Adenyl cyclase of Escherichiacoli

Vol.

36,

No.

1, 1969

BIOCHEMICAL

ADENYL

AND

CYCLASE

BIOPHYSICAL

OF

Escherichia

Misao Shionogi

Research

Received

Laboratory,

Shionogi

RESEARCH

COMMUNICATIONS

coli

Ide 8, Co.,

Ltd.,

Fukushima-ku,

Osaka,

Japan

May 20, 1969

SUMMARY Adenyl Escherichia the extract. cofactor was and oxalacetate. enolpyruvate Two

cyclase activity was found in the cell-free extract of Crooke’s strain of cd. The activity was observed exclusively in the particulate fraction of Adenyl cyclase of E. coli required Mg* for its activity, but no other necessary. The enzyme activity was inhibited by pyridoxal phosphate A weak stimulatory effect wos observed with GMP ond phospho.

species

of bacteria

cyclic

phosphate

(cyclic

in our

laboratory

from

3’,5’-AMP

catalyzes

1967;

Ide

cells

adenyl

the

induction

investigotion will

(1965) and

cyclase Recently,

provide This

of B.

reported

culture in the

media

and

of catabolite of the valuable

communication

in vitro

cyclic

system Pastan

hove

(1968)

been reported

repressible

enzymes

synthesis

of cyclic

information reports

on the

role

the synthesis

adenyl

(Hirata

and

cyclase

Hayaishi,

pyruvate

has been

isolated

in a soluble

is Escherichia which

this

of -E. -Icoli

but

isolated

of cyclic

requires

as a

-coli

form. . Makman

nucleotide

accumulates

attempts

to demonstrate

unsuccessful, that of g.

of this

cyclic &i,

3’,5’-AMP

of cyclic 42

3’,5’-

bacteria

under strain

amount

contains

ATP

3’,5’-AMP

conditions

of Crooke’s

cell-free

Perlman

makes

adenosine a bacterium

a large

extract from

liquefaciens

the

fluid

3’,5’-AMP of this

make

liquefociens,

Its cell-free

cyclase

which

which

in its culture

of cyclic Adenyl

bacterium

Sutherland

in the

synthesis

reported

Brevibacterium

excretes

adenylcyclase

other

been

et al ., 1963).

the

The

The and

sewage,

et al . , 1967).

cofactor.

so far

3’,5’-AMP).

(Gkabayashi

which

have

3’,5’-AMP and

it is expected

by an --E. coli

nucleotide 3’,5’-AMP

participates

cell-free

that

in the system

in bacteria. using

adenyl

cyclase

Vol.

36,

No.

1, 1969

of --E. coli

and

BIOCHEMICAL

describes

same

AND

of the

properties

MATERIALS Preparation was

of E. coli

cultured

glucose,

at 37°C 1%;

under

pended

in 50 mM

forced

mercaptoethanol,

was

and

suspended

and

if any, Assay

wise

Cyclase:

50 mM

2.5

mM

preparation

and

out

the

(pH paper

as the

1.5

7.5) and

(75

for

unit

of enzyme

min

under

mdioactivity was

standard

et al. The validity

defined assay

enzyme

preparation.

standard

medium 0.3%

of

MgS04*7HzO,

and

The

3,000

(pH

containing

This

composed and

MgCIz

cell.

8.0)

8739)

by centrifugation,

between

(pH

assay

9.75),

pCi/)rmole,

sus-

5 mM

extract

x g and

8-

was 30,000

5 mM

x g

8-mer-

preparation

contained

contained,

unless

5 mM

was

Aliquots

of the

cyclic

3’,5’-AMP

using

solvent.

only

to cyclic

a Beckman

as the

The

amount

conditions.

DPM-100 producing

The

protein

was

confirmed

ml.

