Immunochemical evidence that the FITC-labeling site on Na+,K+-ATPase is not the ATP binding site

Vol. 140,No. October

BIOCHEMICAL

1, 1987

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 246-253

14, 1987

Imnunochemical

Evidence

Na+,K+-ATPase

W.J.

Department

that

is

Ball

the

Not the

, Jr.

and M.L.

Cincinnati,

Site

Biophysics,

College

Ohio

on

Friedman

and Cell

of Cincinnati

Site

ATP Binding

of Pharmacology

University

FITC-labeling

of Medicine,

45267-0575

Received August 25, 1987 The covalent labeling of the a subunit of lamb fluorescein 5'-isothiocyanate at Lys-501 has generally at the ATP binding site. We have found that 496HLLVMKGAPER506 serves as the antigenic determinant M&Pl-A3: This antibody binds to both native and while this epitope undergoes ligand-induced changes involved in either enzyme function or the El * E2 monitored by FITC-fluorescence intensity. 0 1987 Academic

Na+,K+-ATPase

is

nit

(-100,DDO

and

the

maintenance

Mr) of

class

of ATP-driven

whose

functioning

ferent

conformations.

(FITC)

has been

has been fic

found

lysine

among

thought

and

to

monoclonal antibody

B subunit

(-50,000

Na+ levels

in

the

of the

The

cycling fluorescent

extensively

inactivate

to the

in a region

antibody

of the

pumps.

ATP blocks

Because FITC

of

the

M&Pl-A3

binds

function

and

enzyme

enzyme's

ATP to

0 I987

by Academic

of reproduction

in any

Press, form

Inc. reserved.

the

246

site

region

a subu-

function belongs

is to a

as ElE2 a series

pumps, of dif-

changes. by labeling is

a 1:l of

the

5'-isothiocyanate

which with

site

ligand-induced

between

activity

reacts

binding

(1)

ligand-induced

100 KD protein

this

enzyme

fluorescein

ATPase

labeling,

This

enzymes

these

FITC

primary

by Racker

probe study

Inc.

whose

cell.

involves

0006-291X/87$1.50 Copyright All rights

the

Press,

of two subunits,

Mr),

designated

be a portion

on

composed

transporters,

ElEp-ATPase

ATP binding

enzyme,

ion

used to

the

K+ and

residue

the

a membrane

kidney Na+,K+-ATPase by been assumed to occur the peptide sequence for monoclonal antibody FITC-labeled enzyme and these changes are not conformational changes

a speci-

highly

conserved

stoichiometry

labeling

is

(2).

We have

of a.

The effect

conformational

FITC

to

generally found

that

of

bound

changes

has

Vol. 148,No.

been

8lOCHEMlCALAND8lOPHYSlCALRESEARCH

1, 1987 The

investigated.

Na+,K+-ATPase

have also

EXPERIMENTAL

PROCEDURES

effects

of

these

ligands

COMMUNICATIONS

on

antibody

binding

to

been determined.

Enzyme preparation and assays. The Na+,K+-ATPase was prepared from frozen lamb kidneys as previously described by Lane et al (3). The Na+,K+-ATPase activity at 37'C was measured at 340 nm using the spectrophotometric, linked enzyme assay system of Schwartz et al (4) while the Kt-dependent p-nitrophenylphosphatase activity was determined by continuous measurement at 410 nm (5). [3H]-ouabain binding to the enzyme was determined at 37'C essentially as The labeling of Na+,K+-ATPase with described by Wallick and Schwartz (6). FITC (Molecular Probes, Inc.) has been described in our previous work (5), measurements were measured at 520 nm as while the fluorescence intensity described by Hegyvary and Jorgensen (7). Antibody M8-Pl-A3 preparation and binding determinations. This antibody was raised against the lamb kidney Na+,K+-ATPase and its isolation, purification and initial characterization was reported previously by Ball et al. (8). The indirect solid-surface adsorption (ELISA) assay used to quantitate antibody binding has been described in detail (8) but we have modified the proceIn this procedure antibody and enzyme were combined for l-2 h dure somewhat. and then the enzyme and any antibody-enzyme complex formed were removed from The unbound M8-Pl-A3 (in solution by centrifugation at 150,000 x g for 1 h. the supernatant) was then exposed to either untreated microtiter plates (Cooke flexible plates), plates previously coated with enzyme (+ 0.1% SDS), or synthetic peptide-BSA conjugates. M8-Pl-A3 binding to these plates was then quantitated using a B-galactosidase sheep anti-mouse IgG F(ab')2 conjugate as Solution phase binding of M8-Pl-A3 to the enzyme under second antibody. (Na +; Kt; Mg2+; Mg2+Pi and Mg2+ATP, all at 5 mM) was various ligand conditions also examined. RESULTS A collection Na+,K+-ATPase synthetic portions

