Proc. Natl. Acad. Sci. USAVol. 85, pp. 7438-7442, October 1988Biochemistry

Receptor-like function of heparin in the binding and uptake ofneutral lipids

(glycosaminoglycans/atherogenesis/cholesterol esterase/intestinal lipid absorption)

MATTHEW S. BOSNER, TOD GULICK, D. J. S. RILEY, CURTIS A. SPILBURG, AND LOUIS G. LANGE III*Department of Medicine, Cardiovascular Division, Jewish Hospital of Saint Louis at the Washington University Medical Center, Saint Louis, MO 63110

Communicated by Bert L. Vallee, June 6, 1988

ABSTRACT Molecular mechanisms regulating the bind-ing, amphipathic stabilization, and metabolism of the majorneutral lipids (e.g., cholesteryl esters, triglycerides, and fattyacids) are well studied, but the details of their movement froma binding compartment to a metabolic compartment deservefurther attention. Since all neutral lipids must cross hydro-philic segments of plasma membranes during such movement,we postulate that a critical receptor-like site exists on theplasma membrane to mediate a step between binding andmetabolism and that membrane-associated heparin is a keypart of this mediator. For example, intestinal brush bordermembranes containing heparin bind homogeneous humanpancreatic "MI-labeled cholesterol esterase (100 kDa) and 1251-labeled triglyceride lipase (52 kDa). This interaction is enzymeconcentration-dependent, specific, and saturable and is re-versed upon addition of soluble heparin. Scatchard analysisdemonstrates a single class of receptors with aKd of 100nM anda Bmaxof approximately 50-60 pmol per mg of vesicle protein.In contrast, enzymes associated with the hydrolysis of hydro-philic compounds such as amylase, phospholipase A2, anddeoxyribonuclease do not bind to intestinal membranes in thismanner. Human pancreatic cholesterol esterase also bindsspecifically and saturably to cultured intestinal epithelial cells(CaCo-2), and soluble heparin significantly diminishes thecellular uptake of the resultant hydrophobic reaction products(cholesterol and free fatty acids). We conclude that a physio-logical role for intestinal heparin is that of a mediator to bindneutral lipolytic enzymes at the brush border and thus promoteabsorption of the subsequent hydrolyzed nutrients in theintestine. This mechanism may be a generalizable pathway fortransport of neutral lipids into endothelial and other cells.

Neutral lipids such as cholesterol esters and triglyceridesmust move into cells from an aqueous milieu, yet thesecompounds are insoluble in water and essentially lack deter-gent properties. They enter various intracellular pools froma variety of stabilized structures. Neutral lipids can besurrounded by a hydrophilic shell composed ofphospholipidsor serum lipoproteins, they can be encompassed by deter-gents and phospholipids (intestinal micelles), or they can bebound to amphipathic proteins (transfer proteins). A centralbiological conundrum has been the molecular bases ofmovement of these lipids into cells from such organizedstructures across the hydrophilic segment of the plasmamembrane. For example, dietary lipid ingested as the fattyacid ester of cholesterol or of glycerol must be hydrolyzedbefore intestinal absorption can occur (1). Hydrolysis pro-duces very hydrophobic compounds in an aqueous environ-ment, and, currently, no cohesive hypothesis exists toexplain transport of these molecules through an unstirred

water layer and across the hydrophilic outer leaflet of theabsorptive membrane after egress from micellar structures.

Therefore, we postulate that a molecular mediator ofamphipathic nature must exist to facilitate juxtaposition ofthese hydrophobic lipids with the hydrophilic outer plasmamembrane under conditions where operation of receptor-mediated endocytosis may be limited. For example, this typeof transport may be required for the internalization of neutrallipids from the intestinal lumen where lipoproteins are notpresent. Homogeneous human pancreatic cholesterol ester-ase binds reversibly to membrane heparin in vitro (C.A.S.and L.G.L., unpublished data), and thus the high concen-tration of heparin in the intestine could serve to concentrateand localize neutral lipolytic enzymes on the intestinalmembrane.