The

mixture

ethanol-1M cyclic

was

in an ice

bath

cochromato-

ammonium

3’,5’-AMP

3’,5’-AMP liquid

reaction

were

(65

eluted

: 16.7 cut

scintillation of cyclic was

acetate

was

were

1 rmole content

enzyme

the mixture

reaction

other-

30 mM

Inc.),

to 0.1

isopropanol-HCI-Hz0

corresponding with

BioResearch

by cooling

of ATP.

using

8-mercaptoethanol,

adjusted

was stopped

as the

medium

Schwarz

volume

and

rechramatographed spots

obtained buffer

buffer

30 min

cold

pressure

Tris-HCl

The

: 30 v/v)

The

was

the final

poles with

solvent.

measured

Lowry

for

on paper

buffer

10 mM

(4.0

ml and

at 30°C

by adding

graphed

containing

in a French

KH2P04,

(ATCC

phosphodiesterase.

glycine

ATP-8-Cl4

0.025

carried

harvested

8.0)

of --E . coli

of the

(20 g) were

as the

of Adenyl

in 5 liters

cells

that

used

strain

The

disrupted

3’,5’-AMP

Craoke’s

0.7%;

and

was

METHODS

phase

(pH

COMMUNICATIONS

enzyme.

K2HP04,

buffer

precipitate

RESEARCH

;

Tris-HCI

cyclic

specified,

MgCIz,

0.1% aeration.

the

AND

logarithmic

were

of this

Cyclase:

in 20 ml of 10 mM

captoethanol little,

to late

(NH&.SO4,

0.01%

centrifuged

Adenyl

BIOPHYSICAL

determined

out

from

: 18.3

v/v)

and

were

counter.

One

3’,5’-AMP

per

according

to

(1951). of this

assay

system

43

by

1) quantitative

recovery

of

Vol.

36,

No.

1, 1969

radioactivity

after

solvent

systems

rabbit

BIOCHEMICAL

brain

rechromatagraphy

and

of the

2) formation

(Drummond

and

tion.

The

a sonic The

1 shows

the

activity

was

disintegrator

rather

and

weak

activity

hibi tory

compounds

between

pH

was

9.5

necessary *

ofMg

.

pyruvate,

for The

a cofactor

et al.,

1967),

any

distinct

effect

About Among

them

one

10.0,

the

hundred

pyridoxal

may

Km for

and

maximum

without

any

other

cyclase

compounds phosphate,

of B.

of adenyl were

tested

Table Distribution

Fraction

activity cofactor

and

for

their

and

malate

effect were

Adenyl units

x ld

observed

observed

at 30 mM such

did

as 1967;

not

show

. on the --E. coli found

Protein 9

Crude

extract

4.0

2.1

3,000

x g supernatant

3.4

1.9

30,000

x g supernatant

0.2

0.8

30,000

x g precipitate

7.1

1 .o

44

in-

of Mg”

compounds

cyclase

cyclase

was

presence

1

of adenyl

of some

enzyme

enzymes,

with

soluble.

and Hayaishi,

(Hirata

of --E. coli

enzyme

was

of mammalian

cyclase

oxalacetate

The

I .O mM.

fractiona-

Treatment

presence

of the

liquefaciens

cofactors

the

to the

activity

to be active

activity

from

during

fraction.

to make

be due

Maximum

obtained

particulate

failed

extract

catecholamines,

on the

in the

was

proceeded

six different

phosphodiesterase

fractions

ATP

of adenyl and

of the

detergents

crude

and

with

with

DISCUSSION

below.

the enzyme

reaction

Ide

of the

hydrolysis

activity

several

as described and

AND

exclusively

with

spots

COMMUNICATIONS

1961).

cyclase

observed

RESEARCH

3’,5’-AMP

after

Perrot-Yee,

adenyl

BIOPHYSICAL

cyclic

of S-AMP

RESULTS Table

AND

inhibitory,

enzyme. whereas

Vol.

36,

GMP

No.

and

1,1969

by

1966;

oxalacetate

the

pyridoxal

Kaldor

and

but

it is also

most

the effects.

cells

is open

to further

cut

that

of various

Compound

et al.,

1967;

the

it is converted

into

have

investigation.

only any

the

have

oxalacetate

fact

that

the

2

on --E. coli

adenyl

added

cyclase*

Enzyme

activity

% None

(100)

Pyridoxal

phosphate,

1 mM

Pyridoxal

phosphate,

10 mM

20.0 0.5

Oxalacetate,

1 mM

78.7

Oxalacetate,

10 mM

10.4

Malate, GMP,

10 mM

38.5

10 mM

Phosphoenolpyruvate, GMP,

10 mM

*

Concentration

155 10 mM

148

+ Phosphoenolpyruvate,

of ATP-8-C14

10 mM

was

45

0.625

186

mM

in this

experiment.

in-

1968),

acids

tested,

an inhibitory

effects.

meaning

ne

(Anderson

during

stimulatory

physiological

However,

The

et al.,

organic

may

weak

inhibition

pyridoxami

enzymes

Shapiro

Among

showed

The

inhibitory.

on several

Malate

compounds

compounds

somewhat

found

2).

phosphate,

inhibition.

