Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P01178 (oxytocin)
 15,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We discovered an enzyme in human platelets that deamidates substance P and other tachykinins. Because an amidated carboxyl terminus is important for biological activity, we purified and characterized this deamidase. The enzyme, released from human platelets by thrombin, was purified to homogeneity by ammonium sulfate precipitation, followed by chromatography on an octyl-Sepharose column and chromatofocusing on PBE 94. The purified enzyme exhibits esterase, peptidase, and deamidase activities. The peptidase activity (with furylacryloyl-Phe-Phe) is optimal at pH 5.0 while the esterase (benzoyl-tyrosine ethyl ester) and deamidase (D-Ala2-Leu5-enkephalinamide) activities are optimal at pH 7.0. With biologically important peptides, the enzyme acts both as a deamidase (substance P, neurokinin A, and eledoisin) and a carboxy-peptidase (with bradykinin, angiotensin I, substance P-free acid, oxytocin-free acid) at neutrality, although the carboxypeptidase action is faster at pH 5.5. Enkephalins, released upon deamidation of enkephalinamides, were not cleaved. Gly9-NH2 of oxytocin was released without deamidation. Peptides with a penultimate Arg residue were not hydrolyzed. Some properties of the deamidase are similar to those reported for cathepsin A. The deamidase is inhibited by diisopropylfluorophosphate, inhibitors of chymotrypsin-type enzymes, and mercury compounds while other inhibitors of catheptic enzymes, trypsin-like enzymes, and metalloproteases were ineffective. In gel filtration, the native enzyme has an Mr = 94,000 while in non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis the Mr = 52,000 indicating it exists as a dimer. After reduction, deamidase dissociates into two chains of Mr = 33,000 and 21,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. [3H]diisopropylfluorophosphate labeled the active site serine in the Mr = 33,000 chain. The first 25 amino acids of both chains were sequenced. They are identical with the sequences of the two chains of lysosomal "protective protein" which, in turn, has sequence similarity to the KEX1 gene product and carboxypeptidase Y of yeast. This protective protein complexes with beta-galactosidase and neuraminidase in lysosomes and is vitally important in maintaining their activity and stability. A defect in this protein is the cause of galactosialidosis, a severe genetic disorder. The ability of physiological stimuli (e.g. thrombin or collagen) to release the deamidase from platelets indicates that it may also be involved in the local metabolism of bioactive peptides.
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PMID:A peptidase in human platelets that deamidates tachykinins. Probable identity with the lysosomal "protective protein". 169 76

From a series of potent cyclic hexapeptide oxytocin (OT) antagonists, a compound that exhibited significant bradykinin (BK) agonist activity was identified. L-366,811 (cyclo[L-proline-D-tryptophan-L-isoleucine-D-pipecolic acid-L-piperazine-2-carboxylic acid-N-Me-D-phenylalanine]) stimulated phosphatidylinositol (PI) turnover in rat uterine slices in vitro (approximately EC50, 2 microM) with a maximal effect (15-fold increase over basal) greater than that obtained for either BK or OT. L-366,811 also elicited dose-related contractions of the isolated rat uterus, producing measurable effects at 100 nM. Several other equally potent OT antagonists from the cyclic hexapeptide structural class were either less potent or inactive as activators of uterine PI turnover or contractility. The stimulatory effects of L-366,811 on uterine PI turnover and contractions were blocked by BK antagonists but not by an arginine vasopressin (AVP)/OT antagonist. In radioligand binding studies, L-366,811 exhibited moderate affinity (IC50, 360 nM) for the [3H]BK binding site in rat uterus, consistent with its potency in the functional models. These results indicate that L-366,811 exhibits BK agonist activity in rat uterus in vitro.
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PMID:Bradykinin agonist activity of a novel, potent oxytocin antagonist. 189 11

A bovine neurophysin II S-methyl-Cys-Tyr-Phe-NH2 complex has been crystallized using ammonium sulfate as the precipitating agent. The crystals are orthorhombic, the space group is either I222 or I2(1)2(1)2(1) with a = 124.9 A, b = 69.6 A and c = 151.5 A. The crystals diffract to at least 3.0 A resolution. Based on one neurophysin tetramer per asymmetric unit, the Matthews coefficient is calculated to be 3.92 with a solvent content of 69%.
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PMID:Crystals of a bovine neurophysin II tripeptide complex. 194 66

