Biological activity of iodinated gastrins

Vol. 69, No. 2, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

BIOKlGICALACTIVITYOF

IODINATEDGAST=NS

Graham J. Uockray, John W. Walsh, and Norton I. Grossman Veterans Administration Wadsworth ~spital Center, Los Angeles, California 90073, and the UCW School of Medicine, Los An9eles, California 90024 Received

January

19,1976

Mono- and di-iodo heptadecapeptide gastrins were prepared by a modifiril chloramine-T technique. The iodinated gastrins did not differ significantly from unmodified gastrins in immunoreactivity and in stimulating acid secretion in gastric fistula dogs. The preparations should prove useful in studies of hormone binding to receptors. Introduction For direct

studies of hormone binding to biological

able to have available with the antral

radio-labelled

hormone gastrin

preparations

ical activity conditions. al activity

Wereport

by oxidation

by difficulties

activity.

in gastric

1,2

fistula

in prepar-

Gastrin biolog-

of methionine under ordinary

now that mono- and di-iodo gastrin

on acid secretion

modification tography.

is inactivated

is desir-

of hormone. Such studies

have been frustrated

ation of labelled hormone with proven biological

receptors it

having full

labelling biologic-

dogs can be prepared by a minor

of the chloramine-T reaction3 and purified

by ion-exchange chroma-

4

Methods Pure natural humanheptadecapeptide gastrin in the unsulphated form (G-37-1) was a gift of Professor R. A. Gregory and Dr. H. J. Tracy. Synthetic [Leu15] G-17-I was a gift of Professor E. Wunsch. Two types of iodination were performed: 1) high specific activity 1251 G-17-I was prepared by adding 2.8 n moles (6 ug) G-17-I dissolved in 6 ul NH HCO 3 (O.OSM) to 25 ul of phosphate buffer (pH7.4, O.ZSM), followed by 10 ul Na 122I (100 mCi/ml) and 9 n moles (2.4 ug) chloramine-T dissolved in 5 ul of phosphatt: buffer. The reaction was stopped after 10 set by addition of 52 n moles (10 )q) sodium metabisulphite dissolved in 20 ul phosphate buffer; 2) larger quantities of low specific activity iodinated gastrins were prepared by adding 480 n moles G-17-I (1.0 mg) in 200 ul of NH4HCOj(O.O5M)to 100 ul phosphate buffer (0.25X, PH 7.41, followed by 480 n moles KI in 10 ul phosphate buffer, 10 ~1 Na 1251 Copyright Q 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

339

Vol. 69, No. 2, 1976

(100 SACS

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AND BIOPHYSICAL

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, 1.42 P moles chloramine-T in 20 ul phosphate buffer, 6.5 )1 moles sodium metabisulphite in 25 ~1 phosphate buffer.

mCi/mf)

and after

30

Both reaction mixtures were applied to COhImn6 (1 x 10 cm) of Sephadex GlG equilibrated and eluted with 0.05M annnoniumbicarbonate. Fractions of 1.0 ml were collected and tubes containing iodinated gastrin (fractions 3-5) were pooled for further purification by ion-exchange chromatography. Amino-ethyl cellulose (Whatman, AZ 41) was equilibrated with 0.05M ammoniumbicarbonate, and packed into columns (1 x 10 cm); the mixture of iodinated gastrins was applied to the Column eluates column and eluted with a gradient to 0.5M ammoniumbicarbonate. were analyzed by absorbance at 28Onm(Zeiss M4 QII U V Spectrophotometer), radioimmunoassay (RIA) and bioassay. Gastrin concentration was estimated from IN absorbance by application molar extinction coefficient (12,261) for G-17-1.'

of

Radioimmunoassayof gastrin was performed by methods already described, 6 , -' using an antibody with specificity for the C-terminal portion of G-17-I (Ab 1296, 1:300,000),7 Specific activity of lz51 G-17 was estimated by caaparison of binding curves for standard G-17-I and labelled preparations (autoinhibition curves). The action of iodinated gastrins on acid secretion was studied in two dogs prepared with gastric fistulas several months previously. After an overnight fast each dog received an infusion of saline into a leg vein for a basal period of 40 min and thereafter graded doses of stimulant which were doubled every 40 min. On separate days the dogs received pentayastrin, standard G-17-I and fractions corresponding to peaks I, II or III of iodinated G-17-I or[Leu15] Gastric acid secretion Was collected every 10 min and acid G-17-I preparations. determined by titration to pH 7.0 with 0.2 M NaOH. Results Figure 1 shows the separation on AS cellulose

of the products obtained when

1 mg G-17-I was labelled with a mixture of 12'1 and reactive

gastrin

(I - III)

could be identified

was-a matching peak of material also corresponded

results

obtained when [Leul']G-17-I

of G-17-I were labelled

in the eluates and for each there

was iodinated,

acid secretion

gastrin

All three fractions

dose-response lines were

The concentration

in the three samples (determined by visual

of biologically

active

comparison of doses of sample

and standard required for 50%maximal response) were similar

340

pooled

in dogs (Figure 2), and over the

range 20 - 60%of maximal response to pentagastrin to that for standard G-17-I.

