Functional organization of the partial reactions of Na+ + K+ - activated ATPase within the red cell membrane

Vol. 36, No. 4, 1969

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FUNclcIONALORGANIZATIONOF THE PARTIAL REACTIONSOF Na+ + K+ -ACTIVATEDATPaseWITHINTREREDCELLMEMBRANE A. Askari and S.N. Rae Dersxtment of Pharmacology, Cornell University New York, New York

College

Medical

10021

Received July 2, 1969 S-y: E+ffe$ts of fslse substrates and modifiers of the partial reactions of the Na , K -ATPase complex on Na+ transport in resealed red cell ghosts were studied. A ouabain-sensitive efflux of Na+ into a Na+-free medim was initiated when p-nitSophenylphosphate (NPP) and K'were added to the medium. Na+-$fflux into a Na -containing mediumrequired the+simultaneous presence of K , NPP, and a nucleotide in the medium. WhenNa -efflux was stimulated, NPP hydrolysis was increased. We conclude that the K+-dependent phosphatase facing the medium-side of the membraneis the primary ion trsnslocator, and that an enzyme on the matrix-side of the membranesupplies a physiologicsl substrate (the "phosphorylated intermediate") for the translocator-phosphatase. The discovery of the membrane-boundNa+ + K'-activated

adenosinetri-

phosphatase (Na', K+-ATPase) by Skou', and the subsequent studies of many laboratories2'3

, have indicated

energy-dependent translocation

that this enzyme complex is related

of Na+ and K' through the plasma membrane.

The mechanismof the intimate relation

of this enzyme activity

movements, however, remains to be determined. studies have been relevant 1.

to the

The following

to ion two groups of

to the proposed mechanismsfor this relationship:

Experiments with "resealed"

red

cell ghosts

4-6

and. internally

perfused

squid axon7-9 have shown that ATP, only when supplied from the inside of the membrane, can be hydrolyzed 2.

and lead to an active

The works of sever&L investigators3

ATPase have shown that ATP hydrolysis a Na+-dependent phosphorylation hydrolysis

of the resulting

extrusion of Na+.

with partially

purified

Na+, K+-

consists of at least two steps:

One

of the enzyme, and the other a K+-dependent

acylphosphate (EtiP).

On the basis of the above

studies most of the proposed mechanismsfor the active transports K' relate the ion movementsto the turnover of E&P within 631

of Na+ and 10 the membrane.

Vol. 36, No, 4, 1969

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

So Par, however, it has not been possible to demonstrate a correlation between this turnover

and the movementsof ions in a functionally

intact

membrane. Recent studies

11

have given strong support to the assmption that

the K+-dependent hydrolysis

of somesimple organic phosphates (e.g. acetyl-

phosphate and p-nitrophenylphosphate) equivalent

by the Na+, K+-ATPase preparations is

to the K+-dependent hydrolysis

of EutP within the ATPase complex.

It occured to us that if this assmption were correct, it may be possible + to supply the Na , K+-ATPase of a functionally intact membranewith one of these false substrates,

instead of EIJP, and determine the correlation

between

ion movementsand at least part of the reactions catalyzed by the ATPase. Inihis

report we present the results of our initial Isotonically

"resealed" red cell ghosts, labeled with Na**, were prepared

as described before'*.

It has been demonstrated that these ghosts sre deficient

in substrates and that the active either lactate

studies in this direction.

extrusion

production is initiated

of Na+ will

not commenceunless

by the addition of appropriate

1*,13 , or ATP is placed inside the ghosts prior to substrates to the medium 22 reseaJ.ingb 6. Figure 1 showsthe time-course of the release of Na from

the ghosts into various Na+-free media.

It is evident that when Kf is

present in the medium, the addition of p-nitrophenylphosphate mediumcauses an increase in Na+ effluxa. dependent efflux

to ouabain indicates

exchange through the carrier the data

of Figure 2

show,

The sensitivity

that the efflux

of the classical

(NPP) to the

of this K+-

is due to Na+, K+-

coupled Na+, K+-pump. Part of

however, that if the incubation mediumcontains

Na+ the addition of NPP is not sufficient

to cause an increase in Na+ efflux

regardless of the presence or absence of K+.

