The effect of human arginine rich apoprotein on rat adipose lipoprotein lipase

Vol. 78, No. 1, 1977

BIOCHEMICAL

AND BIOPH’YSICAL RESEARCH COMMUNICATIONS

THE EFFECT OF HUMAN ARGININE RICH APOPROTEIN ON RAT ADIPOSE LIPOPROTEIN S. H. Quarfordt, Division Center

H. Hilderman,

M. R. Greenfield

LIPASE

and F. A. Shelburne

of Gastroenterology, Department of Medicine, Duke University Medical and Cooperative Lipid Laboratory, Veterans Administration Hospital, Durham, North Carolina 27710

Received

August

1, 1977 SUMMARY

Heterologous human arginine rich apoprotein purified by heparin affinity chromatography from very low density lipoproteins produces a pronounced inhibition of the activity of lipoprotein lipase obtained from rat adipose tissue when the apoprotein is added directly to the assay medium. If, on the other hand, only the triglyceride emulsion bound arginine rich apoprotein is presented to the enzyme, a two-fold increment in the activity of the enzyme is noted. The ratio of the substrate bound arginine rich apoprotein to the free apoprotein importantly influences the effect of this apoprotein on the lipoprotein lipase reaction. These findings suggest a potential receptor role for the protein in this enzyme-substrate interaction. INTRODUCTION The addition lipoproteins, (1)

to assays

or resulted

the arginine

defined

for

plasma

is

7).

this

in triglyceride

with

constituent

(2,

3). for

lipase

(S),

rich

of the

lymph

chylomicron

the addition

of the

arginine

rich

apoprotein

in triglyceride

effect

the

fact

that

heparin

(4),

no role

has been

rich

which

apoprotein

lipoproteins.

events

Yet no function

Despite

The arginine

of triglyceride

low density

been without

affinity

lipoprotein catabolism.

from very

has either

metabolic

is

In fact,

after

one

reaching

to the

the

chylomicron

transport

has been

characterized

an effect

of the arginine

for

apoprotein. The present

apoprotein, affinity

lipase

has a distinct

it

isolated

of the enzyme

apoprotein association

a predominant

apoprotein,

of lipoprotein

close

of the early

rich

in an inhibition rich

may be in

(6,

of arginine

investigation

isolated

from human very

chromatography

enzyme obtained

from

describes

(4), rat

low density

on the activity

adipose

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

tissue.

lipoprotein

of a crude On the basis

(VLDL) lipoprotein

rich by heparin lipase

of the influence

of

302 ISSN

0006-291

X

Vol.

78,

this

No.

