Activation of rat lung particulate guanylate cyclase due to filipin induced fluidity change

Vol.

96, No.

1, 1980

September

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages 203-210

16, 1980

ACTIVATION OF RAT LUNG PARTICULATE GUANYLATE CYCLASE DUE TO FILIPIN INDUCED FLUIDITY CHANGE Pushkaraj John M. Dalton

Received

July

J.

Lad*

Research Center, University of Missouri-Columbia, Columbia, Missouri 65211 U.S.A.

25,198O

SUMMARY: Involvement of cholesterol in the regulation of rat lung particulate guanylate cyclase was studied with filipin. The enzyme was not activated to a great extent by sodium nitroprusside alone; however, in presence of filipin nitroprusside activated the enzyme about 12-16 fold over the basal. Filipin did not affect the soluble enzyme significantly. The changes induced by filipin did not cause solubilization of proteins or enzyme. The Arrhenius plot of filipin-treated particulate enzyme did not have a "break" which was evident with untreated enzyme. The results suggest that the sequestering of cholesterol by filipin can modulate the activity of membrane-associated guanylate cyclase probably by changing the membrane fluidity. INTRODUCTION Guanylate found

in particulate

mammalian soluble

and soluble

tissues.

Lubrol

activity

of the particulate Phospholipid

alamethicin investigation, guanylate

perturbing

other

polyenes,

bilayers

to ions,

membrane

cholesterol

Present

address:

in its

increases

guanylate

the

of the

in the presence X-100

to study

gramicidin cyclase

of cholesterol

hydrophilic

of most

from

Because

is

the

latent of or

regula-

surroundings.

, melittin

the permeability

differs

such as Triton

difficult

native

by utilizing

and small

assayed

detergents

involvement

was examined

lung (1,2).

usually

make it

the particulate

water

is

EC 4.6.1.21

from homogenates

properties

polypeptides

the possible cyclase

it

detergents

enzyme

stimulate

prepared

of nonionic

However,

(cyclizing),

enzyme from rat

and kinetic

concentrations

PX (2,3).

lyase

fractions

The particulate

of the particulate

solubilizing

like

[GTP pyrophosphate

enzyme in physical

nature

tion

cyclase

an antibiotic,

S and

(4,s).

In the present

in the regulation filipin.

of Filipin,

of cholesterol-containing molecules

by interacting

with

(6-8). The Salk

Institute,

P. 0. Box 85800,

San Diego,

CA 92138.

0006-291X/80/170203-08$01.00/0 203

Copyright 0 1980 by Academic Press, Inc. AN rights of reproduction in any form reserved.

Vol.

96, No.

BIOCHEMICAL

1, 1980

AND

MATERIALS

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

AND METHODS

Filipin (Lot #3-1194) was purchased from Polysciences, Inc. GTP and nystatin were purchased from P. L. Biochemicals, and [a-32PlGTP and 13H]cGMP were obtained from New England Nuclear. Cyclic GMP, creatine phosphate, amphotericin B and other unspecified biochemicals were creatine phosphokinase, from Sigma. Bio-Gel A-5m was obtained from Bio-Rad. Male rats weighing in the range of loo-150 g were Enzyme Preparation. decapitated and lungs were perfused in situ with 10 ml of homogenizing buffer (50 mM Tris-HCl, 0.5 mM Na2 EDTA, 10 mM Z-mercaptoethanol, pH 7.6) by injecting it in the right ventricle. The lungs were removed and washed twice, blotted, placed in 3 volumes homogenizing buffer (w/v> and homogenized for 10 set at The 10,000 rev/min with a Willems Polytron equipped with a PT 20 ST generator. homogenate was filtered through a 40-mesh stainless steel wire screen and centrifuged for 10 min at 8000 xg, after which the supernatant solution was The supernatant was used as soluble centrifuged at 160,900 xg for 40 min. enzyme. The pellet (microsomes) was resuspended in 1 ml buffer and chromatoThe column was eluted with graphed on a Bio-Gel A-5m column (1.6 X 15 cm). 50 mM Tris-HCl, pH 7.6 buffer containing 10 mM 2-mercaptoethanol. The void volume fractions were pooled and used as the particulate enzyme.

Assays and Biochemical Determinations. Guanylate cyclase assays were performed by determining the conversion [cr-32P]GTP to [c~-~~P]cGMP. The assay mix contained 1.2 mM GTP, 6 mM MnC12, 5 mM cGMP, 50 mM Tris-HCl, 15 mM Enz e activity creatine phosphate and 10 units of creatine phosphokinase (9). was assayed for 5 or 6 min at 37°C with lo-30 pg protein. The [a-3 TP]cGMP formed was separated from substrate by sequential chromatography on Dowex 50 and neutral aluminum oxide according to White and Karr (10). Proteins were determined by the method of Lowry --et al. (11) after precipitation with silicotungstic acid (12). Bovine serum albumin was used as the standard. Cholesterol was quantitated by the method of Zlatkis and Zak (13). Specific activities are expressed as picomoles cGMP formedlminfmg protein. All assays were in triplicate, and the activities are given as the mean 2 standard error of the mean. Data presented here are representative of at least 2 or 3 experiments.

