The roles of glucose and AMP in regulating the conversion of phosphorylase a into phosphorylase b

Vol. 46, No. 1, 1972

BIOCHEMICAL

THE ROLES REGULATING PHOSPHORYLASE

J.

Martyn

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OF GLUCOSE AND AMP IN THE CONVERSION OF a INTO PHOSPHORYLASE b

Bailey

and

W.J.

Whelan

Department of Biochemistry, The George Washington University Medical Center, Washington, D.C. 20005 and

Department of Biochemistry, University of Miami SchooZ of Medicine, Miami, Florida 33152

Received

November

12, 1971

S UMMARY: The ability of glucose to assist the action of phosphorylase a phosphatase on phosphorylase a, appears to be mediated through conformational changes in the phosphoryl ase. The system is an example of control of enzyme activity by regulation of the conformation of the substrate, and parallels the similar effect of AMP in inhibiting the dephosphorylation. INTRODUCTION Phosphorylase the

“inactive”

The

best

b form studied

inhibits

a phosphatase by

examples

this of

It

was

thereby

of

the

substrate

AMP

effect,

than

lase

a.

to AMP

to

muscle

(3)

and

liver

is

to

an

from favored

have

0 1972 by Academic

AMP

the that

(4)

the

interaction

of

glucose

latter effects

tetramer

on

the

formation

while

Press, Inc.

dephosphory-

the

promotes a.

with since

state

of

AMP

phosphorylase

alter

phosphatase,

possibility

(1).

a.

conformation

(2).

phosphorylases of

the

to

enzyme glucose

rate

from is

residue.

muscle

the

a into

a serine

rabbit

affect

activation

opposing

promotes

of

191 Copyright

not

from

from

isolated

inhibit

reported

due

does

phosphorylase

group

are

action

was

results

also

the

it

evidence

known

that

rather

this

stantial

enzymes

tetradecapeptide

concluded

of

whether

these

a phosphate

but

a phosphorylated

Recently both

of

of

dephosphorylation,

lation

sion

cleavage

“active”

converts

of glucose

the We have or

the

a + b converinvestigated

rather,

like

substrate. AMP

and

Ci rcumglucose

aggregation favors

the

of the

are phosphory-

dimer

(5).

Vol. 46, No. 1, 1972

Furthermore,

BIOCHEMICAL

the

temperature

(5),

at

phosphorylase

form

which

temperatures

of

glucose

cleavage

by

AMP

both

being

or

assisting

variety

glucose

appears

to

have

effects

of

glucose

positive piece

of

is

at

evidence

phosphorylase

chymotrypsin

study

learned

colleagues

have

MATERIALS

confirms

rabbit

AND

is

enhanced

the by

physiological no

effect were

implicating

the

observation

that

glucose

on

(7)

and

noted actions

opposed

by

that

in

an

come

to

a similar

mlJ)

of 1 ml

Pi

protein

(10).

of

and

remaining

by on

I mU of

convert

1 mU

the

enzyme

NaCl was

(approx

25

gradient eluted

53 mU per

phosphatase protamine

in

protease

prepared

treatment

glucose

in

phosphorylase. Hers

and

his

with

(25ug)

dried

on

filter

washing

in

50%

dried

2 X crystallized

phosphorylase

b kinase

activity

with

of

aqueous was

range The activity

(1X3

ethanol,

and

determined

defined

as

phosphorylase

that a

was on

amount into

purified

b per from

salt,

enzyme was

essentially

tested

casein.

192

and

against

the

~1)

were The

liber-

32P-label

liquid

led

scintillation enzyme

which

will

min. rat

1 iver

The

bulk

specific free

by

containing

cm).

of

DEAE-cellulose.

0.28-0.32~

when

by

assayed

8.16, (100

strips

and

phosphatase

pH

Samples paper

phosphatase

was

phosphorylase buffer,

strips is

ng) a

the

from

phosphatase

chromatography

mg protein. and

and

H.G.

