Inhibition of the soluble guanylate cyclase from rat lung by sulfated polyanions

BIOCHEMICAL

Vol. 104, No. 3, 1982 February 11, 1982

INHIBITION

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 957-964

OF THE SOLUBLE GUANYLATC CYCLASE FROM RAT LUNG BY SULFATED POLYANIONS Marcia

A. Liebel

and Arnold

A. White

Department of Biochemistry and the John M. Dalton University of Missouri-Columbia, Columbia, Received

December

22,

Research Center, MO 65217

1981

SUMMARY: Soluble guanylate cyclase was partially purified from rat lung homogenates, and shown to be inhibited by the following sulfated polyanions, in ,,g/ml in parentheses: Polyvinyl sulfate (0.33), 40,000-dalton ate (0.45), polyanetholesulfonate (0.63) 500,000-dalton dextran sulfate (1.8), x-carrageenan (2.9), t-carrageenan (6.1), K-Carrageenan (48.0), heparin (68.0). There was a good correlation between inhibitory potency and sulfate content (as total sulfur). Inhibision by hepariy +and thy+carrageenay$ or !$g (but not the othersj+was potentiated by Mn , but not Ca when [Mn exceeded [GTP]. Mn -potentiation could be blocked by high Na . ieparinagarose shows promise as an affinity matrix for guanylate cyclase. A large

number

of enzymes

have been shown

lytes

(1)

and most of these

by polyanions

showed

that

certain

polyanions,

synthetic

the thyrotropin-stimulated brane.

Salomon

in detail

the effects

clase,

and found

that

inhibitors.

In the

inhibitors

of guanylate

polyanions

differ

excess

Mn2+.

of guanylate MATERIALS

cyclase

(5-7)

extended

We also cyclase

these

the

glycosaminoglycans

report

cyclase

[EC 4*6*1*2];

the synthetic include using

that

ones

preliminary

data

sulfate,

thyroid

inhibited

plasma

observations

mem-

by examining

upon adenylate were

sulfated

however in their

and Cook (4)

dextran

polyanions

we showed

by polyelectro-

Wolff

of bovine

occurring

sulfated

3).

including

of naturally

present

from

(2,

adenylate

and coworkers

to be inhibited

the

the most

potent

polyanions

are

naturally

inhibitory

on the affinity

cy-

also

occurring

response

to

chromatography

heparin-agarose.

AND METHODS

The commercial sources of chemicals used in guanylate cyclase tion and assay have been previously given (10, 11). Heparin-agarose plied by Bethesda Research laboratories, and contained 0.84 mg of heparin per ml of gel. Sigma supplied all of the polyanions except arin (l\la+, 100 unitslmg) which was from Nutritional Biochemicals, polyanetholesulfonate (Liquoid), which was provided by Hoffman-La

957

purificawas supcouplied for hepand sodium Roche.

0006-291X/82/030957-08$01.00/0 Cop.vrighi 0 1982 b-vAcademic Press, Inc. ,411nghrs of reproduction in any form reserved.

]

Vol. 104, No. 3, 1982

BIOCHEMICAL

AND BIOPHYSICAL

POLYANION

d ? 5 0

RESEARCH COMMUNICATIONS

(ugl

706050-

k

40-

5 $

30-

5 a

20 IO i

POLYANION Fi ure 1. Inhibition !a: determined with is shown the effect dextran sulfate (A), (0). In B is shown polyanetholesulfonate

(fig)

of guanylate cyclase by sulfated polyanions. Activity and the indicated amount of polyanion. In A 3 mM MnCls,, of hyalur nlc acid ,lJ), heparin (0), 500,000 dalton40,000-dalton dextran sulfate (A), and polyvinyl sulfate the effect of K(O), I(@) and x-carrageenan (I), (A), and again, heparin (0).

