Bio~hjmjca et B;oph_vsica Artu 834 (1985) 95-102

Elsevier

95

BBA 51882

Antibody against rat adipose tissue lipoprotein lipase

Jacqueline Etienne ap*, Lydie No& ‘, Monique Rossignol ‘, Caroline Arnaud ‘, Nadarajen Vydelingum b and Ahmed H. Kissebah b

11 Departments of Biochemistry and redefine, Centre Hospitalo-Uniuersitaire Saint Antoine - Tenon (Seroires des

FroJesseurs J. Debra-v. P. Laruelfe, J. Poionovski), Paris, (France], and ’ Department of Medicine and Pharmacology,

Medical CoIege of Wisconsin, Milwaukee, WI 53226 (U.S.A.)

(Received October 8th. 1984)

Key words: Enzyme antibody; Lipoprotein lipase; (Rat adipose)

To facilitate detailed studies of rat adipose tissue lipoprotein lipase regulation, a high titre polyclonal antibody was raised against purified rat adipose tissue lipoprotein lipase (in a goat). The first stage of the purification of the lipoprotein lipase was carried out with heparin-Sepharose affinity c~omato~aphy. In the second stage we took advance of the binding property of li~~tein lipase to ampholytes. These ampholytes, used during this second step, do not have to be eliminated prior to injecting the enzyme preparation into the animal. They have neither toxic nor antigenic effects on the anim* moreover, their presence does not affect the antigenic potency of the lipoprotein lipase. When pre-incubated with a constant amount of adipose tissue lipoprotein lipase (8 mU/ 75 PI), an equal volume of the antiserum raised either pure or diluted up to l/50 resulted in complete inhibition of enzyme activity, and half maximal inhibition was observed at a dilution of 1/800. The antibody was effective in inhibiting rat heart li~protein lipase but not salt-resistant hepatic lipase. Immunodiffusion revealed a single line of precipitation between this antibody and the adipose tissue lipoprotein lipase.

Lipoprotein lipase, (triacylglycero-protein acylhydrolase, EC 3.1.1.34) is the primary enzyme involved in the uptake of fatty acids from circulat- ing triacylglycerols. It catalyses the hydrolysis of plasmatic chylomicrons and VLDL triacylglycerols

[Il. The adipose tissue is a major site for lipoprotein

lipase activity. The enzyme is thought to be synthesized by adipocytes and transported to its functions location, the luminal surface of the

* To whom correspondence should be addressed at: Faculty of Medicine, 27 rue Chaligny, 75571, Paris Cedex 12, France, Abbreviations: SDS, sodium dodecyl sulfate: IEF, isoclcctric focusing.

capillary endothelium [2]. Lipoprotein lipase plays an important role in the regulation of plasma lipoproteins metabolism as well as in the differ- entiation and growth of adipose tissue mass. Abnormalities in its synthesis or activity have therefore been implicated in the pathogenesis of some forms of hyperlipide~a and obesity [3].

Metabolic studies on mammal lipoprotein lipase are usually performed first on the rat. An antibody against rat lipoprotein lipase is thus a useful tool for studying lipoprotein lipase metabolism in vivo, as well as in vitro on isolated or cultured adipo- cytes. An antibody against rat heart has been obtained by Schotz et al. [4]. It has been helpful in establishing the location of the functional lipopro- tein lipase [4,5].

Although antibody against rat heart lipoprotein

US-2760/~5/$03.3~ @) 1985 Else-&r Science Publishers B.V. (Biomedical Division)

lipase inhibits lipoprotein lipase from rat adipose

tissue, there are some physico-chemical differences between these two lipoprotein lipases. For in-

stance, these two enzymes have a different K, (61,

which partly explains the differences in lipoprotein

lipase activity observed in heart and adipose tissue

with the nutritional state of the animal [6]. Also,

Ben-Zeev et al. [7] recently showed differences in

thermolability of mouse heart and adipose tissue lipoprotein lipase. Differences in heat stability are

a sensitive indicator of protein structural variation.

According to these authors [7], these two lipopro-

tein lipases appear to differ in structure as well as

in the genes controlling their regulation or processing.

