Alteration of enzymatic activities of rat liver diphosphatase by treatment with papain or urea

Vd. 12, No. 4,1963

ALTWTION

OF ENZYMATIC

DIPHOSPHATASE

BY

ACTIVITIES

TREATMENT

G. Mangiarotti Istituto

di Chimica

Istituto

Received

Universita

Biologica,

fructose-l,

that

Italy,

and

Fsrrara,

of sedoheptulose-1, This

appears

ly described

by Gomori

(2),

and more

(4),

Evidence

but its activity

has been

a single

enzyme

We have with

presented

now

with

Italy

that

in such

toward

the two

substrates.

of the modified

single

enzyme

work

and the Italian

SDP

protein,

protein

however,

was

by grants

R.

activity

Qnpresa

the two

of

also

catalyze

to sedoheptuloseFDPase

studied

original-

by Pogell

(3) and

been

reported.

supports

the conclusion

with

FDP

both

and SDP.

can be modified

as to change

On the basis

for

C. N.

(SDP)

strongly

a manner

is responsible

supported

will

had not previously

the enzyme

or urea

erties

liver

recently

for

papain

preparation

to be the specific

(1) which

is responsible

found

rat

7-diphosphate

(1).

McGilvery

purified

from

(S7P)

This

Genoa,

di Ferrara,

a highly

(FDPase)

‘I-phosphate

activities

OR UREA

di Geneva,

Universita

reported

6-diphosphatase

the hydrolysis

ment

PAPAIN

June 24.1963

We have-previously

that

WITH

LIVER

and S. Pontremoli

Biologica,

di Chimica

OF RAT

it would

by treat-

the relative

of the kinetic still

appear

propthat

a

activities.

from

Enzimologia.

the Rockefeller I1

Foundation

BIOCHEMICAL

Vol. 12, No. 4, 1963 The

enzyme

purification

described

(1) and were

from

and SDP.

FDP

lowed

with

the enzyme creased than

SDP

was

when

to very

the final

from

about Similar

the enzyme

treatment activity The enzyme

was

a change

observed

to those

treated FDP

returned

to about

lO%of

urea

at the levels

at low

of urea diluted

1 M.

was

The

to FDPaee

activities

was

obtained (370)

lacking.

In each

to FDPase

activities

was

at 37O occurred of urea

value with

sufficient

when (Fig.

2).

of urea (0.5M) as the concentration SDP

Again the modified

the original.

toward

temperature

concentration

activity

the activity

that

concentrations

the original

(30 minutes) shown

papain

in-

greater

were

of SDPase

with

activated

to about

of SDPase

with

effect

was

while

SDP

in

activities

papain

results

with

increasing

to approximately

increased

a ratio

with

with

at higher

in ratio

changes

with

was

in activity

were

changes

two-fold

level,

papain

1).

reduced

showed

increase

marked

and SDPase

Similar

with

fol-

com-

Substrates

produced

digestion

levels.

1.0 to 0.1.. (Fig.

was

was

low

was

(1).

the original

incubated

result

activity

of urea

was

the early

case

and then

to nearly

activity

Company.

approximately

longer

phosphate

wa% a recrystallized

FDPaee

levels With

FDPase

of 2O:l

both

out as previously

of inorganic

indicated

at a ratio At 26O,

returned

reduced

that

Chemical

activity.

the enzyme

except

Sigma

reaching

the original FDP

Papain

papain

markedly,

toward

of F6P.

RESEARCH COMMUNICATIONS

carried

studies,

as previously

activities.

were

on the liberation

the kinetic

from

or prepared

Digestion

was

based For

preparation

purchased

The

and assays

by the appearance

mercial

AND BIOPHYSICAL

of 0.1.

