The degradation of corticotropin-releasing factor by enzymes of the rat brain studied by liquid chromatography-mass spectrometry.

The corticotropin-releasing factor (CRF; 41 amino acid residues) is a major regulatory peptide in the response to stress and is distributed over many regions of the brain. We have studied the enzymatic degradation of CRF and related peptides by the CRF-degrading enzyme(s) of the rat brain (CRF-DA) by liquid-chromatographic-mass spectrometric technique and by online tandem mass spectrometric experiments. Peptide fragments of the human/rat CRF (1-41) generated by the CRF-DA of the particulate cell fraction were separated and structurally assigned.

Identification and measurement of beta-endorphin levels in the skin during induced hair growth in mice.

We describe new and effective techniques for extracting proopiomelanocortin (POMC)-derived peptides from mammaliar skin. Using this methodology (hot-acid extraction) and two independent HPLC-controlled RIA systems, we identify beta-endorphin peptide in mammalian skin and demonstrate significant hair cycle-dependent fluctuations in both the skin concentration and the in situ expression pattern of beta-endorphin (sebaceous glands) during the entire murine hair cycle. The observed anagen (growth phase) associated increase in beta-endorphin concentration and its decline during the follicle involution (catagen) or resting (telogen) phase raise the possibility of a regulatory function of this neuropeptide in cyclic changes of skin physiology.

Liquid chromatographic-mass spectrometric studies on the enzymatic degradation of beta-endorphin by endothelial cells.

An on-line HPLC-mass spectrometric procedure with an electrospray atmospheric pressure ionization (ESI-API) ion source was developed to identify the enzymatic degradation products (peptides) generated by incubation of human beta-endorphin (h beta E) with cultured aortic endothelial cells. The samples from the complex incubation mixture were prepurified and enriched using a small reversed-phase (RP) perfusion precolumn. Flow switching was applied to transfer the peptides from this precolumn to the analytical RP column of 2 or 0.

Pathways of degradation of buserelin by rat kidney membrane.

The pathways of in vitro degradation of the gonadotropin-releasing hormone (GnRH) analog buserelin [pGlu-His-Trp-Ser-Tyr-D-Ser(tBu)-Leu-Arg- ProNHEt, B1-9] by the rat kidney membrane fraction was investigated using high-performance liquid chromatography for the separation of the peptide products and electrospray mass spectrometry for their identification. The N-terminal peptides B1-4, B1-3, B1-2, C-terminal peptides B3-9, B4-9, B5-9, B6-9, middle sequence B3-4 and the amino acids Trp, Ser and Tyr were found to be formed. However, due to extreme differences in the stability of the peptides toward the battery of membrane enzymes (B1-2, B6-9 >> B1-3, B5-9 >> B1-9 >> B1-4 > B4-9 > B3-9, B3-4), the final products of buserelin degradation were B1-2, B1-3, B5-9, and B6-9 and the amino acids Ser and, corresponding to the formation of B1-2 and B6-9, Trp and Tyr, respectively.

Liquid chromatographic-mass spectrometric studies on the enzymatic degradation of gonadotropin-releasing hormone.

Gonadotropin-releasing hormone (GnRH) derivatives are used in cancer therapy, but relatively little is known about their metabolic fate in the organism. This paper describes the application of high-performance liquid chromatography combined with electrospray mass spectrometry to identify the degradation products resulting from the incubation of two GnRH analogues, D-Phe6-GnRH and DSer(OtBu)6-desGly10-GnRH-ethylamide (buserelin) with rat kidney membranes. Reversed-phase columns were applied with gradient elution using a flow-rate of ca.

Spontaneous chemical degradation of substance P in the solid phase and in solution.

The instability of the undecapeptide substance P (SP), a neuropeptide implicated in several physiological processes, was occasionally observed when the peptide was stored in the solid state or in solution. The aim of the present study was to identify the decomposition products of SP stored as lyophilized peptide or in aqueous neutral solution. The main pathway of the decomposition of SP acetate consists of the subsequent release of N-terminal dipeptides via their diketopiperazines, cyclo(Arg-Pro) and cyclo(Lys-Pro).

