Release of arachidonate from diglyceride in human platelets requires the sequential action of a diglyceride lipase and a monoglyceride lipase

Vol. 100, No. 4,198l

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages 1688-1695

June 30, 1981

RELEASE OF ARACHIDOHATE FRO!1 DIGLYCERIDE THE SEQUENTIAL ACTIOIJ OF A DIGLYCERIDE Lee-Young North

Received

May 18,

REQUIRES

LIPASE AND A MONOGLYCERIDE LIPASE

Chau and Hsin-t!siung

Departments of Chemistry State University and Texas Denton, Texas

Texas

Iri HUMAN PLATELETS

Tai'

and Biochemistry College of Osteopathic 76203

Medicine

1981

Summary: Release of arachidonate from 2-arachidonyl diglyceridT4by human Diglycerides labeled with C-stearate platelet microsongswas investigated. at sn-1 and with H-arachidonate at sn-2 were usedas a substrate for microsonal diglyceride lipase. Diglyceride was deacylated first at sn-1 as evidenced by the accumulation of 2-arachidonyl monoglyceride but not of 1-stearoyl monoglyceride. Subsequent release of arachidonate from monoglyceride required the action of a monoglyceride lipase. Studies on substrate specificity indicated that diglyceride lipase utilized 2-arachidonyl diglyceride as the best substrate. INTRODUCTION Rapid

turnover

considered appears

to be first

phospholipase Further route

C since

action

from

of a PI-specific an important

of fatty

acid

remains

DG lipase

phospholipase

role

from

to be determined.

catalyzes

in

in releasing

monoglyceride exclusively

by Bell

of a DG lipase

sn-1

1 DG.

and sn-2

Apparently,

the deacylation

to the A novel

the may serve

as a potential

DG lipase

sequence

of deacylation

the

of DG is these at sn-1

not

known.

in subsequent issues position

and found

may Whether deacylathat

and utilizes

'To whom correspondence should be addressed at Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40506. 0006-291X/81/121688-08$01.00/0 Copyrighr AN rights

0 198I by Academic Press, Inc. of reproduction in any form reserved.

1688

the

sequential

Although

participates

We have examined

(5).

(2-4).

--et al (3) who demonstrated which could catalyze the

positions

(MC) lipase

leads

by DG kinase

platelets.

arachidonate,

PI

of DG was observed

C and a DG lipase intact

(1).

by a PI-specific

es on 32P incorporation

recently

has been

stimulation

(DG) catalyzed

catalyzed

2-arachidony

arachidonate

residues

a separate

by stud

fraction

in human platelets

thrombin

accumulation

presumably

was proposed

in the particulate

means of releasing

or not

and transient acid

of arachidonate

(PI)

following

to 1,2-diglyceride

of DG as shown

of catabolism

release

inositol

events

rapid

of phosphatidic

presence

tion

initial

degraded

catabolism

formation

play

of phosphatidyl

one of the

Vol. 100, No. 4,198l

Z-arachidonyl donyl

BIOCHEMICAL

DG as a preferred

MG requires

the

action

AND

BIOPHYSICAL

substrate.

Release

RESEARCH

of arachidonate

COMMUNICATIONS

from

2-arachi-

of a FIG lipase.