Guanylate cyclase purification. (8) purification procedure, filtration and DEAE-cellulose phenylmethylsulfonyl fluoride a 2.6x80 cm Ultragel AcA 34 column was eluted in 1.8 ml 155. The activity peak from

The only change in the previously described (NH ) SO4 precipitation, gel which involved inclusion of (DE 52) chromate 8sr phy was the in the homogenizing medium (50 ug/ml), and that This column was used for the gel filtration step. fractions and activity appeared in fraction 125the DE 52 column was concentrated by precipita-

958

Vol.

104,

No.

3, 1982

TABLE

I:

Correlation

BIOCHEMICAL

between

AND

inhibitory

BIOPHYSICAL

potency

and

RESEARCH

total

sulfur

content Total

I50 Polyanion

(ughl

Polyvinyl

sulfate

Dextran

sulfate

(40,000-dalton)

Polyanetholesulfonate Dextran

sulfate

(500,000-dalton)

COMMUNICATIONS

)

of

poiyaniOnS

S

(%I

0.33

16.7+0.4-

0.45

16.8tO.3

0.63

11.3+0.2-

1.8

15.750.3

A-Carrageenan

2.9

11.Ci+o.2 -

I-Carrageenan

6.1

9.1+0.3 -

K-Carrageenan

48.0

6.7tO.3

Heparin

68.0

7.4+0.5 -

tion with (NH ) SO at 50% of saturation, the precipitate redissolved in 8 ml of 5 mM Tris-fiC?, 8 H 7.6, 15 mM Z-mercaptoethanol and dialyzed 10 hr against 4 1 of this solution. After a second dialysis against 4 1 of 0.5 mM EDTA (Tris), pH 7.6, containing 2.5 mM dithioerythritol, glycerol was added to a concentration of 10:: and the solution stored in 0.5 ml aliquots at -30". Enzyme determination and other methods. Guanylate cyclase activity was determined as previously described (9), using siliconized reaction tubes, 1.2 to unless a different concentration is 1.4 ug of enzyme protein and 3 mM MnCl All determinations were in tri $'. locate, and the Imean + S.E. are renoted. ported. The figures have bars representing the S.E., except-where this was Protein was determined by the method of Bradford smaller than the symbol. Total sulfur content of the using bovine plasma albumin as standard. (lo), polyanions was determined by prompt y-ray neutron activation analysis (11). RESULTS: ronic

Dose-response acid

was

C (not

shown).

l),

Table

in

curves

not

I,

appreciably

The

inhibitors

with

their

anetholesulfonate, sulfate)

of

Inhibition Fig.

2 shows

only

76b

by that

by

2 ng

substitution

of

potentiation. diagrammed groups: and

2+

This in

Fig.

but

or

3,

carrageenans

Ca2+

which

for

show

Mn

(no

2+

rank

be

excess more

that 3A)

order

-potentiated, 959

Mn '+

2+ ),

HyaluA,

(from

Except

an

and

B or

Fig.

for

increase

activity

was

mM excess did

not

Mn

poly-

sulfur

(or

in

polyanions

Mn2+ -potentiated the

2+

in

.

However, a similar

the fell

(Fig. dextran

Mn2+

inhibited

produce

examined

sulfated

while

potency

potency

by

thoroughly the

and

7.2

1.

density.

increased

at

Fig.

content.

change

Mn

in

sulfate

of

between the

88%

shown

chondroitin

sulfur

excess

the was

(Fig. were

to

reached

phenomenon

Mn'+-independent the

found

mM MnGTP

was

correlation hence,

was

1.2

in

total

a good

are

nor

shown

polyanion,

heparin, Mg

are

heparin at

inhibitors

inhibitory,

was

the

the

determined

there

content

for

3B). sulfates,

studies into

two

Heparin poly-

.

Vol. 104, No. 3, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

30. z w k ; 25a

ol

a

0

12

2.4

3.6

EXCESS

4.8

60

72

METAL (mM)

Fiqure 2. Effect of Ca'+, Mg2+ and Mn*+ concentration on the inhibition of guanylate cyclase by heparin. Activity was dets$mined in t5$ presence of Jr2 mM MnGTP plus the indicated concentration of Ca &Al, Mg D,P , or Mn (o,.), in the absence (A,O,O) and presence (A,aO) of 2 pg heparin.

anetholesulfonate

and polyvinyl

obvious

difference

curring

while Wolff

between

M KCl,

examined

that

cyclase. by heparin,

Fig.

x-carrageenan

sulfate

more sensitive polyanions.

