pressinoic-acid and tocinoic-acid

Methodology is described for characterization of the kinetics and equilibria of thiol/disulfide interchange reactions of the disulfide bonds in the neurohypophyseal peptide hormones arginine vasopressin and oxytocin and the related peptides pressinoic acid and tocinoic acid. Thiol/disulfide interchange reaction mixtures are analyzed by reversed-phase high-performance liquid chromatography. The effect of mobile-phase composition and pH on the HPLC capacity factors for the native disulfide and reduced dithiol forms of each peptide was examined. In each case, the capacity factor decreases as the acetonitrile content of the mobile phase increases. For each disulfide/dithiol peptide pair, the capacity factor is larger for the dithiol form of the peptide, indicating that the hydrophobic side chains of the linear peptide are more accessible for interaction with the hydrophobic stationary phase. To illustrate application of the methodology, rate and equilibrium constants are reported for the thiol/disulfide interchange reactions of cysteine with arginine vasopressin at pH 7.0. Cysteine reacts with arginine vasopressin to form two mixed disulfides, which in turn react with another molecule of cysteine to give the dithiol form of arginine vasopressin and cystine. Rate and equilibrium constants were determined for each step by analysis of reaction mixtures by HPLC. The results are compared to rate and equilibrium constants for reaction of cysteine with oxidized glutathione.

Topics: Amino Acid Sequence; Arginine Vasopressin; Chromatography, High Pressure Liquid; Disulfides; Hydrogen-Ion Concentration; Kinetics; Molecular Sequence Data; Oxidation-Reduction; Oxytocin; Pituitary Gland, Posterior; Sulfhydryl Compounds; Vasopressins

To characterize the V2 receptor (for antidiuretic hormone), we have studied the effect of a number of neurohypophysial hormone analogues on cyclic AMP (cAMP) accumulation and short-circuit current in cultured epithelia formed by A6 cells. A6 is the designation of a continuous cell line derived from the kidney of Xenopus laevis. The order of potency for stimulating cAMP accumulation and short-circuit current in A6 epithelia is like that for stimulating water permeability in toad urinary bladder. As anticipated, arginine vasotocin (AVT), the antidiuretic hormone of Amphibia, is more potent than arginine vasopressin (AVP), the antidiuretic hormone of most mammals. The two hormones differ only in the third amino acid (Phe-3 in AVP is a substitution for Ile-3 in AVT). However, there are a number of striking differences in the responsiveness of these amphibian V2 receptors and mammalian V2 receptors to changes in the 7th, 8th, and 9th amino acids where AVT and AVP are identical. 1) Substitution of Lys-8 for Arg-8 in AVP results in marked loss of potency in Amphibia, whereas there is only modest loss of potency in mammals. 2) Desglycinamide AVP is nearly as potent as AVP in Amphibia, whereas it is inactive in mammals. 2) Tocinoic acid, lacking amino acids 7, 8, and 9, has activity in Amphibia, but pressinoic acid, lacking the same three amino acids, is inactive.

Topics: Amino Acids; Animals; Arginine Vasopressin; Cell Line; Cyclic AMP; Dose-Response Relationship, Drug; Epithelium; Kidney; Lypressin; Oxytocin; Peptides; Permeability; Pituitary Gland, Posterior; Receptors, Angiotensin; Receptors, Cell Surface; Receptors, Vasopressin; Vasopressins; Vasotocin; Xenopus laevis