Fate of milk 125I-labeled lipoprotein lipase in cells in culture comparison of lipoprotein lipase- and non-lipoprotein lipase-synthesizing cells

I14

BBA 51071

FATE OF MILK 12’1-LABELED LIPOPROTEIN COMPARISON OF LIPOPROTEIN LIPASE-SYNTHESIZING CELLS G. FRIEDMAN

‘, T. CHAJEK-SHAUL

LIPASE IN CELLS IN CULTURE

LIPASE- AND NON-LIPOPROTEIN

‘, T. OLIVECRONA

‘, 0. STEIN ’ and Y. STEIN ’

” Ltpid Reseurch Luhorcr~oty. Depurtment of Medtcirte B, IIudussuh Utticersi!,~ Ilospitul, Depcrrtmertt of E.vperrntr~rtoI M~&IIW urd Comer Resecrrch, Hebrew Unioersi?.Hudussuh Medical School, Jeruwlen~ (Isruel), ud ’ Depa17n1cwt of C/temi.vt~~~~.Sectiou *))I Physiological Chemistry. Unioersiy of UnteJ, Unteu (Swedeit) (Received

Kq

November

words: Lipoprotein

23rd, I98 I )

lipuse; Cell culture, (Milk)

Radioiodinated lipoprotein lipase, isolated from bovine milk ( ‘251-labeled milk lipoprotein lipase) was shown to retain full hydrolytic activity towards its native substrate, i.e., chylomicron triacylglycerol. The ‘251-labeled enzyme interacted with various cells in culture by being bound to the cellular surface, internalized and degraded. Cellular binding of the labeled enzyme occurred in the presence or absence of substrate and was related to enzyme concentration. Heparin reduced cellular binding by 50% but inhibited uptake and degradation more extensively. Cellular uptake was not affected by chloroquine or NH&I, but degradation of the labeled enzyme was blocked. Uptake and degradation were not inhibited by mannose 6-phosphate. The interaction between the exogenous enzyme and cells which do not synthesize lipoprotein lipase, i.e., fibroblasts and endothelial cells, resulted in a high ratio of surface binding to degradation. In heart cell cultures and preadipocyte cultures, which produce lipoprotein lipase, the ratio of enzyme catabolized to that bound was high at all time points examined. Since in the intact organism lipoprotein lipase acts at the luminal surface of vascular endothelium, it seems expedient that these cells are able to bind the enzyme, but will catabolize it only slowly. The rapid and extensive degradation of the 12’I-labeled lipoprotein lipase in heart cells and preadipocytes may be related to the metabolism of the endogenously produced lipoprotein lipase.

Introduction

[l]. Previously, we have studied the fate of chylomicron and VLDL cholesteryl ester, during lipoprotein lipase -catalyzed hydrolysis of chylomicrons, using its nondegradable analog cholesteryl linoleyl ether [2-41. The results obtained with exogenously added milk lipoprotein lipase provided evidence as to the possible role of lipoprotein lipase in cellular uptake of chylomicron cholesteryl ester [3,4]. Among the many questions raised by the experiments was also the fate of the lipoprotein lipase molecule itself. The t,,, of the lipoprotein lipase, as measured by change in enzyme activity after inhibition of protein synthesis,

The main role of lipoprotein lipase is hydrolysis of the triacylglycerol moiety of chylomicrons and very low density lipoproteins (VLDL). The fatty acids which are released are taken up by the tissues at the site of hydrolysis and the lipoproteins undergo remodulation into remnant particles Abbreviations: VLDL, very low density lipoprotein(s); Hepes, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid; PPO. 2,5-diphenyloxazole; POPOP. l,4-b&(2-(5-phenyloxazolyl)) benzene; TPCK, L-(tosylamido-2-phenyl)ethyl chloromethylketone; TLCK, N-a-P-tosyl-L-lysine chloromethylketone. 0005.2760/82/0000-0000/$02.75

