Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P20366 (substance P)
 21,176 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Our method combines intracellular dye injection and immunohistochemistry. Under optical control, Lucifer Yellow was injected into immunohistochemically identified neurons that reside in fixed tissue. The technique allows visualization of the complete arborization patterns of immunostained neurons. Injections were performed on small neurons (somata < 10 microns in diameter). The technique works on microslices of insect brain. Standard immunohistochemical procedures have only been varied slightly, omitting Triton X-100 treatment. Anti-Lucifer Yellow immunohistochemistry, or alternatively the photoconversion technique, enables extension of the morphological analysis of these cells to the electron microscopic level. In the present study, Lucifer Yellow injections were performed on immunohistochemically pretreated brain microslices (anti-Locusta tachykinin II antiserum) of the beetle Tenebrio molitor.
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PMID:Intracellular dye injection of previously immunolabeled insect neurons in fixed brain slices. 799 May 18

Endopeptidase 24.16 was purified from rat kidney homogenate on the basis of its ability to generate the biologically inactive degradation products neurotensin (1-10) and neurotensin (11-13). On SDS gels of the proteins pooled after the last purification step, the enzyme appeared homogeneous and behaved as a 70-kDa monomer. The peptidase was not sensitive to specific inhibitors of aminopeptidases, pyroglutamyl aminopeptidase I, endopeptidase 24.11, endopeptidase 24.15, proline endopeptidase and angiotensin-converting enzyme but was potently inhibited by several metal chelators such as o-phenanthroline and EDTA and was blocked by divalent cations. The specificity of endopeptidase 24.16 towards peptides of the tachykinin, opioid and neurotensin families was examined by competition experiments of tritiated neurotensin hydrolysis as well as HPLC analysis. These results indicated that endopeptidase 24.16 could discriminate between peptides belonging to the same family. Neurotensin, Lys8-Asn9-neurotensin(8-13) and xenopsin were efficiently hydrolysed while neuromedin N and kinetensin underwent little if any proteolysis by the peptidase. Analogously, substance P and dynorphins (1-7) and (1-8) were readily proteolysed by endopeptidase 24.16 while neurokinin A, amphibian tachykinins and leucine or methionine enkephalins totally resisted degradation. By Triton X-114 phase separation, 15-20% of endopeptidase 24.16 partitioned in the detergent phase, indicating that renal endopeptidase 24.16 might exist in a genuine membrane-bound form. The equipotent solubilization of the enzyme by seven detergents of various critical miscellar concentrations confirmed the occurrence of a membrane-bound counterpart of endopeptidase 24.16. Furthermore, the absence of release elicited by phosphatidylinositol-specific phospholipase C suggested that the enzyme was not attached by a glycosyl-phosphatidylinositol anchor in the membrane of renal microvilli. Finally, endopeptidase 24.16 could not be released from these membranes upon trypsinolysis.
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PMID:Rat kidney endopeptidase 24.16. Purification, physico-chemical characteristics and differential specificity towards opiates, tachykinins and neurotensin-related peptides. 842 55

An enzyme activity capable of hydrolysing the neuroactive undecapeptide substance P (SP) between its Phe7-Phe8 residues was purified from the membrane-bound fraction of human spinal cords. The enzyme preparation yielded was compared with a previously described SP-hydrolysing enzyme from human cerebrospinal fluid (CSF) with regard to inhibition profile, protein chemical properties and kinetics. In addition, the results were compared with those of bovine pancreatic chymotrypsin (a serine protease that cleaves the carboxy-terminal side preferentially at hydrophobic amino acids). The SP peptidase activity was extracted from human spinal cords with 1% Triton X-100 in 20 mM Tris-HCI pH 7.8. After ion exchange chromatography (DEAE-Sepharose) where the enzyme activity was separated from other proteins by gradient elution, the pooled enzyme fraction was further purified by molecular sieving (Sephadex G-50). The enzyme activity was finally recovered by HPLC molecular sieving (Superdex 75 HR 10/30) using a new preparative system, AKTA-purifier, controlled by UNICORN software version 2.20.
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PMID:Purification of substance P endopeptidase (SPE) activity in human spinal cord and subsequent comparative studies with SPE in cerebrospinal fluid and with chymotrypsin. 1007 55

