Retention of the Speract Receptor by Isolated Plasma Membranes of Sea Urchin Spermatozoa1

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Abstract
Membrane vesicle preparations enriched in plasma membrane marker proteins, such as adenylate cyclase, were prepared from spermatozoa of the sea urchin, Lytechinus pictus. These membranes, prepared by nitrogen cavitation and subsequent sucrose gradient centrifugation, retained the capacity to bind [125I]-Bolton-Hunter speract (nonspecific binding was less than 5% of specific binding). Speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly), Tyr-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly, Tyr-Asp-Leu-Thr-Thr-Gly-Gly-Gly-Val-Gly and Gly-Phe-Ala-Leu-Gly-Gly-Gly-Val-Gly caused a 50% decrease in [125I]-Bolton-hunter speract binding at 10, 600, 1260 and 3160 nM concentrations, respectively. One analogue (Phe-Asp-Leu-Asn-Gly-Gly-Gly), which had no biological activity, failed to compete at concentrations as high as 10 .mu.M. To demonstrate that the binding was due to the isolation of membranes with an intact receptor, the speract analogue (Gly-Gly-Gly-Gly-Tyr-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) was synthesized, radiolabeled with 125I at the position of tyrosine, and covalently cross-linked to the receptor with disuccinimidyl suberate. A single radiolabeled band at an apparent molecular weight of 77,000 was detected on Na .cntdot. dodecyl .cntdot. SO4 gels. These studies are the first to identify a receptor for egg-associated peptides in isolated spermatozoan membranes.