .

Kinetic

studies:

Scatchard

plotting

protein (KD

These

x

as

When

the binding

remained Tissue

did

of

any

low

change

of

CAMP

and

were

measured

by

this

competitive

were

also

used

for

The

values

of

cGMP

tigators

using

CAMP are

different

not

change

was

performed

affinity KD

cGMP:

techniques

binding:

one

low

The

x

the

presence the

lo-’

levels

to

those

of

497

Fig.

high (KD

2.

The

affinity

= 0.9-2.0

x

preparations. 40

mM of

presence

of

40

while

that

cGMP

of

in

Mg2+.

mM of

Znzf,

high

affinity

of

several

assay. results

reported

(14-19).

with

by

M).

protein-binding The

in

affinity

disappeared,

(7.4

analyzed

binding-protein

in

determination. similar

example

of in

were

a typical

with

batches

in

levels

cGMP

other

different

experiment

without

the

binding

by for

and

5

in

component

cGMP-piotein

illustrated

M),

constants

binding

of

components

lo-’

tested

binding

are

two

= 5.5-16.3 M),

kinetics

and

contained

IO-’

the

The

are

The

values

tissues

same

tissues

listed

previously The

rat

in by

of

Table

other

CAMP

are

2. invesalso

in

Vol.

67, No.

2, 1975

BIOCHEMICAL

Assay

of

cGMP

AND

Lung

153.1

levels

in

Liver

rat

COMMUNICATIONS

tissues’

CAMP’

cAMP/cGMP

420.0

2 21 .5

2.74

13.4

*

1.5

176.5

f

3.8

3.17

Heart

36.0

+

3.1

395.0

f.

4.2

0.97

Testis

17.2

f

1.0

328.5

f

4.2

9.10

16.9

2

3.0

67.5

+

0.9

3.99

54.3

f

3.8

611.9

f

8.9

11.27

fat

’

same

different The

The

values

range are

pad

cortex

’ Three pl icate.

pad.

CAMP

RESEARCH

+ 10.3

Kidney

tissues

2

cGMP2

Epididymal

the

TABLE and

Tissues

BIOPHYSICAL

results are

the

Compared

to

of each expressed

picomoles/g

reported

on

samples are

by

order other

wet

others.

of

10

were assayed Mean + 1 S.D.

weight

of

I n general, to

organs,

tissue as

20

the

,

with

lung

ratios

exception

contains

du-

tissue.

cAMP/cGMP

the

in

of

more

the

of

various

lung

and

fat

in

many

cGMP.

DISCUSSION Recent mammalian (22))

reports

described

tissues, guinea

including

pig

lung

rat

(23),

adrenal

cortex,

a cGMP-binding

binding

protein

using

increasing

interest

messenger

warranted

quantification cGMP

(14,

measurement.

and

in

cGMP

and bovine

column

of

tissue

19,

26)

appeared

The

radioimnunoassay

partially

samples. to

be

played

and

sensitive

Enzymatic

498

by

Steiner

for

tedious et

cGMP method

methods and

The

by

al.

pancreas

the

from (25).

role

rat

From

separated

time-consuming

developed

21),

(24).

chromatography

a simple

activity

(20,

cortex

physiological

development

kinase

cerebellum

adrenal was

affinity

a possible

in

protein

bovine

protein

CAMP

the of

15,

cGMP-dependent

the for (27)

bovine CAMPeveras

second

for assay routine depended

the of

Vol.

67, No.

on

the

tion

success for

binding

of

protein

several

months.

from

lobster

muscle

kinase

large

quantities

fair

can

number

specific

of for

treatment

of

cGMP,

is The

cGMP-specific

binding that

reported

Mg2+

and

which

Zn

2+

does

ACKNOWLEDGMENTS preparing the

not

has

by

Gill

is

a unique

feature

exhibit

: We wish manuscript.

same

to

thank

The

is

little

the

over

protein

in

procedure

for

method if

a simple

0.2

a

is pre-

The

performed. as

com-

picomole

of

applications. from

rat

sulfate,

is

distinct

of

5.5-16.3

x

constant (25).

The

this

prepared the

as

isolated

of

assay

day.

cGMP

cGMP-

advantages

increased

from

measure

ammonium

Kanstein

protein

further

protein,

a binding

and

be

specific

binding the

cGMP-

comparison,

many

and

a single

general

with

offers

in

CAMP can

for

binding

It

CAMP-binding al.

separating

precipitation protein.

can

us

hours,

finished

specificity

In

immuniza-

using of

cGMP-specific

in

required

activation

sensitive.

of

COMMUNICATIONS

assays

the

by

accomplished

adequate

or

less

extraction

method,

considered

fractionated

developed

sample

the

(16),

assay

be

which

Protein-binding

were

can

and

tissue

sensitivity

since

samples cGMP,

of

be

RESEARCH

antibody,

(171,

The

techniques.

