125I-P12 was used for the quantitative characterization of sperm-P12 binding. To minimize the nonspecific binding of P12 to the glass microfiber filters used in the binding assay, the filters had been blocked with 1.0% skim milk for 30 min. This resulted in low background binding of the radiolabeled P12 to the filters. Consequently, the nonspecific binding of ligand to the cells was sufficiently low to allow the measurement of specific binding. Figure 7 displays the data from one representative determination from the cell incubation (1.0 X 107 cells/ml) in 50 mM Tris-1.0% skim milk (pH 7.4) in the presence of 100 nM 125I-P12. antibiotics levaquin
The equilibrium binding of 125I-P12 to sperm reached a plateau after a 45-min incubation. Therefore, a fixed number of sperm cells (1.0 X 107 cells/ml) were incubated with an increasing ligand concentration for 60 min to determine the steady-state ligand saturation (Fig. 8). The nonspecific binding of 125I-P12 to the cells was determined by addition of a 100-molar excess of unlabeled P12. The amount of 125I-P12 bound to the sperm surface increased with increasing ligand concentration until the available binding sites were saturated. The isotherm saturation suggested 1.2 X 106 P12-binding sites per sperm cell. A Scatchard plot of the data on equilibrium binding due to specific binding yielded a linear plot (Fig. 8 inset) indicating that a single type of binding site on sperm is most likely for P12.
FIG. 7. Equilibrium binding of 125I-P12 to fixed mouse sperm. Mouse sperm (1.0 X 107 cells/ml) in 50 mM Tris, pH 7.4, were incubated in the presence of 100 nM 125I-P12 at 25°C for various times. The 125I-P12 sperm binding at each incubation time was measured. 125I-P12 reached equilibrium binding with a half-time of approximately 8 min. This data set shows results of one representative determination.
FIG. 8. The characteristics of the binding of P12 to sperm. A fixed number of spermatozoa (1.0 X 107 cells/ml) were incubated with various concentrations of 125I-P12 in 50 mM Tris at pH 7.4 at 25°C for 60 min. The total bound ligand (open circles), specifically bound ligand (solid circles), and nonspecifically bound ligand (solid triangles) on the cells were measured as described in the text. A Scatchard plot of data from each ligand concentration is displayed in inset, where B denotes the total amount of specifically bound ligand (nM) and F the concentration of free ligand. The dissociation constant (Kd) and the maximum binding capacity (Bmax) were estimated and listed. Points are means from four determinations. The correlation coefficient for the linear regression fitting of Scatchard plot was calculated to be more than 0.95.