1, 1977

apoprotein

effect reaction

in

when bound

free is

BIOCHEMICAL

form,

a role

AND

BIOPHYSICAL

to the triglyceride for

this

RESEARCH

substrate

apoprotein

in the

COMMUNICATIONS

as opposed lipoprotein

to its lipase

proposed. MATERIALS

AND METHODS

Plasma was obtained from normal and hypertriglyceridemic (Type IV phenocopy) humans after a 14-hour fast and collected in EDTA (.l mg/ml blood). The VLDL were isolated at 4" from plasma by conventional ultracentrifugal methods using a Beckman L5-65 centrifuge and 50 Ti rotor (1.8 x lo8 g-min). They were repurified by an additional flotation through saline under the same conditions and dialyzed against 1,000 volumes of buffered 0.01% EDTA (pH 7.4) the lipoproteins were per volume of lipoprotein. After lyophilization, delipidated three times with 3:l ethanol:ether (50 volumes per volume of lipoprotein) and dried under nitrogen. The lipid free apoproteins were dissolved in a 2mM phosphate buffer The urea soluble (pH 7.4) containing 50 mM sodium chloride and 5 M urea. apoproteins (3 mg per ml buffer) were applied to a heparin affinity column The which had been prepared by a method similar to that of Iverius (8). apoproteins were eluted from the column using a linear salt gradient running The arginine rich apoprotein fraction from 50 r&l to 500 mM sodium chloride. was isolated from all the other urea soluble apoproteins by virtue of its unique affinity for heparin (4). This arginine rich apoprotein was concentrated and repeatedly washed free of urea and phosphate on a PM 10 Amicon and then lyophilized in preparation for Filter system using distilled water, addition to the assay mixtures. The epididymal fat pads of male Sprague Dawley rats were delipidated acetone/ether extractions. exactly as previously described (Y), using repeated The resulting powder was extracted with a buffer (5 mg powder/ml buffer) containing 0.05 M NH40H/NH4Cl pH 8.6 and briefly homogenized at 4" C, and centrifuged in a 2B Sorval for 30 min. at 800 g. The clear enzyme supernatant solution was decanted and used for the assays. The assay of lipoprotein lipase was performed in a manner similar to the procedure of LaRosa, et al. (10). The total assay mixture had the same glyceryl 114 C trioleate concentration, 139 nmoles per 6 ml, and specific activity (3960 dpm/nmole) as previously described, but used a 0.1 M Tris buffer pH 8.6 and the addition of 0.2 ml fasting normal human serum per 6 ml assav The basic assay mixture, containing the solvent free glyceryl l14C mix. trioleate and 0.1 ml 1% triton, was prepared in 6 ml of 0.1 M Tris and sonified For the control incubations, one-half of this mixture (3 ml) was layered under buffer and centrifuged at 30,000 rpm at 8" using a 40.3 Beckman rotor and an L5-65 Beckman ultracentrifuge. The substrate was retrieved in the top 3 ml after centrifugation (spun substrate). The other half of the substrate (3 ml) was assayed without ultracentrifugation (unspun substrate). The arginine rich apoprotein was added to the basic substrate mixture (25 - 100 pg/ml) in the experimental vials, and 3 ml of the substrate was ultracentrifuged prior to assay, and the other 3 ml assayed without centrifugation. The assays were begun by adding 50 ~1 of heparin (100 U/ml), Sigma Chem., 0.1 ml of 1% human albumin, 0.1 ml fasting human serum and either 0.5 ml of enzyme preparation or 0.5 ml of buffer to 3 ml of the basic mixture. The assays were run for one hour at 27", and the hydrolyzed free fatty acids isolated by a conventional method (10) and assayed for radioactivity.

303

Vol. 78, No. 1, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE I The Effect

of Direct

Apoprotein

Concentrations

Concentration

a ARP = arginine b Results

are

within

Additions

Human Arginine

on Rat Adipose

of Added m/ml

Lipoprotein

ARPa

Rich

Lipase

Lipase Activity nmol FFAa/hr/ml

0

0.29

(s)b

25

0.27

(6)

50

0.22

(4)

75

0.16

(6)

rich

apoprotein;

FFA = free

the means of triplicate

the parenthesis

expressed

of Differing

fatty

acid

determinations.

indicates

the

The figure

fractional

standard

deviations

as percent.

RESULTS When arginine concentrations of the

rich

from

crude

The degree

apoprotein

added

assays

with If

the

apoprotein, lower

lipase

to the mixture.

the larger triolein

substrate degree of this

an approximately

rich

apoprotein

acid

analysis)

triton

the

before

adipose

were

mixed

of inhibition apoprotein.

in

activity

I) was of the for

the

of the arginine

(Table

II)

concentration

of the

304

(Table

was noted

100 pg/ml

This

apoprotein

in the

upon the amount

was noted

an appreciable

assay mixture

apoprotein.

with

was centrifuged

rich

tissue

inhibition

of the added

the assay,

of the arginine

rat

to the

a suppression

was dependent

75% inhibition

emulsion

added

per ml,

A greater

contents

a greater

triglyceride

from

of suppression

concentrations

produced

was directly

25 - 75 ng of protein

lipoprotein

observed.

