Physical characteristics of hyaluronate binding to the surface of simian virus 40-transformed 3T3 cells.

C.B Underhill,B.P Toole

doi:10.1016/s0021-9258(19)85527-8

Abstract

The binding of labeled hyaluronate to the surface of Simian virus 40-transformed 3T3 cells was studied as a function of 1) pH, 2) ionic strength, 3) temperature, and 4) molecular weight of the hyaluronate. Binding occurred over a wide range of pH values with optima at pH 7 and at less than pH 4. Binding at low pH was eliminated at high ionic strength whereas that at physiological pH was enhanced, with a maximum at 0.5 M NaCl. The enhancement of binding at pH 7 was reversible and independent of the particular salt used. Scatchard plot analysis showed that increasing the ionic strength resulted in both a decrease in the dissociation constant (Kd) and an increase in the amount bound at saturation (Bmax). Temperature also influenced the binding of hyaluronate to the cell surface. The amount bound at low temperatures (0 degrees C) was 3 to 5 times that bound at high temperatures (40 degrees C) with a sharp transition occurring at 18 degrees C, the temperature of phase transition of the plasma membrane. The temperature effect was primarily a change in the Bmax and was reversible. Finally the molecular weight of the ligand influenced the binding. High molecular weight preparations of hyaluronate had a higher binding affinity (lower Kd) and a lower Bmax than did smaller molecular weight preparations.

Highlights

SV-3T3 cells were suspended in 0.15 M NaCl and 0.6-ml aliquots were mixed with [’Hlhyaluronate (Batch 2, 3 pg/ml final concentration), 0.2 ml of 0.2 M buffer and 0.4 ml of NaCl to give final concentration of either 0.15 or 0.4 M
Effect of pH-To determine the pH optimum for binding, SV-3T3cells were suspended in 0.2 M NaCl at room temper- hyaluronate in solutionosf differing NaCl concentrationsT.he aturean dthen mixed witha set amount of labeled hyaluronate results of two such experimentsare shown inFig. 2, A and B
SV-3T3 cells were suspended in 0.15 M NaCl, 0.02 M Tris, pH 7.3, and 0.5-ml aliquots were mixed with 3 p g / d of ['H]hyaluronate (Experiment 1, Batch 2; Experiment 2, Batch 4) plus 0.5 ml of1.0 M solution of the various substances indicated

Summary

RESULTS

Effect of NaCl concentration on the binding of [3H]hyaluronate to SV-3T3 cells a t p H 4.0 and 7.3. SV-3T3 cells were suspended in 0.15 M NaCl and 0.6-ml aliquots were mixed with [’Hlhyaluronate (Batch 2, 3 pg/ml final concentration), 0.2 ml of 0.2 M buffer (acetate, pH 4, Tris, pH 7.3) and 0.4 ml of NaCl to give final concentration of either 0.15 or 0.4 M. The backgrounds were determined by including 200 pg/ml of unlabeled hyaluronate. The samples were shaken for 15 min at room temperature and centrifuged and processed for scintillation counting

Specific binding

Control NaCl Sucrose

Effect of temperature reversal onlevel of hyaluronate binding

Characteristics of HyaluronBaitnedtoing the Cell Surface

Ionic strength also affects the binding of hyaluronate to the