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)

The proton NMR spectra and role in peptide binding of carboxyl-terminal and NH2-terminal neurophysin residues were studied by preparation of bovine neurophysin-I derivatives from which residues 90-92 had been cleaved by carboxypeptidase or residues 1-8 excised by trypsin. The carboxypeptidase-treated protein showed normal peptide-binding behavior. NMR comparisons of this derivative and the native protein allowed identification of proton resonances associated with residues 89-92, confirmed a lack of functional role for this region of the protein, and permitted new observations on the behavior of neurophysin's aromatic residues. The trypsin-treated protein bound peptide with an affinity only 1/50 that of the native protein at pH 6 but evinced the same binding specificity and pH dependence of binding as the native protein. These results argued against direct interaction of residues in the 1-8 sequence with bound peptide and for a role for these residues, particularly Arg-8, in conformational stabilization of the active site; this role is held to be additional to the reported influence of 1-8 on dimerization. NMR comparisons of the trypsin product and native protein allowed preliminary assignment of a set of alkyl proton resonances to residues within the 1-8 sequence and were compatible with a restricted environment for Arg-8. Conformational differences between native and trypsin-treated proteins were manifest particularly by differences in the NMR spectra of Phe and Tyr-49 ring protons. The behavior of Phe ring protons was consistent with the reported decreased dimerization constant of the trypsin product and suggested participation of Phe-22 or -35 in dimerization. The behavior of Tyr-49 provided the first direct evidence of a change in secondary or tertiary structure associated with excision of residues 1-8. Suggested mechanisms by which this conformational change reduces binding include a direct effect on Tyr-49 and/or a conformational rearrangement of active site residues near Tyr-49.
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PMID:Proton magnetic resonance and binding studies of proteolytically modified neurophysins. 670 74

Carboxamidopeptidase, an enzyme which inactivates neurohypophyseal hormones, has been purified 3800-fold in an overall yield of 22% from toad skin, a neurohypophyseal hormone target organ, by (NH4)2SO4 fractionation, DEAE-Sephadex chromatography, and affinity chromatography on immobilized p-aminobenzamidine and concanavalin A-agrose. The purified enzyme is capable of inactivating both [8-arginine]vasopressin (AVP) and oxytocin by hydrolyzing the Arg8-Gly9-NH2 and the Leu8-Gly9-NH2 bonds, respectively, and can hydrolyze the ester substrates, benzoyl-L-arginine ethyl ester (BzArgOEt) and acetyl-L-trypsine ethyl ester, suggesting that the enzyme has both trypsin-like and chymotrypsin-like activities. Carboxamidopeptidase is maximally active at pH 7.5-8.5 for AVP and BzArgOEt and pH 7.0 for oxytocin. Carboxamidopeptidase is inhibited by ovoinhibitor, ovomucoid, Trasylol. lima bean trypsin inhibitor, concanavalin A, antipain, leupeptin, chymostatin, elastatinal, p-nitrophenyl p-guanidinobenzoate, and 4-methylumbelliferyl p-guanidinobenzoate but not by soybean trypsin inhibitor, alpha 1-antitrypsin, hirudin, pepstatin, bestatin, phosphoramidon, or cysteine. The enzyme is also inhibited by the serine protease inhibitor, diisopropyl phosphofluoridate (i-Pr2PF), and by the chloromethyl ketone derivatives of tosyllysine, tosylphenylalanine, and (benzyloxycarbonyl)phenylalanine, as well as by the sulfhydryl group reagent, p-(chloromercuri)benzoate (PCMB). Inhibition by PCMB is reversed by cysteine. The molecular weight determined by gel filtration in the presence of 1 MNaCl is approximately 100 000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the enzyme is composed of two identical subunits of 48 000 daltons. Each subunit consists of a heavy chain (28 000 daltons) and a light chain (19 000 daltons) joined by a disulfide bond(s). Labeling experiments using [3H]-i-Pr2PF showed that the enzyme active site is located in the heavy chain.
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PMID:Carboxamidopeptidase: purification and characterization of a neurohypophyseal hormone inactivating peptidase from toad skin. 676 14

The effects of binding L-phenylalanyl-L-phenylalanine amide and related peptides on the 220 MHz and 300 MHz proton n.m.r. spectra of bovine neurophysin-I were studied. Throughout both the aliphatic and aromatic proton regions, marked binding-induced changes in the protein spectrum occur which are best explained by invoking conformational change within the neurophysin dimer, in addition to direct perturbation of individual protein protons by bound peptide. In the region downfield from 6 p.p.m., a new resonance, centered at 6.45 p.p.m. was resolved in 300 MHz spectra. This resonance is tentatively assigned to a non-exchangeable -NH and undergoes a reversible binding-induced broadening. Also in this region, the binding-induced chemical shift change in the ortho ring protons of Tyr-49 was used to explore additional aspects of the kinetics of peptide-binding. The results indicate that peptides with affinities greater than or equal to 10(4) M-1 exhibit slow to intermediate exchange rates on the time scale of the Tyr-49 chemical shift change, but that fast exchange can be achieved with peptides having affinities approximately equal to 10(2) M-1.
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PMID:Effects of peptide-binding on the proton n.m.r. spectrum of bovine neurophysin-I. 683 90