In metabolic studies on rat lipoprotein lipase it

would be useful to have an antibody directed against adipose tissue lipoprotein lipase. Besides

the antibody against rat heart obtained by Schotz

et al. [4] and also by Hiilsman et al. [8], other

anti-lipoprotein lipase antibodies have been pre-

pared from different sources such as, for instance,

rat post-heparin plasma [9,10], cow’s milk [ll-151, chicken adipose tissue [16], human milk [17] and

guinea pig milk [IS]. This paper describes the preparation of a poly-

clonal antibody against purified adipose tissue

lipoprotein lipase. We also prepared an antiserum

against cow’s milk lipoprotein iipase, as others

have already done using different methods [ll].

This is, however, the first report on the prepara-

tion of an antibody against rat adipose tissue lipoprotein lipase. A preliminary communication

of part of this work has been given 1201.

Materials and Methods

Purij~cation of lipoprotein iipase

To purify lipoprotein lipase, heparin*Sepharose

4 B chromatography was used in combination with electrophoresis in the presence of ampholytes.

Rat adipose tissue. Male Wistar rats weighing between 300 and 350 g were fed ad libitum. 18 h prior to killing, the drinking water was replaced by glucose (15% w/v). The animals were killed by stunning followed by neck fracture. Epididymal fat bodies were collected within minutes following killing. Acetone-ether powder was prepared without delay. For each preparation, about 50-80

g of adipose tissue were collected from 30 rats

which yielded between 1 and 1.50 g of acetone-ether powder. The crude lipoprotein lipase was extracted with 25 mM NH,OH/NH,CI buffer

tpH 8.5) (35 ml buffer/l g powder). The enzyme preparation was then homogenized by a polytron,

centrifuged for 30 min at 4000 x g at 4°C and the supernatant collected.

Bovine milk. Fresh bovine milk was obtained

from a local farm and transported in ice to the laboratory. It was centrifuged at 3000 x g for 60

min at 4’C to separate off the cream and a small

pellet of insoluble material. It was then stored frozen at - 30°C until use.

First step in pzcrificution: heparin-Sepharose lIj- finit,v chromatography. Affinity chromatography

was performed with BrCN Sepharose 4 B contain- ing covalently linked crude heparin [19].

For rat adipose tissue, the centrifuged am-

monium extract of acetone-ether powder (35 ml)

was diluted with an equal volume of 0.5 M NaCl

buffer (5 mM sodium barbital, pH 7.4) and ap-

plied to a heparin-Sepharose column (2.4 x 7.5 cm). The column was washed with 50 ml of 0.5 M

NaCl buffer, then with 120 ml of 0.75 M NaCl buffer. The enzyme was eluted in 50 ml of 1.3 M

NaCl buffer and dialyzed against 5 mM NH,HCO, buffer (pH 8) for 16 h at 4°C. Glycerol (1.8 ml)

was added to the dialyzed fraction (in order to

protect lipoprotein lipase during the subsequent

steps of purification stages), and the preparation

was concentrated by lyophilization to a volume of 3 ml. The solution obtained was perfectly clear.

For the purification of the bovine enzyme,

skimmed milk (100 ml) was diluted with an equal volume of 0.5 M NaCl buffer, mixed with 35 ml of

heparin-Sepharose gel and incubated at 4°C for 1 h on a magnetic stirrer. The slurry was then col- lected on a sintered giass filter under suction and washed with 300 ml of 0.5 M NaCl buffer, fol- lowed by 500 ml of 0.75 M NaCl buffer. A column (2.4 x 7.5 cm) was packed with this gel and the column was washed with an additional 100 ml of 0.75 M NaCl buffer. Bovine milk lipoprotein lipase was then eluted, dialyzed and concentrated as described above.

Second step in purification: ekectrophoresis in presence of amphalytes (IEF). The granulated gel used was a specially purified dextran gei of Seph-

adex G-75 superfine (Ultrodex’). A slurry was prepared by adding 4 g of the gel to a 2% Ampho- lines@ mixture allowing, after isoelectric focusing, a pH gradient of 4.5-8.2. The slurry was poured on the gel tray (10.5 x 24.2 cm) and evaporated to reduce the water content by 27%. After a 2-3 h pre-run, the partially purified lipoprotein lipase from the heparin-Sepharose step was applied to the gel. Focusing was performed at 0-1°C for 14-16 h with a constant power of 8 W. The focused zones were marked by pressing a fractionating grid onto the gel defining 15 lanes. The pH in each lane was measured with a micro electrode. The 15 discrete gel sections were trans- ferred to small columns and eluted with 10 ml of 25 mM NH,OH/NH,Cl buffer (pH 8.5) at 4°C.