Table Effect

been

Rippa

and GMP

of these

was

COMMUNICATIONS

(Table

Pyridoxamine

undetermined.

clear

possible

effect

pyridoxal

1966;

RESEARCH

activating

has also

is yet

Phosphoenolpyruvate

Whether

however,

Weinbach,

the

BIOPHYSICAL

pronounced.

phosphate

demonstrated

i ncubotion.

most

significance

in itself,

--E. coli

a weak

no effect,

its physiological

effect

showed was

showed

effect

et al., but

phosphate

pyridoxine

hibitory

AND

phosphoenolpyruvate

by pyridoxal and

BIOCHEMICAL

within compounds

the

Vol.

36,

No.

closely

1, 1969

related

flow

around

The

importance in 2.

a recent

report

repression

Adenyl extract,

adenyl

cyclase

relationship

(Oki

Pastan

by glucose

enzyme

to the in the

suggested and

the

(1968)

and

off

.

that

Further

COMMUNICATIONS

suggest

that

of --E. coli mechanism

Dobrogosz,

suggests

in E. -- coli

may

regulation

on and

naka

RESEARCH

metabolic

adenyl

cyclase.

of catabolite 1967).

cyclic

the

re-

Furthermore,

3’,5’-AMP

investigation

reverses

is necessary

to

points. cyclase

culture

of -7 E. coli

and

in this

was

1967).

of this

to be determined

ond

in the

resembles which

Whether

enzyme

found

respect

of B. liquefaciens

Hayaishi,

remains

Perlman

BIOPHYSICAL

affect

metabolism

has been

by

these

function

has some

@i

AND

metabolism

of pyruvate

catabolite

clarify

and

to pyruvate pyrwate

pression

the

BIOCHEMICAL

this of cyclic

fraction

mammalian

has been

implies

particulate

some

3’,5’-AMP

enzymes

isolated

of the --E. coli rather

in a soluble

intrinsic

difference

between

--E. coli

than form

in the and

the (Hirata physiological

-B. liquefaciens

. ACKNOWLEDGEMENTS

I would cussions.

like The

to thank

technical

Dr.

Tadashi

qssistance

Okabayashi

of Mrs.

Miyuki

for

his advice

Mitsugi

and

is gmtefully

stimulating

dis-

acknowledged.

REFERENCES Anderson, B. M., Anderson, C. D., and Churchich, J. E., Biochemistry, 5,2893 (1966). Drummond,G. I. and Permt-Yee,S.,J. Biol. Chem.,z,1126 (1961).Hirato, M. and Hayaishi, O., Biochem. Biophys. Acta, 149, 1 (1967). Ide, M., Yoshimoto, A. and Okabayashi, T., J . Bacterial 3,317 (1967). Kaldor, G. and Weinbach., S., Federation Proc.,z, 641 (1966). Lowry, 0. H., Rosebrough, N. J., Fart, A. L., and Randall, R. J . , J . Biol . Chem., 193,265 (1951). Makman, R. S. and Sutherland, E. W ., J. Biol . Chem. ,%,1309 (1965). Okabayashi, T., Yoshimoto, A. and Ide, M., J. Bacterial . , 86, 930 (1963). Okinaka, R. T., and Dobrogosz, W. J., J. Bacterial ., z,l644 (1967). Perlman, R. L. and Pastan, I., J. Biol. Chem.,z, 5420 (1968). Rippa,M., Spanio, L., and Pontremoli, S., Arch. Biochem. Biophys., =,4B (1967). Shapiro, S., Enser, M., Pugh, E. and Horecker, B. L., Arch. Biochem. Biophys. ,128, 554 (1968).

46