Oligomeric Structure of Solubilized Na+/K+-ATPase Linked to ElmZ Conformation

Y. HAYASHI?' K. KAMEYAMA; T. KOBAYASHIf E. HAGIWARA," N. SHINJI," AND T. TAKAGIb

aFirst Department of Biochemistry Kyorin University School of Medicine

Mitaka, Tokyo 181, Japan bInstitute for Protein Research

Osaku University Suita, Osaka 565, Jupun

The quaternary structure of Na+/K+-ATPase has been studied by solubilizing the membrane-bound enzyme, purified from mammalian kidneys, with nonionic surfac- tants such as C12E8 and Lubrol.'-6 These studies have shown consistently that the two polypeptides, 01 and p, are noncovalently combined in a minimum structural unit, an ap-protomer. The enzyme solution obtained thus, however, contained many other oligomers, such as (01p)2. (LI~)~, and (01p)~, as shown by chemical cross-linking of the solubilized e n ~ y m e . ~ Conflicting conclusions about the structure necessary for Na+/ K+-ATPase enzymatic activity have been obtained using the sedimentation equilib- rium technique, so that whether the ap-protomer (P) or the (ap)2-diprotomer (D) protein unit is the minimum active unit remains to be e~tablished.'-~ We have devised a low-angle laser light scattering photometry coupled with a high-performance gel chromatography (HPGCLALLS) method to study the minimum functional unit of the quaternary s t r~c ture .~ This method has shown so far that P and D are major protein components of the solubilized enzyme and that they are in an equilibrium of association-dissociation (2P * D) at a moderately high temperature.' This suggests that the two oligomeric forms of D and P occur simultaneously during exhibition of ATPase activity, regardless of whether P or D is the minimum active unit. Therefore, to clarify the relation between the structure and function of Na+/K+-ATPase, it is necessary to determine if a specific oligomer of P and/or D works without converting its structure to any other oligomeric form or if such a conversion is essential for its function.

In this paper, by measuring the dependence of M, of the solubilized enzyme on the protein concentration using a conventional, but precise, light-scattering photometer at 20°C, it was established further that the solubilized enzyme is in equilibrium with association constants (K,) which are 100-fold different from the El and E2 conforma- tional states. We also show that P is converted into D on producing phosphoenzyme intermediates at the E2 state (E2-P) by the addition of ATP,9 and that the El and E? conformational states therefore correspond to the protomeric and diprotomeric struc-

'To whom correspondence should be addressed. Tel: +81-422-76-7651; fax: +81-422-76- 7650.

19

20 ANNALS NEW YORK ACADEMY OF SCIENCES

tures, respectively, by simultaneous measurement of fluorescence intensity (conforma- tional state) and M, (oligomeric structure) of solubilized fluorescein 5'-isothiocyanate (F1TC)-labeled enzyme.

STATIC OLIGOMERIC STRUCTURE OF SOLUBILIZED Na+/K+-ATPase

The membrane-bound enzyme was purified from the outer medulla of frozen dog kidney to a specific activity of 40-48 pnol Pi/min/mg protein at 37°C by the zonal rotor method of J@rgensen.8,10 The membrane-bound enzyme was solubilized using octaethyleneglycol dodecyl ether (CI~EB) in a 1:3 weight ratio of surfactant to protein in the presence of either 0.1 M KCl or NaCl at O°C.x The M, and the oligomeric structure of the solubilized enzyme were determined using the HPGCLALLS m e t h ~ d . ~ , ~ The HPGCLALLS system is composed of a main column of TSKgel G3000SWxL and the following three kinds of detectors which are connected in series to the column: a LALLS photometer (TSK model LS-8000), a differential refractom- eter (RI), and an ultraviolet spectrophotometer (W). The column and a flow cell of the LALLS photometer were always cooled to around 0°C by a circulating medium. This cooling was essential for the success of this study. The column was equilibrated using an elution buffer of 0.2 mg . ml-' C& containing 0.05 M NaCUO.05 M KC1/4 mM MgC1211 mM EDTNlO mM imidazolefl3 mM Hepes, at 0°C and at pH 7.0. The solubilized enzyme was charged onto the column and eluted with the same elution buffer as just described. The solubilized enzyme was separated into a minor compo- nent (H) and two major components of D and P. The M, values of the protein moiety itself of the two protein components were estimated to be 302,000 ? 10,000 and 156,000 2 4,000, respectively, by the HPGCLALLS The two major components isolated were composed of the a and p subunits in a molar ratio of 1: 1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).7*x We have also shown that the M, values of the cu and p subunits are 118,000 ? 3,000 and 39,400 ? 900, respectively, by the HPGCLALLS method in the presence of SDS." Therefore, the major protein components of D and P were identified unambiguously as (ap)2-diprotomer and a@-protomer, respectively. The minor component of H was thought to be an associate andor aggregate because its M, values were much higher than those of the major components and it had a nonstoichiometric molar ratio of a:p. The elution pattern with the three peaks of H, D, and P were obtained when chromatography was performed at around O'C, although the relative contents of D to P were altered depending on the kind of ligand included in the elution buffer at neutral pH and on the pH value of the elution buffer.I2 The D and P that emerged from the column contained about 15 mol of phospholipid in common, and 150 and 200 mol of C I ~ E ~ formed part of D and P, respectively, per ap-protomeric nit.^^^ The content of phospholipid decreased with increasing temperature of the chromatography column, but it did not alter when the ligands in the elution buffer were changed between Na+ and K+.8