Reversal

are

naturally

by the

oc-

while

due to Cl - interference

dextran was eliminated,

to 16.5% from

the

Mn2+ -potentiated than may result

electrostatic 960

by

effect.

inhibition

We of

upon inhi-

sulfate. while 30.0% with polyanions

At 80 mM the

in-

no was much

the Mn2+ -independent from

of Mn2+ -independent

reversal with

in the

of the

reversed

of NaCl concentration

NaCl concentration

of Mn"-potentiation

sulfate

was partially

involved

two polyanions

was decreased

inhibition

site,

first

was due to a charge

and 500,000-dalton

first

to increasing

by Nat at a binding probably

that

The most

by dextran

in thyroid

4 shows the effect

by dextran appeared

cyclase

effect

hibition

the

inhibition

of a similar

by the

It

the

inhibition

inhibition

2+ -independent.

is that

the

NaCl the

salt.

that

adenylate

and concluded

Mn

synthetic.

showed

the possibility

guanylate bition

are

and Cook (4)

were

the two groups

the second

thyrotropin-stimulated 0.167

sulfate

binding.

replacement inhibition

of Mnzt is

Vol. 104, No. 3, 1982

BIOCHEMICAL

Figure 3. Effect of by sulfated polyanions. MnGTP plus the indicated no addition (O), and 0.12 pg 500,000-dalton sulfate (A) and 0.02 no addition (0), and (U), 0.1 Vg I-carrageenan

These

results

tography because

it

such

experiment,

with

buffer

taining vity,

readily

while

remaining

activity

was

40%,

the

loss

cular

of

activator

stimulation remains

from

be

these

the

was

polyanions

preliminary

studies

so

column

activity

is

(column

removed

A.

complet

ely

retained

from

recovery

activity due for

(12-14).

to some The

or

loss all

purification

determined. 961

one

equilibrated

buffer

not

with

the

B,

activity about

the

the

Increasing

column

on of

used,

B.

increased the

results

eluted

of

chroma-

shows

was

column

activity The

were other

from

the

IX

the

activity

affinity

heparin-agarose,

B),

detectable

M removed

with

co1 umns

columns

as

Table

heparin-agarose

specific

activity

of

commercially.

the

responsible of

to

0.25

and

total

done

Both

all

none

to

about

A).

nearly

concentration

use

8 mM MnC12

(column

M NaCl,

the

two

containing

however,

0.1

(4.

available

where

Mn*+

concentration on the inhibition of guanylate cyclase Activity was determ@ed in the presence of 1.2 mM concentration of Mn In A is shown the effect with in the presence of 0.04 ~9 polyanetholesulfonate (a), dextran sulfate (A), 0.02 ug 40,000-dalton dextran Pg polyvinyl sulfate (D). In B is shown the effect with in the presence of 2 ug of heparin (0) or K-carrageenan (A) and 0.07 ug A-carrageenan

We have

is

RESEARCH COMMUNICATIONS

Mn*+

suggested

ligands.

AND BIOPHYSICAL

columns

from

applied from

as

well

from 48%.

conacti-

column the

A by

NaCl as

the

one

the

columns

We believe of

the

that

macromole-

concentration-dependent attainable

on

these

columns

Vol. 104, No. 3, 1982

BIOCHEMICAL

O-3

0

AND BIOPHYSICAL

20

40 NaCl

RESEARCH COMMUNICATIONS

60 ktlM1

80

100

Figure 4. Effect of NaCl concentration on guanylate cyclase additional polyanion (Oj, and in the presence of 1 pg heparin 500,000-dalton dextran sulfate Q), or 0.05 ug A-carrageenan reactions contained 6 mM MnC12.

DISCUSSION:

Salomon

aminoglycans

and

adenylate

cyclase

sulfation,

rather

similar

usually

on

therefore

towards

both

guanylate

fate 16 the

we

of

of

synthetic

(7)

became

enzymes.