This anchoring mechanism would facilitate the absorptionof neutral lipids from an unfavorable aqueous milieu byallowing the production of subcritical micelle concentrationsof hydrophobic reaction products at the enterocyte mem-brane. Because of the general nature of this problem, theubiquitous presence of heparin-like molecules in plasmamembranes and the known affinities of other key neutrallipolytic enzymes or binding proteins for heparin [e.g.,lipoprotein lipase (3) and low-density lipoprotein (LDL) (4)],we postulate that a major physiological role of heparin is tofunction as a mediator for facilitating movement of neutrallipids into cells of many types.

MATERIALS AND METHODSMembrane Preparation and Characterization. Brush border

membranes were prepared (5) from the proximal 10 cm ofsmall intestine from male New Zealand White rabbits andwere resuspended in 10 mM Tris (pH 7.4). The preparationcontained 1.0 + 0.2 mg of protein per ml (mean + SD) (6),32 ± 6.0 ,tg of total cholesterol per mg of protein (7), asucrase activity of 1.39 + 0.3 units/mg of protein (8), and 50+ 12 ,g of heparin per mg of protein (9). Vesicles (containingapproximately 50 ,ug of heparin) were incubated at 37°C with1.25 units of bacterial heparinase (Sigma), and the concen-tration of uronic acid in the supernatant and pellet wasquantified as a function of time. Experiments with TritonX-100 (0.1%) showed that approximately 50% of total vesic-ular heparin was unavailable to heparinase cleavage prior tovesicle disruption and thus was likely present on the internalsurface of the vesicle.

Vesicle Binding Studies. Homogeneous human pancreaticcholesterol esterase was 1251I-radiolabeled (10) (" I-choles-terol esterase) to a final specific radioactivity between 1000and 5000 dpm/pmol without affecting enzymatic activity (175,umol/hr per mg of protein). Labeled enzyme (10 nM to 1 ,uM)

Abbreviation: LDL, low density lipoprotein.*To whom reprint requests should be addressed at: CardiologyResearch, Yalem 305, Jewish Hospital of Saint Louis, 216 SouthKingshighway, Saint Louis, MO 63110.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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was added to 0.1 mg of the small intestine vesicle preparation(containing 5.0 ;&g of membrane heparin) in 10 mM Trischloride (pH 7.4) containing milk protein at 1 g/dl for a totalvolume of 0.5 ml per assay. After 15 min at 370C (sufficientfor equilibrium binding), the reaction was quenched on ice byadding 5 ml of cold buffer. The assay mixtures were Milli-pore-filtered (0.2 ,um) to quantitate the concentration of free(unbound) enzyme. Filters were washed with 15 ml of coldbuffer and assayed for 125I. Total binding was defined as dpmof 125I bound to filter paper, and nonspecific binding wasdefined as the amount of 125I-cholesterol esterase bound inthe presence of a 100-fold molar excess of unlabeled choles-terol esterase. Specific binding was defined as the differencebetween total and nonspecific binding. Porcine intestinalheparin was fractionated by using LDL-Sepharose affinitychromatography (11).

Binding of Other Pancreatic Enzymes. Membrane bindingstudies were also conducted with 125I-radiolabeled humanpancreatic triglyceride lipase, bovine pancreatic amylase,phospholipase A-2, and deoxyribonuclease.CaCo-2 Cell Cholesterol Esterase Binding Studies. Colonic

adenocarcinoma cells (CaCo-2; American Type Culture Col-lection) were grown in Dulbecco's modified Eagle's medium(DMEM) containing 10% (vol/vol) fetal bovine serum, 50 ;&gof gentamicin per ml, and 1.5 mM sodium pyruvate. Celldensity was 5.0 x 106 cells per ml, and heparin concentrationwas 19 I&g per 106 cells (9).

