Kinetics of (Na+ + K+)-stimulated ATPase of rabbit kidney microsomes

Vol. 14, No. 2, 1964

BIOCHEMICAL

KINETICS

OF (Na+

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

+ K+)-STIMULATED

RABBIT

KIDNEY

A.L.GREEN

ATPase

OF

MICROSOMES

and C.B.TAYLOR

Smith Kline and French Research Institute, Welwyn Garden City, Herts, England.

Received

October

Skou of crab

(1957)

nerve

Similar

1961),

This

report

kinetic

was

estimated

present

stimulation

various

concentrations

over

is very

This

may

is brought one for

about Nat

high

be qualitatively

and one for

Kt

(Skou,

1).

to that

slightly,

if at all,

The

pattern

obtained

of Kt

in the system 1961). 118

Maximal

of when

of one ion at

Nat

accounted

ATPase

1963).

of concentrations

relative

by the existence

used.

(Taylor,

of curves

(Fig.

mechanisms.

analysis.

markedly.

of the other

known

out more

1962) were

only

1960).

well

proposed

described

a range

is now

kinetic

(Skou,

by the families

concentration pattern

to test

ATPase

t

to be

et al.,

to carry

complete

stimulate

is illustrated is measured

its

stimulate

(Post

and K

attempt

as previously

together

ATPase

little

kidney

and appear

transport

in order

of rabbit

t by Na+ and K .

stimulated

by Nat

a more

by microsomes

distributed

cation

analysis

Na+ and Kt alone but when

widely

has been

summarizes

Microsomes activity

are

of ATP

and

of stimulation

but there

a partial

+t

by Mg

in active

pattern

than

that hydrolysis

systems

concerned

general

(Skou,

showed

is activated

enzyme

intimately The

15, 1963

also

inhibits

(Fig. for

of two

when

2).

if stimulation binding

stimulation

sites, would

Vol. 14, No. 2, 1964

BIOCHEMICAL

NaCl

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

mM

KCI

Fig. 1. The effect of sodium or potassium on constant concentrations of the other cation. 2.5mM ATP, 5. OmM MgC12, 30mM tris:HCl addition to the various concentrations of NaCL ATPase activity is expressed as pmoles Pi/mg

I

I

0. I

I.0 KCI

(mM)

I

I

IO

100

log

mM

ATPase activity at The medium contained buffer (pH 7.5) in and KC1 indicated. N /h.

O!iY%-%-

scale

too

KCI

(mM)

log

scale

on ATPase activity at constant Fig. 2. The effect of potassium concentrations of sodium. a. experimental results - ATPase activity expressed as Fmoles Pi/mgN / h. ; b. theoretical curves, calculated from equation (1) - ATPase activity expressed in arbitrary units.

only more, related there

occur

when

at high

both

sites

concentrations

to the concentration is competitive

inhibition

are

appropriately both

ions

of the other

occupied.

inhibit ion.

to an extent This

by K + at the Na+-site

119

Further-

suggests

inversely that

and by Na+ at

Vol. 14, No. 2, 1964

BIOCHEMICAL

the K+-site. with

The

radioactive

two

ATP,

phosphoprotein this

suggest

the ATP

enzyme,

such a two-step state,

of the intermediate

Kl

ENa

where

(=[E][Na]/[ENa]),

dissociation

Kg

liberation

with

would

constants

i. e. for

the two

constant complex

rate

then

with

of inorganic

be given

by:-

(1) ,2 +k2Kl b2 K4 K2 the Na t and K+ concentrations, K2 is the dissociation klK3

for

the binding

of Kt

instead for

of Nat

K4 is the dissociation

constant

to the phosphoenzyme

and Et is the total

to the free

the binding

of Nat

enzyme

instead

concentration

and

of Kt

of enzyme

phosphoenzyme.

From reached high

be equal,

The

state

stages,

and hydrolysis

dissociation

(=[EP][K]/[EPK]).

the

klk2 ab Et

a and b are

plus

early

is a K+ -phosphoenzyme

(kl+k2)ab+klK3a+k2Klb+-

constant

its

would

the rate

complex

at the steady

v = k2[EPK]=

where

to saturate

during

(EP)

by

phosphate.

of formation

kl and k2 are

and EPK

constant

phosphate

would,

the rates

is a Nat-enzyme

an intermediate

is activated

is sufficient

phosphoenzyme

kl[ENa]=k2[EPK] steps,

where

which

(1963).

in the following

by Nat,

to inorganic

concentration reaction

and Post

occurs

is activated

is hydrolyzed

that

a steady

hydrolysis

in the second,

phosphoprotein

RESEARCH COMMUNICATIONS

of Charnock

that

which

is formed;

Provided

reach

experiments

In the first,

steps.