similar

and when ng quantities

in Figure 1 were separately

and immunochemical activity.

were found to stimulate gastric

Essentially

with 125I only.

activity

Tubes corresponding to peaks I - III

parallel

Three peaks of immuno-

to peaks of radioactivity.

to high specific

for analysis of biological

.

with absorbance at 280 run. Two of the peaks

(II and III) were

127I

to those estimated

Vol. 69, No. 2, 1976

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

?

lb

io

30

40

56

FRACTION

sb

70

80

1oc

60

NUMBER

of iodinated gastrins on AR cellulose. Figure 1. Separation 1 mq IIG-17-T iodinated with KI and tracer amounts of Na 1251 (see Methods). React ion mixture separated first on Sephadex GlO (1 X 10 cm) and tubes corresyondinq to first peak pooled and applied to an AE cellulose column (1 X 10 cm) equilibrated and eluted with 0.05M annnonium bicarbonate. A gradient to 0.5M ammonium bicarbonate was started fran fraction 10. Mixing vessel 125 ml; flow 6 ml/ hr; fractions 1.1 ml. o---e counts per ml; O---O gastrin estimated by radioimmunoassay; A--- A optical density at 280 run. Peaks identified by Roman numerals. For further analysis tubes pooled as follows: 40-51, peak I: 53-59, peak II; 66-71, peak III.

by RIA and by absorbance The W absorbance to the

spectrum

ly lower

In spite W

was essentially RIA inhibition specific

G-17-1,

activity)

of

but

peak

the molar

parallel

II

to that

and III for

341

material

standard

a slight-

to changes

in

induced

or by oxidation

of gastrin

determined

comparable

by

residues

tyrosine

extinction

concentration peaks

spectrum

and tyrosine

concentration

and were

was characterized

atoms onto

using

similar

altered

tryptophan

the

for

were III

the

this,

to the

curves were

I and II

of two iodine

absorbance similar

1).

We attribute of the

substitution

chloramine-T. by

of peaks

of unlabelled

or organization

by the

estimat4

spectra

maximum at 280 nm.

conformation either

at 280 nm (Table

in peak

coefficient by bioassay (prepared G-17-I

with

III

for

by material

C-17-l arid MA.

to high our

C-terminal

Vol.69,No.

2, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

5v 20

40

pmol kg-’ hr.’ ,A kg-’ hr-’

DOSE

2. Bose response relationships for action of standard G-17-I and pooled samplesof peaks I, II, and III (AE column, Fig. 11, or acid secretion in gastric fistula dogs. Abscissa: dose of G-17-I expressed in pool kg-1 hr-1 and of AE peaks in ~1 kg-l hr-1. Ordinate: responses normalised to percent of the peak acid output in response to pentagastrin (2.8 mEq 10 min). l G-17-I; o peak I; A peak If; A peak III. Bach point mean of 6 experiments in 2 dogs. Figure

Table --Concentration

of gastrin

in AE peaks

All

values,

AE peak by UV absorbance

Gastrin Gastrin

II

III 10.0

by RIA

8.7

12.1

13.4

by bioassay

9.4

8.9

11.3

(Figure

3) indicating for

gastrins

of peak for

n mole/ml.

11.8

and labelled

fmol

by 3 methods.

11.4

indistinguishable

activity

estimated

I

Gastrin

antibody

1

peak

II

that

the

standard

G-17-I.

required

for

was

estimated

iodinated

gastrins

By comparing

50% inhibition to be 2,200

III.

342

were

immunochemically

the amounts of binding,

cpn/fmol

canpared

of standard

the

specific

to 4,700

cpn/

Vol. 69, No. 2, 1976

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

fmol /ml cpm/ml

Figure 3. Inhibition curves of standard G-17-I (o), peak II (0) and peak III (A.) samples. In this experiment peaks II and III were obtained from iodination

c-17-1 with 1251 alone. All tubes contained Ab 1296 (1:300,000) and 2000 cpm of mono-iodo [125I] G-17-I as tracer. Abscissa: graded amounts of standard G-17-I expressed in fmol/ml; and graded amounts of 1251 substituted

0f

G-17's

expressed

in c&ml. Ordinate: binding expressed as percentage of bound/free 112511 G-17-I seen with antibody and trace alone.

ratio

maximal

The results in

that

the biological

unreacted

of

was only

with

were 15

[Leu

similar.

]G-17-I

about

[Leu15]G-17-I

were

immunochemical

and

peptides

activity ity

obtained

40% of that

properties

However,

was about

similar

it

is

50% higher

of the

native

to those

of the noteworthy

than hormones

iodinated that

G-17-I

for

and

the biological

whereas

(Table

G-17-I

immunoreactiv-

2).

