Vol. 91, No. 1, 1979 November

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

14, 1979

Pages

ISOLATION

OF HOMOGENEOUS

PHOSPHOLIPASE

63-71

A2

FROM HUMAN PLATELETS Rafael

J.

Apitz-Castro', Mahendra

Manuel A. Masl, Kumar Jain2

Maria

' Centro de Biofisica y Bioqucmica, Instituto Investigaciones Cientificas (IVIC), Apartado (Venezuela), and Department of Chemistry, ware, Newark, DE 19711 (USA) Received

September

The

has

been

ane

Az(TXA2)

ated tal

situ

a variant gation of tially

a key

prostaglandins

from

membrane

of of

is

as

and

deficiency

step

(3). A2

and this

for

in

the the

A2

To its

better

activity

regulation, from

platelets of

arachidonic

in

Also been

which

we have blood

liber-

a congeniobserved

platelet the

human

thrombox-

acid

has

understand

enzyme

blood

(1,2).

syndrome,

has been homogenate the extract. protein of platelet

synthesis

phospholipids

phospholipase

impaired

characterized

A2 activity

from

Hermansky-Pudlak

phospholipase

and

Venezolano de 1827, Caracas 101 University of Dela-

A2 from human blood platelets by treatment of the platelet affinity chromatography of is a soluble, heat-stable enzyme inhibits ADP-induced

phospholipase

implicated

--in

Cruz'

24, 1979

Summary: Phospholipase purified to homogeneity with H2S04, followed by The platelet phospholipase MW 44000. The purified aggregation. Introduction.

R.

putative purified

in

aeererole and

par-

platelets.

Materials and Methods. The following published procedures were used for obtaining the materials used in this study. Egg phosphatidyl-choline was p:epared from hen egg yolks (4): The labelled substrate. 2-1 C linolinoyl-phosphatidylcholine, was prepared by acylation of lysophosphatidylcholine (obtained by hydrolysis of egg phosphatidylcholine by phospholipase A2 from Naja naja venom) with rat liver homogenate (5). The specific activity of the product was ~1 mCi/mmol and all (>98%) the label was released in the fatty acid fraction byphospholipase A2 from The lecithin analog, rac-l-octa-decNaja naja or bee venoms. 9-enyl-2-hexadecylglycero-3-phosphocholine (Calbiochem) was oxidized with potassium periodate and potassium permanganate (6,7) to yield rat-1-(9-carboxy)nonyl-2-hexadecylglycero-30006-291X/79/210063-09$01.00/0 63

Copyright All rights

@ I979 by Academic Press. Inc. of reproduction in any form reserved.

Vol. 91, No. 1, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

phosphocholine, which was coupled to AH-Sepharose 4B (Sigma) The phosphorus content to yield the affinity adsorbant (8). of the affinity gel was ~2.5 nmol/ml of the swollen and packed gel. Platelet rich plasma from freshly drawn human blood was obtained as described elsewhere (9). Phospholipase AZ activity was measured by either of the following two procedures: (a) The radiolabelled substrate dissolved in wet diethylether was incubated with the enzyme (lo), and the products were separated by thin layer chromatography on silica gel plates (Merck) in chloroform + methanol + water (65:35:6), where the fatty acid, diacylphosphatidylcholine and lysophosphatidylcholine migrate at different rates (Rf ^1 0.95, 0.33 and 0.10 respectively. The radioactivity in appropriate spots was visualized with a Beta-Camera LB 290 A (Berthold, Germany) which gave a photographic record. For quantitation of the radioactivity in the spots, the appropriate regions on the t.1.c. plate, after extracting the scraped silica gel were counted. The substrate and products could also be separated by column chromatography on neutral alumina (Woelm) where fatty acid is eluted by chloroform, phosphatidylcholine by chloroform + methanol (7:3), and lysophosphatidylcholine by methanol. (b) Phospholipase A2 activity was routinely measured titrimetrically as described elsewhere (11, 12). Unless stated otherwise the titrations were carried out with 2.5 mM sodium hydroxide at 37'C, pH 9.5 in aqueous solution containing 100 mM KCl, 20 mM CaC12 and 500 uM egg phosphatidylcholine as unilamellar vesicles obtained by sociation in a bath type sonicator (Heat Systems). Control experiments demonstrated that under these conditions there is no detectable proteolytic activity as measured with Azoalbumin (Sigma) (13) even in the whole platelet homogenate. With appropriate precautions we could routinely measure rates of proton release < nmollmin. Results: elets ets