Binding studies were conducted by incubating 10' intesti-nal cells in phosphate-buffered saline containing 5mM CaCl2,1% milk protein (pH 7.4), and 1"I-cholesterol esterase (10nM to 2.5 ,M), with and without a 100-fold molar excess ofunlabeled cholesterol esterase. Mixtures were incubated at37°C for 90 min and then centrifuged at 4°C at 1000 x g for5 min. An aliquot of supernatant was retrieved, and the pelletwas washed thrice with phosphate-buffered saline at 4°C.Radioactivity in the supernatant (free enzyme) and pellet(bound enzyme) was determined and corrected for yield.Total, nonspecific, and specific binding were calculated as

stated previously and expressed as moles bound per 106 cells.CaCo-2 Cell Cholesterol Esterase-Mediated Cholesterol Up-

take. Confluent monolayers of cells (2 x 106) were incubatedwith [3H]cholesterol oleate (5 uM) embedded in phospha-tidylcholine vesicles in DMEM containing 10%o (vol/vol)lipoprotein-depleted serum in the presence and absence of 10nM 100-kDa homogeneous human cholesterol esterase, 2 mMsodium taurocholate, and soluble heparin (5.0 mg/ml). Atserial times medium was aspirated, and lipids were extracted(12) and separated by thin-layer chromatography on silicaplates with 97:52:3 (vol/vol) petroleum ether/diethyl ether/acetic acid to quantitate [3H]cholesterol and [3H]cholesterololeate. The cellular monolayer was washed exhaustively withDulbecco's phosphate-buffered saline and was solubilizedwith 0.1 M NaOH, and then total cellular [3H]cholesterolcontent was quantitated. Similar uptake studies of[4C]oleatewere conducted by utilizing ['4C]triolein (5 ,uM) in phospha-tidylcholine vesicles in the presence and absence of 25 nMpancreatic triglyceride lipase/400 nM pancreatic colipasewith and without soluble heparin at 5 mg/ml.

RESULTSTo demonstrate the biological function of heparin as a

mediator for neutral lipid absorption, a fixed concentration ofsmall intestine vesicles containing surface-associated heparinwas incubated with various concentrations of homogeneous100-kDa human pancreatic 125I-cholesterol esterase, and theextent of binding was quantitated. Total and specific bindingwere concentration-dependent, with saturation occurring atconcentrations of free cholesterol esterase in excess of 400nM (Fig. LA). Nonspecific binding increased linearly with

Eco

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0

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00 m

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3

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00 0.5 1.0

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B

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I0I I

50 100

Specifically bound1251-CholEase, pmol

FIG. 1. Vesicle binding studies. (A) Binding of 1251-cholesterolesterase ('251-CholEase) to intestinal vesicles at 370C. Purified humanpancreatic 25I-cholesterol esterase was added to 0.1 mg of the smallintestine vesicle preparation in 10 mM Tris chloride (pH 7.2), andbinding was quantitated as described. Total binding (0), nonspecificbinding (e), and specific binding (a) are defined in Materials andMethods. (B) Scatchard analysis of specific binding. Kd = 100 nM;Bmax= 78.5 pmol/mg of protein.

respect to concentration and represented 10%ooftotal bindingat Kd. Scatchard analysis (13) suggested a single class ofreceptors with a Kd of 100 ± 35 nM (mean + SD, n 8) andB__ax of 78.5 + 8 pmol/mg (mean + SD) of vesicle protein(Fig. 1B). If an average molecular mass of 12.5 kDa forheparin is assumed, 1.9o of vesicular heparin is bound byenzyme at BmaxCommercially available porcine intestinal heparin (i.e.,

bulk or unfractionated heparin) displaced specific binding ofhuman pancreatic 100-kDa I251-cholesterol esterase to mem-branes in a concentration-dependent manner, with maximaldisplacement at a 1000-fold molar excess over vesicle-associated heparin (Fig. 2). Bulk heparin was separated byutilizing an LDL-conjugated affinity resin (11) into fractionsthat did not bind to LDL and those that bound and wereeluted with 0.5 M NaCl. A 10,000-fold molar excess ofnon-LDL-binding heparin in the assay mixture reducedspecific binding of 1251-cholesterol esterase to vesicles byonly 29%, from 6.3 to 4.5 pmol/100 ,ug of protein (Fig. 2). Incontrast, a 10-fold molar excess of LDL-binding heparinreduced specific binding by 85% (Fig. 2), from 6.3 to 0.9

