Lipoprotein lipase from heart and liver: An immunological study

Vol. 55, No. 1, 1 9 7 3

LIPOPROTEIN H. Jansen,

BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS

LIPASE A. van

FROM HEART Zuylen-van

D e p a r t m e n t of B i o c h e m i s t r y sity Rotterdam, Rotterdam,

Received

September

7,

AND LIVER: Wiggen

AN I M M U N O L O G I C A L

and W.C.

STUDY

Hulsmann.

I, F a c u l t y of Medicine, The N e t h e r l a n d s .

Erasmus

Univer-

1973

SUMMARY: L i p o p r o t e i n lipase a c t i v i t y was p u r i f i e d from rat postheparin serum. An a n t i b o d y against this a c t i v i t y was raised. It was found that this a n t i b o d y inhibits 95% of the p o s t h e p a r i n lipoprorein lipase activity. The same i n h i b i t i o n was found in the perfusate of h e p a r i n - p e r f u s e d rat liver. When m e a s u r i n g l i p o p r o t e i n lipase a c t i v i t y in p e r f u s a t e s of rat hearts, p e r f u s e d with heparin, no i n h i b i t i o n by this a n t i b o d y was found. It is c o n c l u d e d that lip o p r o t e i n lipase a c t i v i t i e s from heart and liver are e a t a l y s e d by d i f f e r e n t enzymes and that the heart enzyme c o n t r i b u t e s not more than 5% to the overall lipase a c t i v i t y in p o s t h e p a r i n serum.

Korn tained

(I)

showed

in 1955

that

acetone

lipoprotein

lipase.

This

enzyme

in lung, venous

adipose

injection

the blood hepatic cently

tissue,

(2).

that

of heparin,

This

organs.

diaphragm

activity

However

lipase

tajn

et al.

(6) had

found

rein

lipase

activity

was

rat heart release

with

of lipase

investigated vity

the

is also

part

of

medium.

liver

also

to be

gland.

from

with

appears

shown

the liver.

of the original

Liver

and heart

in

re-

Borenszlipopro-

perfusion

shows

heparin.

intra-

from extra-

have

after

con-

localized

After

activity

(3,4,5)

the heart

on p e r f u s i o n

of rat heart

to be r e l e a s e d

released

from

containing

contributions

lipase

of authors

that a large

activity

of p o s t h e p a r i n

and m a m m a r y

is assumed

removed

a heparin

is known

lipoprotein

a number

activity

powders

a very

of rapid

Therefore

to the

llpase

we acti-

plasma.

METHODS Rats were 15 min pase

injected

they were

activity

was

with

killed

and

purified

Copyright © 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.

heparin

(I00 l.U./kg

the blood was from

bodyweight).

6-

Lipoprotein

ll-

the serum on a s e p h a r o s e - h e p a r i n

co-

30

collected.

After

Vol. 55, No. 1, 1 9 7 3

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

lumn,

as d e s c r i b e d

crona

and Egelrud

gradient quent

(0-|.25

for

lipoprotein

(7). We r e p l a c e d mg/ml).

calciumphosphate

tion was

obtained.

adjuvant

(1:1),

weeks

The p u r i f i c a t i o n

adsorption

injected

injection

was

collected

y-globulln

was

(50%

saturation)

lowed (not

by a n o t h e r shown)

bulins. This

according

one

for

ammonium

sulphate

that

the

were

10 min

at 37°C.

in addition,

sulphate

After

IO days

4 la-

of

the

precipitation

G-200

column,

fol-

Electrophoresis contained

only y - g l o -

in the

same way.

experiments. narcose

with

Freunds

of the animals,

a modified

Tyrode

medium

replaced

preheparin

was rat

serum

soluby

and 4.5

heparin/ml.

Rat

livers

(about

a Krebs-Henseleit containing ted b e l o w fusion

serum

8 g) were

bicarbonate

5 mM glucose the liver

at a rate

a medium,

of 20 m l / m i n

as well

Lipoprotein substrate, used

80% of

lipase

activity

The

above

(final sprayed was

the

with

liver.

described

vein with

95% 02 - 5% CO2,

caval

medium

v e i n was

After was

above

replaced and

with

(Dow Corning

with

and H u l s m a n n

the m o d i f i e d

method

perby

20% of rat

4.5

Antifoam

liga-

10 min

concentration

estimated

31

the portal

inferior

the p e r f u s i o n

by J a n s e n

substrate,

through

saturated

the m e d i u m

as h e p a r i n

as d e s c r i b e d as the

buffer,

and c a n n u l a t e d

containing

(v/v)

perfused

at 33 + 2°C.

top of the latter was b r i e f l y

was

Nembutal

(v/v)

and

handled

in control

with

Purification

fraction was

purifica-

of a rabbit.

on a S e p h a d e x

the p e r f u s i o n 20%

mixed

punction.

rabbit

In a subse-

two-fold

intramuscularly

technique

Then

pads

by Olive-

by a h e p a r i n

600-fold.

enzyme,

isolated

after

to the L a n g e n d o r f f

was

precipitation.

was used

perfused,

gradient

by a m m o n i u m

filtration

fraction

containing,

I.U.

achieved

skim milk

another

by heart

Serum of a n o n - i m m u n i s e d

Rat hearts

tion

given

and gel

indicated

y-globulin

step

i ~ the foot

ter 30 ml blood was fraction

from

the NaCI

! mg of the p u r i f i e d

was

a booster

lipase

l.U./ml).

palmitoyl-CoA

The Corp.).

as the

(8). W h e n

[|4C]trioleate

of Kelly,

as descri-

Vol. 55, No. 1,1973

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

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Vol. 55, No. 1, 1973

BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS

t30"

130

A IO0-

IOO

~

B

sO"

10

Fig.

|.