DISSOCIATION-ASSOCIATION EQUILIBRIUM OF 2P + D

Computer Simulation of Chromatography Behavior

The two protein peaks corresponding to D and P, separated by HPGC at O'C, merged into a single major protein peak when the temperature of the chromatography

HAYASHI et al.: Na+/K+-ATPase AND El& CONFORMATION 21

was increased to 20°C, regardless of whether K + or Na+ was included in the elution buffer.* The M , of the resultant main component, revealed when the elution buffer contained either 0. I M KCI or NaCl at 20"C, was estimated to be 300,000 or 255,000, respectively, by the HPGCLALLS method. Under different conditions, when the elution buffer contained ligands other than Nat and K+, the single main peak could be reproduced by performing chromatography at 20"C, and the M , values were estimated to be 247.000-258,000 or 298,000-295,000, with the elution buffer containing the ligands favorable for the El or E2 conformation, respectively (TABLE 1) .8 ,13 The M , of around 297,000 coincided with that of D. That of around 253,000 coincided with neither the M , of D nor that of P, but it was intermediate between them. SDS-PAGE still showed that the molar ratio of a:P was approximately 1 : 1 for the merged protein component, indicating that this component was composed of an ap-protomeric unit.

The behavior of the solubilized enzyme thus observed was analyzed using a computer simulation technique developed for a reversibly associating protein by Stevens and Schiffer.I4 As a result, the elution pattern that produced the merged protein peak could be simulated by assuming that D and P were in an equilibrium of dissociation-association with a very fast rate compared with the elution time. By assuming the association constant ( K J of the equilibrium to be either 2 . lo6 M-' or more than I . lo8 M - ' , the best fit could be obtained for the elution pattern observed under conditions favorable for the El or E2, respectively, conformational states of the solubilized enzyme at 20°C.8,13 The ratio of the weight concentration of D:P was revealed at the top of the peak by computer simulation, and it allowed us to calculate a weight-averaged M, (a,) at the same top of the peak as that adopted for the experimental estimation of Mr by the HPGC/LALLS method.* As shown in TABLE I , the calculated 2, value (248,000) was in agreement with the values obtained experimentally. Thus, the different M, of the solubilized enzyme obtained under the conditions favorable for either the E l or E2 state could be attributed to the difference in the K, of the equilibrium of 2P+ D.

Confirmation of Equilibrium by Direct Measurement of the Dependence of M, on Protein Concentration

The solubilized enzyme was run, at 20"C, through the same column as that just described to exclude aggregates larger than the diprotomer, and the protein component that eluted as a single peak exclusively consisting of D and P was isolated as the purified solubilized enzyme without concentrating. After being diluted to various protein concentrations using the effluent eluted through the column before its void volume, the solubilized enzyme thus purified was applied to a conventional, but precise, light-scattering photometer (Ohtsuka Electronics Co., DLS-700) equipped with a cylindrical batch-cell 12 m in diameter and an Ar laser-light source (15 mW output) to estimate a M , value for the enzyme. Measurements weretaken atscattering angles between 30" and 150" and extrapolated to 0" to estimate M,. The M,s of the solubilized enzyme obtained by chromatography using the two kinds of elution buffer, containing either 0. I M NaCl or KCl, are shown in FIGURE 1. By simply assuming the equilibrium of 2P * D for the purified solubilized enzyme, the dependence of M , on protein concentration at values higher than 0.15 mg/ml could be well fitted by association constants of about 2.5 . lo6 and 3.2 . lo8 M-l at concentrations of 0.1 M NaCl and KCI, respectively. The Kas thus obtained were fairly consistent with the values obtained from computer simulation. The discrepancy between the experimental

