The action of phospholipase C on black film bilayer membranes

Vol. 46, No. 5, 1972

BIOCHEMICAL

THE ACTION

Department

Received

of

AND BlOPHYSlCAL

OF PHOSPHOLIPASE

H. Stewart Chemistry,

February

C ON BLACK

Hendrickson St. Olaf

and College,

FILM

E.

RESEARCH COMMUNICATIONS

BILAYER

MEMBRANES

M. Scatter-good Northfield,

Minnesota

55057

11, 1972

Summary : The effects of phospholipase C (Clostridium perfringens) on the electrical and visual properties of black film bilayer membranes are reported. Addition of this enzyme to one side of a phosphatidyl choline : phosphatidyl serine : cholesterol (0.71 : 0.29 : 1 .O, w/w/w) bilayer membrane resulted in a rapid decrease in membrane resistance which leveled off after several minutes at about one-half its initial value. The maximal rate of resistance decrease was proportional to the amount of enzyme added. A maximal effect was observed with phosphatidyl choline : phosphatidyl serine ratios between 1:l to 9:l. No effect was observed in the absence of Ca2+ and an optimal effect was observed at 2 mM Ca2+. A pH optimum of 7.2 was observed. These studies demonstrate the possi bi 1 i ty of forming asymmetric membranes by 1 imi ted phosphol ipase action on one side of a bilayer membrane. Such a system can be used to study the mechanism of enzyme action on a substrate which is part of a bilayer membrane and the possible role of asymmetric membranes in membrane processes such as the control of ion permeability.

Introduction: widely

Phospholipase used

(Roelofsen, Although

ported

et

al.,

action

of

a black

in

this

on

black

of

this

are

asymmetric

and

film

bilayer

1970; bilayer on

has

or on

here.

vesicles, film by

membrane Ohki,

1971)

prompted

The and this

1961 Copyright

@ 1972, by Academic

Press, Inc.

very

little

has

as

effects

membranes.

visual crude

the

The action

control

of

of

has

phospholipase

C

C from of

been

Na+/K+ 1971;

phospholipase

properties

on

of

Reinertsen,

a study

enzyme

re-

involvement

and

of

1971).

been

phospholipase

(Hendrickson

electrical Although

biological

possible

such

Rapport,

intact

bilayer

the

processes

and

with

limited

and

been

membranes

Mcllwain

studied

black

has

biological

1968;

been

membranes. the

of

Singer,

membrane

phospholipases,

functions

membrane,

axonal

nerve

other

membranes

the

reported

and

enzyme

in

perfringens

with

dispersions

membranes

film

along

enzyme

Papahadjopoulos,

Clostridium membranes

of

forming

asymmetric

action

Lenard

action of

and

1971;

the

permeability Ohki

structure

phospholipid

possibility side

the

and on

such

probe

the

membranes

one

to

C,

black shown

film to

BIOCHEMICAL

Vol. 46, No. 5, 1972

contain

phosphodiesterase

activities

phosphoglycerides

(Pastan,

convenience

and

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

does

et

not

specific

al.,

for

19681,

exclude

the

the

sphingomyelin

term

possible

as

phospholipase

involvement

of

well

as

C is

used

than

one

more

for

enzyme.

Experimental of

:

Folch

Phosphatidyl

(Lees,

method

of

tional

1957);

by

was

Phosphatidyl

Singleton,

homogeneous

serine

et

TLC.

al.,

prepared

from

choline

(1965)

and

Phospholipase

was

was

C (C.

beef

brain

by

the

method

from

egg

yolk

by

from

Nutri-

prepared

shown

to

be

perfringens)

the

chromatographically

was

obtained

Biochemicals. Bilayer

cup

in

an

The

thickness

stirred

membranes

apparatus

NaCl,

IO the

and

capacitance

was

calculated

known

teflon

and

were from

Radio

1650-A

flected

light

by

phosphatidyl

and

after

hole

was

The mM tris

and

begun capacitance

several The

1.

to

membrane

in minutes

membrane and

The

series

of

membrane

were

prepared

membrane

was

of

Resistance

membrane

was

with

with

with

a

re-

choline,

n-decane

completely

a

measured

observed

rnkJ

res i stance

series

phosphatidyl in

100

27”+?“.

in

was of

were

otherwise.

Capacitance

Mixtures

(1970).

specified

The

membrane

Tien

consisted

unless

a teflon

(1.5%

black

and

w/w). gave

stable

readings.

