Clostridium perfringens polynucleotide phosphorylase: Two molecular species

Vol. 28, No. 2, 1967

BIOCHEMICAL

AND BIOPHYSICAL

CLOSTRIDIUM

RESEARCH COMMUNICATIONS

PERFRINGENS

POLYNUCLEOTIDEPHOSPHORYLASE: TWO

MOLECULAR

G.W.Dietz,Jr Institut

SPECIES

. , and M. Grunberg-Manago

de Biologic

Physico-chimique

, rue

P . Curie,

an

ubiquitous

Paris,

France

Received May 2, 1967 Polynucleotide among

phosphorylase,

bacteria,

catalyzes

polynucleotides

from

catalytic

different

of the

the

anaerobic

With

this

is stimulated extent activity

towards have

one active during was

the given

a stepwise fered

quite

extent

. It was also

of stimulation been form,

nucleotide exist

elution

of the

structure

enzyme

has

been

prepared

1961,1962).

(Dolin, the formation

of poly

by salt

or by polybases

during

these

studies

A

such that

the

or salt and the phosphorolysis

A changed

during

existence

the

of the of some

purification enzyme

. This

in more

activator

than

or inhibitor

Data

supporting the first alternative two fractions from et al. (1963) w h o obtained

by Knight

of the

markedly

following

enzyme

in their

from

relative

experiments which

for when

demonstrate

activity forms

phosphate activity

polymerization,

phosphorylase

in two different

either

or to the removal

preparation.

examined, diphosphates

details

perfringens

observed

due to the

and polylysine stimulated adenine derivatives. The

of this

by polylysine poly

extensively

phosphorylase

to a large

isolated

unknown.

Clostridium

polynucleotide

as polylysine

of the

remain form

enzyme,

fromnucleoside

most enzyme

of this been

synthesized

A most interesting from

could

have

beencarefullystudied,

and function

of

np + (Np),

properties

bacteria,

and the polynucleotides have

polymerization

C====z==========Eh

the general

several

reversible

:

,ppN Although

the

enzyme

gel

which

dif-

phosphorolysis tested that

with the

poly-

of Cl does indeed - .perfringens now been clearly separated.

have

146

the

Vol. 28, No. 2, 1967

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

3001 ‘ 2 .

1

Polymerization with polylysine\j

I

I

I

I I

I

I

200

15

17 Frartion

L

19 number

21

% : loot

/

\

To r, \

9

\

Ad’

--o---.7

-j

20 Fraction

Figure Activiilrofiles ----

---w--B

25

number

1

forpoly -

-- A -thesis -------------- after centrifugation -------

sucrosesradient-----

0.1 ml of the enzyme preparation was layered on a sr20% sucrose density gradient (Martin and Ames, 1961) made up in a buffer containing 0.02M Tris-HCl, O.OOlM EDTA, and 0.007 M g-mercaptoethanol, pH 8.3 at room temperature, and centrifuged for 12 hours at 36,000 rpm in a Spinco SW 39 rotor at O-5°C. The assay for the enzyme activity by the incorporation of radioactive ADP into acid insoluble precipitate was essentially that of Knight et a1.(1963). The complete system including 0.05 ml of the enzyme fraction from the gradient (final volume 0.150 ml> contained the following reagents in mMolar concentrations . Tris-HCl, pH 8.3 at room temperature, 100; MgC12, 5 ; ADP - l4C (specific activity 23,000 cpm/pmole) ,4; and ,%mercaptoethanol, 1. When included, the polylysine (MW 3,000) concentration was 280 pg /ml, the ammonium sulfate 0.02 M. Insert shows magnified view of low molecular weight region for polymerization with no addition. Fig. centrifugation column

step,

A in the When absence lysine,

1 presents

of the enzyme Fitt

presence

the enzyme

of a sucrose

(purified

et a1.0967))

the more

assayed

through

assayed

of polylysine. was

of polylysine,or only

the results

There for

ADP

in the presence rapidly

sedimenting

147

for

density

gradient

the DEAE-cellulose the

synthesis

are two peaks

of poly of activity.

polymerization of salt material

in instead was

the of polyactive.

BIOCHEMICAL

Vol. 28, No. 2, 1967

In addition, menting

as can component

be seen

AND BIOPHYSICAL

in fig.2,

is active

only

RESEARCH COMMUNICATIONS

the

more

in the phosphorolysis

rapidly of poly

sediA.

/I / I

Polymerization with polylysine

f I I

-/

I I

I

12 Fraction

16 Number

Figure

20

24

28

2

Activixtrofiles ---------- for the synthesis andphosphorolysis ----------------- of25 -- A after sucrosegradient --------m- centrifugtion ---a------Sucrose density centrifugation as in fig. 1 for 12 hours, 30,000 rpm. Phosphorolysis activity was assayed b the standard procedure as reported by Knight et al. (1963 7 The

light

material,

therefore,

has

an absolute

requirement

for

polylysine -- which cannot be replaced by salt _- for the polym merization of ADP, and does not show any phosphorolysis activity. This component also requires &mercaptoethanol: when a gradient was run in the absence of this compound and tested, only the

heavy

peak

appeared

&mercaptoethanol of the further

light peak. investigated

to the incubation The dependence

ethanol-free

gradients,

of the

enzyme

light

g-mercaptoethanol, is slightly

in the

using

pooled

as is

highly

the

heavy

show

activity

fraction in

material

148

Addition

of

mixture restored the activity on this SH compound was fig.3.

