Real-time measurements of the interactions between fluorescent speract andits sperm receptor

Lytechinus pictus sea urchin sperm express receptors for speract, a sperm-a ctivating peptide derived from the homologous egg jelly coat. We found that the fluorescence of fluorophore-labeled, active, speract analogs is quench ed upon receptor binding. This property allowed us to perform real-time mea surements of speract-receptor interactions using intact sperm and to determ ine, for the first time, their association (k(on)) and dissociation (k(off) ) rate constants. The high k(on) (2.4 x 10(7) M-1 s(-1)) and low k(off) (4. 4 x 10(-6) s(-1) (95%) and 3.7 x 10(-4) s(-1) (5%)) can account for the spe rm response to picomolar concentrations of speract. We also examined the in fluence of extracellular ions on speract-receptor interactions using the fl uorescence quenching method described in this study. The association rate o f speract to the receptor is dramatically reduced in Na+-free seawater (NaF SW), divalent cation-free seawater (DCFSW), and high-K+ seawater (HKSW). In seawater speract induces an increase in intracellular pH (pHi), while it i s unable to do so in either NaFSW or HKSW. To test if the lack of this pHi change causes the reduction in the speract association rate, pHi was increa sed with NH4Cl (10 mM) at the time labeled speract was added. Interestingly , this procedure completely (in HKSW) or partially (in NaFSW and DCFSW) res tored the speract association rate to its receptor. These findings indicate that an increase in sperm pHi positively affects the receptor binding acti vity for this peptide and may partially explain the positive binding cooper ativity displayed by the speract receptor. (C) 2000 Academic Press.