Measurement of lipoprotein iipase activity Lipolytic activity was measured with an emul-

sion of glycerol tri[l-‘4C]oleate as substrate [21,22]. The stock substrate contained 25 PCi (926. lo3 Bq) glycerol tri[l-‘4C]oleate, 69 mg unlabelled tri- olein, 3.33 mg soybean phosphatidylcholine and 5 ml glycerol. The mixture was emulsified on ice for 5 min using an Ultraturrax. It was stable for 6 weeks. The assay mixture contained 150 ~1 of the enzyme solution and 100 ~1 of diluted substrate, (40 ~1 of emulsified stock substrate, 40 ~1 of 5% bovine serum albumin solubilized in 0.62 M Tris/0.16 M NaCl and adjusted to pH 8.6, and 20 ~1 of fresh human serum). After incubation at 37°C for 20 min, the labelled fatty acid products released were extracted and the radioactivity was counted. 1 mU of enzyme activity is defined as the amount of the enzyme required to catalyse the release of 1 nmol of fatty acid per min at 37°C.

In some assays, serum was omitted from the incubation medium and was replaced by 0.16 M NaCl. For assays with 1 M NaCl, the incubation was done with 100 ~1 of enzyme + 50 ~1 of 5 M NaCl and 100 ~1 of substrate.

Proteins Protein content was determined with the

Coomassie blue technique [23,24]. Blanks were subjected to the same purification procedure as the test (including affinity chromatography and IEF). Bovine serum albumin was used as standard.

Polyacrylamide gel electrophoresis Disc gel electrophoresis was performed with

SDS as described previously [25]. Prior to electro- phoresis, fractions eluted from lanes 2-4 after IEF were extensively dialyzed against 5 mM NH,HCO, buffer (pH 8) and then lyophilized. Samples were heated at 100°C for 10 min in 10 mM phosphate buffer (pH 7.0) containing 2.5% (w/v) SDS, 7% (v/v) fi-mercapto ethanol and 6 M urea. Electro- phoresis was performed using 5% (w/v) poly- acrylamide. The gels were then stained with R 250 Coomassie blue. A blank was prepared under the same conditions as described in the paragraph above.

Double immuno diffusion Double immunodiffusion was carried out in 1%

agarose gel prepared in 0.04 M Verona1 (pH 8.6) and containing 0.01% (v/v) Nonidet P 40. This uncharged detergent was used to prevent de- naturation and precipitation of enzyme around the sample wells [12]. The gels were stained with R 250 Coomassie blue.

Preparation of lipoprotein lipase antiserum Antiserum raised against rat adipose tissue lipo-

protein lipase. Several purified lipoprotein lipase preparations from lanes 2-4 of the IEF procedure were pooled (about 1 mg of protein), extensively dialyzed against 5 mM NH4HC03 (pH 8), lyophi- lized, then emulsified with 1 ml of complete Freund’s adjuvant. The emulsion was injected in- tradermally at multiple sites into the dorsal surface of a goat. Subsequent injections prepared with incomplete Freund’s adjuvant were given at one month intervals for five months. Ten days after the last injection, blood was collected, and the serum was separated and frozen.

Antiserum raised against bovine milk lipoprotein lipase. Immunizations (about 0.5 mg of protein) were performed in the rabbit under the same con- ditions as described above. But, in order to test the effect of ampholytes on the antigenicity of lipo- protein lipase, two lots of bovine milk purified lipoprotein Iipase were prepared. From the first lot, the purified lipoprotein lipase preparations from lanes 2-4 were used without previous elimination of ampholytes, while, from the second, ampholytes were eliminated, as far as possible, by extensive dialysis.

98

Inhibition tests

75 ~1 of enzyme (8 mu) - either diluted rat adipose tissue fraction, eluted from a heparin-sep-

harose column, or diluted bovine milk - were pre- incubated for 60 min at 4°C with 75 ~1 of different

dilutions containing increasing concentrations of

rat lipoprotein lipase antiserum or bovine lipopro-

tein lipase antiserum. The lipoprotein lipase assay

substrate (100 ~1) was added and incubation per- formed at 37°C for 20 min. Residual lipolytic

activity was measured. Control incubations con-

tained serum from non-immunized animals.

