Differential response of AMP deaminase isozymes to changes in the adenylate energy charge

BIOCHEMICAL

Vol. 85, No. 2, 1978 November

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

29,1978

Pages

DIFFERENTIAL

RESPONSE

CHANGES

IN

THE C.

Department University Received

OF

AMP

ADENYLATE

Solano

2,

ISOZYMES

ENERGY

and

C. J.

of Biochemistry, of Pittsburgh,

October

DEAMINASE

564-571

TO

CHARGE

Coffee

School Pittsburgh,

of

Medicine, PA 15261

1978

Summary AMP deaminase has been prepared from white skeletal muscle fibers, red skeletal muscle fibers, cardiac muscle and liver. The isozymes from skeletal muscle, cardiac muscle and liver can be readily distinguished from one another by the shape of the adenylate energy charge response curve. However, the enzyme prepared from different skeletal muscles which consists of predominately red or white fibers are indistinguishable from one another by this criterion. AMP

deaminase

intracellular

activities

charge tides

is

(1)

and

the

through studies

specific

isozymes

in

in as

of (6)

forms

have

which

Moreover, in

rat

differ

--et

types al.

(6)

B and

three

distinct

one

their in

of muscle suggest

the

another

was that

muscle

to

AMP and

these

their

forms

have to

two

forms

observations of

AMP

this

the Raggi

deaminase

kinetic

of

been

isozymes,

muscle

isozymes

564

of tissue-

The

tissue-specific

These

ooos-29ur/7a/oa52-0564gn.o0/0

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

existence

In addition

of

nucleo-

(2-4).

respect

cardiac

chromatographic

different

cycle

several

energy

inosine

properties.

skeletal

observed.

of

adenylate and

with

C respectively.

distribution

the

the

and

and

regulation

nucleotide

demonstrated

liver

the of

immunological

A,

both

in

guanine

purine

have

fractionated in

to

the

muscle,

further

important stabilization

from

and

skeletal

these

(5)

differ

a difference

various

Raggi

the

isozymes

identification

be

of adenine, in

which

found

al.

the

kinetic

designated

--et

including

participation

chromatographic, enzyme

to

interconversion

its

Previous

believed

into

properties. of

the have

deaminase

enzyme led exist

two

BIOCHEMICAL

Vol. 85, No. 2, 1978

in white

and

identical

to In

red the

muscle

fibers

isozyme

found

this

communication,

white

muscle,

as

these

preparations

adenylate AMP

liver

distinguished on

function

the of

tinguishable muscle

basis

of

energy

fibers

the

identical

the

cardiac have

prepared

results

red

and

the

enzyme

their

in

red

of white

and

these

muscle

found

in by

However,

the

deaminase

found

from

liver,

have

to changes

is

muscle

measuring

the

can

by

this

and

that be

muscle activity

chromatographically red

examined

to the

fibers cardiac

in

and

indicate

either

two

red

and

experiments

skeletal

another

enzyme

response

generated

one

found

the

muscle

in

The

to

type

muscle.

cardiac

profile

of AMP

AND

Materials. and were for these femoris

both

charge.

are

that

differences

from

forms

MATERIALS

killing taken biceps

from

in

from

charge.

deaminase

readily

as

for

energy

and

we

well

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

or as

a

dis-

white

skeletal

criterion.

METHODS

Tissues were taken from an adult rabbit freed of fat and connective tissue. The experiments were the anconeus (predominantly (predominantly white).

