Structural characterization of arginine-vasopressin and lysine-vasopressin by Fourier- transform ion cyclotron resonance mass spectrometry and infrared multiphoton dissociation.

Arginine-vasopressin (AVP) and lysine-vasopressin (LVP) were analyzed by reversed-phase liquid chromatography/mass spectrometry (LC-MS) using Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) electrospray ionization (ESI) in the positive ion mode. LVP and AVP exhibited the protonated adduct [M+H](+) as the predominant ion at m/z 1056.43965 and at m/z 1084.44561, respectively. Infrared multiphoton dissociation (IRMPD), using a CO(2) laser source at a wavelength of 10.6 µm, was applied to protonated vasopressin molecules. The IRMPD mass spectra presented abundant mass fragments essential for a complete structural information. Several fragment ions, shared between two target molecules, are discussed in detail. Some previously unpublished fragments were identified unambiguously utilizing the high resolution and accurate mass information provided by the FT-ICR mass spectrometer. The opening of the disulfide loop and the cleavage of the peptide bonds within the ring were observed even under low-energy fragmentation conditions. Coupling the high-performance FT-ICR mass spectrometer with IRMPD as a contemporary fragmentation technique proved to be very promising for the structural characterization of vasopressin.

Cellular colocalization of diuretic peptides in locusts: a potent control mechanism.

Locust abdominal ganglia are shown to colocalize Locusta-diuretic peptide-, leucokinin I-, and lysine vasopressin-like immunoreactivity in posterior lateral neurosecretory cells. Extracts of abdominal ganglia were partially purified by RP-HPLC then dot immunoassay screened with the same antisera used for immunocytochemistry. Locusta-diuretic peptide-like immunoreactive material coeluted with synthetic Locusta-diuretic peptide, and leucokinin-like immunoreactive material coeluted with locustakinin. Lysine vasopressin-like material eluted in fractions that also showed Locusta-diuretic peptide and leucokinin I immunoreactivity. The diuretic activity of synthetic Locusta-diuretic peptide and locustakinin is demonstrated, and they are shown to act at least additively to promote Malpighian tubule fluid secretion. The immunoreactive neurosecretory cells are assumed to express at least these two peptides, and a model for promoting fluid secretion is proposed.

Neurons in the cockroach nervous system reacting with antisera to the neuropeptide leucokinin I.

Antisera were raised against the myotropic neuropeptide leucokinin I, originally isolated from head extracts of the cockroach Leucophaea maderae. Processes of leucokinin I immunoreactive (LKIR) neurons were distributed throughout the nervous system, but immunoreactive cell bodies were not found in all neuromeres. In the brain, about 160 LKIR cell bodies were distributed in the protocerebrum and optic lobes (no LKIR cell bodies were found in the deuto- and tritocerebrum). In the ventral ganglia, LKIR cell bodies were seen distributed as follows: eight (weakly immunoreactive) in the subesophageal ganglion; about six larger and bilateral clusters of 5 smaller in each of the three thoracic ganglia, and in each of the abdominal ganglia, two pairs of strongly immunoreactive cell bodies were resolved. Many of the LKIR neurons could be described in detail. In the optic lobes, immunoreactive neurons innervate the medulla and accessory medulla. In the brain, three pairs of bilateral LKIR neurons supply branches to distinct sets of nonglomerular neuropil, and two pairs of descending neurons connect the posterior protocerebrum to the antennal lobes and all the ventral ganglia. Other brain neurons innervate the central body, tritocerebrum, and nonglomerular neuropil in protocerebrum. LKIR neurons of the median and lateral neurosecretory cell groups send axons to the corpora cardiaca, frontal ganglion, and tritocerebrum. In the muscle layer of the foregut (crop), bi- and multipolar LKIR neurons with axons running to the retrocerebral complex were resolved. The LKIR neurons in the abdominal ganglia form efferent axons supplying the lateral cardiac nerves, spiracles, and the segmental perivisceral organs. The distribution of immunoreactivity indicates roles for leucokinins as neuromodulators or neurotransmitters in central interneurons arborizing in different portions of the brain, visual system, and ventral ganglia. Also, a function in circuits regulating feeding can be presumed. Furthermore, a role in regulation of heart and possibly respiration can be suggested, and probably leucokinins are released from corpora cardiaca as neurohormones. Leucokinins were isolated by their myotropic action on the Leucophaea hindgut, but no innervation of this portion of the gut could be demonstrated. The distribution of leucokinin immunoreactivity was compared to immunolabeling with antisera against vertebrate tachykinins and lysine vasopressin.

