Pulmonary phosphatidic acid phosphatase: Evidence for a membrane-bound phosphatidic acid-dependent activity associated with the high speed supernatant of rat lung

Vol. 82, No. 2, 1978

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 627-633

May 30,1978

PULMONARY PHOSPHATIDIC

ACID PHOSPHATASE:

EVIDENCE FOR A MEMBRANE-BOUND PHOSPHATIDIC

ACID-DEPENDENT

ACTIVITY

ASSOCIATED WITH THE HIGH SPEED SUPEBNATANT OF BAT LUNG Paul

G. Casola,

Alex

Departments University Received

Yeung,

G. Fraser

of Biochemistry

of Western

February

Fellows

and Fred

and Obstetrics

Ontario,

London,

Possmayer

& Gynaecology

Ontario,

Canada N6A 5A5

lo,1978

The 104,000 x g supernatant fraction from rat lung contains a greater proportion of the phosphatidic acid phosphatase activity toward memhrane-bound phosphatidic acid than the microsomal fraction. The microsomal fraction is more effective in hydrolyzing aqueously dispersed phosphatidic acid. The effects of various ions and chelators, particularly Mg2+ and EDTA, suggest that these two activities are distinct. These results indicate that the supernatant fraction of rat lung contains a phosphatidic acid phosphatase activity which may play an important role in pulmonary glycerolipid synthesis. SUMMARY:

A number acid

of studies

phosphatase

(EC 3.1.3.4)

and phosphoglyceride phosphatase

both induce in

that

during

late

pulmonary

the activity

increased

levels

the principal a number

tissue the

level

a role

in the control

that

experiments

enzyme is

increased

in rabbit

glucocorticoid

acid which fetal

that

of 1,2-dipalmitoyl-sn-glycerol-3-phosphorylcholine

of tissues, with

fraction.

In these

of the pulmonary

including

lung

the particulate

that

(3-6),

when phosphatidic

It

the major

predominantly aqueously acid

increases for

the

(3,5),

surfactant.

fractions,

investigations However,

(6),

to

these

may be responsible

have lung

administration

suggested

phosphatase

glyceride

of phosphatidic

by recent

has been

phosphatidic

of neutral

suggested

It acid

have suggested

The importance

and after

(4,5).

of phosphatidic

component

substrate.

of this (3)

maturation

tissues

(1,Z).

has been

gestation

associated

as the

plays

synthesis

in lung

demonstrated

in different

has been observed enzyme activity

in is

in the microsomal

dispersed phosphatase

PA' was used activities

1

EGTA, ethylene glycol-bis (B-aminoethyl ether) The abbreviations used are: G3P, glycerol-3-phosphate; TLC, thin layer chromaN,N1-tetraacetic acid; tography; PA, phosphatidic acid; tricine, N-tris-(hydroxymethyl)methylglycine. 0006-29lX/78/0822-0627$01.00/O 627

Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

BIOCHEMICAL

Vol. 82, No. 2, 1978

were

measured

in liver

PA formed

biosynthetically

localized

in

activity reports

that

activity

has been

involved rat

lung

activity

suggested

that

and adipose

membranes, being the

contains

the major

found

activity

(7-10).

This

PA and this

PA than

(ll),

using

activity

in the

a phosphatidic

membrane-bound

membrane-bound

tissue

cytosol

synthesis

supernatant

towards with

(10)

little

in glycerolipid

directed

more significant

intestine on microsomal

the cytosol, It

fractions.

(7-9),

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

was

particulate is

the only

communication acid

activity

the microsomal

phosphatase appears

to be

activity.

EXPERIMENTAL PROCEDURES:Most of the methods

and the sources of the materMembrane-bound PA was prepared ials have been previously reported (4,X2,13). microsomes essentially enzymatically from [14C]G3P or [32P]G3P with r at liver as previously described (13), but on a larger scale and with slight modifi(40 mM), G3P concentration cations in the time (30 min), KF concentration acid and 0.05 mM oleic acid (1.0 mM), and fatty acids used (0.05 mM palmitic as the potassium salts). After 15 min, 2 umoles of ATP were added and the The microsomes were isolated by reaction was continued for another 15 min. centrifugation at 104,000 x g for 1 h, redispersed, recentrifuged, resuspended in the original volume of the tissue and heat-inactivated for 5 min at 100°C. This preparation, which was normally used as the membrane-bound PA substrate, contained no residual phosphatidic acid phosphatase activity.

