The amino acid sequence of the fluorescein isothiocyanate reactive site of lamb and rat kidney Na+- and K+-dependent ATPase

Vol. 125, No. 2, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 767-773

December 14, 1984

THE AMINO ACID SEQUENCE OF THE FLUORESCEINISOTHIOCYANATEREACTIVE SITE OF LAMBAND RAT KIDNEY Na+- ANDK+-DEPENDENT ATPase Terence L. Kirley,

Earl T. Wallick,

and Lois K. Lane

Department of Pharmacology and Cell Biophysics University of Cincinnati College of Medicine Cincinnati, Ohio 45267-0575 Received October 31, 1984 Fluorescein 5'-isothiocyanate has been used to label ouabain sensitive and insensitive (Na,K)-ATPases from lamb and rat kidney, respectively. The labeled enzymes were digested with trypsin to generate soluble peptides, which were purified by high performance liquid chromatography and sequenced on a gas phase sequenator. The sequence of the labeled peptide from both species is His-Leu-Leu-Val-Met-Lys-Gly-Ala-Pro-Glu-Arg. Thus, it appears that the primary structure of the fluorescein 5'-isothiocyanate reactive site, and therefore presumably the ATP binding site, is completely conserved in ouabain sensitive and ouabain insensitive (Na,K)-ATPases. 0 1984 Academic Press, Inc. As reported

previously

cyanate (FITC) specifically tivates

the enzyme.

presence of ATP, it

labels

is assumedthat

to detect conformational et al.

chymotryptic In

the

FITC-labeled, small, purified

digestion present

(l),

fluorescein

5'-isothio-

and inactivation

are inhibited

in the

FITC reacts at or near the ATP binding

(2) and Hegyvary and Jorgensen (3) have used FITC

changes induced in (Na,K)-ATPase by various ligands.

(4) have localized

the carboxy-terminal

et al.

the a subunit of (Na,K)-ATPase and inac-

Since both labeling

site of the enzyme. Karlish

Carilli

by Karlish

the site of FITC labeling near the center of

77,000 dalton membrane-boundpeptide obtained by limited of (Na,K)-ATPase. study,

we utilized

extensive

tryptic

digestion

of

membrane-boundrat and lamb kidney (Na,K)-ATPases to release a

soluble FITC-labeled

peptide from the membrane, which was subsequently

by reversed phase high performance liquid

chromatography (HPLC) and

(Na,K)-ATPase, sodium and potassium-activated The abbreviations used are: 5'-isothiocyanate; aclenosine triphosphatase (EC 3.6.1.3); FITC, fluorescein HPLC, high performance liquid chromatography; TPCK-trypsin, toluene sulfonyl phenylalaninechloromethyl ketone-treated trypsin; TFA, trifluoroacetic acid; FTC, fluorescein thiocarbamyl; SDS-PAGE, PMSF, phenylmethylsulfonyl fluoride; sodium dodecyl sulfate polyacrylamide gel electrophoresis. 0006-291X/84 $1.50 767

Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 125, No. 2, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