G 1982 Elsevier Biomedical

Press

was found to be about 35 min in the heart cell cultures [5], but the site of enzyme catabolism has not been elucidated so far. In this work, we have used radioiodinated milk lipoprotein lipase to study the interaction between lipoprotein lipase and various cell types in culture. The cells used were those which synthesize lipoprotein lipase in culture and in the intact animal, i.e., mesenchymal heart cells [5] and preadipocytes [6], and those which so far have not been shown to produce the enzyme, i.e., endothelial cells and fibroblasts. The results obtained indicate that the cells studied behave differently with respect to lipoprotein lipase binding and metabolism, depending on their capacity to synthesize the enzyme. Materials and Methods Cell cultures. The following cultured cells were used in the present study: 1. F, rat heart cell cultures, prepared as detailed in previous reports [5,7], which consisted mainly of non-beating mesenchymal cells. These cultures were used 8 days after isolation of the cells from 2-day-old rat hearts. At that time, the endogenous LPL activity had reached a value of 900-1200 nmol fatty acid released/mg cell protein per h. 2. Cultured rat preadipocytes, which had undergone adipose conversion. The cells were isolated from epididymal fat pads of 3-week-old rats of the Hebrew University strain, as described by Bjorntorp et al. [6]. In brief, the pads were removed under aseptic conditions, rinsed in several changes of 0.02 M Hepes buffer/O.12 M NaCl/O.OS M KCl/O.OOl M CaCl, /O.OOS M glucose/ 1.5% (w/v) b ovine serum albumin, pH 7.4, cut into small pieces and incubated in sterile plastic test tubes at 37°C with collagenase, 3 mg/ml of buffer, for 30 min with intermittent agitation. Thereafter, the cell suspension was filtered through a metal screen with a pore size of 250 pm, to remove clumps, and then filtered through 25 pm pore-size nylon screen. Following centrifugation for 10 min at 1000 rev./mm, the cells were pelleted, resuspended in 30-40 ml F,, culture medium, supplemented with 10% calf serum, 10% horse serum and 40 munits/ml of insulin. 2 ml of the cell suspension was plated in 35-mm petri dishes (cells from one pad were used for one dish) and the cells were cuirured till confluency was reached

in 8-10 days. The medium was changed every 3 days. Accumulation of large lipid droplets in clusters of cells was evident after 3 days in culture and their number increased with longer times of cultivation. After 8 days lipoprotein lipase activity in the medium was lOOO- 1200 and in the cells 400600 nmol fatty acid released/mg cell protein per h. 3. Human skin fibroblasts and endothelial cells were used as examples of cells devoid of endogenous lipoprotein lipase activity. Human skin fibroblasts were isolated from adult skin as described before [8] and cultured in modified Dulbecco-Vogt medium, containing 5% fetal calf and 5% new-born calf serum. The cells were plated at lo5 tells/35-mm petri dish and used after 5-7 days, when confluent. Endothelial cells were isolated from bovine aorta [9] and cultured in 35-mm petri dishes with Dulbecco-Vogt medium containing 10% new-born calf serum in the presence of fibroblast growth factor (donated by Dr. Denis Gospodarowicz of the Cancer Research Institute, University of California, San Francisco), till confluent. Isolation and iodination of lipoprotein lipase. Milk lipoprotein lipase was isolated from bovine milk and purified as described before [lo]. The iodination of the enzyme was carried out with lactoperoxidase [ 111, the enzyme was reisolated on a heparin-Sepharose column and stored at - 20°C in the presence of 1% bovine serum albumin [ 121. Preparation and labeling of chylomicrons. Mesenteric lymph duct chylomicrons were isolated after intraduodenal infusion of Intralipid and were labeled with 25 PCi of [l-‘4C]palmitic acid complexed to albumin [3]. Determination of lipoprotein lipase activity. The enzyme activity was determined on aliquots of homogenates of cells which had been released from the petri dish with a rubber policeman in 1 ml 0.025 M NH,/NH,Cl buffer (pH 8.1) containing 1 unit/ml of heparin. The assay system consisted of 0.1 ml cell homogenate (50-70 and 40-60 pg protein in heart cells and preadipocytes, respectively) and 0.1 ml of substrate, prepared according to the method of Nilsson-Ehle and Schotz [13]. Incubations were carried out at 37’C for 60 min. The reaction was stopped by addition of methanol/chloroform/heptane (1.4 : 1.25 : 1,