We prepared nine analogues (1-9) of MCPA-D-Phe-Phe-Ile-Asn-Cys-Pro-Arg-Gly-NH2, [MCPA1, D-Phe2, Phe3, Ile4, Arg8]oxytocin (MCPA = beta-mercapto-beta,beta-pentamethylenepropionic acid), a potent antagonist of the rat uterotonic action of oxytocin (OT). We replaced D-Phe with D-Trp and made [MCPA1,D-Trp2,Phe3,Ile4,Arg8]OT (1), which had OT pA2 of 7.51, somewhat higher than that of the D-Phe2 antagonist which has OT pA2 = 7.35 in our rat uterotonic assay. Both compounds are equipotent as antagonists of [Arg8]vasopressin in the rat antidiuretic assay, with pA2 = 8.1. Other substitutions gave [MCPA1,D-Trp2,4-Cl-Phe3,Ile4,Arg8]OT, (2), OT pA2 7.44; [MCPA1,D-Trp2,Phe3,Ile4,3,4-dehydro-Pro7,Arg8]OT (3), OT pA2 = 7.42; [MCPA1,D-Trp2,Phe3,Arg8]OT (4), OT pA2 = 7.58; [MCPA1,D-Trp2,Phe3,Arg8,Gly9-NHEt]OT (5), OT pA2 = 7.49; [MCPA1,D-Trp2,Ile4,Arg8]OT (6), OT pA2 = 7.46; [MCPA1,D-Trp2,Val4,Arg8]OT (7), OT pA2 = 7.58; [MCPA1,D-Trp2,Thr4,Arg8]OT (8), OT pA2 = 7.48; and finally, [MCPA1,D-Trp2,Arg8]OT (9), which was a more potent and more selective OT antagonist, with OT pA2 = 7.77 in the uterotonic assay and ADH pA2 less than 5.9 in the antidiuretic assay and hence is an important lead for the design of OT antagonists.
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PMID:Design of potent oxytocin antagonists featuring D-tryptophan at position 2. 199 88

Analogs of oxytocin containing tetrahydroisoquinoline carboxylic acid (Tic) of L or D configuration in position 2 were synthesized and their biological activities were tested. Both analogs showed negligible agonist activity in uterotonic, galactogogic, and pressor assays, but they are in vitro uterotonic inhibitors. In comparison with oxytocin analogs containing L- or D-phenylalanine in position 2, the analog with the D-configuration of the conformationally fixed aromatic residue has significantly increased inhibitory activity which suggests that the proper conformation for the interaction with the receptor, but not for its activation, was stabilized. 1H NMR and CD studies, supported by theoretical calculations, suggest that the conformational properties of the analog containing D-tetrahydroisoquinoline carboxylic acid are similar to those of [2-D-phenylalanine]oxytocin.
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PMID:Conformationally restricted analogs of oxytocin; stabilization of inhibitory conformation. 207 87

Nanosecond time-resolved tyrosinate fluorescence lifetimes were compared for oxytocin (OXT) and vasopressin (AVP) in propylene glycol. Long-lifetime tyrosinate fluorescence (LTF), characteristic of stable intramolecular hydrogen bond formation of the Tyr hydroxyl group, was present for OXT but not AVP in propylene glycol. The Tyr OH proton was also found to be labile for OXT but not AVP in DMSO by 1H-NMR. The spectroscopic data illustrate that the Tyr hydroxyl in OXT participates in an intramolecular hydrogen bond in certain receptor-simulating environments; the absence of potent LTF for [Ala5] OXT suggests that the Tyr hydroxyl of OXT forms an H-bond with the Asn5 carboxamide side-chain. The lability of the Tyr OH proton of OXT, but not AVP, is in accord with the biological activities of the peptides (OXT 100%, AVP 1%) in the rat uterus assay, suggesting that propylene glycol and DMSO mimic the environment at uterine receptors. 1H-NMR studies in DMSO demonstrate that for AVP there is a perpendicular-plate ring pairing interaction between the Tyr and Phe side-chains in which the hexagonal axis of the Tyr ring interacts with the face of the Phe ring. The present findings are discussed in terms of the proposed "cooperative model" for neurohypophysial hormone action.
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PMID:Tyrosinate fluorescence lifetimes for oxytocin and vasopressin in receptor-simulating environments: relationship to biological activity and 1H-NMR data. 217 14

