A particulate arachidonate lipoxygenase in human blood platelets

BIOCHEMICAL

Vol. 76, No. 2, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A PARTICULATE ARACHIDONATE LIPOXYGENASE IN HUMAN BLOOD PLATELETS Peter

P. K. Ho, C. Patricia

Lilly

Research

Received

April

Walters,

Laboratories, Eli Indianapolis,

and Hugh R. Sullivan

Lilly and Company, Indiana 46206

P.O.

Box 618,

4,1977 SUMMARY

The microsomal fraction of human platelets contains a lipoxygenase activity in addition to the thromboxane-synthesizing The enzymatic activity was stimulated by tryptophan, activity. but inhibited by catecholamine, methemoglobin, and hydroquinones. Hamberg and Samuelsson converted

(1) found

added arachidonic

acid

that

into

three

12L-hydroxy-5,8,10,14-eicosatetraenoic 5,8,10-heptadecatrienoic derivative

of

acid

acid

acid

(HHT),

(thromboxane

by the action

of a novel

enzymes involved

in prostaglandin

TXB2 were formed

by pathways

oxygenase

in the

initial

mediate.

This

preincubation methacin

platelets at least

positions

into

Recent in vitro --

Copyright All rights

for

12L-hydroxy-

with

unrelated

the

fatty

with

of the

acid-induced

that

release

of

aggregation

(2).

isolated

eicosapolyenoic 8- and ll-

(3).

shown that

human polymorphonuclear

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

bonds at the

inter-

or indo-

was subsequently

acids

cyclo-

G2 as the

aspirin

and was shown to convert

have also

acid

HHT and

by the observation

stimulation

double

to the

whereas,

prostaglandin

lipoxygenase

two cis

HETE was

biosynthesis,

suspensions

L-12-hydroperoxy

studies

lipoxygenase

or arachidonic

arachidonate

with

i.e.,

hemiacetal

B2 or TXB2).

was supported

in a 3-fold

thrombin

from bovine acids

conclusion

resulted

A soluble

(HETE),

and the

involving

step

of platelet

HETE during

metabolites,

8-(1-hydroxy-3-oxopropyl)-9,12L-dihydroxy-5,10-

heptadecadienoic formed

human platelets

HETE was chemotactic

leukocytes

and eosinophils

398

1SSN0006-291X

Vol. 76, No. 2, 1977

These findings

(4,5).

lipoxygenase that

this

fraction. the

BIOCHEMICAL

from

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

have prompted

human platelets.

enzyme activity

is

In this

found

The characterization

identification

of the

an attempt

in the

of this

reaction

paper

isolate

a

we report

particulate

particulate

products

to

enzyme and

are described.

EXPERIMENTAL PROCEDURES Materials [l-l4 Cl-Arachidonic acid (sp. act. = 55 mCi/mmole) and [l-14C]-8,11,14-eicosatrienoic acid (sp. act. = 57 mCi/mmole) were obtained from New England Nuclear. The out-dated platelet concentrates were provided by the Community Blood Bank of Marion County (IN). The lyophilized platelet microsomal powder was prepared by the previously described procedure (6). Analytical precoated layers of silica gel G glass plates (LQGD) for thin-layer chromatography (TLC) were products of Quantum Industries, Fairfield, New Jersey. Whatman Extraction Thimbles (10 x 50 mm) were purchased from Sargent-Welch Scientific, Cincinnati, Ohio. Enzyme Assay The reaction mixture contained 50 mM Tris-HCl buffer, pH 8.0, 5 mM L-tryptophan, 0.3 mM indomethacin, and 0.1 mg of lyophilized microsomal powder in a total volume of 0.2 ml. The reaction mixture was initiated by the addition of 10 1.11 of 100 uM 14C-arachidonic acid (sp. act. = 55 mCi/mmole). After 15 min incubation at 37"C, the reaction was terminated by the addition of 10 ~1 of 1 M citric acid. The products were separated by TLC with the solvent system of ethyl acetate-2, 2,4-trimethylpentane-acetic acid (1OO:lOO:l). Preparation

of

the Product

of Lipoxygenase

A large-scale reaction mixture (200 ml) was prepared with 50 mM Tris-HCl buffer, pH 8.0, 5 mM L-tryptophan, 0.3 mM 1 mg of arachidonic acid (1.5 uC of 14C-arachidonic indomethacin, 500 mg of lyophilized microsomal acid was added as a tracer), powder and the incubation was performed at 37°C for 60 min. The mixture was extracted twice with 1 liter of ethyl acetate. The residue obtained after the evaporation of the ethyl acetate The solution was dissolved in a small amount of methanol (2 ml). was removed by was then cooled to -2O“C and the precipitate This procedure was repeated until the solution centrifugation. It was then applied onto two silica gel was clear at -2O'C. plates and subjected to TLC. Zones with an Rf value of 0.41 were collected into Whatman After Extraction Thimbles and extracted with methanol (50 ml). the product was dissolved in 5 ml of evaporation to dryness, chloroform and subjected to silica gel column chromatography (2 x 45 cm) as described previously (6). HETE was eluted with v/v) and showed a single 500 ml of chloroform:methanol (95.5, spot with an Rf = 0.41 in TLC. The methyl ester of this The product was then prepared by treatment with diazomethane.