MATERIALS

AND METHODS

Materials: Snake venom (crotalus atrox), phospholipase C (Bacillus cereus, 400, lysophosphatidyl choline (LPC), phosphatidylcholine (PC), indomethacin and dibucaine were purchased from Sigma Chemical Co. Lysophosphatidyl inositol (LPI), phosphatidyl inositol (PI), phosphatidyl ethanolamine (PE), phosphatidyl ethanolamine plasmalogen, dioleig and monoolein y$re obtained from '@-dary Research.Laboratoriet4 Inc.3[le - C] Stearate (lC] linoleate, ClC] 8,11,14eicosatrienoate, [lC] ar chidonate and [9,10-3H] oleate were obtained from New England Nuclear. [5,6H] arachidonate was synthesized as described previously by Tai --et a1.(6). Precoated silica gel G plates were products of MC6 manufacturing Chemists, Inc. Outdated human platelet concentrates were supplied by the Wadley Central Blood Bank of Dallas, Dallas, Texas. Preparation of human platelet microsomes: Human platelet concentrates were centrifuged at 200 x g for 10 min to remove contaminating red blood cells. The platelet were collected by centrifugation at 2,000 x g for 20 min. The pellet was washed twice with saline and centrifuged each time at 2,000 x g for 20 min. The washed platelets were suspended in tow volumes of 10 mF1 Tris-HCl, pH 7.0 and lysed by a Polytron homogenizer operated at full speed for 2 min with cooling. Disrupted platelets were centrifuged at 8,000 x g for 20 min and the supernatant was further centrifuged at 105,000 x g for 60 min. The final pellet was suspended in two volumes of 10 mM Tris-HCl, pH 7.0 and was designated as microsomal fraction. This fraction was used as the source of diglyceride and monoglyceride lipases. Preparation of labeled substrates: 2-[3H] and 2-[14C] diacylphosphoglycerides were prepared biosynthetically from rat liver microsomes by using I-acyl LPC or LPIl$nd respective labeled fatty acids as described by Robertson and Lands (7). l-[ C] Stearoyl PE was phepared biosynthetically from rat liver microsomes by using 2-acyl LPE and [lC] stearate according to Lands and Merkl (8). 2-acyl LPE was generated from PE plasmalogen as described by Norton (9) just befor use to keep3the isomerization of 2- to 1-acyl migration to a minimum. Both l-[ 74 C] and 2-E H] phospholipids were found to possess greater than 98% of positional purity as examined by snake venom phospholipase A treatment of the labeled phospholipids followed by thin layer chromatograp ii y of the reaction products. Labeled DG were obtained by treating the respective labeled phospholipids with phospholipase C followed by isolation of the DG by thin layer chromatography using petroleum ether/ethyl ether/acetic acid (60:40:1) as the solvent system. Labeled DG was eluted form the gel by chloroform/methanol/water (1:2:0.8) and the solvent was then removed by a stream of N Labeled DG was redissolved in petroleum ether and used immediately or store $' at - 20°C to prevent isomerization to 1,3-DG. The assay mixture for DG lipase contained: l-['4C]DG or 2-['4C]DG wi.5 to 10 nmol ; NaCl, 500 nmol; and platelet microsomes in a final volume of 0.5 ml of 50 mM sodium citrate buffer pH 3.5. Labeled DG was first dispersed in the buffer containing NaCl by vigorous vortex mixing for 1 min. The reaction was initiated by adding microsomal protein and terminated by adding 0.1 ml of 1 N HCl after incubation at 37°C for 20 min. The reaction mixture was extracted with 1 ml benzene. The benzene layer was removed after phase separation by centrifugation and dried under 112. The residue was dissolved in chloroform/methanol (1:2) and spotted on a silica gel G plate (2 x 20 cm) with monoolein and fatty acid standards. The plate was developed in the solvent system of petroleum ether/ethyl ether/acetic acid (60:40:1) to a height of 8.5

1689

Vol. 100, No. 4,1981

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Cm. Both substrate and products were3localized by exposure to iodine vapor. The labeled MG (from 2-C 4C]DG or 2-C H]DG) or the labeled fatty acid (from l-[14C]DG) region was scrapped into a scintillation vial and the radioactivity was determined by liquid scintillation counting. MG lipgse activityltas assayed by using 2-[3H]DG or 2-['4C]DG to generate The reaction was carried out in C]MG as a substrate. --in situ 2-[ H]MG or 2-[ 0.5 ml 50 mM sodium acetate buffer, pH 5.5. Incubation, extrgction and chromatography were carried out as described above. The release of [ H] arachidonate represented EIG lipase activity.

RESULTS AND DISCUSSION Previously oleyl

Bell

that --et al. (3) reported DG was hydrolyzed by platelet particulate

[1-14C]oleate

without

DG lipase

detectable

catalyzes

donate

and that

ficity

of this

catalyze

direct

appeared

to reexamine

the

release

products

and substrate

same reaction

mixture

of various ratio

of the

stearoyl pH 5.0,

the

products

may also

yield

DG lipase. kinetics

valuable

of the

of both

off

was also

earlier increased

that

in a time

l-[l-14C]steroyl

MG was not

clearly

that

indicate

position.

Similar

observed

findings

were

l-[l-14

MG again

Clstearoyl

diglyceride substrate at 5,10, the mixed

at sn-2 before

the

and 20 min, substrate

position reaction

microsomes

during

substrate with

MG, 1-[l-14C]

are

shown

in Fig

to the concept not occur. with

that

direct

ratio

that

to

[l-14C]stearate

the

formation

These that

of

results

at sn-2

DG was mixed

with

of formation

of

The lack

deacylation

of

ratio

of the mixed

of the products

of DG lipase

was found

DG and l-[l-14C]stearoyl

1690

of

precedes

When the 3H/14C

in product

2-[5,6-3H]arachidony1

1B.

