Purification of a leucine-specific binding protein from Escherichia coli

Vol. 38, No. ~$1970

PURIFICATION OF A LEUCINE-SpECIFIC BIWDING PROTEIN FROM ESCBEBICBIA COLI* Clement E. Furlong Section Cornell

and Joel H. Weiner+

of Biochemistry

and Moleculsx

University

Ithaca,

Biology

New York

14850

Received February 9, 1970 summmy: A new binding

protein for leucine was purified from E_. coli and crystallized. It appears to be similar in eize to a protein, presly isolated from E. coli, which binds leucine, isoleucine and valine. The new protein, however, binds only leucine. Furthermore, the amino acid analogue trifluoroleucine effectively inhibits the binding of leucine to the leucinespecific binding protein whereas , it is without effect on the protein that binds s.XL three amino acids. The KD for leucine binding is 0.7 @I. The nature of the inhibition of leucine transport by trifluoroleucine snd isoleucine suggests that there is more than one transport system for leucine. Two laboratories

have reported

which binds leucine, the LIV-binding protein

isoleucine

protein.

peak isolated

proteins.

and v&Line (1,2);

of a binding

we shall

Anraku (1) observed a shoulder by DEAE cellulose

We have now resolved

chromatography,

the crystallization

chromatography

two leucine-binding

the second of which is highly

refer

protein

to it

as

on the LIV-binding of shock fluid

proteins

specific

by DEAE cellulose

for leucine

(L-binding

protein). M&CERIALSANDMEl!HCDS Binding containing

Assays - The binding two wells

(0.2 ml each),

In the assay for column fractions (Assay 1).

Side A contained

pH 7.0, in a total

assays were carried

out in plexiglass

separated by a disc of dialysis a sub-saturating

protein,

level

0.05 M NaCl, O.OlM

volume of 0,lO or 0.05 ml.

*

of leucine

tubing. was used

phosphate buffer,

Side B contained

2 x lo-$

This investigation was supported in part by United States Public Health Service Grants ~~-33058 and AM-U789, and by Grant GB-7093x frm the National Science Foundation. +Predoctorsl

student. 1076

cells

BIOCHEMICAL

Vol. 38, No. 6, 1970

14C-leucine, that

0.05 M NaCl and 0.01 M phosphate buffer When a saturating

in side A.

studies

AND BIOF’HYSICAL RESEARCH COMMUNICATIONS

level

(Assay 2), side B contained

equilibration

at 2' on a rotating

to the one described

locating

peaks of binding

ml of 2 x 10m5M leucine

B-6 filter

activity

23'.

After

solution

(3).

A filter

In this

of colurmn fraction,

was filtered

on a 25 mm Scbleicher

(T ra?'isport filters

were treated

for 5 minutes

phase cells in minimal

level

of leucine

medium (5) supplemented

which contained

9 1 x 10B5M l4 C-labeled

volume of 0.5 ml.

To determine

were taken at 15 and 30 set, filtered 10 ml. of O.OlM

Tris-HCl,

(6) (temperature,

23').

amino acid and minimal the initial

with 10 mM

pH 7.3, containing For determination

7 (derived

cold deionized

medium to a sliquots

and washed with

0.15 M NaCl and 0.5 mM MgC12 of Km the nxrnber of cells was taken up at sll

from KlO, fkxn P.E.

The cells

were harvested

(1) except that the concentration

added to a final

filter,

used

concentra-

C. C. Lin) was grown to

phase in minimaJ mediun (5) supplemented

acid (Baker and Adsmson). described

40 ~.rgper ml

used.

Strain stationary

was then

rate of transport,

on a Millipore

was such that less than 1% of the substrate of substrate

at

twice washed with minhnal. medium

10 mM glucose,

mixture

were

similarly).

added to the reaction

early

&

of 15 g PEO and 0.2 g KZQP (Packard Inst.

A ssmple of cells

tions

assay,, 0.05 followed by

glucose and 40 H per ml of cblorsmphenicol.

final

in

and washed with 0.5 ml of 0.2 M MgC12. The filters

A suspension of mid-log

chlorsmphenicol

assay

(4) is also useful

Assays - These were done at a saturating

was incubated

overnight

were removed from each

in colwnn fractions.

was added to O.lml

of toluene.

Transport

counting

by Riggs and Bourgeois

dried and counted in a solution Co.) per liter

was used for quantitative

aliquots

apparatus,

0.05 ml of 1 M MgC12. The mixture Schuell

of leucine

2 x 10s5M leucine.

side and counted in a Triton-toluene sMlar

in a volume equal to

concentration

of 1mM within

water. 1077

tith

1% succinic

snd shocked as previously

of MTA was 2 mM and&r+ one minute

after

was

shocking with

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 38, No. 6, 1970

Radioactive

isotopes

obtained

from New England Nuclear

specific

activities

from 20 to 275 mc per m mole.

obtained

from Dr.J.