N

N

TABLE

1. M

,s an

d A

ssoc

iati

on C

onst

ants

(Ka)

of So

lubi

lize

d N

a+/K

+-A

TP

ase i

n th

e Tw

o C

onfo

rmat

iona

l St

ates

of E

l and

E2

Rev

eale

d by

the

HPG

CL

AL

LS

Met

hod

and

the

Con

vent

iona

l Lig

ht-S

catt

erin

g M

etho

d U

sing

a B

atch

-Cel

l at

2O

OC"

HPGGiLALLS M

etho

ds

Con

vent

iona

l Lig

ht-S

catte

ring

Met

hodb

Lig

ands

in

Con

form

atio

nal S

tate

Mr

M

r Ka

M

r Ka

El

utio

n B

uffe

r (e

xpec

ted)

(e

xper

imen

t)

(sim

ulat

ion)

(s

imul

atio

n)

(exp

erim

ent)

(s

imul

atio

n)

0.1

M N

aCl

255,

000

263,

00@

290,

000

2.5

X 1

0 M

-I 0.

1 M

NaC

l +

9.8

pg/m

l

0.1

M N

aCl +

4 m

M

0.1

M K

CI

-

300,

000

-

0.05

M K

CI +

0.05

M

0.05

M K

CI +

0.05

M

"The

sol

ubili

zed

enzy

me

was

sub

ject

ed to

get

chr

omat

ogra

phy

on a

TSK

gel

G30

00SW

xL co

lum

n w

ith a

n el

utio

n bu

ffer

con

tain

ing

the

ligan

ds

indi

cate

d in

the

tabl

e as

wel

l as 0

.2 m

g/d

Cj2

Es a

t 20°

C. T

he M

, of t

he m

ain

prot

ein

com

pone

nt e

lute

d w

as m

easu

red

usin

g th

e H

PGC

LALL

S m

etho

d,

and

the

K, f

or th

is c

ompo

nent

was

obt

aine

d by

com

pute

r sim

ulat

ion,

assu

min

g th

at th

e en

zym

e was

in a

n eq

uilib

rium

of 2

Pro

tom

er --

L D

ipro

tom

er. T

he

mai

n pr

otei

n co

mpo

nent

was

iso

late

d an

d th

en d

ilute

d w

ith t

he e

fflu

ent c

onta

inin

g no

pro

tein

. The

M, o

f th

e pr

otei

n co

mpo

nent

at

vario

us p

rote

in

conc

entra

tions

was

mea

sure

d di

rect

ly by

the

con

vent

iona

l, lig

ht-s

catte

ring

met

hod

with

a n

ovel

pre

cisi

on a

ppar

atus

equi

pped

with

bat

ch-c

ell a

nd A

r-la

ser

light

sour

ce. T

he K

, was

obt

aine

d fr

om th

e de

pend

ence

of M

, on

the

prot

ein

conc

entra

tion

obta

ined

(FIG

. 1) b

y as

sum

ing t

he s

ame

equi

libriu

m a

s abo

ve.

olig

omyc

in

El

247,

000

248,

OW

2

X

106M

-'

-

-

MK

12

258,

000

-

-

2.

P

z P

320,

000-

327,

000

3.2

x 10

* M-I

Fr; 8 8 e 4 29

8,00

0 -

21

X 1

0RM

-' -

-

NaC

l E

2

NaC

l + 4

mM

MgC

I2

295,

000

-

-

*:

9 5

hDat

a obt

aine

d at

pro

tein

con

cent

ratio

ns hi

gher

than

0.1

5 m

g/m

I wer

e us

ed.

'Cal

cula

ted

usin

g th

e co

nten

ts of

D a

nd P

obta

ined

by

com

pute

r v)

HAYASHI et al.: Na+/K+-ATPase AND El& CONFORMATION 23

(solid line in FIG. 1) and the calculated (dotted line) curve shown at protein concentrations of less than 0.1 m g h l might be attributable to a release of phospholip- ids from the enzyme, because phospholipids would make the protein components of the solubilized enzyme associate.13

1 0.1 0.2 0.3 0.4

[ PROTEIN (mg/ml) ]

FIGURE 1. Dependence of reciprocal weight-averaged molecular weight (z,) of the solubi- lized enzyme on protein concentration, estimated using an elution buffer containing 0. I M NaCl (0) or K C l ( 0 ) at 20°C. The M,s were measured as described in the text. Dotted lines show the dependence calculated by assuming that the enzyme is in the equilibrium of 2P + D with various values of association constant (&).