1OmM mercaptoethanol.

phosphatase

Phosphorylase by

of

(9).

tris-glycerophosphate and

removed

counting.

effect with

Dr.

a was

Phosphorylase

0.02M

intervals

was

study,

phosphorylase

acetate

at

the

interaction

conclusion

a by

labelled

3mM magnesium removed

that

by

phosphorylase

ATP

in

indeed

independent

phosphorylase

y-32P-labelled incubation

inference

METHODS

muscle

ated

the is

32 P-Labelled

was

on

dephosphorylation

We have

ates

exclusive

(8). This

(24

The

A final

30”.

trypsin

the

(6).

below and

is

temperature

inactivation

at

AMP

dimeric

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

from

phosphorylated

homogenof

the

activity non-specific histone,

Vol. 46, No. 1, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RESULTS When was

inhibited

the 90%

phosphatase by

0.1

acted

mM AMP,

on

phosphorylase

16.7 mM glucose

while

15” the

a at doubled

react

the

ion

rate

of

200+ AMP

I 40

0

I 80

I 120

I 160

TIME (minutes)

Fig. 1. Influence of glucose and AMP on the action of phosphorylase j2P-Labelled phosphorylase a was incubated phosphatase on phosphorylase a. with 16.7 mM glucose or 0.1 mM AMP at 15” with phosphorylase phosphatase, added as indicated. Samples were removed at intervals for determination of the 32P-labelled protein remaining (see Materials and Methods).

reaction

(Fig.

I).

Other

During ratio

of

its

constant

the

glucose

effect

was

When charcoal at

treatment,

37”.

AMP

still

(Fig.

2).

Only

at

overcome

levels

the

AMP

Paralleling phorylation is

tetrameric

were at

Is”,

no

on

absence

was

effect.

of

DEAE-cellulose, glucose

unlikely

a was by the

the

freed

glucose

enzyme fact

at

that

10 uM AMP,

that

the

system. completely

was

maximal

37”

and

the

the

remained

therefore

phosphatase-activating

below

sugar however,

from at

15-20’

a glucose

by and

absent

effect

opposed was

AMP

the

at

AMP

glucose

37”

inhibition

able

complete

inhibition. the

the

It

phosphorylase

from

and

profile.

a separate

or

phosphatase

presence

inhibited

demonstrated

1 ittle of

stimulation

be

to

the

enzyme on

the

could

ly

in

the

had

fractionation

activity

across

sugars

effects

ultracentrifugal wholly

of

glucose

and

observations dimeric

at

35”,

193

AMP that

and

dimer

on

the

rate

of

phosphorylase appears

at

dephosa

15”

if

Vol. 46, No. 1, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

240

4

6

AMP (lo-’

M)

Fig. 2. Restoration of the glucose sensitivity of the dephosphorylation of phosphorylase a at 37” by addition of AMP. The activity of the phosphatase was measured in the presence and absence of 28 mM glucose, with increasing amounts of AMP. The j2P-labelled phosphorylase had been treated with charcoal to remove AMP. See Materials and Methods for conditions.

Table

1.

Influence behaviour

of of

added a

glucose

on

the

Component(s) (dimer)

S1

sedimentation

observed S2 (tetramer)

15"

none

none

15.3

15"

g 1 ucose

9.8

14.7

23

g 1 ucose

9.8

13.9

35"

none

9.0

none

35"

g 1 ucose

9.1

none

35"

glucose

9.5

none

The solvent plus 0.01 coefficients

glucose

the

and

Additions

Temperature

in

temperature phosphorylase

was the phosphatase assay M NaCl, with 55 mM glucose in are-corrected to 20’

is

added

concentrations

(Fig. at

phosphorylase

was

32P-Labelled labelled

+ AMP

phosphorylase

3,

Table

which wholly

peptides a with

At 35",

1).

