Vol. 82, No. 3, 1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

June 14,1978

Pages

USE OF AMP SPECIFIC

ANTIBODIES

TO DIFFERENTIATE

865-870

BETWEEN

ADENYLYLATED AND UNADENYLYLATED E. COLI GLUTAMINE SYNTHETASE Robert J. Hohman and Earl of Biochemistry, NIH, NHLBI,

Laboratory Received

April

R. Stadtman Bethesda, Maryland

20014

18,1978

Anti-AMP specific antibodies were purified by affinity chromatography SUMMARY: of serum from sheep immunized with adenylylated bovine serum albumin. Results of immunotitration experiments and light scattering measurements show that these antibodies can be used to separate adenylylated from unadenylylated forms of E. coli glutamine synthetase and to detect variations in protein configurations elicited by partial adenylylation of the enzyme or by allosteric interactions with divalent cations. These results suggest that the reaction of anti-AMP antibodies with variously adenylylated forms of glutamine synthetase can be used to investigate the dependence of immunoprecipitability on the density, absolute numbers, and possibly, the spatial distribution of multiple identical antigenic sites on a given macromolecule. INTRODUCTION:

Previous

that:

(a)

cyclic

adenylylation

regulation

the enzyme's under

investigations of --E. coli

growth

subunits

per molecule;

subunits

within

the

(c)

configuration,

depending

various

Prompted used 3),

to detect

adenylylated between it

different

form

influence

both

can exist

states

are

by allosteric

elicited

unsuccessful

antibodies

and unadenylylated and stability taut

metal

or

tightened

cations;

interactions

(e) of the

inhibitors. that

conformational

with

adenylylated

catalytic

divalent

by

produced

to twelve

in a relaxed,

with

shown

in each of

cells

adenylylated

interactions

and unadenylylated

that

between

residue

from

zero

have

is mediated

tyrosyl

isolated from

review)

activity

on its

feedback

enzyme species

was reported

can contain

by the demonstration

we made several

enzyme

interactions

each enzyme

conformational

enzyme with

synthetase

same enzyme molecule

(d)

1 for

of a unique (b)

subunits; conditions

characteristics;

different

glutamine

and deadenylylation

12 identical

different

(see reference

enzyme-specific

antibodies

have been

states

allosteric

enzymes

attempts forms

of other

to see if

of glutamine

different

states

capable

antibodies synthetase

of adenylylation.

of reacting

specifically

raised could

(2,

against distinguish

In the meantime, with

AMP moieties

0006-291X/78/0823-0865$01.00/0 865

Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 82, No. 3, 1978

could

be obtained

present with

study

BIOCHEMICAL

by immunization

shows that

anti-AMP

BSA-AMP can discriminate

glutamine

synthetase

enzyme due to subunit

and also

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of rabbits

with

antibodies

prepared

between detect

and divalent

AMP derivatized

adenylylated differences

cation

by immunization and unadenylylated

in conformational

BSA'

(4).

The

of sheep

species of states

of the

interactions.

METHODS AND MATERIALS: Preparation of antiserum: Adenylylated bovine serum albumin (BSA-AMP) containing 16 moles of AMP per mole of protein was prepared by the reaction of BSA with AMP in the presence of a carbodiimide as described by Halloran and Parker (5). To prepare adenylyl group specific antibodies, the sheep was injected initially at multiple sites with 0.5 mg of BSA-AMP emulsified in complete Freund's adjuvant. Booster injections of 0.5 mg of BSA-AMP in incomplete Fruend's adjuvant were made at 2 week intervals. One week after the 3rd and final injection, 600 ml of blood were collected from the sheep. The serum was separated from the clot by centrifugation and stored at -2O'C. purified against

For immunoprecipitation tests, the y-globulin fraction was partially from the serum by (NH4)2SO4 fractionation (6) and after dialysis buffer (10 mM Imidazole, 150 mM KCl, pH 7.3) was stored at -2O'C.

Immunoprecipitation Tests: Innnunoprecipitation tests were carried out in a total 100 mM KCl, 1 mM MnC17, volume of 500 ul containing buffer (10 mM Imidazole, pH 7.0), a fixed amount of glutamine synthetase and decreasing-amounts of& paritally purified gamma globulin. Total glutamine synthetase activity and the average state of adenylylation (i.e., the average number of AMP groups per molecule) were determined by means of the y-glutamyl transferase reaction as previously described (7). Immediately after addition of the enzyme and antibodies, glutamine synthetase activity in each reaction tube was measured and the samples were incubated for 20 min at 37°C followed by incubation at 4'C for 18 hours. Tubes were then centrifuged and residual GS activity in the supernatant solution was determined. Purification of antibodies: AMP specific antibodies used in light scattering studies were purified by preferential absorption and elution from an affinity resin prepared by the covalent attachment of GS= to AH Sepharose-4B as described by the manufacturer (Pharmacia). Light scattering experiments: Changes in light scattering elicited by the interaction of antibodies with glutamine synthetase were observed by exposing the reaction mixture in a 1 cm cuvette to incident light at 400 nm and measuring emitted light of the same wavelength at 90" angle by means of a HPF-2A spectrofluorometer equipped with a Hewlett-Packard 700 4B X-Y recorder. Typically, millipore filtered buffer plus purified antibodies were added to the cuvette and after a baseline was established glutamine synthetase was added and the light scattering was followed with time. The initial scattering due to addition of glutamine synthetase was subtracted from all light scattering values. RESULTS:

The ability

of the antibodies

to react

with

adenylylated

glutamine

' Abbreviations: GS;j, refers to glutamine synthetase preparation containing on the average ii adenylylated subunits per dodecameric molecule. The value of ii can vary from 0 to 12 and is sometimes referred to as the state of adenylylation. BSA, bovine serum albumin, BSA-AMP, bovine serum containing covalently bound AMP moieties.

866

Vol. 82, No. 3, 1978

O

20

BIOCHEMICAL

AND BIOPHYSICAL

40

loo

60

--80

ANTISERUM

Immunoprecipitation 'Experiments were

Figure 1. antiserum. Percentage

residual

61 ug/ml

GQ

(0);

(b)

of natural precipitated

GS(0). P raction.

synthetase

could

precipitation (Fig.

la and 2a,

unadenylylated Figure (obtained

of a native adenylylated about

80

loo

200

1~11

of glutamine as described

either

contained:

46 ug/ml

The numbers

(a)

GSF

by light

2b) show that

plus

in brackets

as described

synthetase with in Methods.

anti-m

either 46 ug/ml

scattering

GS=

react

Gy

or

(0);

or 50 ug/ml of the non-

measurements

with

(a)

the ii value

Measurements

in Methods. antibodies

46 ug/ml

indicate

x 100

tube

and by immuno-

by either

adenylylated

method but

not with

enzyme. lb

shows

by mixing

precipitation

mixtures

be demonstrated

tests

60

enzyme activity = non-precipitated total enzyme activity per reaction

activity

reaction

For each point,

curves performed

I40

20

RESEARCH COMMUNICATIONS

that

antibody

titration

equal

amounts

of pure

of the GQ

fraction

preparation

G-5

enzyme preparation;

the same number

but led

of an artificial GS-l- and GSE)

GSc led

moreover,

of adenylylated

to quantitative

In contrast,

none of the GSr.

to precipitation

of only

([6.5]

titration

60% of the partially

the nonprecipitable

subunits

preparation

vs [7.5])

fraction

contained

as the untreated

enzyme. Figure

that

when compared

and enzyme the rate

of light

scattering

is

is

increased.

number

2b shows

This

of antigenic

increases

not due solely

sites

with

at a fixed

concentration

of antibodies

as the state

of adenylylation

to a progressive

increased

867

state

increase

of adenylylation

in the since

total qualitatively

Vol. 82, No. 3, 1978

BIOCHEMICAL

-0

5

AND BIOPHYSICAL

10 0

5

10

0

RESEARCH COMMUNICATIONS

5

10

TIME Iminutes)

Figure 2. Increase in light scattering as a function of time, enzyme concentration and state of adenylylation. 200 ng of anti-AMP antibodies purified by affinity chromatograph (see Methods) were used for each time course. Buffer for all experiments was 10 mM Imidazole, 100 mM KCl, 1 mM MnC12, pH 7.0. In addition, reaction mixtures contained: (a) 17, 50, 67, or 83 ug of GSE as indicated; (b) 67 ug of either GSF, Gv, or GS-8-, or GQ, as indicated; (c) 70 1-g GS=, 105 ug GSF, or 210 ug GSr, as indicated.

similar

results

are

are adjusted

so that

groups

2C).

(Fig. As noted

in the

configurations show that rate

relaxed

DISCUSSION:

already

1978,

glutamine

synthetase

free)

for Mn

2+

anti-AMP

interactions.

each

in different

Data in Table concentrations,

configuration.

1 the

The relative

> Mgzf-tightened

enzymes, that

by adenylylation

> Mg2+-taut

described

in various

relaxed,

here

Y>

taut

Am.

Sot.

and tightened

can also

of

They have

synthetase Proc.

should

the regulation

organisms.

Siedel,

the antibodies

868

in

the glutamine (J.

used with that

antibodies of adenylylation

in demonstrating

indicate

can exist

of AMP

enzyme.

specific

The studies

synthetase

-taut

, GS-8 and GSQ

the same number

enzyme and antibody

as follows:

and other

regulated

in press).

at identical

of GSc

contains

cation

on the participation

synthetase,

is

to divalent

is different

cation

been useful

vinelandii

glutamine

are

The highly studies

glutamine

Introduction,

scattering

(divalent

facilitate

mixture

when compared

of reaction

when the concentrations

each reaction

in response

of light

rates

obtained

detect

of Azotobacter Biol.