I I6

v/v) an the extraction of fatty acids was performed according to the method of Belfrage and Vaughan [ 141 as modified by Nilsson-Ehle and Schotz [ 131. Enzyme activity was calculated according to the formula of Nilsson-Ehle and Schotz [ 131 and was expressed per mg cell protein/h. Experimental procedure. On the day of the experiment the complete growth medium was removed from the cultured cells, the cell layer washed with phosphate-buffered saline and 1 ml of growth medium F,a without serum, containing 4% bovine serum albumin, was added. All further additions were made to this serum-free medium. The incubations were carried out at 37°C in a CO, /air incubator. To measure the metabolism of ‘251-labeled lipoprotein lipase the following procedure was used. At the end of incubation the medium was drawn off and an aliquot was taken for deter~nation of radioactivity. After removal of the medium the cell layer was washed three times with 0.2% albumin in phosphate-buffered saline and three times with phosphate-buffered saline at room temperature, 0.5 ml of 0.05% trypsin in Versene buffer was added to each petri dish and incubated at 37’C for 5 min. The cells were collected and, following inactivation of the trypsin by addition of serum-containing medium, the cells were separated by centrifugation at 2500 rev./mm for 20 min and an aliquot of the supernatant fluid was assayed for radioactivity. The cell pellets were washed twice by suspending in 4 ml of phosphatebuffered saline and recentrifuging. Cellular “‘1 radioactivity was measured directly on the cell pellet following trichloroacetic acid precipitation. To determine catabolism of ‘251-labeled lipoprotein lipase, ‘251-labeled protein degradation products were determined in the culture medium, as trichloroacetic acid - soluble, chloroform non extractable “‘1 [ 151. Chemical and radiochemical determinations. Protein was determined according to the method of Lowry et al. [16] using bovine serum albumin as standard. In some experiments ‘251-labeled lipoprotein lipase was added to culture medium containing chylomicrons labeled with ji4C]palmitic acid, mainly in their triacylglycerol moiety. The enzymic hydrolysis of triacylglycerol was determined after extraction of the lipids [ 171 and

separation of the labeled fatty acid on thin-layer chromatography using TLC ready plastic sheets F1500 (Schleicher and Schtill, Dessel, F.R.G.) and hexane/diethyl ether/acetic acid, 83 : 16 : 1 (v/v) as solvent. The lipid areas were visualized with iodine vapor, identified with the help of reference standards, cut out and counted. 14C radioactivity was determined in a /? scintillation (TRI-CARB 2660) and ‘*‘I in a y scintillation spectrometer (Kontron MR 480C). The scintillation fluid used was 20% Triton X-1~/0.005~ POPOP/O.4~ PPO in toluene. Materials [ l-14C]Palmitic acid, specific activity 59 Ci/mol, and glycerol tri[9,10(n)-3H]oleate, specific activity 544 Ci/mol, were obtained from The Radiochemical Centre, Amersham, U.K. Trypsin, collagenase type I, chloroquine, TPCK, TLCK, were obtained from Sigma. Culture media were obtained from Gibco (Grand Island, NY). Insulin (Actrapid, Novo Industrias, Copenhagen, Denmark). Heparin, thrombolique, from Organon, Oss, Holland.

Red ts To study the fate of milk lipoprotein lipase added to cultured rat heart cells we have used radioiodinated enzyme. To compare the enzymic activity of ‘251-labeled lipoprotein lipase to that of the nonlabeled molecule, both enzymes were incubated with chylomicrons, labeled in their triacylglycerol moiety with (‘4C]palmitic acid and the rate of release of the fatty acid in the presence and absence of apolipoprotein C-II (added in the form of high density lipoprotein) was determined. The data presented in Fig. 1 show that both the native and radioiodinated enzyme catalyzed the degradation of [‘4C]triacylglycerol at the same rate and were affected by apolipoprotein C-II to the same extent. When ‘251-labeled lipoprotein lipase was added to rat heart celi cultures three events were observed (Fig. 2). The enzyme became bound to the cell, and could be removed from its surface by trypsin, which was added to the cultures after three washes with albumin-containing buffer and three washes of buffer alone. The labeled enzyme which was retained with the cells after trypsiniza-

