THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 254, No. 21, Issue of November 10, pp. 10830-10838, 1979 Prrnted in U.S.A.

Adhesion of Hepatocytes to Immobilized Sugars A THRESHOLD PHENOMENON*

(Received for publication, April 26, 1979)

Paul H. Weigel,$ Ronald L. Schnaar,g Mark S. Kuhlenschmidt,l Eli Schmell,(J Reiko T. Lee, Yuan C. Lee,** and Saul Roseman

From the McCollum-Pratt Institute and the Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218

We have previously reported that hepatocytes ad- here specifically to sugars covalently linked to flat polyacrylamide gels. Rat hepatocytes bind to the galac- tosyl gel whereas chicken hepatocytes bind to the cor- responding N-acetylglucosaminyl gel. In the present studies gels were varied with respect to the concentra- tion of sugar and the nature of the ligands; five galac- toside aglycon groups and three N-acetylglucosaminide aglycon groups were used to link the sugars to the gels. In addition, two different chemical procedures were used to synthesize these gels.

When the glycoside concentration in the gels was varied, a threshold response was observed for the bind- ing of cells to the gels. With either cell type or any of the ligands tested, cell binding was detected only at or above a particular concentration of ligand (designated the critical concentration). When the concentration was increased above this critical concentration by 10 to 20%, maximal binding occurred. This threshold bind- ing response did not depend on the way in which the gels were synthesized or the procedure used to remove the nonbound cells from the gels. However, the exact value of the critical concentration depends on the type of gel used and the aglycon group of the ligand. The critical concentrations for binding ranged from 50 to 200 nmol of N-acetylglucosaminide/gel (cm” X 0.25 mm) with chicken hepatocytes, and 875 to 2500 nmol of galactoside/gel (cm” x 0.25 mm) with rat hepatocytes. Critical concentrations for binding were also deter- mined indirectly by examining the ability of free sugars in solution to prevent cell-gel binding, and these values were within 10% of those obtained directly.

The results with this model system suggest that, if the cell-gel binding in vitro mimics phenomena in duo, then slight changes in the quantities of complex car- bohydrates on the cell surface or in the extracellular

* This work was supported by National Institutes of Health Grants CA 15161, CA 21901, AM 09851, and Contract NOl-CB43985. Contri- bution No. 1030 from the McCollum-Pratt Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 USC. Section 1734 solely to indicate this fact.

f Postdoctoral Fellow of the National Cancer Institute, 5F22 CA 01501-03. Present address, Division of Biochemistry, Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch, Galveston, Tex. 77550.

6 Present address. National Heart, Lung and Blood Institute, Na- tio&l Institutes of Health, Bethesda; Md.20205.

1 Olive V. Levin Postdoctoral Fellow of the Leukemia Society of America.

I( Postdoctoral Fellow of the Leukemia Society of America. ; * Supported by National Institutes of Health Research Grant AM

9970 and National Institutes of Health Research Career Development Award AM 70,148.

matrix may have profound effects on the behavior of neighboring cells.

The mechanisms by which cells recognize and interact with one another have been widely studied but have not yet been elucidated. We have previously suggested that cell recognition and adhesion may be mediated via the carbohydrates present on cell surfaces (1). This hypothesis is supported by evidence from studies with prokaryotic (2, 3) and both lower (4-6) and higher (7-14) eukaryotic organisms.

As a method for probing the mechanism(s) underlying intercellular recognition and adhesion, we have devised tech- niques by which carbohydrates are covalently linked to solid

surfaces. Our fast studies used sugars covalently linked to Sephadex beads (15). More satisfactory and reproducible re- sults were obtained with carbohydrate ligands covalently im- mobilized to flat polyacrylamide gels (16-19). The advantages of the polyacrylamide gels were described in previous reports (16, 18). The use of different gels containing a variety of carbohydrates showed that hepatocytes bound in a sugar- specific manner to certain carbohydrates; chicken hepatocytes bound to gels derivatized with N-acetylglucosaminides (16, 18), whereas rat hepatocytes adhered to gels containing ga- lactosides (19).

The results presented in this report show that the binding

of hepatocytes to the sugar-derivatized gels is a threshold phenomenon. There is no binding until a critical concentration of glycoside in the gel is reached; increasing the concentration in the gel by 10 to 20% above this value permits maximal binding. These results suggest that studying interactions of intact cells with a chemically defined synthetic matrix may increase our understanding of cell surface phenomena and cell behavior. Preliminary accounts of this work have been pre- sented (17, 19).

EXPERIMENTAL PROCEDURES

Materials

The following compounds were purchased from commercial sources, and further purified, as indicated: acryloyl chloride, Polysci- ences; ZV,N’-methylenebisacrylamide (Gold Label), acrylamide (three times recrystallized from ethanol), and ethanolamine (vacuum-dis- tilled), Aldrich Chemical Co.; N-hydroxysuccinimide, Pierce Chemical Co.; 6-aminohexanol (chloroform-extracted, Norit-decolorized, and recrystallized from ethanol), N-acetylglucosamine, N-acetylgalacto- samine, N-acetylmannosamine, bovine serum albumin (Fraction V), and Hepes,’ Sigma Chemical Co.; collagenase CLS III and CLS IV,

’ The abbreviations used are: Hepes, 4-(2-hydroxyethyl)-l-pipera- zineethanesulfonic acid, bisacrylamide, N,N’-methylenebisacrylam- ide: TEMED. N.N.N’.N’-tetramethvlethylenediamine. Abbreviations for’the glycoiides’used are summ&zed in Table I. All mono- and disaccharides were n-pyranosides.

10830

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TABLE I

10831

Glycosides used in this study

The numbers in parentheses are references to the methods of synthesis and characterization of the compounds. The effective length of the spacer arm (the aglycon portion of the glycoside) is from the polyacrylamide gel amide nitrogen atom (indicated by the dashed line) to the sugar. The abbreviations are based on the structure or origin of the spacer arm: AH = 6-aminohexyl; BA-(l)- = N,N’-methylenebisacrylamide; BA-(6)- = N,N’-hexamethylenebisacrylamide; AHAT = aminohexamidotris.

