Purification and characterization of guanylate cyclase from Caulobacter crescentus

Vol.61,No.1,1974

BIOCHEMICAL

PURIFICATION

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

AND CHARACTERIZATION

OF GUANYLATE CYCLASE FROM

CAULOBACTER CRESCENTUS*

Irene C. Sun, Lucille

Shapiro

and Ora M. Rosen

Department of Molecular Biology, Division of Biological The Department of Medicine, Albert Einstein College Bronx, New York 10461

Received

September

Sciences, of Medicine,

and

18,1974

cyclase has been purified 60-fold from cell extracts SUMMARY: Guanylate It h$f a molecular weight of of the bacterium Caulobacter crescentus. for activity. Enzymic approximately 140,000 and is dependent upon Mn activity is unaffected by cyclic AMP cyclic GMP or N6,0*'-dibutyryl cyclic AMP but is stimulated by N*,Oi'-dibutyryl cyclic GMP. The partially purified preparation of guanylate cyclase does not contain detectable adenylate cyclase activity. The cell with

a series

temporal

cycle

of morphogenetic

sequence

(1,2).

of gene expression implicated

of the bacterium

the

AMP and cyclic

cyclic

sole

carbon

cultures source

to the formation the addition derivative prevented

*

grown (3).

This

GMP.

the formation

formation

without

Address all correspondence Biology, Albert Einstein Bronx, New York 10461.

cycle

block pili

, and cell

flagella

Furthermore,

dibutyryl

affecting

the rate

Copyright 0 1974 by Academic Press, Inc. Ail rights of reproduction in any form reserved.

division,

lactose

(cyclic

just

by

as the prior

can be overcome

by

GMP or the dibutyryl

cyclic

GMP, however,

spontaneous

rescue

of growth

cyclic

GMP blocked

surface

of growth

of mid-log

phase

Irene C. Sun, of Medicine,

193

have

can be arrested

not cyclic

during

the timing

(3,4).

and development,

of dibutyryl

and

products

monophosphates

cycle

AMP but

The addition

to: College

of gene

to medium containing

in growth

associated

regulate

of C. crescentus

cyclic

of polar

cultures.

of the cell

in glucose

of flagella,

of cyclic

and guanosine

is spatial

which

and assembly

cell

of N6,0*'-dibutyryl

in the blocked structure

localization

the

in a fixed

of the mechanisms

the control

We have shown that

crescentus

occurring

3',5'-adenosine

GMP) in

transferring

events

Studies

and the

Caulobacter

Department 1300 Morris

of Molecular Park Avenue

Vol. 61, No. 1,1974

cultures

using

Schmidt

mannose

as carbon

and Samuelson

tion

in mutants

both

cyclic

In view are

BIOCHEMICAL

(4)

involved

studies

of both

lished)

in this

cyclase

from

that

cyclic

cyclic which

in regulating

the presence

(Kurn

that

AMP and dibutyryl

of the cited

source

observed

of C. crescentus

AND MOPHYSICAL

form

5'-triphosphate

and Nuclear Schwarz

Corp.,

cyclic

(yeast)

were

was from Miles

Assay

described

30nM Tris-HCl

buffer,

phosphokinase

gift

from

International

Chemical

of New England

Nuclear.

cyclic (human), Alumina

from Creatine

GMP, N6,02'-dibutyryl beef

heart

(neutral)

l&diphosphatase

Dr.

by White pH 7.6,

were

was carried

out

bath,

Philip

Enzyme activity

system

ice-acetone

GTP and [(x-~~P]

purchased

cyclic

were

and alcohol

Corp.

phosphodiesterase

et al

15mtj

and bovine

dehydro-

serum albumin

followed

from

was determined The incubation

(5).

MnC12, 3.6mM_[a-32P]

for

optimal

194

mixture

phosphate,

contained cpm/pmole),

15ug creatine

2-mercaptoethanol

activity

for

by a modification

GTP (20-40

15 min at 379 and then by boiling

Blastocladiella

M. Silverman.

volume ,of 100~1.

required for

purified

3.7mMMcreatine

and enzyme in a final

GTP regenerating

in a dry

the guanylate

AMP were

Biochemical

GMP, 1OmM mercaptoethanol,

The reaction

unpub-

Inc.

Cyclase

of the method

cyclic

transferrin

Fructose

Co.