The

resulted

the

1970).

scope.

the

7.2

in

compartments

solution

across

cholesterol

and

electrodes.

bridge.

after

Both

a temperature

Ag-AgCl

hole

Huemoeller

mm.

pH

Tien,

binocular

and

0.3

at

drop

impedance

by

electrolyte

out with

1.5 mm diameter

a

buffer,

voltage

Discussion:

Figure the

the

addition

choline-phosphatidyl

W/W/W)

off

described

carried

the

a 40X

were

phosphatidyl 1.0,

that

measured

serine,

and

to

(Huemoeller

General

resistance

IO

were

resistance

Experiments

across

stirrers.

experiments

Results

at

magnetic

mM CaC12,

All

formed

similar

of

with

were

of

phospholipase

C to

serine-cholesterol a rapid at

decrease about

resistance resistor

in

membrane its

measured

and

recording

1962

side

membrane

one-half was

one

(0.71

resistance

initial

value

continuously the

voltage

of

: 0.29

which as by

a

leveled

shown applying

drop

:

across

in 50 the

mv

BIOCHEMICAL

Vol. 46, No. 5, 1972

AND BIOPHYSICAL RESEARCH COAMUNICATIONS

c-o.7 * 4 w 0.6. # g 0.5. $I 0.4'

OL 0

2

I

5

4

3

ys

6'

c II

IO

TIME (mid Figure

I.

Membrane Concentration

(3) 1.76

membrane

resistance of pg/ml. broke .

versus enzyme: Stirring

time (I) rate:

after

addition &ml; (2) 740 rpm.

0.44

of phospholipase 0.88 w/ml; f - indicates

C. time

n

Figure

2.

membrane tance did to

was not

With

break,

before

high

leveling

a value

increased initial

.

Membrane

concentrations off,

but

the

sometimes , as value.

the

low

with

resistance, slightly

enzyme

of

of

while

resistance At

addition

greater decreased, levels,

enzyme,

enzyme

capacitance

the

low

concentrations

after

decreasing than to after

1963

its

the

membrane of and

initial

a value

= 0.42

broke enzyme

the

leveling

resistance

The

30%

the

increased capacitance

greater

had

resis-

membrane

off,

value. about

as

uF/cmz.

decreased

than

the and

Vol. 46, No.. 5, 1972

BIOCHEMICAL

Figure

Membrane capacitance

3.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

15 minutes = 0.51

after uF/cm2.

addition

of

enzyme,

0.44 a40

8

t

ENZYME

Figure

leveled creased) after

4.

off, to addition

Rate (1) (2) (3) (4)

the

of resistance 1 week old 1 week old 5 week old 10 week old

capacitance

a value of

CONCENTRATIONJB@I)

sometimes the

enzyme

decrease versus enzyme lipid solution, stirring lipid solution, stirring lipid solution, stirring lipid solution, stirring

then less many

decreased than small

(as its light

1964

the

initial spots

concentration. 740 rpm. rate: 580 rpm. rate: 740 rpm. rate: 580 rpm. rate:

resistance value. appeared

slowly Several on

inminutes

the

membrane

Vol. 46, No. 5,1972

which

appeared

to

Figures

2 and

enzyme.

It

is

not

of

can

The

maximum

an

added of

the

shown

in

serine

not idyl nor

age

of

of 5.

after

break

in

broke

ratios

of

were

not

the

of not

the

of no

Ca

change

break

as

constant

the

rate

seen

after

.

9:l

time.

addition

0.42

in

of

of

to

0.51

to

the

the pF/cmz

phosphatidyl

choline

:

With

no

little

phosphatidyl

decrease and

addition

of

of

the

the

or

re-

enzyme. since

enzyme, but

in

capacitance

however,

capacitance, of

subse-

is

serine,

resistance,

In

possible.

appearance

phosphatidyl

a

decrease

was

addition

also

stirring.

hydrolyzed,

after

was

amount

resistance

(w/w).

after

addition

of as

the

still

curve

rate

there

Also,

With

this

between

and

apparently

of the

significantly

2-k

in

proportional

and

to

stable

12 minutes

absence

membranes

1:l

enzyme.

change was

was

is

about as

after from

was slope

on

effect

within

there

choline,

kept

composition

choline

the

were

A maximum

did

phosphatidyl

did

lipid

The

solution

factors

out

area.