WhenNPP hydrolysis

was also

measured in the course of the above experiments it became apparent that in the a. Our preliminary studies showedthat organic phosphates other than ATP when placed inside the ghosts had no effects on Nt$ efflux. This is in agreement with the findings of Brinley and Mullins who showed th$t acetylphosphate perfused through the squid axon did not affect Na efflux. 632

Vol. 36, No. 4, 1969

Nat Free Media

A K+ A NPP l K+ + NPP + ouabain q No additions

0

medium the presence

no K+-dependent (Table 1). K+-dependent containing

I 20

I I I 40 60 Time (minutes)

I

I 80

1 100

ST~ION OF Na+ EF'FLUX INTO Nat-F'FZE MEDIUM BY K+ AND NPP. The Na+-free medim contained 160 mM choline chloride, 10 mM Tris-HCl (pH 7.4). The indicated compounds were added to this mediun at the folJ.owing final concentrations: KCl, 6 It&Ii TY.‘iS salt of Npp, 3 mM; ouabain, O.lmM. In each experiment lml of ghosts was added to 50 ml of the medium, and the release of Na** at 370 was measured by the procedure described before=.

Figure1

Na+-free

4

This

hydrolysis

of K+ caused an increase could be detected

correlation

NPP hydrolysis medim

was perhaps

dependent phosphatase 14,15 .

between suggested

in the Nat-containing

the existence that

of Na',

but that mediwn

K+-exchange

and

the absence of exchange in the Na+-

due to the inhibitory Since nucleoside 633

in hydrolysis,

effects

triphosphates

of Nat on the Ktare able not only

Vol. 36, No. 4, 1969

Table 1.

BIOCHEMKAL

AND BlOfflYSlCAL

RESEARCH COMMUNICATIONS

CO-ION l3EJxms THESTIMJMTIONOFNa+EF'FLUXAND I3YDFK&YSIS OF NPP. Composition of the media, and the concentrations of added eds were the sameas indicated in legends to Fi es 1 and 2. p-nitrophenol was measured by the calorimetric methodET after the deproteinasation of aliquots of the reaction mixtures. Additions media

Na+-free medium:

Na+-containing medium:

to

$ of NP2 released inonehr.

Nitrophenol ( p moles/mljhr)

NPP

35

0.12

NPP +K+

70

0.31

NPP + K+ + ouabain

31

0.13

NPP

33

0.14

NFF + K+

36

0.12

to overcome this inhibitory

effect

of Na+ but also cause an extra activation

of the K+-dependent phosphatase11,15 , we determined the effects

of addition

of nucleotides to the medilnn on Naf efflux

The results

of Figure 2 clearly

show that in a Na+-containing mediumwhere neither NPP alone

nor ATP alone affect initiates

Na+ efflux,

a ouabain-sensitive

the simultaneous presence of both compounds and K+-dependent efflux

Frcnn the data of Table 2 the following sll

and NPPhydrolysis.

cases there is a direct

additional

correlation

of Na' from the ghosts.

points are evident:

between the stimulation

and an increase in the production of p-nitrophenol.

2.

of Na+ efflux.

3.

can also stimulate Na' efflux

NaS-containing medium. 4. of Naf, indicating

of Na+ efflux

which is

CTP, which is not a substrate

for Na+, K+-ATPase but has the samemodifying effect phosphatase as ATPll,

In

It is the hydrolysis

of NPP, and not the presence of the products of this hydrolysis, related to the stimulation

1.

Iodoacetate has no effect

on the K+-dependent and NPP hydrolysis

in the

on the stimulated efflux

that the efflux

is not due to the metabolism of traces of 12 substrates that may have remained within the ghosts . The data of this paper show that when a membrane-boundphosphatase, which is apparently oriented toward the medium-side of the membrane, is 634

Vol. 36, No. 4, 1969

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Na+-ContainingMedia

A

20

l

A K+ t+ NPP A NPP t ATP OK+ tATP OK+ t NPP+ ATP + ouabain

Time(minutes) Figure2

N OF Na* EFFLUX7NTONa+-CONTAININGMEDIUMBY THE -US ZSU3SBiCE OF K+, NPP, AND&l!P IN THE INCUB&!ION MJZDIUM. Each mediumcontained 160 mMNaCl, 10 mMTris-HCl El and the other added components as described for . ATP concentration w&s 0.5 nt4.

supplied with a substrate and a nucleotide modifier is capable of catalyzing This extrusion

in its

glycosides is similar on the matrix

of Na+ against a concentration

gradient.

dependence on external. Ki and its sensitivity

to cardiac

to thd

of ATP

caused by the presence and hydrolysis

4,6 . side of the membrane

The following 1.

the extrusion

(both from the medium) it

two important questions are raised by these findings:

under the conditions

described in these experiments, which are not likely 635

Vol. 36, No. 4, 1969

Table 2.