AMP-specific

of Guanylate

the

N2,02'-dibutyryl

from Worthington

was a generous

8mM cyclic

and

and'shapiro,

[v-~*P]

from

and chromatographic

Laboratories,

The cyclic

(GTP),

obtained

triphosphates,

of Sigma Chemical

emersonii

GMP

AND METHODS

was a product

phosphokinase,

phosphodiesterase

genase

GMP.

of C. crescentus,

and characterized

GMP and [3H]

Na432P207

AMP, nucleoside

products

were

C3H] cyclic

creatine

nucleotide

of cyclic

AMP and cyclic

GMP (Kurn

5'-triphosphate (ATP)

BioResearch.

phosphate,

and that

C. crescentus.

[cx-~' P] guanosine

adonosine

elonga-

stalks,

effect

cyclic

and development

we have purified

stalk

long this

that

AMP (3) and cyclic

bacterium,

unpublished).

GMP stimulated

abnormally

suggest

MATERIALS Materials

and Shapiro,

AMP inhibited

the growth

cyclic

RESEARCH COMMUNICATIONS

3 min.

(see Table stopped After

and a 4).

by freezing cooling

on

BIOCHEMICAL

Vol.61,No.1,1974

ice,

0.8 ml of 2mM sodium

cyclic

[3H]

mately

buffer,

Triton-toluene

of cyclic

eluted

between

(50 cpm/pmole)

Cyclase guanylate

in a reaction

mixture

with

was added

Triton,

7 parts

in each assay Purified

cyclic

was measured

(a)

60%).

and

approxi-

had been equilibrated

GMP formed

cyclase,

except

AMP were

used

0.054

to 9 ml of

toluene

and

was measured

was corrected.

for

re-

GMP was generally

boiling were

and cooling,

added

Cyclic

E3*P]

graphy

as described

legend

to Table

creatine

for

for

from the

In the

second

cyclase

(0.25mM)

fluid

ATP

15ug creatine assay

assay

the

except

and Ba(OH)*

was removed

nucleotide

GMP,

3.61@ [WEEP]

the guanylate

supernatant

ATP

of GTP and cyclic

of 50~1.

precipitate

the cyclic

the conditions

[c,-~*P]

phosphate,

50~1 each of ZnS04

and the resultant

AMP was isolated

3.6r@

instead

volume

as described

that

under

ti-dithiothreitol,

AMP, 3.7@

and enzyme in a total

was terminated

(0.25mM)

centrifuged

of 3H and 32P in the aliquot

containing

8mPJ cyclic

phosphokinase

after

which

Activity

and 8mlj cyclic

(50 cpm/pmole),

that

vigorously,

cpm of

15 and 18 ml.

above for

reaction

r3*P]

80,000

(8 x 1lOmm) containing

(3 parts

C3H] GMP (about

Assay of Adenylate described

oxide

The amount

of cyclic

covery

mixed

a column

fluid

Omnifluor).

and the amount

were

containing

One ml of each 3 ml fraction

scintillation

4 gm/liter

or (b)

onto

alumina

pH 7.6.

pH 7.6,

The tubes

was decanted

3 gm of neutral

Tris-HCL

pyrophosphate,

GMP was added.

each supernatant

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

by centrifugation.

by paper

chromato-

phosphodiesterase

assay

in the

2. RESULTS

Purification phase

of Guanylate

in nutrient

wet weight, containing MgS04,

were 20@

0.05mlj

min at 10,000

Cyclase

broth

(1)

ground

with

Tris-HCl,

and harvested alumina

pH 7.6,

EDTA and 20% glycerol rpm in a Sorval

C. crescenhus

(1:2,

CB13 was grown

by centrifugation. w/w)

GSA rotor,

195

G). the

After

supernatant

60 gm

Cells,

and resuspended

7mM_mercaptoethanol, (buffer

to mid-log

in a solution

3mMMnC12,

5ti-

centrifugation (step

1, Table

for

15 1) was

Vol.61,No.1,1974

BIOCHEMICAL

Table

Step

1.

Purification

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of Guanylate

Fraction

Volume

Protein

(ml 1 I

Extract

II

100,000 x g supernatant

III

fluid

Ammonium sulfate

IV V

G-200 gel eluate

from

C. crescentus

Units

pecific ctivity units/m:

(pmoleslmin)

(ms)

Recovery (%I

120

6,240

118,560

19

100

100

3,000

96,000

32

81

fractior

DEAE-cellulose

Cyclase

eluate

35

980

64,680

66

55

82

87

17,574

202

15

11

27

27,720

filtration 1,026

23

One unit of guanylate cyclase activity is defined as that amount of enzyme required to catalyze the formation of 1 pmole of cyclic GMP in 1 min at 37". The specific activity is the number of units per.milligram of protein; protein was measured by the method of Lowry et al (13).

