360

Biochimica et Biophysics Acta, 409 (1975) 360-366 @ Elsevier Scientific Publishing Company, Amsterdam

-

Printed

in The Netherlands

BBA 56687

DETECTION AND PARTIAL LIPASE IN BOVINE AORTA

LINDSEY

C. HENSON

CHARACTERIZATION

and MICHAEL

OF LIPOPROTEIN

C. SCHOTZ

Research Service, Veterans Administration Wadsworth Hospital Center, Los Angeles, Calif. 90073 and Department of Medicine, School of Medicine, and Division of Environmental and Nutritional Sciences, School of Public Health, University of California at Los Angeles, Los Angeles, Calif. 90024 (U.S.A.) (Received

May 28th,

1975)

Summary Extracts of acetone-ether powders of bovine thoracic aorta contain lipase activity which has an alkaline pH maximum (7.8-8.4) and is stimulated 4---lo-fold by adding serum or isolated apolipoprotein-glutamate* to the assay mixture, Serum activation is completely reversed by isolated apolipoproteinserine or apolipoprotein-alanine. Lipolysis is strongly inhibited by NaCl (0.5 M) and protamine sulfate (1 mg/ml) and partially inhibited by heparin. Based on these characteristics, the lipase is identified as lipoprotein lipase.

Introduction Hydrolysis of plasma very low density lipoproteinand chylomicron-triacylglycerols is catalyzed by lipoprotein lipase in a variety of tissues, in which the physiological site of action of the enzyme is believed to be the endothelial surface of the capillaries [l] . Zilversmit [2] has proposed that lipoprotein lipase is also present in arterial endothelial cells and may play a role in atherogenesis. While a number of investigators have demonstrated lipolytic activity in various preparations of aortic tissue [ 3-101, the activity has not been well characterized as lipoprotein lipase. In this communication, evidence is presented for a lipolytic activity in bovine aorta which satisfies the usual in vitro criteria for identification of lipoprotein lipase.

* Delipidated acid.

apolipoproteins

are

designated

by

“apolipoprotein-”

plus

the carboxyl-terminal

amino

361

Materials and Methods Preparation of tissue. Pieces of bovine thoracic aorta (approx. 30 cm) were obtained immediately after evisceration of the animals (Vernon Meatland, Los Angeles, Calif.). Subsequent cleaning and dissecting procedures were carried out at 4°C. Adhering fat was removed from the outer surface of the blood vessel which was cut lengthwise and opened flat with the endothelial surface exposed. The intimal-medial layer was separated from the adventitial layer by pulling the layers apart with forceps and the pieces of intima-media were stored in fresh 0.15 M NaCl for no longer than 1 h. Intimal-medial samples (lo-20 g) were homogenized for 1 min in 200 ml acetone (room temperature), using a Polytron homogenizer (Type PT 10-20-3500, Brinkman Instruments) with the sawtooth generator (PT 20 ST) and rheostat (PCU-2) at setting 4.5. The homogenates were filtered under vacuum through Whatman No. 1 filter paper using a Buchner funnel and the precipitate was washed with 100 ml acetone and 100 ml diethyl ether (room temperature). The resulting powders were stored in airtight containers at -20°C for up to 2 months with no measurable loss of lipolytic activity. Enzyme assay. Acetone-ether powders were extracted by homogenizing in 0.05 M NH4 OH/HCl buffer, pH 8.5, at 4°C (75 mg powder/ml buffer) using the same Polytron homogenizer at setting 4.5 for lo-20 s. The homogenates were centrifuged at 4°C for 540 000 X g * min (Rotor SS-34, Servall Refrigerated Centrifuge RC-2) and the supernates (3-4 mg protein/ml) were assayed for lipolytic activity by a modification of the method of Nilsson-Ehle et al. [ll] . The triacylglycerol substrate emulsion was prepared as previously described [ll] using a Sonifier Cell Disruptor (Branson Model W185) at setting 2.5 (1.0-1.5 g force). The assay was started by adding 0.1 ml substrate to 0.1 ml enzyme source and/or buffer and the complete mixture was incubated at 37°C generally for 1 h. Each assay tube contained 0.25 mg glycerol trioleate, 1.2 . lo6 cpm glycerol tri[9,10-3H2 ] oleate, 0.01 mg lysolecithin, and 0.04 mg crystallized bovine albumin in 0.083 ml 0.2 M Tris * HCl buffer, pH 8.0; 0.017 ml of rat serum previously heated to 60°C for 10 min (see Results); and enzyme source and/or buffer. Fatty acids released during the incubation were extracted [11,12] and radioactivity was measured in a Beckman LS-250 liquid scintillation counter. One munit of lipase activity was defined as release on 1 nmol fatty acid per min. In most experiments, incubations with lipoprotein lipase activity extracted from acetone-ether powders of rat adipose tissue were performed for comparison. Protein in aortic and adipose extracts was determined by the method of Lowry et al. [13]. Materials. Glycerol trioleate (stated purity >99%) was obtained from NuChek Prep and glycerol tri[9,10-3H2 ]oleate (radiopurity >99% as determined by thin-layer chromatography) from Amersham-Searle. Lysolecithin (egg lecithin, Grade 1) and bovine serum albumin (Fraction V powder) were obtained from Sigma. Crystallized bovine albumin (Grade A, Pentex), sodium heparin (160 I.U./mg), and protamine sulfate were obtained from Calbiochem. Apolipoproteins isolated from human very low density lipoproteins [14] were the kind gift of Dr Virgil Brown, School of Medicine, University of California at San Diego.