sequenced in a gas phase sequenator. presented at the 4th International

A preliminary

report

of this work was

Conference on (Na,K)-ATPase, August 5-10,

1984, in Cambridge, England. EXPERIMENTAL PROCEDURES Materials. The (Na,K)-ATPase was prepared from kidneys as described preThe activity of the lamb enzyme preparations varied from viously (5). 800-1100 umole/mg/hr and the activity of the rat enzyme was approximately 500 nmole/mg/hr as measured by the linked enzyme spectrophotometric assay (6). All HPLCgrade solvents used were obtained from Fisher Scientific, fluorescein Probes, Inc. and sequenal grade 5'-isothiocyanate was from Molecular trifluoroacetic acid (TFA) was from Pierce Chemical Company. Ampholines were purchased from LKB and all other electrophoresis grade chemicals were purchased from Bio-Rad. The HPLCcolumn used for reversed phase separation of peptides was a Vydac 15 cm Cl8 300 A pore size protein and peptide column (cat. 8218TP5415) from the Separations Group. TPCK-trypsin was purchased from Worthington (Millipore). Methods. The labeling of (Na,K)-ATPase with FITC was carried out at room temperature (22°C) for 30 min with stirring under the following conditions: 1 mg/ml (Na,K)-ATPase, 5 pM FITC, 2 + EDTA, 100 mM_ Tris-Cl, pH = 9.2. The reaction was stopped by addition of 8mercaptoethanol to 15 ~2, centrifuged at 150,000 xg for 30 min, washed with 1 @ EDTA (pH = 7.3), and recentrifuged. The pellet was resuspended in 1 roM_ EDTA (pH = 7.3), and the labeled enzyme was stored at 4'C, protected from light, until used. The FITC-labeled (Na,K)-ATPase was digested with TPCK-trypsin under the following conditions: 2 mg/ml (Na,K)-ATPase, 1 mMEDTA, 25 UM imidazole-Cl, PH = 7.2, for 1.5 hr at 37°C. Lamb enzyme was digested with OTO2mg/ml trypsin, while rat enzyme was digested with 0.1 mg/ml trypsin. The reaction was stopped by adding phenylmethanesulfonyl fluoride (PMSF) to 0.5 u&Jand centrifuging at 150,000 xg for 30 min. The supernatant was collected by pipet and lyophilized, and the amounts of protein and FITC in the pellets were determined. FITC was quantitated by dissolving SDS and 0.4 M NaOH, heating for 10 min 650 MI to 350 nm. The concentration of calculated assuming E~SS = 75,000 M-lcrn-l

the labeled protein (1 mg/ml) in 2% at lOO'C, cooling, and scanning from FITC in standards run in parallel was (4) in 50 mMTris-Cl, pH = 7.4.

The LDC-Milton Roy HPLC system utilized in this study consisted of two Constametric pumps, a Rheodyne injector, a Spectromonitor III variable wavelength W monitor, a Fluoromonitor III fluorescence flow monitor with a 440 nm filter, and a 500-700 nm emission filter, a 2 pen law, a 440 nm excitation thermal printer-plotter and a dual disk drive, all controlled by a LDC chromatography control module. Since the lyophilized tryptic supernatant was not very soluble in 0.1% TFA, it was dissolved in HPLCgrade H20 and 0.1% TFA in H20 was then added to bring the final concentration of TFA to 0.067% in the injected sample. The solvent gradient was formed by mixing 0.1% TFA in H20 (the "A" solvent) and 0.1% TFA in acetonitrile (the "B" solvent) at a flow rate of 0.7 ml/min through the 300 ?! pore size Cl8 Vydac column. Isoelectric focusing wbs carried out on both disc and slab gels containing 6% acrylamide and 0.16% bisacrylamide, as described by O'Farrell (7). Fluorescent bands on the isoelectric focusing and electrophoresis gels were visualized using a Spectroline Model TS-365 transilluminator, and photographed through a Promaster Spectrum 7 red filter. Protein was determined by the method of Lowry et al. (8), with bovi ne serum albumin as standard. 768

Vol. 125, No. 2, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The purified peptides were sequenced on a gas phase sequenator (Applied Biosystems Protein Sequencer 470A) in the laboratory of Dr. Thomas Vanaman at the Biochemistry Department in the University of Kentucky's Medical Center. The peptides were coupled in a single step (initial coupling yield of 63 and 33% for the lamb and rat (Na,K)-ATPase derived peptides, respectively) and analyzed for 15 cycles (65 min each). Repetitive yields of 97-99% were obtained.

RESULTS AND DISCUSSION Following activity

incubation

drops

to

titative

labeling

2.7

nmole

FITC/mg

1.1

nmole

fluorescence

xg

of

with total

but

the

due to slight the

time

supernatant. lamb

1.

variations

there

80-85%

resides

in

peak

peptide

from

concentration

these

the

single

both or

enzymes evaporation

gradients. repurifications

The

total

peptides

the

of the FITC, eluting

was via

at

then

a 1.0

ml

and sample

lamb

peptide, 769

are

FITC

and rat

varied

around

of

(Na,K)-ATPase. on the

50X,

amount

into

released are

contain

the from

shown

small

in

amounts

by 440 nm absorbance, The

major

rechromatographed loop)

along

by

of lamb

column

29% acetonitrile.