treated

with

DNase (lOug/ml)

90 min at 30,000

for

II).

rpm (step

25% saturation

with

and sufficient

ammonium sulfate

solution

two changes (1.7 washed

ammonium sulfate.

of buffer

was added

with

III)

collected.

trated

by precipitation

plied,

in 3 ml of buffer

precipitate

rate

(-0.08M)

with

amnonium

G (step

IV),

containing sulfate

196

to bring

overnight

to a DEAE-cellulose After

gradient

to

the

and fractions activity

(60% saturation) G-200

against column

the column

of NaCl

enzymic

to a Sephadex

brought

was discarded

G, dialyzed

was 10 ml/hr

for

was then collected

same buffer.

a 400 ml linear

The flow

The eluate

the

was slowly

to the supernatant

and applied

with

150 ml of buffer, G was applied.

fluid

in 15 ml of buffer

G (step

by centrifugation

The precipitate

The resulting

dissolved

followed

The supernatant

x 30 cm) pre-equilibrated

in buffer were

solid

to 50% saturation.

by centrifugation,

30 min at 4",

column

was

(0.02-0.255) of 4 ml was concenand then (Fig.

apl)-

BIOCHEMICAL

Vol. 61, No. i, 1974

IO

30

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

50

70

!a0

II0

FRACTION

130

IJO

I70

(ml)

of guanylate cyclase from a calibrated column Fig. 1. Elution The Sephadex G-ZOO column was caliof Sephadex G-ZOO (1.7 x 46 cm). brated with proteins of known molecular weight [see Methods). Alcohol dehydrogenase and fructose 1,6-diphosphatase activities were assayed according to Vallee and Hoch (16) and Pontremoli (17), respectively. VE, elution volume.

Fractions

containing

against

buffer

G containing

(2 mg/ml)

could

A sumnary

of the

cation

(step

be stored

was about

reported

guanylate

V, Table

with

(step

for

pooled

V).

loss

in Table

of 23%.

performed

1.

All

with

and dialyzed

The concentrated

a month without appears

were

V, Table

phosphodiesterase

activities

(Table

l'ined

(step

a recovery

communication

nucleotide

purified

at -70"

were

eluate

of activity. The final

of the

the most

purifi-

studies

to be

purified

fraction

1).

Gel filtration

the

activity

60% glycerol

enzyme purification 60-fold

in this

cyclase

2).

preparation

tn Methods.

1) yielded

an enzyme preparation

but containing

We could

not detect

of guanylate

The guanylate

GTPase and pyrophosphatase any adenylate

cyclase

cyclase

197

low in cyclic

eluted

using from

either

cyclase

activity

of the assays

the Sephadex

column

in outin

Vol.61,No.1,1974

Table

BlOCHEMlCAL

2.

Other

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Enzyme Activities

in Guanylate

Cyclase

Preparations

Fraction

(nmoles/mg/min) 1 II III

Extract 100,000 fluid

V

DEAE-cellulose G-200

--

0.01

0.46

1.0

--

0.15

0.56

0.84

--

0.03

0.09

1.5

120.8

0.09

0.09

6.4

0.09

0.09

x g supernatant

Ammonium sulfate

IV

0.06

gel

fraction eluate

filtration

fraction

0.06

L

i

Activities of GTPase and pyrophophatase were determined by measuring the release of inorganic phosphate from GTP and PPi. respectivsiy. The reaction mixtures contained either O.lmM (Y-~~P]GTP (48 cpm/pmole), or 0.5mM.Na P 07 (660, cpm/nmole) and 0~0lM MgS04, 2.5mM DTT, 0.015M Tris-HCl pH 7.4, in a Tota 4 vo ? ume of 200~1. The GTPase assays were i! cubaTed for 5 min at 37" and the pyrophosphatase assays were incubated for 10 min at 37': Pi (0.5&i) was added as carrier and the released inorganic phosphate was measured using the solv&t extraction method of Martin and Doty (14). Phosphodiesterase activity was The reaction mixture contained 1.5uM cyclic GMP: measured as described previously (15). (1,436 cpm/pmole) or 1.5uM cyclic AMP (3,458 cpm/pmole), 1OmM Tris-HCl pH 8.0, 5mM MgSO4; The incubxtions were carried out at 7.5mM mercaptoethanol in a total volume of 100~1. 30" For 40 min and were stopped by boiling in a water bath for 2 min. An aliquot was applied to Whatman No. 1 paper. Nucleotides were separated by descending chromatography: The area containing cyclic GMP or cyclic, for 16 hr in ammonium acetate/ethanol (30:70). AMP, 5'-GMP or 5'-AMP, guanosine or adenosine were detected by UV adsorption, cut out and counted in the Triton-toluene scintillation fluid.

a volume

consistent

Identification reaction with

with

of Cyclic was eluted

marker

cyclic

a molecular

GMP The radioactive

from alumina GMP in

titative

with

beef

heart

appearance

and further

two solvent

and isopropanol/concentrated duct

weight

NH40H/O.lM cyclic

nucleotide

of 5'-[32P]

systems

of approximately product

of the guanylate

identified (ammonium

acetate/ethanol Treatment

phosphodiesterase

198

treatment

(Fig.