of (9)

peptides

were

This

sequence

has

been

isolated,

(2,ll).

site

of

holoenzyme nM antibody, was about

Figure peptide

this the 2 6-BSA

respectively. 12-fold

lower

residues

a soluble

tryptic

and

to

shows

of

was achieved affinity

enzyme, 247

(Fig.

this

of the

the maximum

the

region

binding at

of

labeling of

levels

for

further

M8-Pl-A3

for of

a that

ATP binding

approximately antibody

peptide

site

was

of

of

sequence

1,

fragment

a portion

kidney

various

to the

(BSA)

covalent

to

lamb

a series

to bind

half-maximal

conjugate

for

be the

the

representing

digestion

M8-Pl-A3

that

the

length)

albumin

may comprise

binding

to recognize

was found

serum

region

to

in

M8-Pl-A3,

shown

raised

ability

bovine

While than

their

acid

to

sequenced

Na+,K+-ATPase,

and

amino

represents

antibodies

for

One antibody,

Because

characterized.

tested

conjugated

6).

FITC

monoclonal

(11-15

of a (10).

4g6HLLVMKGAPER506

five

to

7 and 85 the

bound

peptide antibody

BIOCHEMICAL

Vol. 148, No. 1, 1987

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

I.07

cE

.8 -

: E T B 0 2 0 Li 0

.6 -

.4 -

.2 -

rl 123456709 PEPTIDE

NUMBER

Determination of Monoclonal Antibody M8-Pl-A3 Binding to Synthetic ps. ntibody binding to peptide-BSA conjugates was determined using the solidsurface adsorption assay and a B-galactosidase sheep anti-mouse IgG F(ab')p conjugate as second antibody. Monoclonal antibody concentration was 1 uM. The sequence regions for each of the a peptides were: (1) 1-12; (2) 16-30; (3) 111-122; (4) 303-314; (5) 366-377; (6) 496-506; (7) 823-833; (8) 932-943; and (9) 1,003-1,013. achieved

were

that

antibody

body

binding

Competition

similar. binding to both

to

holoenzyme

plate-adsorbed

binding "in

studies solution"

enzyme

ANTl6ODY

[M8-P

(Fig. effectively

and peptide

1 -A3)

3) then

6-BSA

demonstrated

prevented conjugate.

antiThese

nk&

Determination of M8-Pl-A3 Binding to Antigen as a Function of iii&&Y Concentration. bpen circles (0) and closed circles (0) represent antibody binding to Na+,K+-ATPase and peptide 6-BSA conjugate, respectively. All values are given as the percentage of antibody binding relative to the maxlmum amount bound to Antibody binding to antlgen was detected using a each particular antigen. solid-surface adsorption assay. Antigen was adsorbed to the plates at 50 pg/well (Na+,K+-ATPase) and 5 uglwell (peptide 6-BSA conjugate) and bound antibody quantitated using B-galactosidase sheep anti-mouse IgG F(ab')2 conjugate. 248