&em.,

forms

of

conformational

Vol. 82, No. 3, 1978

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE 1 Reactivity

of antibodies

as a function

of divalent

with cation

GSz environment

Initial Rate hA 400 nm /min

Enzyme Form

1.45 Mg

2+ tightened

1.69

Relaxed

0.62

Light scattering experiments performed as described in text. 200 pg anti-AMP antibodies and 75 ug GSn used in each experiment. Values given in table represent the initial slope of the light scattering reaction. Gs1-2 had been dialyzed against 10 mM Imidazole, 100 mM KCl, pH 7.0 plus either 1 mM MgC12, Buffer used in the experiments was the final dialysate. 1 mM MnC12 or no metal. Relaxed enzyme was dialyzed against 5 mM EDTA followed by 10 mM Imidazole, 100 mM KCl, pH 7.0. The enzyme was tightened by incubating relaxed enzyme with 10 nM MgC12 at 37°C for 20 min before running the experiment.

changes

elicited

noteworthy

is

the

adenylylated GSE

fact

they

that

are not

of GSF

GSmight 7.5 in the distribution

(Intra-molecular adenylylated

reflect

molecular bivalent

subunits

would

are

reactions reaction preclude

other

composed

subunits.

conformational

subunits,

precipitate

subunits

of hybrid

Incomplete

its

869

involvement

with

(l),

bivalent

lattice

this

containing

precipitation

proximally in

enzyme in

molecules

variants

enzyme with

and

of adenylylation

attributable

in hybrid

of an antibody

of GSy

of evidence

or be due to differences of the

of the

adenylylated

states

variations

all

a mixture

lines

at intermediate

but

of adenylylated

will

enzyme from

with

Particularly

to the enzyme.

60% of the paritally

enzymes

and G%

of adenylylated

vs inter-

only

and unadenylylated

natural

same number

antibodies

In agreement

occuring

adenylylated

effecters

of the unadenylylated

precipitate

naturally

mixtures

of allosteric

anti-AMP

GSpreparations. 7.5

shows

intra-

that

and none

, whereas

native

both

by the binding

of the to differences

containing in

the rates antibodies. positioned

formation).

the of

BIOCHEMICAL

Vol. 82, No. 3, 1978

Similar

considerations

on the state scattering

explain

of adenylylation

the rate

of glutamine

dependence

synthetase

of the antibody as measured

reaction

by the light

technique.

Since

many previous

enzyme by a variety worthy

might

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

that

a quantitative

in reactivity

of antibodies

of its

glutamine chemical ability

unique

molecular

synthetase level.

sites

multimeric

the

it

is

note-

be achieved

the observed

by variations

of adenylylation

separation

of glutamine

synthetase

of adenylylation. subunit

structure in

designed

for

Among other

problems

the relationship

number,

macromolecule

and the extraordinary

the electron

well

on a single

failed,

Hopefully,

for

as viewed

absolute

from unadenylylated

could

of the state

states

imagery

is

and the density,

antigentic

procedures

have

and GSE

antibodies.

intermediate

of its

of Gv

as a function

to develop

at different

Because clarity

anti-AMP

the adenylylated

techniques

separation

with

can be exploited

to separate

of physical-chemical

immunoprecipitation

species

efforts

fundamental

microscope,

studies

at the

between

and spatial

immuno-

immunoprecipit-

distribution

can be studied

coli

--E.

of identical

using

the anti-AMP

antibodies. ACKNOWLEDGMENTS We wish to thank Drs. Andrew Shrake and Brian discussions during the course of these experiments.

Hemmings

for

many helpful

REFERENCES 1.

Stadtman, E. R., Press, New York.

2.

Lazdunski, 309-335.

3.

Cowie, (1973)

4.

Drocourt,

J.-L.

5.

Halloran,

M. J.,

6.

Campbell, D. H., Garvey, J. S., Cremer, N. E., and Sussdorf, D. H. (1970) Methods in Immunology, pp. .189-191, W. A. Benjamin, Inc., Massachusetts.

7.

Stadtman, E. R. (1969) The Role of Nucleotides for the Function and Conformation of Enzymes (eds. H. M. Kalckar, H. Klenow, M. Ottensen, A. Munch-Peterson and J. H. Thaysen), pp. 111-137, Munksgaard Press, Copenhagen, Denmark.

C. J.,

and Ginsburg, Pages,

A. (1974)

J. M.,

D. B., Truff-Bachi, P., BBRC, 53 (l), 188-193. and Leng, and Parker,

The Enzymes,

and Louvard, Costrejean,

M. (1975)

Eur.

C. W. (1966)

870

D. (1975) J. M.,

755-807,

J. Mol.

Py, M-C,

J. Biochem. J.

pp.

56,

of Immunology

Academic

Biol.

97 (3),

and Cohen,

G. N.

149-155. 96 (3),

373-378.