The chemical structure of the hormone binding region of the neurophysins has been investigated by photoaffinity labeling with the photolabile tripeptide, L-[methyl-3H]Met-L-Tyr-p-azido-L-Phe amide. Photolysis of the photoaffinity tripeptide in the presence of bovine neurophysin I and II and a human neurophysin II led to approximately equal extents of covalent incorporation of radioactivity into protein. Photolabeled bovine neurophysin II was fractionated into binding site derivatized protein and nonbinding site derivatized protein by affinity chromatography, with results of amino acid and radiolabel analysis of the hormone binding site blocked protein indicating that 1 mol of tripeptide was covalently incorporated/mol of protein. Tyrosine 49 was the only protein amino acid modified in the binding site photolabeling reaction as assessed by peptide mapping of the performic acid oxidized and trypsin-digested photolabeled protein using reverse phase high performance liquid chromatography. Modification of the single neurophysin tyrosine also was found by amino acid analysis of performic acid oxidized photolabeled bovine neurophysin II. The covalent bond formed in neurophysin upon photolysis was cleaved by either exhaustive acid hydrolysis or reduction-carboxymethylation without loss of the protein amino acid residues and by performic acid oxidation with loss of both protein and tripeptide tyrosine residues. These overall data indicate that tyrosine 49 is the probable site for specific covalent attachment of the photoaffinity tripeptide. Assuming that the tripeptide binding site is the high affinity hormone binding site reported for the neurophysins, this conclusion argues that tyrosine 49 is close to or within this site.
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PMID:Photoaffinity labeling of the hormone binding site of neurophysin. 706 22

Bovine neurophysin II was partially digested by chymotrypsin and by chymotrypsin followed by carboxy-peptidase B to produce large fragments collectively representing deletions of residues 1-5 and 91-95. All such fragments were capable of binding peptides to the principal hormone-binding site of neurophysin with normal or near-normal affinity, indicating that residues 1-5 and 91-95 do not directly participate in binding. In addition, preliminary results with thermolysin-derived fragments suggested that residue 6 does not participate in peptide binding. During the course of chymotrypsin studies, it was demonstrated that bovine neurophysin II behaves as a transient competitive inhibitor of chymotrypsin; for neurophysin-peptide complexes, Ki congruent to 8 x 10(-6) M. This inhibition is dependent on neurophysin conformation and is relieved by the anomalous preferential splitting by chymotrypsin of Arg-Arg and Phe-Pro bonds near the carboxyl terminus of neurophysin II. It is suggested that this phenomenon might reflect the interaction of neurophysin II with a chymotrypsin-related enzyme in the pituitary. One approach used in the study of binding properties of proteolytically modified neurophysin was affinity chromatography; the preparation and properties of a conveniently prepared affinity column for neurophysin are described.
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PMID:Partial digestion of neurophysins with proteolytic enzymes: unusual interactions between bovine neurophysin II and chymotrypsin. 707 53

The reciprocal modulation of neurophysin self-association and noncovalent peptide--protein interaction between neurophysin and the hormones oxytocin and vasopressin has been assessed by quantitative affinity chromatography. Competitive elutions of radiolabeled bovine neurophysin II (NPII) from the affinity matrices Met-Tyr-Phe-omega-(amino-hexyl)- [and (aminobutyl)-] agarose were performed with increasing concentrations of either of the soluble ligands oxytocin or lysine-vasopressin. Also, the dependence of NPII retardation by the same adsorbents on the concentration of applied protein was investigated in the absence of soluble ligand. The affinity constant of NPII for the immobilized peptide increased markedly with increasing amounts of applied protein and with the addition of small amounts of soluble ligand, the latter being more pronounced at higher protein concentrations. The affinity constant of the protein for the soluble ligand showed a smaller increase. The variation of l/(V - V0) (where V = the NPII elution volume and V0 = the elution volume of noninteracting control protein) with soluble ligand concentration was linear except near [ligand] = 0. The quantitative affinity chromatographic results on the tripeptidyl affinity columns are consistent with the view that NPII exists in a monomer in equilibrium dimer equilibrium, with the dimer exhibiting a stronger interaction with both neuropeptide and tripeptide analogues. The data also indicate that the self-associated protein dimer itself exhibits cooperativity, that is, stronger binding of the immobilized ligand at one site when a second site is occupied with a molecule of the soluble ligand than when no soluble ligand is bound. The deduction from the above of ligand-induced dimerization is evident also in the increased retardation of NPII on neurophysin--Sepharose when the eluting buffer contains soluble peptide hormone.
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PMID:Interdependence of neurophysin self-association and neuropeptide hormone binding as expressed by quantitative affinity chromatography. 708 36

