BIOCHEMICAL

Vol. 69, No. 3, 1976

LYSOLECITHINASE

ACTIVITY

ZELINA

AND BIOPHYSICAL

IN SUBCELLULAR

LEIEOVITZ-BEN

GERSHONl,

Laboratory of Neurochemistry, Hebrew University - Hadassah Received

January

7,

RESEARCH COMMUNICATIONS

FRACTIONS

OF RAT

and SHIMON

GATT

ORGANS

Department of Biochemistry, Medical School, Jerusalem,

Israel.

1976

Summary: Lysolecithmase activity was measured in subcellular fractions of rat liver, kidney, lung and intestine and compared to similar findings in brain. To obtain optimal assay conditions, each fraction was subjected to a kinetic analysis in the absence and presence of albumin. Among the particulate preparations, lysolecithinase activity of the intestine exceeded by far similar fractions of other organs. Among the soluble fractions, the 100,OOOxg supernatant of lung had the highest activity. Under the assay conditions used, most of the lysolecithinase activity of all organs was particulate.

The

activity

of lysolecithinase

an enzyme

which

is widespread

organisms

(summarized

(lysolecithin in nature,

in (1))”

The

was also

studied

(2,3,4)

D Several

inhibited

by its substrate

(2, 4-8,

by lysolecithin, resulted

which

in non-hyperbolic,

Ben Gershon

et al.

the specific

activities

values,

but varied

enzyme

or an added

obtain

optimal

under

differing

1

Present

Copyright All rights

belongs

markedly, protein

tissue

Therefore,

“Irregular”

kinetics

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

Using

Unit,

592

fractions

inhibition

lipids,

rat brain

were

was

Leibovitz-

inhibition, not fixed

of the substrate, in order

to

had to be assayed

was also

Hadassah

in rat organs

substrate

amphiphilic

on the concentrations

subcellular

Research e

This

of

the enzyme

of the substrate

of this

the various

Pediatric Jerusalem

(7).

because

such as albumin.

conditions.

:

of soluble,

fractions

depending

that

in 9).

V/S curves that,

enzyme

reported

summarized

EC 3.1 .l. 5),

in a variety

of this

investigators

of subcellular

activities,

address

distribution

biphasic

hydrolase

was determined

to the class

(7, 10) showed

acyl

obtained

University

using

Hospital,

purified

Vol. 69, No. 3, 1976

BIOCHEMICAL

enzyme

preparations

(11).

parable

investigation

of lysolecithinase

well

as a reevaluation

investigated several bumin The

The

on the kinetics

vity values

were for

observations

activity effects

of lysolecithin

used to assess

on rat brain

activity

of the data reported

lysolecithinase

rat organs.

results

These

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of other

organs

of substrate hydrolysis

In this

and subcellular

concentration of these

conditions

a com-

of the rat,

in the literature.

of homogenates

optimal

warranted

paper

fractions

and of serum fractions

of assay

as

were

and specific

we of

alstudied. acti-

each fraction.

EXPERIMENTAL PROCEDURE Subcellular fractionation. Rats were killed by cervical dislocation, the organs were removed and chilled in 0.32 M sucrose. was Each organ, except intestine, homogenized in 9 volumes of 0.32 M sucrose and the subcellular fractions were prepared by differential centrifugation, as follows: Fraction 1, debris which sediments at 800 xg for 10 min; Fraction 2, particles which sediment by subsequent centrifugation at 8400 xg, for 10 mm; Fraction 3, particles which sediment by subsequent centrifugation at 35,000 xg for 15 min; Fraction 4, microsomes which sediment by subsequent centrifugation at 100,000 xg for 60 min; Fraction 5, the supernatant of the above. Each of the sediments was suspended in 0.32 M sucrose and stored at -20’. The intestinal homogenate was prepared according to the procedure of Epstein and Shapiro (12) with the following two modifications: Microsomes were sedimented at 100,000 xg and the sediments of all fractions were suspended in 0.32 M sucrose. This procedure yields four fractions, debris (fraction l), particles (fraction 3), microsomes (fraction 4) and supernatant (fraction 5) 1 Assay of the reaction: Incubation mixtures, in volumes of 0.2 ml each, contained 15 pmoles of Tris-HCl, pH 8.1, l- c9, 10-3H2] pal mitoyl-sn-glycero-3-phosphoryl choline, enzyme and sucrose. In those experiments in which albumin was added, its concentration equalled half ,the molar concentration of the lysolecithin. The mixtures were incubated at 37 for 15 min and the radioactivity of the released fatty acid was determined (13). A control tube without enzyme was added to each experiment D In those experiments which measured the effect of substrate concentration on the reaction rates, a control tube without enzyme was used for each concentration of lysolecithin. MATERIALS AND METHODS. Tritium-labelled lysolecithin was prepared from livers of rats which had been injected with [9, 10 3H2] palmitic acid (13). The specific activity of the substrate was 3.8 x lo6 dpm/pmole. Albumin was Cohn’s fraction V, fatty acid poor (Pentex). Protein content was determined according to the procedure of Lowry et al. (14) and phosphorus according to the procedure of Bartlett (15). One unit is defined as that amount which released 1 nmole of fatty acid in 1 hr.