The 220 nm absorbances, of the

which

the

origi-

fraction

supernatant

as determined

collected

into

amount

effect

300 8, Vydac present

of the FITC

the FITC released

tryptic

on the

kidney.

calculated

for

had little of

rat

released

preparations

1 hour

of the

other

approximately

different

and

all

from

released

no case was all

(Na,K)-ATPase are

pellet

protein

beyond

and in

of FITC,

for

in

of digestion

kidney

Although

total

lamb kidney

were

(Na,K)-ATPase.

of

The chromatograms

40 mg of

FITC

digestion

in the

was

shown).

and

trypsin

that

quan-

labeling

for

was 83% and 61% respectively

amount

solubilized,

not

protein

FITC-labeled

supernatant

for

a. subunit

(data

(Na,K)-ATPase

nearly of

ca subunit

FITC/mol

the OL subunit

remaining

the

indicating

SDS-PACE reveals

amount

(Na,K)-ATPase,

constrained

(1,4),

the measured

the

of material

results

mol

by

into

Increasing

0.15

(Na,K)-ATPase

stoichiometry

FITC/mol

fraction

in

possibly

or

mol

inhibitable

original,

The

supernatant

present

released

Fig.

protein

ouabain

the

enzyme.

or 0.37

previous

amounts

subtracting nally

protein

the

10% of

active

comigrates

The 150,000

of

with

FITC,

approximately

FITC/mg

Consistent

with

(without

on successively with

shown

labeled

the

gradients

in Fig.

2.

more used Approxi-

Vol. 125, No. 2, 1984

0

8lOCHEMlCAL

10

01

AND 8lOPHYSlCAL

RESEARCH COWvtUNICATIONS

20

Time

02

bnh)

Time

(mid

HPLC chromatograms of the supernatant resulting from 1.5 hrs of tryptic digestion of native lamb kidney (Na,K)-ATPase labeled with FITC. Panels A through D represent the absorbance at 220, 280, and 440 nm and the fluorescence intensity (emission 500-700 nm) of the eluant, respectively. The The arrows indicate the extrema of the linear 20-35X acetonitrile gradient. amount of total protein injected was 0.2 mg in Panels A, B and D, and 20 mg in Panel C. Figure

1.

Figure 2. HPLC chromatograms (220 nm) of the repurification labeled tryptic peptide from Figure 1 (Panel C). The arrows extrema of the linear acetonitrile gradients. Panel A - 28-31X Panel 8 - 28-292 acetonitrile; Panel C - 28.2-28.6X acetonitrile.

mately

18 nmoles

labeled

lamb

was

then

of

and rat

purified (Na,K)-ATPase,

characterized

sequencing.

by

As shown

in Fig.

fraction

revealed

other

purified

peptide

contains

Identical

results

peptides

were

were also

peptide

determined

obtained

respectively.

isoelectric

bands

only

a for to

from This

focusing,

3, isoelectric

fluorescent

obtained

were

beside

single

770

major

70 mg of peptide

analysis,

one,

band

enzyme. (Table

acid

of the total

fluorescent

lamb and rat be identical

the

45 and

HPLC purified

amino

focusing

of the main indicate the acetonitrile;

supernatant but

of

the

p1

The sequences I),

and

and are

=

HPLC 5.32.

of the

completely

Vol. 125, No. 2, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

6.0 -

PH SO-

4.0Figure 3. Slab isoelectric focusing gel showing the fluorescence seen by W illumination (see Experimental Procedures) of the gel of a total tryptic supernatant fraction derived from 4 mg of labeled enzyme (lane A) and the HPLC purified PITC-labeled peptide (1.5 nmoles - lane B) from lamb kidney (Na,K)-ATPase.

homologous There

is

sequences

to also of

dog

(Na,K)-ATPase

some homology the

labeled

Sequences

as with

peptides

of FITC Peptides

reported

recently

the

Ca2+-ATPase

isolated

in

Table Derived

this

1 by Tryptic

by Farley FITC work

peptide

et

al.

(10).

and by Farley

(9). The et al.