The

MG appeared

when 2-[9,10-3H]oleyl in Fig.

1A.

was increased

30 min incubation. position

speci-

l-[1-14C]

in 50 mM Tris-citrate,

On the contrary,

the

as shown

was compared

the increase

the

The release

fashion.

label of the

determination

of 2-[5,6-3H]arachidonyl

obtained

attests does

kinetics

DG was mixed

of DG at sn-1

DG as substrate

the possible

MG and [5,6-3H]arachidonate

dependent

l-[1-14C]stearoyl

the mechanism l{l-14C]stearoyl

the

regarding

of [5,6-3H]arachidonate.

deacylation

We decided

of 2-[5,6-3H]arachidony1

The formation

than

speci-

which

Furthermore,

and [1-14C]stearate

arachi-

The use of dual

of following

experiment.

2-[5,6-3H]arachidony1

in a timedependentmanner.

(10).

of DG lipase.

human platelet

formation

lipases

and to explore

information

that

The positional

We employed

When 2-[5,6-3H]arachidony1 with

to release

DG to investigate

advantage

in a single

MG, [5,6-3H]arachidonate

production level

specificity

or

They proposed

other

linkage

of DG lipase

has the

DG and incubation

stearoyl

ester

DG or 2-[9,10-3H]oleyl

reaction

of 3H/14C

from

DG in human platelets.

in the

ficity

position.

specificity from

DG and 2-[5,6-3H]arachidonyl

formation

sn-1

at sn-1

MG.

position

to be different

deacylation

positional

of arachidonate

cleave

DG or 2-[l-14C]

to [l-14C]arachidonate

of labeled

of DG at sn-2

the enzyme may also

preferentially

fraction

accumulation

deacylation

DG lipase

2-[l-14C]arachidonyl

to be greater DG than

with with

the

BIOCHEMICAL

Vol. 100, No. 4,198l

16

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

IA

-

TIME, min. Fig.

1:

Time course jf the release of monoglycerides from mixed [ H]- and [14C]diglycerides (DG). was incubated with human Flatelet microsomes of 51) mM Tris-citrate, pH 5.0 at 37°C for the Extraction and chromatography of products were in Haterials and Methods. A.

2-[5,6-3H]Arachidony1 (15,000 cpm) 2-[g,lO-3H]Oleyl

6.

mixed

substrate

The higher higher

ratio

2-[3ti]HG;

-u--o-,

1-[14c]~~;

of using

DG is

analysis

trienoyl

acid of the

specificity, Using we examined

indicates arachidonyl the mixed the release

and

l-[l-14C!stearoyl

with

DG as shown

DG in the mixed higher

for for

specificity either

substrate MG.

DG lipase

than

of DG lipase 2-[1-14C]1inoleyl

exhibits

DG > eicosatrienoyl substrate of fatty

acids

1691

sn-1

Z-oleyl

DG.

shown

Comparison

DG > linoleyl

from

the % conversion

The results is

I.

indicate A more

in Table

II.

DG, or 2-[l-14C]eicosa

the following

2-[5,6-3H]arachidony1

in Table

was not due to

In fact,

2-oleyl

DG as a substrate. DG lipase

cpm)

[~~c]FA

substrate

substrate

DG (12,000

[3H]FA

P1G to arachidonate.

a better

DG

1-[1-l4C]stearoyl

DG and l-[l-14Clstearoyl

was slightly

that

and

a---d-,

DG or 2-[l-14C]arachidonyl ratios

cpm)

2-arachidonyl

DG was mixed

cpm)

--A-+-,

of 2-arachidonyl

fatty

2-arachidony

2-[9,10-3H]oleyl 3tl/14C

-&-&-,

conversion

that

3G (37,SO0

2-[9,10-3HJoleyl

of MG to free detailed

DG (40,000

(t1G) and fatty acids (FA) Mixed labeled OG (6.7 nmot) (15 ug protein) in 0.5 ml indicated period of time. carried out as described

of the

order DG = oleyl

DG.

DG and l-[l-14C]stearoyl and sn-2

positions

product

of substrate DG,

at pH ranging

Vol. 100, No. 4,1981

Table

I.

Mixed

A.

BlOCHEMiCAL

Substrate

3 to

or l-[l-

table

at pH 5.5.

