resact and speract

Topics: Adult; Amino Acid Sequence; Animals; Caenorhabditis elegans; Culture Media, Conditioned; Diarrhea; Egg Proteins; Guanylate Cyclase; Helminth Proteins; Humans; Ligands; Male; Membrane Proteins; Nerve Tissue Proteins; Oligopeptides; Peptides; Protein Structure, Tertiary; Receptors, Atrial Natriuretic Factor; Receptors, Cell Surface; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Sea Urchins; Species Specificity; Spermatozoa

Speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly), a peptide obtained from the culture medium of Strongylocentrotus purpuratus eggs, stimulates the respiration and motility of S. purpuratus spermatozoa under appropriate conditions. Resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-LeuNH2), a peptide obtained from Arbacia punctulata eggs also stimulates the metabolism and motility of A. punctulata spermatozoa, however, it fails to stimulate S. purpuratus spermatozoa. Early biochemical responses of the spermatozoa to the egg peptides include a net H+ efflux and elevations of cyclic AMP and cyclic GMP concentrations. In addition, in A. punctulata spermatozoa, a major plasma membrane protein is modified in response to resact such that its apparent molecular weight shifts from 160,000 to 150,000. If cells are incubated with 32P, the 160,000 molecular weight form of the protein becomes radiolabeled; subsequent addition of resact causes a rapid loss of 32P from the protein. The plasma membrane protein appears to be the enzyme, guanylate cyclase; coincident with the shift in apparent molecular weight, enzyme activity decreases by as much as 90%. Since speract fails to cause these responses in A. punctulata, it can be concluded that the events are receptor-mediated.

Topics: Animals; Female; Guanylate Cyclase; Male; Oligopeptides; Oxygen Consumption; Peptides; Receptors, Cell Surface; Sea Urchins; Sperm-Ovum Interactions

Topics: Amino Acid Sequence; Animals; Cross-Linking Reagents; Female; Male; Molecular Sequence Data; Oligopeptides; Peptides; Receptors, Peptide; Sea Urchins; Sperm Tail; Sperm-Ovum Interactions; Succinimides

Fragmentary evidence indicates that intracellular [Ca2+] (Cai) mediates sperm chemotaxis. However, neither correlations of swimming responses to chemoattractant-induced alterations of Cai nor explanations of how chemoattractant gradients control Cai exist. Here Cai increases produced by the egg peptide speract-not previously known to cause flagellar responses--were prolonged by treatment with 3-isobutyl-1-methylxanthine (IBMX). Flagellar waveform asymmetry then increased 40% and swimming paths became tightly circular. Moreover, both responses required external Ca2+ (as does sperm chemotaxis to eggs and egg products). Cai increases by the established chemotactic peptide resact also required external Ca2+ and were enhanced by IBMX. Therefore, diverse egg peptides may use fundamentally similar mechanisms to control Cai and thereby swimming behavior in chemotaxis. Repetitive increasing additions of speract produced adaptive membrane potential and Cai responses indicating that sperm can detect increasing gradients of egg peptide over a broad concentration range. We offer a model in which shallow or decreasing gradients elevate Cai and redirect swimming paths but sufficiently steep gradients keep Cai low and swimming linear until the egg is reached. A negative-feedback loop, initiated by cGMP-mediated activation of sperm K+ channels and terminated by subsequent inactivation of guanylyl cyclase, may coordinate gradient detection with control of Cai. Continued stimulation of more receptors by steeply increasing gradients of egg peptide thus maintains membrane hyperpolarization and suppresses Ca2+ entry and Cai elevation. The molecular basis for chemotaxis therefore is explained as translation of the spatial gradient of peptide concentration into changes in K+ channel activity in the time domain.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Calcium; Chemotaxis; Cytosol; Egg Proteins; Male; Models, Biological; Oligopeptides; Peptides; Sea Urchins; Sperm Motility; Sperm Tail

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.

Topics: Animals; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Guanylate Cyclase; Guanylyl Imidodiphosphate; Male; Membrane Proteins; Molecular Weight; Oligopeptides; Peptide Mapping; Peptides; Phosphates; Phosphorylation; Sea Urchins; Spermatozoa; Thionucleotides

Resact, a peptide obtained from eggs, causes a change in the Mr, and a loss of 32P from a plasma membrane protein identified as guanylate cyclase. Here, a resact analog (125I-[Tyr1, Ser8] resact) was synthesized and shown to bind to isolated sperm membranes. Resact, but not speract, competed with the radiolabeled ligand for binding. When membranes were prepared under appropriate conditions, guanylate cyclase remained at Mr 160,000; the incubation of membranes with gamma-32P-ATP resulted in the formation of 32P-labeled guanylate cyclase. The addition of resact to the membranes caused a shift in the Mr, a complete loss of 32P, and a 70% reduction in guanylate cyclase activity within 1 min; resact had an ED 50 at 100 nM concentration. Speract failed to cause any of these effects. This represents the first demonstration of receptor-mediated responses of isolated sperm membranes identical to those seen in the intact cell.

