The effect of temperature on the activity of the adenylate cyclase system of liver plasma membranes

Vol. 80, No. 3, 1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 609-615

February 14, 1978

THE

EFFECT OF TEMPERATURE ON THE ACTIVITY

OF THE ADENYLATE

CYCLASE SYSTEM OF LIVER PLASMA MEMBRANES J. Antonio Biochemistry

Pliego*

Department,

Avanzados

de1 Instituto

Received

November 4,1977

and Boanerges Centro

PolitGcnico

Rubalcava'

de Investigaci6n

National,

P.O.

y de Estudios

Box 14-740,

Mexico

14, D.F.

SUMMARY The rat liver adenylate cyclase system shows a discontinuity in the Arrhenius plots at 20°C in the nonstimulated activity (basal) with activation energies of 16 and 28 Kcal/mole. The discontinuity disappears when the enzyme is stimulated either by glucagon, sodium fluoride, 5'guanylyl-imidodiphosphate or glucagon plus 5'guanylyl-imidodiphosphate and the energy of activation was the same with all the compounds tested. If the activator was initially in contact with the membranes at 0°C the energy of activation was similar to that observed below the break (26 Kcal/mole) but it changed to that above the break if the compound contacted the membranes at temperatures above the break (22-24°C). We discuss the possibility of two different conformations of the enzyme; both conformations can be "frozen" by any of the compounds tested, "isolating" the enzyme from any subsequent physical change of the membrane due to temperature. A model

has been proposed

In such a model, proteins liquid

the phospholipids

it

is

and the structure

phase

in which

the

operate

independently

thought rather

proteins

properties

system it

that than

membranes

their

is an intrinsic

There

activity effect

of the

several

cations

adenylate

is due to actions

on the enzyme lose

are

component

by the lipids

to be influenced

of the membrane. on the

although

(1).

globular but a

influenced

of the lipids.

cyclase expect

membrane

in a bilayer

It is not a rigid

of hormones

dispersed

arranged

plasma

phase.

protein

membrane

of the

in the lipid

we would

effect

are

structure

immersed

The adenylate

other

the

are

by the physical

therefore

for

the hormonal

itself.

It

response

cyclase on the

of membranes just

that

like

structure

retain

(2,3),

and

detergent

the ability

*Fellow of CONACYT Mexico. Presented as partial fullfilment for at Centro de Investigaci6n y de Estudios Avanzados de1 Instituto National. -!-To whom correspondence should be addressed.

the

of the

has been shown that but

any

influence

system

and

to re-

M. SC. degree Polit6cnico

OOO6-291X/78/0803-9609$01.0010

609

Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.

BIOCHEMICAL

Vol. 80, No. 3, 1978

act

to the

specific dence

lipids that

affect ship

fluoride

on the

acidic

the

rat

(4-8).

liver

of this

of these

enzyme system.

studies

the discontinuities

enzyme activities and separations)

relationship

with

The present

ious

(8);

lipids

adenylate

changes study

and the

activity

with

either

to

relationof the

that

controls

the ac-

in the Arrhenius

plots

of mem-

phase

changes

with

lipid

to establish

activation

effect

a clear

binding

to a site

and have intended

the

of

and nucleotides

we showed

have been correlated

describes

the participation

glucagon

also

in the Arrhenius

cyclase

suggest

for

or of nucleotides

(transitions

membrane

required

cyclase

RESEARCH COMMUNICATIONS

We have presented sOmeevi-

(8-11).