apoprotein

amount floated

for

the

of apoprotein

enzyme activity.

away from

than

rich

the

added

When the arginine

(10 - 20%) by amino up with

the substrate

Vol. 78, No. 1, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE II The Effect Arginine

of Triglyceride

Rich

Apoprotein

Experiment Control

on Rat Lipoprotein

Lipase

Lipase

Activitya Centrifuged Substrate

nmol/hr/ml

(No ARPb)

0.31

0.34

ARP 33 (100 ug/ml)

0.09

0.59

ARP 426 (100 ug/ml)

0.06

0.48

ARP 518 (100

0.09

0.54

0.07

0.61

us/ml) ug/ml)

a The activity

figures

lipoprotein b

1).

had been

with exposed

When this

centrifuged

with

(Table

II).

of the

control

the

lymph

These

containing increment

substrate substrate unbound

triglyceride

when it

arginine

of

infranate emulsions

to take enters

rich

demonstrated

the plasma

protein,

almost eight

had the bulk

(6).

was incuwas observed

twice times

which

up the

in enzyme activity

and was almost

which

in the

appeared

chylomicron

substrate

centrifuged

substrate

determinations

text.

remained

apoprotein

a reproducible

The centrifuged

in the

rich

substrate,

uncentrifuged

in the

(>80%)

radioactivity.

the native

the enzyme

as described

of the apoprotein

to the arginine

as does

the means of duplicate

apoprotein.

glyceride

apoprotein

bated

assayed

rich

The bulk

unassociated

represent

lipase

ARP = arginine

(Figure

protein

and Unbound

Uncentrifuged Substrate

ARP 21 (100

than

Bound

the activity more

of the arginine

active

rich

apo-

form. DISCUSSION

A number has a function

of observations in

the plasma

have transport

suggested

that

the arginine

of triglycerides.

305

It

rich

apoprotein

has been noted

Vol. 78, No. 1, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Figure 1. The sodium dodecyl sulfate - 10% polyacrylamidc elcctrophorctic A) the pure arginine rich apoproteins added to tire incubations; pattern of: E) the ultracentrifuged triglyceride emulsions wilich had been exposed to the arginine rich apoprotein; C) the ultracentrifuged control triglyceride emulsions.

that

in humans

triglyceride group.

(11)

rich It

apoprotein

ide

lipoprotein

it

has been

micron tein

of the

apoprotein revealed

it

(4)

apparent

that

suggestive

is predominantly

little

that

on any other

the arginine

soluble

group

is

the addition

on the

lipoprotein

rich

apoprotein

of proteins

affinity

one of the early

observations

of plasma

for

of the

suggest

some role

indicating

a role

on triglycer-

heparin.

metabolic

Thirdly,

events

arginine for

this

is

of a chylorich

apopro-

apoprotein

triglyceride. data transport,

of the apoprotein

of activity is

very

relatively

the plasma

triglyceride

no effect

suppression here,

these in

(6)

these

in the metabolism Despite

with

apoprotein

to have an appreciable

upon entering All

rich

been observed

recognized

to it.

arginine

lipoproteins

has also

the only rich

the

from the why a differing

assays

for

the

of this

apoprotein

on the lipoprotein

lipase

apoprotein assay

306

(2, result

3).

arginine have (1)

From the data might

rich

be anticipated

either

or a presented depen-

Vol. 78, No. 1, 1977

ding

BIOCHEMICAL

upon the conditions

the arginine

rich

apoprotein

effect

of this

lipase

activity.

amount

of apoprotein

apoprotein

assay:

bound

The data

to the

assay

in

triglyceride

appears

the activity

direct

addition

an inhibitory

phenomenon

observed

similar

These

are compatible

data

apoprotein lipase

associated reaction.

of substrate

The affinity

likely

is

tein

lipase.