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10 100 1000 10,000Heparin, fold molar excess

FIG. 2. Heparin displacement of bound cholesterol esterase. AnLDL-conjugated affinity column was prepared by the procedure ofCardin et al. (11). Binding experiments were performed with brushborder membranes, 1251-cholesterol esterase, and a heparin fractioneluted from the resin with 0.5 M NaCl (A; heparin bound to LDL),a heparin fraction that did not bind to the resin (e; heparin not boundto LDL), and unfractionated heparin (o). Total binding, specificbinding, and nonspecific binding were calculated as described inFig. 1.

Biochemistry: Bosner et al.

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Proc. Natl. Acad. Sci. USA 85 (1988)

pmol/100 ixg of protein. Thus, a subfraction of intestinalheparin that binds to LDL has an IC50 for cholesterol esterasedisplacement that is lower than that of bulk heparin by afactor of 100.To further assess the specificity of this binding, we per-

formed experiments in the presence of other potential dis-placing agents. Chondroitin sulfate (5 mng/ml), sodium chlo-ride (up to 0.2 M), and sodium taurocholate (5 mM) were allineffective, decreasing specific binding of human pancreatic1251-cholesterol esterase from 6.3 pmol to 5.4, 5.9, and 6.0pmol/100 ,ug of protein, respectively. We also examined thebinding of 125'-cholesterol esterase to small intestine vesiclestreated' with purified bacterial heparinase, an enzyme thatreleased approximately 50% of total vesicular heparin intothe supernatant (i.e., that heparin most likely associated withthe external surface of the membrane vesicle). 'Specificbinding ofhuman pancreatic 1251-cholesterol esterase to thesevesicles depleted of surface heparin was reduced by 94%o,from 6.3 to'0.4 pmol/100 ,g of protein. Collectively, theseresults indicate that cholesterol esterase' binds to smallintestine membrane vesicles by an interaction that is specificfor a subfraction of intestinal heparin.To evaluate the generality of brush border heparin as a

specific adaptor molecule for neutral pancreatic lipolyticenzymes, we assessed binding of amylase, deoxyribonu-clease, phospholipase A2, and triglyceride lipase to smallintestine vesicles. Of these pancreatic hydrolytic enzymes,only 'human pancreatic 125I-labeled triglyceride lipase (52kDa) bound specifically and saturably to small intestinemembranes in a concentration-dependent fashion (Fig.' 3).Binding was reversible and heparin-specific. Scatchard anal-ysis revealed that Kd was 86 nM and Bm. was 50 pmol/mgof vesicle protein, values almost identical to those found forcholesterol esterase. In contrast, no saturable binding wasnoted either for 125I-labeled amylase or 125I-labeled deoxy-ribonuclease, enzymes that act on hydrophilic substrates toproduce hydrophilic products, or for 1251-labeled phospholi-pase A2, an enzyme that acts on an amphipathic substrate toproduce a polar and neutral lipid. Though other mechanismsof facilitating uptake of these more complex mixtures ofproducts may exist, mediation by heparin thus appears to beminimal.

Intact intestinal cells (CaCo-2) that possess small intestineepithelial microanatomy and physiology (14) also bind humanpancreatic 'cholesterol esterase in a saturable and specificmanner. Scatchard plots were linear and'indicated the pres-ence of a single binding' affinity with a Kd of 114 nM and aBmax of42 pmol per 106 cells at 370C (data not shown). Addedheparin also displaced enzyme.

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,3 2-ac

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.