RESULTS Rat lung cyclase.

homogenate

A vasodilator,

cyclase

20-25

fold

(5).

2-4

fold,

fold

and the optimum

with

higher

concentrations

than

that

nitroprusside

achieved plus

was greater

filipin than

the particulate 1, fillipin

activity

appeared

was about

the activity

activated

observed

204

mg/ml

A decrease

activated

synergistically

soluble

with

guanylate

activated

only

the particulate

at 0.2-0.4

However,

PX, which

guanylate

enzyme is activated

one.

of filipin. Lubrol

and particulate activates

in Fig.

ratio

with

soluble

nitroprusside,

however,

As illustrated

to filipin

both

sodium

(14);

cholesterol

which

contain

enzyme

filipin

where

in activity

was less fold.

the enzyme about Lubrol

PX.

the

was seen

the maximum activity the enzyme 7-9

2-3

Sodium 24-fold,

Vol.

96, No.

1, 1980

BIOCHEMICAL

FILlPIN

BIOPHYSICAL

CONCENTRATION

filipin on particulate tubes containing various at 37"C, then guanylate

for 6 min at 37°C with

( 0 ) and without

in

Filipin soluble

soluble

medium

enzyme activators activate

soluble

like

organic

the particulate

enzyme Km (with

with

disrupted

or without

nitroprusside

205

about

with

Other

hypo-

soluble

and arachidonic

enzyme treated

PX but

or

and column

shown).

0.2 mM, which

(not

contaminating

vesicles (not

the basal

by nitroprusside

lipoxygenase

the particulate

or without

and

assayed

cGMP formed/min/mg

by solubilization

peroxides,

Lubrol

was

not activate

not express

sensitive

with

picomoles

did

membrane

GTP of about

Enzyme

of filipin

activity

activation

enzyme treated

had a Km for enzyme treated

cyclase

its

was evident

nitroprusside

cyclase.

concentrations 1127.1

did

Furthermore,

GTP was determined,

nitroprusside particulate

This

guanylate

- 0.5 mg/ml

treatment

COMMUNICATIONS

( 0 ) 1 mM sodium nitroprusside.

PX was

or potentiated

studies. remained

Lubrol

of 0.001

filipin

enzyme.

chromatography

Km for

0.9%

cyclase

In addition

trapped

of

in the range

guanylate

shown).

tonic

presence

RESEARCH

(mg/ml)

Fig. 1. The effect of aliquots were added to preincubated for 5 min Activity protein.

not

AND

filipin. with

treatment).

higher

did

When the

filipin

was identical 4 times

acid

plus to the than

the

Vol.

96, No.

BIOCHEMICAL

1, 1980

AND

I. Effects of filipin rat lung particulate

Table

BIOPHYSICAL

RESEARCH

and detergents on the guanylate cyclase Guanylate

Treatment

- SNP

105.0 385.0 836.8 737.0 302.3

None

Tween 20 Triton X-100 Lubrol PX Filipin

4.0 4.0 1.2 0.4

Particulate and

fraction

assayed

for

sodium nitroprusside

Treatment activators zation did

(Table

I>.

of filipin

filipin

were

activity was found observations cyclase

activity.

filipin

treatment suggest Effect

preparation

that

volume. where filipin

was activated filipin

of temperature treated

there that

with

caused

the

filipin

speed

by sodium

was no significant

non-sedimentable preparations

did

nearly

amount

not

solubilize

cyclase

all

with

of the and enzyme

eluted. guanylate

rechromatographed better

These

than

after Lubrol

PX

change.

cyclase

206

used

treated

of protein

is normally

nitroprusside

(microsomes

centrifugation was no significant

enzyme

an irreversible

of protein

and there

a trace

guanylate

detergents

majority

the particulate

soluble

treatment

on guanylate

done,

A-5m column,

Only

in solubili-

guanylate

When microsomes

on a Bio-Gel

The particulate

that

Also

II),

enzyme contamination.

fractions

support

8.6 7.7 a.6 a.9 22.5

known

to filipin,

upon high

(Table

its

results

were

the pellet

suggesting

in the void

in the

2 i + 2 +

or 1 ti

with

of the particulate

fraction

chromatographed

preparation

studies

into

particulate

soluble

eluted

detergents and without

with

In contrast

activation

in control,

from

various

activity

(15).

enzyme activity.

enzyme activity free

activity

sedimented

non-sedimentable

with

413.6 408.3 859.8 714.4 1684.2

and lysolecithin

When solubilization

treated

2 1.6 + 9.8 t 2.6 f 12.5 2 10.3

cyclase

detergents

nitroprusside

and enzyme activity

which

guanylate

cyclase

not potentiate

cyclase

+ SNP

(SNP).

of particulate

of guanylate

were

(30 ug) was treated guanylate

such as non-ionic

cyclase

Cyclase Activity

Concentration bg/ml)

filipin

COMMUNICATIONS

activity

chromatographed

in particulate on Bio-Gel

A-5m

Vol.

96, No.

BIOCHEMICAL

1, 1980

Table

II.

AND

Solubilization

of by

BIOPHYSICAL

particulate

A.