However,

make

inhibitor

of

it

cyclase B),

essentially

(15U=2 was no

of

Mg

of

adenylate

500,000-dalton

,,g/ml ,,g/ml)

on dextran

glycosovary

with

the

of

Mn

fAn

cyclase

heparin

at

3 mM Mn2*)

activity is

inhibition,

is

no and

as

guanylate

cyclase. had

weak

compared

Similarly, cyclase

it

potency a relatively

adenylate

sulfate

potentiate

, there

inhibitor

that

(6).

2+

to

guanylate

cyclase

than

of

to

of

come

on

2+

adenylate

rather

degree

We had polyanions

comparison

a potent effect

rat

correlate

ability

2+

no ug

sulfated of

sulfated

the

appears 68

the

with 0.15 The

polysaccharide.

the

a direct

(150=

sulfate

polyanion

to

Since

potentiation to

with

aware

of

inhibitors

the

of

presence

difficult

adenylate

of

effects

the

some

potent

polysaccharides.

in

(chrondroitin ug/ml,

the

that

appeared

structure the

a similar

cyclase

inhibition

the

about

determined

information is

than

some

were

Inhibition

until

by

showed

sulfate,

(5-7).

activity

inhibition

coworkers

dextran

conclusions

cyclase

is

and

activity (O), (A).

with

dermatan inhibitor, In similar

its sul-

15U= contrast, inhibitory

Vol. 104, No. 3, 1982

TABLE

II:

BIOCHEMICAL

Effect

of

MnCi,

on

Fraction number

AND BIOPHYSICAL

the

NaCl buffer CM)

binding

of

guanylate

cyclase

Guanylate (nmol cyclic

in

Column

Applied

0

36.7+0.2 (looTloo)

1

0

(0,LO)

2

0.1

53.0+0.2 (40.5,

3

RESEARCH COMMUNICATIONS

0.25

by

heparinagarose

cyclase activity GMP/min/mg protein) A

Column

B

34.5+2.2 (100;100)

28.0)

23.4+0.4 (6.2T9.8)

(0,

li.7)

(0,

l/).4)

52.6+9.0 (43.4, F8.4)

Two 0.7x2 cm heparin-agarose columns were equilibrated respectively with 50 mM Tris-HCl, pH 7.6, 15 mM P-mercaptoethanol (buffer A) and the same solution with 8 mM MnC12 (buffer B). To each column was applied 0.5 ml enzyme, followed by three 2 ml applications of buffer containing the indicated concentrations of NaCl. Activity of the applied enzyme, and in the collected fractions, was determined with 3.2 mM MnCl and 0.1 M NaCl. In parentheses are shown recoveries of activity and prote ? n, consecutively.

potency

for

adenylate

cyclase

(15D

= 1.8

electrostatic structure

cyclase

are

,,g/ml). while

involved.

The

ternate

B-D-galactose-4-sulfate

sulfate

residues X-ray

geenan

have

been

ever,

double

the

x-ray

data

helix.

The

the

inverse

fore, is

though

it

fraction has

helix

can patterns

shown

fects

in

high

degree

of

that circular of

their

in

terms

have

be

shown of

the to sodium

is

inhibited

potency may

have

helical

order

helical

blue

that

of

a random structure

salts

are

complexes which solution.

963

rank of

in

order,

A‘.l:'k

k-

r-carra-

and

How-

but

thus not

a double

(k>liX)

is

cyclase, the

coil

in

when

x-ray

extrinsic that

in

there-

enzyme.

solution

(17).

al-

additional

sulfation,

with

indicated

of

structure.

guanylate

display

of

largely

helices

recorded

to

aspects

structure double

only

with

increase

interaction

spectroscopy, in

an

form

of

calcium

the

a double

inhibitors

a helical

or

in

guanylate

due

consist

fibers

decrease

conformation

to

a helical

as

be

other

oriented

to

with

D-galactose, is

by

carrageenans

heparin-methylene dichroism

of

did

may

known

content of

it

polyanions

anhydro-

suggest

formation

to

3,6

as

inhibition

are

patterns

the

(7)

natural

sulfate

A-carrageenan of

order

considered

Their

formation

for

the

carrageenans

interpreted

ability

double

with

diffraction

helix

,g/ml)

sulfate

and

(15).