Vol. 148, No. 1, 1987

8lOCHEMlCALAND8lOPHYSlCALRESEARCH

200

COMMUNICATIONS

400

[N~+.K+-ATPw~J

pglml

;ig. 3.1 Competition Binding Study. pen c rcles (0) and closed circles (9) show decrease in M8-Pl-A3 binding to plate adsorbed Na+,K+-ATPase and peptide 6-BSA conjugate, respectively, after exposure to varying concentrations of Na+,K+-ATPase. Antibody concentration was 10 nM. Plate adsorbed antigen concentrations were 50 pg/well, Na+,K+-ATPase and 5 ug/Well, peptide 6-BSA conjugate. Binding of antibody to Na+,K+-ATPase in solution phase occurred before exposing antibody to either solid-surface bound peptide 6-BSA conjugate or Na+,K+-ATPase.

results this

showed binding

gens.

Having

dies

a

high

(pNPPase) binding

data In

binding

addition,

its

effects studies

FITC-modified

of

> native

ligand

to both were

enzyme

(9)

antibody

M8-Pl-A3

various not

data

and

holoenzyme.

also

plate-adsorbed

native

enzyme

determined.

(>50:1)

was;

was

no

conditions

that anti-

and

peptide

In these

stu-

because

our

curves

plate-adsorbed Excess

showed

isolated

antibody

was

a >

found

to

p-nitrophenylphosphatase

effect

(Mg2+;

used

binding

Kt-dependent had

and

the

competition

affinity

or

Na+,K+-ATPase

of

function

to

Na+,K+-ATPase

activity. under

Mg'+Pi,

FITC,

on

on enzyme

titer

holoenzyme effect

binding

antibody

order

plate-adsorbed no

of

"native"

recognition

antibody

binding

relative

recognized

to antibody

effects

ratio or

the

antibody

established

antibody

"affinity"

have

the

corresponded

conjugate,

that

that

on

the

Mgz+ATP;

rate

of

ouabain

K+Na+Mgz+ATP;

and

shown). since

the

on FITC were enzyme

done,

antibody

was binding

fluorescence

were

as before,

exactly

as

which it

249

did

to

the

investigated. showed

that

to

unlabeled

same region First, M8-Pl-A3

of a as

competition bound

enzyme.

to the Next,

Vol. 148,No. fluorescence enzyme

spectroscopy

(in

bound

BIOCHEMICAL

1.1987

Tris-HCl

measurements

buffer)

Further,

probe. fluorescence

intensity

bound

antibody.

These

enzyme

moiety not

of

and

(since

epitope

and

on the

it

by

clear

Na+ were that

environment

to

addition

of

the

of

of the in

effected

by

was the

regulatory

the

decreases

not

M8-Pl-A3

the

to

intensity

dependent

perturbing

responses

binding

fluorescence

reversal

made

nor

antibody

and Mg*+* ouabain

their

results

its

it

for

is antibody

mational

changes.

antibody

and enzyme

ligands

ditions the

where

and

of

little

state

neither

fluorescent ligands

(data

that

centration

bound

the

different

binding

in

the

changes.

shows

of enzyme

presence than

Mg*+Pi

20X,

was

M8-Pl-A3

the

not

4 clearly in

free

conditions, Mg*+Pi

was required

that

binding

solution

the

we found

that

Nat

as

a reduction

data

from this the

the

absence

conforenzyme

by

binding

sen-

concentration

of

To achieve a

in

demonstrated

enzyme's

illustrates

increased.

if

the

displaced

had

contrast,

caused

to

required

in the

we found

These

antibody is

bound,

addition, Mg*+ATP

addition con-

In

sensitive

was

Figure

of

to

respectively.

enzyme

the

Using

antibody.

and

conforin which

was determined.

to antibody

Mg*+,

decrease ligand

the

prior

region

enzyme's

or after

was enzyme

bound

the

exposed

of experiments

before

antibody

of

be an in

by a series

or Mg*+ATP

level

to

involved

of M8-PI-A3

18 and to

not

1 h either

Mg*+Pi

on the

conformational It

Mg*+,

but

but

for

70-753

35,

is

of unbound

effect

binding

under

level

combined

M8-Pl-A3

sitivity.