The

to stabilize

in 1 M urea enzyme time

of

the enzyme

in the figure. found

in the absence

to be reversible. of urea 306

When

the activities

the urea-treated returned

to es-

-0

MINUTES

40

60 MOLARITY

OF UREA

l

on diphosphatase activity. Aliquots d the enzyme Fig. 2. Effect of urea treatment preparation were incubated in the presence of different concentrations of urea for 30 The incubation minutes at 26’ and then assayed for FDPase (x) and SDPase ( ) activities. 0.1 M, pH 9.0; urea at the indicated concentmixtures (0. 5 ml) contained: TEA buffer, out in the same mixture by adding rations, angenzyme 15 y. The ?,says were carried FDP, 1x10M and MnClz, 1x10 M, or SDP, 1x10e3M and M&l2 3~10’~M.

Fig. 1. Effect of papain digestion on FDPase and SDPase activities. A purified preparation of diphosphatase (original activity - 740 units of FDPase and 770 units of SDPase pzr mg of protein) was incubated in the presence of papain at 37O (dotted lines) and tt.226 (full lines.) The incubaion mixture (0.15 21) contained: acetate buffer, 8x10 M, pH 5.5; cysteine, 5x10 M; versene, 3x10 M; papain, 15 Y/ml, and diphosphatase, 0.3mg/ml. Aliquots were collected and analyzed for FDPase and SDPase activities. Solid circles represent activities with SDP; x’s represent activities with FDP.

20

Vol. 12, No. 4, 1963 se&ally was

BIOCHEMICAL

the original

again

1.0.

values

This

AND BIOPHYSICAL

and the ratio

reversal

required

RESEARCH COMMUNICATIONS

of activities

with

incubation

for

SDP and FDP

lo-20

minutes

at

26’. Kinetic view

that

These

identical

witb

a single

studies

possessed

with

studies

protein

were

that

the modified

ratio

was

We have

previously

with

as a competitive

SDP when

Native

in the enzyme with

urea.

with

0. 85x10-5M

KS (SD@

0. 31x10-4M

Ki

0.34x1

which

found

The

to be

Ki for

SDP

on the hydrolysis the untreated enzyme,

enzyme. identical

as a substrate

or

of FDP.

of the Protein Urea Treated

0.90x1

0.32x1

to determine

by digestion interest 308

o-5h4

w--..-

0-4M

in progress

It is of particular

1).

the native

Enzyme

KS (FDP)

produced

was

effect

Constants.

Native Enzyme

protein

activities.

I

-Menten

(SDP)

FDP

with

of the hydrolysis

and Modified

are

its

the previous

enzyme

it is employed

constant

studies

enzymic

the Ks determined

for

Michaelis

the two

(Table

from

(1) that

inhibitor

support

Ks for

enzyme

Table

Further

The

of 0.1.

reported

obtained

for

protein

the urea-treated

of the untreated

identical

are

out with

enzymqcalculated

of FDP,

con$tants

enzyme

is responsible

carried

an activity with

the modified

that

with

0-*M

the nature papain

the affinities

of the changes

or by treatment for the substrates

BIOCHEMICAL

Vol. 12, No. 4, 1963

appear The

to be unchanged encyme

appears

but to be capable litic

control

these

the marked

to possess

of being

activities.

hexose,

despite

AND BIOPHYSICAL

Since

FDPase

changes

suggest

changes

a unique

modified

RESEARCH COMMUNICATIONS

binding

differentially

is a key enzyme that

in ensyme

it may

site

for

with

respect

activities.

FDP

and SDP to its

in the biosynthesis

be a site

for

cataof

a metabolic

mechanism.

3

1.

Bonsignore,

A. i Pontremoli,

and De Flora, 2.

Gomori,

3.

Pogell,

A.,

G.,

J.

J. Biol.

B. A&.,

Biol.

S. , Mangiarotti, Chem.,

Chem.,

148,

and McGilvery,

R.

G. , Mangiarotti,

M. ,

in press. 139 (1943). W.,

J.

Biol.

Chem.,

208,

149

(1954). 4.

Mokrasch,

L.

C.,

and McGilvery,

R.

(1956).

309

W.,

J.

Biol.

Chem.,

221,

909