0 0.2 0.6 1.0

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FIG. 3. Vesicle binding of pancreatic enzymes. Binding of 25I-radiolabeled human pancreatic cholesterol esterase (o) and triglycer-ide lipase (e), bovine pancreatic amylase (A), phospholipase A2 (°),and deoxyribonuclease (m) to vesicles was quantitated as described.

To demonstrate the biological significance of the receptor-like interaction of pancreatic neutral lipolytic enzymes withintestinal cells, CaCo-2 cells (2 x 106) were incubated with[3H]cholesterol oleate (5 AM) embedded in phosphatidyl-choline vesicles in the presence or absence of 2 mM sodiumtaurocholate and 10 nM homogeneous human pancreatic100-kDa cholesterol esterase with and without added solubleheparin (5 mg/ml). In the absence of cholesterol esterase,uptake occurred at a rate of 1.3 pmol/hr per 106 cells,probably as a result of vesicle fusion with cell membranes(Fig: 4). An identical rate was seen in the presence ofcholesterol esterase without taurocholate (i.e., under condi-tions where the enzyme is not active). In the presence ofenzyme and taurocholate, uptake occurred at a rate of 8.0pmol/hr per 106 cells. Importantly, [3H]cholesterol uptake bythe cells in the presence of enzyme, taurocholate, and bulkheparin (5 mg/ml) was decreased to 1.6 pmol/hr per 106 cells.Thus, heparin decreased the cholesterol esterase-dependentcholesterol uptake from cholesterol oleate by >95%. Underthese conditions, the quantity of [3H]cholesterol in themedium at each time-point was unaffected by heparin (Table1), supporting the importance of enzyme proximity to themembrane for absorption ofthe hydrolytic reaction products.Similarly, the uptake of [14C]oleate from [14C]triolein vesiclesin the presence of 25 nM triglyceride lipase was dependentupon the presence of colipase and was'markedly diminishedwith the addition of heparin to the medium (data not shown).Thus, both cholesterol esterase and triglyceride lipase bind tointestinal cell membranes at a membrane site containingheparin, and exogenous heparin markedly suppresses theuptake of their respective hydrolytic products,'cholesteroland fatty acid.

DISCUSSIONExperiments reported here show that human pancreaticcholesterol esterase and triglyceride lipase bind to intestinalmembranes and/or cells in a receptor-like manner and thatheparin or a heparin-like substance functions as the centralcomponent of this binding site. This interaction is saturable,concentration-dependent, and specific for a subfraction of

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Incubation time, hr

FIG. 4. Heparin inhibition of cellular uptake of [3H]cholesterol.Confluent monolayers of CaCo-2 cells (approximately 2 x 106) wereincubated with 5 1AM [3H]cholesterol oleate embedded in phospha-tidylcholine vesicles in medium containing 10% lipoprotein-containing serum, 2 mM sodium taurocholate, and 10 nM cholesterolesterase with (A) or without (o) 5 mg of heparin per' ml. Cell viabilitywas unaffected by any of these agents. At serial times the mediumwas aspirated, and the cellular layer was washed exhaustively withcold buffer. The cellular fraction was then solubilized with 0.1 MNaOH, and the dpm of [3H]cholesterol was quantified as pmol per 106cells. Controls were incubations in parallel without cholesterolesterase ( x).

7440 Biochemistry: Bosner et al.

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Table 1. Appearance of free cholesterol in culture mediaCholesterol, nM

Time, hr No heparin Heparin

o 0 00.5 160 1201 192 1802 220 2204 252 2448 260 256

18 318 326

Confluent monolayers of CaCo-2 cells (2 x 106) and 5 AM[3H]cholesterol oleate embedded in phosphatidyicholine vesicleswere incubated together in T-25 flasks in medium containing 10olipoprotein-depleted serum, 10 nM 100-kDa homogeneous humanpancreatic cholesterol esterase, and 2 mM sodium taurocholate withand without intestinal heparin (5 mg/ml). At serial times, an aliquotof medium was aspirated from the flasks, and [3H]cholesterol and[3H]cholesterol oleate were determined as described.