II7

Time yfm~;;ubation

-0 Fig. I. Comparison of lipolysis of chylomicron [ “C]triacylglycerol by native and radioiodinated milk lipoprotein lipase. Conditions: rat mesenteric duct chylomicrons, labeled in their triacylglyeerol moiety with ]l-‘4CJpalmitic acid, 1.86 mg triacylglycerol, were incubated at 37°C with 5 gg protein of native lipoprotein lipase with (0) or without HDL (0), or 5 pg ‘251-labeled lipoprotein lipase with (A) or without (a) HDL (0.96 mg protein) in 4% bovine albumin in 0.1 M Tris-HCI buffer, pH 8.2. Liberation of ‘“C-labeled fatty acid and decrease in [ “C]triacylglycero1 was determined by thin-layer chromatography after extraction of the lipids according to the method of Bligh and Dyer [ 171.

tion indicated intracellular uptake (Fig. 2). The culture medium was examined for ‘*‘I-labeled protein degradation products and it can be seen that four times as much enzyme had undergone proteolytic degradation as had been retained in the cells (Fig. 2). The uptake and degradation were linear with the enzyme concentration between 0.1 and 1 pg protein/ml. No saturation was seen when the experiment was repeated and the concentration curve extended up to 10 pg lipoprotein lipase protein/ml. In the next experiments, the interaction between the ‘251-labeled lipoprotein lipase and various cultured cells was compared. While in all cell types studied the enzyme was bound, internalized and degraded, the ratio of enzyme bound to that metabolized (taken up and degraded) varied (Table1). In the endothelial cells and fibroblasts, surface binding exceeded by far the amount de-

0.1 0.2

‘~$I-Lipoprotein

0.5 Lipase

1.0 (pg protein/ml)

Fig. 2. Interaction between ‘251-labeled milk lipoprotein lipase and rat heart cells in culture. Conditions:cells were cultured in complete culture medium containing 10% calf and 10% horse serum for 8 days. On the day of the experiment the medium was removed, the cells were washed with phosphate-buffered saline and F,, medium containing 4% bovine serum albumin was added. ‘251-labeled lipoprotein lipase and chylomicrons, 50 ng triacylglycerol/mi, were added to this medium and the cells were incubated for 6 h. At the end of incubation, the medium was collected, the cells washed three times with 0.2% albumin and three times with phosphate-buffered saline and released from the dish with trypsin. After addition of barn-cont~~ng medium, the cells were centrifuged and the supematant trypsinreleasable label was used for the determination of surface binding (A). The cell pellet was washed twice by resuspension in phosphate-buffered saline and the cell-associated ‘25I represents cellular uptake (0). The medium was anaIyzed for “‘1 degradation (a) products, and the data presented are after subtraction of values obtained in media incubated without cells, Values are means* SD. of triplicate dishes.

graded, while the opposite was true for the heart cells and preadipocytes. The latter two cell types are known to synthesize lipoprotein lipase, while the fibroblasts and endothelial cells do not. In the LPL-producing cultured preadipocytes, the rate of degradation of ‘251-labeled lipoprotein Iipase was more rapid during the first 6 h and the total amount metabolized was 45% in 24 h (Fig. 3). In the fibroblasts, the amount of enzyme degraded was linear for 24 h and only at that time did it exceed the amount bound to the cell surface. To confirm

TABLE I METABOLISM SYNTHESIZE

OF ‘251-LABELED MILK LIPOPROTEIN LIPASE

LIPOPROTEIN

LIPASE

IN CULTURED

CELLS

WHICH

DO AND

DO NOT

Conditions: All cell types were grown in their respective media (see Materials and Methods) until fully confluent, The cell protein (mg’dish) was 0.56 for bovine aortic endothelium and rat heart cells. 0.53 for rat preadipocytes and 0.47 for human skin fibrobiasta. On the day of the experiment, the culture medium was removed, the cells were washed with phosphate-buffered saline and “‘I-labeled lipoprotein lipase, 5 pg protein, and chylomicrons, 50 r.r(gtriacylglycerol. were added to I ml F,,, medium containing 4%) bovine serum albumin. Values are means of duplicate dishes. Binding, uptake and degradation values are cpm/dish per 6 h. Cell type

