Vol. 90, No. 1, 1979 September

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

12, 1979

Pages

PHOSPHOLIPASE ACTIVITY OF BOVINE MILK LIPOPROTEIN ON PHOSPHOLIPID VESICLES: INFLUENCE OF APOLIPOPROTEINS H.G.

Muntz,

Departments

N.

of Biological

University

Received

July

Matsuoka,

and

Pharmacology Chemistry of Cincinnati Cincinnati,

R.L.

LIPASE C-II AND

15-21

C-III

Jackson

and Cell Biophysics, and Medicine College of Medicine Ohio 45267

23,1979

SUMMARY The effect of human plasma apolipoproteins C-II and C-III on the hydrolytic activity of lipoprotein lipase from bovine milk was determined using dimyristoyl phosphatidylcholine (DMPC) vesicles as substrate. In the absence of apoC-I I or C-III, lipoprotein lipase has limited phospholipase activity. When the vesicles were preincubated with apoC-It and then phospholipase activity determined, there was a time dependent release of lysolecithin; activity was dependent upon both apoC-II and lipoprotein lipase concentrations. The addition of apoC-I II to DMPC did not stimulate phospholipase activity. We conclude that apoC-II has an activator effect on the phospholipase activity of lipoprotein lipase and that the mechanism is beyond that of simply altering the lateral compressibility of the 1 ipid. INTRODUCTION Lipoprotein cerides

(VLDL)

plasma

~

(Ref.

human

plasma

two

lipoprotein

I

of

the

(apoC-I

I)

crons,

VLDL been

ma and

and

shown

hydrolysis tion

At

least

by to

phosphatidylchol substrates

the

extra-hepatic (2,3),

by

78

amino

density

the

ine

in

(Y-12);

the

other

is

very

is

hepatic

that

it

lipase

--in

is

lipoprotein bond

chylomicrons,

VLDL

the

HDL

ipids

of

and are

(4)

to

from

triglylipopro-

post-heparin

on

the

vasculature

The

characteristic

by

apolipoprotein

present

in

apoprotein,

inhibit

presence

of

density in

localized

Another

lipase

acyl

present

peptide

in

low

stimulated

(s-8)

vitro

hydrolysis

origin.

(HDL).

investigators

phosphol

and

enzymes

residue

of

primary

the

are

of

is acid

hydrolysis,

hydrolyzes

of

in

lipases

lipoproteins

a number

lipoprotein

involved

chylomicrons

enzyme a

high

enzymes

One

whereas

triglyceride milk

the

review),

lining

feature

lipid

in

1 for

endothelial

has

are

transported

teins

C-l

lipases

chylomiapoC-

triacylglycerol of

apoC-Il.

both

In

not

addi-

post-heparin

plas-

phosphatidylethanolamine

artificial

I I I,

and

triglyceride-phosphohydrolyzed

(13).

Using

an

0006-291X/79/170015-07$01.00/0 15

Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 90, No. 1, 1979

isolated

perfused

particles vity

as of

not

enhances

phospholipid

dylcholine results

MATERIALS

and

Groot

which

inhibit the

is

phosphol

tivity

heart

substrate,

did

apoC-II

The

rat

heparin-releasable

apoC-III

on

BIOCHEMICAL

have

(DMPC)

as

substrate

of

these is

reported

apoC-II

activation.

of

the

show

that

that

the

phosphol

dependent

on

mechanism toward

by

action

vesicles

of

dimyristoyl

lipoprotein

lipase lipase

actiand

has

of

that

which

triglycerides

of

lipoprotein

ipase

apoC-II

mechanism

sonicated purified

triglyceride

The lipase

understand

and

on

was

lipoprotein

utilized

studies

dependent

(12)

lipase

To

we

al.