20

30

jut ~-6LOBU LI N g

--

1C

2'0

30

~'O

ST0

6'0 INCUBATION TIME( MIN )

I n f l u e n c e of d i f f e r e n t a m o u n t s of a n t i b o d y and d i f f e r e n t i n c u b a t i o n times on p o s t h e p a r i n l i p o p r o t e i n l i p a s e a c t i vity. 20 UI p o s t h e p a r i n p l a s m a was e i t h e r i n c u b a t e d for ] h w i t h d i f f e r e n t a m o u n t s of a n t i b o d y - or c o n t r o l p r e p a r a t i o n at 3 7 ° C (Fig. |A) or at d i f f e r e n t i n c u b a t i o n times, as s h o w n in Fig. ]B. The m i x t u r e s w e r e c e n t r i f u g e d for 2 m i n at |5 000 x g. F r o m the s u p e r n a t a n t s a m o u n t s e q u i v a l e n t to 5 UI o r i g i n a l p o s t h e p a r i n s e r u m w e r e t e s t e d . The a m o u n t s of c o n t r o l - or a n t i b o d y p r e p a r a t i o n u s e d w e r e r e l a t e d to 5 ~I p o s t h e p a r i n serum.

bed

in ref.

leased min,

8, was

per m i n

when

used.

in the

trioleate

Activities

are

palmitoyl-CoA

was

the

expressed

assay,

and

as n m o l e s

as n m o l e s

CoASH

FFA

re-

released/

substrate.

RESULTS The

antibody

concentration fraction. time

we

preparation

of

By v a r y i n g

found

that

ty of

5 UI w h o l e

60 m i n

at 37°C,

(Fig.

]A and

rates

measured

enzyme trol

or

3.35

labile

of

during

the

control

same was

0.9%

done

concentration

15 UI

antibody

inhibited

The

during at

the

with

antibody

followed

B).

diluted

the

postheparin

antibody,

amount

mg/ml,

was

the

and

incubation

95%,

values

first

10 min.

inactivated

incubated antibody at O°C

are

at 0 °,

for

the

control

and

the

incubation

incubated

for

2 min

based

during

the

on

lipase between

at

|5 000

linear

serum

activi30 and x

reaction

to e x c l u d e

that

an

incubation

with

con-

serum with

37 ° and

3.5 h no

33

with

In o r d e r

postheparin

to a p r o t e i n

lipoprotein

when

centrlfugation

presented

37°C was we

by

serum

NaCI

40°c.

activity

a non-saturating

It was was

lost

found and

that that

Vol. 55, No. 1, | 973

with

this

amount

Incubation remaining for of

at

enzyme.

be

antibody gave

activity

antibody. could

of

37°C

| h at 4 0 ° C the

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The

control

the

to e x c l u d e

activating

heparin

human

serum

and

added

human

serum

addition

of h u m a n

does

influence

not

purified rat

by

with

was

again

was

in

70%

inhibited.

activity;

antibody.

a heat-labile

the

reaction

preheparin

75%

of

the

Incubation

inactivation

inhibited

by

the

non-lnhibited

obtained

From

this

from Table

precipitation In o r d e r

of

serum,

enzyme

containing

medium

activity

released

but

the

postheparin

liver serum

37°C)

the

(Table from

(not the

found

plasma

concluded the

source

of

II).

It was

found

is

did not

activity

of

the

par-

serum

precipitate were

re-

puri-

preparation

the

the

fraction.

antibody

is not

After liver

34

lipase

caused

that

to

activity

in v i t r o

with

inby

the

with same

incubation perfusate

the

lipase antibody,

extent

for

in p o s t -

a heparin

lipoprotein

interact

inhibited

activity.

the

proteins.

perfused

perfusate

that

the

antiserum

~-globulin

inhibition

were

heart

that

whole

this

li-

antiserum

y-globullns

that w i t h

that

by h u m a n

resulting

the o r i g i n a l

that

I shows

shown

shown)

pre-

antiserum)

with

same w a y

heart

rat

lipase

It is also

and

the p r i n c i p a l and

Table

with

precipitation

the

incubation

antibody

together no

with

enzyme.

in the

I it can be and

showed

supernatant,

as w i t h

activating

liver

the

It was

directly

to d e t e c t

rate.

from

antiserum

assay

after

serum

From

results

enzyme

or

resulted

serum

experiment

rat

same

the

human

is a c t i v a t e d

In a n o t h e r

were

incubated

inhibited

before

before.