regulatory

In several bound

are

adenylate

residue

studies

action

phospholipids

histidine

tivity

Previous

hormonal

between the presence

glucagon

brane

ion

AND BIOPHYSICAL

a physiological

energy

of temperature

and without

the

(12-15). on the

stimulation

rat

liver of var-

compounds. MATERIALS

AND METHODS

Glucagon was supplied as a gift by Lilly.[a-32P]ATP, Gpp(NH)p' and cyclic AMP were supplied by the International Chemical and Nuclear Corporation Creatine phosphate and creatine and used without any further purification. phosphokinase were purchased from Sigma Chemical Co. All other reagents were of analytical grade. Partially purified plasma membranes from rat liver (male Wistar rats) were prepared according to Neville's procedure (16) with a modification described by Pohl et al. (17), and frozen and stored in liquid nitrogen. For further use the membranes were suspended in 20 mM Tris-HCl, pH 7.5. Adenylate cyclase activity was measured by the method described by Salomon, Londos and Rodbell (18). Unless specified otherwise, the assay medium contained 3.2 mM 25 mM Tris-HCl pH 7.5,1 mM cyclic [CP P]ATP (20-40 cpm/pmole), 5 mM MgC12, AMP and an ATP regenerating system consisting of 20 mM creatine phosphate and 100 U/ml creatine phosphokinase. Either glucagon, sodium fluoride, Gpp(NH)p or Glucagon plus Gpp(NH)p was added to stimulate the enzymatic activity. Incubations were carried out for ten minutes at the temperature indicated. Unless stated otherwise, the reaction was initiated by the addition of the membranes which were measured by the procedure of to give 30-40 micrograms of proteins, Lowry et al. (19) using bovine serum albumin as standard. The slopes of the Arrhenmots and the existence and locations of break points were determined by linear regression analysis, and the activation energies of the basal and stimulated activities were compared to each other by the Student test. RESULTS AND DISCUSSION Under

the conditions

=The abbreviations AMP, adenosine

of the assay

used are: Gpp(NH)p, 3':5'monophosphate.

for

basal

and stimulated

5'guanylyl-imidodiphosphate;

610

activities Cyclic

BIOCHEMICAL

Vol. 80, No. 3, 1978

with

10w6M glucagon

cyclase the

activity

specific

(5,12).

and 10 mM sodium is

linear

activity

We were

AND BIOPHYSICAL

in time

fluoride, during

to measure

the

we found

that

the ten minutes

was of the same order

able

RESEARCH COMMUNICATIONS

of the

basal

adenylate

of incubation

values

activity

the

and

previously

reported

at temperatures

as

low

as 5°C. The temperature as shown in Fig. activity the

1, where

presents

activities Gpp(NH)p

ally

the

the break

by adding

stimulated performed

point described

activities

the membranes

already

the plot

of

or glucagon energy

to that

is essenti-

of the basal

activi-

I).

obtained

through

initiating

medium,

contained

the

activators,

activity

the

break

basal

change

the conformation

this

hypothesis,

that

the reac-

in the case of

and this

addition

was

these

conditions

between

similar

point

(Fig

2) (Table

To assure

that

this

experiments:

I5 minutes,

then

the break

I,

but

also

two different first, we added

we kept

found

energy for

of the

the basal

added

and then reaction

activity

under above

II).

to the

stabilizing

active

conformations,

the membranes

611

in

but keeping

15 minutes

not have a different

the activator

seems

point.

the above experiments,

Condition did

plot

the activator

22 and 24°C for

to that

reaction

to the temperature

activities

The activation

solution.

was very

on an enzyme with

below

of the Arrhenius

of the enzyme due to a change

we repeated

at temperatures

to the activator

for

similar

incubation

of a conformational

added

following

to the

fluoride

The activation

condition

of the basal

2O"C, whereas

sodium

and in the case of the stimulated

To test

tors

I,

plot

differently,

at 0°C.

seems to "freeze"

due only

and very

above were

the membranes

to be the result

the break

such a break.

(Table

In the case of the

the lipids

by glucagon,

each compound

activities

the Arrhenius

at approximately

either

do not present

same for

and stimulated

is seen that

a discontinuity

The results tion

the basal

it

stimulated

plus

ty below

affected

activation action

of the activa-

we performed

above

and returned

energy

20°C

the

(22-24'C)

the mixture

to an

BIOCHEMICAL

Vol. 80, No. 3, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

20 I 0

IO “C I Glucogon

+ GPP IN H jp

Fig. 1 Arrhenius plot of the adenylate cyclase activity (nmoles cyclic AMP/mg protein in ten minutes) Effect of 10 mM Sodium fluoride, lO-'jM glucagon and lO-'jM glucagon combined with 10-5M Gpp(NH)p. The membranes at 0°C were added to the incubation medium (see methods) containing the activators (Condition I). The different symbols for the basal activity indicate 3 different experiments with membranes frozen in liquid nitrogen one year ( D ) 6 months ( A ) and one week ( ) before the experiment.

l

ice bath

and at this

point

The activation

energy

basal

above the

tion

activity III-A

(Table

the substrate ice-bath; observed

Finally,

was,

substrate

break

point

is a control

an experiment

conditions

(Table

I,

in which

the

the activation below

was performed

612

to start

the reaction.

