A simple and specific locust protein binding assay for cyclic 3′5′ GMP

BIOCHEMICAL

Vol. 71, No. 4, 1976

A SIMPLE

AND

SPECIFIC

Peter Department

of

June

The

g x

fifth

instar

binding

Wood,

binding

procedures. agree

The

second

suitable

assay4

has

a protein cost of

is

desirable.

cyclic

GMP5

have

not

assays4

*Cyclic Cyclic

binding

achieved

GMP

Surrey.

provides

the

to its

is is

measurement

assay

for

is protein

the

directly

radioimmunoassay.

role

cyclic

of

AMP*l

cyclic

GMPk

2 has

led

to

as

an

a search

measurement.

well

established3 The

on

the

cyclic

Published 6 7 8 require

of

be measured

by

a drawback. impact

a

published

may

in

whole

Specificity

obtained

for

a major

for

levels

messenger

antisera had

Gui Idford,

homogenised

previously

interest

Radioimmunoassay of

3’5’

CYCLIC

Marks

Surrey,

hoppers

used

in

results

methods

purchase

FOR

Vincent

purification.

Urine with

from

be

reported

increasing

alternative for

may further

that

well

and

gregaria

which

than

ASSAY

of

supernatant

Schistocerca

greater

Hartman University

15 min

GMP without

and

Gordon

BINDING

22,1976

protein

cyclic

PROTEIN

Biochemistry,

Received 10,000

LOCUST

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

receptor

of

similar

of

comparable

production receptor cyclic

for

the to

or

AMP

simplicity

procedures

purification

the

bovine-adrenal

measurement GMP of

a specificity

but

that

and the

binding

and

measurement protein

shown

low

by

and

cyclic

AMP

q.

GMP is

used as the abbreviation

AMP as the abbreviation

Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

for

for

guanosine-3'S'-cyclic

adenosine-3'S'-cyclic

1139

phosphate, phosphate.

Vol.71,No.4,

1976

We have using,

as

whole

desert

tested

in

with

that

which

investigate for

crickets

cyclic

than

excess

and,

of

locusts

of

GMP over I1 as

instar

1969 that

in

confirmed8

GMP from

fifth

nanomoles

cyclic

been later,

the

range

two

cyclic

fraction

at

wide

COMMUNICATIONS

for

reportedlO the

more

recently

specificity

gregaria)

group

contained

have

high

RESEARCH

a supernatant

from

three-fold

BIOPHYSICAL

protein,

different

it to

binding

Sutherland’s

markedly

findings,

of

assay

(Schistocerca

of

a two-

an

of

locusts

Members was

AND

developed

a source

tissue

BIOCHEMICAL

stage.

“Cricket”

mammalian

tissues

cyclic

GMP

cyclic

AMP.

led

a source

us

first

of

binding

per

gram, These

to protein

GMP.

EXPERIMENTAL

Preparation

of

Fifth rapidly

frozen

(250

mM-sucrose; two

blender

ground

extracted 50

15 minutes

decanted

through

stored

at

frozen

preparation

three

months.

Assay

procedure

The

at

10,000

nylon

cloth

-2O’C.

Aliquots maintaining

(at

incubation

at

nylon

liquid

25

2.

10,000

and

(125

5.0

were

9

thawed its

on

cooling for

u pore

resulting ml

The

a

per

an

immediately

and

the

for

was

again

frozen

for

super-

respun

a

and

before

properties

gram,

ice-bath.

supernatant were

cold

‘Polytron’ in

size)

were

medium

15 minutes,

aliquots

binding

g,

15 mM-MgC12)

speed with

The

1.5-2

nitrogen.

mM-KCI;

maximum

cloth

weighing

homogenisation

Limited)

centrifuged

through

7.4; at

Supply

in

ice-cold

pH

periods

was

decanted

further

mM-Tris,

Media

each

a powder

6.0 ml

thirty-second

homogenate

gregaria,

to

with

(Northern

natant

protein

Schistocerca

and

was

using

binding

instar

powder

The

the

use, at

the

least

4’C)

mixture

comprised

1140

100

ul

assay

buffer

(50

mM-HEPES,

Vol.

71,

No. 4,1976

pH

7.4;

BIOCHEMICAL

8 mM-theophylline;

albumin

(Armour))

binding

protein

the

range

One in

mmol),

at

cyclic

of

dissolved

GMP)

[8-3H]

assay and

cyclic

3.8

ice-cold

buffer, the

and

made

up

pmol 100

in

GMP

was

bovine

cyclic

ul

the

of assay

after

100

or

standard

ul

Centre;

for

of (in

buffer.

a thirty

continued

serum

AMP; test

(Radiochemical

added

incubation

COMMUNICATIONS

5 g/l

200

cyclic

GMP was

M-ammonium

‘Whirlimixer’,

thirty

minutes

at

ml

I ,800

distilled

was

transferred

toluene

at

supernatant

1.0

-

water to

a

19

minute

a further

a counting

RESULTS

AND

DISCUSSION

two

to

three

antisera cyclic the

standards

times

to

range

setting-up alter

(MSE

of these

4L’

and

mixed.

the An

Ci/

pre-

two

I2

in GMP

and

a

150-300%

in

standards

in

recovery

sensitivity

taken

up

from

each

Company), the

in tube

four tritium

AMP was in

with

Addition of

1141

by

ml

of

content

not

urine

of

the in

the

assay.

in

were using

The

to

of

assay The

buffer. urine

AMP

amount

locust

assay

pre-treated cyclic

which

a known

with

standards

of

included

radioimmunoassay

addition values

phosphodiesterase

values. the

cyclic

obtained

comparison

for

35%.