However,

% arachidonate

consistent formation

7.8

9.2

7.7

DG

3.0

5.8

4.6

3.g

based

that

results

formation

indicated

in

of either

of 3.5,

and the

from

60% at pH 5.5 that

the

1.

release

MG

of [5,6-3H]arachidonate

as the pH optimum

that

Clstearoyl

to 93% at pH 7.0 at limiting

pH optimum

of MG lipase

MG lipases

the

is

have alkaline

MG was not observed

of DG lipase.

than

pH optima

t

7.0 which

Lipase Ratios

again

as

c3h]FA + L'4CJFA

0 min

-10 min

-20 min

3.1

2.8

3.0

3.2

2.6

2.5

2.9

1.8

1.9

DG t DG

DG

DG (10 nmol) was incubated with human platelet in 0.5 ml 50 ml1 sodium citrate buffer, pH 3.5 period of time. Extraction and chromatography out as described in Materials and r!ethods. The of labeled DG used was: DG, DG, DG,

54,500 64,600 75,700

corn; cpm; cpm;

E-[14C]Linoleyl 2-[14C]Eicosatrienoyl 2-[14C]Arachidonyl

1692

DG,

the

MG)

(11,lZ).

at any pH indicating

of Human "latelet DigIyceride Substrate and Product 3t!/l4C of Mixed Labeled Diglycerides

since

MG concentra-

higher

[3H]:lG ['4C,jMG

DG Mixed labeled 15 pg protein) the indicated were carried radioactivity

Fig.

can -not be regarded as the pH optimum of MG lipase from the total MG (free arachidonate + accumulated

B. 2-[3H]Oleyl DG + 2-[14C]Eicosatrienoyl

2-[3H]Oleyl 2-[3H]Dleyl 2-[3H]Oleyl

23 min

2-[5,6-3H]arachidony1

Substrate

2-[3H]01eyl 2-[14C]Linoleyl

A. B. C.

on the

the

II. Substrate Specificity Studied by Determininq During Deacylation

Mixed

A.

27.,

the reports

of l-[l-l4

Table

DG

had a pH optimum

indicates with

10 min

2 shows

release

This

of

5 min ---

The pH of 3.5 can be considered

increased

tions.

Deacylation

0 min

prepared

the pH of 5.5

generated

During

2-[3~1~G + [~H]FA L'4CJFA

was

14C]stearate

peaked

and Product 3tl/14C Patios Mixed Labeled Diglycerides

COMMUNlCATlONS

DG +

Fig.

9.

RESEARCH

DG +

B. 2-[3H]Dleyl 1-[14C]Stearoyl

from

BIOPHYSICAL

Substrate

2-[3H]Arachidonyl 1-[14C]Stearoyl

The

AND

microsomes at 37°C for of products amount of

17,400 cpm. DG, 20,200 cpm. DG, 25,900 cpn.

The that

is

Vol. 100, No. 4.1981

Fig.

the

2:

direct

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Effect of pH on the release of monoglycerides (YG) and fatty acids from mixed labeled diglycerides (DG). Nixed labeled DC, (10 nmol; 2-[5,6-3H] arachidonyl DG, 61,500 cpm; 1-[1-14C]stearoyl DG,(14,600 cpm) was incubated with human platelet microsomes (15 pg protein) in 0.5 ml of 100 ml1 Tris-citrate buffer at the indicated pH at 37°C for 20 min. Extraction and chromatography of products were carried out as described in Flaterials and Methods. --e--C-,

2-[%]arachidonyl

MG;

-C--C--,

[3H]arachidonate;

d--d-,

deacylation

arachidonate

of

from

DG in

which

DG at human

sn-2

was

platelets

of

a DG lipase

utilizes

at

sn-1

position

with

a pH optimum

at

sn-2

position

with

an

-L--L-.

not

permitted.

DG as

of

3.5,

requires

as

by

the

the

a preferred

followed

pH optimum

1%

Apparently,

microsames

arachidonyl

alkaline

[14C]stearate 2-[14C]stearoyl

sequential

substrate MG lipase

depicted

in

release action

and which

the

deacylates

hydrolyzes

following

scheme.

0 HZO-:-STA

CH20H

0 5 CH-O-;-ARA

Diglyceride

CH20H

I ;,,s~,,

Lipase

I roH

:Y;zY;det

-x+

ARA

L H20H

In not

to

contrast

include

properly the

CH20H

GSH after

reaction

STA:

Stearic

ARA:

Arachidonic

Acid

to

the

incubation

in

the

assay

the

addition

mixture

Acid conditions

mixture of

either

CH20H

used

since

it

acidic

GSH.

since

neither

1693

was We did

by

Bell

--et

inactive not

2 mM EDTA

al if

(3), pH was

add

any

nor

1 mM Cat+

we

did

controlled

exogenous

Cat+ affected

of

Vol. 100, No. 4,1981

either

lipase

activities

was no effect Both

BIOCHEMICAL

significantly.

of EDTA or Ca++

DG and MG lipases

curibenzoate MG lipase

was found Similar

lipases

were

on platelet

(12)

to be much more sensitive have

also

COMMUNICATIONS

reported

2-MG or l(3)-MG

to sulfhydryl

that

lipase

there

activity.

inhibitors.