In-vivo release of a GnRH agonist from a slow-release poly(lactide-glycolide) copolymer preparation: comparison in rat, rabbit and guinea-pig.

Different batches of 50:50 poly((+-)-lactide-glycolide) copolymer (PLG) were used as biodegradable carriers for D-Phe6-gonadotropin-releasing hormone (GnRHa) in the form of injectable long-acting implants loaded with 10% GnRHa and tracer amounts of [125I]GnRHa. After their injection subcutaneously into rats, rabbits, and guinea-pigs, the release kinetics of the peptide were determined by counting the radioactivity remaining in the implants (i) after recovery from the rats after death or (ii) directly on the skin above the injection site of rabbits and guinea-pigs in-vivo. No significant differences in the release pattern of the peptide amongst the three species whether the release process was controlled by diffusion or by degradation of the polymeric matrix were found.

Gonadotropin-releasing hormone (GnRH) analogs: relationship between their structure, proteolytic inactivation and pharmacokinetics in rats.

There are two types of superactive agonists of gonadotropin-releasing hormone (GnRHa-I: (D-amino acid)6-GnRH and GnRHa-II: (D-amino acid)6-(desGly)10-GnRH- ethylamide) the high hormonal activity of which is understood to be due to their higher receptor affinity and their higher proteolytic stability as compared with the native GnRH sequence. Using the soluble fractions of various rat tissues in studies on the inactivation of GnRH peptides, we confirmed the higher proteolytic resistance of GnRHa-II, but not of D-Phe6-GnRH (GnRHa-I) and of another analog, D-Trp3-D-Phe6-GnRH, as compared with GnRH. The exact behaviour of the peptides during degradation was found to be dependent on the peptide concentrations used, showing the importance of using conditions as near to the physiological ones a possible.

Insulin aggregation in solution.

The process of insulin aggregation in neutral solutions was studied by dynamic light scattering. Solutions of different concentrations were subjected to thermal and mechanical stress (37 degrees, rotation) for a period of 4 weeks. The starting solutions contained exclusively one particle distribution of insulin in the association equilibrium with hexamers as the largest structures.

Proteolytic inactivation of luteinizing hormone-releasing hormone (LHRH) by the whole rat ovary in vitro.

Using 3H-labeled luteinizing hormone-releasing hormone (LHRH) at low concentrations, the in vitro proteolytic inactivation of the peptide hormone by whole rat ovaries was studied and compared with that by the soluble and particulate rat ovarian fraction. Whole rat ovaries were found to express the three proteolytic activities that were, according to their properties, also observed in rat ovarian homogenates: (1) soluble intracellular activity which was released into the medium, (2) released activity of membrane-bound origin, and (3) firmly membrane-bound activity. It is suggested that in vivo LHRH is largely inactivated extracellularly at least by enzymes that are located in the plasma membrane although the membrane-bound activity comprises only about 1% of the whole LHRH-inactivating capacity of the ovary.

Gonadotropin-releasing hormone (GnRH) pharmacokinetics: peptide hormone pharmacokinetics needs clarification.

The plasma level curves of the peptide hormone gonadotropin-releasing hormone (GnRH) after its intravenous, intramuscular, and intraperitoneal administration into rats were fitted according to a two- (i.v.) and one-compartment model (i.

[The purification and characterization of Cordemcura].

3-Amino-5-(4-pyridinyl)-1,2-dihydro-pyrid-2-one (1) is an amphoteric compound and forms one crystalline sodium salt and two hydrochlorides. Physicochemical properties UV, NMR and MS are described. TLC has been used mainly and is the most sensitive method for estimation of 1-byproducts.

Effect of dextran on the release of gonadotropin-releasing hormone (GnRH) injected into rats: plasma GnRH and gonadotropin response.

