Abstract
Human serum spreading factor (SF) is a blood glycoprotein that promotes attachment and spreading and influences growth, migration, and differentiation of a variety of animal cells in culture. SF purified from human plasma or serum by chromatographic methods reported previously (Barnes, D. W., and Silnutzer, J. (1983) J. Biol. Chem. 258, 12548-12552) does not bind to heparin-Sepharose under conditions of physiological ionic strength and pH. In a further examination of the heparin-binding properties of human serum SF, we found that exposure of purified SF to 8 M urea altered several properties of the protein, including heparin affinity, and these alterations remained after removal of the urea from SF solutions. Urea-treated SF bound to heparin under physiological conditions, and salt concentrations of 0.4 M or higher were required for elution of urea-treated SF from heparin-Sepharose at pH 7.0. The alteration of heparin-binding properties of SF also was observed upon exposure of the protein to heat or acid. Treatment of SF with urea, heat, or acid resulted additionally in greatly decreased cell spreading-promoting activity of the molecule. The decreased biological activity was associated with a reduced ability of the treated SF to bind to the cell culture substratum, a prerequisite for the attachment-promoting activity of the molecule. Experiments examining the heparin-binding properties of native SF in unfractionated human plasma indicated that the major portion of SF in blood did not bind to heparin under conditions of physiological ionic strength and pH.
Highlights
Human serum spreading factor (SF) is a blood gly- concentrations of 1pg/ml or less [12, 14]
We have reported previously that SF does not exhibit physiologically significantinteractions with either collagen or heparin [5, 14, 25], and experiments leading to similar conclusionshave been published concerningthe spreading factorof Knox [39]
The recent report that human serSuFmexhibits an affinity graphed on DEAE-Sepharose, suggesting that for heparin at physiological pH andionic strength [13]led us urea treatment did not result in a major change in the net to re-examine the heparin-binding propertiesof the protein. charge of the molecule
Summary
The recent report that human serSuFmexhibits an affinity graphed on DEAE-Sepharose (not shown), suggesting that for heparin at physiological pH andionic strength [13]led us urea treatment did not result in a major change in the net to re-examine the heparin-binding propertiesof the protein. charge of the molecule. Incubation of SF solutions in 1.5 M NaCl did not purified SF to 8 M urea, as well as other treatments, led to result in a major alteration in heparin affinitywhen that salt alterations inseveral properties of the molecule, including the concentration subsequentlywas lowered to physiological ionic heparin-binding properties. SF pretreated with 8 M urea and chromatographed on heparin-Sepharose in phosphatebuffered saline in the absence of urea was retained by the inhibitor of heparin binding that mightcopurify with the SF molecule and whose effects might be reversed by urea, acid, or heat treatment of the SF preparations. Because identical results were obtained if the SF was used directly upon pretreatment and removal of the pretreatment conditions, or if the pretreated SF was first isolated by chromatography on heparin-Sepharose as shown in Fig. 3 and returned to physiological ionic strength for the assay, it is unlikely that the residual activitiesintheseexperiments were dueto residual nonheparin-binding SF.
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