DG and MG lipases

properties

RESEARCH

p-Hydroxymer-

40% and 100% respectively.

to sulfhydryl

been described

for

inhibitors

rat

brain

than

DG and MG

(13).

Our conclusion different

from

on the mechanism that

reported

from

DG requires

lipase,

they

claimed

whereas

catalyzed

by DG lipase.

incubation. mulated

Z-arachidonyl albeit

from

the rate

The pathway MG lipases

We are

able

0.3 mM. to support

from

that

includes a novel

In fact

very

release

and a FIG

could

be directly

pH used

in the

of arachidonyl

membrane

role

MG was acculittle

MG was seen

for

inositol

indomethacin provides

the

first

novel

pathway

(14) inhibit

evidence

platelets.

C coupled

with

DG and

of release that

indome-

by inhibiting DG lipase for

of

following

reported

platelets

does

in the

pH.

the mechanism

Rittenhouse-Simons

at neutral

step

in human platelets

of DG in activated

stimulated

30% of the optimum

the rate-limiting

phospholipase

hypothesis

phosphatidyl

that

of this

becomes

PI-specific

Recently,

observation

DG was only

DG at physiological

working

accumulation

in normal the

of MG from

of arachidonate

to confirm

This

DG is the

studies. Apparently, --et al (3) in their as soon as it was formed from 2-arachidonyl DG

by DG lipase

stimulation. induced

be due to the

amount

from that

of a DG lipase

of arachidonate

could to 7.0.

catalyzed

from

thacin

DG lipase

the optimum

of formation

provides

arachidonate

action

the release

2 decreasing

MG was deacylated

release

We have shown

was the pH used by Bell

The reaction

thrombin

that

in Fig.

as pH changed

of arachidonate

--et al (3). the sequential

The discrepancy

As shown

at pH 7.0 which

of release

by Bell

of arachidonate

PH. overall

Fielding

sensitive

at 0.1 mM inhibited

DG lipase.

AND BIOPHYSICAL

with

DG lipase. an I5o of

the functioning

Undoubtly,

more evidence

in releasing

arachidonate

of

is

required

in activated

platelets. ACKNOWLEDGEMENTS This

work

was supported

in part

by grants

Health (GM-25247), the American Heart Asscoiation Research Fund of the Texas College of Osteopathic

from

the National (78-865) Medicine.

Institutes

and the

Faculty

REFERENCES 1. Mitchell, R.H. (1975) Biochim. Biophys. Acta 415:81-147. 2. Rittenhouse-Simmons, S. (1979) J. Clin. Invest. 63:580-587. 3. Bell, R.L., Kennerly, D.A., Stanford, N. and Majzus, P.W. (1979) Natl. Acad. Sci. U.S.A. 76:3238-3241. Smith, A.D. (1980) Biochem. Biophys. 4. Homa, S.T., Conroy, D.M .,and Commun. E:1321-1327.

1694

Proc. Res.

of

Vol. 100, No. 4,198l

5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Lapetina, E.G. and Cuatrecasas, P. (1979) Biochim. Biophys. Acta 573:394-402. Tai, H.H., HSU, C.T., Tai, C.L., and Sih, C.J. (1980) Biochemistry E:1989-1993 Robertson, A,F. and Lands, W.E.H. (1962) Biochemistry 1:804-810. Lands, W.E.M. and Merkl, I. (1963) J. Biol. Chem. 238:898-903. Horton, W.T. (1960) Biochim. Biophys. Acta 38:340-34 . Brockerhoff, H. and Jensen, R.G. (1974) Lipgytic Enzymes (Academic Press, New York) pp. 55-58, 107. Bry, K., Anderson, L.C., Kuusi, T., and Kinnunen, P.K.J. (1979) Biochim. Biophys. Acta 575:121-127. Fielding, C.J.7981) J. Biol. Chen. 7256:876-881. Cabot, M.C., and Gatt, S.(1976) Biochim. Biophys. Acta 431:105-115. Rittenhouse-Simmons, S. (1980) J. Biol. Chem. 255:2259-2262.

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