Cslvo,

The unlabeled

Corp. had

Trifluoroleucine

was

acids were Mann "Assayed"

mino

grade. Protein

was determined

(7), with bovine

by a modification

of the method of Lowry --et al.

selrmt aLbumin as a standard. RESULTS

Purification chromatograp& senting

of the leucine-specific - Concentrated

369 g wet cell

CO~UUI equilibrated protein

binding

shock fluids*

protein.

DEAE-ceUulose

from three preparations,

paste were pooled and passed through

with deionized

water.

The protein

repre-

a Biogel-P

peak (1,344 mg of

in 320 ml) was loaded on a 2.5 cm x 54 cm column of DE&-cellulose

(Brown and Co.) which had been prepared by the usual washin@; procedure followed

by equilibration

was eluted

ltith

HCl buffer,

pH 7.6.

bound isoleucine only leucine.

with 5 mM Tris-RCL

a 7 liter

the DEAE-cellulose

linear

column (Figure l), and valine,

Electrophoresis apparatus

peaks were found to elute Fractions

from the first

gel electrophoresis were pooled,

a 1:4 dilution

was carried

in that

The 3 cm high resolving

from

peak also

from the second peak bound from E. coli

- The fractions

concentrated

of stacking

out in a CansJ.co preparative

a buffer

fluids were concentrated with a DM-10 filter.

(see below). electrophoresis

A published

system developed by Jotin

gel contained

0.083 M Tris-HCl,

procedure

(10) was used.

pH 7.4; the 4 ml

in an Amicon model 401 ultrafiltration

1078

frcnn the

by ultrafiltration,

gel buffer

(in two runs) using column nxanber PD-2/320.

(9) was modified

The column

was also observed when extracts

[email protected]

against

pH 7.6.

in the ssme way.

peak which bound only leucine and dialyzed

buffer,

(8),

from 0 to .15 M N8l.X in 5 ml4 Tris-

while fractions

The same pattern

Prenarative

gradient

Two leucine-binding

XL2 were chromatographed

*hock fitted

10

cell

Vol. 38, No. t&l970

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

?I654I

3-

i

2-

Fraction

Fig. 1. DEAE&cellulose Closed circles, protein activity (Assay 1).

stacking buffer

gel contained contained

and elution

fractionation of the two leucine binding proteins. absorbance at 225 rnp; open circles, leucine binding

0.069 M Tris base-O.052 M H$04,

0.05 M Tris

buffers

base-O.06 M Tricine,

contained

0.06 M Tris-HCl,

59 fold over the crude desalted yield

number

shock fluid

pH 6.7; the upper

pH 7.9; pH 7.3.

and the lower A purification

was obtained

of

with an overall

of 58%. CrystsXSeation

zation

(Fig.

not alter

of the leucine-specific

2) by dialysis

the specific

gel electrophoresis

of materi&

It

is interesting

step.

and dine

a solution

activity

the s8me ss that reported isoleucine

against

for the crystalline

(1,2).

This material

judged by disc gel electrophoresis

binding

protein

of mmnium obtained that

-Crystallisulfate

did

from the preparative

this value is essentially

protein

that binds leucine,

appeared to be homogeneous as

at two pH values and at different

gel

concentrations. Estimation indicated

of size - The disc gel method of Hedrick

that the L-binding

protein

was identical 1079

and Smith (U)

in size to the LJZV-

BIOCHEMICAL

Vol. 38, No. 6,197O

Fig.2.

binding

Crystals

protein

of the leucine-specific

which has a reported

The Sephadex gel filtration molecular

priate

binding

molecular

The molecular

protein

protein

x 420.

weigbt of about 36,ooO

method of Andrews (13)

weight of the L-binding protein.

binding

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

sJ,so indicated

w&s identical

weight determined

(2,X?).

that

the

to that of the LIV-

by this

method with appro-

standards was 37,000. Specificity

of bincUng - Table 2 shows that

binding

protein

is not inhibited

binding

of leucine

leucine

binding

protein

trifluoroleucine

ia not inhibited

to the LIV-binding

port of isoleucine, binding

by isoleucine

leucine

and valine.

is, however, qtite @FL).

(2), TFL did not inhibit

of leucine

to inhibition of the results

of leucine

to the L-

Further,

aa well as to inhibit

sensitive

1080

binding

the

which we found to inhibit

The binding

In corroboration the binding

or valine.

by threonine protein,

leucine

the transto the L-

by the analogue

of Penrose -a et al. to the LTV-binding protein.

Vol. 38, No. t&1970

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table I Purification

of the leucine-specific

binding

protein

(L-BP).

Data for the LIV-binding protein are also included since this makes it possible to estimate specific activities of both proteins in the shock fluid. One unit equals 1 mwle of leucine bound under conditions of saturation (Methods).

Fraction Desalted

Volume ml

Shock Fluid

units per ml

Total units

Protein mg/ml

3.6

1.153

4.2

320

DEAFI LIV -BP DEAE L - BP Prep Gel L - BP

19.5

7*9

13.2 1.4

Specific activity o . & -6(Lm-BP) c .26(~-BP)

58

of Leucine transport and of Leucine binding protein and the L-binding protein.

