Orthophosphate and histone dependent polyphosphate kinase from E.coli

BIOCHEMICAL

Vol. 53, No. 3, 1973

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ORTHOPHOSPHATEAND HISTONE DEPENDENT POLYPHOSPHATE KINASE FROM -E. COLI Heng-Chun Li and Gregory Department School

of Biochemistry,

of Medicine

New York,

Received

June

of the

New York,

G. Brown Mount

City

Sinai

University

New York

of

10029.

11,1973

SUMMARY: A polyphosphate

kinase

has been purified

over

lOO-fold

from an extract of E. It requires both orthpphosphate -- coli K-12. and a basic protein (histone or protamine) for maximum activity. Because its activity is stimulated kinase may easily lead to an error tein

kinase

in the

stimulatory be important

cell

by histone, polyphosphate in the determination of pro-

extract.

Our data

suggest

that

the

effect of orthophosphate on polyphosphate kinase may in the regulation of phosphate metabolism in the

microorganism. INTRODUCTION Long chain

polyphosphate

(poly

P) is widespread

in many

biological

systems, ranging from bacteria to mammalian cell Polyphosphate kinase (poly P kinase), the enzyme (1). is responsible for the biosynthesis of poly P, has been

nuclei which

detected in many microorganisms (1,2). However, enzymes has been purified to a homogeneous state

none of these or well charac-

terized.

the

effects

purified

poly

In this

orthophosphate from E,.. -- coli.

communication,

and histone

Enzyme Assay:

we describe

on a partially

METHODS AND MATERIALS The activity of the poly

P kinase

of P kinase

was mea-

volume of 0.1 ml containing 20 mM sured at 30° in an incubation potassium phosphate (KPi) buffer, pH 7.0, 10 mM MgC12, 0.2 mg histone (calf thymus Type II, Sigma), 0.4 mM y- 32 P-ATP (specific activity

from 5 to

an appropriate was isolated Copyright All rights

28 dpm/pmole,

Nuclear)

and

32 P-poly

amount of the enzyme preparation. and determined by the paper chromatographic

@ 1973 by Academic Press. Inc. in any form resewed.

of reproduction

New England

875

P method

Vol. 53, No. 3,1973

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

for protein kinase (3) except that the development of the paper chromatograms was performed with cold 5% trichloroacetic acid. the Also, developing filtration One unit

sample spots

were not

dried

before

being

placed

solvent. A camparison of this method with millipore was carried out and identical results were obtained. of poly

enzyme which

P kinase

catalyzes

activity

is

the transfer

from ATP (gamma position) incubation mixture.

into

Enzyme Purification:

as the

peptone, 5 g NaCl and Fifty grams of frozen na powder with This

with crude

P per min at

P kinase

30'

was purified

in a 0.1 ml from E. coli grown to broth, 5 g

1 g glucose in a liter of distilled water. cells were disrupted by grinding in alumi-

a mortar

and pestle.

5 mM Tris-Cl

buffer

extract

amount of

of 1 nmole of phosphate

poly

Poly

defined

Crookels strain (obtained from Dr. H. V. Rickenberg), a late log phase in a medium containing 8 g nutrient

tracted

in the

was then

The broken containing

incubated

with

cells

were ex-

2 mM EDTA, pH 7.5. DNase for

1 hr at

0%

After the removal of particulate matter by centrifugation at 35,000 x g for 30 min the soluble proteins were fractionated by the

aaaition

was dissolved

of solid in

dithiothreitol

(NHq)2S04.

5 mM KPi buffer

(DTT),

pH 7.0,

The O-451 (NH4)2S04 fraction containing 1 mM EDTA and 10 bM

dialyzed

overnight

against

this

and applied to buffer, previously equilibrated adsorbed dient buffers kinase

to the

of

a DEAE-cellulose column (2.2 x 20 cm.), with the same buffer. Proteins that DEAE-cellulose were eluted by a 500 ml linear gra-

increasing

KPi concentration

(0.005-0.5

M) with

both

at pH 7.0 containing 1 mM EDTA and 10 yM DTT. The poly P came off at 0.25 M KPi. The active fractions were pooled

and concentrated by precipitation with solid (NH,,)2S04 at 60% saturation. The precipitate was dissolved in 5 mM KPi buffer, 10 ~JMDTT, dialyzed overnight against this pH 7.0, containing and applied to a Sephadex G-200 column (2.5 x 40 cm) buffer, The active fracequilibrated and eluted with the same buffer. tions were pooled, concentrated by (NH42) SO4 precipitation and dialyzed against 5 mM Tris-Cl buffer, pH 7.4, containing 10 FcM The enzyme so obtained DTT. the following experiments.

was stored

at -20°

for

use in

RESULTS AND DISCUSSIONS Column chromatographic

separation

of a poly

P kinase

ac-

BlOCHEMlCAl

Vol. 53, No. 3, 1973

7

40 c

;

30

> t

20

5 c5

10

;

2.0

1

1.0

2 g N tQ

0 40

SEFHADEX

G -200

0

0.4

% $

0.2

2 s: 2

20

a 3

RESEARCH COMMUNICATIONS

DEAE -CELLULOSE

z 30 z” 2

AND BIOPHYSICAL

10

B

HISTONE

0

20

Fig.