shortly skeletal

after muscles red) and

the

The method used for the purification of Purification of the Enzyme. rabbit liver AMP deaminase was identical to that reported by Ogasawaea (7). The purification of the enzyme from skeletal and cardiac muscle --et al. followed the procedure described by Raggi et al. (6) with a few minor Samples of muscle were m=edand homogenized in 2.5 modifications. volumes of cold buffer containing 20 mM potassium phosphate, 0.1 M KC1 and 1 mM 2-mercaptoethanol (pH 7.0). After standing on ice for 50 min, the homogenate was centrifuged at 20,000 x g for 30 min. The supernatant was absorbed onto a column (1 x 15 cm) of cellulose phosphate which had been previously equilibrated with 40 volumes of 20 mM potassium phosphate buffer (pH 7.0) containing 0.2 M KC1 and 1 mM 2-mercaptoethanol. After loading the sample, the column was washed with 100 ml of the equilibration buffer prior to elution. The enzyme was eluted with a linear gradient consisting of 50 ml each of 0.2 M KC1 and 2.0 M KC1 both in buffer containing 20 rnM potassium phosphate and 1 mM 2-mercaptoethanol (pH 7.0). Enzyme

Assay.

Unless

otherwise

indicated,

enzyme

activity

was

determined spectrophotometrically using a Gilford 240 spectrophotometer The amount of AMP converted equipped with an expanded scale recorder. to IMP was monitored by following the increase in absorbance at 285 nm as a function of time. The absorbance changes were converted to units of

565

BIOCHEMICAL

Vol. 85, No. 2, 1978

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

concentration using a A mM of 0.23 (8). The standard assay used in monitoring the enzyme from cardiac and skeletal muscle was performed cuvettes of 1 cm light path and with a total volume of 1.0 ml containing 50 mM imidazole-HCl, 100 mM KC1 and 2 mM AMP (pH 6.5). For monitoring the enzyme from liver throughout purification, the generation of ammonia was measured by coupling it to the glutamate dehydrogenase reaction and following NADH oxidation at 340 nm as previously described

in

(1).

Generation of Energy Charge Ratios. Mixtures of ATP, ADP and AMP corresponding to varying energy charge ratios from 0.9 to 0.1 were generated as described below. Mixtures of AMP and ATP were made in proportions ranging from 1 part AMP/9 parts ATP to 9 parts AMP/l part ATP respectively. The total concentration of adenine nucleotides was kept constant at 10 mM, and MgCl2 was added to give a final concentration of 25 mM. Adenylate kinase (1.0 wg, Sigma Grade III, 1400 units/mg) was added to 10 ml of each of the AMP/ATP mixtures, and the reaction was allowed to proceed for 2 hr at 30”. This amount of time was more than sufficient to achieve an AMP/ADP/ATP equilibrium mixture. The concentrations of AMP, ADP and ATP at equilibrium were determined directly, and the individual nucleotide concentrations were found to be within S% of those predicted from the initial AMP/ATP ratio and the equilibrium constant for the adenylate kinase reaction, RESUL

I’S AND

The red

elution

skeletal

cellulose which

profiles

muscle

consists

(Fig.

the

elution

considerably muscle. distinct from results

Not from

is the enzyme

that

in white muscle by Raggi

deaminase

The

muscle, isozyme. et al.

in red

by 0.64 anconeus

from

the enzyme

from

properties

These

data

are

they

In

both precede

of activity

muscle

peak

muscle

concentration

skeletal

femoris

M KCl.

a salt

but its

566

and on

biceps

which

peak

M KCl.

(6) in which

from

shoulders

to elute

white

as a symmetrical

from

major

0.56

required

only

the cardiac reported

that

enzyme

the resin

shows

from

by chromatography

elutes

from

fibers,

with

The

fibers

for AMP

is eluted than

1.

is released

in red

preparations obtained

in Fig.

peak of activity.

muscle lower

muscle

of white

profile

is rich

the main

anconeus

deaminase

shown

1A) which

1B) which

and follow

are

predominantly

(Fig.

contrast,

of AMP

and cardiac

phosphate

of activity

red

DISCUSSION

the

white

chromatographically are

different concluded

also

different from

that

the the major

BIOCHEMICAL

Vol. 85, No. 2, 1978

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

1.0

160

05

40

60

60

.

