Covalent coupling of a resact analogue to guanylate cyclase.
GGGYG-resact (Gly-Gly-Gly-Tyr-Gly-Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg -Leu-NH2) was synthesized and shown to possess the same respiration-stimulating activity and receptor-binding ability as resact. The incubation of intact sperm cells with radioiodinated peptide, 125I-GGGYG-resact, and the chemical cross-linking reagent, disuccinimidyl suberate, resulted in the appearance of a single, major radioactive band of apparent molecular weight 160,000 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The interaction was specific since 150 nM nonradioactive resact but not speract (200 nM) blocked formation of the radioactive band. The radioactive, cross-linked protein co-migrated with 32P-labeled guanylate cyclase and could be immunoprecipitated with a polyclonal antibody raised in rabbits against the sperm guanylate cyclase. The incubation of intact cells with NH4Cl resulted in the partial dephosphorylation of guanylate cyclase and a change in its apparent molecular weight from 160,000 to 150,000; NH4Cl also caused the same conversion in the apparent molecular weight of the cross-linked protein. These data demonstrate that an analogue of resact can be covalently coupled to guanylate cyclase with the specificity predicted for the peptide receptor.
D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D003432:Cross-Linking Reagents / Q000494:pharmacology; D006162:Guanylate Cyclase / Q000378:metabolism*; D007163:Immunosorbent Techniques; D008297:Male; D008970:Molecular Weight; D010455:Peptides / Q000378:metabolism*; D010766:Phosphorylation; D012617:Sea Urchins; D013094:Spermatozoa / Q000187:drug effects / Q000378:metabolism; D013388:Succinimides / Q000494:pharmacology
D003432:Cross-Linking Reagents; D010455:Peptides; D013388:Succinimides; D000643:Ammonium Chloride; C042019:resact; D006162:Guanylate Cyclase; C019358:disuccinimidyl suberate