40 FRACTION

1.

GO NUMBER

Column

00

chromatography

of

on DEAE-cellulose and

on

~1

described Fig.

2.

G-200 Poly

10

Effect

of

kinase

was

coli

)

under

"Methods

histone

on

except

of

activity

was

indicated

and

Materials".

the

determined fractions,

P kinase

described

collected)

2 ml were

the

poly

as

P kinase

6 ml were

(fractions from

assayed

poly

of

P kinase

aliquots

Materials"

--E.

(fractions

Sephadex

collected). with

1 Mg

0

as

activity. under

concentration

Poly

P

"Methods

and

histone

was

of

varied. tivity ity

from was

than The

tion

is

their

shown and

ators. substrates kinases

from

one

can

for acyl easily

specific

units

per

histone

in

Fig.

to

stimulate

the found

proteins

be

misled

void of

to are

the

the

enzymatic

proteins the

were

generally

(4)

from

2C to

and

E . coli

crescentus consider the

87’7

for

(Table

effective the

(5) (6). poly

the

reac-

tested

activity most

of

G-200

extract.

P kinase

activ-

represented

crude on

be

enzyme

volume

This

the

of

poly

The

most

monophosphate-dependent

tissues

kinase

the

activity from

3' ,5'-adenosine animal

at

1.

mg protein.

A variety

were basic

Fig.

concentration

2.

histone

in

peak

purification

of

two

shown

sharp The

150

a lOO-fold

These

a protein

column.

effect

is

a single,

around

ability

Protamine

extract

as

G-200 was

more

coli

eluted

Sephadex eluate

--E.

I). stimul-

effective protein

as well Because P kinase

as

for

of this, from

Vol. 53, No. 3,1973

Table

1.

BIOCHEMICAL

Effect

of

various

incubation

proteins

conditions

except 0.2

AND BIOPHYSICAL

for mg of

the the

for

5 min,

0.1

ml

were

replacement indicated

25

Protein

on

poly

as

described

p kinase in

of

0.2

histone

mg of

proteins.

1.11 of

incubation

RESEARCH COMMUNICATIONS

aliquots

mixture

After were

for

added

Poly

p

-

Serum

Albumin

tration.

Protamine

2,932

effects

are

summarized for

was

protein

sistant action 7.0,

activity phosphate

of

phosphate

acyl was

activity

sodium

in

low

was

or was

kinase.

In

when

the

potassium

in in-

phosphate

obtained

in

pre-

assayed

proportionally

concentration

20

concen-

mM phosphate.

caused

activity

enzyme

MgC12, with

the those

exhibited MnC12

could

could

poly

P kinase

sedimented

containing properties

reported

on phosphate Phosphate

the

be

catalytic

previously

a bell-shaped replace

Both

activity.

protamine), mixture

histone

2.

When or

a reaction

and

Table

inhibitory.

enzyme and

in

maximum

(histone In

ATP

maximum

protein

activity

the

either

The required

the

raising

or

a decrease

in

P formation.

vity alone

However,

increase

poly

1-11

645 2,092

The

Formation mini25

Histone

E. as a protein kinase -- coli sence of histone, the enzyme

Further

the

620

Bovine

by

from

245

Casein

creased

by

incubation

withdrawn

None

buffer.

text

analysis.

cpml.5

Tris-Cl

activity the

MgC12,

was

solution

878

P kinase and

histone

or

histone

mixed became

by

actiwere

with

a basic

turbid

and

centrifugation.

phosphate of

the

(7,8).

pH profile the Km of

poly

with ATP

buffer,

histone,

enzyme were The enzymatic optimum was 1.4

conre-

pH around mM, and

Vol. 53, No. 3, 1973

Table

BIOCHEMICAL

Effect

2.

of

orthophosphate

activity. in

and

Incubation

the

text.

aliquots ture

AND BIOPHYSICAL

for

histone

conditions

After

were

RESEARCH COMMUNICATIONS

were

incubation

withdrawn

on

for

from

0.1

poly

as

describe&

5 min, ml

P kinase

25

~1

incubation

of mix-

analysis.

Buffer

Poly

P Formation

(cpm/5

-Histone 20 mM Tris*Cl,

pH 7.5

20 rni

pH 7.2

Tris'Cl, + 20 mM potassium

min)

+Histone

1,703

762

981

6,490

797

6,455

phosphate,

PH 7.2 20 mM potassium

phosphate,

pH 7.2

14

C-ATP

could

CC13COOH

not

precipitable

and

NaF

the

enzymatic

at

0.2

ture

the

radioactive

absence

of by

assay

mixture

column

that

M KCL,

by

reverse product

histone

was

incubating for was 10

of

adding

enzymatically

inhibited

isolated

absence

ADP,

ADP to

30 min

mM EDTA

and

completely. the

filtered

The of

amount through

a solution

mM HCl.