Korn

lipoprotein

lipase

conditions

has not (12)

arginine

of tying

competing

with

of the enzyme

apoprotein

reasonable

arginine

lipase

up the enzyme heparin

complex

heparin

(12)

amounts

of the arginine

triglyceride

unbound,

or bound

rich

substrate

to non-triglyceride

307

rich

is

role

under

(4) most for

lipoprotissue

physiologic

of the enzyme

here

could

effect

be on the

substrate"

or by

'The much greater

explanation apoprotein

somewhat that

to that

substrate,

for

of an

The inhibitory

a "false

as opposed

when

then

between

affinity

sites.

makes the second

influence

as the mediator

with

3).

rich

heparin

association

observed

binding

(2,

whereas

effect

interaction.

apoprotein

for

solution;

receptor

heparin

there

previously

such an association

to propose

heparin

system,

for

the strong

arginine

a positive

apoprotein

an intimate

rich

the enzyme for

free

in the lipoprotein

in bulk

proposed

by

lipoprotein

the arginine

inhibitory

its

However,

been proven.

free

even

although

for

to substrate,

rich

determines

and heparin

The relative the appropriate

that

noted

lipoproteins

anticipated

apoprotein-lipoprotein

of the substrate basis

the

(5) has suggested

makes it

rich

is bound

of the arginine

the property

role

of

is an approximate

to the incubation

rich

is noticed

is substrate-free,

observed.

heparin

triglyceride

relative

species

of predominantly

a receptor

When the protein

on the hydrolysis the protein

with

with

on the

When the

to that

an

and the amount

there

of the enzyme.

of

indicate

in the bound

protein,

as seen with

amount

lipoprotein

dependent

made rich

in a milieu

apoprotein

reaction

tissue

substrate

is

the unbound

the

strongly

adipose

triglyceride

When the substrate

stimulation

described

on rat

on the

elminating

in particular,

occurs

rich is

the

apoprotein

The effect

ultracentrifugally

lipase

for

added.

heterologous

unbound.

two-fold

chosen

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

affinity unlikely.

are bound amount

of

may importantly

to

Vol. 78, No. 1, 1977

govern

BKXHEMKAL

the activity

of the lipolytic

of substrate

and enzyme is

conditions.

Most

associated

with

in plasma. critical

quite

system --in vivo. different

of the enzyme appears acidic

A receptor for

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the association

these

to be fixed

glycosaminoglycans protein

from

The --in viva

(5),

on the substrate

but

in tissue, the substrate

would

of enzyme and substrate

--in vitro

potentially

relationship

bulk

solution

probably circulates be even more

--in vivo.

AEFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Ganesan, D., Bradford, R. H., Ganesan, G., McConathy, W. J., Alaupovic, P ., and Bass, H. B. (1975) J. Applied Phys. 39, 1022-1032. Ganesan, D., Bass, H. B., McConathy, W. J. and Alaupovic, P. (1976) Metabolism 25, 1189-1195. Clin. Chim. Acta 63, 29-35. Ekman, R. and Nilsson-Ehle, P. (1975) In Press. Shelburne, F. A. and Quarfordt, S. H. (1977) J. Clin. Invest. (1957) 3. Biol. Chem. 226, 827-832. Korn, E. D. Robinson, S. and Quarfordt, S. H. Manuscript submitted. Imaizumi, K., Fainaru, M., and Havel, R. J. (1976) Circulation 54 supplement II, 134. Lverius, P. H. (1971) Biochem J. 124, 677-683. Korn, E. D. and Quigley, T. W., Jr. (1957) J. Biol. Chem. 226, 833-839. LaRosa, J. C., Levy, R. I., Herbert, P., Lux, S. E., Fredrickson, D. S. (1970) Biochem. Biophys. Res. Comm. 41, 57-61. Shelburne, F. A. and Quarfordt, S. H. (1974) J. Biol. Chem. 249, 14281433. Dolphin, P. J. and Rubinstein, D. (1974) Biochem. Biophys. Res. Comm. 57, 808-814.

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