Binding

Uptake

Degradation

Uptake + degradation (% medium label)

Endothelial cells Fibroblasts Preadipocytes Heart cells

4886 7 go7 1556 I 949

I 982 2391 6 84X 9414

492 I268 426 791

that indeed degradation of ‘251-labeled lipoprotein lipase occurred by cell-associated proteolytic enzymes rather than by enzymes released into the medium, use was made of media conditioned with the various cell types for 18 h. The amount of degradation of “‘I-labeled lipoprotein lipase which occurred during incubation with conditioned media was the same as that seen in nonconditioned media,

3cll

,’

20

I’

I’

.

.I

,,’

,$

10

,,,’

&..

o-.-._._.

_/

0

Time

of

which has been used as a base-line value and was subtracted from the experimental values. So rar all experiments were carried out in the presence of chylomicrons. We next evaluated the role of the substrate in the interaction between the enzyme and the cultured cells. The results shown in Table II indicate that addition of chylomicrons had no effect on the metabolism of ‘*‘I-labeled lipoprotein lipase in either the rat heart cells or in human skin fibroblasts, which are devoid of endogenous lipoprotein lipase activity. The rate of uptake and degradation was proportional to the enzyme concentration at 5 and 10 pg protein/ml in both types of culture. Addition of heparin to the culture medium resulted in a 50% reduction in ‘251-labeled lipoprotein lipase binding to the rat heart cells or to human skin fibroblasts. At the same time, cellular uptake and degradation of the labeled enzyme fell 85% in the heart cells and 70% in the fibroblasts (Table II). Even if the cells had been exposed to heparin for 3 min only and the compound was washed away prior to addition of the labeled enzyme, the uptake and degradation was only 25% of that seen in the absence of heparin. Addition of chylomicrons to the medium did not change the effect of heparin. The effect of heparin on the uptake and degradation of ‘251labeled lipoprotein lipase was concentrationdependent and could be demonstrated even at 0.05 units/ml.

.\ ,,* L* Lo c FIBROBLASTS

lncubatmn

6

Lm 12

AL

2L

(hours)

Fig. 3. Time course of interaction of ‘251-labeled milk lipoprotein lipase with cultured preadipocytes and fibroblasts. Conditions: as in Table I. Values are means of duplicate dishes. A, Binding: 0, uptake, 0, degradation.

9.4 12.9 26.4 34.9

II

OF CHYLOMICRONS

AND

HEPARIN

ON THE METABOLISM

OF “‘I-LABELED MILK

LIPOPROTEIN

LIPASE

IN CULTURED

CELLS

5 5 5 5 5 10 10

0 50 0 50 0 0 50

(ag/ml)

lipase Chylo micron triacylglycerol

Lipoprotein

(ag/ml)

medium

Incubation

0 0 5 5 5* 0 0

Heparin (units/ml)

1066 7 250

4139 4621 2263 2150 _

Binding

Rat heart

Metabolism cells

Ill3 1902 641 684 1014 2968 2670

Uptake

of ‘251-labeled

1623 8 149 782 192 I 203 13940 13109

Degradation

lipoprotein

8480 8953 3 144 3252 3844 13577 14955

Binding

Human

lipasc

2892 3 334 549 510 785 4237 5 498

Uptake

skin fibroblast

3231 3 208 1356 1461 I893 5 364 4921

Degradation

19.4 20.2 3.0 3.0 5.0 19.9 19.8

Rat heart cells

13.1 13.9 4.0 4.2 6.2 12.5 13.0

Human skin fibroblasts

Uptake + degradation (% of medium label)

Conditions: All experimental conditions as in Fig. 2. In some dishes, heparin was present in the culture medium throughout the entire period of incubation; in others (*) the cells were exposed to heparin for 3 min. washed extensively and ‘251-labeled lipoprotein lipase and chvlomicrons (where appropriate) were added to medium devoid of heparin. Values are means of triplicate dishes: the cell protein (mg/dish) was 0.54 for rat heart cells and 0.52 for human skin fibroblasts. Unless stated otherwise. values are cpm/dish per 6 h.

