Associative properties of the Escherichiacoli galactose binding protein and maltose binding protein

Vol. 105, No. 2, 1982 March 30, 1982

BIOCHEMICAL

ASSOCIATIVE

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 476-481

PROPERTIES OF THE ESCHERICHIA COLI

GALACTOSE BINDING PROTEIN AND MALTOSE BINDING PROTEIN Gilbert Institut

Received

February

Richarme

de Microbiologic, Universite 91405 Orsay, France.

4,

Paris-Sud,

1982

SUMMARY The periplasmic galactose binding protein and maltose binding protein of Escherichia coli are recovered mostly in dimeric form when purified, from osmotically-Eked bacteria, in the presence of protease inhibitors and 2-mercaptoethanol without dialysis and concentration of the shock fluid. The specific ligands, galactose (but not glucose) for galactose binding protein, and maltose for maltose binding protein, provoque the monomerisation of the dimeric native forms. These results are discussed in relation to the function of both binding proteins in transport and chemotaxis.

INTRODUCTION

Most

have been described to 42,000

(1)

periplasmic

as monomers

; however,

after

protein storage

evidence protein nomers

that

protein

when the

of molecular form

inhibitors

galactose

(but

protein which

and dimers protein

(GBP),

(mostly

dimeric),

when purified

of the dimeric

native

In this

rapidly

for

the

in

study,

(4,5)

binding

are

in the the

specific

MBP, provoque

buffer,

we present

by others,

(MBP)

Furthermore,

GBP, and maltose

for

of the galactose

have been purified

(GBP) and 40,000

for

22,000

and the maltose

35,000

not glucose)

from

was dissolved

(3).

weight

and Z-mercaptoethanol.

ranging

bacteria

have been reported

precipitate

binding e,

(2)

of Gram negative

weights

properties

as an ammonium sulfate the galactose

proteins

molecular

have been described,

protease

risation

binding

(MBP) of Escherichia

in multimeric

with

aggregating

leucine-isoleucine-valine binding

binding

as morecovered

presence

of

ligands, the monome-

forms.

MATERIALS AND METHODS D-glucose, D;galactose and D-maltose were from Merck. ["C] sugars Materials S sulfate 25 Ci/mg was from Amersham. Phenylmethylwere from C.E.A., France. markers were from Calbiochem. sulfonyl fluoride was from Sigma. Protein 0006-291X/82/060476-06$01.00/0 Copyright 0 1982 by Academic Press, Inc. A II rrghrs of reproduclion m my form reserved.

476

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Vol. 105, No. 2, 1982

AND BIOPHYSICAL

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Purification of galactose binding protein and maltose binding protein A single procedure was developed for purification of both proteins. E. coli K12, (from Dr. M. Schwartz, Institut Pasteur, strain ~0~3325, maltose constitutive, Paris), was grown, at 35"C, in minimal medium, containing 10 mCi/l of [35S] sulfate at a final concentration 0.2 mM, and 10-3 M fucose. The cells were har; they were osmotically shocked at vested at the end of the exponential phase O'C in distilled water containing 5x10-4 M MgC12 as described by Nossal and after shock, the supernatant shock fluid was adjusted Hewe1 (6) ; immediatly to pH 7.3 with buffer containing 0.01 M sodium phosphate pH 7.3, 2 mM 2-mercaptoethanol, 10-4 M phenylmethylsulfonyl fluoride (PMSF). The shock fluid was immediatly loaded on a hydroxylapatite (from Or. G. Bernardi, Paris) COlumn, equilibrated in the same buffer ; elution was made with a linear gradient of 0.01 M to 0.5 M sodium phosphate buffer containing 2 mM Z-mercaptoethanol, 1O-4 M PMSF. Both proteins were eluted in the same fractions ; the pooled fractio s were dialysed (4 hours) against 10-2 M Tris pH 8.1, 2mM mercaptoethanol, on a QAE-Sephadex column, equilibrated in the same buflo- 9 M PMSF and loaded fer ; elution was made with a linear gradient of 0 to 0.5 M KC1 in the same buffer ; galactose binding protein and maltose binding protein containing fractions were pooled, dialysed against 10-2 M Tris pH 8.1, 2 mM 2-mercaptoethanol, 10-4 M PMSF and loaded on a DEAE-cellulose (DE 52 Whatman) column equilibrated in the same buffer ; elution was made with a linear gradient of 0 to 0.3 M NaCl in the same buffer. The galactose binding protein was eluted at 0.07 NaCl and the malt se binding protein at 0.15 M NaCl. The proteins were dialysed against lo- 3 M Tris pH 8.1, 2 mY Z-mercaptoethanol, and stored at -7O'C. Detection of [35S] GBP and [35S] MBP in the first steps of the purification was made with specific antibodies prepared as described in (5). Galactose and maltose binding activities were measured by the filtration procedure described previously (7) : twenty volumes of a saturated ammonium sulfate solution were added to one volume of reaction mixture (binding protein f '14C] sugar) ; the samples were filtered on cellulose esters filters and b rinsed three times with saturated ammonium sulfate at 0°C. The filters were counted in a dioxane-based scintillation mixture ; a blank was made without the [14C] sugar. Both binding proteins were fully active in binding their specific ligands. Biogel P-100 chromatography Columns (80 cm x 0.7 cm) of Biogel (IOO-200 mesh) were equilibrated at 22°C with the column buffer, pH 8.1, 2 mM 2-mercaptoethanol (other constituents were added in buffer as described in the text) ; 150 111 of the purified protein was applied to the column ; the flow rate was 2 ml/h ; fractions were collected. The void volume of the column was determined with blue ; protein markers serum albumin (68,000) and chymotrypsinogen were included in each experiment.