Compound Abbreviation structure

6-Aminohexyl glycopyranoside (22) AH-O-sugar H2;-CH2CH2CH2CH2CH2CH2-O-sugar

GAminohexyl l-thio-1-deoxy-glycopyranoside (21) AH-S-sugar H2N-CH2CH2CH2CH2CH2CH2-S-sugar

N-Acrylamidomethyl 2-aminocarbonylethyl l-decay-1-thio-glycopyranoside (23)

0; HO BA-(1)~S-sugar CH2=CH-e-k-CH2-hT-kCH2CH2-S-sugar

1

N-(6-Acrylamidohexyl) 2-aminocarbonylethyl 1-deoxy-l-thio-glycopyranoside (23)

OH HO II I I I/

BA-(6)~S-sugar CH2=CH-C-y-(CH2)6-:l-C-CH2CH2-S-sugar

6-(Aminohexanamido)tris(glycopyranosyl- oxymethyl)methane (24)

I

I CH2-O-sugar

I I AHAT-(O-sugar)3

OY H2N-CH2CH2CH2CH2CH2-k-N-C-CH2-O-sugar

I I \ I CH2-O-sugar

Worthington Biological Corp.; dry powder tissue culture media (Cat- alogue No. 420-1400), Gibco, Inc.; N-succimidyl acrylate was synthe- sized as described previously (18,20). All other chemicals were reagent grade.

Glycosides-The following 0- and S-glycosides containing w-amino groups in the aglycon were synthesized as described: 6aminohexyl I- thio-l-deoxy-P-n-galactopyranoside and 6-aminohexyl 2-acetamido- 1,2-dideoxy-l-thio-/3-n-glucopyranoside (21); 6-aminohexyl /I-n-galac- topyranoside, and 6-aminohexyl 2-acetamido-2-deoxy-p-n-glucopyr- anoside (22). The synthesis of the BA-(1)~S-glycosides (see Table I) of a-mannose, P-glucose, P-galactose, and /3-N-acetylglucosamine, and the BA-(6)~S-glycoside of /3-galactose has been reported (23). Briefly, these glycosides were prepared by unimolar Michael addition of fully acetylated 1-thio sugars to N,N’-methylenebisacrylamide or N,N’- hexamethylenebisacrylamide, followed by de-0-acetylation. The de- acetylated products are w-acrylamido derivatives of thioglycosides (i.e. these compounds have acryloyl functional groups at the aglycon termini and can be co-polymerized into polyacrylamide gels). The “cluster” glycosides designated as AHAT-(O-sugar)3 in Table I were synthesized as reported (24). These ligands have a terminal free amino group in the aglycon and contain three glycosidically linked sugars and one spacer arm per molecule. 6-Acrylamidohexyl glyco- sides (which are also acrylamide derivatives) were synthesized and purified as described (18, 25).

Methods

Synthesis of Polyacrylamide Gels Containing Covalently Linked Glycosides-The methods used to synthesize the polyacrylamide gels, as well as the properties of these gels and previous studies with intact cells have been described (18). Modifications of these procedures and further details are given below.

Two methods are used for synthesizing derivatized polyacrylamide gels. In the N-hydroxysuccinimide substitution procedure, the acry- late ester of N-hydroxysuccinimide is co-polymerized with acrylamide and bisacrylamide. This activated gel is then treated with the desired ligand containing an amino group that displaces the N-hydroxysuc- cinimide in the gel. Gels made by the N-hydroxysuccinimide substi- tution procedure were previously designated type I gels (18). In the present studies, although the basic method of preparation is similar, we have modified the original procedure (described below) and the gels are designated type I*. In the co-polymerization procedure, an acrylamidyl derivative of the ligand (e.g. an acrylamidohexyl glyco- side) is co-polymerized with acrylamide and bisacrylamide (type II gels (18)). Unlike some of the earlier work (la), in which type I and

type II gels with different physical dimensions were prepared, only thin (0.25 mm) gels were cast for the present studies.

The following method was used to obtain a set of type I* gels containing different concentrations of glycoside: 3.0 g of acrylamide, 0.3 g of bisacrylamide, 0.27 g of NaHzPO+ and 15 ~1 of TEMED were dissolved in distilled water (final volume, 13.5 ml). N-Succinimidyl acrylate (320 mg, 1.89 mmol) was dissolved in 3.6 ml of this solution. Different proportions of the two solutions were then mixed such that the N-succinimidyl acrylate concentration was varied while the con- centrations of the other components were kept constant. Polymeri- zation was started by adding 0.2 ml of 10% (w/v) ammonium persul- fate solution to 1.8 ml of the mixture. The final concentrations in the polymerizing solution were 20% (w/v) acrylamide, 2% (w/v) bisacryl- amide, 0.15 M NaH2P04, 0.10% (v/v) TEMED, and 1.0% (w/v) am- monium persulfate. Immediately after the addition of ammonium persulfate, 1 to 2 ml of the well mixed polymerization solution were placed between two acid-cleaned glass plates (8.3 x 10.2 cm; Kodak projector slide cover glass, Catalogue No. 140 2130), which were separated by 0.25-mm plastic spacers and clamped together in a vertical position. After 1 h, the resulting gels were cut into squares (8 x 8 mm). Each set of gel pieces was washed three times at 4°C for 10 min in 40 ml of distilled water, placed into capped glass tubes (13 x 100 mm), and washed twice for 15 min at 4°C with anhydrous dimethyl sulfoxide (0.3 ml/gel) by shaking the tubes on a reciprocal shaker. Each set of dehydrated N-hydroxysuccinimide gels was then gently shaken at room temperature with a solution (75 pi/gel) of the appropriate ligand (30 mrvr) dissolved in dimethyl sulfoxide. The substitution of N-hydroxysuccinimide by ligand was followed kineti- cally by determining the amount of N-hydroxysuccinimide (20) re- leased from the gels into the supernatant solution as a function of reaction time. The reaction was allowed to proceed for 21 h (80 to 90% of the N-hydroxysuccinimide groups were released within 5 h) and then ethanolamine (0.5 pi/gel) was added to displace any un- reacted N-hydroxysuccinimide groups. After shaking for another 3 h, the gel pieces were washed thoroughly with water at room tempera- ture for 30 min in 15-ml polystyrene capped tubes filled with water and continuously rotated at 6 rpm on a rotating drum. The axis of the tube was perpendicular to the axis of the drum. Gel pieces were handled carefully with a thin-bladed spatula. Whatman No. 50 filter paper was used for blotting the gels when necessary. The gels were stored in 10% isopropyl alcohol at 4°C. Before use, the gels were washed in the 15-ml tubes as described above, twice with 0.15 M NaCl and twice with the medium used for the experiment.