593

Vol. 69, No. 3, 1976

RESULTS.

BIOCHEMICAL

Subcellular

fractions

as described

in Methods.

lysolecithinase

activity

drolysis

concentration

experiments

in which

at a fixed

concentration

tions

when shown

using

(v vs.

in accord

with

This

the maximal

hydrolysis

microsomes

of liver,

results

which

were

S curves

biphasic.

occurred kidney

of albumin

The

depended

to the incubation

v vs. S curves

to rectangular

hyperbolas

ratio

of 0.5.

and pancreas effect

This

was

was

was however

which

were

was variable.

animals

on the microsomal

protein

The

increased

not true

inhibited

for

enzyme

at an albumin particulate

by albumin.

activity

while

observed

of concen-

increased

(7) r Fig. of liver

and

1B

and kidney;

to lysolecithin

molar

of intestine

lung

microsomes,

enzyme

of lungs

was decreased

It

of rat brain

preparations

Using

of the microsomal

that of others

concentration

mixtures

the biphasic

of this

prepara-

and lung but not intestine.

converted

effect

with

on enzyme

preparations

the optimal

obtained

of similar

by particulate

of albumin

by similar

was varied,

had been

of lysolecithin

effect

of hy-

concentration,

fraction

of hydrolysis

a similar

measuring

was then followed

the rates

shows

prepared

the rate

of substrate

(7) the v vs.

and lung

that addition

for

and fractions,

S) D

were

(v vs ~ E).

intestine

previously

rats

conditions

of the subcellular

of rat brain

kidney,

at which

tration

that,

of adult

as a function

of substrate

preparations

of liver,

organs

the concentration

1A shows

substrate

was

of the individual

of enzyme

organs the optimal

was measured

at a fixed

particulate

of several

To determine

of lysolecithin

Fig.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the of some

in the presence

of

albumin. Fig. Most

curves

concentration brain thmase

(7).

2 shows were

v vs.

S curves

not hyperbolic

of the substrate; Addition

of all rat

of albumin organs

tested;

of 100,000 but showed

the same

xg supernatants a discontinuity

was true

using

decreased

the reaction

albumin

was therefore

594

of four

rat organs.

above a certain the soluble

rate

fraction

of soluble

not added

of rat

lysoleci-

to the assay

sys-

BIOCHEMICAL

Vol. 69, No. 3, 1976

AND BIOPHYSICAL

01

02

RESEARCH COMMUNICATIONS

01

LYSOLECITHIN

02

(mM)

Fig. 1: Effect of substrate concentration on the hydrolysis of lysolecithin by microsomal preparations of rat organs. Standard assay conditions were used. was added. A kidney (61pg), 0 intestine (0. *g) , Fig. IA - no albumin 0 liver (lO@g), CJ lung (31pg). Fig. 1B - albumin was added, at half the molar concentration of the substrate. A kidney (6pg), 0 liver (108hg).

r

I 005

I

01

LYSOLECITHIN

015 (mM)

Fig. 2: Effect of substrate concentration on the hydrolysis of lysolecithin by soluble fractions of rat organs. Standard assay conditions were used, albumin was not added. 0 intestine (72. @g), Cl lung (l+g), 4 kidney (15$g), 0 Iiver (133pg, right ordinate).

tern

in which

the soluble

To determine (Table

I),

lysolecithinase

particulate

the presence xg supernatants

preparations

fractions

of albumin, were

while

assayed

were activity

of rat similar

used. of the subcellular

liver

and kidney

fractions

in the absence

595

and lung

of intestine

of albumin.

fractions were

as well Each value

of rat organs assayed as all

in 100,000

in the Table

Vol. 69, No. 3, 1976

Table

BIOCHEMICAL

I: Lysolecithinase

Activity

AND BIOPHYSICAL

in Subcellular

RESEARCH COMMUNICATIONS

Fractions

of Bat Organs.

Subcellular fractions were prepared and the enzymatic activity was determined as described under Experimental Procedure. Lysolecithin concentration 0.15 mM. 0.075 mM albumin was present in each reaction mixture, except in those specified in the footnote to the table.

Specific

activity

of subcellular

fraction

(u/mg

protein)

Total Activity @As)

Heavy particles

Liver

23780

120

460

195

70

Kidney

41976

300

800

750

40

LuJx

37800

90

340

700

740

Intestine2

Light particles

1 58804

412086.