4.

lipid

spread

increase

decrease

Figure

more

15 minutes

capacitance

resistance

the

and

and

membrane

in

addition

membranes

all

of

larger

before

the in

shown

membranes

membranes

that

as

serine

sistance

The

seen

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

become

membrane

these

Figure

the

the

the

rate

effect

phosphatidyl

and

increase

experiments The

of

be

to

function

coalesce

3 show

due

enzyme

quent

BIOCHEMICAL

but no

the did

phosphat-

appearance,

enzyme.

8

WEIGHT

Figure

5.

Rate of centration:

resistance 1.76

FRACTION OF PHOSPHATIDYL TO TOTAL PHOSPHOLIPID

decrease versus &ml , stirring

1965

lipid rate:

CHOLINE

composition 740 rpm)

(enzyme .

con-

BIOCHEMICAL

Vol. 46, No. 5, 1972

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

,a*.

I-

E

026

-

Co 1

024

.

g

a20-

1

0.16 .

z

0.12 .

i

aoa.

z

ao40

65

60

65

x.5

20

SOLUTION

6.

Figure

The This

is

the

C.

enzyme for

in

and

observed vesicles change

at

since concentration

Figure

7. The

effect

Ca

of 1 and in

the

pressure

exact

same

Ca2+ nature

by

MacFarland

and

phosphatidyl

of

lipids

on

the Ca

2+

membrane.

effect

on

required

for

lower

in ESR

and activity

the

potential

Knight C.

the

Knight

they

(1968)

as

between

simiiar and

--al

observed

of

phosphatidyl

serine

and

capacitance

changes

Bangham

’

myelin an

that

2 mM Ca

-et

the

resistance

1966

2+ may

(1970)

lipid

organizational a change monolayers

concentration. of

is

0.2

to

1 and

as

ob-

activity

Butler,

for

necessary was

(1941)

interpreted

(1941)

was

seen

spin-labeled

6.

perfringens

optimum

2 mM is

at

Figure

membrane

between

of

Papahadjopoulos

surface

and

Ca2+

membrane.

signal

, which

in

was

of

MacFarland in

shown

the

activity

concentration

increase

for

concentration: buffer used

choline.

no

of

as

(1962)

Dawson

2 mM was

by

10 rn!

and

reported

Cazi

sharp

2+

pH 7.2

plateau

optimal

The

at

absence

of

a reorganization

lipids

its

versus pH (enzyme 740 rpm; Acetate

response

of

dispersions

(1962).

this The

in

A second

micellar

surface about

Bangham

dispersions

between of

pH optimum

A Ca2+

an

decrease rate:

optimum

and

activity

in

to

the

micellar

Dawson due

an

enzyme

1 .O rnfi Ca2’.

observed

the

to

welchii

enzyme

and

gave

similar

served.

be

enzyme

with

shown

Rate of resistance 1.76 vg/ml , stirring pH = 5.6).

as

6.0

ptl

is

not

in

Vol. 46, No. 5, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I’ 9

CALCIUM Figure

7.

known. any of

Rate of resistance concentration:

These great

results

serine

incubation choline

resulting

in

in

the

5 potential

diglyceride enzyme

at

, after

membrane

.

breakage

of

the of

indicates

that

Mcllwain

myelin into

the and

interior

a second

This

enzyme

Rapport

does (1971)

Their of

one

results the

micelle

leveled

of

not

the

in

their can and

had to

be

the that

the

lipid

explained

its

1967

replacement

of

leveling

off

on

the

side

no

further

same

the

other.

no

rapid

as

by

the

was

phosphatidyl

in

redisalso suggested

C action of

the

resulted

This

bilayer

migration

of on

side is

of

of

effect

there

of

cessation

phospholipase by

repulsion

accumulation

the

opposite

phos-

A decrease

the

for

the

enzyme

of

choline

resistance.

from

to

study

14%

only

serine in

membrane

penetrate

of

Hydrolysis

by

indicate

to

phosphatidyl

account

off, then,

would

side

may

of

enzyme,

of

result

evidenced

portion

of

92%

may

hydrolyzed

hydrolysis

a decrease

This

had

membrane.

and

as

report

(enzyme .

not

inter-phosphatidyl

values

point

, addition

from

film

is

perfringens).

in

binding.

resistance

lipids

vesicles. the

Ca2+

of

increase

concentration 740 rpm)

serine

versus

C (C.