%

SERUM

IN ASSAY

7.0 -

k g SO-

; 2 30. >” F 4 l.O* U K

1 1 t 012345

1

I

1

I IO

Apo{ipopro~e~n - glutamate

IS IN ASSAY

t 30

(Pg/ml 1

c r”

ki 4 ~.o(~+=t-_-_z_y_-_-_-_ E 5

IO I

P

f =f- -- - - - -40

- --A -__A 1

60

Apo)ipoprotein-setine (-+a) or apolipoprotein-olanine

80

(0% A1

IN ASSAY (Pg/ml) Fig. 1. Effects of serum and specific serum apolipoproteins on aortic lipase activity. Protein in all assays was equalized by replacing albumin usually contributed by rat serum v&b bovine serum albumin (Fraction V powder, 4% (w/v) in 0.2 M Tris . HCl, pH 8.0). In B and C, peptides were preincubated with aortic extract at 4’C for 5 min before addition of substrate emulsion. All enzymatic activities are expressed relative to control assays, containing aortic extract and substrate emulsion without serum, incubated as described in Materials and Methods. Results are means of duplicate determinations. (A) Effect of coneentration of rat serum in complete assay mixture. Data from one of seven experiments is presented. Serum stimulation of aortic llpase varied from 4- to lo-fold in separate experiments. (B) Effect of concentration of apolipoprotein-glutamate in complete assay mixture. (C) Effect of concentration of apolipoproteinserine (‘*,A) or apolipoprotein-alanine (0,~) in complete assay mixture on lipase activity measured with or without serum activation. land O-L assays containing 4.15% (v/v) rat serum as Jipase activator: I- - - - - -I and A- - - - - -h, assays containing no serum activator. Data from one cf two eXk3‘iments is presented.

363 TABLE

I

EFFECT

OF HEATING

AND RAT EPIDIDYMAL

SERUM

ON THE STIMULATION

ADIPOSE

OF LIPASE

ACTIVITY

OF BOVINE

AORTA

TISSUE

Extracts of acetone-ether powders of aorta or adipose tissue were prepared and assayed for lipase activity as described in Materials and Methods. Results are means of duplicate determinations: representative data from one of three experiments is presented. ___ __-II_ __Addition

Blank (cpm)

Lipase Activity

Aorta

Adipose

0.079 0.662 0.705

1.60 3.87 4.66

-0.017 0.017 0.017 ~-.-

ml bovine serum albumin* ml non-heated rat serum ml heated rat serum**

391 2259 551

____~___

(munitslmg

extract

protein)

-_____

.--__-_

* Fraction V powder, 4% (w/v) in 0.2 M Tris * NCl, PH 8.0. ** Fresh serum from fasted rats was heated in a shaking water bath at 60°C for 10 min. cooled at 4OC, and frozen in aiiquots. Protein concen~ations of heated and non-heated serum samples were identical as determined by method of Lowry et a3. 1131.