Prolonged release of the peptide gonadotropin-releasing hormone (GnRH) from its aqueous solution was achieved by addition of the polymer dextran (Mw ∼ 500,000). This effect observed in an in vitro system was caused by a decrease of the diffusion coefficient of the peptide. When GnRH was intramuscularly injected into male rats, the addition of dextran to the injected peptide solution led to a prolongation of the GnRH plasma level at the expense of its peak value.

[The influence of viscosity-increasing pharmaceutic aids on the liberation of the peptide gonadotropin releasing hormone (GnRH) in solution].

The in vitro liberation of the peptide hormone GnRH from polymer solutions was studied as a function of the viscosity of the polymer solutions. The liberation experiments were performed with a flow through dialysis apparatus. From solutions of dextran, carboxymethylcellulose, hydroxyethylcellulose, and polyvinylpyrrolidone the release of GnRH is prolonged whereas the release of GnRH from highly viscous solutions of methylcellulose and polyacrylic acid is not.

[Synthesis of cyclic and non-cyclic tachykinin partial sequences. 2. Synthesis of the homocyclic substance P(6-11) hexapeptide].

The authors describe the synthesis of Gln-Phe-Phe-Gly-Leu-Met by cyclization of H-Leu-Met-Gln-Phe-Phe-Gly using three different methods. The linear sequence was obtained by a (2+4)-segment condensation. The resulting cyclopeptide showed only a small kinin activity on isolated guinea pig ileum compared to substance P, but it is a full agonist.

Direct tritium-labelling into histidine of luteinizing hormone-releasing hormone agonists and use of the tracers in studies on proteolytic breakdown.

Analogs of luteinizing hormone-releasing hormone (LHRH) having higher biological activity than LHRH itself are being mainly used to study the biological effects and the mechanism of action of LHRH. In the present study, conditions for the direct 3H-labelling at the histidine residue of analogs of LHRH were worked out, circumventing the synthesis of precursor peptides for labelling. [D-Phe6,desGly10]-LHRH ethylamide and [D-Ser(But)6,desGly10]-LHRH ethylamide were tritiated by tritium gas and a 10% Pd/Al2O3 catalyst to high specific radioactivities.

Proteolytic degradation of gonadotropin-releasing hormone (GnRH) by rat ovarian fractions in vitro.

GnRH in physiological concentrations is highly degradable by both soluble and particulate fractions of rat ovarian homogenate in vitro. The two proteolytic enzyme activities differ strongly by the soluble activity showing a dithiothreitol optimum, high inhibition by diisopropyl fluorophospate (ki = 0.7 microM), and a relatively high affinity (Km = 1.

In vitro degradation of substance P: new rapid assay and distribution in rat brain.

An ion exchange batchwise procedure was developed for the estimation of substance P degradation by rat brain fractions using 3H[Nle11]substance P as tracer. With this very sensitive and rapid method an especially high degradative activity was found among others in the microsomal fractions of the striatum.

Circular dichroism studies of substance P and its C-terminal sequences. CD spectra in aqueous solution and effects of hydrogen ion concentration.

CD spectra of substance P (SP) and its C-terminal partial sequences have been measured in diluted aqueous solution including variation of hydrogen ion concentration. In the far u.v.

[Serological identification of spectrin in the 2 dimensional immunoelectrophoresis].

Using polyvalent antisera directed against solubilized human erythrocyte ghosts in the immunoelectrophoresis we obtained some precipitation arcs. Antisera against the purified spectrin allowed the identification of spectrin in the crossed immunoelectrophoresis.

[Studies on the mechanism of action of peptides attacking smooth muscles. III. The effect of N-azylation on the activity of C-terminal partial sequences of eledoisin, physalaemin and substance P on the guinea pig ileum].

C-terminal pentapeptides of eledoisin, physalaemin, and the substance P, when N-substituted with acetyl, halogenacetyl and other acyl residues, are increased in their action more than 100fold, reaching the activity of acylated hexa- and heptapeptides. The effect found with a number of compounds is interpreted as the influence of predominantly hydrophobic substituents upon the peptide sequence essential for the action. Polar groups in the acylic residue seem to cause additional increase in action.