The response to small peptides such as Arg-vasopressin, oxytocin and tachykinins was investigated in cultured porcine aortic endothelial cells. The production of endothelium-derived nitric oxide was assessed indirectly by the accumulation of cyclic GMP, a response that is due to the increased activity of soluble guanylate cyclase of the endothelial cells after release of the mediator. Arg-vasopressin, oxytocin, substance P and physalae-min (an analog of substance P, pGlu-Ala-Asp-Pro-Asn-Lys-Phe-Tyr-Gly-Leu-Met-NH2) markedly and transiently stimulated the production of cyclic GMP without affecting that of cyclic AMP. Treatment of endothelial cells with either hemoglobin or methylene blue reduced significantly both the basal and stimulated level of cyclic GMP. The production of cyclic GMP evoked by Arg-vasopressin and substance P was inhibited selectively by NG-monomethyl-L-arginine but not by its D-enantiomer. The neurohypophyseal hormones and related peptides stimulated the accumulation of cyclic GMP in a concentration-dependent manner, with the following relative order of potency: oxytocin greater than Lys-vasopressin greater than Arg-vasopressin much greater than [deamino-Cys1, D-Arg8]-vasopressin. The production of cyclic GMP evoked by oxytocin was inhibited selectively by [d(CH2)5, Tyr(OMe)2, Orn8]-vasotocin, an oxytocin antagonist. The production of cyclic GMP evoked by Arg-vasopressin and Lys-vasopressin was inhibited by [beta-mercapto-beta, beta-cyclopentamethylene-propionyl1, O-Me-Tyr2, Arg8]-vasopressin, a selective V1-receptor antagonist. The moderate production of cyclic GMP evoked by [deamino-Cys1, D-Arg8]-vasopressin was inhibited significantly by the V1-receptor antagonist. The peptide antagonists affected only minimally or not at all the production of cyclic GMP evoked by a donor of nitric oxide, SIN-1 (3-Morpholino-Sydnonimine). These observations indicate that 1) neurohypophyseal hormones and tachykinins stimulate the accumulation of cyclic GMP in cultured porcine aortic endothelial cells by increasing the production of endothelial-derived nitric oxide, which in turn enhances the activity of soluble guanylate cyclase; 2) the production of cyclic GMP in response to oxytocin is due to activation of oxytocinergic receptors; and 3) the production of cyclic GMP evoked by Arg-vasopressin and Lys-vasopressin is due mostly to activation of V1-vasopressinergic receptors.
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PMID:Neurohypophyseal peptides and tachykinins stimulate the production of cyclic GMP in cultured porcine aortic endothelial cells. 217 9

Goose VLDV-neurophysin (mesotocin-associated neurophysin) has been purified from posterior pituitary glands through molecular sieving on Sephadex G-75 and high-pressure reverse-phase liquid chromatography on Nucleosil C-18 columns. Despite apparent molecular mass of unreduced VLDV-neurophysin measured by polyacrylamide gel electrophoresis with sodium dodecylsulfate appeared near 17 kDa, this value fell to 11 kDa after reduction with mercaptoethanol, suggesting the existence of a homodimer. Complete amino acid sequence (93 residues) of goose VLDV-neurophysin has been determined. N- and C-terminal sequences of the protein have been established by Edman degradation (microsequencing) and use of carboxypeptidase Y, respectively. Peptides derived from oxidized or carboxamidomethylated neurophysin by trypsin or staphylococcal proteinase hydrolyses have been isolated by high-pressure liquid chromatography and microsequenced, allowing determination of the complete sequence. Comparison within the vertebrate VLDV-neurophysin lineage, namely goose VLDV-neurophysin to mammalian VLDV-neurophysins and to deduced toad VLDV-neurophysin, reveals a residue insertion between positions 66 and 67 in the nonmammalian VLDV-neurophysins. When goose MSEL-neurophysin (vasotocin-associated neurophysin) and goose VLDV-neurophysin are compared to their bovine counterparts, identical substitutions are found in positions 17 (Asn in both goose neurophysins instead of Gly in both ox neurophysins), 18 (Arg instead of Lys), 35 (Tyr instead of Phe), and 41 (Thr instead of Ala). Identity of the sequences 10-74 in both ox neurophysins has been explained by partial gene conversion between oxytocin and vasopressin genes, and identical substitutions in both goose neurophysins might reveal a similar gene conversion between mesotocin and vasopressin genes in birds.
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PMID:Complete amino acid sequence of goose VLDV-neurophysin. Traces of a putative gene conversion between promesotocin and provasotocin genes. 227 74

