SPERACT RECEPTORS ARE LOCALIZED ON SEA-URCHIN SPERM FLAGELLA USING A FLUORESCENT PEPTIDE ANALOG

In two species of sea urchins, Strongylocentrotus purpuratus and Lytec hinus pictus, the egg jelly-associated decapeptide, speract, binds to specific sperm surface receptors resulting in increased sperm motility and respiration rate. Previously, a peptide analog, GGG[Y2]-speract, was used to identify a 77-kDa receptor on intact sperm cells using che mical cross-linking. In this paper we describe the synthesis and chara cterization of a fluorescent derivative of GGG[Y2]-speract for use as a probe for the sperm receptor. Fluorescein isothiocyanate (FITC) was conjugated to the amino terminus of GGG[Y2]-speract and the resulting analog (FITC-GGG[Y2]-speract) was purified by size exclusion chromatog raphy and reverse-phase HPLC. Competition binding studies with the flu orescent peptide and intact spermatozoa yielded IC50 values which were indistinguishable from native speract and GGG[Y2]-speract (approximat ely 20 nM). FITC-GGG[Y2]-speract half-maximally stimulated sperm respi ration at a concentration nearly identical to that of the native pepti de (EC50 approximately 50 pM). Using digitally enhanced video imaging fluorescence microscopy, FITC-GGG[Y2]-speract was used to localize the speract receptor on the flagella of intact sperm. Excess concentratio ns of both unlabeled speract and GGG[Y2]-speract abolished the binding of the fluorescent analog, yet unrelated peptides did not. Further, r esults of cross-linking experiments using I-125-GGG[Y2]-speract and pu rified sperm flagella and heads were consistent with the fluorescent l abeling results on whole cells. The finding that the speract receptor is localized exclusively to the sperm flagella may reveal its role in the regulation of flagellar motility. (C) 1994 Academic Press, Inc.