(60 m i n

the

resulted

diffusion

serum

as d e s c r i b e d

that

of

activity

initial

by

inhibition

we

to the

centrifugation.

and

the

immuno

fied

heparin

the

inhibited

existence

that

enzyme

serum.

mixed

hibits

|9% of

activity

(preheparin

in the O u c h t e r l o n y

moved

initial

y-globulins

protelnsj

nes

was

of

the

excluded.

against

as by

of

again

remaining

Therefore

In o r d e r

tially

a loss

70% was

with

70%

as

a longer

is c o m p l e t e l y

the time inhi-

Vol. 55, No. 1, 1973

BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS

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Vol. 55, No. 1, 1973

bited

(not

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

shown),

is i n h i b i t e d

95%

whereas

(Fig.

the

lip~se

activity

of p o s t h e p a r i n

serum

|B).

DISCUSSION From that

the data

are

released

immunologieally heparin

obtained

by h e p a r i n

different

injection,

inhibitory

it can be

seen

that

antibody,

when

measured

than w h e n

palmitoyl-CoA

hydrolase fusate. heart

contributes

liver. 95%

is the

released

[|00 I.U. Another

by

vs.

the

250

organs

would

be e q u a l l y

enzyme

that

l.U./kg

would

showed gel

be

to the by

already

only

that, serum

the liver

enzyme

by

heart

conclusion

activity

that

(5)

found,

than

the

from

even

lipase

13%

that a b o u t

derived that

find

per-

in p o s t h e p a r i n

and not more

of the

the

up to

activity

a somewhat

is

higher

a smaller

amount

of h e p a r i n

by Kraus

et al.

(5)].

used

in a d d i t i o n activity

been

found,

band with

had a d i f f e r e n t

and

This

that

(pH 8.9)

36

the

is 8% less

can be e x p l a i n e d

serum was

antibody.

electrophoresis

by

with

the

the

one p r o t e i n

lipase

table

compared

that we

we used

body w e i g h t

inhibited

It has

polyacrylamide found

liver b e c a u s e

serum

from our results

substrate)

In this

trioleate

et al.

in p o s t h e p a r i n

as the

contribute

investigation.

Kraus

It is p o s s i b l e

explanation

other

I allow

in respon-

over

substrate,

to c o n c l u d e

trioleate

when

II).

after

activity

5% to the lipase is the

activity

by the liver.

contribution

in Table

substrate.

It is t e m p t i n g

hydrolase

enzyme

substrate,

This

by

in serum,

to the liver (Table

activities

eatalysed

activity

as the

substrate.

perfusate,

maximally

lipase

(87% with

serum

in liver

lipase

lipase are

of p o s t h e p a r i n

trioleate

is the

palmitoyl-CoA

trioleate

65% of the

with

that

liver

the a n t i b o d y

inhibition

data o b t a i n e d

when

of

The

and

similarly

of p a l m i t o y l - C o A

activity

The

plasma, when

ratio

very

action the

from heart

enzymes.

behaves

se to the

the h i g h e r

it can be c o n c l u d e d

to heart that

this

hydrolase shown).

substrate

liver,

activity

is p r e s e n t l y

the p u r i f i e d

(not

and

under

postheparin

activity

in

Fielding

(4)

specificity

Vol. 55, No. 1,1973

than

the a c t i v i t y

that

this

rent

enzymes

cussed

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

released

difference

before

from o t h e r

(at least

and not

only

by L a R o s a

for heart

to the

et al.

organs.

present

and liver)

influence

(3)

The

is due

of c o f a c t o r s ,

and Kraus

et al.

paper

shows,

to diffe-

as was

dis-

(5).

ACKNOWLEDGEMENTS We wish and help This lands aid

to thank Dr.

in the p r e p a r a t i o n investigation

Foundation

from

H.G.

was

Research

and Mr.

P. B a c k h u y s

for advice

of the antibody.

carried

for Chemical

the N e t h e r l a n d s

Scientific

van Eijk

out

Research

Organization

under

the auspices

(S.O°N.) for

and with

the A d v a n c e m e n t

of the Netherfinancial of Pure

(Z.W.O.).

REFERENCES I. Korn, E.D. (1955) J. Biol. Chem. 215, 1-14. 2. Hahn, P.F. (1943) Science 98, 19-20. 3. LaRosa, J.C., Levy, R.I., W i n d m u e l l e r , H.G. and F r e d e r i c k s o n , D.S. (1972) J. Lipid Res. 13, 356-363. 4. F i e l d i n g , C.J. (1972) Biochim. Biophys. Acta 280, 569-578. 5. Kraus, R.M., W i n d m u e l l e r , H.G., Levy, R.I. and F r e d e r i c k s o n , D.S. (1973) J. Lipid Res. 14, 286-295. 6. B o r e n s z t a j n , J. and Robinson, D.S. (1970) J. Lipid Res. II, 111-117. 7. O l i v e c r o n a , T. and Egelrud, T. (1971) Biochem. Biophys. R--es. Commun. 4__33, 524-529. 8. Jansen, H. and Hulsmann, W.C. (1973) Biochim. Biophys. Acta 296, 241-248.

37