Type II gels containing different concentrations of glycoside were

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10832 Adhesion of Hepatocytes to Immobilized Sugars

ACRYLOYL GLYCOSIDE SOL”TlON (m,,

FIG. 1. Effect of acryloyl glycoside concentration on incor- poration of glycoside into type II polyacrylamide gels. The free amino group of the 6-aminohexyl glycoside (300 pmol/ml) was acryloylated and the product, the 6-acrylamidohexyl glycoside, was mixed with acrylamide and bisacrylamide and purified by treatment with ion exchange resins as described previously (18, 25). Polyacryl- amide slab gels were prepared from solutions containing various amounts of the column eluate (abscissa) and constant concentrations of acrylamide and bisacrylamide as described in the text. After polymerization, the large gels were cut into smaller squares (0.64 cm”), washed thoroughly, and analyzed for sugar content. Open symbols, AH-O-Gal-gels; closed symbols, AH-0-GlcNAc-gels. A set of gels of different ligand concentration prepared on a given day is illustrated by a specific symbol.

synthesized in a similar manner by varying the concentration of the acrylamido glycoside, while the concentrations of the other compo- nents were kept constant. The final concentrations in the polymeriz- ing solutions were the following; 20% (w/v) acrylamide, 1% (w/v) bisacrylamide, 50 mM sodium phosphate, pH 7.3, 0.17% (v/v) TEMED, and 0.2% (w/v) ammonium persulfate. Gels were polymer- ized, cut into squares, washed thoroughly as described above, and stored at 4’C in 30% ethanol and 0.15 M NaCl.

Fig. 1 shows the linear correlation between the amount of N- acryloyl-6-aminohexyl galactoside or the corresponding N-acetylglu- cosaminide in the polymerizing solution and the amount of glycoside covalently incorporated into the type II gel. This correlation was also obtained with co-polymerized gels containing the BA-(I)- or BA- (6).S-glycosides. The proportionality was observed at all glycoside concentrations tested (up to 3000 nmol/unit gel piece’). The concen- tration of active ester in type I* gels made by co-polymerizing N- succinimidyl acrylate can also be varied linearly (as in Fig. 1) by changing the amount of N-succinimidyl acrylate in the polymerizing solution. Quantitative substitution of the N-hydroxysuccinimide in these gels by the amino-containing glycoside yields a series of gels with final glycoside concentrations directly proportional to the amount of active ester initially present. Thus, it is consistently pos- sible to synthesize gels with predictable sugar concentrations.

Glycoside Content of Polyacrylamide Gels-The 0-glycoside con- tent of derivatized gels was determined as described (18, 25). Gel pieces containing S-glycosides were first hydrolyzed with mercuric ions by incubation with 0.5 ml of 20 mM Hg(OAc)s in 50 mM acetic acid at 55°C for 1 h (26). Galactose levels were assayed directly by the phenolsulfuric acid procedure (27), but N-acetylglucosamine sam- ples were further treated with an equal volume of 8 N HCl for 5 to 6 h at 105°C and the free glucosamine was then determined with an automated sugar analyzer (28) or the ninhydrin procedure (29).

Structure of the Two Gel Types-Type I* and type II gels contain glycosides covalently linked to the polymer backbone of the gel by an amide bond at the terminal amino group of the aglycon. However, these gels differ substantially in the amount of anionic charge they contain (18). Type II gels contained virtually no cationic or anionic charge (lees than 1 nmol/unit gel piece’ (18, 25)), whereas type I* gels made by the N-hydroxysuccinimide substitution procedure contain significant amounts of anionic charge because of hydrolysis of the N-

’ All concentrations for ligands immobilized in gels are expressed as nanomoles per gel piece (1.0 cm X 1.0 cm X 0.25 mm) (a unit gel piece).

hydroxysuccinimide ester (18). In the latter gels, the molar ratio of anionic charge to ligand concentration varied with the type and concentration of ligand used. In the extreme case, when the highest concentration of AHAT-(O-sugar):> as the ligand was used, the charge and ligand content were equivalent.

Qualitatively identical results were obtained with the two gel types, but quantitative differences were observed and are presented below. These differences may relate to the surface structure of the gels. At this time, only the total glycoside composition of each gel piece is known; important parameters, such as the concentration, distribution, and orientation of the sugar ligands at the surfaces of each gel type are not known. There is no reason to suppose that these parameters are identical in the two different gel types even if the total quantity of ligand in each gel piece is the same.

Cell Preparation-Chicken hepatocytes were prepared and char- acterized as described (18, 30). Modifications for the preparation of rat hepatocytes were as follows: Sprague-Dawley rats (150 to 250 g, CFE line) were purchased from Charles River Breeding Laboratories, Inc., Wilmington, Mass. The livers were perfused with 6000 units of Sigma type IV collagenase for 17 min. Cells were futered successively through IOO-, 35-, and 25.pm mesh Nitex nylon (Tetko, Inc.). Medium A (in which cells were normally kept), perfusion and lysis buffers, and lactate dehydrogenase assay buffer have been described (18, 30).

Measurement of Cell Adhesion to Glycoside Gels-Cell adhesion to a derivatized polyacrylamide gel is operationally defined by the conditions used for washing the gels. For these studies, two sets of conditions were used. Although we have no quantitative measure of the shear forces exerted on the cells bound to the gels in the two methods, Procedure A was designed to minimize the shear forces while Procedure B is a vigorous method for washing the gels. Rou- tinely, Procedure A was used to wash many gels in a Petri dish simultaneously, i.e. different gels in contact with cells for one incu- bation time, whereas Procedure B was used for washing individual samples incubated for different periods of time. Modifications of these previously described procedures (18, 19) are given below. The same volume of cell suspension, 60 ~1, was used on all gel squares (8 x 8 x 0.25 mm).