The

gave

in

centrifuge).

ml)

and

of

mixed

centrifuged

(0.5 and

ml

were

precipitate

concentrations

laboratory.

tubes

1.0

refrigerated

added

which

those

of

then

Searle

of

than

this

addition

aliquot

(G-5.

cyclic

samples

increased

‘Mistral

aspirated;

urine

recovery

minutes,

was

higher in

ten

efficiency

gave

raised GMP

for

experiments,

buffer,

the

The

scintillant

with

assay

2

by

solution.

‘Minivial’

measured

the

4’C

‘Synperonic-NXP’

Preliminary

separated

sulphate

kept

The

in

is

mg protein);

of

4’C

RESEARCH

hours. Bound

a

pmol

picomole

incubation half

which

(3.5-4.0

50 ul

BIOPHYSICAL

6 mM-Z-mercaptoethanol;

in

O-50

AND

the effect

did cyclic was

not GMP

Vol.

BIOCHEMICAL

71, No. 4, 1976

AND

BIOPHYSICAL

TABLE

Cyclic

GMP

Nine by

levels

random the

in

urine

locust

different

rabbit

radioimmunoassay

LOCUST

binding

based

ASSAY

SAMPLE Cyclic

pm01 /ml

were

I

GMP

Ilmol/!il creatinine

diluted

1 in

method

anti-cyclic is

COMMUNICATIONS

urine

samples

protein

RESEARCH

and

by

GMP antisera on

the

method

I

20

analysed

in

radioinvnunoassay

of of

and

high

with

et

two The

specificity.

Steiner

duplicate

al

(1972).

RADIOIMMUNOASSAY

ANTI SERUM

pm0 1 /ml

1

ANT I SERUM Cyclic

lJmo1/g creatinine

2

GMP

pmol /ml

urn01 /cl creatinine

240

1.18

200

0.99

240

1.18

400

1.22

420

1.28

420

1.28

560

0.92

600

0.98

540

0.89

320

1.14

300

1.07

340

1.21

90

0.50

90

0.50

100

0.56

180

0.64

200

0.71

160

0.57

680

1.19

700

1.23

640

1.12

860

1.26

860

1.26

1000

1.47

400

0.80

360

0.72

380

0.76

1142

BIOCHEMICAL

Vol. 71, No. 4,1976

detectable and

with

was

maximal

affects AMP

per

the (Table

binding

tube

to

of both

in

the

The pmol

excess

ox-heart

content

to

cyclic

in

GMP

the

less

than

than

one-thousandth.

than

one

fi.ftieth

than

that

of

interpreted constants

= 2.4-5.2

x

The in

plasma

replacing resulted

in

use

agreed

well,in

radioimmunoassay

ten

urine

specimens

independent

of

urine

assay

was

pH 7.4

calculated13

to

be

of reduced

urine

samples

the

cyclic

the

assay

with

GMP

fifteen

nucleotides results

avidity

of

cyclic

of

the

as

an

obtained

Cyclic

IMP GMP

nucleotides

of

methods

data

KD,

three

cross-reacted

with

cyclic

AMP

of

= 1.5-2.5

x

with

to

the

considerably

for

revealed

populations

order

with

therefore,

is,

binding

two

and

are

cross-reacted

Specificity

binding

terms

being

cyclic

protein

the

in

cyclic

GMP.

smooth

curves

binding

sites

10mgM

which with

and

10-8M.

of

cyclic

EDTA AMP

theophylline in

to

cyclic

limit.

AMP.

of

pm01

by

added

tube,

AMP

which

GMP were

locust

at

existing

plots

GMP

preparations.

cyclic

200

results

Preincubation

similar

the

cyclic

determined

cyclic

tube.

Other

less

dissociation KD2

the

of 1,

of

per

l:32.

per

protein

Scatchard be

cyclic

AMP

Thus,

gave those

of

detection

Figure

binding

higher

of

of

reactivities

illustrated

or

with

cyclic

addition

tests

phosphodiesterase

Cross

extent

and

to

limit

below

different

the

and

RESEARCH COMMUNICATIONS

5 pmol

GMP

Measurements

1:2

as pmol/tube.

recovery

range

little

100-200

investigated,

detection

0.2-0.3

as

standards

98%.

dilution

of

cyclic

Moreover,

averaged

of

a level

samples

1).

could

addition

at

the

urine

now

the

AND BIOPHYSICAL

a 20-30s

l4

studies by increase

anticoagulant

this

led

us

and to

compound in

zero

investigate in

standard

1143

phsphodiesterase

the

the assay binding

inhibitor effect

This

buffer. but

of

also

markedly

less same

Vol.