Materials

Tri[l-‘4C]oleylglycerol, 50 Ci/mol (1850. lo3 Bq/pmol) was obtained from the Commissariat a

1’Energie Atomique. The unlabelled trioleylg- lycerol, L-a-phosphatidylcholine and Tris were

purchased from Sigma; SDS and Coomassie blue

(R250 and G) were from Bio-Rad, and bovine

serum albumin (fraction V) was from Armour. Sepharose 4B, and the low molecular weight pro-

tein standard kit used for polyacrylamide gel elec- trophoresis, were obtained from Pharmacia. Ultrodex@, Ampholines@ and elution columns came from LKB. Freund’s adjuvant was obtained

from Difco Lab.

Results

Preparative IEF eiution profiles

Fig. la shows a representative elution profile following preparative IEF of lipoprotein lipase

from rat adipose tissue.

The lipolytic activity was recovered close to the

origin of IEF plate in lanes 2-4. The eluate from this region exhibited characteristic properties of

lipoprotein lipase: lipolytic activity was optimum when incubation was carried out in the presence of serum and with low molarity NaCl, and reduced to 5-25% and O-10%, respectively, by omitting serum or adding 1M NaCl.

Two protein peaks were detected. The first pro- tein peak was eluted from lanes 2-4 and corre- sponded to the lipoprotein lipase activity peak. The second peak was eluted from lanes 12-14 and had no lipolytic activity.

Fig. lb shows the similar elution profile ob- tained from the bovine milk lipoprotein lipase. An

2 :

RAT ADIPOSE TISSUE (a)

? b

BOVINE MILK (b) ‘h 3

5 2

C... 9‘ .I..

. ..\ ‘.

Fig. 1. Preparative Isoelectric focusing elution profiles. Rat

adipose tissue (a) or bovine milk (b) lipoprotein lipase (LPL)

partially purified by affinity chromatography on heparin-Sep-

harose was deposited on a flat bed of pre-focused polydextran

gel UltrodexB containing 2% (w/v) Ampholines@ (pH 4.5-8.2).

To apply the sample, 3 ml of the gel (corresponding to lane 3.

pH 7.7) were removed, mixed with an equal volume of sample

and the gel sample mixture was returned to the slab. Focusing

was continued for 14-16 h at 0-1°C with constant power of 8

W. The slab gel was then separated into 15 lanes with a grid

and the pH (0 - - - - - - 0) of each lane was measured with a

microelectrode before elution with 5 ~2 ml of 25 mM

NH,OH/NH,Cl buffer (pH 8.5). Protein (0- - - - -0) and

lipoprotein lipase activity (O- 0) activity were determined

in each fraction. Values are from one representative experiment

among the four that were done.

additional third protein peak, with no lipoprotein

lipase activity, was recovered in lanes 9-11. In 12 preparations from rat adipose tissue and

10 preparations from bovine milk, the recovery of lipoprotein lipase activity after IEF was about 80%. The specific activity of the enzyme eluted from the IEF plate increased by about 1.552-fold. A typical experiment is shown in Table I.

Polyacrylamide gel electrophoresis Rat adipose tissue lipoprotein lipase purified by

heparin-Sepharose affinity chromatography. fol-

99

TABLE I

PURIFICATION OF RAT ADIPOSE TISSUE AND BOVINE MILK LIPOPROTEIN LIPASE

Extractum refers to acetone-ether powder extracted with NH,OH/NH,Cl buffer. ‘Before IEF’ refers to enzyme preparations purified by hep~in-Sepharose affinity chromatography alone (enzyme solution was then dialyzed and concentrated by lyophili~tion) ‘After IEF’ refers to enzyme prepared by affinity chromat~raphy followed by preparative IEF (the enzyme solution was eluted from lanes 2-4). The results represent values from four separate experiments.

Rat adipose tissue Extraction Before IEF After IEF

(lanes 2-4)

Vol

(ml)

35 3

30

Protein

(mg)

260 0.339 0.186

Enzyme Spec. activity act.