Discussion Three ing peak

main

iodination I)

components of gastrin

corresponds

derivatives.

efficiency

calculated

specific

presented

in Figure

indistinguishable active ical

to the

activities

in

to immunochemical

original was

of

in the reaction One

of these

gastrin,

the

other

unreacted

iodinated

125

with

I the

2200 and 4700 cpn/fmol observed

specific

activities

of mono-

and

3 indicate

that

iodinated

unlabelled gastric potencies

the G-17-I.

acid

were

broadly

343

two are

respectively; activities

di-iodo

(Figure

well

are

with

the Results

immunochemically were

2) and the ratio

comparable

for

respectively.

peptides

iodo-

products

allowing agree

G-17-I

follow-

(designated

two iodinated

The two derivatives

secretion

mixture

method.

of 60% the

from

stimulating

identified

by the chloramine-T

When gastrin

had specific counting

have been

to those

also

fully

of bioioq-

of unlabelled

VoL69,No.

BIOCHEMICAL

2, 1976

AND BIOPHYSICAL

Table Comparison

of biological

and Leu15

G-17-I.

2

and immunochemical

Concentrations

potencies

calculated

from

Dose required for 50% maximal H+secretion G-17-I

172 pmol

Mono-iodo Leu15

G-17-1

G-17-I

Mono-iodo

Leu15

peptides.

Morley'

15 in G-17-I widely gastrin

showed

abolished

believed

ratios

present

study

oxidizing

and under

The results

suggest

also

be used

using

which for

monoiodo

peptide

An alternative radio-labelled molecule inactivation

full

et al' molar

binding solution

the methionine by oxidation.

activity

only

is the

when

of activity.

a 3-fold

In the

molar

excess

and di-iodo

of

products

activity.

conditions

iodinated

used

in RIA and which

currently

specific

receptors.

gastrins could

The advantages

and uncontaminated

with

of

unlabelled

obvious.

are

of the problem

is provided

loss

and immunochemical

biological

it

of chloraminc-T.

the mono-

appropriate

at position

and so inactivates

greater)

using

both

to those

50% binding

of G-17-I

of activity

not cause

with

systems

(or

lodinated

by using

studies

iodination

loss

excess

did

residue

methionine

found

conditions

similar

of high

During

oxidizes

biological

that

binding

gastrins lacks

Stagg

these

are

G-17-I

in these

activity.

was successfully

to have

be prepared

of the methionine

to chloramine-T

been shown

may

7.1

oxidation

at 280 nm.

2.4 pm01 1-l

100

to a four-fold

G-17-I

agent

hr"

6.0

that

G-17-1

Dose required for inhibition of antibody

115

However,

of G-17-I

UV absorbance

2.3

the chloramine-T

was exposed

Lower

kg-'

of iodinated

180

biological

that

molecule.

G-17-I

have

G-17-1

RESEARCH COMMUNICATIONS

by the

residue

of preparing [Leul']G-17-I

(position

[Leul']G-17-I

was

344

biologically

15)

analogue, it

is

less

active since

susceptible

up to 50% more active

than

this to G-17-I

Vol. 69, No. 2, 1976

in stimulating than

that

shown

acid

of the

of preparing of

2

3H G-17-I

the is

radio-labelled speed

employed

for

was about

RIA.

pattern

The present of biological

40% less study

has

and immuno-

peptide.

preparation

likely

and our

RESEARCH COMMUNICATIONS

immunoreactivity

has a similar

altered

for It

its

the antibody

to the

a method

AND BIOPHYSICAL

although

[Leu15]G-17-I

properties

has been described.

terms

with

monoiodo

Recently

erties

secretion

of G-17-I

that

chemical

BIOCHEMICAL

that 125

gastrins

of high

specific

the biological

I G-17-I which

are

3H G-17-I

and immunochemical

similar.

was used

activity

here

However, offers

prop-

the method

advantages

in

and simplicity.

References 1. 2. 3. 4. 5. 6. 7. 8.

Stagg, B. H., Temperley, J. M., Rochman, H., and Morley, J. S. (1970) Nature, 228, 58-59. Ginna, J. P., Morgat, J. L., Fromageot, P., Lhrbrasguet, M., Accary, J. P., Vatier, J., and Bonfils, S. (1974) Biochimie, 56, 763-767. Hunter, W. M., and Glover, Greenwood, F. C., J. J. (1963) Biochem. J, 89, 114-123. Stadil, F., and Rehfeld, J. F. (1972) Stand. J. Clin. Lab. Invest, 30, 361-368. Walsh, J. H., Debas, H. T., and Grossman, M. I. (1974) J. Clin. Invest, 54, 477-485. S. A. (1970) Gastroenterology 58, 1-14. Yalow, R. S., and Berson, Walsh, J. H. (1974) Nuclear Medicine in Vitro, pp 231-248, J. B. Lippincott, Philadelphia and Toronto. Morley, J. S. (1968) Proc. Roy. Sot. B, 178, 97-111.

345