THE JOURNAL OF BIOLOGICAL CHEMISTRY Val. 258, No. 20, Issue of October 25, pp. 12548-12552,1983 Printed in U. S. A.

Isolation of Human Serum Spreading Factor*

(Received for publication, June 7, 1983)

David W. Barnes and Janet Silnutzer From the Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260

Serum spreading factor (SF) was isolated from hu- man serum by a four-step procedure employing affinity chromatography on glass beads, concanavalin A-Se- pharose, DEAE-agarose, and heparin-agarose. The fi- nal product was purified approximately 260-fold from the starting material and was maximally active in assays of cell spreading-promoting activity at 300 ng/ ml. The isolated human SF preparation consisted of two proteins of apparent molecular weights approxi- mately 65,000 (SF65) and 75,000 (SF75). Both SF65 and SF75 have been shown previously to exhibit cell spreading-promoting activity and to bind monoclonal antibody to human serum SF.

SF’ is a glycoprotein component of human serum that promotes the attachment and spreading of a wide variety of both fibroblastic and epithelial cells in vitro (Barnes and Sato, 1979; Barnes et al., 1980,1981,1982a). In studies using serum- free hormone-supplemented cell culture medium, Barnes and Sat0 (1979, 1980a, 1980b, 1980~) observed that this factor mediates cell spreading-promoting effects that in some cases are not mimicked by fibronectin or cold-insoluble globulin (Hynes and Yamada, 1982), another spreading-promoting protein in serum. Subsequently Barnes et al. (1980, 1981, 1982a, 1982b, 1983) reported that serum SF is distinct im- munologically, biochemically and in biological activities from fibronectin, laminin, and chondronectin, all glycoproteins capable of stimulating attachment and spreading of some types of cells in culture (Kleinman et al. 1981), and that serum SF contributes a significant portion of the total spreading- promoting activity of human serum in serum-supplemented culture medium. In addition to effects on cell attachment, spreading, and gross morphology, serum SF also stimulates growth and affects differentiation of a number of cell types in culture in hormone-supplemented serum-free media (Barnes and Sato, 1980b, 1980c; Barnes et al., 1980, 1981, 1982a). Experiments using monoclonal antibody that inhibits serum SF-promoted cell spreading established that serum SF exists in two forms in human serum, one form migrating in polyac- rylamide gel electrophoresis in a manner consistent with M, = 65,000-70,000 (SF65) and the other in a manner consistent with M , = 75,000-78,000 (SF75) (Barnes et al., 1982b, 1983). Experiments with monoclonal antibody to serum SF also

* This work was supported by American Cancer Society Grant BC- 368 and National Institutes of Health Grant CA-35214-01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adver- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: SF, spreading factor; SF65, form of serum SF of molecular weight approximately 65,000; SF75, form of serum SF of molecular weight approximately 75,000; PBS, phosphate- buffered saline; ELISA, enzyme-linked immunosorbent assay; SDS, sodium dodecyl sulfate.

”

identified the factor in soluble extracts of washed human platelets (Barnes et al., 1983).

Holmes (1967) originally described a spreading-promoting activity present in a serum fraction isolated by glass bead affinity chromatography. This fraction contains both spread- ing-promoting and growth-promoting activity when assayed on cells plated in serum-free media without additional sup- plementation. The spreading-promoting activity in this par- tially purified preparation, which was found to be separable chromatographically from the growth-promoting activity (Barnes and Sato, 1980a; Barnes et al., 1981), is the material that we have termed “serum spreading factor” (Barnes and Sato, 1980a, 1980b, 1980c; Barnes et al., 1980). In this paper we describe the isolation of serum SF (SF65 and SF75) using as a first step glass bead affinity chromatography, followed by chromatography on concanavalin A-Sepharose, DEAE- agarose, and heparin-agarose. Because serum SF exhibits a strong tendency to adsorb to the surfaces of glass or plastic vessels or dialysis tubing, the procedure was developed in such a way that dialysis or other manipulations between chromat- ographic steps were unnecessary. Serum SF was eluted a t each step in this procedure in a solution that allowed direct loading of the material onto the next column in the series, and the isolated material was obtained at the end of the procedure in a solution of approximately physiological pH and salt concen- tration. This approach resulted in a purification procedure that can be carried out fairly rapidly with reasonable yields.

