ABSTRACT
Vasopressin, oxytocin as well as other active nonapeptides (vasotocin, etc) are difficult to isolate from tissues. Traditionally they were identified using cumbersome biological assays or immunoassays, commercially unavailable, and with some cross reactivity. Based on the fact that all these peptides have two Cysteines in their molecules we developed a simple, sensitive and specific method to detect them by HPLC after pre-column fluorescent derivatization with monobromobimane (mBBr). The peptides were separated on a Vydac C18 column after reduction with Tris (2-carboxyethyl) phosphine (TCEP) and derivatization with mBBr for 5 minutes in dark. Using this method we were able to detect specific peaks for arginine-, lysine-vasopressin, and vasotocin at levels as low as 10 pmol. The method can be used to detect other active peptides with cyst(e)ins in their molecule, as well.
Subject(s)
Biochemistry/methods , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Cysteine/chemistry , Peptides/chemistry , Peptides/isolation & purification , Arginine Vasopressin/isolation & purification , Chromatography, High Pressure Liquid , Disulfides , Lypressin/isolation & purification , Phosphines/pharmacologyABSTRACT
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.
Subject(s)
Artiodactyla/metabolism , Lypressin/analysis , Animals , Arginine Vasopressin/analysis , Female , Humans , Lypressin/isolation & purification , Male , Mammals/metabolism , Oxytocin/analysis , Pituitary Gland, Posterior/analysis , Placenta , Species SpecificityABSTRACT
Vasopressin (VP)-like immunoreactivity (IR) has been located in the testes of several species of mammal. There is evidence that most of this IR in the rat does not represent authentic arginine vasopressin (AVP) and that a second AVP-like peptide may exist. We have studied testis samples from the pig, which produces lysine vasopressin (LVP) in its pituitary, and have found both LVP- and AVP-like IR. High-performance liquid chromatography (HPLC) of testis extracts showed two peaks of VP-IR. The first peak co-eluted with authentic LVP and was recognized only by antisera which cross-reacted with LVP. The second peak co-eluted with authentic AVP and was recognized by antisera raised against AVP. Both VP-like peptides bound to a neurophysin affinity column and the HPLC elution profiles of the bound peptides were similar to those of the authentic hormones. When the LVP-like material was oxidized with performic acid, a peak of IR running in the same position as oxidized authentic LVP on HPLC was produced. Similarly, the performic acid-oxidized AVP-like material co-eluted with oxidized authentic AVP. The presence of both LVP- and AVP-like peptides in the pig testis may mean that more than one gene is involved. A second VP-like gene could also explain the anomalies of VP-IR in other species.
Subject(s)
Peptide Fragments/isolation & purification , Swine/physiology , Testis/metabolism , Vasopressins/isolation & purification , Animals , Arginine Vasopressin/isolation & purification , Chromatography, Affinity , Chromatography, High Pressure Liquid , Lypressin/isolation & purification , MaleABSTRACT
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.
Subject(s)
Arginine Vasopressin/analysis , Lypressin/analysis , Opossums/metabolism , Oxytocin/analysis , Pituitary Gland, Posterior/analysis , Amino Acids/analysis , Animals , Arginine Vasopressin/isolation & purification , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Lypressin/isolation & purification , Oxytocin/isolation & purification , Species SpecificityABSTRACT
The neurohypophysial hormones of the quokka wallaby (Setonix brachyurus) have been isolated through preparative high pressure liquid chromatography (HPLC). One oxytocin-like and two vasopressin-like peptides have been found. These peptides have been characterized by their amino acid composition and by their retention time in HPLC. Mesotocin ([I1e8]-oxytocin) has been identified by amino acid composition, polar partition chromatography, high pressure liquid chromatography with a reversed phase column, and pharmacological properties. Lysine vasopressin and phenypressin ([Phe2]-arginine vasopressin) have been characterized by amino acid composition, ion-exchange chromatography, and high pressure liquid chromatography. Lysine vasopressin is about twice as abundant as phenypressin. These three peptides have previously been found in three species belonging to the genus Macropus, namely the red kangaroo, the Eastern gray kangaroo, and the tammar wallaby; they are therefore current in the family Macropodidae. Comparison with other native Australian mammals is discussed.