399

Vol. 76, No. 2, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

DISTANCE (CM 1 Fig.

1.

Thin-layer chromatography of labeled products in reaction mixture. Human platelet microsomes (0.1 mg) were incubated with 14C-arachidonic acid in the presence of L-tryptophan and indomethacin and the reaction was performed as described in METHODS. Solvent system = ethyl acetate-2,2,4trimethylpentane-acetic acid (1OO:lOO:l v/v/v).

use of column chromatography the colloidal gel particles during extraction.

was necessary which filtered

Combined Gas Chromatography-Mass

in order to remove through the thimbles

Spectrometry

The separation and identification of the trimethylsilyl (Me3Si.O) derivative of HETE methyl ester were accomplished utilizing an LKB-900 combined gas chromatograph-mass spectrometer. Separations were accomplished using a 1.2 M siliconized glass column (1.2 mm i.d.) packed with 1% UV-W98 methylvinyl silicone gum rubber on 800-100 mesh Gas Chrom Q (Applied Science Lab., State Collete, PA). The column temperature was maintained isothermally at 210°, while the flash heater and separator were held at 250'. The carrier gas (helium) flow was 30 ml/min. The ionization potential and trap current were The entrance and collector 70 eV and 60 uA, respectively. slits of the MS were adjusted to 0.1 and 0.3 mm. The preparation of the trimethylsilyl derivative of the HETE methyl ester was accomplished by reaction of the product with bis(trimethysilyl)trifluroacetamine (Regisil-Regis Chemical Co.) in the presence of triethylamine (1:l).

400

BIOCHEMICAL

Vol. 76, No. 2, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

w

r

x 15

310

320

Fig.

2.

Mass spectrum recorded on the Me3SiO derivative of the methyl ester of the product isolated from the reaction mixture by TLC.

RESULTS Products

of the

Since

the

complex, (61,

acid

indomethacin

was included

and HETE.

The identity

chromatography,

the major to that ion

ester peak

reported

of

Cofactor Table formation

the

reaction

of the

peaks which

mixture

to

As shown in Fig. incubated

1,

reaction

correspond

to arachidonic Rf value,

and mass spectrometry. with

the Me3SiO derivative

time

and ions 316,

295,

(Fig.

= 2.5 min)

by Hamberg and Samuelsson

335,

synthetase

of HETE was based on its

(90%, retention

375,

enzyme

and thromboxane

HETE and the mass spectrum

at m/e 406 was obtained

seen at m/e 391,

contain

in the

radiochromatogram two mjaor

methyl

also

the cycle-oxygenase.

Two peaks were observed the

Mixture

cycle-oxygenase

contains

gas-liquid

Reaction

microsomes

inactivate

thin-layer

mixture acid

platelet

fatty

completely the

Incubated

(1).

of high 229,

is

2) of

identical A molecular

intensity

225,

of

205,

were and 173.

Requirements 1 shows the of HETE.

effect

of various

L-tryptophan

components

stimulated

401

the

on enzymatic

enzymatic

BIOCHEMICAL

Vol. 76, No. 2, 1977

Table

Cofactors

1.

AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

Requirements

for

HETE Formation HETE (pmoles)

Cofactors

134

None

57

2 uM Methemoglobin 5 mM L-tryptophan

200

1 mM Epinephrine

61

5 mM L-tryptophan

+ 1 mM Epinephrine

94

1 mM L-tryptophan

+ 2 uM Methemoglobin

52

1 mM Epinephrine

16

+ 2 uM Methemoglobin

5 mM L-tryptophan + 1 mM GSH

+ 2 uM Methemoglobin 26

The reaction mixture contained 5 uM 14C-arachidonic acid, 0.3 mu indomethacin, and 0.1 mg of lyophilized microsomes in 0.2 ml of 50 mM Tris buffer, pH 8.0. After incubation for 15 min at 37V, the HETE was assayed as described in METHODS.

reaction,

while

reaction. either

epinephrine

and methemoglobin

Reduced glutathione alone

or in combination

or epinephrine. pH = 6.0

had no effect

The rates

with

inhibited

on HETE synthesis

methemoglobin,

of HETE formation

the

L-tryptophan,

were maximal

at

- 9.0.

Time Course of HETE Formation At 10 uM arachidonic only

linear

a cofactor. upon the Substrate

for

the

acid,

first

The degree

the

formation

15 min using

L-tryptophan

of HETE synthesis

amount of microsomal

protein

of HETE was as

was also

added

dependent

(Fig.

3).

Specificity

14C-8,11,14-Eicosatrienoic mole)

was substituted

mixture

and product

for formation

acid arachidonic

at

5 uM (sp. acid

was examined

402

act.

in the by TLC.

= 57 ?JCi/

reaction A product

Vol. 76, No. 2, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I 30

20

10

Fig.