(mu) (mU/mg)

10329 39.73 1255 3 702 1028 5 527

Purifi- cation

93 139

Yield

(%)

100 12 10

Bovine milk Skimmed milk Before IEF After IEF

(lanes 2-4)

100 2500 1~~ 64 100 3 0.562 27456 48 854 763 I7

30 0.275 21965 79 872 1248 14

Fig. 2. Disc polyacrylamide gel electrophoresis of rat adipose tissue lipoprotein lipase. Samples for SDS/polyacrylarnide gel electrophoresis (5%) were prepared as described in Methods.

lowed by IEF, exhibited a single Coomassie blue stainable band on disc SDS polyacrylamide gel electrophoresis (Fig. 2).

Anti-lipoprotein iipase antiserum Fig. 3 shows the inhibitor profiles of the anti-

sera raised against rat adipose tissue lipoprotein lipase (a) and against cow milk lipoprotein lipase

(b, 4. With the antiserum raised (in a goat) against rat

adipose tissue (curve a), complete inhibition of 8 mU of rat adipose tissue lipoprotein lipase oc- curred at antiserum dilution of l/50 (93% inhibi- tion was still obtained with antiserum diluted l/100) and half maximal inhibition was observed with dilution of l/800.

With the antiserum raised (in a rabbit) against bovine milk lipoprotein lipase, complete inhibition of 8 mU of bovine lipoprotein lipase occurred at antiserum dilution of l/15, and half maximal

The left gel shows the migration pattern of marker proteins. The 6 main bands are, from top to bottom, phospho~l~ b (jw, 94000). bovine serum albumin (Mr 67ooO), ovalbumin (JW, 43000) carbonic anhydrase (M, 30000), trypsin inhibitor (M, 20100) and a-lactalbumin (M, 14400). The right gel shows the band of purified lipoprotein lipase from rat adipose tissue (eluted IEF fraction from lane 2-4, approx. 40 pg of protein).

100

- rat (a) A---A bovine 1 (b)

:.----I bovine 2 (c)

AntIserum : pL

Fig. 3. Immune-titration of rat adipose tissue and bovine milk

lipoprotein lipase. Rat adipose tissue (a) or bovine milk (b.c)

with a constant lipoprotein lipase (LPL) activity (8 mU in 75

~1) was preincubated for 60 min at 4°C with 75 ~1 of different

dilutions containing increasing concentrations of either rat (a)

or bovine (b.c) lipoprotein lipase antiserum. The antiserum

dilutions were prepared with the serum of non-immunized

animals such as goat (a) or rabbit (b.c). Substrate (100 ~1) was

then added and the incubation was continued at 37°C for 20

min. Lipoprotein lipase activity was measured. Results are

expressed as percentages of remaining lipoprotein lipase activ-

ity. Controls, in which non-immunized serum (from goat or

rabbit) was used instead of antiserum. did not alter the original

lipoprotein lipase activity. In bovine 1 (b) ampholytes were

removed as far as possible by extensive dialysis prior to

immunization, whereas in bovine 2 (c), the enzyme sample was

injected without the dialysis step.

inhibition with dilution of l/60 (curves b and c).

There was no difference in antibody potency if

ampholytes were removed (curve c) or not re- moved (curve b) from the enzyme prior to im- munization of rabbits. Pre-incubation of rat

adipose tissue lipoprotein lipase, or cow’s milk lipoprotein lipase, with the serum of a non-im-

munized animal (goat or rabbit, respectively), did not alter the original lipoprotein lipase activity.

In analytical IEF, lipoprotein lipase is known to

form one [26,27] or several peaks [28], depending on experimental conditions. Bengtsson and

Olivecrona [28] have suggested that lipoprotein

lipase binds ampholytes to form complexes with an isoelectric point intermediate between that of the enzyme and that of the ampholytes which it had bound [28]. Here, we used the property of lipoprotein lipase to bind ampholytes to our ad- vantage in order to separate lipoprotein lipase

from protein impurities which had co-eluted with it during affinity chromatography.

The antiserum raised against rat adipose tissue The nature of the protein eliminated by IEF as also inhibited lipoprotein lipase from rat heart. the final step in the purification has not yet been Complete inhibition of 8 mU of rat heart adipose identified. When lipoprotein lipase is obtained tissue lipoprotein lipase occurred at antiserum di- from post-heparin plasma it is known that the lution of l/50, and half maximal inhibition was main protein co-eluted with this lipoprotein lipase observed with dilution of l/1000. No inhibitory during heparin-Sepharose affinity chromatography effect was detected with the salt-resistant hepatic is essentially antithrombin III [29]. In adipose lipase isolated from rat post-heparin plasma. This tissue. protein co-eluted could also be antithrom-