P-100 IO-2 M Tris the column solution of 10 drops dextran (25,000)

Glycerol gradients ultracentrifugation Glycerol gradient ultracentrifugatlon was performed with a Splnco SW/50 rotor, in a Spinco model L2-65B ; isokinetic glycerol gradients (5 to 20%) of 4.8 ml were prepared in IO-2 M Tris pH 8.1 buffer containing 2 mM 2-mercaptoethanol, and other constituents as indicated in the text. Samples and standards of 100 111 were applied to the gradients and centrifuged at 4°C for 17 hours at 45,000 rpm. Fractions of 200 ~1 each were collected. S20,w was calculated on the basis of a linear relationship of the sedimentation coefficient to the distance migrated in the gradient. Bovine serum albumin was used as a standard for these calculations. RESULTS

Fig.

1A shows

Bio-Gel

P-100

column

binding

protein

was

void

volume

of

the

chromatography

equilibrated eluted

as

column

and

of in

buffer

a peak, the

at

elution

the

binding

containing an

elution

volume 477

maltose

no maltose. volume

of

protein

the

lying serum

on

The

maltose

between

the

albumin

(68,000)

a

Vol. 105, No. 2, 1982

BIOCHEMICAL

AND BIOPHYSICAL

10

20

30

RESEARCH COMMUNICATIONS

LO

FRACTION

50

60

NUMDER

figure 1, Bio-Gel PlOO chromatography of maltose binding protein in the absence and in the presence of maltose. 150 ~1 of a mixture containing MBP and chymotrypsinogen (1 mg each) was loaded on a (50 !J3), serum albumin column eouilibrated in buffer containina no maltose (0). 150 ul of a mixture containing MBP (50 ;1g), IO-3 M maltose -serum albumin and chymotrypsinogen (1 mg each) was loaded on a column equilibrated in buffer containing 10-3 M maltose (X). 50 ;11 of each fraction was counted for radioactivity. Arrows indicate 1 : void volume, 2 : serum albumin elution volume, 3 : chymotrypsinogen elution volume.

; this

marker

peak corresponds

in some experiments

a small

volume

of the

column,

shown).

Fig.

protein

on a Bio-Gel

40,000, (5).

indicating

P-100

binding

at an elution

The results

from

in equilibrium

with

in

tose

binding

sence

of galactose

sence

of

10e3

in the presence

protein

sedimented

M glucose,

ligand

maltose

of

binding

binding

protein

protein,

forms,

ligand,

protein

mostly

maltose,

dimeric, pro-

form. in three

linear

glycerol

: no galactose

no glucose,

2, in the first

two gradients,

the galac-

third

in the

than

in the

of galactose

a sedimentation

of 10s3 M galactose,

the

was sedimented

faster

; in the absence

a specific

weight

in multimeric

native

binding

to a molecular

exists

coefficient

a sedimentation 478

;

(not

of maltose

binding

the maltose

and that

forms

the maltose

show that

form,

; as shown in fig

protein

multimeric

the monomeric

protein

at the void

M maltose,

experiment,

for

study,

respectively

10-3

corresponds

of the dimeric

binding

containing

10s3 M galactose

columns

the monomeric

voque the monomerisation The galactose

which

reported

this

of higher

containing

binding

was eluted

of the same preparation

In this

volume

both

as described

gradients,

of the protein

column

weight

form of the maltose

the presence

protein.

the molecular

purified

fraction

1B shows chromatography

of the maltose was eluted

to a dimeric

gradient

and glucose, of

10m3 M glucose,

6.3

coefficient

pre-

or in the preS was

determined

of 3.7 S was

;

Vol. 105, No. 2. 1982

BIOCHEMICAL

AND BIOPHYSICAL

t

RESEARCH COMMUNICATIONS

10

WTTOM

20

FRACTION

T

TOP

NUMl3ER

figure 2 Sedimentation of galactose binding protein on glycerol radients containing : no galactose, no glucose (+), 10-S M glucose (o), lo- !I M galactose (X). 100 ~1 of GBP (120 1-19) was loaded on each gradient. GBP was preequilibrated with the ligand contained in each gradient. 50 ~1 of each fraction was counted for radioactivity.