Oxytocin (OT) has been shown to play a role in the control of physiological PRL release and has been demonstrated to have a direct effect on the pituitary to stimulate PRL secretion. Administration of OT into the third ventricle, however, lowers PRL levels. This reduction could be mediated by either an inhibition of the release of endogenous OT into the hypohysial portal circulation or via an alteration in the release of some other PRL releasing (PRF) or PRL release-inhibiting (PIF) factor. In order to determine if centrally administered OT lowers PRL levels by increasing secretion of dopamine (DA) into the portal circulation, endogenous dopaminergic tone was blocked by injection of the DA antagonist domperidone (DOM). Subcutaneous administration of DOM resulted in elevated PRL levels which could be further augmented by iv infusion of OT (at 0.01 or 0.1 microgram OT/kg.min) or partially, but significantly, reduced by pretreatment with anti-OT antiserum (0.75 ml) indicating that under conditions of DA blockade, OT (which has little PRF activity during conditions of normal dopaminergic tone) can stimulate PRL secretion by a direct pituitary action. Treatment with DOM did not prevent, however, the reduction in PRL levels produced by central administration of OT (2 micrograms). This suggests that the effect of OT to alter PRL secretion when administered into the third ventricle was not mediated via an increase in DA release into the portal circulation. Furthermore, central administration of the OT antagonist CAV-259 (1-deamino-2-D-Trp-4-Val-8-Orn-OT) after DOM treatment resulted in a significant increase in PRL secretion indicating that endogenous levels of OT within the hypothalamus inhibit PRL secretion through a nondopaminergic mechanism. This stimulatory effect of the OT antagonist was not blocked by pretreatment with anti-OT antiserum (iv) which had been demonstrated previously to reduce the PRL surges in lactating mothers and steroid-primed ovariectomized rats, as well as to block the increase in PRL secretion seen after central administration of vasoactive intestinal peptide (VIP). Thus the central effect of OT to alter PRL secretion was probably not due to a change in the release of OT into the portal circulation. Intravenous administration of a VIP antagonist (D-4-Cl-6-Phe-17-Leu-VIP, previously demonstrated to be capable of reducing the PRL surge seen in lactating mothers) into DOM-treated rats does not alter PRL levels but blocks the ability of central administration of the OT antagonist CAV-259 to increase PRL levels under these conditions.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Interactions of dopaminergic and peptidergic factors in the control of prolactin release. 229 69

1. The effect of caffeine on mechanical activity was studied in pregnant rat myometrium. 2. In muscle cells with intact plasmalemmae, caffeine (0.1-50 mM) produced no contraction whatever the experimental conditions. 3. Caffeine (0.1-10 mM) inhibited, in a concentration-dependent manner, contractions induced by electrical stimulation, potassium-rich (60 mM K+) solution, sodium-free solution or oxytocin (22.5 nM). 4. In Ca2(+)-free solution, various substances (oxytocin, sodium orthovanadate and prostaglandin E2) evoked sustained contractions that were suppressed by caffeine (5-10 mM). When caffeine (greater than 5 mM) was applied during Ca2(+)-loading of the tissue (2.1 mM Ca2+, 5 min) in the presence of a K(+)-rich solution, the subsequent transient contraction induced by a short application (10s) of oxytocin (22.5 nM) in Ca-free solution was reduced (63 +/- 3.5% reduction for 20 mM caffeine, n = 4). 5. In saponin-skinned strips, application of caffeine (5-10 mM) during loading of the Ca2(+)-store increased the subsequent contraction induced by myo-inositol 1,4,5 trisphosphate (IP3, 10 microM). Caffeine (10-30 mM) decreased calcium-activated contractions in skinned fibres lacking a functional internal Ca-store. This effect was reduced by the cyclic AMP-dependent protein kinase inhibitor Thr-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile- His-Asp (8 microM). 6. In conclusion, it is suggested that the inability of caffeine to cause spasm of rat myometrium is due to the absence of a caffeine-sensitive calcium-release channel in the sarcoplasmic reticulum. Relaxant effects of caffeine can be explained by mechanisms leading to a decrease in both the cytoplasmic free Ca2+ concentration and the Ca2 +-sensitivity of the contractile machinery.
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PMID:Caffeine acting on pregnant rat myometrium: analysis of its relaxant action and its failure to release Ca2+ from intracellular stores. 232 93


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