Cleavage of ATPase

Source

Sequence

Reference

Lamb Kidney (Na,K)-ATPase

His-Leu-Leu-Val-Met-Lys-Gly-Ala-Pro-Glu-Arg I FTC

This work

Rat Kidney (Na,K)-ATPase

His-Leu-Leu-Val-Met-Lye-Gly-Ala-Pro-Glu-Arg I FTC

This &rk

Dog Kidney (Na,K)-ATPase

His-Leu-Leu-Val-Met-Lys-Gly-Ala-Pro-Glu-Arg I FTC

Farley et al.

Rabbit Skeletal Ca-ATPase

Met-Phe-Val-Lys-Gly-Ala-Pro-Glu-Gly-Val-Ile-Asp-Arg I FTC

771

(9)

Mitchinson et al. (10)

Vol. 125, No. 2, 1984 (9)

are

in our

identical

the

rat

in

the

primary

work the

putative

one

ouabain

that

the

al.

with

that

site

(rat)

the

of

isolated

lOOO-fold

and sequenced

(see

is

is

(Na,K)-ATPases

for

are

identical

and

indicate

and

sensitive

affinities nearly

results

This

and

and dog)

conserved

site.

work difference

(lamb

ouabain

affinity

this

structure

These

highly

between

the

from

sensitive I).

in

due to a difference

apparent

in the ATP binding

that

is

no primary

Table

(Na,K)-ATPase

occur

observation

It is

difference

ouabain

two ouabain

differences

do not

insensitive

first

for

there

species of

structure

the

and ouabain

(9)

site

species

that

(Na,K)-ATPases

ATP binding

primary

peptides

of the a subunit.

insensitive

insensitive sistent

kidney

et

ATP binding

possible

(12-14)

structure Farley

tryptic

as T14 (11).

suggested

and lamb

of

the

identified

has been

of

in

to one of

laboratory, It

the

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

that

any

and ouabain result

is

ATP of ouabain

con-

sensitive

(14).

ACKNOWLEDGMENTS This

work

PO1 HL-22619 HL-07382

was (Mission

Department

like

in

promptness to

I,

by United Core

2),

States

Public

ROl HL-25545,

Health and

Service

Training

Grants

Grant

T32

(TLK).

We thank

and

supported

express

encouragement

Dr.

Thomas

the

University

of

Kentucky's

in

sequencing

the

peptides

isolated

to

Arnold

our

Vanaman

appreciation

and

Mr.

Dr.

Steven Medical

Gathy

at

Center in

this

Schwartz

the for

Biochemistry their

work. for

insight We should

his

ongoing

and interest.

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

Karlish, S.J.D., Beauge, L.A., and Glynn, I.M. (1979) Nature 282, 333-335. Karlish, S.J.D. (1980) J. Bioenerg. Biomembr. 12, 111-136. Hegyvary, C. and Jorgensen, P.L. (1981) J. Biol. Chem. 256, 6296-6303. Carilli, C.T., Farley, 'R.A., Perlman, D.M., and Cantley, L.C. (1982) J. Biol. Chem. 257, 5601-5606. 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. O'Parrell, P.H. (1975) J. Biol. Chem. 250, 4007-4021. Lowry, D.H., Rosebrough, N.J., Farr, A.L., and Randall, R.G. (1951) J. Biol. Chem. 193, 265-275. 772

Vol. 125, No. 2, 1984

9. 10. 11. 12. 13. 14.

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. Mitchinson, C., Wilderspin, A.F., Trinnaman, B.J., and Green, N.M. (1982) FEBS Lett. 146, 87-92. Collins, J.H., Zot, A.S., Ball, W.J., Lane, L.K., and Schwartz, A. (1983) Biochim. Biophys. Acta 742, 358-365. Periyasamy, S.M., Lane, L.K., and Askari, A. (1979) Biochem. Biophys. Res. Commun.86, 742-747. Sweadner, K.J. (1979) J. Biol. Chem. 254, 6060-6067. Periyasamy, S.M., Huang, W.-H., and Askari, A. (1983) Comp. Biochem. Physiol. 76~, 449-454.

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