lipoprotein

activity

ipids,

phosphatidylethanolamine-coated

et

the

unknown.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and the

enzyme phosphati-

from

bovine

phospholipase

milk. ac-

apoC-II,

AND METHODS

Lipoprotein lipase. Bovine milk lipoprotein lipase was purified to homogeneity by affinity chromatography on heparin-Sepharose as described by Kinnunen (14). The enzyme preparation had a specific activity of 87 mmol fatty acid released/h/mg protein using glycerol [14C]triolein in 0.02% Triton X-100 as substrate. The enzyme was stored in 50% glycerol, 10 mM Tris-HCl, pH 7.4, I M NaCl, at -7O”C, and thawed at -2O’C prior to its addition to the react ion mixtu res . Preparation of phosphol ipid vesicles. Dimyristoyl phosphatidylcholine (Sigma) was purified on Silica gel and yielded a single species upon two-dimensional TLC on Silica gel 60 F-254 (EM Reagents) in chloroform:methanol:25% ammonia:water, 180:108:11:11 and chloroform:methanol:acetic acid:water, 180: 80:24:4. To prepare phospholipid vesicles, the DMPC was dissolved in chloroform, evaporated under a stream of N2, and lyophilized for 15 min. Phospholipid dispersions were then prepared by adding a standard buffer (0.1 M NaCl, 0.1 M Tris-HCl, pH 7.4) to give 10 mg DMPC/ml. The lipid was suspended in the buffer by mechanical agitation and was sonicated at 25’C for 30 min using a Heat Systems Ultrasonics, Inc. Cell Disrupter (Model W-225R). Large phosphol ipid structures were then removed from the sonicate by ultracentrifugation at 150,OOOxg for 1 h at 15°C. The vesicles were prepared daily and stored at room temperature Isolation of apol ipoproteins. ApoC-I I and apoC-I II (with 2 residues of sialic acid) were isolated from VLDL which was obtained from plasma of fastinq individuals with endogenous type IV hyperlipoproteinemia, as described previously (4). The isolated proteins were homogeneous as determined by isoelectric fo[1251]apoC-ll was prepared by the method of Roholt and Pressman ;y;;ing (15). . Phospholipase activity. The activity of 1 ipoprotein lipase toward DMPC vesicles was assayed under a variety of conditions of temperature, apoprotein concentrations, and inhibitors, as described in the legends to the Figures and Table. In all experiments the reactions were initiated by the addition of the enzyme and were terminated by the addition of 600 ul MeOH and 300 ~1 CHC13, followed by the lipid extraction (17). The lipid was redissolved in CHC13 and The spotted on Sil ica gel TLC plates. in chloroform:methanol :water (70:30:4), visualized with I2 staining; the lipids phol ipid determined (18).

plates and were

16

were developed one-dimensionally the lecithin and lysolecithin scraped from the plates and

spots phos-

Vol. 90, No. 1, 1979

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

/ .;/’ .’ .’ _ -o!. 0

30

60 Time

90

0

120

2

(minutes)

Lipoprotein

Lipose

(pg)

FIGURE I. Time and enzyme dependence of hydrolysis of dimyristoyl phosphatidylchol ine (DMPC) in the absence (-o-o-) and presence ( -e-o-) of apoC-I I. (A) Each reaction mixture contained in a final volume of 0.2 ml of 0.1 M TrisHCl, 0.15 M NaCl, pH 7.4, the following additions: DMPC vesicles (700 ug), apoC-II where appropriate (0.8 ug) and fatty acid-free bovine serum albumin Each reaction mixture was preincubated at 23.4”C for 15 min prior to (5 mg). the addition of lipoprotein lipase (1.4 vg). At the indicated reaction times, the mixtures were extracted and the % hydrolysis determined as described in the text. (B) The reaction conditions were the same as those described in (A), with the exception of variable amounts of enzyme and a final incubation time of 90 min.

RESULTS In vine to

the

absence

milk

The

was

was with

tested

for

and

its

lipoprotein

the

effect However apoC-II

ug,

apoC-Ill at ,

high

activated

ug

of

I be

enzyme

activate.

Table

the I

to

Table

next shows the

1%

and

all

enzyme

us/ml)

lipoprotein I.

increased

lipase

experiment,

and

50%

apoC-I

apoC-III

possibility thus

15%

35%

with

that

by

Approximately

compared

However,

preparation

(4

at

bo-

dependent.

in

apoC-II,

of

1)

apoC-II

of

shown

In

activity.

slightly that

account

II

for

was stimu-

apoC-II the

hydro-

may

slight

discounted.

was low

1.6

as

respectively. to

apoC-II

cannot When

90

and

(Fig.

of

activity

dependent,

lipase the

time

activity

negligible

addition

phospholipase

with

phospholipase was

The

was

the

ability

stimulation,

I).

of

observed

20

contaminate

ble

reaction

concentration

hydrolysis

DMPC

tested.

stimulation

apoC-II

cofactor,

against

concentrations

The

lated

apoprotein

lipase

activity.

lysis

any

lipoprotein

substrate

enzyme

of

added

ratios

on

apoC-III lipase

to the levels activity

mixtures

of

apoC-II

activated

(60

pg by

17

apoC-II,

apoC-III approximately

apoC-III

had

phospholipase to

20

no

apprecia-

activity pg

20%.

apoC-II)

(Table inhibited

Vol. 90, No. 1, 1979

BIOCHEMICAL

AND BIOPHYSICAL

TABLE EFFECTS

OF APOLIPOPROTEIN PHOSPHOLIPASE

C-II, ACTIVITY

ApoC-

I

APOLIPOPROTEIN OF BOVINE MILK

I I

ApoC-

C-III AND LIPOPROTEIN

I I I

reaction

0.1

M Tris-HCI,

fatty

acid-free

apoC-II

or

MIXTURES LlPASEa

!Jg

P9

%

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