EXPERIMENTAL PROCEDURES

Materials-Glass beads (Number 1014, Class IV-A) were obtained from Ferro, Cataphote Division, Jackson, MS. Chemicals and bio- chemicals were obtained from Fisher and Sigma. Materials for gel chromatography and SDS-polyacrylamide gel electrophoresis were obtained from Bio-Rad and Pharmacia Fine Chemicals. Immuno- chemicals were obtained from N. L. Cappel Laboratories Inc. Human plasma fibronectin (cold-insoluble globulin) was obtained from Meloy Laboratories Inc. and was judged to be greater than 98% pure upon analysis by SDS-polyacrylamide gel electrophoresis.

Monoclonal Antibody-binding Assay for Measurement of Serum SF-We have previously described procedures for measurement of serum SF by this assay, as well as the derivation, characterization, and isolation of the monoclonal antibody to serum SF used in the assay (Barnes et al., 1983). The standard assay procedures are based in general on the methods of Rennard et al. (1980). Briefly, a fixed amount of monoclonal antibody (0.2 pg) in 100 pl of PBS containing 1 mg/ml of bovine serum albumin was incubated 16 h with samples (100 pl) to be assayed for serum SF, diluted to various concentrations in PBS with 1 mg/ml of bovine serum albumin. The amount of antibody remaining free at the end of this incubation was determined by transfer of the incubation mixtures to serum SF-coated microtiter wells and subsequent measurements by standard ELISA, using per- oxidase-conjugated goat anti-mouse IgG as the second antibody and 2,2’-azino-di(3-ethylbenzthiazelinesulfoni~ acid) as the peroxidase- dependent chromogen (Barnes et al., 1983). Extent of the enzymatic reaction was determined by measuring the absorbance of the peroxi- dase reaction mixture at 415 nm. In this assay, samples containing relatively high amounts of serum SF bound a relatively larger amount of anti-SF and thus contained less free anti-SF at the end of the

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Isolation of Human Serum Spreading Factor 12549

incubation period. ELISA of these samples gave rise to lower absorb- ance at 415 nm at the end of subsequent incubations than ELISA of samples containing relatively less serum SF in the initial incubation mixtures. One unit of anti-SF antibody-binding activity was defined as the amount of activity in the initial incubation mixture necessary to bind half of the total anti-SF antibody in the standard assay as described above. Protein concentration was measured by the method of Bradford (1976) using bovine y-globulin as a standard.

Cell Spreading Assay-Procedures for assay of spreading-promot- ing activity were as described previously (Barnes et al., 1980, 1983). Briefly, cells were plated in serum-free medium containing 1 mg/ml of bovine serum albumin onto tissue culture dishes, either pretreated with the samples to be assayed (1-h incubations at room temperature) or onto control plates preincubated with PBS. The number of spread cells was determined 90 min after plating. For the assay presented, Hela cells were used; we also have used other epithelioid and fibro- blastic cell types in this assay. One unit of cell spreading-promoting activity was defined as the amount of activity necessary to elicit an effect that was 50% of the maximum effect under the conditions of the assay.

Purification of Human Serum SF-Fresh frozen human plasma was obtained from the Pittsburgh Central Blood Bank, dialyzed overnight against 0.15 M NaCl, and clotted by the addition of 1 mg/ ml of CaC12. The resulting serum was stored frozen at -20 “C in 25- ml aliquots. Serum SF levels in serum do not differ markedly from those in plasma (Barnes et aL, 1983), and we have used serum as the starting material in our isolation procedures. Both SF65 and SF75 are present in serum and plasma. Human serum SF was purified in a four-step procedure described below.