Limited tryptic fragmentation of disulfide-intact bovine neurophysins I and II (NP-I and -II, respectively) has been found to cause selective disruption of both hormone binding and neurophysin self-association. Loss of binding interactions, measured as a loss of ability to stimulate retardation of 125I-labeled neurophysin on Met-Tyr-Phe-amino-butylaminoagarose, is complete within 3 h at 37 degrees C. Reverse-phase high-performance liquid chromatography (HPLC) analysis of tryptic digests of neurophysin I allows detection of two major protein products and the peptide fragment 1-8. Release of the latter N-terminal piece occurs at about the same rate as loss of binding interactions. Reverse-phase HPLC elution behavior before and after performic acid oxidation and amino acid composition of the protein products led to their identification as NP-I-(9-93) (the 9-93 sequence) and [des-19,20]NP-I-(9-93) (the 9-93 sequence with the dipeptide 19-20 missing) for the more rapidly and more slowly formed species, respectively. NP-I-(9-93), unlike intact neurophysin I, is not retarded strongly by either Met-Tyr-Phe-amino-butylaminoagarose or neurophysin II-Sepharose. In contrast, both NP-I-(9-93) and [des-19,20]NP-I-(9-93) are equally as effective as intact NP-I in binding neurophysin I antibodies. The role of amino-terminal residues in promoting hormone binding, self-association, and antigenic recognition interactions is considered.
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PMID:Effects of limited tryptic proteolysis of bovine neurophysins on molecular properties of hormone binding, self-association, and antigenicity. 715 May 67

Subcutaneous injections of 30 mg atropine into lactating cows induced a 20--40% decrease of free amino acid (AA) levels in arterial plasma. Minimum levels were observed after 30--50 min. The decline persisted for more than 6 h. The greatest fall in concentration was noted for tyrosine, methionine, lysine, arginine, phenylalanine and threonine. Arterial glucose levels remained unaffected. The effect of atropine on milk secretion was studied in 2 cows which were milked every hour with the aid of oxytocin. Maximal effects were observed after 3--4 h. They included reduction in concentration of casein and alpha-lactalbumin (alpha-la) and a decline in production of milk (20%), casein (35%), alpha-la (45%) and lactose (18%). Uptake by the lactating udder over a period of about 1 h after injection of atropine was studied in 2 cows. Mammary blood flow and glucose uptake remained unaffected. There was a positive correlation between arteriovenous differences of essential AA and arterial plasma concentrations. The uptake of essential AA decreased by approximately 50%. There was no evidence that atropine has a direct inhibiting effect on the udder. It is suggested that the decrease of alpha-la synthesis might induce an inhibition of lactose synthesis and milk production.
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PMID:Effect of atropine on plasma amino acid levels and milk secretion of cows. 719 9

Recent investigations on marsupial neurohypophysial hormones have revealed that species belonging to the Australian family Macropodidae and the American family Didelphidae have, apart from an oxytocin-like hormone, two vasopressin-like peptides which can be separated either by ion-exchange chromatography or chromatoelectrophoresis. The major pressor hormone of two Australian species, the red kangaroo (Macropus rufus) and the tammar (Macropus eugenii), has been identified as lysine vasopressin by its amino acid sequence anda its pharmacological properties. We report here that the minor pressor hormone, which chromatographs on Amberlite CG-50 like arginine vasopressin, differs from it in sequence only at position 2 where phenylalanine replaces the tyrosine of arginine vasopressin.
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PMID:Phenypressin (Phe2-Arg8-vasopressin), a new neurohypophysial peptide found in marsupials. 743 83

1. Modulatory effects of the four molluscan neuroactive peptides. FMRFamide (Phe-Met-Arg-Phe-NH2), APGW-amide (Ala-Pro-Gly-Trp-NH2), oxytocin and [SER2]-Mytilus inhibitory peptide ([SER2]-MIP) (Gly-Ser-Pro-Met-Phe-Val-NH2) were examined on the inward current (Iin) caused by achatin-I (Gly-D-Phe-Ala-Asp), which has been isolated from the Achatina ganglia. 2. Two Achatina giant neurone types, v-RCDN (ventral-right cerebral distinct neurone) and PON (periodically oscillating neurone), were used. Achatin-I was applied locally to the neurone tested by brief pneumatic pressure ejection, and the other molluscan neuroactive peptides were perfused around the ganglia. 3. FMRFamide, perfused at 3 microM, suppressed markedly the Iin elicited by the achatin-I of both v-RCDN and PON. APGW-amide at 3 microM also suppressed the Iin of v-RCDN, but did not affect that of PON. Oxytocin at 1 microM suppressed the Iin of PON, but did not affect that of v-RCDN. [Ser2]-MIP at 3 microM did not affect the Iin of v-RCDN. 4. The dose-response curves of FMRFamide, APGW-amide and oxytocin, indicated that their respective suppressive effects on the Iin of achatin-I were dose-dependent, and that APGW-amide was slightly more potent than the other peptides. The dose (pressure duration)-response curves of achatin-I (1 kg/cm2, 10(-3) M, 5 min interval), obtained by varying the duration of the achatin-I pressure ejection, were measured in the presence and absence of each of the three peptides.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Suppressing effects of neuroactive peptides on the inward current caused by achatin-I, an Achatina endogenous peptide. 754 26


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