calculated

which

galactose in

the

binding

These in

and

form

same

of

show

study,

cose,

DISCUSSION tose

binding

(for

MBP),

tion

of

with

these

pendence

artificial

conditions,

galactose

of on

the

of

state

of

be

galactose GBP dimer

(3),

monomer

aggregation

under and of

dimer the

479

protein

not

the

glu-

could

the

maltose

monomerisa-

experiments

of be

ammonium and described.

a de-

a prefe-

form only

made

show

reflects

identical, was

galac-

ligands,

elsewhere),

saturated was

of

binding

monomeric

dimers

described

but

and

This

the

as

provoque

published

for

galactose. columns.

specific

concentration.

precipitation for

The GBP),

of

form.

ligand

(to

protein

and

after

galactose

(for

of

galactose,

protein

study.

glucose

existence

prepared

native

binding

this

monomeric

chromatography

that

dimeric

; furthermore,

on

report

and

the

absence

protein,

maltose

proteins

maltose

previous

affinity

forms

affinity of

the

the

coefficients, with

the

P-100

form,

in not

binding

binding

the

but

native

multimeric

the

described

galactose,

affinity In

are

dimeric

of

rential

the

the

dimeric

of

in

binding

of

forms

compatible

Bio-Gel

for

sedimentation

protein

galactose

reported

the

is

binding

in

value

of

galactose,

monomerisation

protein

the

ratio

using

the

Dimeric

and

of

recovered

the

than

The

obtained

that

is

provoque

(4).

galactose

were

results

higher

absence the

results

this

slightly protein

presence

a dimeric The

is

MBP andGBP. seen

under

sulfate no effect

; of

Vol. 105, No. 2, 1982 The ability lower

of periplasmic

affinity

transport

of the

of the triggers

contact

binding

equilibrium

transport

of the galactose

tor

maltose)

galactose)

proteins,

ducts

(14);

tion

tetramers

aggregating that

of aggregation several

tar

systems

could

the

protein

mediated

12)

exists

might

be important

phenomena

for

in membrane

favor

(13)

if

lower

induced

transfer

for

glucose

(W. Boos, protein

for

two methyl

membrane,

the -tar

and trg

protein

(15)

the chemotactic receptors

function

accepting

binding

an aggregation

per-

(chemorecep-

with

promote

ligand

on the mono-

(chemoreceptor

and the maltose

of

of the

efficiency

binding

by its

of affinity

transport

the

membrane

and galactose

as tetrameric

gene product

properties

decrease

protein

in the cytoplasmic

tar

possibly

explain

(9,

gene product

protein,

should

the

In transport,

of glucose

binding

for

the cytoplasmic

the maltose

galactose

respectively

of the tetrameric

wed with

influence

In chemotaxis,

located the

ligand

binding

and the

interact

taxis

its

and the

with

the consequent

of GBP might

communication). for

for

The different

mer-dimer

binding

dimers,

be important

proteins.

complex

(lo),

RESEARCH COMMUNICATIONS

to form

should

of these

of the pore

proteins ligands

protein-ligand

protein

cytoplasm.

the

functions

a multimeric

the dimeric

sonal

for

a dimerisation

with

to the

binding

AND BIOPHYSICAL

binding

dimers

and chemotactic

contact (8,9)

BIOCHEMICAL

chemogene pro-

; interac-

protein of several

response.

endo-tar

The importance

has been reported

in

(16-18).

ACKNOWLEDGEMENTS

We thank

J.P.

Waller

and M. Arrio

for

helpful

discussions.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Boos, W. (1974) Ann. Rev. Biochem. 43, 123-146. Antonov & al. (1975) In advances in enzyme regulation 14, 269. Rashed, I., Shuman, H. & Boos, W. (1976) Eur. J. Biochem. 69, 545-550. Anraku, Y. (1968) J. Biol. Chem. 243, 3123-3127. Kellerman, 0. & Szmelcman, S. (1974) Eur. J. Biochem. 47, 139-149. Nossal, G.L. & Heppel, L.A. (1966) J. Biol. Chem. 241, 3055-3062. Richarme,G. & Kepes,A. (1974) Eur. J. Biochem. 45, 127-133. Richarme, G., Meury, J. & Bouvier, J. (1982) J. FernsMicrobial. Lett. 13, 35-37 Richarme, G. (Feb. 1982) 3. Bacterial. (in press). Singer, S.J. (1974) Ann. Rev. Biochem. 43, 805-830. Ferro-Luzzi Ames, G. & Nikaido, K. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 5447-5451. 12. Koiwai, 0. & Hayashi, H. (1979) J. Biochem. 86, 27-34. 480

Vol. 105, No. 2, 1982 13. 14. 15. 16. 17. 18.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Strange, P.G. & Koshland, D.E. Jr. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 762-766. Springer, M.S., Goy, M.F. & Adler, J. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3312-3316. Chelsky, D. & Dahlsquist, F.W. (1980) Biochemistry 19, 4633-4639. Segal, D., Taurog, J.D. & Metzger, H. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 2993-2997. Kahn, C.R., Baird, K.L. & Flier, J.S. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4209-4213. Schlessinger, J., Shechter, Y., Cuatrecasas, P., Willingham, M.C. & Pastan, I (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 5353-5357.

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