Immunological and chemical identification of a neurophysin-containing protein coded by messenger RNA from bovine hypothalamus.
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The biosynthetic origin of the 10,000 molecular weight neurophysins, carriers of the peptide hormones oxytocin and vasopressin, has been studied by cell-free synthesis, Poly(A)-RNA was isolated from bovine hypothalamus and translated in a wheat germ system containing (35)S- or (3)H-labeled amino acids. A number of unique [(35)S]cysteine- but few [(35)S]-methionine-labeled proteins were coded by hypothalamic mRNA. A single, major, isotopically labeled protein (molecular weight 23,000-25,000) was immunoprecipitated from these translation mixtures by addition of purified antibodies against bovine neurophysin II and subsequent addition of Cowan I strain of Staphylococcus aureus. Specificity of the immunoprecipitation was demonstrated by competition with unlabeled authentic neurophysins and the absence of competition with structurally unrelated ovalbumin. Furthermore, neither nonimmune serum nor purified antibodies against ribonuclease immunoprecipitated the protein. The [(35)S]cysteine-labeled protein that was specifically immunoprecipitated was oxidized with performic acid and digested with trypsin in the presence of unlabeled, authentic bovine neurophysin II. Peptide mapping revealed that most of the major [(35)S]cysteine-labeled peptides (of the translation product) were identical to major cysteine-containing peptides of authentic neurophysin. The data show that hypothalamic mRNA directs the translation of several unique cysteine-rich proteins in an in vitro cell-free system. Furthermore, one of these proteins, which has a higher molecular weight than authentic neurophysin, is recognized by purified antibodies to bovine neurophysin II and has cysteine-containing tryptic peptides in common with those of authentic neurophysin. The data suggest that this protein is the primary translation product, pre-pro-neurophysin. (+info)
Complete assignment of neurophysin disulfides indicates pairing in two separate domains.
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The pairing of the 14 half-cystine residues of bovine neurophysin was established by sequential proteolytic digestion. Purified released peptides and the residual disulfide-linked core were monitored at each step by use of amino acid analysis, gas-phase sequencing, and mass spectrometry. The approach included application of gas-phase sequencing to assign disulfide pairs in peptides containing multiple disulfides. The results demonstrate that neurophysin disulfides are paired in two distinct domains--an NH2 domain (residues 10-54) containing four disulfides and a COOH domain (residues 61-85) containing three disulfides. The specific disulfide bridges are Cys-10 to Cys-54, Cys-13 to Cys-27, Cys-21 to Cys-44, Cys-28 to Cys-34, Cys-61 to Cys-73, Cys-74 to Cys-79, and Cys-67 to Cys-85. The results place the internally duplicated segments of neurophysin (residues 12-31 and 60-77) in separate domains. Disulfide-pairing patterns within each domain are homologous with the exception of the Cys-10 to Cys-54 bond, which is unique to the NH2 domain and which links the two ends of this domain together. The potential role of the Cys-10 to Cys-54 bond in organizing the hormone-binding site is discussed. (+info)
Trypsin liberates an arginine vasopressin-like peptide and neurophysin from a Mr 20,000 putative common precursor.
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Although the hypothesis that vasopressin and its associated neurophysin are synthesized together in one macromolecular common precursor was put forward more than a decade ago, direct conformation of this hypothesis has been lacking. A [35S]cysteine-labeled putative precursor for vasopressin-related neurophysin (Mr 20,000, pI 6.1) has been isolated from the supraoptic nuclei of rats. This precursor was subjected to limited proteolysis with trypsin which produced a Mr 10,000 protein and peptide products. The former was identified as neurophysin on the basis of its pH-dependent affinity for vasopressin and its behavior in isoelectric focusing systems (pI 4.6-4.8). The tryptic peptides proved to be vasopressin-like because they: (i) were rich in cysteine, (ii) comigrated with vasopressin on gel filtration columns in 6 M guanidine HCl, (iii) bound to a neurophysin-Sepharose affinity column at pH 5.7, and (iv) were recognized by antibodies against vasopressin. These data on the Mr 20,000, pI 6.1 protein represent direct experimental evidence for a candidate for the common precursor of vasopressin and neurophysin. We propose that this common precursor be called "propressophysin." (+info)
The human vasopressin gene is linked to the oxytocin gene and is selectively expressed in a cultured lung cancer cell line.
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The human genes for prepro-arginine-vasopressin-neurophysin II (prepro-AVP-NPII) and prepro-oxytocin-neurophysin I (prepro-OT-NPI) were cloned from a human genomic library and the nucleotide sequence of both genes was determined. The two genes are similar in their intron-exon structure, linked together with 12 kilobases intervening, and transcribed from opposite DNA strands. A human small cell lung cancer cell line, H378, produces significant quantities of pre-pro-AVP-NPII mRNA using a transcription unit predicted from the genomic DNA sequence. Despite the proximity of the actively transcribed prepro-AVP-NPII gene, transcription of prepro-OT-NPI is not detected in this cell line. (+info)
Inhibition of ubiquitin-dependent proteolysis by non-ubiquitinatable proteins.