For step 1, serum (25 ml) was adjusted to pH 8.0 with 1 N NaOH and chromatographed on a glass bead column (2.5 X 40 cm; 50 ml/h) previously equilibrated with 0.6 M NaHC03 (pH 8.0). This and all subsequent procedures were carried out at 4 “C. Fraction volumes of 1-3 ml, depending on the expected sharpness of the peak, were collected in polypropylene tubes. Serum on the column was followed by 100 ml of 0.6 M NaHC03, 50 ml of 0.6 M NaHC03, 0.2 M Na&O, (pH 9.3), 50 ml of water, 50 ml of 0.6 M KHCO,, 0.2 (pH 9.5), and 100 ml of 0.2 M K2C03 (pH 11.0). Elution with 0.2 M K2C03 (pH 11.0) allowed regeneration of the column for reuse. Elution of protein was followed by measurement of absorbance at 280 nm, and peak fractions eluting with 0.6 M NaHCO,, 0.2 M Na2C03 were pooled. For step 2, the pooled peaks from four to six glass bead column runs were combined and chromatographed directly on a concanavalin A-Se- pharose column (2.5 X 9 cm; 15 ml/h). Following sample application, the column was eluted sequentially with 90 ml of 25 mM NaH2P0,/ Na2HP04 (Na/P,) at pH 6.0, 75 ml of 25 mM Na/Pi (pH 6.0) contain- ing 50 mM mannose, and 25 ml of PBS containing 1 M a-methylman- noside. A total of approximately 75 mg of protein could be loaded onto a concanavalin A-Sepharose column (2.5 X 9 cm) under the conditions described without exceeding the glycoprotein-binding ca- pacity of the column.

Serum SF was also present in the peak eluting from the glass bead column with 0.6 M KHC03, 0.2 M K,CO,. This material could be chromatographed in a manner identical with that described for the peak eluted with 0.6 M NaHC03, 0.2 M Na2C03; however, we found that better yields were obtained if the peak eluted from the glass bead column with KHC03/K2C03 was dialyzed against PBS before loading onto the concanavalin A column. This is because the glycoprotein- binding capacity of the lectin column was reduced significantly if the sample was loaded in the presence of KHC03/K,C03, compared to the capacity of the column if the sample was loaded in NaHCO,/ Na2C0,. Approximately 70% of the serum spreading factor retained by the glass beads was recovered in the peak eluting with 0.6 M NaHC03, 0.2 M Na2C03. The remaining portion of the serum spread- ing factor eluted from the column in the later portion of the 0.6 M NaHC03, 0.2 M Na2C0, wash (about 10% of the total retained) and in the 0.6 M KHCOs, 0.2 K&03 wash (about 20% of the total). No serum spreading factor was recovered in the water wash, but inclusion of this step allowed a sharp demarcation between the sodium carbon- ate and potassium carbonate washes that was useful in subsequent processing of the fractions for concanavalin A-Sepharose chromatog- raphy. Although all of the bound serum spreading factor could be eluted eventually with extensive washing with 0.6 M NaHC03, 0.2 M Na2C03, changing to potassium carbonate after an initial elution with sodium carbonate allowed the recovery of the remaining portion of the serum spreading factor in reasonably concentrated peak fractions. Elution of serum spreading factor with potassium carbonate after elution with sodium carbonate was first reported by Holmes (1967),

and the chemical basis for this phenomenon is unclear, although the relative chaotropic natures of sodium carbonate and potassium car- bonate in solution may be involved.

In step 3, fractions constituting a broad peak eluting from the concanavalin A-Sepharose column with 50 mM mannose in 25 mM Na/P, (pH 6.0) were pooled and chromatographed directly on a DEAE-agarose column (1.5 X 5 cm; 15 ml/h) previously equilibrated with 25 mM Na/Pi (pH 6.0). After loading the sample, the column was eluted with 20 ml of 25 mM Na/Pi (pH 6.0), 20 ml of 100 mM NaCl in 25 mM Na/Pi (pH 6.0), and 20 ml of 1.5 M NaCl in 25 mM Na/Pi (pH 6.0). In step 4, fractions containing material eluting from the DEAE-agarose column in a sharp peak with 100 mM NaCl in 25 mM Na/P, (pH 6.0) were pooled and loaded directly onto a heparin- agarose column (0.7 X 14 cm; 7.5 ml/h) previously equilibrated with 100 mM NaCl in 25 mM Na/Pi (pH 6.0). The sample was followed on the column by 15 ml of 100 mM NaCl in 25 mM Na/Pi (pH 6.0), 15 ml of 100 mM NaCl in 50 mM Na/Pi (pH 7.2), and 10 ml of 1.5 M NaCl in 50 mM Na/P, (pH 7.2). Most of the serum SF initially bound to the column eluted in a sharp peak with 100 mM NaCl in 50 mM Na/P, at pH 7.2 (“step 4” material). A relatively small amount of SF remained bound to the column under these conditions and was eluted with 1.5 M NaCl in 50 mM Na/Pi (pH 7.2).