combined

K+,

appeared

was tested

were

75% to

enzyme,

concept

the

from

mational these

that

no effect

Kt had

that

accessible)

were

Na+,

and ligands

binding

therefore,

approximately

addition

enzyme

M8-Pl-A3,

This

and then

essentially

body

Mg*+Pi

that

shown). The

of

K+,

function,

FITC

showed

had no effect

the

FITC

altering

AND BiOPHYSlCALRESEARCHCOMMUNlCATlONS

four-fold of added

50% antihigher

con-

ligands.

DISCUSSION These represents ligand-induced

data

demonstrate

the

epitope conformational

that for

the

monoclonal or

tertiary 250

sequence

region

antibody structural

496-506

M8-Pl-A3,

of

a,

undergoes

rearrangements

which some which

Vol. 148,No.

1. 1987

8lOCHEMlCALAND8IOPHYSlCALRESEARCHCOMMUNICATlONS

Effect of ligands on antibody binding to Na+,K+-ATPase. w e ecreasing curves show the ability of Na+,K+-ATPase under various ligand conditions to compete for MB-Pl-A3 binding to plate adsorbed (SDS-treated) enzyme. Open circles (0) show Na+,K+-ATPase concentrations in Tris-HCl buffer only; Open triangles (A) buffer plus Mg2+; Open squares (Cl) buffer plus Mgz+ATP; and Closed triangles (A) buffer plus Mg2+Pi. Antibody (1.5 pg/ml) and enzyme binding was done for 1 hr at 4OC before the centrifugation step was done.

alter

M8-Pl-A3

binding.

bound

antibody.

displace effect

on either

the

M8-Pl-A3

but not with enzymes

the

are

for

are

of

native

rat in

not.

are

or

the

this

that

(9).

The

these

rat

we

to

has

which

kidney

acid

their

two

antibody

Further,

amino

while

however,

changes

denatured

region,

bound

FITC.

with

Nevertheless,

sufficient,

ligand-induced

enzyme-linked

enzyme

not

we find

cross-reacts

identical

figurations

function,

intensity

that

changes

In addition,

enzyme

fluorescence

interest

These

no

alter

find

it

of

Na+,K+-ATPase

sequences of these

apparent

enzymes

have

is

involved

tertiary

similar

con-

affinities

ATP (12). We therefore

binding the

or

hydrolysis.

Lys-501

(and

fluorescein

appears

to the

group. fluorescein

must site)

group result

of from

ATP-binding This

this

region

given

the

in

the

proximity

order

for

it

to serve

FITC.

The

region

rather also

is

fixing

is

than

consistent

a competitive

inhibitor 251

the

in

either

adenine-binding

site

reactive

site

of

ATPase

activity

the

in

the

a position modification

with

the with

ATP

ATP and FITC,

as the

FITC from

between

of

inactivation

irreversibly

not

interactions

reside in

hypothesis

derivative,

that

However,

residue

thiocyanate

block

conclude

fact respect

for

then

to

sterically

of

the

that

the

lysine

eosin,

to ATP,

and it

a

Vol. 148,No. inhibits

ATPase

seems

likely

emission This

that

to

-SH groups

(14).

of

effected

by the in

important

regions

shift the

using

is

is

the but

not

Lys-719 actual

sequence

at

These

difference

5'iodoacetamido-

with

intensity

higher

several

to

con-

different

changes

results

not.

and the

a 20-fold

reacts

does

this

the

least

techniques

regions

two ATP analogues distant

a

(18).

portions

of

require

from

It

eosin's

repositioned

results to

yet

FITC

by ATP and enzyme

(15,16).

involved

have been

from

the

ATP

shown

analogous

activity

identify

to

is

emphasize

not

the

dif-

functionally

The

ATP binding

be any

site

they

but

alkylating

(FSBA)

may not

have

at amino

(ClRATP)

adenosine regions

binding

to react

benzylamide-ATP

These the

in

Lys-501.

