Brian T. Miller

Control of Gonadotropin Secretion by Follicle-Stimulating Hormone-Releasing Factor, Luteinizing Hormone-Releasing Hormone, and Leptin

Fractionation of hypothalamic extracts on a Sephadex G-25 column separates follicle-stimulating hormone-releasing factor (FSHRF) from luteinizing hormone-releasing hormone (LHRH). The FSH-releasing peak contained immunoreactive lamprey gonadotropin-releasing hormone (lGnRH) by radioimmunoassay, and its activity was inactivated by an antiserum specific to lGnRH. The identity of lGnRH-III with FSHRF is supported by studies with over 40 GnRH analogs that revealed that this is the sole analog with preferential FSH-releasing activity. Selective activity appears to require amino acids 5-8 of lGnRH-III. Chicken GnRH-II has slight selective FSH-releasing activity. Using a specific lGnRH-III antiserum, a population of lGnRH-III neurons was visualized in the dorsal and ventral preoptic area with axons projecting to the median eminence in areas shown previously to control FSH secretion based on lesion and stimulation studies. Some lGnRH-III neurons contained only this peptide, others also contained LHRH, and still others contained only LHRH. The differential pulsatile release of FSH and LH and their differential secretion at different times of the estrous cycle may be caused by differential secretion of FSHRF and LHRH. Both FSH and LHRH act by nitric oxide (NO) that generates cyclic guanosine monophosphate. lGnRH-III has very low affinity to the LHRH receptor. Biotinylated lGnRH-III (10(-9) M) labels 80% of FSH gonadotropes and is not displaced by LHRH, providing evidence for the existence of an FSHRF receptor. Leptin has equal potency as LHRH to release gonadotropins by NO. lGnRH-III specifically releases FSH, not only in rats but also in cows.

Peptide Biotinylation with Amine-Reactive Esters: Differential Side Chain Reactivity

N-hydroxysuccinimide (NHS) esters of biotin are reported to react specifically with amino groups of peptides and proteins. However, we have found that these reagents can readily acylate other functional groups in specific peptide sequences under relatively mild conditions. We have extended our inquiry of sequence-dependent acylation by evaluating the reactivity of a variety of commonly employed biotinylation reagents typically used for amino group modification. These included the p-nitrophenyl ester of biotin, NHS-esters of biotin containing aminohexanoic acid spacer arms, and a sulfonated NHS-biotin ester that contained a disulfide bond within its spacer. The decapeptide [D-Lys6]gonadotropin releasing hormone was employed as a model peptide. Reaction products were characterized by high-performance liquid chromatography, amino acid compositional analysis, reaction with hydroxylamine, and mass spectrometry. In addition to the O-acylation of Ser4 and Tyr5 in this peptide, we have also identified a novel biotinylation of the Arg8 side chain.

Hypothalamic control of gonadotropin secretion

Publisher Summary This chapter discusses the hypothalamic control of gonadotropin secretion. The control of gonadotropin secretion is extremely complex as revealed by the research of the past 40 years since the discovery of luteinizing hormone releasing hormone (LHRH), commonly called gonadotropin-releasing hormone (GnRH). This was the second of the hypothalamic-releasing hormones characterized. It stimulates follicle-stimulating hormone (FSH) release, albeit in smaller amounts than luteinizing hormone (LH). For this reason, it was renamed GnRH. Overwhelming evidence indicates that there is a separate follicle-stimulating hormone releasing hormone (FSHRH) because pulsatile release of LH and FSH can be dissociated. In the castrated male rat, roughly half of the FSH pulses occur in the absence of LH pulses, and only a small fraction of the pulses of both gonadotropins are coincident. The hypothalamic areas controlling LH and FSH are separable. Stimulation in the dorsal medial anterior hypothalamic area causes selective FSH release, whereas lesions in this area selectively suppress the pulses of FSH and not LH. Conversely, stimulations or lesions in the medial preoptic region can augment or suppress LH release respectively without affecting FSH release. The putative FSHRH is synthesized in neurons with perikarya in the dorsal anterior hypothalamic area with axons that project to the mid and caudal median eminence to control FSH release selectively.

Binding of cationic cell-permeable peptides to plastic and glass

Cell-penetrating peptides derived from hydrophilic regions of the homeoprotein Antennapedia (Antp) or the transcription-regulating factor Tat have been used to transport several peptide and oligonucleotide cargoes into the interior of cells. Such vector peptides penetrate cells, in part, because they contain multiple lysine and arginine residues. Using radiolabeled peptide cargoes covalently linked to Antp- or Tat-related vectors, or to D-Arg heptamers, we found that a significant amount of the label remained tightly bound to plastic and glass surfaces. Binding of the labeled conjugates was due entirely to the cationic vector moieties. Under certain conditions, such non-specific binding could be mistaken for cellular penetration.

Biotinylated peptides/proteins: I. Determination of stoichiometry of derivatization

A method is described for the determination of the stoichiometry of biotinylation of peptides and proteins after reaction with an N-hydroxysuccinimide ester of biotin containing the extended spacer arm 6-aminohexanoic acid (NHS-epsilon Ahx-biotin). The method of analysis, based on the quantification of phenylthiocarbamyl derivatives of 6-aminohexanoic acid, is able to measure low picomolar amounts of biotinyl derivative. Analyses were performed using an automated on-line hydrolyzer-derivatizer followed by high-performance liquid chromatography. Compositional analyses determined for known peptides were in excellent agreement with analyses obtained by mass spectrometry. Procedures are also described for the production of biotinylated protein probes that can be labeled reproducibly to contain specific amounts of biotin. The analytical advantage and steric freedom provided by the 6-aminohexanoic acid spacer arm argue strongly for the NHS-epsilon Ahx-biotin reagent to be a reagent of choice for the biotinylation of peptides and proteins.

Biotinylated peptides/proteins. II. Identification of biotinylated lysyl phenylthiohydantoins

The identification and characterization of biotinylated lysyl residues in a polypeptide chain by automated sequence analysis is described. An in depth analytical study was conducted for the delineation of N epsilon modification of lysyl residues with N-hydroxysuccinimide esters of biotin and 6-aminohexanoic biotin. Confirmation of the structure of the phenylthiohydantoin derivatives of N epsilon biotinylated lysine was achieved by mass spectrometry. The analytical study focused on the identification of biotinylated lysine-4 of neuropeptide Y which served as a model peptide for the analytical procedures detailed.

Full-length, unprocessed, progastrin peptide (PG) exerts proliferative effects on colonic mucosa of mice via high affinity binding sites, that are specific for gastrin-like peptides with negligible affinity for cholecystokinin

fluid secretion by 71.7 % (p<O.05), epithelial cell damage by 56%, (p<O.05), mucosal congestion and edema by 26% (p<O.O5), and neutrophil infiltration by 60.8%, (p<O.O01). None of the CRH antagonists had any effect on basal fluid secretion. Conclusions: CRH plays a significant proinflammatory role in TxAinduced intestinal secretion and inflammation and the CRH receptor 1, at least in part, participates in the mediation of these responses. These findings may be important in the treatment of gut inflammation in view of the recent development of CRH receptor antagonists. Supported by the Crohns and Colitis Foundation of America, Inc., and the National Institutes of Hea~th (DK 33506).