879

enzyme

further. larger

with 10

reverse

synthesized

examined

and

the

reaction

a lo-fold

of

pyrophosphate, inhibited

a standard 32 P-poly

by

formation

and KC1 were also did not stimulate

histone

formed

presaturated

the

respectively,

NaCl monophosphate

the

for

product.

20 mM,

completely.

demonstrated

The

0.2

In

containing

Histone and

1 mM and

3',5'-Adenosine activity. be

y- 32 P-ATP

for

radioactive

mM, reaction

inhibitory. enzyme could

substitute

The

incubation

in

mix-

P and

enzyme.

the

presence

product was the standard

a Sephadex containing

elution

the

reaction

patterns

G-25 8 M urea, of

Vol. 53, No. 3,1973

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

12 l t

$0 - HISTONE

‘0

x i?

0

+ HISTONE \

8-

: I

, z 5 i= 24

x

I’;

-

i

’ 1 1 ;
z 55 I= 2

, ,

:

0

z

P 0 d b

4

FRACTION

Fig.

0

0

20

0

25 o,s5TANCE’OFROM’50RIGIN21)CM)

NUMBER

32 P-poly P by Sephadex G-25 column. 3. Isolation of The open circle (o-o-o) represents the chromatogram of the complete reaction mixture, while the closed circle (o-o-e) represents the chromatogram reaction mixture minus histone.

Fig.

4. Paper

chromatogram

product

after

(2)

N NaOH at

0.5

of the

treating

with

the

reaction

mixture

Fractions at the against distilled

with

pronase

paper

2 hrs (o----o), and The arrow 15 min (OF). of the 32 P-orthophosphate

chromatogram.

and without

histone

are shown in Fig.

void volume were pooled and extensively water at 4". Both products, containing

or not, showed the were non-dialyzable, did not of both

on the

(1)

move on the paper products remained

chromatogram. at the origin

spread

out

chromatogram. No radioactivity These results indicate position.

orthophosphate chain polyphosphate was hydrolyzed to heterogeneous The product was readily other than orthophosphate.

880

They and

All the radioactivity of the chromatogram As shown in solvent (9).

in the

3.

dialyzed histone

characteristic properties of poly P (7). bound tenaciously to ion-exchange resin

after 20 hrs development with Ebel's Fig. 4, Pronase digestion of the histone-containing not change the mobility of the radioactivity. After the radioactivity with 0.5 N NaOH at 40" for 2 hrs, to

32P (O---a),

40" for

(3) 1 N HCl at 90' for indicates the position standard

histone-containing

of the

product did treating was found

was in the that the long fragments and com-

BIOCHEMICAL

Vol. 53, No. 3,1973

pletely

hydrolyzed

to

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

orthophosphate

at 90'

in 1 N HCl for

All radioactivity was found, to have an Rf value identical thophosphate standard. These data exclude the possibility the stimulatory effect of histone on poly P kinase activity a result

of the

boxy1 residue (4) or protein for

phosphorylation

finding

that

maximum activity

poly

the

of poly flow

P kinase

of phosphate

activity into

involved

in the

regulation

requires

the

poly

in turn P pool

It

or-

that is

or car-

protein

kinase orthophos-

enzyme may be in-

cellular level.of level will result

phate level relatively constant. P serves as a phosphorus reservoir is

that

which the

in the

P kinase

suggests

volved in the regulation of the Since the increase of phosphate tion

of a hydroxyl

on the histone as reported acyl kinase (6) reaction.,

The present phate

covalent

15 min. to

phosphate. in the stimula-

will

the

phos-

has been suggested that (l), and the poly P cycle

poly

of the

cellular

thus

accelerate keeping

level

of phosphate

in microorganisms (10). Our data on the control of polyphosphate kinase activity by phosphate tends to support that hypothesis. REFERENCES 1. 2. Z:

2: 7. 8. 9.

10.

Harold, F. M., Bacterial. Rev., 3l, 772 (1966). Muhlradt, P. F., J. Gen. Microbial., 68, 115 (1971). Li, H-C., and Felmly, D. A., Anal. Biochem. 52, 300 (1973). Krebs, E. G., Current Topics in Cellular Regulation, 2, 99 (1972). Kuo, J. F. and Greengard, P., J. Biol. Chem., 244, 3417 (1969). Agabian, N., Rosen, 0. M., and Shapiro, L., Biochem. Biophys. Res. Comm., 49, 1960 (1972). Kornberg, A., Kornberg, S. R. and Simms, E. S., Biochim. Biophys. Acta, 20, 215 (19561. McConnell, D. J. and Bonner, J., Biochem., ll, 4329 (1972). Ohashi, S. and Van Wazer, J. R., Anal. Chem., 35, 1984 (1963). Kaltwasser, H., Arch. Mikrobiol. 41, 282 (1962).

881