Membrane-bound phosphatidic acid phosphatase was routinely assayed in a standard system containing (final vol. 100 ~1): 2.4-3.0 nmoles [32P]PA, 0.4-0.9 mg cytosol protein and 50 mM tricine buffer (pH 7.4). The reaction was terminated by the addition of 200 ~1 of cold 0.4 M perchloric acid. After centrifugation, an aliquot was counted in Aquasol (4). Under the standard assay conditions, the release of 32Pi was proportional to the amount of protein up to 0.9 mg and time to 30 min. This activity, which was stable to freezing and thawing, had a broad pH optimum from pH 7.4-8.0. Concomitant experiments with membrane-bound [ 14C]PA demonstrated that the release of 32Pi closely paralleled the formation of [14C] neutral glycerides. 14C-Labelled lipids were extracted, washed and separated by TLC on silica gel G plates impregnated with 0.35 N oxalate using petroleum ether:acetone:formic acid (76:24:0.25) as previously described (13). Microsomal phosphatidic acid phosphatase was determined using aqueously dispersed PA as previously described (4), except that tris-maleate buffer (pH 7.4) was used. The release of Pi was proportional to the amount of microsomal protein up to 1.0 mg and with time to 120 min. The supernatant fraction demonstrated only a slight activity with aqueously dispersed PA.

RESULTS: Esterification of fatty

acids,

activity

in PA (74%)

incubated percent only

ATP,

to measure of the

increased

of

[ 14C]G3P by rat lung microsomes 2+ CoA and Mg , results in an accumulation

in

(Fig.

reisolated

la).

endogenous

total

radioactivity

from

12 to 16%.

When these phosphatidic migrating However,

628

microsomes

were

acid

phosphatase

with

the

when lung

the presence of radio-

activity,

diacylglycerol

supernatant

and the fraction

was added,

the

Vol. 82, No. 2, 1978

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

b

50 t

SUFERNATANT

0

ORIGIN

02

0.4

06

RELATIVE

MOBILITY

FIGURE1:

00

IO SOLVENT FRONT

(a) TLC separation of the products of the esterification of [14C]G3P by lung microsomes. The incubation conditions were the same as the G3P esterification system for preparation of membrane-bound PA described in the Experimental Procedures, except KF was eliminated and 1.5 ml of a lung microsomal suspension (3.66 mg protein) were added to the incubation medium Microsomes were reisolated and resuspended to their (final volume, 2.5 ml). original volume. The lipids were separated by TLC as described in the (b) Phosphatidic acid phosphatase activity of Experimental Procedures. Aliquots rat lung microsomes and the effect of addition of supernatant. (500 ~1) of the resuspended lung microsomes described above in (a) (0.825 mg microsomal protein) were incubated alone (O---O) or in the presence of supernatant (O----C). The incubation medium contained in a 1.0 ml final volume: 2.67 mg lung supernatant protein and/or 0.825 mg microsomal protein, 50 mM After incubation for 40 min at 37"C, the radiotricine buffer (pH 7.4). activity in the lipids was determined as above.

629

Vol. 82, No. 2, 1978

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TIME ( min) Membrane-bound phosphatidic acid substrate utilization by rat lung supernatant (G----O) and microsomal (H) fractions. Aliquots of the respective subcellular fractions corresponding to 60 ~1 of a 20% rat lung homogenate were incubated in the standard assay system using membrane-bound [32P]PA described under Experimental Procedures.

FIGURE2:

radioactivity

in diaclyglycerol

corresponding

decrease

purposes,

tions

corresponding

bated

with

membrane-bound

tidic

acid

phosphatase

natant,

supernatant

tures. the

activity

1 summarizes

cations

activities and anions

concentrations.

Under

32P-label

the effects

these

could

with

exhibited

an inhibition except

Generally

Mg 2+ , which

the supernatant

630

and cytosol homogenate

were

not

and chelators (aqueously

activity

shown).

on the

dispersed

PA)

some common fea-

to a varying showed

2).

of super-

(data

share

incu-

phospha-

(Fig.

amounts

be released ions

frac-

the major

the cytosol

larger

PA) and the microsomal two activities

lung

was a

lb).

conditions

with

of various

of these

tested

whole

substrate

the total

There

of the microsomal

was associated

the membrane-bound

The properties Both

at low

[32P]PA.