The was

from

lo-40%

overall

essentially 200

ml

linear

stilled homogenate

After

23000

g)

with

and

water. by

residue

KCl)

and

all

up

to

of The

0.16

N H2SOs

was

applied

to

the

in

and

homogenized

pH 7.5,

the

in

affinity

column

64

I

platelon

a

in

dis-

total

The volume

centrifuged

(20

(36

dialyzed

hrs)

min,

and

finally

against

the

dialysis

bag

supernatant

solution

plat-

B homogenizer. 40 ml

was

precipitate and

separated

in

from

Typically,

extensively

1 mM Tris,

A2

were

type

homogenate

white

dissolved

14)

Kontes

centrifugation, was

steps.

glass

the

phospholipase

plasma (9,

supernatant

changes

distilled

The

an

two

rich gradient

made

the

in

platelet

30 minutes

several

removed

of

with was

of

achieved

glycerol

water

(15).

purification

was

(20

mM CaC12

(40

ml

packed

was

lyophilized. and

100

swollen

mM

Vol. 91, No. 1, 1979

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

06

Fig.

l.-

volume)

Elution profile obtained from the affinity column obtained as described in Methods. In this particular experiment, 1.2 ml fractions were collected. Rate is arbitrarily expressed as divisions per minute.

pre-equilibrated

washed

with

solution

at

nm.

The

280

with

the

KCl,

pH 8.0).

and

for

moving

the

in I

till

adsorbed front

the

same

the

solution.

effluent

showed

phospholipase of

the

A typical phospholipase

II

profile

A activity

is

column

no

A activity

solution

elution

The

was

absorption

was

eluted

(40

mM EDTA,

100

for

the

protein

presented

in

mM

(A280) Fig.

1.

B

A

d’

5 3 ‘4 0” -3 I

I

I

I

I

I

123456 MIGRATION (cm) Fig.

2.-

SDS-polyacrylamide pattern of purified platelet phospholipase (PPL) in a 4-40X acrylamide gradient. 15 ugr. protein were loaded on the gel (right). Comparative migration of PPL with regard to known MW protein standards (4-40% acrylamide): cytochrome C (12.5 K), soybean trypsin inhibitor (21.5 K), ovalbumin (43.5

K),

and

bovine

serum

6.5

albumin

(68

K).

BIOCHEMICAL

Vol. 91, No. 1, 1979

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

0.90

0.33

Fig.

The

3.-

Hydrolysis of C-2 radiolabelled phosphatidylcholine by purified platelet phospholipase. Reaction was performed in diethylether and the products separated by thin layer chromatography, as described in Methods. Rf for fatty acid is 0.87, for phosIn this system, phatidylcholine is 0.35 and lysophosphatidylcholine do not migrate from the origin (16).

fractions

dialyzed

gradient

showed of

ular

weight

was

iour

of

enzyme

the

MW,

constituting

was

sometimes

The

rate

of

enhanced

tain

2-position,

in

the and

gel

when

the

1% of

and

The

of

in

band

purified

the

ab-

molec-

the

behav-

of

higher

protein,

excluded.

additives

n-hexanol.

The in

pro-

estimated with

total

On

purified

presence

phosphatidylcholine

presence

no

the

coincides

the

pooled,

lyophilised.

A minor

SDS was

dissolved

then

2).

which

were

SDS,

filtration.

of

and

in

(Fig.

than

hydrolysis in

both

dalton,

in

seen

detergents

the

44000

and

gel

dodecylsulfate

less

phatidylcholine in

band,

activity

water,

polyacrylamide

a single

sodium

enzymatic

against

sence

is

the

extensively

a 4-40% tein

containing

by like

labelled

is

66

found

diethylether,

A2 cer-

2-linolenoylphos-

diethylether

label

phospholipase

releases in

the

the

fatty

lysophosphatidyl-

acid

Vol. 91, No. 1, 1979

choline

fraction

have

(Fig.

isolated

is

phospholipase

shown

in

of

rich

Table

I

is

te

the

the

rate

of

It

enzymatic

activity

has

obtained

preparation. the

Table

530

to

be

rate of

et of

al.

I.