Fig. 4. Double immunodiffusion of purified rat adtpo\c ti\\ue

lipoprotein lipase and lipoprotein lipaae antibody. Adipose

tissue lipoprotein lipase was purified by heparin-Sepharo\c

chromatography and IEF. Lanes 2-4 (from ILF) uere elutcd.

dialyzed against 5 mM NH,HCO, (pH 8) and freeze-dried

The right well contained purified hpoprotem Itpase (10 fig

protein) and the left well contained rat lipoprotetn Iipa\e

antiserum (5 ~11).

antiserum also inhibited mouse adipose tissue lipo-

protein lipase. Fig. 4 shows interaction between rat adipose

tissue antiserum and rat adipose tissue lipoprotein

lipase. A single line of precipitation was observed

in the double immunodiffusion system.

Discussion

An antibody was raised against purified rat adipose tissue lipoprotein lipase. To our knowl-

edge this is the first time such an antibody has ever been obtained. Purification of the lipoprotein

lipase injected was achieved by combining affinity

chromatography (as the main purification proce- dure) and preparative IEF (as a complementary

procedure). The antiserum obtained had a high titre.

bin III, as suggested by Parkin et al. [30]. It might possibly originate from capillary blood contained in adipose tissue.

It has been reported that a~tithrombin present in partially purified lipoprotein lipase (from post- heparin plasma) could be removed by a second affinity chromatography on heparin-Sepharose with low affinity for antithrombin [31], or by gel filtration on Rio-Gel A-4m [32], The procedure described here, to eliminate protein impurities co- eluted with lipoprotein lipase during affinity chro- matography, has the advantage of being easy to perform. Furthermore, it gives a high recovery (about 80% of the lipoprotein lipase applied to the gel). This high recovery is possible partly because lipoprotein Iipase is stationary, and losses due to trailing are avoided. Also, IEF is performed at 0-1°C and in the presence of glycerol; hence, lipoprotein lipase is retained in a protective en- vironment in lanes 2-4 throughout the run.

The presence of ampholytes in the purified lipoprotein lipase sampIes after IEF did not affect the antigen potency of the lipoprotein lipase, as seen in Fig. 3. Furthermore, it is convenient that ampholytes have no antigenic properties of their own. When using concanavalin (as Sepharose con- canavalin) in a second stage purification [l&33,4,30], bleeding of Iectin from the column occurs. This makes it necessary either to eliminate the concanavalin before the lipoprotein lipase pre- paration is injected into the animal, or to eliminate the ~ti-concanavalin ~mmu~oglob~ns from the antiserum raised against the lipoprotein lipase pre- paration containing concanavalin [4].

The antibody against rat adipose tissue pre- pared here has already been used in the following studies: (i) in an immunotitratiun assay, in order to clarify the role of insulin in enhancing lipopro- tein lipase activity in mature adipocytes, by Vyde- lingum et al. [34]. The inhibitory profiles obtained with adipose tissue extracts, treated with or without insulin, were superposable, revealing an increase in lipoprotein lipase enzyme protein units. Also, in- sulin enhanced iuco~oratio~ of radioactive amino acids into immuno-precipitable lipoprotein lipase; (ii) in an immunofluorescence study, to visualize lipoprotein lipase in preadipocytes cultures by Vannier et al, [35]. There is no available method for identifying preadipocytes before the accumula-

tion of cellular triacylglycerols characteristic of mature adipocytes occurs. Now, lipoprotein lipase is an early marker of adipocyte conversion preced- ing the accumulation of t~ac~IgI~cerols; therefore, preadipocytes can be identified by fluorescent anti-lipoprotein lipase techniques. The high titre of the antibody makes it possible to work with l/100-l/2000 diluted antiserum, and thus to eliminate the nonspecific immunofluorescent reac- tions; (iii) in inhibition tests, to characterize lipoprotein Iipase_ after rats had been injected with cholera toxin (a potent stimulator of adenylate cyclase and lipoprotein lipase), by Knobler et al.

1361. In conclusion, this specific and high titre anti-rat

adipocyte Iipoprotein lipase antibody is a prom- ising tool for further studies of Iipoprotein lipase at the cellular level.

Acknowledgements

These studies were supported by a grant from the Institut National de la Sante et de la Re- cherche Medicale, Paris, France (CRL-82 7002).

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