BlOCHEMlCAL

AND BlOF’HYSlCAL RESEARCH COMMUNICATIONS

STIMULkTORY EFFECTSOF NUCLEUCIDNS ON Na+ EFFLUX AND NPP HYDROLYSIS Concentrations of ATP, NPP and ouabain INANa'-CONTAINLNGMEDIZM. were the same as described in Figures 1 and 2. Other compounds CTP, 0.5 mM; p-nitrophenol, 1mM; were added as follows: orthophosphate (Ns2HP04-HCl, pH 7.4), 1 mM; iodoacetate, 1 mM.

Additions to the medium

$ of Na22 released in one br.

None

32

m-m

NaCl, 160 n&l;

NPP

37

0.10

KCl, 6 mM;

ATP

31

SW-

Tris-HCl

NPP+ATp

84

0.40

NPP+ATP+ouabain

30

0.12

NPP+A!l!P+U

81

0.36

NPP+CTp

78

0.39

p-nitrophenol+Pi

29

0.06

Incubation Medim:

(pH 7.4)

10 n!M

to be duplicated The simplest phosphatase

under physiological

way of explaining is the "primary

between the chemical of its activator hydrolysis

reaction

cation

conditions,

how is the plop operating?

the data would be to assume that the anisotropic translocator JO , and that there is no distinction catalyzed

by this

(K+) from outside

enzyme and the translocation

to inside.

The sufficiency

of NPP

for the occurance of Na+, K+-exchange in the absence of external

Na+ suggests that when the enzyme (or the active is freed of substrate

and products,

In the presence of relatively

reaction

high concentrations

does not proceed.

site by a nucleotide

center region of the enzyme)

it can move Na+ from inside

ion exchange does not occur because Na+ inhibits chemical

Nitrophenol &fmles/ml/hr )

In this

becomes necessary.

to outside.

of Na+ in the mediun the the phosphatase

case the occupation

The similar

effects

and the of a modifying

of ATP and CTP,

both in the experiments partially

purified

modifying

site

described in this paper and those dealing with the phosphatase ll , and the apparent accessability of the

from the medium-side

to the idea that this

site

of the membrane, give further

is different

from that involved 636

support

in the hydrolysis

Vol. 36, No. 4, 1969

of internal

BIOCHEMICAL

ATP.

2.

Fran the findings

drawn about the physiological validity

di&inct

the over-all

(or proteins)

discussed above.

paper what conclusions of the punp?

of the RPP effects,

Ra+, K+-ATPase activity

enzyme activities:

protein

of this

mode of operation

to our interpretation

to consider

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

One involved

translocator-phosphatase.

as a combination

in the formation

of the first

of two

of phosphorylated

enzyme, which is facing the matrix

Since under physiological

substrate

conditions

for the

(high external

site of the phosphatese must be occupied by a nucleotide,

one must assune that this

additional

is

it is perhaps more logic&L

side of the membrane, would be to supply a phosphoprotein

phosphatase,

If.there

.X'rcsaATP, snd the other the trsnslocator-phosphatase

The function

Ha+) the modifying

site,

in contrast

to the active

site of the

can be reached from both sides of the membrane; snti that physiological.

Acknowledsment:

may be

role of ATP is the occupation

of this modifying

This work was supported by U.S. Rzblic

Research Grant Rob. 10884 frcm the Rational

Health

site.

Service

Heart Institute.

1.

Skou, J.C.,

Biochim.

2.

Skou, J.C.,

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3*

Albers,

4.

Hot'fbmn, J.F.,

5.

Gordas, G., Rxperientia,

6.

Garrahan,

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Brinley,

F., Jr.

and Mullins,

L.J.,

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a

2303 (lg6V).

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Brinley,

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Baker, P.F., Foster, R.F., Gilbert, Biophys. Acta, &$ 560 (1968).

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R.W., Ann. Rev. Biochem.,

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BIOCHEMICAL

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Askaxi, A. and Koyal, D., Biochem. Biophya. Res. Cammun.,&

227 (1968).

12.

Askari,

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Hoffman, J.F.,

14.

Fujita, M., Nakao, T., Tashima, Y., Mizuno, N., Nagana, K. and Nakao, M., Biochim. Biophys. Acta, 1l7, 4.2 (1966).

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Yoshida, H., Nagai, T., Ohashi, T., Acta, In, 178 (1969).

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Bessey, O.A.,

A. and Baa, S.N., J. Pharmacol., 163, 40'7 (1968). J. Gen. Physiol.,

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837 (1962).

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3U (1946).