Phospholipase

substrate

of

activator

(%

None

Similar

a partially found

enzyme,

of

Human

to

be

after

% of

Initial

Activity* nmol*min-l*rng-l

100

100

0.4

10

58

2.3

0.03

40

530

column

is in

these

measured with the presence

of

unilamellar egg 45 mM n-hexanol

67

condiwith

is the

at maximal

results

independent cofactor

Steps. Phospholipase

Initial)

of

purified

substrate,

Platelets

The

nature

enzyme

Ca 2+ , and .

phosphatidylcholine at pH 9.5.

phos-

linearly

and

dialysis

Activity vesicles

with was

the

the

2+

the egg

(n-hexanol).

Activity

of

varies

pH for

(16)

the

Under

y 5 mM Ca

of

10 mM CaC12. to

for

replatelet

sonicated

published).

hydrolysis

addition

the

at

with and

requirement

the

activity

sensitive

the

overall

from

-1

smin

optimum

achieved

The

be

of

absolute

Franson

Treatment

*

homo-

phospholipase

= 40%

specific

quite

(to

The

is

-1

Protein

Affinity eluate

in

the

with

mM n-hexanol

hydrolysis

an

The

nmolemg

activator

or

N H2S04

we both

identified

purification

Purification

1.6

enzyme

however,

of

fold

overall

45

found

by

sequence

(Ca2+ 1,

is

> 3000

fold.

concentration.

9.4-9.5.

of

be

purification

was

500,000

is

and

protein

been

overall

containing

lipid

the

Az,

could

The

protein

activity

tions

the

over

phatidylcholine

the

A2 activities

activity.

lyophillized

enzymic

that

phospholipase

platelets

the

plasma

suggests

RESEARCH COMMUNICATIONS

(3).

homogenized

covery

AND BIOPHYSICAL

This

exclusively

platelets

from

3).

A1 and

genized As

BIOCHEMICAL

Various

have

Vol. 91, No. 1, 1979

Discussion.

In

procedure

for

human

platelets.

(44000

dalton)

from

BIOCHEMICAL

the

preceding

a complete The

molecular

differs

membranes it

is

modified

by

n-hexanol,

or

For

the

on

than

a soluble

the

zymes

for

compared

to

hundred

fold

specificity,

the

the

purified

and

other

implicated

in

action

or

pig

diethyldetergents.

fold

to

to

a difference the

en-

In is

contrast, several

ADP-induced due

lower

pancreatic

enzyme

is

the

1000

(11,12).

platelet

a difference

in

in

enzyme.

A:! may

4.

The

data

The

second

bating

the

platelets

one

first

amounts

platelet

that

inhibits wave

help

the

state

Studies to

resolve

wave of

the

(17,18).

with these

the

second

wave

of

aggregation

is

u 0.0015

units

3 minutes)

aggregation.

enzyme.

68

contrast,

effect

shown (=

is

by

the

or

aggrega-

inhibited of

of

in

3 ug

ADP-induced

periods which

In

is enzyme

of

A2 has The

aggregation platelet

(1

of

phospholipase

the

with Longer

endogenous

aggregation on

shows

minute.

the

of

platelet

phospholipase

tion.

larger

venom

to

other bilayers

in

about

presented

sheep

from

in

is

phospholipase

the

units)

block

is

due

from

bilayers

platelet

this

obtained

phospholipid

enzyme

from

enzyme

or

dissolved

inhibiting

and

platelet

0.0015

than

in

A2

enzyme

on

first

possibilities.

platelet Fig.

the

that

substrate

Physiologically, been

acts

conditions

effective

the

which

snake

the

enzyme

as micelles

platelet

enzymes,

We suspect

in

it

identical

aggregation. substrate

the

the

substrate

venom,

other

more

for

(13)

Like

dispersed

the

bee

under the

purified

egg-phosphatidylcholine of

measured

the

the

25000)

(8).

substrate

the

of

(


that

not

Interestingly

(20)

These

such

As

due

platelets. ic

at

phospholipase

likely

i,

6

The effect of purified platelet ADP-induced platelet aggregation A) Concentration dependence, Bee venom phospholipase (BVPL) comparison.

naturally

of

5

(min.)

aggregation

fatty

effect

4

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

outer

the

platelet

sources.

