PEPTIDE RECEPTORS IN SEA-URCHIN SPERM FLAGELLA

Egg peptides have been isolated from echinoderm species and grouped into families based on their primary structure. Two peptides that have been extensively characterized are speract and resact, isolated from the jellycoats of Strongylocentrotus purpuratus and Arbacia punctulata, respectively. They do not cross-react in any detectable manner with sperm from the other species, but appear to possess nearly identical biological properties and potencies in homologous species. Receptors for the peptides were identified using classic chemical crosslinking techniques. Radiolabeled peptide analogs were synthesized and characterized and used to label putative receptor proteins. A critical component is the development of crosslinkable ligands that resemble closely the natural product. For peptide ligands this is accomplished by synthesizing structural analogs and characterizing their potencies in the appropriate bioassay and in competition binding experiments. Alterations to the primary structure of the peptide may be required to introduce reactive groups or radioiodination sites that may alter the binding kinetics and specificity of the ligand. Different ligands for the same receptor can lead to different patterns of crosslinked proteins. The choice of crosslinking agent and Ph can alter the pattern of tagged proteins when the same radiolabeled ligand is cross-linked to intact spermatozoa. The different patterns may reflect conformational changes in the ligand or receptor that alter the accessibility of the crosslinker to reactive sites. Long or short crosslinking agents may be able to span the distance between reactive groups on the ligand and the receptor. The procedures described in this chapter are collection and washing of sea urchin spermatozoa, radioiodination and purification of ligand, crosslinkage of iodinated peptides to intact sperm and analysis of labeled proteins. The examples have used certain crosslinkers that need to be selected on an individual basis. The amount of radiolabeled ligand added is determined by the binding characteristics of the ligand. © 1995, Academic Press Inc.