Determination of the amino acid sequence of cystine-containing peptides by tandem mass spectrometry.

A method has been developed for the determination of the amino-acid sequence of a cyclic peptide containing cystine. It is based on the reduction of the peptide in a reductive matrix prior to ionization by fast-atom bombardment. The amino-acid sequence of the resulting linear peptide is then determined by tandem mass spectrometry from the spectrum produced by the collision-induced decomposition of the [M + H]+ ion of the peptide.

Isocratic high-performance liquid chromatography-photodiode-array detection method for determination of lysine- and arginine-vasopressins and oxytocin in biological samples.

A simple, isocratic, sensitive (1 ng), and specific high-performance liquid chromatographic (HPLC) method based on photodiode-array detection (PAD) is described for simultaneous quantitation of the bioactive peptides, lysine vasopressin (LVP), arginine vasopressin (AVP) and oxytocin (OXY). Acidified pig plasma and left ventricular (LV) tissue samples were first extracted with Sep-Pak C18 columns, and the bioactive peptides were eluted with methanol, then dried at 37 degrees C and reconstituted with HPLC mobile phase. The bioactive peptides were separated by HPLC on a Dynamax 3009-A C8 column with a mobile phase of 0.1% trichloroacetic acid-50 mM heptanesulfonic acid-30mM triethylamine-20% acetonitrile in water, pH 2.5 and identified with a Waters 990-PAD system (spectrum index plots in the range 200-400 nm). Standards of LVP, AVP and OXY and their mixtures showed a linear increase in the range 5 to 100 ng and were eluted at 6.1, 6.9 and 4.6 min, respectively. Spectrum analysis showed a distinct absorption peak at 280 nm, corresponding to peptide bonds. The reproducibility of the method coefficient of variation for standards is 6.9, 5.8 and 4.7% for LVP, AVP and OXY, respectively. In plasma and tissue it is much higher: 12.9% (LV tissue) and 18.6% (plasma) for LVP. Pig plasma contains negligible amounts of AVP and OXY; LVP is much higher (0.28 +/- 0.19 ng/ml). In pig tissue, LVP predominates (6.95 ng/g wet weight) compared to AVP (1.45) and OXY (1.50). Spectral analysis is necessary to identify the bioactive peptide peaks among interfering substances and to increase the sensitivity four-fold. The method described here is useful for the simultaneous determination of LVP, AVP and OXY in the nanogram range and can be extended to picogram levels by employing PAD spectral analysis techniques.

Effect of available surface water on levels of antidiuretic hormone (lysine vasopressin) and water and electrolyte metabolism of the Rottnest Island quokka (Setonix brachyurus).

A sensitive radioimmunoassay was developed to measure circulating levels of the neurohypophysial peptide lysine vasopressin (LVP) in the marsupial quokka (Setonix brachyurus), which is abundant on Rottnest Island off the coast of Western Australia. Animals from locations on the island where free water is completely absent were compared in midsummer with animals from sites where brackish water is available and utilized by the quokkas. In the animals from West End, where free water is absent, circulating levels of LVP averaged 89.2 +/- 19.6 pg/ml, which was significantly higher than the mean level of 35.6 +/- 15.8 pg/ml measured in individuals collected from the Lakes site with access to brackish drinking water. Rates of water and sodium turnover, measured with isotopes, were significantly greater in Lakes than West End animals, as were renal clearances of sodium, chloride, urea, and total osmolytes. Despite an obvious osmotic diuresis resulting from the ingestion of salty water, the Lakes animals were in better physical condition at the end of summer than the West End animals which lack free water, and these latter individuals showed signs of slight dehydration with elevated plasma and urinary electrolyte concentrations and osmolalities.