Analysis by SDS-polyacrylamide gel electrophoresis indicated that the step 4 material consisted of proteins migrating in two bands corresponding to molecular weights of approximately 65,000 (SF65) and 75,000 (SF75). Protein staining of overloaded gels indicated that some preparations appeared to contain only SF65 and SF75. Other preparations, although greater than 98% SF65 + SF75, contained trace contaminants, primarily a protein of apparent molecular weight approximately 50,000. These preparations could be freed of trace contaminants by direct gel filtration chromatography of the step 4 material.

RESULTS

Isolation of Serum SF-Table I summarizes the relative specific activities calculated from the antibody-binding and cell spreading-promoting assays, total amount of protein re- covered, and the percentage of total activity recovered at each step in the isolation procedure. Analysis by SDS-polyacrylam- ide gel electrophoresis of the product obtained at each step is shown in Fig. 1. The greatest purification in a single step was obtained in the glass bead affinity column (step 1) procedure. Although in previous work we have used partially purified serum SF isolated by glass affinity chromatography as a first step (Barnes et al., 1980, 1981), the ratio of serum volume to glass bead column volume was about four times higher in these earlier procedures than that of the step 1 procedure described here. In attempts to maximize total yields, we found that best results were obtained if the amount of serum chro- matographed, as well as the volume of the eluting buffers, was decreased from those of our earlier methods. The adhesion of serum proteins to glass represents rather complicated inter- actions (Haas and Culp, 1979), and the amount of serum protein loaded on the glass bead column affects to some extent which proteins predominate in the fractions subsequently eluted.’

When step 1 material was passed through a concanavalin A-Sepharose column (step 2), over half of the total protein passed through the column essentially unretarded. These proteins were primarily of molecular weight less than 30,000 with some additional material of molecular weight approxi- mately 88,000. Serum SF was eluted from the convanavalin A-Sepharose column with 50 mM mannose in pH 6.0 buffer. Elution with mannose at 50 mM gave more consistent results with fewer contaminating proteins present than did elution with a-methylmannoside at 50 mM or less. Elution of serum SF from the lectin column at pH 6.0 in 25 mM Na/Pi allowed direct loading of the eluted material onto a DEAE-agarose column (step 3). Serum SF was eluted from the column by

D. W. Barnes, L. Mousetis, B. Amos, and J. Silnutzer, submitted for publication.

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12550 Isolation of Human Serum Spreading Factor

raising the salt concentration (100 mM NaCl in 25 mM Na/Pi

Elution of serum SF from DEAE-agarose in the indicated buffer solution allowed direct application of the eluted mate- rial onto a heparin-agarose column (step 4). Serum SF was

TABLE I Isolation of human serum spreading factor

Procedures for assay of specific monoclonal antibody-binding ac- tivity (immunoassay) and cell spreading-promoting activity are de- scribed under “ExDerimental Procedures.”

(pH 6.0)).

Specific activity

Purification stage Immuno- spread- protein recove@ Cell Total Activity

assaj ing assayb

units fpg mg % Human serum 0.012 0.062 8750 Starting

material Glass bead column (step 0.661 3.12 71.8 45

Concanavalin A-Sephar- 1.66 NDd 19.0 30

DEAE-agarose (steu 3) 2.21 ND 4.59 10

1)

ose (step 2)

Heparin-agarose ( sbp 4) 3.23 19.6 2.48 8 Anti-SF monoclonal antibody-binding assay as illustrated in Fig.

3. * Cell spreading-promoting assay as illustrated in Fig. 4. Calculated from specific activity determined by immunoassay. ND. not determined.