(membrane-bound

activities.

aliquots

in PA (Fig.

to 60 1~.1of a 20% (w/v>

up to 90% of Table

to 40% of total.

in the radioactivity

For comparative

By incubating

increased

extent

a slight

with

stimulation

was more sensi-

all

Vol. 82, No. 2, 1978

l'ABLE 1.

BIOCHEMICAL

The effect microsomal

of various phosphatidic

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ions and chelators acid phosphatase

Relative

on the supernatant and activities from rat lung

Activities

(% control)

Microsomal

Addition 0

--it-2.5

Supernatant

r

10

5

10

82.0

32.0

27.0

38.7

16.6

5.8

78.0

58.5

58.5

67.2

43.4

11.6

104.3

64.0

36.3

115.1

117.0

105.7

F-

81.0

84.0

43.0

93.1

81.3

70.8

EDTA

98.0

93.7

110.6

0.0

0.0

EGTA

91.0

83.0

91.0

107.0

98.7

2+

Mn

Ca2+ 2+

Mg

--~2.5

0.0 83.1

supernatant The specific activities with the standard assays were: (membrane-bound [32P]PA) activity, 6.0 + 0.5 pmoles 32Pi releasedjminfmg protein; and microsomal (aqueously dispersed PA) activity, 18.7 +_ 1.2 nmoles Average values from 3 separate experiments are Pi released/min/mg protein. presented.

tive

to Ca

F- and Mg found

2+

2+

with

and Mn2+ , whereas The most

. Mg

2+

tions. the

The Mg complete

tions

2+

of EDTA.

activity

requirement and explained

could for

were

a slight

effect

inhibited

be reversed in the

by the presence

the

at higher

activity

was noted

of endogenous

at 2.5 mM

EGTA at these was essentially

in

by

of low concentra-

of EDTA has been 2+

were

was illustrated

of Mg 2+ (data

Mg

same conunaffec-

of the not

noted

the microsomal

with

investigations

631

conducted

in other

super-

shown). previously fraction

(8). DISCUSSIOi~: In agreement

to

concentra-

of EDTA treatment

by addition presence

with

activity

effect

two activities

stimulated

in the presence

the microsomal

The inhibitory

was more sensitive

slightly

of the supernatant

of PA hydrolysis

Mg2+ only

activity between

was markedly

In contrast,

ted up to 10 mM EDTA.

activities

activity

dependence

(Only

differences

both

inhibition

centrations.)

natant

striking

While

.

2+ Mg , the microsomal

the microsomal

tissues

A

BIOCHEMICAL

Vol. 82, No. 2, 1978

(7-11,13),

these

microsomes

in the

lation

studies

microsomal lipid

that

of fatty

acids,

This

la).

phosphatidic

absence

from

the

system

the cytosol.

associated

with

the supernatant

lism

is

related

phosphatidyl

These

two separate

striking

differences

dependencies

that

1);

suggest is

ion

these

two phosphatidic

should

be noted

altered

forms

that

when bound

activities

will

subcellular

fractions

tidy1

choline

ellar

bodies

tidy1

choline

of lung

synthesis in lung de

phosphatase

different

tissue

is not tissue

no~o

(14,15),

activity

the

previously

(16,17)

must

be questioned.

clusion

that

the bulk

of

in their

a distinct

that

requirement

there

were

However, could

at it

he drastically

purification they

Mg 2+

On the basis

tissue.

whether

activities

of the are

different

have been detected

(3-6), With have

but

their

the

recent

the capacity

importance

evidence choline

632

is

relation

in many to phospha-

suggestion to synthesize

and role

demonstrated

The present

of phosphatidyl

terms

proteins.

clear.

do not

in

can be found

protein

distinguish

phosphatase

surfactant.