Brain3

microsomes

20300

550

2117

66

776

17147

s upernatantl

1. All the 100,000 supernatants were assayed without albumin. 2. All mtestinal fractions were assayed without albumin. 3. Values for brain are derived 4. This fraction from a previous publication (7). contained both heavy and light particles.

was derived

from

at 0.15 mM

lysolecithin

the reaction

mixture)

rate

a curve

of hydrolysis

respective

describes

(and 0.075 mM as a function

of lysolecithin

the rate albumin,

of hydrolysis

when

of concentration was directly

this

of lysolecithm,

protein

was added

of the respective

proportional

to

fraction.

The

to the concentration

of

fractions.

Table that of all

I shows other

lowest

while

of all

organs

that the activity

fractions

brain

had lower Lung

equalled

or exceeded

mlity

of the enzyme activity

when

of the particulate

many-fold.

had the highest

parations.

full

which

specific

Among specific

activities

was an exception

stored

for at least

the other

The

liver

The 100,000

than the corresponding activity

preparations 100,000

4-7 months

596

of intestine

organs,

activities.

in that the specific

that of the particulate preparations.

fraction.

showed

the

xg supernatants particulate

pre-

of the supernatant

of these

xg supernatants at -20’.

exceeded

organs. retained

In contrast,

almost

the micro-

Vol. 69, No. 3, 1976

somal

preparations

preparation lung

from

kidney

substrate

kedly

liver

retained

from

of different

This

that

et al. their

(11) purified

present

activities

Acknowledgements: Part of this work to this laboratory. acknowledged.

of the soluble

conflicting

two lysolecithinases

that more

than

thereby

fractions.

with

fractions

particulate

the above

which

enzymes

Furthermore,

as well

properties

De Jong of (7,16).

activity

to the varying some

as

in some

of rat brain

lysolecithinase

mar-

data of

(2-6).

resemble

of

fractions

The kinetic

contributes

both the kinetic

of enzyme,

differed

in the literature

and soluble

and that this

changing

lysolecithinase

liver

one enzyme

organ

of the soluble

found from

of the particulate

by subcellular

be used to analyze

reports

and that

2 months.

in the effects

variability.

The

the preparations

within

Furthermore,

considerable

at -2O’C.

4 months

2 months;

activity

of lysolecithin

could

sucrose after

variability

fractions.

(7,lO)

of the subcellular

may be solubilized relative

papers

in each mammalian

tic behaviour

all their

considerable

showed

in isotonic

and 20% after

practically

The properties

those

It is thus possible

one month

shows

also

stored

25% of its activity

on the hydrolysis

and frequently

properties

when

of the particulate

organs

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

about

lost

paper

organs.

and of former

variable

retained

30% after

or albumin rat

not stable

and intestine

DISCUSSION:

several

were

from

from

this

BIOCHEMICAL

is kine-

of the enzyme (4,7)

and the

enzyme.

This paper was supported in part by NIH grant NS02967. during a summer visit was done by William S. Weintraub The expert technical assistance of Mrs. H. Gaaton is

REFERENCES 1. 2. 3. 4. 5. 6. 7.

McMurray, W.C., Magee: W.L. (19’72) Ann. Rev. Biochem. 41, 129-160. Dawson, R.M.C. (1956) Biochem. J. 64, 192-196. Marples. E.A., Thompson, R.H.S. (1960) Biochem, J. -’74 123-127. Ottolenghi, A. (1967) Lipids 2, 303-307. Shapiro, B. (1953) Biochem. J. 53, 663-666. Van den Bosch, H., Aarsman, A. J., Slotboom, A.J., and Van Deenen, L. L.M (1968) Biochim. Biophys. Acta 164. 215-225. Leibovitz-Ben Gershon, Z.: KobG, I., and Gatt, S. (1972) J. Biol. Chem. 247, 6840-6847.

597

Vol. 69, No. 3, 1976

8. 9. 10. 11. 12. 13. 14. 15. 16.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Kunze: H., Nahas. N., Wurl. M. (1~74) Biochim. Biophys. Acta 348, 35-44 Gatt, S. (1972) In Metabolic Inhibitors (Hochster, R. M., Quastel, Gy.. Kates, M. , eds.) 3, 349-387, Academic Press, New York. Leibovitz-Ben Gershon, Z., Gatt, S. (1974) J. Biol. Chem. 244, 1525-1529. De Jong, J.G.N., Van den Bosch, H., Rijken, D., Van Deenen. L.L.M. (1374) Biochim. Biophys. Acta 369, 50-63; De Jong, Thesis, U. Utrecht. Epstein, B., Shapiro, B. (1959) Biochem. J. c, 615-619. Gatt, S. (1968) Biochim. Biophys. Acta 159: 304-316. Lowry, 0. H. Rosebrough, N. J., Farr. AT. , and Randall, R. J. (1951), J. Biol. Chem. 193, 265-275. Bartlett, G.R. (1959) J. Biol. Chem. 234, 466-468. Leibovitz, Z. , Gatt, S. Q968) Biochim. Biophys. Acta 164, 439-441.

59%