Additions None Isoleucine

100 200

VaUne

100 200

Trifl~roleucine

100

200

Isoleucine + V&line Isoleucine + Trifluoroleucine

100 100 100 100

Valine + Trifluoroleucine The KD for leucine

a KD of 1 J for leucine

to the L-binding binding

other 16 amino acids tested binding

3g

l

binding

protein

to the LIV-binding

inhibited

the binding

is 0.7 @l compared to protein.

l?one of the

of leucine

to the L-

protein. system - The finding

Evidence formorethanonetrensport leucine-binding for leucine. inhibit

to

Relative Leucine Transport Leucine Binding (Relative rate) LIV-BP L-BP 100 . . . . . . . . . 100 . . . . . . . ..lc)o ClM 39 Nf . . . . . . . 29 . . . . . . . . . . 10 . . . . . . . . . gy m CrM. . . . . . . ti . . . . . . . . . . 13 . . . . . . . . . 91 @4 49 l-@+f. . . . . . . 39 . . . . . . . . . lof) . . . . . . . . . . 6 w . . . . . . . .15 IJMI & m I . . . . . . . 18

100 w 100 @l1 ******

protein

prcmrpted us to look for another transpoti

Figure 3 shows that

the initial

rate of leucine

"cold"

isoleucine

fransport 1081

of a second system

does not ccnnpletely

and further

100

95

2," I 15.6

Table II Inhibition the LTV-binding

Yield %

that the residual

BIOCHEMICAL

Vol. 38, No. 6, 1970

D--O--O--O

AND BlOPHYSlCAL RESEARCH COMMUNICATIONS

Trifluorolaucine, 0 20

pM (Isoleucine

40 8 "

1 "

0

50

100 Isoleucinc,

= IOOflM)

60 60 100 2 I 'I "1

150 ph4

l

200

-•-o-*

Inhibition of leucine transport by isoleucine and isoleucine plus Fig.3. TFL. Solid circles solid line, leucine transport at the indicated level of isoleucine; open circles (broken line) leucine transport at 100 ~ isoleucine plus the indicated level of trifluoroleucine. transport true,

i.e.,

sidual line

is very sensitive

to inhibition

TFL does not completely

transport

is very sensitive

Further,

transport

the cells

by isoleucine.

of more than one transport

A second system for

transport,

isoleucine,

0.2 and 2 j&

we found egain two

were still

able to transport

a considerable

amount of

I& These results

than one transport

are consistent

system for leucine.

of Penrose --et alu. (2) strongly implicate In addition, unpublished data frcm this inhibitors

and the re-

0.2 I.&%and 23 I.&!. Even In the presence of 200 p unlabeled

DiSCUSSi0Il

constants

leucine

in the presence of 20 @4 unlabeled

leucinetithaI&of2

tive

The reverse is also

is that two Km values were found for leucine

Km's for leucine, isoleucine

inhibit

to inhibition

of evidence for the existence

leucine

by TFL.

of the LIV-binding

and Ki for the inhibition

The results

laboratory

of transport. 1082

of more

of Auraku (1.,6) and

the LIV-binding

protein

have shown a good correlation

with the existence

in leucine

with a family

transport.

of competi-

verying widely in inhibition

between Ki for inhibition It

is taapting

of binding

to speculate

that

Vol. 38, No. 6, 1970

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the leucine-specific

binding

system for leucine.

Most of the inhibition

concept. inhibit

protein

However, it is difficult

may be involved

data are consistent

to explain

the sane component of leucine

in the second transport

why valine

transport

with this

and TFL appear to

(Table II).

This matter

is under investigation.

Acknowledgements: whose laboratory stimulating

The authors are most grateful. these investigations

discussions;

were carried

to Dr. L. A. Heppel in out;

for his many

and for his help in the preparation

of this

manuscript.

1. 2.

Anraku, Y., J. Biol. Chem., 243, 3116 (1968). Penrose, W. R., Nichoa;Lds, G. E., Piperno, J. R., and Oxender, D. L., J. Biol. Chem0, 243, 5921 (1968). Patterson, M. S., and Green, R. C., Anal.yt. Chem., a 854 (1965). Riggs, A. D., and Bourgeois, S., J. Mol. Biol., 3& 361 (1963). Tanaka, S., Lerner, S. A., and Lin, E. C. C., J. Bsct., B 642 (1967). Anraku, Y., J. Biol. Chem., 243, 3128 (1968). 7. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J., J. Biol. Chem., Q& 265 (1951). 8. Peterson, E. A., and Sober, H. A., in Methods in Enzymology V, 3, (1962), Acad. Press. Colowick, S. P. and Kaplan, N. O., Eds. 9. Furlong, C!. E., and Preiss, J., J. Biol. Chem., 244, 2539 (1969). 10. Wilson, D., personal. communication. XL. He&rick, J. L., and Smith, A. J., Arch. Biochem. Biophys., 126, 155 (1968). 12. Anraku, Y., 5. Biol. Chem., 2& 3123 (1968). 13. Andrew's, P., Biochem. J., & 222 (1964).

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