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The effect in reticulocyte lysates of proteins with blocked amino groups on the ATP-dependent degradation of casein and serum albumin was studied in order to assess the extent to which proteins with blocked and with free amino groups share common paths of proteolytic degradation. Completely acetylated or succinylated casein and acetylated or succinylated serum albumin (reduced and carboxymethylated), in addition to other amino-modified proteins, inhibited the ATP-dependent proteolysis of both casein and reduced carboxymethylated serum albumin. Inhibition of serum albumin degradation by acetylated serum albumin was competitive, whereas inhibition of casein degradation by acetylated casein was largely competitive with evidence of mixed kinetics. The different amino-blocked proteins studied, which were largely unfolded under assay conditions, were similarly effective as inhibitors on a weight basis, with Ki values in the range 0.2-0.6 mg/ml; there was no correlation between the ability of the blocked proteins to serve as proteolysis substrates and their effectiveness as inhibitors. Studies of the effects of acetylated proteins on the conjugation of ubiquitin to serum albumin and casein demonstrated that the acetylated proteins blocked formation of ubiquitin-albumin conjugates and of selected casein conjugates; the steady state concentration of selected conjugates of endogenous lysate proteins was increased in the presence of amino-blocked proteins. The results suggest that proteins with blocked amino groups, which cannot serve as substrates for ubiquitin conjugation, can compete for binding to those ubiquitin conjugation factors that recognize and ubiquitinate potential substrates of the ubiquitin pathway. The similar inhibitory properties of the different blocked proteins in turn suggest that a common factor in binding to these conjugation factors may be recognition of the polypeptide backbone. (+info)
Presence of neurophysins in the human pituitary corticotrophs, Cushing's adenomas, and growth hormone-producing adenomas detected by immunohistochemical study.
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Neurophysins have been recognized as the carrier proteins of vasopressin and oxytocin. The distribution of neurophysins is immunohistochemically confirmed in the hypothalamus, median eminence, and posterior lobe of the pituitary gland. The authors detected neurophysins in the human corticotrophs and pituitary adenomas with the use of the immunohistochemical method with antiserum to human neurophysins, which did not cross-react with adrenocorticotropic hormone (ACTH), beta-endorphin, and corticotropin-releasing factor. All of ten pituitary glands obtained by autopsy revealed the presence of neurophysin-positive cells in the anterior, intermediate, and the posterior lobes. The neurophysin-positive cells were similar to the corticotrophs in shape and distribution. Simultaneous staining for ACTH and neurophysins in the serial sections revealed that neurophysin-positive cells were also ACTH-positive. One hundred twenty-four cases of pituitary adenoma operated upon were investigated. All of 7 Cushing's adenomas were composed of neurophysin-positive cells. Six tumors with giantism showed sparsely distributed neurophysin-positive cells. No neurophysin-positive cells were observed in any other cases. This study is the first reported evidence of the presence of neurophysins in the human corticotrophs and pituitary adenomas. (+info)
Evidence for the presence of oxytocin in the ovine epididymis.
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The testes of several species contain oxytocin and/or neurophysin, but the content or localization of oxytocin in epididymal tissue has not been studied. The present study was undertaken to localize oxytocin and neurophysin in epididymal tissue of the ram, and to quantify oxytocin in the ductus epididymidis and fluids entering and leaving the ductus epididymidis. Neurophysin was not detected in the epididymis; thus, synthesis of oxytocin by the epididymis is unlikely. Immunohistochemical localization of oxytocin was confined to the epithelium and capillaries. Oxytocin immunostaining was most intense for epithelium of the caput and declined in corpus and cauda regions. However, based on radioimmunoassay, no difference in oxytocin concentration was detected among regions of the epididymis. Since rete testis fluid entering and cauda epididymal fluid leaving the epididymis contained at least fourfold more oxytocin than testicular venous plasma, it was concluded that regional differences in epithelial concentration of oxytocin may have been masked by oxytocin contained in the luminal fluid. It was concluded further that the epididymis of the ram does not synthesize oxytocin, but about 22 ng/day enters the epididymis in rete testis fluid. Most of this luminal oxytocin apparently is absorbed by the epithelium of the caput epididymidis, with additional adsorption in the corpus and cauda. Although a role for oxytocin in ductal contractility cannot be excluded, it is more likely that the luminal oxytocin influences epithelial or sperm function. (+info)
An amphibian two-domain 'big' neurophysin: conformational homology with the mammalian MSEL-neurophysin/copeptin intermediate precursor shown by trypsin-sepharose proteolysis.
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A 'big' frog (Rana esculenta) neurophysin, encompassing sequences homologous to mammalian MSEL-neurophysin and copeptin, has been passed through a trypsin-Sepharose column in order to compare its conformation with that of the two-domain intermediate precursor isolated from guinea pig. Whereas the polypeptide possesses 8 arginine residues, only two cleavages were observed located in a putative inter-domain sequence (at Arg-94 and Arg-114). Because free vasotocin has been isolated from the frog, it is assumed that pro-vasotocin has a three-domain conformation similar to that of pro-vasopressin but processing in amphibians involves only one step rather than two steps as in mammals. (+info)