1).

cyclase

by co-chromatography

H3B03 68:10:30).

GMP,whereas

140,000

resulted with

a cyclic

30:70 of the pro-

in the quanAMP-specific

BIOCHEMICAL

Vol.61,No.1,1974

Table

3.

Identification

of the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Product

Treatment

of the Guanylate

Cyclic

e*P]

Phosphodiesterase

177

Phosphodiesterase Blastocladiella

(bovine

29

heart)

204

emersonii

GMP

Reaction

T 5'-[3*P]

GWP

(cm)

(WI

None

Cyclase

44

203 48

Standard guanylate cyclase reactions (see Methods) were carried out with lO@g of enzyme and then applied to alumina columns as described in Methods. The eluate (500~1) containing the 32P:labeled product was incubated with 7ti-MgSO4 and 289 of beef heart cyclic nucleotide phosphodiesterase (0.2-0.5 units/mg) for 30 min at 30'. One unit of enzyme converted lnmole of the 3',5'-cyclic nucleotide to the 5'-mononucleotide per min at 30". The reaction was terminated by boiling, the precipitate removed by centrifugation, and 2501~1 of the supernatant fluid applied to paper and chromatographed as described in the legend to Table 2. Areas corresponding to carrier cyclic GMP and 5'-GMP were identified by their adsorption of UV light, cut out and their radioactivity determined. One ml of the same eluate from alumina oxide was concentrated by evaporation to 0.5 ml and incubated with 1OOng of B. emersonii phosphodiesterase (15-20 units/mg) for 30 min at 37O. One unit is defined as that amount of enzyme required to convert 1 nmole of cyclic AMP to 5'-AMP per min at 37'. The reaction was terminated and and processed as described above. The recovery of 3*P after chromatography was 95% of the radioactivit applied. Under the conditions described, 80% of [ J H] GMP (2&l) was hydrolyzed by the bovine heart added standard cyclic diesterase and ~0.1% by the diesterase from B. emersonii. The enzyme from B. emersonii completely hydrolyzed cyclic [3H] AMP (2@) under identical conditions.

a

MnCIZ (mM1

Fig. 2. Effect of Mn*+ on the synthesis of cyclic GMP. Assays were performed as described in Methods using 1OOpg of protein in each reaction.

199

Vol.61,No.1,1974

BIOCHEMICAL

Table

4.

Requirements

Reaction

for

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Cyclic

GMP Formation

mixture

Cyclic GMP formation (pmoles/l5 min)

Complete

963

Minus

MnC12

Minus

MnC12,

<9 plus

MgC12 (15mM)

Plus

MgC12 (15mM)

578

Plus

CaC12 (15mM)

323

Plus

ZnC12 (15@)

<9

Plus

NaF (2mM)

575

Plus

NaF (l(mM)

<9

Plus

sodium

pyrophosphate

Plus

sodium

pyruvate

Minus

GTP regenerating

Minus

2-mercaptoethanol

(2njrl)

205

system

phosphodiesterase

from

chromatographic

behavior

Characterization the reaction

of the reaction

was constant

with

upon enzyme concentration pH 7.6 and pH 8.3.

optimal

rates

Ca2',

Catalyzed time

between

between

Mg2',

was as described

Blastocladiella

of the Reaction

obtained

462 255

mixture

isolated

338

(5mM)

The complete reaction contained 1OOpg of enzyme.

ions,

<9

for

emersonii

product

(6)

the

200

reaction

not alter

did

T he rate

Cyclase

10 and 50ug of protein.

the

Activity

Other

of

and was dependent

was dependent 2).

and

3).

25 min at 3.6mtjGTP

10 and 15mM (Fig.

and Zn2+ inhibited

(Table

by Guanylate

The enzyme activity

between

in Methods

was optimal

on Mn2+ with divalent

when present

metal

in concentra-

Vd61,No.

tions

equal

effect

BIOCHEMICAL

1,1974

to Mn2+(Table

upon enzymic

an activator

during

of the adenylate

the formation of the

The addition

4).

activity

of cyclic

adenylate

(4mfJ) inhibited

activity

from

40% (data

tion

whereas

(Table

cyclic

5).