Procedure A-The dish containing gel pieces was placed on a level gyratory platform shaker (model G2, New Brunswick Scientific Co.). A Cole-Parmer variable speed peristaltic pump (Catalogue Nos. 7545 and 7014) was used at a pumping rate of 15 ml/min to add 10 ml of Medium A at 37°C to the dish. The dish was then swirled at 88 rpm for 0.4 min (which caused a circularly traveling wave to pass over the gels), after which the medium and nonadherent cells were pumped out at 15 ml/min. Gels were then transferred to lysis buffer.

Procedure B-Medium A (0.5 ml) at 37°C was added to the well of a Linbro dish (2 cm’, 3.5 ml/well, No. FB-16-24-TC from Linbro Chemical Co., Inc.) by pipetting the medium into a plastic l-ml disposable pipette tip held to the side of the well; the medium flowed out of the tip unassisted, i.e. by gravity. The medium and any nonadherent cells were then removed from the well by aspiration (which caused a liquid meniscus to sweep rapidly over the gel) with a Pasteur pipette attached to a water aspirator. At full aspiration, the pressure was less than 1 mm Hg. The washing operation was repeated once and 1 ml of lysis buffer was added to the well.

The number of cells adhering to a derivatized gel was determined by the lactate dehydrogenase method (16, 18).

RESULTS

Sugar-specific Adhesion of Hepatocytes to Gels--We have previously reported that chicken hepatocytes bind specifically to N-acetylglucosaminides linked to both gel types (17, 18); 70 to 100% of the cell population (depending on the cell prepa- ration) bound to GlcNAc-gels, whereas less than 5% bound to gels containing acrylamide alone, aminohexanol, or seven other &aminohexyl-n-glycosides. When thin (0.25 mm) gels were used, the rate of cell adhesion was rapid (10 to 15 min) and no lag period was detected. Adhesion required calcium ions, occurred at a slower rate (and less specifically) in the cold, and was inhibited by free N-acetylglucosamine (or sol- uble molecules containing terminal nonreducing N-acetylglu- cosaminide groups), but was not inhibited by other sugars. Moreover, chicken hepatocytes were released from GlcNAc- gels after incubation with free N-acetylglucosamine or EDTA.

In a preliminary report (19) we showed that rat hepatocytes

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Adhesion of Hepatocytes to Immobilized Sugars 10833

adhered in a sugar-specific manner to polyacrylamide gels containing galactosides. From 65 to 100% of the cells adhered, but only after a significant lag period (19).

As shown in Fig. 2, the adhesion of rat hepatocytes to Gal- gels required Ca’+, but not Mg”+. In the absence of Ca’+, less than 2% of the added cells adhered to the Gal-gel, whereas the addition of 1.8 mM Ca2’ (the normal concentration in serum and Medium A) completely restored both the rate and the extent of cell adhesion (80% for the cell preparation in Fig. 2). Moreover, in a manner analogous to that for chicken hepatocytes, inhibition of the adhesion of rat hepatocytes to Gal-gels was also sugar-specific, as shown in Table II. Free galactose and all the galacto derivatives tested, regardless of their anomeric configuration (or substitution at C-2, namely N-acetylgalactosamine), completely inhibited cell-gel binding at 10 mM concentrations, whereas the gluco and manno deriv- atives had little or no effect. As observed with chicken hepa- tocytes, rat cells bound to Gal-gels were released by incubating these gels in medium containing galactose or other galacto- sides but not other sugars (data not shown).

Structures of Glycoside Ligands-Since both rat and chicken hepatocytes showed sugar-specific adhesion to deriv- atized gels, new ligands containing the appropriate monosac- charides (GlcNAc and Gal) were synthesized. The structures and abbreviations for these compounds are shown in Table I. These glycosides differed structurally in three respects. (a) Either 0- or S-glycosides were used. (b) The structure and length of the aglycon spacer arm was varied. (The spacer, or linker arm, connects the sugar from C1 to the amide nitrogen of the polymer in the gel.) The shortest spacer arm used was

OH H

II I I -S-(CH2)2-C-N-CHe-N-

while the longest was

OH H II I I

-S-(CH&-C-N-(CH&-N-. (c) The ratio of glycose units to spacer arm (moles per mol) was either 1 or 3. The AHAT-(O-sugar):i molecules are trivalent and have three sugars linked 0-glycosidically to hydroxymethyl groups at- tached to the same carbon atom in the spacer arm.

We emphasize that the sugar-specific binding of chicken and rat hepatocytes to gels containing N-acetylglucosamine and galactose, respectively, was observed not only with AH- 0- and AH-S-glycosides, as previously reported, but with all the glycosides used in this study. That is, sugar-specific cell binding was observed regardless of the nature and length of the spacer arm in the gel (i.e. the aglycon of the ligand).

Effect of Gel Glycoside Content on Adhesion-Chicken and rat hepatocytes were tested for their ability to adhere to a series of gels containing different concentrations of N-acetyl- glucosaminide or galactoside, respectively (Fig. 3). The rate and extent of chicken hepatocyte adhesion to type II gels containing AH-0-GlcNAc (Fig. 3A) did not change when the glycoside concentration was varied from 278 to 1075 nmol/ unit gel piece. However, these cells did not bind to a gel which contained 149 nmol/unit gel piece. This suggested that a change in the glycoside concentration from 149 to 278 nmol/ unit gel piece permitted adhesion of chicken hepatocytes to these type II gels containing AH-0-GlcNAc.