*O

CONVERSION OF P TO D BY ATP-INDUCED PHOSPHORYLATION

The solubilized enzyme was incubated with various concentrations of ATP under conditions expected to produce a phosphoenzyme intermediate of Ez-P. The resultant enzyme was charged onto a column equilibrated with an elution buffer containing 0.1

24 ANNALS NEW YORK ACADEMY OF SCIENCES

0.5 1 1.5 " a c l l ( M )

FIGURE 2. Change in the relative contents of (cYP),-diprotorner (D), a@-protomer (P), and higher oligomer (H) involved in the solubilized enzyme incubated with ATP under conditions producing phosphoenzyme intermediates in the presence of the various concentrations of NaCl indicated in the horizontal axis. Solubilized enzyme was incubated with 1 mM ATP to produce a phosphoenzyme intermediate at 0°C and was subjected to gel chromatography using an elution buffer containing 0.1 M NaCl at 0°C. Contents of the protein components were estimated by calculating the area under the peaks corresponding to the respective protein components. The relative contents of D, P, and H compared to those of all the protein eluted were plotted against the concentration of NaCl used in the incubation.

M NaCl at 0°C and eluted with the same elution buffer. Chromatography allowed us to estimate the relative contents of D and P of the solubilized enzyme from the areas under the two resulting protein peaks of D and P.l2 With increasing ATP concentra- tions from 0 to more than 1 mM, the content of P decreased from 56 to 26%, whereas that of D increased from 37 to 64%. Thus, it was concluded that ATP induced the conversion of P into D.9 ADP, however, did not have this effect. In another series of experiments, incubation of the solubilized enzyme with ATP was followed by incubation with a stoichiometric amount of [3H]ouabain for 5 minutes at O"C, and then the enzyme was subjected to the same chromatography as that just de~cribed.~ The conversion of P to D occurred in the same way as in the case without [3H]ouabain, as already mentioned. The amount of [3H]ouabain bound to D increased with increasing

HAYASHI et al.: Na+/K+-ATPase AND El& CONFORMATION 25

ATP concentration in parallel with the conversion of P to D. The K0.5 values of [ATP] for an increase in D and a decrease in P were consistently 0.17 mM. The K0.5 values of [ATP] for ["H] ouabain binding to D were 0.19 mM. Thus, both K0.5 values were consistent with each other. Therefore, it was concluded unambiguously that the phosphoenzyme intermediate of E2-P would have been produced first, that it induced the conversion of P to D, and that ouabain bound to the resultant D.

The solubilized enzyme was incubated with 1 mM ATP in the presence of various concentrations of NaCl ranging between 43 mM and 1.6 M to produce a phosphoen- zyme intermediate of El-P as well as E2-P and then subjected to the same chromatog- raphy as that just described. As shown in FIGURE 2, with increasing concentration of NaCI, the content of D decreased and that of P increased. But the content of the higher oligomer (H) did not change. The ADP sensitivity of the phosphoenzyme intermedi- ates produced under the same conditions was investigated. The intermediate produced in the presence of 83 mM NaCl was not sensitive to ADP, showing that it was E2-P. With increasing NaCl concentration, the phosphoenzyme became more sensitive to ADP, and at 1.3 M NaCl the sensitivity reached 93%, showing that the intermediate of El-P had been produced. Thus, conformation of the phosphoenzyme was converted from E2-P to El-P with increasing NaCl concentration from 83 mM to 1.3 M in parallel with the curve of decreasing D or increasing P versus NaCl concentration. Ouabain is known to bind to the membrane-bound enzyme in its E2-P form."-" Our data strongly suggest the following relation between conformation of the phosphoenzyme and the oligomeric structure: El-P had a protomeric structure, and it was dimerized to D on changing the conformation to E2-P, and ouabain bound to the E2-P form and kept the structure diprotomeric.