The most

in adipose

Only

of

be guarded.

concluded

membrane.

of the same enzyme or are acid

(11)

of a soluble

to a biological sources

effects

metabo-

precursor

is Mg 2+ -independent.

phosphatases

the properties

from both

Phosphatidic

acid

acid

of diacyl-

of the pulmonary

exhibits

for

least

enzyme activity

the direct

and chelator

2+ the microsomal activity Mg , whereas 2+ dependencies, Jamdar and Fallon of Mg

the

to

activity

to pulmonary

is

activity

glycero-

phosphatase

effector

observation

two activities

the soluble

for

that

the major

the

of PA appears

acid

must necessarily

between

(Table

results

component

activities

conditions,

the accumulation

diacylglycerol

of the various

defining

for

of this

the principal

Interpretation

optimal

may be rate-limiting

fraction

to the fact

under

of the phosphatidic

The relevance

choline,

esterification of G3P by lung 2+ leads to an accumuCoA, and Mg

that

one reason

with

synthesis.

the

phosphatase

associated

glycerol

suggests

acid

At least

synthesis.

be the

demonstrate

presence

of PA (Fig.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in

that

lam-

phospha-

of the phosphatidic these

inclusion

supports

the general

synthesized

in the

bodies conendo-

Vol. 82, No. 2, 1978

plasmic

reticulum

Because

lung

by the

activity,

endoplasmic

from

rate-limiting

however,

the

in

reticulum.

activity

AND BIOPHYSICAL

are

of PA formed

has a high PA is

and transported

microsomes

phosphatase hydrolysis

BIOCHEMICAL

relative

used as substrate.

there

This

to the microsomal this

of PA formed

on the endoplasmic

surfactant

synthesis

the lung.

Acknowledgements: This Research

Council

remains

communication

hydrolysis

Medical

regard

investigation of Canada

bodies

(18,19).

to phosphatidic

acid

to account

ViVO as a membrane-bound

We suggest

in

to the lamellar

with

problem

RESEARCH COMMUNICATIONS

biosynthetic has shown

fraction

activity reticulum

was supported and the Ontario

for

the intermediate

that

the

cytosol

when membrane-bound

could

be important

and therefore

for eventual

by grants from the Ministry of Health.

REFEREKES 1. 2. 3. 4. 5.

6. 7. 8.

9. 10. 11. 12. 13.

Hcbscher, G. (1970) in Lipid Metabolism (Wakil, S.J., ed.), Academic Press, New York, pp. 279-370. Schacht, J. and Agranoff, B. (1973) Biochem. Biophys. Res. Comm. 50, 934-941. Schultz, F.M., Jimenez, J.M., MacDonald, P.C. and Johnston, J.M. (1974) Gynecol. Invest. 2, 222-229. Possmayer, F., Duwe, G., Metcalfe, R., Stewart-DeHaan, P.J., Wong, C., Las Heras, J. and Harding, P.G.R. (1977) Biochem. J. 166, 485-494. Brehier, A., Benson, B.J., Williams, M.C., Mason, R.J. and Ballard, P.L. (1977) Biochem. Biophys. Res. Comm. 77, 883-890. Mavis, R.D., Finkelstein, J.N. and Hall, B.P. (1977) ABSTRACT Fed. Proc. 36, 790. Smith, M.E., Sedgwick, B., Brindley, D.N. and Hiibscher, G. (1967) Eur. J. Biochem. 3, 70-77. Mitchell, M.P., Brindley, D.N. and Hibscher, G. (1971) Eur. J. Biochem. 18, 214-220. Lamb, R.G. and Fallon, H.J. (1974) Biochim. Biophys. Acta 348, 166-178. Johnston, J.M., Rao, G.A., Lowe, P.A. and Schwarz, G.E. (1967) Lipids 1, 14-20. Jamdar, S.C. and Fallon, H.J. (1973) J. Lipid Res. I& 517-524. Possmayer, F. and Strickland, K.P. (1967) Can. J. Biochem. 45, 53-61. Hendry, A.T. and Possmayer, F. (1974) Biochim. Biophys. Acta 369, 156-172.

14. 15. 16. 17. 18. 19.

Tsao, F.H.C. and Zachman, R.D. (1977) Pediat. Res. ll, 849-857. Barazska, J. and Van Golde, L.M.G. (1977) Biochim. Biophys. Acta 488, 285-293. Spitzer, H.L., Rice, J.M., MacDonald, P.C. and Johnston, J.M. (1975) Biochem. Biophys. Res. Comm. 66, 17-23. Meban, C. (1972) J. Cell Biol. 2, 249-252. Chevalier, G. and Collet, A.J. (1972) Anat. R&a. 174, 289-310. Rooney, S.A., Page-Roberts, B.A. and Motoyama, E.K. (1975) J. Lipid Res. 16, 418-425.

633

the