Identification of neurohypophysial peptides in the ovaries of several mammalian and nonmammalian species.

Ovarian tissue from a variety of mammalian and nonmammalian species were extracted in acid. All extracts contained both oxytocin- and vasopressin-like immunoreactivites as determined by radioimmunoassay. Analysis by high performance liquid chromatography revealed the presence of oxytocin in all ovarian extracts examined. This was in contrast to the corresponding posterior pituitary gland which other workers have shown do not necessarily contain the oxytocin peptide. It is suggested that oxytocin may play an important role in ovarian function in species of differing phylogeny.

The distribution of lysine vasopressin (lysipressin) in placental mammals: a reinvestigation of the Hippopotamidae (Hippopotamus amphibius) and Tayassuidae (Tayassu angulatus) families.

The neurohypophyseal hormones of the hippopotamus (Hippopotamus amphibius) and collared peccary (Tayassu angulatus) were isolated by molecular sieving and preparative high-pressure liquid chromatography (HPLC). Oxytocin and arginine vasopressin have been identified by their amino acid compositions and their retention times in HPLC. Lysipressin (lysine vasopressin) was not detected in posterior pituitaries of two hippopotami and nine peccaries (less than 2% of arginine vasopressin in molar ratios). Among the suborder Suiformes of Artiodactyla, the families Hippopotamidae and Tayassuidae do not seem to possess lysipressin, in contrast to the family Suidae in which the pig has lysipressin in place of arginine vasopressin.

Dual duplication of neurohypophysial hormones in an Australian marsupial: mesotocin, oxytocin, lysine vasopressin and arginine vasopressin in a single gland of the northern bandicoot (Isoodon macrourus).

Neurohypophysial hormones of an Australian marsupial, the Northern bandicoot (Isoodon macrourus), have been identified by their retention times in high-pressure reverse-phase liquid chromatography using two solvent systems and by their molar pressor or uterotonic activities. Two pressor peptides, arginine vasopressin and lysipressin, and two uterotonic peptides, mesotocin and oxytocin, have been characterized. Because mesotocin and arginine vasopressin have been identified in three other Australian marsupial families, it is assumed that a duplication of each ancestral gene occurred in Peramelidae and subsequent mutations in one copy led to the additional oxytocin and lysipressin. A similar dual duplication of neurohypophysial hormones has previously been discovered in the North-American opossum (Didelphis virginiana) so that the duplication propensity seems peculiar to marsupials in contrast to placental mammals.

Development of an immunoassay for glypressin, an N-terminal extended vasopressin analogue.

The development and evaluation of a radioimmunoassay for N alpha-tri-glycyl-lysine8-vasopressin is described. The site of hapten conjugation of the immunogen has been controlled and the use of various radiolabelled tracers has been evaluated with special reference to the site of iodination. The most extensively studied antiserum showed specificity for the N-terminal triglycyl-extension as well as for several amino acid residues of the vasopressin ring. It crossreacted 27%, 28%, and 0.3% with Lys8-vasopressin, arg8-vasopressin and oxytocin respectively, and it was used to quantify triglycyl-lysine8-vasopressin in human plasma after SepPak C18 extraction. The sensitivity of the assay was 5 pg/tube with an intra-assay CV of 5-6% at 17 and 70 pg/tube. The identity of the immunoreactivity was studied by reversed phase chromatography.

Neurohypophyseal hormones as evolutionary tracers: identification of oxytocin, lysine vasopressin, and arginine vasopressin in two South American opossums (Didelphis marsupialis and Philander opossum).