EFFECT

TABLE

120

The data presented so far indicate that when i251-labeled lipoprotein lipase is added to cultured cells it undergoes extensive proteolytic degradation. Therefore, in the next experiments, we tested the possibility that the degradation is mediated by lysosomal enzymes. Heart cell cultures and human skin fibroblasts were preincubated for 2 h in the presence of chloroquine or NH,Cl, the best evaluated lysosomotrophic agents, and the ‘251labeled lipoprotein lipase was added subsequently, for 6 h. As can be seen in Table III, treatment of either the heart cell cultures or human skin fibroblasts with 10 mM NH,Cl or 100 pM chloroquine did not reduce the sum of uptake and degradation of ‘251-labeled lipoprotein lipase. Both

TABLE

lysosomotrophic agents inhibited completely the degradation of the enzyme in human skin fibroblast. In the rat heart cell cultures 90% inhibition of degradation occurred in the presence of chloroquine and 75% in the presence of 10 mM NH,Cl. Two additional proteinase inhibitors, TLCK and TPCK, caused also a concentration-related inhibition of ‘Z51-labeled lipoprotein lipase degradation. In order to test whether the uptake of lipoprotein lipase might occur via the mannose &phosphate receptor, uptake and degradation of the labeled enzyme were determined in the presence of 5 and 10 mM mannose &phosphate. As shown in Table III, the phosphomannoside did not affect the uptake and degradation of the labeled enzyme.

III

EFFECT OF PROTEINASE INHIBITORS TEIN LIPASE IN CULTURED CELLS

ON THE

UPTAKE

AND

DEGRADATION

OF “‘I-LABEL.ED

MILK

LIPOPRO-

Conditions: The cells were cultured as described in Materials and Methods and legend to Fig. 2. On the day of the experiment, the cuiture medium was removed, the cells were washed with phosphate-buffered saline and 1 ml of F,,, medium containing 4% bovine serum albumin was added. Chloroquine and NH,CI were added to this medium and the cells were incubated for 2 h. Thereafter. rat mesenteric duct chylomicrons, 50 pg triacylglycerol/ml and 5,pg ‘ZSI-labeled milk lipoprotein lipase were added and incubations were carried out for 6 h. TLCK, TPCK and mannose 6-phosphate were added together with the chylomicrons. At the end of incubation the medium was collected and used for the determination of trichloroacetic acid-soluble, chloroform-nonextra~table ‘“fI-labeled protein degradation products. The cells were washed three times with 0.2% albumin in phosphate-buffered saline and three times with phosphate-buffered saline and released with trypsin. After addition of serum-containing medium. the cells were centrifuged and the trypsin supernatant was used for determination of surface binding. Cellular uptake of ‘2s1-labeled protein was determined on the cell pellet after two washes with phosphate-buffered saline and precipitation dishes. Values for metabolism are cpm/dish per 6 h. Cell type

Heart

cells

Additions

Metabolism

with trichloroacetic

of ‘251-labeled

acid. Values

lipoprotein

lipase

are means

Binding

Uptake

Degradation

Uptake + degradation (% of medium

167X

None NH&l (IO mM) Chloroquine ( 100 p M)

2515 2521

2077 3 724 3 885

2126 556 234

17.3 19.7 21.0

Fibroblasts

None NH,CI (10 mM) Chloroquine (100 pM)

4360 4587 4 284

2936 3751 3x17

934 0 0

I x.3 19.1 18.4

Heart

None NH,CI (IO mM) TLCK (28 PM) TLCK (280 pM) TPCK (140 PM) Mannose &phosphate Mannose 6-phosphate

10x1 1366 1581 2011 2460 I293 1371

9X2 3547 2259 4 so2 2094 I OX6 1026

3712 103X 2313 0 939 4107 4290

21.2 21.1 2 I .2 21.7 14.0 21.2 22.9

cells

(5 mM) (IO mM)

of triplicate

label)