the same as in the case of the

membrane-activator

as we expected, the basal

was added

as we expected,

to the mixture

again, under

I)

the

Condition

The condi-

reaction

was started

which

had been kept

energy

the break that

III-B).

was similar

by adding in an to that

point.

probed

that

the

change

in

BIOCHEMICAL

Vol. 80, No. 3, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE

THE EFFECT OF VARIOUS

I

COMPOUNDS ON THE ENERGY OF ACTIVATION

OF A~ENYLATE COMPOUND ADDED

CONDITION

NONE (BASAL)

27.9

+ 2.5(12)

15.G

+ 0.98(11)*’

GLUCAGON

26.0

t 2.8(11)

FLUORIDE

25.5

GPP(NH)P

24.9

GLUCAGON+ GPP(NH)P

26.4

(Kcal/mol)

CYCLASE

I*

CONDITION

II*

CONDITION

III*

A

17.0

+ 0.73(10)

+ 1.2(E)

17.1

+ 1.6(6)

+ 2.0(4)

18.5

+ 0.12(3)

r 1.6(7)

18.5

T 0.91(5)

The results are expressed as mean + S. D. results that qave a determination coefficient The range of femperature studied was 10" l For definition of conditions I, II and ** The basal activity showed a change in

The figures in parentheses < 0.9 were discarded. - 30°C. III see the text.+ the slope at 19.7"0.99'.

the enzyme due to temperature

is

activator

15 minutes,

standed

at 23°C for

returned

to the

and the

experiments

(not

shown)

ice-bath

were

was similar

those

expected

(27 Kcal/mole)

and after

as described for

to that

the

number

that the

such a condition: observed

for

performed.

slowly was added The results

activation

basal

(1)

the

were

I.

the

the

without

activator

+ 0.63(3)

17.5

of experiments

time

in condition

17.7

(1)

the membranes

Once there,

performed

i 0.50(Z)

24.7 are

reversible:

temperature.

were

25.5

8

below

energy

the break

point. The Student dicative

that

III-A

and the

being

identical.

vation for

This animal

the

with

results

basal

all

the

of the

activation

data

of the experiments

of the activation

experiments

energy

The probability

energies

activity

test

done under

below

was also

energies conditions

20°C had a high high

done under

was in-

to be the conditions

I and

probability same for

II

of the acti-

and III-B

and

above 20°C. study

presents

shows that a biphasic

the adenylate behavior

cyclase

in the Arrhenius

613

enzyme from a homeothermic plots.

This

is not

The

Vol. 80, No.3,

BIOCHEMICAL

1978

A SODIUM 0 0

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FLUORIDE

GPP(NHIP GLUCAGON

:

w

Fig. 2 Arrhenius plot of the adenylate cyclase activity (nmoles cyclic AMP/mg protein in ten minutes). Effect of 10 mM sodium fluoride, 10-6M glucagon, lo-'M Gpp(NH)p and 10-6M glucagon combined with lo-'M Gpp(NH)p. The plasma membranes were incubated at 23°C for 15 minutes and then were added to the medium (see methods) containing the activators (Condition II).

surprising

since

various

shown the same behavior a result

of a change

viscosity covalent

probes labeling

membrane (20).

enzymes

This

lipid

of the membrane

the features

of that

the changes

observed

behavior

in the membrane

of the membrane

change

in studies

lipid core

components

in the membrane

on the enzyme.

Although

614

already

has been usually phase (21)

(12-15,

20).

and some kind

(22)

could

help

and its

possible

all

the activators

published

have

interpreted

as

The use of of photoactive to elucidate relationship used here

with act

BIOCHEMICAL

Vol. 80, No. 3, 1978

at different energy,

but

tivation the

sites instead

energy;

components

subsequent

of the complex all this

physical

In conclusion, to cyclic

AMP with

to a conformational tion

of the

physical

(5,

23)

they

of them "stabilize" suggests

of the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

system,

changes

and this

the adenylate two activation change state

activators

tested

are able

when added

to the membranes.

the

the existence

of the

of the

do not modify system

of a high

the activation

with

the very

cooperativity

"stabilization"

same acamong

isolates

it

from

lipids. cyclase

catalizes

energies.

It

is

enzyme possibly

of the membrane. to stabilize

Also,

the conversion proposed

that

as a result it

is

this

is

due

of a modifica-

proposed

the enzyme conformation

of ATP

that

the

they

find

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