I

W il ii

a b c d e f g FIG. 1. SDS-polyacrylamide gel electrophoresis of human

serum SF preparations. The four-step method for isolation of serum SF is described under “Experimental Procedures.” Samples of material isolated at each step were reduced with mercaptoethanol, denatured at 100 “C, and subjected to electrophoresis using a 7.5% polyacrylamide slab gel with a 2.5% stacking gel. a, molecular weight standards: 5 pg each of (top to bottom) myosin (200,000), @-galacto- sidase (116,000), phosphorylase b (92,500), bovine serum albumin (66,700), and ovalbumin (43,000); b, 50 pg of human serum; c, 40 pg of preparation from glass bead column affinity chromatography (step 1); d, 20 pg of preparation from concanavalin A-Sepharose chroma- tography (step 2); e, 20 pg of preparation from DEAE-agarose chro- matography (step 3); f , 15 pg of isolated human SF (SF65 and SF75) from heparin-agarose chromatography (step 4); g, molecular weight standards as in a above.

FRACTION

FRACTION FIG. 2. Heparin-agarose column chromatography of iso-

lated human fibronectin (A) and human serum spreading fac- tor (B) . Methods are described under “Experimental Procedures” and “Results.” Arrows indicate change of elution conditions: Fraction 17, change from pH 6.0 to 7.2 (100 mM NaCl, 50 mM Na/Pi); Fraction 42, change from 100 mM NaCl to 1.5 M NaCl (50 mM Na/Pi, pH 7.2).

subsequently eluted from the heparin column by adjusting the eluting solution to 100 mM NaCl and 50 mM Na/Pi (pH 7.2). The heparin column procedure removed several contaminat- ing proteins from the preparation, particularly a protein of molecular weight approximately 50,000 that co-purified with serum SF through the first three steps, but remained bound to the heparin column under conditions that eluted most of the bound serum SF.

Like serum SF, fibronectin also binds to heparin (Stathakis and Mossesson, 1977; Yamada et al., 1980), although this binding is maintained under conditions that elute serum SF from the column. This difference between the two spreading- promoting serum proteins is illustrated in the experiment of Fig. 2. Human serum SF (step 4 material) and human fibro- nectin were loaded onto identical heparin-agarose columns (0.7 X 2.5 cm) in 50 mM Na/Pi (pH 6.0) containing 100 mM NaC1. The columns were eluted with 15 ml of 100 mM NaCl in 50 mM Na/Pi (pH 6.0), 20 ml of 100 mM NaCl in 50 mM Na/Pi (pH 7.2), and 15 ml of 1.5 M NaCl in 50 mM Na/Pi (pH 7.2). Although serum SF eluted from the column under con- ditions of approximate physiological salt concentration and pH, human fibronectin remained tightly bound to the column under these conditions and was eluted with 1.5 M NaCl, as previously reported by others (Stathakis and Mossesson, 1977; Yamada et al., 1980).

Total yield in the four-step isolation procedure from 1 unit of human plasma was approximately 2.5 mg of serum SF, representing an overall recovery of about 8% of the total activity in the starting material. The step 4 material consisted of a mixture of SF65 and SF75 in about equal proportions. The total yield and relative amounts of the serum SF forms varied somewhat among different preparations, the total yield varying roughly in relation to the concentration of serum SF in plasma. Our measurements3 indicate that serum SF con- centration in normal adults ranges from about 100 pg/ml to about 400 pg/ml, with average concentration approximately

M. C. Shaffer, E. D. Avner, T. P. Foley, and D. W. Barnes, submitted for publication.

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Isolation of Human Sej w m Spreading Factor 12551

200 pg/ml. Immunoblots of the final preparations indicated that monoclonal antibody to serum SF recognized both the SF65 and SF75 bands; no fibronectin was detected in the step 4 material assayed by ELISA using rabbit antiserum to human fibronectin (not shown). SDS electrophoresis of the isolated serum SF preparations in 12 and 15% polyacrylamide gels did not detect additional lower molecular weight protein bands that might not have been detected in the 7.5% acrylamide gels of Fig. 1.

Amino acid compositions of two independently isolated step 4 serum SF preparations are given in Table 11. Also included in Table I1 for comparisons are the amino acid compositions of a spreading-promoting factor of M, = 62,000 isolated from fetal calf serum (Whateley and Knox, 1980) and a partially purified spreading-promoting preparation isolated from fetal calf serum that contains some components in the molecular weight range from 65,000 to 80,000 (Grinnell et al., 1977). The amino acid compositions of the two preparations of serum spreadlng factor were quite similar and considerably different from those of the other two spreading factor preparations, particularly in relative percentage of tyrosine, proline, and arginine. NHz-terminal analysis of isolated serum spreading factor identified aspartic acid (asparagine) as the single NHz- terminal amino acid in the preparation. The NHP-terminal amino acid of the spreading factor of Whateley and Knox (1980) is reported to be alanine.