The neurohypophyseal hormones of two South American opossums (Didelphis marsupialis and Philander opossum) were isolated by molecular sieving and preparative high-pressure liquid chromatography (HPLC). One oxytocin-like and two vasopressin-like peptides were found in each species. These peptides have been identified by their amino acid composition and by their retention time in HPLC. Oxytocin, lysine vasopressin, and arginine vasopressin have been characterized in both species. Lysine vasopressin is roughly as abundant as arginine vasopressin. Comparison is made with Australian marsupials Macropodidae and Phalangeridae, and possible evolutionary mechanisms are discussed.

Identification of mesotocin, lysine vasopressin, and phenypressin in the eastern gray kangaroo (Macropus giganteus).

The neurohypophysial hormones of the eastern gray kangaroo (Macropus giganteus) have been isolated through molecular sieving and paper chromatoelectrophoresis. One oxytocin-like and two vasopressin-like peptides have been found. These peptides have been characterized by amino acid analysis. Mesotocin ([Ile8]-oxytocin), has been identified both by amino acid composition and by behavior in partition chromatography. Lysine vasopressin has been characterized by amino acid composition and by partial amino acid sequence determination. Phenypressin ([Phe2]-arginine vasopressin) has been identified by amino acid composition. Lysine vasopressin is about twice as abundant as phenypressin. These three peptides have previously been identified in two other macropodids, namely, the red kangaroo and the tammar wallaby, and seem to be present in all the family Macropodidae. The evolution of neurohypophysial hormones is discussed in regard to these results.

Synthesis and biological activities of [7-(azetidine-2-carboxylic acid)]-oxytocin and -lysine-vasopressin.

[7-(Azetidine-2-carboxylic acid)]-oxytocin and -lysine-vasopressin have been synthesised by a (6 + 3) strategy using protected hexapeptide acids with preformed disulphide bridges, and their biological activities have been investigated. All activities were reduced but not to the same extent. In assays of pressor and antidiuretic activity it was observed consistently that the responses to the vasopressin analogue were of shorter duration than responses to lysine-vasopressin of the same amplitude.

The relationship of radioimmunoassay to bioassay: in vitro studies with synthetic lysine vasopressin in aqueous solution inactivated by heat.

The relationship of radioimmunoassay to pressor assay and antidiuretic assay was investigated in a simple in vitro system of synthetic lysine vasopressin in aqueous solution inactivated by heating at 100 degrees C for 9, 18, 27, 36, 54 and 72 h. An apparent dissociation between radioimmunoassay and bioassay was demonstrated, with biological activity being lost more rapidly than immunological activity. The half-times were 32 h for radioimmunoassay, 23 h for antidiuretic assay and 22 h for pressor assay. However, ion-exchange chromatography showed immunological heterogeneity but biological homogeneity of the lysine vasopressin used, and indicated that the presence of impurities in the vasopressin might to some extent explain the discrepancy between assay results. Synthetic arginine vasopressin and arginine vasopressin of pituitary origin showed a similar immunological heterogeneity by ion-exchange chromatography.

Post-column fluorescence derivatization of peptides. Problems and potential in high-performance liquid chromatography.

Some critical parameters such as the pumping system, mixing devices and detector design in instrumentation for post-column derivatization in high-performance liquid chromatography are discussed. The derivatization was studied with pharmaceutically important nona-peptides containing primary amino groups which react with Fluram and reaction parameters such as pH, solvent and reagent concentration were investigated. Both adsorption systems and reversed-phase systems were used to separate the peptides prior to the post-column reaction. Reversed-phase chromatography has the advantage of simpler sample preparation, better reaction control and optimization of solvent conditions. As a result, detection limits of between 5 and 10 ng per injection can be obtained and the reproducibility of the results is better than +/- 2% (relative standard deviation). The method has been applied to the analysis of injection solutions (ampoules).