Rat hepatocyte adhesion to Gal-gels was also markedly dependent on the concentration of the glycoside in the gel. Preliminary studies (19) showed that an increase of the AH- O-Gal concentration in type II gels affected both the extent of cell binding and the lag period before the formation of stable cell-gel adhesive bonds. The previous results were confirmed

fm 0 IO 20 30 40 50 60

TIME (MINUTES)

FIG. 2. Effect of calcium and magnesium ions on the bindin’g of rat hepatocytes to galactoside gels. Sixteen type II gels con- taining 1047 nmol of AH-O-Gal/unit gel piece were washed twice in 0.15 MNaCl and once in Ca’+- and M?+-free Medium A. The 16 gels were then divided into four sets (four gels/set) and each group was washed with Medium A containing: 1.8 mM Ca”+, A; 1.8 mM Mg*‘, e, 1.8 mM of both Ca2+ and Mg’+, a; or neither cation, 0. To minimize the lag period for attachment of cells to gels, rat hepatocytes (84% viable, 96% single cells, 2.8 x 10” cells/ml) were fist incubated for 30 min at 37°C in complete Medium A under conditions which did not permit intercellular adhesion (19). Cells were then chilled on ice and 2-ml portions of the suspension were added to four tubes, each containing 10 ml of one of the four media described above. The cell suspensions were mixed gently, centrifuged, the supernatant solutions were discarded, and the cell pellets were gently resuspended on ice in 10 ml of the appropriate cold medium and recentrifuged. The final cell pellets were resuspended in 2 ml of the same ice-cold medium. Cells washed in Medium A with or without Cal+ or Mg2+ were then placed on gels equilibrated in the same medium at 37”C, and cell binding was measured at different times by Procedure A as described under “Experimental Procedures.” Cell viability, determined on all the 60-min samples by the lactate dehydrogenase procedure, was 89%

TABLE II

Effect of sugars on binding of rat hepatocytes to galactoside gels Sugar solutions (20.0 ITIM) were made in Medium A and stored in

ice. A rat hepatocyte suspension (1 ml; 88% viable, 73% single cells, and 4 x 10” cells/ml) in ice-cold Medium A was placed in each of 13 tubes on ice. An equal volume of each sugar solution was then carefully mixed with each cell suspension by gentle pipetting. These sugar-containing cell suspensions were then placed on type II gels and cell binding was measured after 60 min (Procedure A) as described under “Experimental Procedures.” The gels used contained 1836 nmol of AH-S-Gal/unit gel piece. Note that Medium A contains 5.5 mM glucose.

Suear or elvcoside (10 ITIM) Cells bound

None Galactose N-Acetylgalactosamine Methyl a-galactoside Methyl P-galactoside

Melibiose (Gal 13 6 Glc)

Lactose (Gal 14 4 Glc) Glucose N-Acetylglucosamine

Maltose (Glc 13 4 Glc) P Cellobiose (Glc 1 + 4 Glc)

Mannose N-Acetvlmannosamine

R cells added to gel

loo 1 1 1 1

1 94

101

86

80 101 107

by using type II gels containing BA-(l)-S-Gal (Fig. 3B). (See Table I for a comparison of the glycoside structures.) Both the extent of cell binding and the lag period preceding binding were affected by changes in the glycoside concentration in the gel. Rat hepatocytes bound with only a 2- to 4-min lag to gels

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10834 Adhesion of Hepatocytes to Immobilized Sugars

i- . L . 149

I 1 I I I I IO 20 30 40 50 60

TIME (MINUTES)

FIG. 3. Effect of concentration of glycoside in the gel on binding of rat and chicken hepatocytes. A, effect of 6-aminohexyl iV-acetylglucosaminide concentration in type II gels on the rate of chicken hepatocyte binding. Type II gels containing the following final AH-0-GlcNAc concentrations (in nanomoles per unit gel piece) were prepared: 0, 149; 0, 278; q , 552; A, 681; X, 895; and n , 1075. After being equilibrated with Medium A, the gels were transferred to polystyrene Petri dishes so that each dish contained six gels in a circular array, and each of the six gels in one dish contained a different ligand concentration as indicated above. Aliquots (60 ~1) of an ice- cold chicken hepatocyte suspension (91% viable, 74% single cells, 7 x IO6 cells/ml) were placed on each gel in a dish and the dish was

containing 2422 nmol of BA-(1)-S-Gal/unit gel piece. When the concentration was decreased to 2141 nmol of BA-(l)-S- Gal/unit gel piece, the lag period increased to 17 min. A further decrease in galactoside concentration to 1953 nmol/ unit gel piece did not prolong the lag period, but decreased the extent of binding at 65 min. Finally, less than 2% of the hepatocyte population bound to gels that had 1875 (or fewer) nmol of galactose/unit gel piece. Therefore, at a glycoside concentration between 1875 and 1953 nmol of BA-(l)-S-Gal/ unit gel piece, the cells became capable of binding to these gels.”

Although rat and chicken hepatocytes showed different kinetic behavior, there is a glycoside concentration below which both cell types can no longer bind to the glycoside gels. These phenomena were investigated further in an extensive series of kinetic experiments of the type shown in Fig. 3, with the use of gels prepared by both synthetic procedures, and with glycosides containing the various aglycon groups shown in Table I. Typical results are shown in Fig. 4, and the results with all the combinations of gel type and aglycon structure tested are summarized in Table III. The essential findings were as follows.

A striking threshold response was observed for cell-gel binding with all the glycosides tested. The concentration at which the cells began to bind is designated the critical con- centration of ligand. An increase of 10 to 20% above this critical concentration resulted in the binding of 60 to 100% of the hepatocytes in a given cell preparation. A further increase in the concentration of glycoside in the gel by as much as an order of magnitude did not increase the number of cells that would bind. In other words, the maximum number of cells that were capable of binding in a given preparation adhered to gels when the ligand concentration in the gel exceeded the critical concentration by only 10 to 20%.

The results shown in Figs. 3 and 4 and summarized in Table III indicate that the threshold binding response of hepatocytes to the variation of glycoside concentration in the gels is indeed a general phenomenon. The effect was observed with the two

3 It is, of course, possible that cells are capable of binding at 1875 nmol/gel piece, but that the lag period is so long (>65 min) that binding was not observed within the limitations of the experiment.