BIOPHYSICAL

cGMP-specific

protein-binding other

AND

of

protein

petitive

et

producing

a period

dependent

the

BIOCHEMICAL

2, 1975

particular

from effect

rat of

Miss

enhancement

I iver divalent

Constance

lungs

by from

IO-’ of

CAMP-

M, cGMP

cGMP-binding by

the

Balint

similar

to

binding

by

protein,

method

cations

simple

of

Kumon

(12).

for

her

help

in

REFERENCES 1. 2.

3. 4. 5. 6. 7.

Sutherland, E. W., Oye, I., and Butcher, R. W. (1965) Recent Prog. Horm. Res., 21, 623-646. Schultz, G., Hardman, J. G., Schultz, K., Baird, C. E., and Sutherland, E. W. (1573) Proc. Nat. Acad. Sci. USA, 70, 3888-3883. Hadden, J. W., Hadden, E. M., Haddox, M. K., and Goldberg, N. D. (1972) Proc. Nat. Acad. Sci. USA, 69, 3024-3027. Clyman, R. I., Sandler, J. A., Manganiello, V. C., and Vaughan, M. (1975) J. Clin. invest., 55, 1020-1025. Illiano, G., Tell, G. P. E., Siegel, M. I., and Cuatrecasas, P. (1973) Proc. Nat. Acad. Sci. USA, 70, 2443-2447. White, A. A., and Aurbach, G. D. (1969) Biochim. Biophys. Acta, 151, 686-697. Kuo, J. F., and Greengard, P. (1970) J. Biol. Chem., 245, 2453-2498.

499

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8. 9. IO. 11.

12. 13. 14. 15.

17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Miyamoto, E., Petzold, G. L., Kuo, J. F., and Greengard, P. (1973) J. Bio?. Chem., 248, 179-189. Lowry, 0. H., Rosebrough, N. J., Fart-, A. L., and Randall, R. J. (1951) J. Bio?. Chem., 193, 265-275. Kumon, A., Nishiyama, K., Yamamura, H., and Nishizuka, Y. (1972) J. Bio?. Chem., 247, 3726-3735. Gilman, A. G. (1970) Proc. Nat. Acad. Sci. USA, 67, 305-312. Kumon, A., Yamamura, H., and Nishizuka, Y. (1970) Biochem. Biophys. Res. Commun., 41, 1290-1297. and Guidotti, A. (1974) Anal. Biochem., 59, 63-68. Mao, C. C., Ishikawa, E., Ishikawa, S., Davis, J. W., and Sutherland, E. W. (1969) J. Biol. Chem., 244, 6371-6376. Goldberg, N. D., Dietz, S. B., and O'Toole, A. G. (1969) J. Biol. Chem.,

244, 16.

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4458-4466.

Murad, F., Manganiello, V., and Vaughan, M. (1971) Proc. Nat. Acad. Sci. UsA, 68, 736-m. Kuo, J. F., Lee, T. P., Reyes, P. L., Walton, K. G., Donnelly, T. E. Jr., and Greengard, P. (1972) J. Bio?. Chem., 247, 16-22. Steiner, A. L., Pagliara, A. S., Chase, L. R., and Kipnis, D. M. (1972) J. Bio?. Chem., 247, 1114-1120. Shibuya, M., Arai, K., and Kaziro, Y. (1975) Biochem. Biophys. Res. Commun., 62, 129-135. and Sold, G. (1972) Biochem. Biophys. Res. Commun., 49, ??OOHofmann, F., 1107. Takai, Y., Nishiyama, K., Yamamura, H., and Nishizuka, Y. (1975) J. Bio?. Chem., 250, 4690-4695. Van Leemput-Coutrez, M., Camus, J., and Christophe, J. (1973) Biochem. Biophys. Res. Commun., 54, 182-190. Kuo, J. F. (1974) Proc. Nat. Acad. Sci. USA, 71, 4037-4041. Shima, S., Mitsunaga, M., Kawashima, Y., Taguchi, S., and Nakao, T. (1974) Biochim. Biophys. Acta, 341, 56-64. C. B. (1975) Biochem. Biophys. Res. Commun., Gil?, G. N., and Kanstein, 63, 1113-1122. Schultz, G., Hardman, J. G., Schultz, K., Davis, J. W., and Sutherland, E. W. (1973) Proc. Nat. Acad. Sci. USA, 70, 1721-1725. Steiner, A. L., Parker, C. W., and Kipnis, D. M. (1972) J. Bio?. Chem.,

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500