J. Biol. Chem., Vol. 259, Issue 22, 13712-13716, 11, 1984

Identification and partial characterization of the receptor for speract

LJ Dangott and DL Garbers

Here, we report the first identification of a sperm receptor for an egg- associated peptide (speract). Gly-Gly-Gly-Gly-Tyr-Asp-Leu-Asn-Gly-Gly- Gly-Val-Gly (GGG[Y2]-speract), was chemically synthesized and shown to possess equivalent respiration-stimulating activity and the same receptor binding characteristics as speract (Gly-Phe-Asp-Leu-Asn-Gly- Gly-Gly-Val-Gly). The incubation of Strongylocentrotus purpuratus intact sperm cells with radioiodinated GGG[Y2]-speract and the chemical cross-linking reagent, disuccinimidyl suberate, resulted in the appearance of a single radiolabeled band with an estimated Mr = 77,000 (determined by Na dodecyl SO4-gel electrophoresis under reducing conditions). The covalent coupling of 125I-GGG[Y2]-speract was prevented by biologically active analogues of speract but not by inactive analogues and the 125I analogue was not detectably coupled to any protein in Arbacia punctulata spermatozoa, a species with which speract does not cross-react. Comparisons of the amount of 125I-GGG[Y2]- speract bound in the presence of variable concentrations of unlabeled GGG[Y2]-speract showed that about 1 nM unlabeled peptide reduced 125I- GGG[Y2]-speract binding by 50% when estimated as either specific binding to intact cells or as the reduction in radioactivity found in the 77,000 molecular-weight band on autoradiographs after covalent coupling. The radiolabeled receptor could be solubilized with 0.5% Lubrol PX and was bound to wheat germ lectin-Sepharose. It could be subsequently eluted with N-acetylglucosamine or diacetylchitotriose but not by methyl-alpha-D-mannoside or NaCl, suggestive that the receptor is a glycoprotein.
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