IO

c; / El 2

05:

IO

0.5 c20

40

VOLUME

OF

Fig. 1. of AMP muscle Methods.

Comparison deaminase (C). Details

of chromatographic from rabbit biceps of the experiment

form

enzyme

red

As

of can

from the

be red

seen

( 10%)

a salt

concentration

cardiac

muscle. order

of

muscle

is

comparing is

found

portion

representative

by

muscle

enzyme

In

in

to

to

in

cardiac

muscle.

the

total

insure of

the

that total

AMP

(ml)

properties femoris (A), are described

to and

cellulose

the

on cellulose phosphate anconeus (B) and cardiac under Materials and

heart

Fig.

muscle

lC,

most

phosphate We

do,

deaminase to that

the

EFFLUENT

1B

adsorbed

identical

1 60

identical

Fig.

AMP

I 60

however,

activity

required

elution

567

in

red the

shown activity

the

more

in in

the

enzyme tightly

observe

to elute

profiles

deaminase

much

of

isozyme.

that

muscle

elutes

enzyme

Fig. crude

from

1 were extracts,

than a small with

BIOCHEMICAL

Vol. 85, No. 2, 1978

AND BIOPHYSICAL

TABLE RECOVERY

OF

DEAMINASE

Red Skeletal (Anconeus)

each

in each

when

the fractions

each

column,

40

35

87

on the same generated

column for

Therefore,

1.1

muscle cellulose

all

enzyme

and under three

samples

were that

muscle,

red

muscle

and cardiac

another

with

respect

to their

it seems tissue

likely

energy

that

to tissue

a particular been

of the presence

suggested charge

for within

the AMP

this

enzyme the cell.

Therefore,

568

shown

profiles in Fig.

activity

from

distinguishable

isozyrnes of this

from

of AMP

enzyme

metabolic

physiological

is to regulate

for

1. white one

properties.

by unique

One of the several

in the

separately

the elution

to those

all

of tissue-specific role

for

and rechromatographed

deaminase

are

of the

Furthermore,

pooled

conditions,

ahromatographic

be dictated

be accounted

were

strength,

muscle

carefully

chromatography.

identical

the physiological

and may

tissue.

ionic

was

80 and 90 percent

could

activity

the same

it can be concluded

In view

extracts

91

columns

1, between

phosphate

to the proper

1.0

phosphate

in Table

containing

diluted

Percent

Recovery

42

of the cellulose

from

Units Recovered from Column

to

83

of the crude

recovered

PHOSPHATE

50

As can be seen

fractions

Units Applied Column

1.5

from

monitored.

CELLULOSE

60

Muscle

the recovery

activity

FROM

80

Skeletal Femoris)

Cardiac

1

Total Units in Crude Extract from 10 g Tissue

Type of Muscle

White (Biceps

AMP

RESEARCH COMMUNICATIONS

vary

patterns functions

or stabilize we decided

may

deaminase, from within which

the adenylate to examine

the

has

Vol. 85, No. 2, 1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

100

WHITE MUSCLE RED MUSCLE CARDIAC MUSCLE

0

0.2

04

06

ENERGY

08

I

CHARGE

Response of AMP deaminase isozymes to variations in the adenylate Fig. 2. The assay mixture contained 50 mM imidazole-HCl (pH 6.5), energy charge. 100 mM KC1 and adenine nucleotides at a total concentration of 4.0 mM and at the energy charge values indicated on the abscissa. The enzyme used was prepared from rabbit white skeletal muscle (A), red skeletal muscle (o), cardiac muscle (D) and liver (e).

activity the

of

the

enzyme

adenylate

energy

summarized

in

skeletal

muscle,

another

by

presence

Fig.