0 II375

0 IO 20 30 40 50 60

TIME (MINUTES)

placed in a 37°C water bath. At the indicated times a dish was removed, nonadherent cells were washed off by Procedure A, and the number of adherent cells was determined by the lactate dehydrogen- ase assay. B, effect of BA-(1)~S-Gal concentration in type II gels on the rate of rat hepatocyte binding. Experiments were conducted as in A, except that the ligand used in preparing the gels was BA-(l)-S- Gal. The rat hepatocyte suspension used was 83% viable, 86% single cells, and 2 x 10” cells/ml. The numbers to the ri&t of the panel represent the BA-(I)-S-Gal concentration of the gels in nanomoles per unit gel piece.

cell types, with gels synthesized by two chemically distinct procedures, and with three to five glycosides differing in their aglycon structures. Quantitative differences were observed, however. That is, the critical concentration for a given cell type varied with the aglycon or type of gel.

For the present studies we arbitrarily selected AH-O-gly- coside type II gels as a “standard” to which the other combi- nations of ligands and gel preparations could be compared (Table III). Interestingly, the rat and chicken hepatocytes responded in opposite ways to the same change in the aglycon of the ligand. With type II gels, for example, the critical concentration for the binding of chicken hepatocytes shifted to a lower value when the spacer arm was changed from AH- 0- to BA-( 1)-S-, whereas the opposite effect was observed for the rat hepatocytes. In fact, in all the cases tested (five in the rat hepatocyte Gal-gel system and three in the chicken he- patocyte GlcNAc-gel system) any change in the structure of the ligand or the type of gel used resulted in opposite responses in the binding ability of the two hepatocyte systems. The critical concentration for rat hepatocyte binding always in- creased whereas that for chicken hepatocyte binding de- creased, compared to the standard.

Effect of Wash Procedure on Cell-Gel Binding-Since adhesion is an operational term defined by the conditions of washing the gels, it was important to determine whether the critical concentration phenomena described above were de- pendent on the particular shear forces generated when non- adherent cells were washed from the gels. Therefore, we devised a gentle and a harsh procedure for removing nonad- herent cells from gels (see “Experimental Procedures” for details).

Good reproducibility (+5%) was obtained with Procedure A or B when the glycoside concentration in the gel was 20% or more above the critical concentration. However, much poorer reproducibility of cell binding was observed when the glyco- side concentration in the gel was closer to the critical concen- tration (e.g. compare the duplicates at 940 versus 1797 nmol/ unit gel piece in Fig. 4B). The rate of hepatocyte binding to gels with glycoside concentrations close to the critical concen- tration was also more variable (not shown). Thus, cell binding to gels seemed to be more sensitive to slight variations in the forces generated during either wash procedure when the gels

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Adhesion of Hepatoq tes to Immobilized Sugars 10835

OO- 0 1002003co4005006007006009001w 2oco2100

Glc NAc CONCENTRATION ( nmoles/cm2 x0.25mm gel)

I g , y--y--+$ , , , , 4 0 200 400 600 SO0 loo0 1200 1400 1600 I600

Gal CONCENTRATION (nmoles/cd x0,25mm gel)

FIG. 4. Threshold binding responses of hepatocytes to gly- cosides linked to polyacrylamide gels. A, chicken hepatocyte binding to AH-O-GlcNAc type II gels. Five separate experiments were conducted with five different celI preparations and type II gels prepared as described in Fig. 1. The kinetics of cell-gel binding was observed as described in Fig. 3, with the use of wash Procedure A. Cell preparations (0, 0, A, 0, W) ranged in viability from 91 to 96%, contained 67 to 87% single cells, and were used at concentrations of 5.5 to 20 x 10” cells/ml. The ordinate represents the 60-min incubation time points. In each experiment the maximal number of cells bound was normalized to 100%; the actual values in the five experiments (maximal number of cells bound x lOO/number of cells added to gels) were all greater than 77%. B, rat hepatocyte binding to AH-O-Gal type II gels. Four experiments with four different preparations of rat hepatocytes (0, 0, 0, n ) and type II gels were performed as described in A. The cell preparations were 80 to 90% viable, 75 to 90% single cells, and contained 2 x lo6 cells/ml. The ordinate represents the number of cells bound after 60 min of incubation. The maximal number of cells bound in each experiment was normalized to 100%. The actual values (maximal number of cells bound x lOO/number of cells added to the gels) depended on the cell preparation and the wash procedure. With Procedure A (---) more than 80% of the cells added to the gels were capable of binding (in each experiment). In one experiment, two sets of gels were washed by Procedure A (0) and one set by Procedure B (A- - -A). In this case, maximal cell binding

TABLE III Critical concentrations of ligands required for binding of rat and

chicken hepatocytes Experiments were performed as described in Figs. 3 to 5 and under

“Experimental Procedures.”

Ligand Gel type

Standard AH-O-sugar

Other AH-O-sugar AHAT-(O-sugar)~ BA-(1)~S-sugar BA-(6)~S-sugar AH-S-sugar

a N.D., not determined.

II

1* 1* II II II

Critical concentration for hepato- cyte binding

Rat Chicken

nmol glycoside/unit gel piece

875 200

2500 50-100 1800 50 1900 100 1900 N.D.” 1100 N.D.

I 1 I I 5. I 0 50 100 150 200 250 3cxJ’< 500 5:o+ik

LIGAND CONCENTRATION (nmoles/cm2 xQ25mm gel)