Activity of Isolated Serum SF-Measurement of the relative anti-serum SF monoclonal antibody binding activity (Barnes et al., 1983) for human serum, Cohn fraction IV from human plasma, a step 1 preparation eluted from the glass bead column, and step 4 material eluted from the heparin-agarose

TABLE I1 Amino acid composition

Amino acid composition of human serum spreading factor was determined on a Durrum D-500 analyzer after hydrolysis of samples in 6 N HCl for 24 h at 110 “C. Values shown are from single deter- minations of two different serum spreading factor preparations (A and B). Hydrolytic losses of serine and threonine were not corrected. Values for the fetal calf serum spreading factors of Whateley and Knox (1980) and the mixed factor of Grinnell et al. (1977) are calculated from data in the publications cited.

Amino acid

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Tryptophan Ornithine

spreading Serum

factor prep- aration A

107 38.6 62.9

78.7 82.6 63.4

149

11.7 52.0 8.76 29.6 65.3 46.2 49.8 23.3 53.8 77.1 ND“

0

spreading factor of Serum Spreading

factor Whateley prepara- and Knox tion B (1980)

mo1f103 mol amino acid 110 85.8 37.8 50.1 61.0 73.7

83.9 49.1 85.0 94.8 67.1 75.6 19.5 31.9 51.1 64.0 11.2 6.69 28.4 32.0 61.2 81.3 46.6 25.2 49.8 39.2 20.3 48.3 47.4 64.2 75.7 40.8

0 10.5

144 127

ND ND

Spreading factor of

Grinnell et al. (1977)

105 76.0 71.6

58.6 73.8 56.5 28.6 78.3 0 40.9 76.6 33.5 36.8 24.0 46.9 47.2 32.2

0

113

Total 1000 1000 1000 1000 ND, not determined.

P R O T E I N ( p q l m l 1

FIG. 3. Monoclonal antibody-binding assay for measure- ment of serum SF. Methods are described under “Experimental Procedures.” 0, isolated human SF (SF65 and SF75) from heparin- agarose column chromatography (step 4); X, human SF preparation from glass bead column chromatography (step 1); ., Cohn fraction IV from human plasma; 0, human serum.

I I I I 1 I 1

j 40 W

I I

I I I

I I

I

I I

I % / I / e /

I I

0 .01 0. I 1.0 10 I00 c“-.-

P R O T E I N (pqlrnl)

FIG. 4. Assay of cell spreading-promoting activity of iso- lated human SF (0) and human serum (0). Methods are described under “Experimental Procedures.” Hela human carcinoma cells were used in the assay.

column is shown in Fig. 3. Cohn fraction IV was enriched approximately %fold in serum SF over human serum; other Cohn fractions tested contained less serum SF/mg of protein than did human serum. The protein concentration of step 4 serum SF isolated from the heparin-agarose column necessary to reduce the amount of free antibody in the assay mixture by 50% was approximately 260-fold lower than the protein concentration of human serum necessary to produce the same effect in the assay. Similarly, the step 4 serum SF preparations were about 300-fold more active in promoting cell spreading in culture than was the starting material (Fig. 4). Maximal effect of the isolated serum SF preparations on cell spreading was seen at about 300 ng/35 mm-diameter plate (8 cm’), and a detectable effect was observed at 10 ng/plate.

DISCUSSION

Using anti-serum SF monoclonal antibody-binding activity and cell spreading-promoting activity in serum-free culture medium as an assay, we have purified human serum SF approximately 260-fold from human serum by a four-step procedure. The greatest purification in a single step occurred in the initial glass bead affinity column chromatography. Analysis by SDS-polyacrylamide gel electrophoresis indicated that the final product of the procedure was a mixture of SF65

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12552 Isolation of Human Serum Spreading Factor

and SF75. We have reported previously that both SF65 and SF75 are active independently in assays of monoclonal anti- body-binding and cell spreading-promoting activity (Barnes et al., 1982a, 1983; Hayman et al., 1982). Yields of serum SF isolated by the procedure described from 1 unit of human plasma were sufficient to provide about 2.5 mg of material maximally active in the biological assay at 0.3 pg/ml.