intestinal heparin. Once binding has occurred, secondarybiological events occur that promote lipid absorption, andthis uptake can be modulated by manipulation of the heparinconcentration in the medium. This system describes a mo-lecular mechanism by which hydrophobic compounds arelocalized to the intestinal membrane to facilitate absorptionof these nutrients in an otherwise thermodynamically unfa-vorable aqueous environment.These findings also directly relate to current views on the

mechanism of intestinal neutral lipid absorption in particularand lipid binding and transmembrane transport in general.For decades it has been recognized that the intestinal mucosacontains an abundance of heparin, but its physiologicalsignificance has remained obscure. As shown here, the abilityof intestinal vesicles to bind neutral lipolytic enzymes via areceptor-like complex mediated through membrane-boundheparin shows that this glycosaminoglycan plays a centralrole in neutral lipid absorption. Moreover, this implies that adistinct physiological role for heparin is to localize neutrallipolytic enzymes where their hydrophobic cleavage prod-ucts can be readily absorbed.

O Pancreas

Proc. Nati. Acad. Sci. USA 85 (1988) 7441

The physiological advantage of this system can be readilyappreciated. If cholesterol ester hydrolysis were to occurwithin the intestinal lumen, sterol and fatty acid would haveto diffuse through the unstirred aqueous layer to the apicalmembrane of the mucosal cell before absorption could occur.Diffusion of these reaction products would be hindered by alarge effective diffusion constant. Thus, cholesterol absorp-tion would be dependent on random movement through theaqueous intestinal lumen and unstirred water layer.An alternate explanation exists for the observed efficient

absorption of neutral esterified lipids (Fig. 5). In this hypoth-esis, neutral lipolytic enzymes are secreted by the pancreasinto the intestine, where they are anchored to the intestinalmembrane through a receptor-like interaction with heparin.When these immobilized enzymes hydrolyze dietary esters,free cholesterol, fatty acids, and/or 3-monoglycerides areformed at the absorption site, obviating the need either forpassive transport of hydrophobic reaction products throughan aqueous milieu or for further packaging of these productsin micelles. The hydrolytic capacity of the enzyme is thuscoupled to absorption so that hydrophobic products do notexceed their critical micelle concentration. This model pro-vides an efficient, generalizable, ATP-independent pathwayfor juxtaposing a hydrophobic compound and a hydrophilicpart of the cell membrane that lacks an intrinsic cholesterolor lipoprotein binding protein, and it provides a mechanismfor promoting lipid absorption.These results provide a wider context for understanding

previously recognized binding of enzymes to heparin bysuggesting a role for it in neutral lipid metabolism. It has beenpostulated that heparin-like molecules are responsible forlipoprotein lipase binding to the endothelial cell surface, sinceheparin releases this enzyme into plasma (15). The modelproposed here suggests that membrane-bound heparin an-chors lipoprotein lipase at the endothelial-blood interface toallow cleavage of triglycerides and subsequent transport ofhydrophobic fatty acid reaction products to occur in anorderly, efficient, and ATP-independent manner. In yetanother system, it has long been recognized that glycosami-noglycans bind lipoproteins in the presence of divalent

M=MicelleH=HeparInC-FA=Cholesterol-Fatty AcidG-(FA)3=TrigiycerideG-FA=P-Monoglyceride

(.7)(FA)3

2FA+G-FAFA+Cl

Binding Hydrolysis AbsorptonFA+'C2FAG-FA

FiG. 5. Proposed mechanism for intestinal absorption of neutral lipids. Neutral lipolytic enzymes (Ease) are secreted into the intestinal lumenfrom the pancreas. They bind tightly to brush border cell surface receptors containing heparin (H). Mixed micelles (M) transport neutral esterifiedlipids, cholesterol esters (C-FA), or triglycerides [G-(FA)3] from the intestinal lumen to the active site of the respective immobilized hydrolase,and the esters are cleaved to produce submicellar concentrations of free cholesterol (C), free fatty acids (FA), and f3-monoglycerides (G-FA),which can then be efficiently transported across the enterocyte membrane.