SIMULTANEOUS DETERMINATION OF QUATERNARY STRUCTURE AND CONFORMATIONAL STATE

USING A SOLUBILIZED FITC-ENZYME

FITC acts as a selective label for the ATP binding site, and the El and Ez conformational states are easily distinguished by large changes in the fluorescein fluorescence ernission.l8 To establish whether D or Pcan exhibit a fluorescence signal that allows the distinction between El and E2 conformation, the membrane-bound enzyme was first labeled with FITC by the method reported by Carilli et al., I9 and then the FITC-labeled enzyme was solubilized with Cl2E8 in the same way as just described. As shown in the upper portion of FIGURE 3, the solubilized FITC-enzyme as well as the original membrane-bound FITC-enzyme could distinguish the El from the El conformation by emitting a 20-30% higher intensity of fluorescence in 0.1 M NaCl than in 0. I M KCl. The reversibility of the conformational change was also confirmed with both types of the FITC-enzyme (the lower portion of FIG. 3). The solubilized FITC-enzyme was subjected to the LALLS system additionally equipped with a spectrofluorometric detector. FIGURE 4 shows the four kinds of elution patterns of LS, the fluorometer (FL), UV, and RI obtained with an elution buffer containing 0.1 M NaCI. The two major protein components were identified as D and P according to their M, value. As shown in the inset of FIGURE 4, the patterns of FL and UV were superimposed on each other, thus making the heights of the peaks of D in the two patterns the same. For P then, the height of the new peak of FL was 1.22-fold higher than that of the UV peak, whereas for D the FL peak was 1 .OO-fold higher than the UV

26 ANNALS NEW YORK ACADEMY OF SCIENCES

FITC-Na'/K'-ATFase MEMBRANE-BOUND SOLUBILIZED

E

WAVE LENGTH (nmj 4oNa'

4 8

4

3K'

1; 0

3' -

b -l 8 TIME (min)

FIGURE 3. Preservation of the capacity to report the conformational change between E, and E, states after solubilization of membrane-bound FITC-enzyme. Membrane-bound enzyme was bound by FITC and then solubilized to produce the solubilized FITC-enzyme. Upper porrion: Fluorescence emission spectra were scanned for the two kinds of FlTC-enzyme in 0.1 M NaCl (-) or KCI (--) at pH 7.0 with a fixed excitation wavelength of 485 nm. Lower portion: Fluorescence intensity at a fixed emission wavelength of 520 nm was estimated when NaCl or KC1 was added to the two kinds of FITC-enzyme. Numerical values within the bars represent the concentration (mM) of cation involved in the initial protein solution and those above the bars represent the concentration of cation added to the solution.

peak. In other words, when the ratio of fluorescence intensity to absorbance at 280 nm, (output)m/(output)uv, for D was normalized to 1 .OO, that for P was 1.22. The ratio thus normalized is equivalent to the difference in fluorescence intensity per unit of protein concentration between D and P. Ratios were 1 .OO and 1.3 1 for D and P, respectively, with the elution buffer containing 0.1 M KC1 (FIG. 5, upper portion). Almost the same values were obtained with NaCl (lower portion). If each D and P can take the conformation of E2 with KCl and that of El with NaCl, the fluorescence intensity of the two protein components with NaCl should become 20-30% higher than that with

HAYASHI et nl.: Na+/K+-ATPase AND EIEz CONFORMATION 27

KCI. This was the case neither for D nor for P. A difference of about 30%, however, was found between D and P regardless of whether NaCl or KCI was included in the elution buffer (FIG. 5). This implies that D itself is E2 and, in reverse, that P itself is El. The contents of D and P changed according to which monovalent cation of KC1 and NaCl was included in the elution buffer (compare the upper UV pattern with the lower one in FIG. 5). We presented a paper on this subject elsewhere in this volume.12 Therefore, it was concluded that KCI and NaCl increased the content of D and P, respectively, and that this alteration was followed by a change in fluorescence intensity that was proportional to the content in the solubilized enzyme.

CONCLUSIONS

Solubilized Na+/K'-ATPase was in an association-dissociation equilibrium of 2P * D at 20°C. The Ka under the ionic environment favorable for the E2 state was about 100 times higher than that for the El state. NaCl and KCI shifted the equilibrium to P and D, respectively. ATP-induced formation of E2-P dimerized P into D and then i t promoted ouabain binding to each P of the D produced. With the solubilized FITC-enzyme, P emitted 30% higher fluorescence intensity than did D, in 0.1 M KCl

RETENTION TIME (min)

FIGURE 4. Elution patterns of solubilized FITC-enzyme obtained with an elution buffer containing 0.1 M NaCl using the HPGCLALLS method. A spectrofluorometric monitor was added to the usual system of the HPGCLALLS method to estimate the conformational state simultaneously with the molecular weight of the protein component separated. Inset shows the elution pattern of FL (-) and UV (..-..) which are superimposed on each other, making the heights of the peaks of D in the two patterns the same.