3.

similar rate the

4.

of reaction substrate,

arachidonic

to hydroxy

however,

was found.

was approximately

The

acid

40% of

that

as for

and Inhibitors

by the

could

technique

enzyme activity

not

it

lost

of 4 M KCl.

The heat

most of stability

The lipoxygenase

be released

of freezing

was stable

and thawing,

4.

acids

acid.

The lipoxygenase

Fig.

fatty

14 C-8,11,14-eicosatrienoic

using

Enzyme Stability

the

0.2 mg 0.1 mg 0.05 mg

Effect of temperature on the stability of the lipoxygenase. The assays were performed under standard assay conditions, and the lipoxygenase activity was expressed as pmoles of HETE formed per 15 min.

in Rf value

component

70

TEMPERATURE

Time course of HETE formation. The assays were performed under standard assay conditions with 10 uM arachidonic acid and various amounts of microsomal powder per 0.2 ml of reaction mixture. OW x-x M

Fig.

60

Q

TIME(MIN)

to the its

activity of

and thawing. treatment

Although

of freezing

in the presence

lipoxygenase

was incubated

403

from the membrane

is

at various

shown in temperatures

BIOCHEMICAL

Vol. 76, No. 2, 1977

for at

5 min and then 60'

and lost.

Certain donors with

a I5o

at

50% activity

transition

(e.g.,

systems

assayed

metal

ions

The enzyme is very

unstable

Cu 2+ ) and hydrogen

(e.g.,

were inhibitors

those

to

37°C.

at 5OoC.

hydroquinone) similar

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

found

of

in the

lipoxygenase

thromboxane-synthesizing

(6,7). DISCUSSION

The present oxygenase finding with

paper

was based on an earlier Rf values

incubated

between

with

fatty

acid

amount of

as the

It

our results difference

is not

the

of

(8).

acid

mixture We, therefore, inhibitor mixture,

synthesized

and

by the

and inhibition

of

mechanisms

(3)

is due to

up the

tryptophan

(6). than

of lipoxygenation

between

species

platelets.

a method employed

the cycle-oxygenase

supernatant

Nevertheless,

from the membrane component

lipoxygenase

rather

in the

human platelets

the discrepancy

in breaking

by Nugteren

from bovine as well

by hydroquinones

an inhibitor

prepared

activity

whether

be released

soluble

freshly

Nugteren

and thawing,

The effects

is

clear

or methods

enzyme cannot

with

any enzymatic

and that

by freezing

with

reaction

a potent

product

compounds

arachidonic

to the reaction

experiments

not detect

fraction.

obtain

indomethacin,

This

preparation.

In separate we could

of

microsomes

only

a lipo-

in which

esters in the

cycle-oxygenase,

HETE was isolated microsomal

observation

the methyl

human platelet

of

of human platelets.

B2 were obtained

added an excess

the

the presence

in the membrane fraction

and thromboxane

of

demonstrates

as the

are similar However,

an activator

platelets. rapid

fact

suggests

and cycle-oxygenation

404

inactivation

to those

the

to

that

observed epinephrine

that are different.

Vol. 76, No. 2, 1977

BIOCHEMICAL

More recently, of arachidonic of rabbit

Borgeat acid

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

et -- al.

(9) reported

and homo-y-linolenic

peritoneal

neutrophils

acid

led to the

5-L-hydroxy-6,8,11,14-eicosatetraenoic 9,11,14-eicosatrienoic a lipoxygenase

activity

activity

in the

nuclear

leukocytes

results).

acid,

acid respectively.

as well

microsomal

that

as the

preparation

to a suspension

synthesis

of

of

and 8-L-hydroxyWe have also

detected

thromboxane-synthesizing of human polymorpho-

(P. K. Ho and C. P. Walters,

The characterization

addition

this

unpublished

lipoxygenase

is now

in progress. REFERENCES (1974)

Nat.

Acad.

Hamberg, M. and Samuelsson, (USA) 71, 3400-3404. Sci.

2.

Hamberg, M., Svensson, Proc. Nat. Acad. Sci.

3.

Nugteren, 299-307.

4.

Turner, Nature,

S. R., Tainer, 257, 680-681.

5.

Goetzl, J. Clin.

E. J., Woods, J. M., and Gorman, Invest. 59, 179-183.

6.

Ho, P. P. K., Walters, (1976) Prostaglandins

7.

Ho, P. P. K., and Walters, C. P. (1976) Res. Comm. in and Pathol. 15, 673-687. Chem. Pharmacol.

8.

Ho, P. P. K., Walters, C. P., and Herrmann, R. G. (1976) Biochem. Biophys. Res. Comm. 69, 218-224.

9.

Borgeat, J. Biol.

D. H.

(1975)

B.

Proc.

1.

J., and Samuelsson, (USA) 71, 3824-3828. Biochim. J. A.,

Biophs. and Lynn,

B.

Acta

405

380,

W. S. R. R.

C. P., and Sullivan, 12, 951-970.

P., Hamberg, M., and Samuelsson, Chem. 251, 7816-7820.

(1974)

(1975) (1977)

H. R.

B.

(1976)