Inhibition of human red cell NaK-ATPase by magnesium and potassium

VoL66,No.

2, 1975

INHIBITION

BIOCHEMICAL

AND BIOPHYSICAL

OF HUMAN RED CELL NaK-ATPase

RESEARCH COMMUNICATIONS

BY MAGNESIUM AND POTASSIUM

G. H. Bond and P. M. Hudgins Departments of Physiology Medical College of Virginia Cononwealth Richmond, Virginia

Received

July

and Pharmacology Virginia University 23298

17,1975

SUMMARY: The inhibition of NaK-ATPase (EC 3.6.1.3) from human red cells by MgL+ is markedly dependent on the relative concentrations of Na+ and K+. with increasing Nat. Inhibition increases with increasing Kt and decrease distinct The inhibition appears to be a combined effect of MgSt and Kt at sites J!e kinetics of from the sites at which these cations activate the enzyme. and Kt are activation of the enzyme by Nat with inhibitory levels of Mg biphasic, indicating both low and high affinity Nat sites. At noninhibitory Nat sites are seen. The results are levels of Mg2+ and Kt only high affinit inconsistent with any model in which MgSt and Kt compete with Nat at a single si e A kinetic model is proposed to explain the mechanism of inhibition by RI Et* and Kt.

INTRODUCTION: a function of

about

Magnesium

ion

has a biphasic

of Mg2+ concentration 1.0

(l-3).

Since

passes

by Mg2+ has been studied

inhibition

at Previous

competing not found

Nat or Kt at

to have

that

inhibition

influence

suggested

on the

of relative

of these

that

their

been studied

little

activity,

of

(4);

on inhibition

the mechanism but

the

mechanism

of

attention. at

sites

systematically.

concentrations

as ratio

Mg2+ inhibits,

from

activity

a maximum at a Mg2'/ATP

for

respective

NaK-ATPase

cations

on NaK-ATPase;

in some detail

Mg2+ has received

workers

appear

dependent the

with

through

Mg2+ is required

activation

higher

effect

least

(5-6),

but

In preliminary

in part, this

by

point

does

experiments

human red cells

by Mg2+ was strongly

of

This

Nat and Kt. of

the

enzyme

report

we

concerns

by Mg2+.

METHODS: Red cell membranes were prepared from freshly outdated human blood accarding to standard procedures (7). The protein content of each preparation was measured by the method of Lowry et al. (8). Activity was assayed by hate (Pi) according measuring the production of inorganicphos to a modification of the method of Fiske and SubbaRow (9 P as described previously (7). The following conditions were comm n to all experiments: ATP, 2.0 mM; Tris.HCl (pH 7.4), 63 mM; and TrisvEGTA P 0.25 mM. The incubation temperature was 38". Other conditions are described in the text.

Vol. 66, No. 2, 1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

All activities reported represent NaK-ATPase. In each case Mg-ATPase, measured in the presence of ouabain (0.5 mM), was subtracted from activity in the presence of Na+ and Kt to obtain the NaK-dependent ATPase activity. Activity is expressed as umoles Pi/mg protein/hr. All figures represent the average lEthy?eneglycol-bis(B-aminoethyl ether)N,N'-tetraacetic acid. of two or more

experiments

run in duplicate. Inhibition

RESULTS AND DISCUSSION: dependent

on the

shows that

with

of

at

higher

others

(l-3),

when Kt was increased (curve

D),

was more Mg2+. inhibitory of

It

Mg2+ is

who used

With

pronounced.

thus

that

for

NaK-ATPase monovalent curve B D - 30 mM

Kt media B),

inhibit,

2

3

activity as a function cation concentrations. 160 mM Nat, 25 mM Kt; Nat, 25 mM Kt.

646

C).

Kt must bind

3 ,

1.0

is optimal,

assay.

at

the

However,

30 mM Nat

lower

complete

A)

with

or with

in the

4 ION

1 (curve

Mg*+ and with

6 mM

Mg2+ had virtually

mM (curve

to activation

Mg2+ to

for

was nearly

(30 mM), however, to 0.5

strongly

is consistent

was evident

respect

I

1.

low

is

of about

inhibition

MAGNESIUM

Fig.

result

30 mM Nat inhibition

with

ratio

(curve

when Kt was reduced

appears

Nat,

Figure

160 mM Nat

same Nat concentration

half-saturating

This

high

was sharper,

NaK-ATPase

of Nat and Kt.

gradual.

to 25 mM with

optimum

effect

Kt is

Nat.

the

At the

concentrations

cell

160 mM Nat and 8 mM Kt a Mg2+/ATP

and inhibition findings

relative

of human red

This presence

no

concentration of 30 mM

to a site

in

6

mM

of Mg2+ concentration with varying Curve A - 160 mM Nat, 8 mM Kt; curve C - 30 mM Nat, 0.5 mM; curve

Vol.&No.