I).

= 4-6

Dextran

binding,

(Table

(ISO

Heparin (16),

fiber

al-

dif-

Stone Cotton heparin

(18) efhas

a

Vol. 104, No. 3, 1982 It quired

is

not

for

Mn

independent strong

2+

up the

potentiated (Table

imnediately

between random

inhibitors

I),

obvious

-potentiated

polyanions

repulsion

and open

BIOCHEMICAL

and therefore

tion

for

Mnzt-potentiation,

form

of a new affinity

AND BIOPHYSICAL why the

We are

inhibition. are

not

the coil

helical ionized

(19).

have a lower

groups

chromatography

conformation assuming

at these

ionic

would

that

charge

tend

hoping

density form

medium for

helices.

the

the Mn

that

the other Whatever

the

phenomenon

purification

be re2+

since

to stretch

than

to exploit

would

strengths,

It may not be coincidental

may more readily we are

helical

RESEARCH COMMUNICATIONS

the

the molecule the Mnztpolyanions the

explanain the

of guanylate

cyclase. to M.D. Glascock and T. Spaulding of the ACKNOWLEDGEMENTS: We are grateful for the determinations of total University of Missouri Research Reactor, This work was supported by Grant No. sulfur by neutron activation analysis. USPH 5 SO7 RR05387 awarded by the National Institutes of Health, Biomedical Research Support Grant Programs, and the John M. Dalton Research Center. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. E: 12. 13. 14. 15.

K: 18. 19.

Elbein, A D. (1974) in Advances in Enzymology (Meister, A., ed.) Vol. 40, pp. 29-61 Academic Press, New York. Bernfeld, P. (1963) in Metabolic Inhibitors (Hochster, R.M. and Quastel, J.H., eds ) Vol. 2, pp. 437-472, Academic Press, New York. Bernfeld, P. (1966) in The Amino Sugars (Balazs, E.A. and Jeanloz, R.W., eds.). vo ZB, pp. 213-228, Academic Press, New York. Wolff, J. and Cook, G.H. (1975) J. Biol. Chem. 250, 6897-6903. Salomon, Y. and Amsterdam, A. (1977) FEBS Lett. 83, 263-266. Salomon, Y., Amir, Y., Azulai, R. and Amsterdam, A. (1978) Biochim. Biophys. Acta 544, 262-272. Amsterdam, A., Reches, A. Amir, Y., Mintz, Y. and Sa lomon, Y. (1978) Biochim. Biophys. Acta 544, 273-283. White, A.A., and Karr, D.B. (1980) J. Cyclic Nucleo tide Res. 6, 221-282. White, A.A. and Karr, D.B. (1978) Anal. Biochem. 85, 451-460. Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. Hanna, A.G., Brugger, R.M. and Glascock, M.D. (1981) Nucl. Instr. and Meth., in the press. Tsai, S.C., Manganiello, J.C. and Vaughan, M. (1978) J. Biol. Chem. 253, 8452-8453. Liebel , M.A., Rapp, N.S. and White, A.A. (1980) Biochim. Biophys. Acta 616, 310-318. Nakazawa, K. and Kitajima, S. (1980) Biochim. Biophys. Acta 612, 171-177. Rees, D.A. (1969) in Advances in Carbohydrate Chemistry and Biochemistry (Wolfram, M.L. and Tipson, R.S., eds.) Vol. 24, pp. 267-332, Academic Press, New York. Stivala, S.S. (1977) Fed. Proc. 36, 83-88. Atkins, E.D.T. and Nieduszynski, I.A. (1977) Fed. Proc. 36, 78-83. Stone, A.L. (1977) Fed. Proc. 36, 101-106. Katchalsky, A. (1964) Biophys. J. 4;Suppl., 9-41.

964