Topics: Animals; Binding, Competitive; Cell Membrane; Chemotaxis; Cyclic GMP; Egg Proteins; Guanylate Cyclase; Male; Membrane Proteins; Molecular Weight; Oligopeptides; Peptides; Phosphorylation; Receptors, Cell Surface; Sea Urchins; Spermatozoa

The sea urchin egg peptides speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) and resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Arg-Leu-NH2) bind to spermatozoa of the homologous species (Lytechinus pictus or Arbacia punctulata, respectively) and cause transient elevations of cyclic GMP concentrations (Hansbrough, J. R., and Garbers, D. L. (1981) J. Biol. Chem. 256, 1447-1452). The addition of these peptides to spermatozoan membrane preparations caused a rapid and dramatic (up to 25-fold) activation of guanylate cyclase. The peptide-induced activation of guanylate cyclase was transient, and the subsequent decline in enzyme activity coincided with conversion of a high Mr (phosphorylated) form of guanylate cyclase to a low Mr (dephosphorylated) form. When membranes were incubated at pH 8.0, the high Mr form was converted to the low Mr form without substantial changes in basal enzyme activity. However, the peptide-stimulated activity of the low Mr form of guanylate cyclase was much less than the peptide-stimulated activity of the high Mr form. Activation of the low Mr form by peptide was not transient and persisted for at least 10 min. In addition, the pH 8.0 treatment that caused the Mr conversion of guanylate cyclase also caused an increase in the peptide-binding capacity of the membranes. We propose a model in which activation of the membrane form of guanylate cyclase is receptor-mediated; the extent of enzyme activation is modulated by its phosphorylation state.

Topics: Animals; Cell Membrane; Egg Proteins; Enzyme Activation; Female; Guanylate Cyclase; Kinetics; Male; Oligopeptides; Peptides; Receptors, Cell Surface; Sea Urchins; Sperm-Ovum Interactions; Spermatozoa

Two peptides, speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) and resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-Leu-NH2), which activate sperm respiration and motility and elevate cyclic GMP concentrations in a species-specific manner, were tested for effects on guanylate cyclase activity. The guanylate cyclase of sea urchin spermatozoa is a glycoprotein and it is localized entirely on the plasma membrane. When intact sea urchin sperm cells were incubated with the appropriate peptide for time periods as short as 5 s and subsequently homogenized in detergent, guanylate cyclase activity was found to be as low as 10% of the activity of cells not treated with peptide. The peptides showed complete species specificity and analogues of one peptide (speract) caused decreases in enzyme activity coincident with their receptor binding properties. The peptides did not inhibit enzyme activity when added after detergent solubilization of the enzyme. When detergent-solubilized spermatozoa were incubated at 22 degrees C, guanylate cyclase activity declined in previously nontreated cells to the peptide-treated level. The rate of decline was dependent on temperature and protein concentration. When spermatozoa were first incubated with 32P, the decrease in guanylate cyclase activity was accompanied by a shift in the apparent molecular weight of a major plasma membrane protein (160,000-150,000) and a loss of 32P label from the 160,000 band. Other agents (Monensin A, NH4Cl) which were capable of stimulating sperm respiration and motility also caused decreases of guanylate cyclase activity when added to intact but not detergent-solubilized spermatozoa. The maximal decrease in guanylate cyclase activity occurred 5-10 min after addition of these agents. The enzyme response to Monensin A required extracellular Na+ suggestive that the ionophore caused the effect on guanylate cyclase activity by virtue of its ability to catalyze Na+/H+ exchange. These studies demonstrate that guanylate cyclase activity of sperm cells can be altered by the specific interaction of egg-associated peptides with their plasma membrane receptors.

Topics: Ammonium Chloride; Animals; Cell Membrane; Cyclic GMP; Guanylate Cyclase; Hydrogen-Ion Concentration; Male; Molecular Weight; Monensin; Oligopeptides; Peptides; Polyethylene Glycols; Receptors, Cell Surface; Sea Urchins; Sodium; Spermatozoa; Time Factors

At an extracellular pH of 6.6, a peptide (resact) isolated from the egg jelly of Arbacia punctulata increased the respiration rates of A. punctulata spermatozoa but did not activate sperm cells from Lytechinus pictus. In contrast, speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly), elevated the respiration rates of L. pictus but not A. punctulata spermatozoa. At normal seawater pH (7.6-8.0) egg jelly from A. punctulata, or egg jelly from L. pictus purified free of speract, inhibited L. pictus sperm respiration rates. Similarly, the egg jelly from L. pictus inhibited the respiration rates of A. punctulata spermatozoa. The jelly component responsible for the inhibition of respiration was nondialyzable. The inhibition of respiration induced by jelly could be reversed by the addition of speract to L. pictus spermatozoa and by the addition of resact to A. punctulata spermatozoa. Speract stimulated L. pictus sperm respiration half-maximally at about 1 nM in the presence of either heterologous or homologous (speract-free) jelly. Monensin A, an ionophore which elevates sperm intracellular pH, reversed the jelly inhibition of respiration. These results demonstrate that two peptides associated with eggs (speract and resact) can stimulate sperm motility and metabolism in the face of inhibitory components present in the egg jelly. Additionally, the peptides demonstrate species specificity.

Topics: Animals; Extracellular Space; Female; Gels; Hydrogen-Ion Concentration; Male; Oligopeptides; Ovum; Oxygen Consumption; Peptides; Sea Urchins; Species Specificity; Sperm Motility; Spermatozoa; Stimulation, Chemical