dependent

inhibited.

profile.

from

g-mercapto-

While upon

the activity

the presence

is not and,

if anything,

of

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 28, No. 2, 1967

2

0

4

Concentration

of

6

8

p-mercaptoethanol

10

(M x 10m3)

3

Figure

Effect of /3-mercaptoethanol on the activity -----------------------------------fractions ---me--

of the two

enzyme ---

The enzyme preparation was fractionated on a sucrose density gradient as in fig. 1, except that the buffer in the gradient lacked B-mercaptoethanol. The light and heavy fractions were separately pooled and tested in the presence of polylysine, 280 pg/ml, for poly A synthetic activity. heavy light These

two poly

peaks

of ADP

into

is run

on the

menon

is not an artefact

sucrose

Furthermore, enzyme

A have

these

fraction fraction

of polylysine-stimulated

incorporation

also

crude

been found

density

gradient,

resulting two

preparations

when the suggesting

from

peaks

have

extract

that

the purification been

originating

from

weights

of the two

shown

different

the

pheno-

procedure. in two

batch

different

cultur.es

of

the bacteria. The ated

from

included heavy has

molecular a gradient

as marker species

a molecular

species enzyme

is quite (Williams

centrifugation with

gives weight

the enzyme

a value

with

The

have

alcohol

(Martin

of 192,000

of 62,000.

peaks

been

calcul-

dehydrogenase

and Ames,

while

the

light

value

for

the

1961). The material heavy

similar to those calculated for Escherichia coli and Grunberg-Manago ,I9641 and for the enzyme

149

Vol. 28, No. 2, 1967

BIOCHEMICAL

isolated

from

1963).

Although

molecular

Micrococcus

to the light

lysodeikticus

there

weight

AND BIOPHYSICAL

has

been

found

(Thang

unpublished). lead

here,

weight the

report

this

a study

of a low

corresponding

with

the

enzyme

of the

recently,

enzyme may

smaller

from

exist

as the

species

retain-

and Grunberg-Manago of a mutant

,

of --E.coli has a molecular

of an altered

enzyme

which

but

which

in this

case

(Thang

, Thang

and Grunberg-Manago

100,000,

phosphorolysis

that some

Schenkein

discovery

of about

studies

, Godefroy,

Most

to the

and O’Brien,

phosphorylase

suggest the possibility --E.coli aggregate of sub-units, with ing activity

(Singer

no previous

polynucleotide

material

RESEARCH COMMUNICATIONS

reaction

has

catalyzes

only

1967). Seen

in the light

that

the

from

may vary -Cl .perfringens molecular species present.

two

properties

of the

(1961,1962)

phoresis,

Dolin properties

ponent

that

his

relative

possible

block

light

peak

electrowhich

found

preferentially

for

observed

Whether sub-unit

that

affinity

two fractions

of the heavier experiments

had

here,

yielded

of activity,

corresponding

and it

this

derived

from

com-

tions

for

component it will Although

protected

be important

the

into

an open

point.

material

was gave

in dilute may

as yet

whether

in the

effect

sub-units

exposes

structure

150

have

the

optimal

found.

the

the

and anysub-

therefore

lf the weight

it is active to form

re-

one peak

However,

200 000 molecular

reaction

pooled,

solution

been

a Prelimi-

only

material.

the above.

represents

or alternatively, not

in

explain

species

as such

heavier

been condismall enzyme, or whether

component.

of fLmercaptoethano1

be taken as evidence it is possible that the

tertiary

would

gradient,

material

of the

difference

by the

on the

to show

during

molecule

the heavy

have

two components might sub-unit hypothesis, large

remains

heavy

is a sub+nit

the

molecule

unstable

dissociation

it aggregates

weight

handling,

differ

et al. (1963) andmentioned

to the heavy

the

during

components

gel,which

molecular

again

is rather

inactivated

two

phosphate

in which and run

material

these

by Knight

the low

concentrated

unit

starch

of the

.

their

light

preparation

a preparation

to the

obvious

on the proportions

After

procedure

it becomes

phosphorylase

depending obtained

similar

It is also

nary

results,

of a polynucleotide

catalytic is probable

above

on the

against the one enzyme dissocation of the

a sensitive of the multimer

group form.

previously

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 28, No. 2, 1967

This work was supported by the following grants : C-04580 of United States Institutes of Health; Convention 6600 020 ef D. G . R . S . T . (Commission Biologie Moleculaire); French National Research Council(RCP N”24); Comite de la Seine de la L.N.F. C . C ; F . R . M. F . and French Atomic Energy Commission. G.W.Dietz is a N.S.F. fellow. REFERENCES Dolin,M.I., Dolin, Fitt,P.S.,

M.I.

Biochem. , J. Biol.

Biophys Chem.

.Res .Comms.

,a,1626

,6,11

(1961)

(1962)

and M. Grunberg?Manago

, Biochim.

Xnight,s?$‘ilc? g??ere-Manaeo .M.. Biochem. Aioahvs _Res .&&IX. Martintd Ames,B.N Singer, M. F . and O’Brien, B .-r Thane.M.N., ‘IThana,D.C,, -*Biophys .ResTComms.. , in press (1367) . d Grun berg-Manago , M. , Biochim. Biophys . Williams ,F R Acta; &,an66 (196-O

151