Abstract
These studies are the first to report egg peptide-mediated stimulation of protein phosphorylation in spermatozoa. Speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) or resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-Leu-NH2) stimulated the incorporation of 32P into various proteins of isolated spermatozoan membranes in the presence, but not absence, of GTP. The Mr of three of the phosphorylated proteins were 52,000, 75,000, and 100,000. GTP gamma S (guanosine 5'-O-(3-thiotriphosphate] but not GDP beta S (guanosine 5'-O-(2-thiodiphosphate] or GMP-PNP (guanylyl imidodiphosphate) also supported the peptide-mediated stimulation of protein phosphorylation. The peptides markedly stimulated guanylate cyclase activity, and GTP gamma S or GTP but not GMP-PNP served as effective substrates for the enzyme. The accumulation of cyclic AMP was not stimulated by the peptides. Subsequently, it was shown that added cyclic GMP or cyclic AMP increased 32P incorporation into the same membrane proteins as those observed in the presence of peptide and GTP. The amount of cyclic GMP (up to 3 microM) formed by membranes in the presence of peptide and 100 microM GTP equated with the amount of added cyclic GMP required to increase the 32P content of a Mr 75,000 protein selected for further study. 32P-Peptide maps of the Mr 75,000 protein indicated that the same domains were phosphorylated in response to cyclic nucleotides or to egg peptide and GTP. Intact cells were subsequently incubated with 32P to determine if the radiolabeled proteins observed in isolated membranes also would be obtained in intact cells. The 32P contents of proteins of Mr 52,000, 75,000, and 100,000 were significantly increased by the addition of resact. Peptide maps confirmed that the increased 32P incorporation obtained in a Mr 75,000 protein of isolated membranes occurred on the same protein domains as the 32P found on the Mr 75,000 protein of intact cells. These results suggest that a GTP or GTP gamma S requirement for peptide-mediated protein phosphorylation in spermatozoan membranes is mainly due to the enhanced formation of cyclic GMP, and it therefore is likely that peptide-induced elevations of cyclic nucleotide concentrations in spermatozoa are responsible for the specific increases in 32P associated with at least three sperm proteins, all apparently localized on the plasma membrane.
Highlights
From the $HowardHughes Medical Institute and the Departmentsof §Pharmacology and VMolecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville
Guanylate cyclase activity can be stimulated by the egg peptides in isolated sperm membranes, and dephosphorylation of the enzyme coincides with a decrease in the rate of formation of cyclic GMP [9]
Cyclic Nucleotides Increase 32PIncorporation into the Same Protein as Egg Peptide and GTP-Subsequent experimentation demonstrated that added cyclic AMP or cyclic GMP, in Membranes Resembles That Found in the Intact Cell-To assess whether or nothe same domains were phosphorylated on the MI 75,000 protein and to compare the L. pictus sperm cell M, 75,000 proteinto the A. punctulata spermatozoan protein, proteolytic maps were performed (Fig. 8)
Summary
Materials-Arbaciupuntuhta sea urchins were obtained from Gulf Specimen Co., Inc., Panacea, FL. The abbreviations used are: GTP& guanosine 5’-0-(3-thiotriphosphate; GDPDS, guanosine 5’-0-(2-thiodiphosphate);MES, 4morpholineethanesulfonicacid; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; EGTA, [ethylenebis(oxyethylenenitrilo)] tetraacetic acid; GMP-PNP, guanylyl imidodiphosphate Samples taken for electrophoresis were further processed as described below Conditions were such that linear incorporation of 32Pinto protein was observed as a function of protein concentration. Proteolytic Maps-Coomassie Blue-stained M, 75,000 protein was excised from gels, rinsed three times for 15 min each in 0.1 M Tris, pH 6.5, with 0.1% sodium dodecyl sulfate, and subjected to proteolysis during electrophoresis as described [21]
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