AMP

inhi

dimeri

buffer (see and 1 mM AMP water.

bition c were

of

and

AMP is

present manifested,

1 1.

prepared

194

glucose

and Methods) Sedimentation

dephosphorylation

(Table

chymotrypsin

with

Materials as noted.

by or

trypsin.

brief

treatment Neither

of glucose

nor

Vol. 46, No. 1,1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Influence of temperature and added glucose on the sedimentation Fig. 3. A, sedimentation of phosphorylase of phosphorylase a in the ultracentriguge. Lower curve, plus 55 mM glucose. Speed a at 15”. Upper curve, control. Movement from left to right. D and T 60,000 rev./min. Time 18 min. indicate the positions of the dimeric and tetrameric forms of phosphorylasee a. B, Sedimentation of phosphorylase a at 35”. Upper curve, phosphorylase Lower curve, phosphorylase b as reference. Note that phosphorylase u a. behaves as a tetramer at 15” but is completely dissociated to dimer at 35”. Addition of glucose at 15” causes the appearance of dimer (see also Table 1).

AMP is

affected

their

already

rates

known

(2,

of

dephosphorylation.

This

lack

of

effect

of

AMP

11).

DISCUSSION The that

the

effect

like

that

of

not

AMP

the

This

dimer

opposition at

ultracentrifuge

the

tetramer

37”

se

of

the (Fig.

on

on

the

was

that

the

is

2), shows

glucose

well

not

a substrate.

when

no

only

the

to

produce

glucose effect dimeric

was

was

and

shown

AMP on

of

glucose form

195

phosphorylase,

to

produce

dimer,

2,

is Table

a

a substrate.

cannot the

was

fact

be

the

case

that

dephosphorylation alone

(Fig.

not it

from

is,

however,

tetramer,

a substrate,

This

inference

inference,

effect

be

original

of

Another

was

of

our

dephosphorylation

the

very

effects

confirmed

substrate.

AMP effect may

per

experiment

glucose (2),

while

While

seen

of

sustained.

substrate,

that

last-mentioned

an can

noted, 1).

and

be

when

We have

the to

Vol. 46, No. 1, 1972

conclude and

that

there

stabilized

else

effect

of

ble.

When,

AMP,

the

effects

any

physiological

range In

early

AMP of

it

from

as

one

expose

of

can the

in and

(Fig.

inhibiting

trypsinolysis

include

the

resistant

opposing for

the

the

regulation

a substrate. of

glucose

reaction

is

glucose phosphate

the

b,

negligi-

We

producing in

the

mM).

serine

explaining

The

a and

envisage

It

2).

any is

mechanism

concentration

action.

a parallel

enhancing

products

terms

that form

phosphorylase

(4-5

rapid

of

a significant

the

At susceptible

stabilization

dephosphorylation glucose

to

in

so

the

susceptible

its

phosphatase,

glucose. in

is

phosphorylases

the

blood

phosphatase

finds

to

of

the

the

37")

point

level,

all

forms

to

to

by

demonstrated

changes

for

a so

be

levels

molecular

phosphorylase

respectively

to

resistant

either

two

change at

metabolites

given

stimulation

also

effect

is

the

present

relative

half-maximal

shield

is

one

stabilized a

the

conformational

at

dimer,

susceptible,

promoting

two the

on that

model

one

the

between

AMP

these of

depending

in

a glucose

regulation

to

and

of

phosphorylase

however,

of

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

forms

equilibrium

glucose

permits

noted

two

temperature

or

form

are

by

physiological form,

BlOCHEMlCAL

is

significant

the

effects

effects

as

reacting

group,

and that

of of

AMP

so

this

as

crude

glucose

trypsin,

phosphorylated

with

and since

AMP the

(8).

peptides

ACKNOWLEDGEMENTS This AM

10082

of

absence

and

work

was

AM 12532, at

the

supported and

was

University

by

U.S.

carried

of

Public

out

while

Health J.M.

Service Bailey

Grants was

on

leave

Miami.

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E.H.,

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and

Saari,

J.C.,

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3.

Holmes,

4.

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Novoa,

W.B.,

Krebs,

E.G.

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E.H.,

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Mansour,

P.E.,

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Acta

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H.,

Lederer,

B.

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196

and

Hers,

H-G.,

E~rop.

J.

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5.

Wang,

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Shonka, ~8:

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H.N.

and

Graves,

A.J., Met&oZism,

J.M.,

M.L.

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M.,

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H.G.,

Biochem. Biophys. Res. Cornmun., in E.G. N-0.

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