1

-I 0

i---- ----+> :

lu---- 2._J!--+>----qj

0 I

’ 50 100 150 200 250 3Ao w5Th-4

Glc NAc CONCENTRATION ( nmoles/cm2 X0.25 m m gel )

was 83 and 88% for the duplicate set of gels washed by using Procedure A, and 68% for the set washed by Procedure B. C, chicken hepatocyte binding to type I* gels containing AH-0-GlcNAc or AHAT-(O- GlcNAc)s. A series of gels containing various concentrations of N- hydroxysuccinimide was prepared as described under “Experimental Procedures” and the gels were then treated with AH-0-GlcNAc (0) or AHAT-(O-GlcNAc)z (0). Ligand concentrations (abscissa) are presented as nanomoles of ligand rather than as glycose units. Kinetic experiments were conducted as described under A, with the use of wash Procedure A. Each point is the average of two 60.min incubation values obtained in separate experiments with two different cell prep- arations (after normalizing the maximal cell binding to 100% as described under A). The cell preparations were 86 and 85% viable, 90 and 83% single cells, and were used at densities of 6 x lo6 cells/ml, respectively. Maximal cell binding was 85 and 82% with the two cell preparations. D, effect of the wash procedure on the threshold binding response of chicken hepatocytes to BA-(l)-S-GlcNAc type II gels. Type II gels containing various concentrations of BA-(1)~S-GlcNAc were prepared as described under “Experimental Procedures.” The cell preparation, 92% viable and 77% single cells, was used at a density of 6 x 10” cells/ml in kinetic experiments of the type shown in Fig. 3. The numbers of adherent cells present after 60 min of incubation are shown on the ordinate as the percentage of cells added, not normalized as in A to C. Values for duplicate gels are shown. U, wash Procedure A; 0- - -0, wash Procedure B.

contained ligand concentrations close to the critical concen-

tration. When Procedure B was used, the critical concentration for

rat hepatocyte binding to AH-O-Gal-gels was shifted by about 15% to a higher glycoside concentration and the extent of binding was about 15% less than when Procedure A was used (Fig. 4B). The critical concentrations for chicken hepatocyte binding to BA-(1)-S-GlcNAc-gels were virtually identical with either wash procedure (Fig. 4D), although the extent of bind-

ing was slightly less (about 13%) when Procedure B was used. In summary, slight quantitative differences were observed

when cell binding to gels was measured with the use of vigorous versus mild washing conditions. However, the essen- tial finding was that a threshold binding response occurred at a critical concentration of glycoside in the gel and was not dependent on the particular wash conditions used to measure

cell-gel binding.

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10836 Adhesion of Hepatocytes to Immobilized Sugars

Inhibition of Cell Binding to Gels by Free Sugars--We previously reported (17, 18) that chicken hepatocyte binding to GlcNAc-gels is inhibited by derivatives containing terminal N-acetylglucosaminide residues or by the free sugar itself. Similar results were obtained for the effects of galactose derivatives on the binding of rat hepatocytes to Gal-gels (Table II). The effects of the concentration of glycoside in the gel on the inhibition of cell-gel binding by the appropriate free sugars were therefore examined.

As the concentration of glycoside in the gel was increased, higher concentrations of free sugar were required to inhibit chicken (Fig. 5A) or rat (not shown) hepatocyte binding. For example, when the gel had 256 nmol of N-acetylglucosamin- ide/unit gel piece, 50% inhibition of chicken hepatocyte bind- ing was obtained with 0.7 mM free sugar, whereas if the gel concentration of N-acetylglucosaminide was increased to 895

I 1 / 1 I I I 0 I.0 2.0 3.0 4.0 5.0

GlcNAc IN SOLUTION (mM)

FIG. 5. Inhibition of binding of chicken hepatocytes to AH- O-GlcNAc type II gels by free N-acetylglucosamine. A, type II gels were prepared containing the following concentrations of AH-O- GlcNAc: 256 (0, l ), 385 (A, A), 552 (0, n ), and 895 (V, V) nmol/unit gel piece. Experiments were performed with two different cell prep- arations (open and closed symbols); these were used at final densities of 6.8 x 10” cells/ml, and were 92 and 958 viable, and 90 and 91% single cells, respectively. Portions (1.0 ml) of ice-cold N-acetylgluco- samine solutions in Medium A were gently mixed with LO-ml aliquots of the cell suspension; final concentrations of N-acetylglucosamine are shown on the abscissa. No correction has been made for the aqueous volume of the gels, which had been equilibrated with Medium A but not with the N-acetylglucosamine solution. If the sugar com- pletely equilibrated with the gel during the course of the experiments, then the maximal error in the abscissa is about 18% (the values shown being too high). Cell binding to gels was measured at 60 min by wash Procedure B. The maximal percentage of cells bound at 60 min as defined in Fig. 4A (in the absence of free sugar) was 68 and 83% for the two cell preparations. The dashed line represents 50% of maximal cell binding. B, the relationship between the concentration of AH-O- GlcNAc linked to the gel and the concentration of free N-acetylglu- cosamine required to inhibit cell binding. The data are taken from A. The ordinate represents the concentration of covalently bound AH- O-GlcNAc in the gel. The abscissa represents the intercept between the dashed and s&id lines; this gives the free N-acetylgiucosamine concentration reauired to inhibit cell-gel binding bv 50%. The extrao- olation of the line-predicts the critical concentration of AH-0-GlcNAc required in the gel in order to observe hepatocyte binding (see text).

nmol/unit gel piece, then 50% inhibition required 4.5 mM free sugar (Fig. 5A). There was a direct correlation between the concentration of free sugar required for 50% inhibition of cell- gel binding and the concentration of glycoside in the gel (Fig. 5B).

An interesting feature of Fig. 5B is the extrapolated inter- cept on the ordinate (where the free sugar concentration is zero) which should represent the minimum concentration of glycoside in the gel required for cell binding. This experiment would predict a threshold binding response for chicken hepa- tocytes as the N-acetylglucosaminide content of the gel was increased. In fact, with the AH-0-GlcNAc-gels used in these inhibition experiments with chicken hepatocytes, the inter- cept was at 180 nmol of N-acetylglucosaminide/unit gel piece (Fig. 5B). This value agrees well with the directly determined value of 200 (Fig. 4A). Similar results were obtained when the binding of rat hepatocytes to AH-O-Gal-gels was studied, with free galactose as the inhibitor (data not shown). In this case, the extrapolated intercept was about 800 nmol of galactoside/ unit gel piece, a result close to the critical ligand concentration determined by direct measurement to be 875 (Fig. 4B).

We have noted (18) that the inhibitor titration curves, of the type shown in Fig. 5, can be very sharp (at least at lower concentrations of glycoside in the gel). These cell binding curves in the presence of free sugar resemble (as mirror images) the cell binding curves on gels containing different ligand concentrations (Fig. 4). In a sense, therefore, a critical concentration of sugar in solution is required before cell-gel binding is inhibited, and increasing this concentration by 1.5- to 2-fold completely prevents the binding of cells to gels.