121

Discussion

The aim of the present study was to determine the fate of bovine milk ‘*‘I-labeled lipoprotein lipase added to cultured cells, since in previous studies the addition of this enzyme resulted in enhancement of the uptake of chylomicron cholesteryl ester [3,4]. The results obtained presently provide additional evidence that the radioiodinated enzyme, which was shown to retain its hydrolytic activity towards synthetic substrate 1121, hydrolyzed chylomicron triacylglycerol at rates similar to the nonlabeled preparation. The labeled lipoprotein lipase interacted with the cultured cells and the interaction can be subdivided into three phases. Firstly, the enzyme is bound to the surface of all cells so far tested. In our previous study [4] such binding was suggested by the enhancement of cholesteryl ester uptake into heart cell cultures pulsed for 60 min with milk lipoprotein lipase and postincubated with chylomicrons labeled with cholesteryl linoleyl ether, a nondegradable analog of cholesteryl ester. Binding of milk lipoprotein lipase to endothelial cells and retention of hydrolytic activity was demonstrated also by WangIverson et al. [ 18). The binding of the radioiodinated enzyme to the cultured rat heart cells was not saturable up to 10 pg enzyme protein/ml, and it was also proportional to the enzyme concentration between 5 and 10 pg protein/ml when added to cultured fibroblasts. The fact that when using such a wide range of enzyme concentration no saturation of binding was found would suggest that no specific receptors were involved in this process. The concentrations used can be compared to those of other ligands, such as epidermal growth factor [19], or low density lipoprotein [20], the uptake of which is mediated by specific receptors. When compared on a molar basis, the concentraton of 10 pg protein/ml of lipoprotein lipase would be above saturating levels in the examples mentioned (19,201. The binding of ‘251-labeled lipoprotein lipase to the cultured cells was reduced to about 50% of control value by addition of 5 units of heparin and the interference in binding persisted when the cells were in contact with heparin for only 3 min. Since the decrease in surface binding was accompanied by a reduction in cellular uptake and degradation of ‘*‘I-labeled

lipoprotein lipase, it seems that surface binding is a necessary step for subsequent cellular uptake. The amount of surface-bound enzyme which will undergo interiorization was found to vary with the cell type used. Since the ratio of degradation to binding was much higher in preadipocytes and heart cell cultures than in human skin fibroblasts or endothelial cells, it seems that the cells which produce lipoprotein lipase possess a more efficient mechanism for enzyme degradation than those which do not synthesize the enzyme. Studies are in progress to determine whether the difference in interaction between milk lipoprotein lipase and fibroblasts or preadipocytes is indeed specific for lipoprotein lipase or is due to a more general difference in endocytosis. In the present study we have equated intracellular uptake with the sum of the radioactive enzyme taken up and degraded. The assumption that degradation of ‘251-labeled lipoprotein lipase represented an intracellular event was validated by the lack of degradation in conditioned media and by its inhibition by chloroquine and NH,Cl, which are well-known inhibitors of intralysosomal degradation [21-231. In recent years, these amines were shown to exhibit yet another activity, i.e., to prevent dissociation between lysosomal enzymes bearing the phosphomannosyl recognition marker and a specific receptor [24]. Such interference resulted in a depletion of unoccupied receptors, the function of which is to direct the transport of lysosomal enzymes to lysosomes, both from the site of their intracellular synthesis and from the cell surface 1241.The process of uptake of ‘251-labeled lipoprotein lipase studied presently was quite different from that of lysosomal enzymes added to cultured fibroblasts [24,25]. While the uptake of fi-glucuronidase was inhibited by lysosomotrophic amines, the uptake of ‘251-labeled lipoprotein lipase was not. The failure of mannose &phosphate to compete for the uptake of ‘*‘I-labeled lipoprotein lipase indicates further that lipoprotein lipase, which is a glycoprotein, is not recognized by the phosphomannosyl receptor. The finding that only a brief exposure to heparin was sufficient to reduce cellular uptake and degradation of ‘251-labeled lipoprotein lipase is reminiscent of our previous finding [S] that in heart cell cultures a brief exposure to heparin sufficed to