K+,

recent

AND BIOPHYSICAL

the Figures,

at much

lower

concentrations,

If equation and b

2

must

concentrations inhibition

(1) is to reproduce be much

it is clear

greater

that maximal of Kt

is much this than

pattern, those 120

than

greater

activity

is

Na t , and that by Kt

than

the coefficients 2

of a and a , i.e.

at

by Nat. of b

BIOCHEMICAL

Vol. 14, No. 2, 1964 k2Kl

klK3

>

and k2Kl/K2

satisfied

>>

if the binding

tude

and if the rate

than

the apparent

possibility kinetic

that grounds

rate

constant

Kl = K2 = 20mM,

assigned Na+

possible

K3 = K4 = 40mM imperfect

stimulation (1).

lead

this

at high

when

are

is much

greater

The be excluded

one. between equal

Secondly,

on

agreement

even

if it is assumed steps,

between

both

that

160k2,

results.

values

gives

inserted,

fair

quantitative

nature

of the

that when

concentration

the Na+ state

rates

may

were those

kinetics

are

consistent

as pointed

>>

kl,

the rate-limiting upon

121

out above,

and that

seriously,

This

with

it is possible and calculated

the relative

(1).

the using is the

the kinetics

the binding

in which

step

from

about

mechanism

in which

been

calculated

be added

particular

is low

measured.

than

must

be reproduced

not have

lower

fit.

in the values

cannot

experimental

depending

the constants

be due to errors

the situation but k2

all

the sigmoidal

that this

and more

similar

the following

of warning

Firstly,

for

K+ concentrations

rates

as evidence

approximately

sequential

be

magni-

(1) to the experimental

the reaction

a word

distinguish

K3 tt Kl’

values

the steady

the observed

mechanism,

permissible

cannot

high,

to experimental

agreement

only

to derive

in part

It is possible

Although assumed

but cannot

However,

curves

at the time

would

of the same

phosphorylation.

and kl=

fit may

and the K+ concentration reached

may

dephosphorylation

2, (1) with

in Fig.

to the constants.

by equation

all

likely

of equation

as shown

The

requirements

are

(kl) for less

RESEARCH COMMUNICATIONS

alone.

application

However,

Kl-K4

(k2) for

Kl seems

t

These

constants

It has not been by rigorous

klK3/K4.

constant

K3

AND BIOPHYSICAL

klti

constants k2 but

to obtain activities

is not one of two concentrations

BIOCHEMICAL

Vol. 14, No. 2, 1964 of Nat

and

but

Kt,

requiring

Nat

the

requiring

other

hydrolysis by

type

stage

(dissociation

of

involving

constant

Kt

under

the

a single

AND BIOPHYSICAL

(K3)

these

and

Kl)

by

Laidler

k ab

=KlK3+(lt$Jabt

term

of Nat in

(3)

and

ab,

Kl-

K4

b and

b‘

Kg

= 0.175mM.

all

concentrations

will

give

alone

both,

to

those

in

(3)

in

fact

that

values

for

cation

sites.

equal

affinities

Kl-

can K4 If for

Socquet

example

(1950),

to

be:-

(3)

(1) only

by

high

the

presence

Numerical

values

for

by

equating

values be

very

and

equations

Thus

the

of

Charnock

at

almost

and

(3)

pattern

and

a two-stage

Post

account assuming

that

and

of

K

t

may

one

, the

kinetics

approximately

there

only

the

site same

122

not, with result

in

(1963)

of

the

equal fact,

be

two

approximately would

a, and

(1) and

kinetic

a one-stage

the

con-

Kl=K2=K4=20mM small

studied,

the

coefficients

are

of

concentrations

(1).

of

given

were +

for

possibility.

suggests

Na

of

identical

rates.

evidence

there

rate

curves

between

be

The

theoretical

Kt

a satisfactory

mechanism

and

give

would

and

latter

as

At

These

KlK3

the the

from

obtained

(3).

distinguish

favours The

by

identical

although

strongly

will

be

of Nat

not

form

given may

shown,

(K2)

E

denominator. (3)

Thus,

does

in

the

(1) and

virtually

reaction,

stage

or

Kt,

appearance

stants

in

(KJ).

Kt

(Klt~)bt~a2t~b2

differs

KlK3

one by

Nat

and

(K3ty)a:

Equation constant

be

sites,

blocked

by

can

given

and

blocked

conditions

analysis

two

RESEARCH COMMUNICATIONS

be

found

two-

a

2

,

Vol. 14, No. 2, 1964 provided

that

BIOCHEMICAL phosphorylation

by Na+ and hydrolysis possibility of sodium

may

only

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

only when

be of importance

occurs

when

it is occupied in considering

the site by Kt.

is occupied This

the mechanism

transport. REFERENCES

Charnack, J.S., and Post, R.L., Nature Laidler, K. J., and Socquet, I.M., J.Phys. 530 (1950). C.R., Kinsolving. Post, R. L., Merritt, J. Biol. Chem., 235, 1796 (1960). 23, Skou, J. C., Biochimxiophys.Acta, “Membrane Transport and Metabzsm”, A and Kotyk, A. , Academic Press, Pharm., Taylo;,’ C. B., Biochem. -12, 539

123

(London), 199, Colloid.Chem., C.R.,

910 (1963). 2,

and Albright,

C.D.,

394 (1957); -58, 314 (1962); edited by Kleinzeller, New York, 228 (1961). (1963).