DISCUSSION

In earlier work (16-18), we showed that chicken hepato- cytes adhered specifically to polyacrylamide gels containing covalently bound N-acetylglucosaminides, whereas rat hepa- tocytes adhered to gels containing galactosides (19). This report describes a study of the effect of the concentration of glycoside in the gel on these cell-binding processes. It was found that below a critical concentration of glycoside, no cells adhered to the gels, whereas increasing the concentration above this critical value permitted all cells in a preparation capable of forming stable cell-gel bonds to adhere to the gel. This binding response was elicited, in an all-or-none manner, by very slight changes in the concentration of glycoside in the gel. An increase of 10 to 20% (depending on the cell type and glycoside used) above the critical concentration resulted in maximal cell binding.

The threshold binding response was observed with both rat and chicken hepatocytes and with polyacrylamide gels in which ligands were incorporated by two different chemical procedures. Qualitatively identical results were obtained with the use of three to five different ligands for each cell type (Table III), and when the gels were washed either gently or vigorously to remove nonadherent cells.

The existence of a critical concentration of glycoside for the

binding response of hepatocytes was shown both directly, by measuring cell binding to gels of different glycoside concen- trations (Figs. 3 and 4), and indirectly, by examining the efficiency of free sugar in solution to inhibit the binding of cells to these gels (Fig. 5). The critical concentration values obtained from these independent experiments agreed within 10%.

The quantitative value for the critical concentration (moles of ligand per gel piece (cm’ X 0.25 mm) varied with the structure of the aglycon linking the sugar to the polyacryl- amide and with the type of gel used. Interestingly, the chicken and rat hepatocytes displayed contrasting behavior with re-

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Adhesion of Hepatocytes to Immobilized Sugars 10837

spect to these variables. For example, changing the aglycon structures of the glycoside from H

I -O-(CH&-N- to

0 H H

II I I -S-(CHz)z-C-N-CHZ-N- (using type II gels in both cases) lowered the critical concentration of N-acetylglucosa- minide for chicken hepatocyte binding by 50% (from 200 to 100 nmol/unit gel piece), but raised the critical concentration of galactoside for rat hepatocyte binding by more than 100% (from 875 to 1900 nmol/unit gel piece) (Table III). These results imply that the spacer arms as well as the particular sugar in the gel can influence the binding of the cell surface receptors, and that the receptors responsible for chicken and rat hepatocyte binding respond differently to the same aglycon groups. The two cell types also respond differently when the type of gel (i.e. type I* or type II) is changed.

To our knowledge, the present study is the first investiga- tion and demonstration of a cellular threshold response to a ligand immobilized on a surface. In general, threshold phe- nomena are fundamentally important to the functioning of biological systems. For example, the allosteric regulation of enzyme activity and the self-assembly of viral coat proteins (31) represent molecular threshold phenomena, and many hormones elicit a threshold response from sensitive cells at a critical concentration of the hormone (32).

Two major questions are raised by the results presented here and previously (16-19). What is the cell surface receptor responsible for cell-gel binding? What is the mechanism un- derlying the threshold binding response? We have not yet identified the cell surface receptors, but one obvious possibil- ity is that they are the membrane proteins which bind specific carbohydrates described by Ashwell and co-workers (33-36). These binding proteins are found in liver (e.g. on the surface of hepatocytes), have an absolute requirement for Ca’+, and are specific for molecules terminating in an N-acetylglucosa- minide for chicken (33, 34) or in a galactoside for rat (35, 36). These sugar and ion requirements are the same as those in the systems we have described, but our experiments (to be presented elsewhere) have not yet confumed or refuted the conclusion that these proteins are involved.

Whatever the nature of the cell surface receptor, the thresh- old effect remains to be clarified at a molecular level. Some possible explanations (19) for the cellular threshold binding behavior include the following: (i) a minimum number of bonds between cell surface receptors and glycosides in the gel may be needed for stable cell binding: (ii) a particular arrange- ment of glycosides in the gel may be required before binding by cell surface receptors can occur; (iii) successful cell-gel binding may reflect a cooperative interaction involving cell surface components, such as the sugar. receptor complex. The probability of satisfying any of these conditions would increase as the glycoside concentration in the gel was increased, until it became probable that a cell would bind to the gel (i.e. the critical concentration was exceeded).

The occurrence of a critical concentration effect and a lag period, at least for rat hepatocytes, suggests that a cooperative process of some kind is involved in the ability of these cells to bind to a glycoside gel. Any nucleation events required to initiate such a cooperative process could occur during the lag period. We noted previously (19) that the results in toto indicate that a significant change involving the cell surface receptor(s) or its (their) complexes may occur after the cell makes contact with the immobilized sugar. This change pre- sumably occurs only above the critical glycoside concentration in the gel and could be the insertion of additional receptors

into the cell surface, the redistribution or modification of existing receptors (or complexes), a morphological alteration of the cell, or some combination of these phenomena. It is also possible that the mechanisms underlying the threshold re- sponses of the two cell types may be different.

Whatever the nature and mechanisms underlying the bind- ing phenomena described in this report, these studies have potential physiological significance. Cells are normally con- fronted in vivo with carbohydrate-containing molecules on neighboring cells and in the extracellular matrix. I f a cell were responsive to such a molecule which was present at a concen- tration at, or close to, the critical concentration for the re- sponse, then slight changes in the quantity of that molecule could profoundly affect the behavior of that celI (as evidenced by the model systems presented here). Thus, the small quan- titative differences observed in the levels of some cell surface components between normal and transformed cells (37-40) could be much more important to the differences in behavior exhibited by these two cell types than is currently appreciated.

Acknowledgments-We are grateful to Dr. Pamela Talalay for her expert editorial heln. We also wish to thank Mrs. Dorothy Regula for her help in preparing this manuscript.

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Adhesion of hepatocytes to immobilized sugars. A threshold phenomenon.

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