Results Assay system. Lipase activity increased linearly with increasing found of enzyme source until approx. 6 nmol of fatty acid had been released from the substrate; the reaction was also linear with time for up to 90 min. It was necessary to incorporate at least 0.1% crystallized bovine albumin (w/v in complete assay mixture) into the substrate during sonication in order to achieve linearity in the system and maximal enzymatic activity*. Serum obtained from rats fasted ove~ight was heated to 60°C for 10 min routinely before use in the assay. This procedure resulted in a substantial decrease in blank values (Table I), possibly due to denaturation of lipase activity present in the serum. Heating did not reduce the ability of serum to activate lipolytic activity of aorta or adipose tissue (Table I). Enzyme c~~~uc~e~~s~~cs. Addition of small amounts of rat serum to the assay mixture markedly increased aortic lipase activity; a lo-fold stimulation was observed with addition of serum at a final assay concentration of 4.15% (v/v) or higher (Fig. 1A). Addition of increasing amounts of the polypeptide cofactor apolipoprotein-glutamate produced a similar hyperbolic curve of increased lipolysis (Fig. IB), with an apparent K, of 6 fig apolipoprotein-glutamic acid/ml assay mixture. Addition of increasing amounts of either apolipoprotein-serine or apolipopro~in-alanine had no effect on lipase activity measured using triacylglycerol substrate without serum activator (Fig. IC). However, when 4.15% (v/v) serum was used as lipase activator in the assay, stimulation of lipolysis was reversed by preincubation of enzyme source with either apolipoprotein-serine or apolipoprotein-alanine (80 &g/ml in complete assay mixture) (Fig. 1C). The pH maximum of the lipase activity was found to be between 7.8 and * The

substrate albumin concentration required for maximal lipolysis and linear fatty acid release is 3-fold higher for aortic lipase activity than for adipose lipoprotein lipase activity (ref. II. unpublished), despite the fact that the specific activity (munits/mg extract protein) of adipose extracts is from 6 to 15 times as great as that of aortic extracts.

364

TABLE

II

RESPONSE Effecters assay

OF were

AORTIC combined

was carried

mixture

are given.

Addition

to assay

out

LIPASE with

ACTIVITY

aortic

as described

Number

extracts

in Materials

in parentheses

Lipase

activity

TO

IN

at 4’C:

VITRO substrate

and

Methods.

indicates

number

(percent

EFFECTORS

OF

emulsion

was added

Concentrations of separate

LIPOPROTEIN immediately

of effecters

in complete

LIPASE and

the

assay

observations.

of control)

NaCl 0.2

M

0.5

M

1.0

M

Protamine

53.0

+ 3.61*

22.0

i

13.8

? 2.18

(6) (6) (6)

3.11

sulfate

0.25

mg/ml

99.8

t 2.83

(6)

0.50

mg/m1

64.5

f 3.61

(4)

1.00

mg/ml

31.5

+ 4.84

(6)

2.50

mg/m1

14.2

k 4.41

(6)

(4)

Heparin 0.01

unit/ml

94.5

F 1.29

0.05

unit/ml

90.5

* 2.08

(4)

0.10

unit/ml

85.0

* 0.82

(4)

1.00

unit/ml

63.0

t 4.08

(4)

units/ml

47.0

i 2.58

(4)

100.00

*

Mean

i-S.D.

8.4; decreasing or increasing the pH beyond this range caused a rapid fall in activity. NaCl strongly inhibited lipolysis (Table II). About 20% of control activity was measured when NaCl was present at a concentration of 0.5 M in the assay mixture. Protamine sulfate also inhibited lipolysis, although at concentrations