GMP was added

Table

5.

cyclic

N2,02' -dibutyryl Maximal

not

stimulation

at concentrations

Effect

liquifaciens

of Nucleotides

effect

GMP stimulated

(2-3

fold) equal

(9).

The nucleoside

AMP had little

occurred to GTP.

on Cyclic

Fluoride,

(7,8),

and pyruvate,

shown).

cyclic

GMP had no

purification. cells

pyrophosphate

Brevibacterium

cyclic

of the

of eukaryotic

GMP, as did

cyclase

of carrier

any phase

cyclase

at 3.6mM- and N6,02' -dibutyryl formation

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

inhibited an activator Caffeine triphosphates

on cyclic the cyclic

GMP GMP forma-

when N2,02'-dibutyryl The stimulatory

effect

of

GMP Formation

Addition

A)

Cyclic GMP formation (pmoles/l5 min)

None

986

N6,02(-dibutyryl Cyclic

Sodium

cyclic

AMP

4.0

1,083

2.0

1,080

:-': 316

868 1,786 1,300

T:i

1,115 1,153

AMP

NB,02'-dibutyryl

B)

Concentration h!!)

cyclic

GMP

butyrate

None

306

ATP N2,02!dibutyryl

1.8 cyclic

A) Enzyme assays of enzyme-

were

224

GMP

performed

1,074 1,070

as described

in Methods,

using

1OOug

B) The amount of [c~32P] GTP in the reaction mixture was decreased from 3.6mM to 1.8mM. The concentration of the other components were as described-in Metho&.

201

Vol.61,No.1,1974

BIOCHEMICAL

N2,02'-dibutyryl

effect

GMPwas apparent

cyclic

was present

during

the assay.

on the crude

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

whether

or not

N2,02'-dibutyryl

guanylate

cyclase

carrier

cyclic

activity

cyclic

GMP

GMP had no stimulatory

I and II,

(step

Table

1).

DISCUSSION Cyclic cells

GMP is

GMP in eukaryotic is

cells

known about

Escherichia

coli,

AMP-receptor

cyclases.

Clark

studied (or

adenylate

(ll).Even

lichenformis

it

cyclases

is

that

cyclase. exogenous by the

our

for

but did

purify

conditions

raises

dibutyryl

endogenous

by F',

it

requires

of cyclic

cyclic synthesis

GMP on assembly of cyclic

in comnon with

the

it

cyclase enzyme

is

soluble

an agent

known to activate

Mn2+ for

activity,

the

and it

the purified

physiological

of polar

the does

It is interesting

reaction.

that

fraction

The guanylate

breakage

of

guanylate

in a particulate

GMP activates

the possibility

In

synthesis

bacterial

it.

of cell

the cyclization

derivative

This

activity

AMP (10).

AMP to the cyclic

AMP-induced

known about

not

by cyclic

of cyclic

to identify

of eukaryotes,

the dibutyryl

is

of cyclic

GMP in bacteria.

the cyclic

not stimulated

not use ATP as substrate

of cyclic

has many properties

under

promoted

the binding

less

(12),

crescentus

solubilized),

role

and prokaryotic

some of the effects

to those

antagonizing

able

of eukaryotic

at least

GMP inhibits

thereby

in eukaryotes;

that

antagonistic

et al were

Caulobacter

variety

biological

cyclic

in vitro

Bacillus

from

are the

protein

B-galactosidase

from

in a wide

It has been suggested

(10).

Little

found

structures

guanylate

effects

of

may be mediated

GMP.

ACKNOWLEDGMENTS * This Science from no.

work

Foundation,

the National 19100-02.

Institute

was supported grant

nos.

Institutes This

of Molecular

by grant GM 11301,

of Health,

is Cormnunication Biology.

no. GB 42545X AM 09038

from

and 3201-GM

and the NIH Genetics No. 326 from

O.M.R.

202

the

is a recipient

the National 2209-02

Center grant

Joan and Lester of a Career

Avnet

Develop-

Vol.61,No.1,1974

ment Award L.S.

BIOCHEMICAL

from

is a Faculty

the United Research

States Associate

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Public

Health

Service

of the American

(5K03

Cancer

GM 22345)

and

Society.

REFERENCES

:: 3. 4. 5. 6. 7. 9": 10. 11. 12. 13. ii: 16. 17.

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