Activity

Water

Protein

guanylate

COMMUNICATIONS

cyclase

filipin Enzyme

Fractions

RESEARCH

1.0% SA

in

Lubrol

Presence

of

PX

TA

1 mM SNP

SA

TA

SA

(4

TA

Original

1.96

260

132?

6

2239

1137

+ 15

1046

Pellet

2.6

196

752

1

2234

898

t 26

627

Supernatant

0.1

22

204

_+ 60

312 + 20

45

428

+ 12

2222

996 + 13

2449

1098

2161

795 2 29

1405

517

2 13

920

+ 76

CONTROL

B. FILIPIN

33

531

2 13

2412

4 ? 56

TREATED

Original

2.23

465

208

Pellet

2.7

339

125 t

Supernatant

0.1

15

8

140 ? 64

16

152 + 80

98

?

2

About 1.8 ml of a particulate enzyme preparation was mixed with 0.2 ml buffer (control) of 5 mg/ml filipin (filipin treated). About 0.25 ml from each tube was removed (original) and assayed, remaining suspensions were centrifuged at 139,000 xg for 60 min. Pellets and supernatants were also assayed for guanylate cyclase activity in presence of various treatments. TA = total activity, picomoles cGMP formed/min in each fraction, SA = specific

activity,

column

picomoles

after

filipin

without

filipin

control

enzyme

for

studies activates

membrane been

showed

with

lung

Kimelberg

In membranes, average

area

phospholipid at 37"C,

the

and Paphadjopoulos the effects

above

the transition

per molecule

and reduced

bilayer

(19-20).

membrane

matrix

The Arrhenius but

guanylate

The activity upon

During is

chromatographed plot

such a break

of the

was not

evident

filipin.

The Arrhenius

to describe

"viscotropic,"

2.

22-25"C,

particulate

changes.

(microsomes

DISCUSSION that the cholesterol

demonstrate

by nitroprusside. fluidity

shown in Fig.

treated

shown to be dependent

(16-18).

ation

is

around

rat

protein.

and untreated

a break

enzyme

These

activation

treatment)

treatment)

particulate

filipin,

cGMP formedlminlmg

plots of several

fluidity

binding cyclase

and potentiates

suggest

that

membrane

have introduced

of membrane

fluidity

temperature, the motion guanylate

in a liquid

207

filipin bound

of the phospholipid

(18)

the

antibiotic,

the

enzymes

has

term

on enzyme activity.

of hydrocarbon

crystalline

causes

environment

cholesterol

cyclase

its

condenses chains

activity state

thus

the

of determinfilipin

Vol.

96, No.

1. 1980

BIOCHEMICAL

Fig. 2. The effect Microsomal preparation were chromatographed for 2 min at various lines were determined

probably

increases

supported

by spin

label

fluidity

studies

by sequestering

which

in the

hydrophobic

core

However,

another

polyene

amphotericin

did

not activate

interacts

with

in nature,

(not

of an aminosugar

altered

"break"

other attached

alter

COMMUNICATIONS

fluidity

in Arrhenius

B did

not affect

cyclase for

to the macrolide of membranes of rat

208

ring

polyenes

amphoteric

and nystatin its

activation

that

filipin

and is neutral

properties

because

(22-23).

of various lung

is

(21).

(21).

B, candicidin

thanother

used possess

membrane

or potentiated

is

increased

fluidity

the difference

more effectively

This

filipin

containing

amphotericin

A reason

plot

that

of a cholesterol

studied,

polyenes

cholesterol.

demonstrated

guanylate

shown).

cholesterol

whereas

Alcohols

polyenes

particulate

by nitroprusside

RESEARCH

temperature on the particulate guanylate cyclase. treated with 0.3 mg/ml filipin ( 0 ) and buffer ( 0 ) on Bio-Gel A-5m column. The enzymes were preincubated temperatures and activity was assayed for 4 min. The by the method of least squares.

membrane

other

BIOPHYSICAL

of

fluidity

Interestingly,

AND

particulate

origin

(24-25). guanylate

Ethanol cyclase,

Vol.

96, No.

1, 1980

however, (Lad,

it

did

P. J.,

mechanisms induces

BIOCHEMICAL

not potentiate

paper

disorganization site

cyclase

is

submitted enzyme

hydrophobic It

site(s)

[26]).

This

altered

membrane

fluidity

changes

bound with

adrenergic cytochrome

postulated

that

a conformational

freedom

that

fluidity. is

receptor

in adenylate

(28,29).

the rate-limiting

of guanylate

cyclase

A conformational

change step

in the enzyme

in lipid for

fluidity

A. A.,

paper particulate complex

by detergent cyclase

molecules

activity

change

activation

by

due to

by catecholaminehas been observed

(30).

an enzyme-catalyzed

It

has been reaction

can be

(31).

ACKNOWLEDGEMENT This work was supported in part by a USPHS Grant I-IL 15002, from the National Institutes of Health, and by the John M. Dalton Research Center. I am grateful to Dr. Arnold A. White, in whose laboratory this work was done, for his generous support, advice and criticism. REFERENCES 1.

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COMMUNICATIONS

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