2, 1975

addition

BIOCHEMICAL

to the

site

at which

it

AND BIOPHYSICAL

activates;

RESEARCH COMMUNICATIONS

and higher

K+ concentrations

are

required. Figure presence were

2 shows more

of 2.5

varied

increased

mM Mg2+.

reciprocally with

a plateau

respect

to both

abruptly the

relative likely

Kt from

a site

compete

with

shown

the

Kt at

fig.

2.

the At the

the

enzyme a second

Here

site,

about

range lower

inhibit

over

with

and this is

the

second

A”,

30

were

occupied with

of activity

appeared

portion

exceeds

that

resulted excess

Nat and ti

When Nat

In this

5.

in the

to be saturated

increment

from Mg2+,

of the in Nat.

the

20

of Nat with

of activity

experiment 1.0

2.

NaK-ATPase activity as a function are described in METHODS.

647

Other

(6 mM)

Michaelis-

0

of Na/K ratio.

mM

appeared

mM

Fig.

of

Nat could

in the

by Nat obeyed

IO

thus

dissociation

The Kt concentration

increment

and

curve It

Clearly,

was tested

activation

40

of

as Nat sites

mM Mg*+ (inhibitory).

which

Na/K ratio

strength.

as a function

Mg2+ concentration,

(Kl60

ionic

possibility

plotted

the

concentrations

appeared

activity

activity

and 3.0

the

greatly

additional can

of varying

increased

exceeded

it

this

Mg2+ (noninhibitory) was within

activity

in Kt concentration

at which

3.

effect

a constant

However,

Na/K ratio

that

in fig.

experiment

which

cations.

change

appeared

In this

Kt,

through

when the

the

to maintain

saturating

reached

clearly

conditions

in

Vol.66,No.2,

1975

BIOCHEMICAL

= \ z

0.5

c k it

0.4

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

0.3 E \ c

0.2

v) Y

0.1

z a I

40 SODIUM

Fig.

3.

Menton

with

an apparent

Mg2+,

however,

activation

classes

of

for

sites

low affinity

low Kt. which

are

when occupied is

Nat with

ION.

mhl

by Nat.

Mg2+,

higher

followed

Nat and with concentration Present

2, the

only

inset).

biphasic,

(see

inhibitory

low affinity

sites

by Kt (with Kt from

these

3.0

suggesting

affinities

with

With

levels are those excess sites

mM

two

inset).

The

of Mg2+ and on the

Mg2+), only

right;

but

not

when the

Na/K

high. pattern

activation

of activation

increased

activity

then

4 B shows a similar Nat delayed the suggest

two curves that

to follow

peaked

up to 10 mM Kt;

3.0 mM Mg2+.

by K" at

30 mM Nat and at

(3 mM) and one noninhibitory

by Kt appeared

however,

Figure

results

7 mM (see

different

Nat can displace

by inhibition

concentration.

widely

one inhibitory

Mg2+,

about

when occupied

4 A shows the

lower

Km of

evident

inhibitory

Mg2+ concentrations,

At the

were

In fig.

sufficiently

Figure

the

I

100

by Nat was distinctly

Nat sites

relatively

ratio

I

60

NaK-ATPase activity as a function of Na+ concentration at two Mg2+ concentrations. The Kt concentration was 6 mM. The inset is a modified Lineweaver-Burke plot of the same data.

kinetics

sites

I

60

0

20

the

typical

sharply activity

of

at

mM).

saturation

1.0 mM Kt.

was independent

experiment onset

at

(0.5

two fixed

inhibition,

but

two At

kinetics This

was

of Kt levels as the

of k

merged.

inhibition

648

of red

cell

NaK-ATPase

by Mg2+ is

Vol. 66, No. 2, 1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

94

mM

34

I

3mM

Fig.