122

induce a prolonged leak of endogenously synthesized lipoprotein lipase into the culture medium. Both phenomena are due apparently to the rapid and poorly reversible binding of heparin to cellular surfaces as evidenced by the use of [ 35S]heparin (unpublished data). The use of ‘251-labeled milk lipoprotein lipase has provided evidence that in culture systems in which this enzyme was shown to enhance cellular uptake of chylomicron cholesteryl ester, there is rapid and extensive interiorization and degradation of the enzyme molecule. It still remains to be shown if lipoprotein lipase synthesized by rat heart or preadipocyte cell cultures is indeed subject to a similar retrieval and degradation system and whether the uptake of the lipoprotein cholesteryl ester is linked to the uptake of the enzyme molecule. Acknowledgements The excellent assistance of Mrs. M. Ben-Naim, Mrs. A. Mendeles, Mrs. Y. Dabach, Mrs. E. Bardach and Mr. G. Hollander is acknowledged gratefully. References I Stein, Y., and Stein, 0. (1980) in Metabolism

of Plasma Lipoproteins, in Atherosclerosis V, Proceedings 5th Internal. Symp. on Atherosclerosis (Gotto, A.M. Jr., Smith, L.C. and Allen, B., pp. 653-665, eds. Springer-Verlag. New York 2 Stein, 0.. Halperin, G. and Stein, Y. (1980) B&him. Biophys. Acta 620, 247-260 3 Chajek-Shaul, T., Friedman, G.. Stein, 0. and Stein, Y. (1981) Biochim. Biophys. Acta. 666, 147- I55 4 Friedman, G., Chajek-Shaul, T., Stein, O., Olivecrona, T. and Stein, Y. (1981) Biochim. Biophys. Acta. 666, 156-164

5 Chalek, T., Stein, 0. and Stein, Y. (1978) Biochim. Act; 528. 456-465

Biophya. . _

6 BjGrntorp, P., Karsson, M., Pertoft, H.. Pettersson, P.. SjGstrem, L. and Smith, L.L. (1978) J. Lipid Res. 19. 3 16-324 7 Friedman, Cr., Stein, 0. and Stein, Y. (1978) Biochim. Biophys. Acta 53 I, 222-232 8 Stein, O., Halperin, G.. and Stein, Y. (1979) Biochim. Biophys. Acta 573 I - I I 9 Coetzee, G.A., Stein, 0. and Stein, Y. (1979) Atheroaclerosis, 33, 425-43 I IO Bengtsson. G. and Olivecrona. T. (1977) Biochem. J. 167, 109-l 19 II David, G.S. and Reisfeld, R.A. (1974) Biochemistry 13, 1014-1021 I2 Bengtsson. G. and Olivecrona, T. (1980) Eur. J. Biochem. 106, 549-555 13 Nilsson-Ehle. P. and Schotz, M.C. (1976) J. Lipid Res. 17, 536-541 I4 Belfrage, P. and Vaughan, M. (1969) J. Lipid Rcs. IO, 341-344 15 Bierman, E.L., Stein, 0. and Stein, Y. ( 1974) Circ. Res. 35. 136-150 I6 Lowry, O.H., Rosebrough. N.J.. Farr, A.L. and Randall. R.J. (1951) J. Biol. Chem. 193, 265-275 I7 Bligh, E.G. and Dyer, W.J. ( 1959) Can J. Biochem. 37. 91 I-917 I8 Wang-Iverson, P., Jaffe, E.A. and Brown, W.V. (1980) in Atherosclerosis V, Proceedings. 5th Int. Symp. (Gotto, A.M. Jr, Smith, L.C. and Allen. B., eds.). pp. 375-378, SpringerVerlag, New York I9 Carpenter, G. and Cohen, S. ( 1976) J. Cell Biol. 7 I, l59- 17 1 20 Brown, M.S. and Goldstein, J.L. (1976) Science 191. l50154 21 Fedorko, M.E., Hirsch, J.G. and Cohn. Z.A. (196X)J. Ccl1 Biol. 38, 392-402 22 Wibo, M. and Poole, B. (1974) J. Cell Biol. 63, 430-440 23 Seglen, P.O. (1977) Exp. Cell Res. 107, 207-217 24 Gonzalez-Noriega, A., Grubb, J.H.. Talkad. V. and Sly. W.S. (1980) J. Cell Biol. 85, 839-852 25 Fischer, H.D., Gonzalez-Noriega. A. and Sly, W.S. (I 980) J. Biol. Chem. 255. 5069-5074