the

calcium rate:

(1971)

vesicles

negative

a certain

the

However

tribution

by

more

Addition

membrane

an of

reduced

activity

resistance.

cause

to

and

Rapport

myelin

expansion

versus stirring

phosphatidyl

phospholipase

may an

and

in

with

phatidyl

that

Mcllwain

extent.

phosphatidyl

after

decrease

1.76 ug/ml,

indicate

IO

CCYKENTRATION(mM)

on

diglyceride choline

Vol. 46, No. 5, 1972

migrating

from

ganization

of

membranes

could

remains

the

interior

the

the

bilayer

caused

by

membrane

and

Dawson,

structure.

the

The

accumulation

(Lenard

and

1962), spots

of

Singer,

or

by

which

and

appear

diglyceride

1968,

lysis

which

Ottolenghi

reor-

on

the

probably

and

Bowman,

* Although

which

Ohki

differ

in

potential,

these

rea ted

enzyme-t

change

the

calcium

net

the no

applied resistance

used

that

one

membrane

.

the

potential

loss

give

of

choline

current

in

the

to

measure

the

should

could

be measured

in

lipid

membrane

a with does

not

and

sufficiently There

potential. the

polarity

resistance

had

not

packing

may

transmembrane since

exhibit

however,

phosphate

membrane

membranes

other

Changes

a measurable

of

bilayer

C treatment,

choline,

by

to

to

Phospholipase

phosphatidyl about

side

asymmetric

of

no

the

effect

on

the

measured. These

branes

studies

by

limited

This

system

can

which

is

part

demonstrate enzyme

be of

action

in

nerve

axonal

other

phospholipases

Dr.

Orin

Lofthus

study

such Studies

on

(6822809)

on

bilayer

function

This

Foundation

to

a lipid

membrane.

the

action

used

membrane

Acknowledgments:

thank

on

charge

voltage

such

membranes

rectification

postulated from

no

brought

surface

has

charge

charge

binding

alter

(1971)

surface

transmembrane

was

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(Bangham

micelle be

with

1970)

BIOCHEMICAL

work and

The for

of

and

also

as

possible

bilayer

action role

control

of

now

being

conducted

Na+/K+

membrane. on

of

mem-

a substrate

phospholipase

permeability on

the

in

effects

the

of

membranes.

by Institutes with

asymmetric

film

enzyme

the

supported

assistance

forming

a black of

the

bilayer

National

of

side

mechanism

film

was

one

the

are

black

possibility

grants

from of

the

Health

The

National

Science

(NS09210-02).

We

photography.

REFERENCES Bangham, Butler,

A.D., K.W.,

comn. Hendrickson,

and R.M.C. Dawson, Biochim. Biophys. Acta H. Dugas, I.C.P. Smith, and H. Schneider, 40, 770 (1970). H.S., and J.L. Reinertsen, Biochem. Biophys.

(1971).

1968

59, IO3 BEchem. Res.

(1962). Biophys.

Comm.

2,

Res. 1258

Vol. 46, No:5,

1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Huemoeller, W.A., and H.T. Tien, J. Chem. Ed. 2, 469 (1970). Lees, M.B., in S.P. Colowick, and N.O. Kaplan ed., Methods in Enzymology Academic Press Inc., New York (1957). and S.J. Singer, Science 159, 738 (1968). Lenard, J., MacFarland, M.G., and B.C.J.G. Knight,iochem. J. 35, 884 (1941). Mcllwain, D.L., and M.M. Rapport, Biochim. Biophys.-&ta 239, 71 (1971). Ohki, S., J. Colloid Inter. Sci. 37, 318 (1971). Ohki, S., and D. Papahadjopoulos FM. Blank ed., Surface Chemistry of Biological Systems, Plenum Press, New York (1970). Ottolenghi, A.C., and M.H. Bowman, J. Membrane Biol. 2, 180 (1970). Papahadjopoulos, D., Biochim. Biophys. Acta. 163, 240 (1968). Pastan, I., V. Macchia, and R. Katzen, J. Biol. Chem. 243, 3750 (1968). Roelofsen, B., R.F.A. i'waal, P. Comfurius, C.B. Woodward, and L.L.M. van Deenan, Biochim. Biophys. Acta 241, 925 (1971). Singleton, W.S., M.S. Gray, M.L. Brown, and J.L. White, J. Am. Oil Chem.

2,

53 (1965).

1969