In

Vol. 91, No. 1, 1979

our

experiments,

outer of the

besides

surface, the

other

enzyme

and

membrane,

could

Indeed,

preliminary

substrate

and

lyzed

BIOCHEMICAL

by

the

the

factors also

from

AND BIOPHYSICAL

action arising the

contribute

platelet

activator enzyme

the

from

enzyme

to

this

changes

the

of

that rate

the

at

least

the

substrate behaviour

depending of

on

specificity

particular

suggest used,

only

substrate

organization

experiments the

of

RESEARCH COMMUNICATIONS

hydrolysis several

in (21).

upon

the

catahundred

fold.

Acknowledgments. Work supported in part by CONICIT (Grant i/ 0665 to R.A.C.) and by a travel grant from U.S. National Science Foundation (to M1K.J.). We-wish to thank the personnel of the Blood Bank of the Hospital Universitario (Caracas) for the generous supply of fresh platelet-rich plasma. One of US (M.K.J.) would like tQ thank Atala Apitz for a generous hospitality and useful discussions. We acknowledge excellent technical assistance of F. Triana, T. Cardenas, V. Michelena and Mrs. A. Istok, Highly purified Naja Naja phospholipase was a gift of Dr. Flor Barnola.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11. 12. 13.

Blackwell, G.J., Duncombe, W-G., Flower, R.J., Parsons, (1977) Br. J. Pharmac. 59, 353-366. M.F., and Vane, J-R. Silver, M.J., Bills, T.K., and Smith, J.B. (1978) In: Platelets, Ed. G. de Gaetano, and S. Garattini, pp. 213225, Raven Press, New York. Breton-Gorius J,, Trugnan, G., Castro-Mamaspina,, Rendu, F., H ., Andrieu, J.M., Bereziat, G., Lebret, M., and Caen, J.P. (1978) Amer. J. Hematology 4, 387-399. Singleton, W.S., Gray, M-S., Brown, M.L., and White, J.L. (1965) J. Amer. Oil Chemist's Sot. 42, 53-56. Waite, M., and Van Deenen, L.L.(1967) Biochim. Biophys. Acta 137, 498-517. Shimojo, T., Abe, M., and Ohta, M. (1974) J. Lipid Res. 15, 525-527. Rock, C.O., and Snyder, F. (1975) J. Biol. Chem. 250, 6564-6566. Kramer, R.M., Wcthrich, C., Bollier, C., Allegrini, P.R., and Zahler, P. (1978) Biochim. Biophys, Acta 507, 381-394 Apitz-Castro, R., Ramirez, E., Maingon, Rh., Murciano, A., Biochim. Biophys. Acta 455, 371-382. and Ribbi, A. (1976) Biochim. Coles, E., McIlwain, D.L., and Rapport, M.M. (1974) Biophysic. Acta 337, 68-78. Biochem. Biophys. Upreti, G.C., and Jain, M.K. (1978) Arch. 188, 364-375. Jain, M.K., and Apitz-Castro, R. (1978) J. Biol. Chem. 253, 7005-7010. Proc. Natl. Kaplan, L., Weiss, J., and Elsbach, P. (1978) Acad. Sci. 75, 2955-2958.

70

Vol. 91, No. 1, 1979

14. 15. 16. 17. 18. 19. 20. 21. 22.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Murata, Y., Stake, M. and Suzuki, T. (1963) J. Biochem. (Tokyo) 53, 431-437. Barber, A.J., and Jamieson, G.A. (1970) J. Biol. Chem. 245, 6357-6395. Kates, M. (1972) In: Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 3. Ed. T.S. Work and E. Work, North Holland Publishing Co., Amsterdam. PP. 551-553, Beckerdite, S., Mooney, C., Weiss, J., Francon, R., and Elsbach, P. (1974) J. Exp. Med. 140, 396-409. Circulation 57 (Supp. II), Franson, R., and Jesse, R.(1978) 11-124. Marcus, A.J. (1978) J. Lipid Res. 19, 793-826. Vincent, J.E., and Zijlstra, F.J. (1976) Prostaglandins 12, 971-979. Iatridis, S.G., Datridis, P.G., Tsiala-Paraschou, E., Markidou, S.G., and Ragatz, B.H. (1976) Haemostasis 5, 165-175. Verger, R., and De Haas, G.H. (1976) Ann. Rev. Biophys. Bioengg. 5, 77-117.

71

Isolation of homogeneous phospholipase A2 from human platelets.

Vol. 91, No. 1, 1979 November BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 14, 1979 Pages ISOLATION OF HOMOGENEOUS PHOSPHOLIPASE 63-71...
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