28 ANNALS NEW YORK ACADEMY OF SCIENCES

FL+\ 0.1 M KCI( E,)

(Ou tpu t)FJ(OU tpu t ) w Diprotomer 1.00

I

Diprotomer 1. 06 1.1 Protomer 1. 37

RETENTION TIME ( m i d

FIGURE 5. Ratios of fluorescence intensity, (output)a, to absorbance at 280 nrn, (output),", for D and P in an elution buffer containing 0.1 M KCI or NaCl at pH 7.0. Solid and dotted lines were obtained as described in the legend for the inset of FIGURE 4. The ratio of (output),/ (output),, for D in the elution buffer containing 0.1 M KCI was normalized to 1.00; the other ratios are represented as relative to it.

as well as in 0.1 M NaCl, indicating that P and D structures correspond to El and E2 conformational states, respectively. Therefore, the interconversion of oligomeric structure between D and P is essential for enzymatic function. The molecular structure of Na+/K+-ATPase in the membrane is consequently thought to be as follows: the El and E2 conformations correspond to a loosely associated diprotomer and a tightly associated diprotomer, respectively; two-dimensional crystals of the membrane- bound enzyme grown at pH 4.8 in sodium citrate buffer20 can be expected to reveal a three-dimensional structure of the enzyme in E2-conformation for reasons described elsewhere.12

REFERENCES

1. HASTINGS, D. F. & J. A. REYNOLDS. 1979. Biochemistry 18: 817-821.

HAYASHI et al.: Na+/K+-ATPase AND Elm2 CONFORMATION 29

2 .

3. 4. 5.

6 .

7. 8.

9.

10. 11. 12.

13.

14. 15. 16. 17.

18. 19.

20.

ESMANN, M.. C. CHRISTIANSEN, K. A. KARLSSON, G. C. HANSSON & J. C. SKOU. 1980.

CRAIG. W. S. 1982. Biochemistry 21: 2667-2674. CRAIG, W. S. 1982. Biochemistry 21: 5707-5717. BROTHERUS, I. R., L. JACORSEN & P. L. JBRCENSEN. 1983. Biochim. Biophys. Acta

HAYASHI, Y., T. TAKAGI, S. MAEZAWA & H. MATSUI. 1983. Biochim. Biophys. Acta

HAYASHI, Y., H. MATSUI & T. TAKAGI. 1989. Methods Enzymol. 172: 514-528. HAYASHI. Y., K. MIMURA, H. MATSUI & T. TAKAGI. 1989. Biochim. Biophys. Acta

983: 2 17-229. HAYASHI, Y., T. KOBAYASHI, T. NAKAJIMA & H. MATSUI. 1994. In The Sodium Pump,

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JBRGENSEN, P. L. 1988. Methods Enzymol. 156: 2943 . TAKACI, T., S. MAEZAWA & Y. HAYASHI. 1987. J. Biochem. 101: 805-81 1. KORAYASHI, T., E. HAGIWARA, N. SHINJI & Y. HAYASHI. 1997. Ann. N.Y. Acad. Sci., this

MIMURA, K., H. MATSUI, T. TAKAGI & Y. HAY ASH^. 1993. Biochim. Biophys. Acta

STEVENS, F. J. & M. SCHIFFER. 1981. Biochem. J. 195: 213-219. CHARNOCK, J. S. & R. L. POST. 1963. Nature 199: 910-91 I . MATSUI, H. & A. SCHWARTZ. 1968. Biochim. Biophys. Acta 151: 655-663. ASAMI, M., T. SEKIHARA, T. HANAOKA, T. GOYA, H. MATSUI & Y. HAYASHI. 1995. Biochim.

KARLISH, S. J. D. 1980. J. Bioenerg. Biomembr. 12: 1 11-136. CARILLI, C. T., R. A. FARLEY, D. M. PERLMAN & L. C. CANTLEY. 1982. J. Biol. Chem.

TAHARA, Y., S. OHNISHI, Y. FUJIYOSHI, Y. KIMURA & Y. HAYASHI. 1993. FEBS Lett.

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