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7442 Biochemistry: Bosner et al.

cations, and, indeed, the majority of LDL bound to theextracellular matrix can be accounted for by its binding toheparin-like molecules isolated from aortic extracts (2, 16).The ubiquitous existence ofheparin on the plasma membraneof many cell types (Table 2) emphasizes the potential impor-tance of this mechanism in other organs where neutral lipidmetabolism is important. Our results suggest that membraneheparin interacts with a wide variety of neutral lipid-rec-ognizing proteins and thus may be important in regulatingdiverse metabolic fates of these lipids.

In summary, we have demonstrated that (i) pancreaticneutral lipolytic enzymes (cholesterol esterase and triglycer-ide lipase) bind to small intestine membranes in a receptor-like manner to mediate the efficient cleavage and transport ofneutral lipids into the cell, (ii) heparin or a heparin-likesubstance is a key component of the mediator, and (iii)heparin modulates the uptake of the hydrophobic reaction

Table 2. Heparin concentration of membrane preparations fromvarious organs

OrganJejunumLiverTestisAdrenalIleumLungDuodenumKidneyFatHeartAortaMuscleSpinal cord

Heparin, ,ug of uronic acidper mg of membrane protein

56.345.944.940.039.027.023.020.820.717.613.213.011.3

Proc. Natl. Acad. Sci. USA 85 (1988)

products of neutral lipolytic enzymes in isolated intestinalcells in culture. Therefore, we propose a general model forthe binding and transport of neutral lipids in an ATP-independent and thermodynamically efficient manner inwhich membrane heparin plays a mediative role in themovement of neutral lipids destined for intracellular metjab-olism.

This manuscript is dedicated to the memory of Louis G. Lange II.

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3. Jackson, R., McLean, L., Ponce, E., Rechtin, A. & Demel, R.(1987) Adv. Exp. Med. Biol. 210, 73-77.

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5. Hauser, H., Howell, K., Dawson, R. & Bowyer, D. (1981)Biochim. Biophys. Acta 602, 567-577.

6. Lowry, 0. H., Rosebrough, N., Farr, L. & Randall, R. (1951)J. Biol. Chem. 193, 265-275.

7. Zlatkis, A., Zak, B. & Boyle, A. J. (1963) J. Lab. Clin. Med.41, 486-490.

8. Dahlquist, A. (1964) Anal. Biochem. 7, 18-25.9. Bitter, T. & Muir, M. (1962) Anal. Biochem. 4, 330-334.

10. Fraker, P. J. & Speck, J. C. (1978) Biochem. Biophys. Res.Commun. 80, 849-857.

11. Cardin, A., Barnhart, R. L., Witt, K. & Jackson, R. (1984)Thromb. Res. 34, 541-550.

12. Bligh, E. G. & Dyer, W. J. (1959) Can. J. Biochem. Biophys.37, 911-917.

13. Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 51, 660-672.14. Pinto, M., Robine-Leon, S., Appay, M.-D., Kedinger, M.,

Triadou, N., Dussaulx, E., Lacroix, B., Simon-Assmann, P.,Haffen, K., Fogh, J. & Zweibaum, A. (1983) Biol. Cell 47, 323-330.

15. Olivecrona, T., Bengtsson, G., Marklund, S., Lindahl, U. &Hock, M. (1977) Fed. Proc. Fed. Am. Soc. Exp. Biol. 36, 60-65.

16. Steele, R., Wagner, W., Rowe, H. & Edwards, I. (1987)Atherosclerosis 65, 51-62.

Organs were removed from a rabbit fed a regular diet, and heparin(uronic acid) (9) aid membrane protein (6) concentrations weredetermined in a 100,000 x g membrane preparation.

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