5'-(p-fluorosulfonyl)benzoyl of

is

(13).

of a,

while

FITC

clearly

blocked

enzyme

same region

contrast

requires

modification

the

binding,

fluorescence

y[4-(N-2-chlorethyl-N-methylamino)] and

in

COMMUNiCATlONS

of proteins.

considerably

(17)

upon

and

labeling

chemical

the

effects

which

shows

labeling

at

Lys residue

This

it

modifying

functional

Na+,K+-ATPase

covalent

Currently established

a red

derivative

its

ficulties

bind

labeling

Though

FITC,

chemically

locations.

label

AND BIOPHYSICALRESEARCH

probes

two probes'

(5'-IAF)

centration

dues

upon

specific

fluorescein

two

undergoes

in the

their

without

these

spectra

suggests

those

activity

that

differences in

BIOCHEMICAL

1, 1987

are

acid

ATP reacts

at

more

not

analogue

Cys-656 to

additional

resi-

at Asp-710

the

likely

been

and

represent

regions

that

investigation.

ACKNOWLEDGEMENTS We thank

Purabi

assistance.

This

ROl-HL-32214

(WJB).

Dey

work

was

WJB is

and

Chuck

Loftice

supported

by

an Established

for NIH

grants

Investigator

their

excellent T32-HL-07382 of

the

technical (MLF)

American

Heart

Association.

REFERENCES 1.

Racker, E. (1985) Reconstitutions Pathological St!

of Transporters

252

and Receptors,

and

and

Vol. 148, No. 1, 1987

2. 3. 4. 5. 6. 7. 8. 9.

10. 11. 12. 13. 14. 15. 16. 17.

18.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Farley, R.A., Tran, C.M., Carilli, C.T., Hawke, D, and Shively, J.E. (1984) J. Biol. Chem. 259, 9532-9535. Lane, L.K., Potter, J.D, and Collins, J.H. (1979) Prep. Biochem. 9, 157-170. Schwartz, A., Allen, J.C. and Harigaya, S. (1969) J. Pharmacol. Exp. Ther. 168, 31-41. Ball, W.J. (1986) Biochemistry 25, 7155-7162. Wallick, E.T. and Schwartz, A. (1974) J. Biol. Chem. 249, 5141-5147. Hegyvary, C. and Jorgensen, P.L. (1981) J. Biol. Chem. 256, 62966303. Ball, W.J., Schwartz, A. and Lessard, J.L. (1982) Biochim. Biophys. Acta 719, 413-423. Bal1,W.J. and Lane, L.K. (1986) Biochim. Biophys. Acta 873, 79-87. Ball, W.J. and Loftice, C.D. (1987) In press. Kirley, T.L., Wang, T., Wallick, E.T. and Lane, L.K. (1985) Biochem. Biophys. Res. Comnun. 130, 732-738. Periyasamy, S.M., Huang, W.H. and Askari, A. (1983) Comp. Biochem. Physiol. 76B(3), 449-454. Skou, J.C. and Esmann, M. (1981) Biochim. Biophys. Acta 647, 232-240. Steinberg, M. and Kirley, T. Unpublished results. Kapakos, J.G. and Steinberg, M. (1986) J. Biol. Chem. 261, 2084-2089. Kapakos, J.G. and Steinberg, M. (1986) J. Biol. Chem. 261, 2090-2095. Ovchinnikov, Yu.A., Dzhandzugazyan, K.N., Lutsenko, S.V., Mustayev, A.A. and Modyanov, N.N. (1987) FEBS Letters 217, 111-116. Ohta, T., Nagano, K. and Yoshida, M. (1986) Proc. Nat'1 Acad. Sci. USA 83, 2071-2075.

253