2.

the

shape

variation other

in hand,

and

size

activity

with

the

enzyme

linearly

as

then

curve

from

continues

zero

a function

these

these

liver

data

can

generated ADP

and

be

by AMP

which

muscle

enzyme

shows

energy

charge

between

zero

energy

charge

to increase

and

decreases more

569

slowly

in

the

of 4.0

mM.

virtually and

from

approximately maximum

no On

0. 9.

muscle

the

energy

experiments,

white

until

one

activity to

a concentration

red

from from

correspond

of these

in

enzyme

the

cardiac

both

the

distinguished

the

from

at

that

kept

the

constant

alterations are

measuring

In all

to one.

of

experiments

was

activity the

of

from and

of ATP,

conditions,

increases to 0. 7,

the

as

results

clear

muscle

of

pool

these

It is

ranging

adenylate

tissues

The

cardiac

values

Under

various

charge.

of mixtures

charge total

from

the

fibers 0.9 activity

BIOCHEMICAL

Vol. 85, No. 2, 1978

is

reached

at

preparations throughout liver

energy

of

enzyme

the is

response

muscle

isozyme

showing

These

the

to be

muscle

and

skeletal

liver..

On

one

clearly,

fibers

behave

The

muscle

0. 65.

very

charge

values 0. 5 and

from

energy

shape

of

from

below

identically

at

the

different

the

isozyme

isoeyme

However,

is

at

Quite

values.

skeletal

that

0.6,

with

decreasing

shape

of for

the

other

can

phosphate,

they

the

rapidly

energy

the of

the

the

liver

at

lower

although

the from

functionally

from

fibers

and

or in

white

predominantly the

energy

muscle

AMP

on

columns

of

similar

or

identical

another very

curve cardiac

preparations

red

either

response muscle,

alterations

one

distinguished

charge

red to

immunologically

easily

skeletal

enzyme

respect

the

and

from

the

separated

which

quickly

predominately

that

are

be

enzyme

with

be

by

adenylate

hand,

either

suggest

activities

can

the

identically

data

criterion

deaminase

containing

These

cellulose

the

additional

different

behave

deaminase

an

The very

fibers

charge.

muscle

charge

activity

of AMP

muscle

white

energy

energy

0.2.

white

isozyme,

at

provide

another.

appears

and

about

RESEARCH COMMUNICATIONS

values.

isozymes one

liver

maximum

data

distinct

of

approximately

enzyme

charge

from

of from

for

skeletal

red

range

than

curve

value

from

entire

greater

energy

charge

indistinguishable

charges

to

an

AND BIOPHYSICAL

another.

ACKNOWLEDGMENTS This Association Institute

work

was supported in part by a grant of America and by Research Grant of Arthritis, Metabolism and Digestive

from the AM 16728 Diseases.

Muscular from the

Dystrophy National

REFERENCES 1.

Chapman, 8309-8312.

2.

Setlow, 1245.

A.

B.

and

G.

and

Atkinson,

Lowenstein,

D.

E.

J.M.

(1973)

(1967)

570

J.

J.

Biol.

Biol.

Chem.

Chem.

248,

241,

1244-

BIOCHEMICAL

Vol. 85, No. 2, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

3.

Tornheim, 3241-3247.

K.

and

Lowenstein,

J.M.

(1974)

J.

Biol.

Chem.

249,

4.

Tornheim, 6304-6314.

K.

and

Lowenstein,

J.M.

(1975)

J.

Biol.

Chem.

250,

5.

Ogasawara, Acta 403,

6.

Raggi,

7.

Ogasawara, Biochem.

8.

Smiley, J. Biol.

N., 503-437. A.,

Goto,

H.

Bergamini, N., Biophys.

K. L., Chem.

C.

Goto,

H., Res.

Jr., 242,

and

and

Watanabe,

Ronca,

Yamada, Comm. 79,

Berry, A. J. 2502-2506.

and

T.

G. Y. and 671-676. Suelter,

(1975)

(1975)

Biochim.

FEBS

Yoshino,

C.H.

Biophys.

Ltrs. M.

(1967)

58, (1977)

19-23.