Although serum SF exhibits some similarities to serum fibronectin or cold-insoluble globulin (Hynes and Yamada, 1982) in biological properties and heparin-binding activity, the two glycoproteins are biochemically and immunologically distinct by several criteria (Barnes et al., 1980, 1981, 1982a, 1983). Serum SF does not bind to gelatin-Sepharose (Barnes et al., 1980), and we have made serum SF preparations by procedures described in this paper using serum derived from human plasma that had been passed over gelatin-Sepharose to remove fibronectin, and found no reduction in yield of serum SF. Including the gelatin-Sepharose step in the begin- ning allowed the isolation of both of these spreading-promot- ing proteins independently from the same plasma sample. Furthermore, because plasma fibronectin and serum SF eluted from heparin-agarose under different conditions, the combi- nation of gelatin-Sepharose and heparin-agarose chromatog- raphy makes it unlikely that SF preparations contained con- taminating fibronectin or fibronectin fragments. We have confirmed this by ELISA for fibronectin under conditions that would detect less than 0.05% fibronectin contamination in our preparations. Our data indicate that, unlike fibronectin binding to heparin (Stathakis and Mossesson, 1977; Yamada et al., 1980), strong interaction between heparin and the major portion of the serum SF initially bound to the column at pH 6.0 did not occur at physiological pH and salt concentration. This observation suggests that the heparin-binding phenom- enon that we found useful in devising an isolation procedure for human serum SF may not reflect an interaction of impor- tance between plasma or tissue SF and heparin or heparin proteoglycans in vivo.

Recently Hayman et al. (1983) also have observed binding of serum spreading factor to heparin-agarose and reported that monoclonal antibody identifies serum spreading factor at the cell surface of cultured fibroblasts and in tissues. In our experiments with the monoclonal antibody to serum spreading factor used in this and previous studies (Barnes et al. 198213, 1983), we have been unable to identify cell- or tissue-associated serum spreading factor in cultured human cells or frozen sections of human kidney and liver.3 In the latter studies, however, immunostaining with our antibody clearly identified serum spreading factor in Bowman’s space in kidney and sinusoids of liver, both plasma-containing areas of these tissues.

Cell spreading-promoting activity has been reported previ- ously in preparations containing proteins in the molecular weight range from 60,000 to 80,000 isolated from fetal calf serum (Grinnell et al., 1977; Whateley and Knox, 1980). Although the “mixed factor” of Grinnell et al. contains several spreading-promoting molecules, including fibronectin, the spreading factor of Whateley and Knox is immunologically distinct from fibronectin. This preparation contains a single

protein of M, = 62,000, somewhat smaller than our SF65, and contains no material comparable to SF75 in our preparations. Furthermore, comparisons of amino acid composition and NH,-terminal point out significant biochemical differences between human serum spreading and the two cell spreading- promoting preparations from fetal calf serum.

Epibolin is a M, = 65,000 protein isolated from human serum (Stenn, 1981) that promotes spreading of epidermal cells in culture. Spreading-promoting activity of epibolin in the concentration range effective for serum spreading factor ( ie . 1 pg/ml or less) requires the presence of a second factor synergistic with epibolin and termed co-epibolin (Stenn, 1981). In collaboration with Dr. Stenn, we have observed some immunological cross-reactivity between epibolin prepa- rations and serum spreading factor preparations (Barnes et al., 1983). Our serum spreading factor preparations appear to be unique, however, in the presence of two forms of the spreading-promoting factor (SF65 and SF75) and the absence of requirement for a synergistic cofactor. More detailed bio- chemical comparisons of epibolin and serum spreading factor are in progress.

Acknowledgments-We thank Dr. M. Sussman for suggestions and M. Shaffer for help and advice. We also thank Dr. C. Coffee for NHZ- terminal amino acid analysis of isolated serum spreading factor.

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D W Barnes and J SilnutzerIsolation of human serum spreading factor.

1983, 258:12548-12552.J. Biol. Chem. 

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