4.

effect

inhibitory

interaction whose

of

I

I

I

0

5

IO

15

any model

for

Nat site.

is

of

,

I

I

1

20

25

3(

mM

account

of Mg2+ does problem

not

could

for

the

The following

an inactive

fact

tentative

a regulatory complex,

low Mg2+;

2)

Nat-site.

The complex

partially

overcome

Nat sites

appears

is susceptible that

and k

Nat can also with

the

Nat is

fully

649

with

at

a single

to displacement

of Nat by k

and Mg2'.

by Kt at an inhibitory (fig.

account

site the

for

the

active

but

but

results

are:

has not Kt-site;

4 A),

level leaves

Kt is complete

Mg2+ can combine

with

3).

of

site

combine

(fig.

two populations

The assumptions

to this

inhibition

competition

of enzyme.

bound

Nat

to be incompatible

by Mg2+ at high

before

this

by postulating

as K+ increases

could

of

of low affinity

involves

inhibition

model

A - 30 mM N + and 3 mM Mgf!+ .

One consequence

of a new class

be explained

why inhibition

two populations with

of

go to completion

of explaining

requiring

least

sites.

by Kt and Mg2+ which

one of which

could

separate

appearance

two classes

inhibition

only

the

can at

The results

enzyme,

combine

ION

Mg2+ and Kt at

occupancy

The co-existance

This

Na

NaK-ATPase activity as a function of Kt concentration. and two Mg2+ concentrations; B - two Nat concentrations

a combined

sites

mM

Mg

L

POTASSIUM

Na

1).

without k

its

site

effect this

(fig.

1)

with

the

must to form

on activity is

the

at

low affinity

has a low affinity

for

Mg2+.

Vol. 66, No. 2,1975

According long

BIOCHEMICAL

to this

as Mg*+ is

limited

by the

less

than

inhibitory

because

Mg*+ secondary

only

then

kinetic

the

described

saturating

Mg*+,

of

the

on the

overall

because

complex

combination

as

will

inhibition

be will

go

when Mg*+ and Kt Nat with

reaction,

i.e.

this to

site

displace

Kt. could

be unique

NaK-ATPase

from

sheep

kidney.

They

(10) state

to NaK-ATPase obtained that

from

similar

red

cells,

results

Mg2+ inhibits

but

with

in the

presence

Kt.

These

results

levels,

could

cellular

Kt,

be abruptly

suggest

regulate the

overcome

from

the

that

colloid

intracellular

Mg*+,

Na,K pump in red

pump would

way a reserve cell

inactive

would

Wang and Lindenmayer

the

With

of

complete,

only

that

this

amount

not

are evident

appears

of high

is

RESEARCH COMMUNICATIONS

Na+ sites

effect

to displacing

The results

by ti

of Mg*+.

The low affinity

have any observable

it

inhibition

saturating,

availability

to completion. are

scheme,

AND BIOPHYSICAL

be partially

when the pump activity osmotic

Na/K ratio could

cells.

at moderate

inhibitory

With

high

normal

inhibited,

but

exceeded

a critical

be available

this

intra-

inhibition

could

threshold.

In

when needed,

to protect

lysis.

ACKNOWLEDGEMENTS: We wish to thank criticism of this work. This study

Dr. John DeSimone'for constructive was supported by NIH grant AM-17190.

REFERENCES

:: :: 2 7. 8. 1::

Dunham, E.T. and Glynn, I.M. (1961) J. Physiol. (London), 156, 274-293. Schwartz, A., Laseter, A.H., and Kraintz, L. (1963) J. Cell. Comp. Physiol., 62, 193-203. Wheeler, K.P. and Whittam, R. (1962) Biochem. J., 85, 495-507. Glynn, I.M. and Karlish, S.J.D. (1975) Physiol. Rev., 37-, 13-54. Wilkes, A.B. and Bader, H. (1969) Pharmacol. Res. Comm., 1, 211-217. Robinson, J.D. (1972) Biochim. Biophys. Acta, 266, 97-102. Bond, G.H. and Green, J.W. (1971) Biochim. Biophys. Acta, 241, 393-398. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J.(1951) J. Biol. Chem., 193, 265-275. Fiske, C.H. and SubbaRow, Y. (1925) J. Biol. Chem., 66, 375-400. Yang, T. and Lindenmeyer, G.E. (1975) Biophys. J. (AEtracts), j!j, 244a.

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