BIOCHEMICAL

71, No. 4, 1976

AND

BIOPHYSICAL

NUCLEOTIDE

FIGURE

COMMUNICATIONS

(Ptxd~tUk)

1

Specificity

The

RESEARCH

of

cross

the

reactivities

incubation

of

procedure

A:-

3’5’

D:-

dibutyryl

3’5’

G :-

dibutyryl

3’5’

dTMP,

Method

5’

cyclic

GMP-PNP,

and

(M

and

(W)

not

plotted

increased of

and

specificity

B:-

the

cyclic

cyclic

GMP;

E:-

3’5’

cyclic

AMP;

H:-

Mean

5’

monobutyryl

the

GTP,

2’3’

3’5’

cyclic

means

determined

using

the

test.

3’5’

ATP,

show

in

were

IMP;

cyclic

C:-

3’5’

cyclic

cyclic

AMP;

F:-

result

(2

AMP,

2’3’

SEM)

CMP; 3’5’ for

cyclic

UMP;

cyclic

3’5’

cyclic-

GMP,

AMP-PNP,

UMP.

(+SEM)

of

results

for

AMP.

In

nucleotides

which

are

individually.

the

use

5’

nucleotides

described

GMP;

AMP,

fifteen

cross-reactivity

isobutylmethyl

of xanthine

results

in

cyclic

(8mM) good

in

agreement

1144

the

assay with

contrast buffer

those

to gave

found

for

this,

the

binding theophylline.

Vol.

71, No.

BIOCHEMICAL

4,1976

Binding

protein

differences

in

Tiss.ue

fifth may

specificity

showed instar

much

of

cyclic

The the

Urine be

of

may

to

prior

nucleotide

We thank of

the

or

isolated

than

fourth

or

phenomenon

endocrinology.

fifth

instar will

Locusta

also

be

migratoria

suitable

as

a

protein.

binding

the

advantages

protein

with

without out

of high

on

procedure

specificity

preliminary

measurements

a simple

for

purification, plasma

cyclic and

and

tissue

it

extracts

fractionation.

Shell

Research

locusts

cross-reactivity this

distinct

stages.

insects

of

COMMUNICATIONS

showed

juvenile

whole

species

combines

carry

and

insect

this

analysed

possible

without

gifts

be

AMP

with

binding

method

preparation

GMP . may

locust

adult (either

on

that

GMP

gregaria

investigation

investigations indicated

of

the

studies

RESEARCH

Schistocerca

cyclic

Further in

BIOPHYSICAL

males

greater

interest

migratorioides source

adult

hoppers.

prove

from between

from

Preliminary

for

extracts

preparations

abdomens)

AND

used

Limited

in

this

(Sittingbourne,

Kent,

UK)

for

study.

REFERENCES

1.

‘News

and

2.

Goldberg, Kuehl,

3.

F.A. New

Steiner,

A.L.,

Brown, (1571)

Nature

N.D.,

307-330,

111:

4.

Views’

Haddox,

Jnr.,

and

York:

Albano,

Biochem.J.,

Nature

M.K.,

Nicol,

Estensen,

Raven

Wehman,

Adv.Cyc.Nuc.Res., B.L.,

(1373)

S.E.,

186-187. Glass,

(1975)

In:

D.B.,

Sanford,

Adv.Cyc.Nuc.Res.,

C.H., 5,

Press. R.E.,

2, J.D.M., -121,

R.

246,

Parker,

51-61,

New Ekins,

C.W.

and

Kipnis,

York:

Raven

R.P.,

Sgherzi,

561-562.

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D.M.

(1872)

Press. A.M.

and

Tampion,

W.

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5.

6.

Murad,

BIOCHEMICAL

F.,

USA,

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Kuo,

J.F.,

Manyaniello,

AND

V.

and

BIOPHYSICAL

Vaughan,

RESEARCH

M.

(1971)

COMMUNICATIONS

Proc.Nat.Acad.Sci.

736-739.

(4)

Wyatt,

G.R.

and

R.

Fang,

Greengard,

P.

(1871)

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246,

7159-7167. 7.

Kobayashi,

and

V.S.

(1875)

Biochem.Biophys.Res.Comm.,

G.R.

(1975)

Anal.Biochem.,

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305-312.

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(2)

493-500.

8.

Fal!on,

A.M.

and

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Gi Iman,

A.G.

(1870)

10.

Ishikawa, J.Biol

11.

Fallon,

12.

Wood,

E., .Chem., A.M. P.J.,

Lab.Practice Ekins,

R.P.

14.

Tovey,

K.C.,

6,

Proc.Nat.Acad.Sci.USA,

Ishikawa,

S.,

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G.R. J.,

(11)

(1874) Oldham,

Davis,

J.W.

and

614-619.

Sutherland,

(1969)

E.W.

6371-6376.

Wyatt,

English, 24

13.

Wyatt,

(1975)

Biochim.Biophys.Acta,

Chakraborty,

J.

and

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Hinton,

R.

(1875)

(1874)

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