Rab3 proteins (isoforms A, B, C and D) are low molecular weight GTP-binding proteins proposed to be involved in regulated exocytosis. In the present study, Rab3 protein expression and localization was examined in rat parotid gland by reverse transcription (rt) PCR, Western blotting and immunocytochemistry. An approximately 200 bp PCR product was obtained from parotid RNA by rtPCR and this fragment was cloned and sequenced. Nucleotide and deduced amino acid sequences obtained from five clones were identical to rab3D. Membrane and cytosolic fractions prepared from parotid acini were immunoblotted with antisera specific for each of the four Rab3 isoforms. A 28 kDa protein was detected with Rab3D-specific antisera in both fractions with staining being more intense in the membrane fraction. No other Rab3 isoforms were detected by immunoblotting, a result consistent with those obtained by rtPCR. Rab3D was enriched in zymogen granule membranes and Triton X-114 extraction revealed that this isoform is predominantly lipid-modified in parotid. Localization of Rab3D was done on frozen sections of parotid gland by immunofluorescence microscopy. Staining was observed primarily in the acinar cells and was adjacent to the acinar lumen. Incubation of dispersed acini with isoproterenol and substance P stimulated amylase secretion 4- and 2-fold above basal, respectively. Isoproterenol, but not substance P, induced redistribution of Rab3D from the cytosol to the membrane fraction in dispersed parotid acini. Consistent with these findings, isoproterenol injections into fasted rats also resulted in increased membrane-associated Rab3D in the parotid acini. These results indicate that Rab3D is: (1) the major Rab3 isoform expressed in rat parotid gland; (2) localized to zymogen granule membranes; and (3) involved with regulated enzyme secretion in acinar cells.
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PMID:Expression and localization of Rab3D in rat parotid gland. 1039 46

The sensitivity to cholesterol depletion of calcium handling by rat submandibular glands was investigated. The glands were digested with collagenase. After homogenization, the lysate was extracted at 4 degrees C with 0.5% Triton X-100 and the extract was submitted to an ultracentrifugation in a sucrose discontinuous gradient. A population of detergent-resistant membranes (DRM) was collected at the 5%-35% interface. The DRM had a higher content of cholesterol, saturated and long-chain fatty acids. Caveolin-1 and alpha(q/11) were located in these membranes. They were more ordered than vesicles from total cellular lysate as determined by anisotropy measurement. They disappeared after cholesterol extraction with methyl-beta-cyclodextrin (MCD). Exposure of the cellular suspension with MCD nearly abolished the response to carbachol, epinephrine, and substance P and inhibited the activation of phospholipase C (PLC) by these agonists and by sodium fluoride. MCD did not affect the mobilization of intracellular pools of calcium by thapsigargin. It increased the uptake of extracellular calcium or barium and did not inhibit the uptake of calcium after depletion of the intracellular stores of this ion. From these results, it is concluded that Triton X-100 can extract a fraction of membrane resistant to detergents. Treatment of the cells with MCD disrupts these membranes. The coupling between the heterotrimeric GTP-binding protein G(q/11) and poly-phosphoinositide-specific PLC is affected by disruption of these membrane fractions. At the opposite, the store-operated calcium channel (SOCC) is not affected by DRM-disruption.
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PMID:Cholesterol depletion perturbs calcium handling by rat submandibular glands. 1552 Oct 67

The possible existence of endogenous substances other than ?-aminobutyric acid (GABA), that can also bind to rat brain GABA receptors, has been investigated in synaptic membranes derived from whole rat brain, or from cerebral cortex; as well as in isolated synaptic vesicles obtained from cerebral cortex, striatum, hypothalamus, cerebellum and spinal cord and in the superfusion fluid of electrically stimulated brain cortex slices, where a GABA-like substance is released by a calcium-dependent process. The detector used to study the presence of such presumed non-GABA endogenous ligands, were frozen and thawed rat brain synaptic membranes, that had been treated with 0.05% Triton X-100 and thoroughly washed. With this highly sensitive preparation, at least 5 pmol of GABA/ml could be detected. The extracts of the different preparations where these hypothetical ligands were looked for, were analyzed by means of gel filtration on Sephadez G-10, paper chromatography and high voltage electrophoresis. In a very great number of experiments performed, the only endogenous ligand detected was GABA itself. The possible influence of a number of peptides on binding of GABA to its receptor, was also looked for. No significant effect was found for substance P, neurotensin, cholecystokinin octapeptide sulfated, somatostatin, thyrotropin releasing hormone, luteinizing hormone releasing hormone, methionine enkephalin (all 10(?5) M), angiotensin II (10(?4) M), ACTH (3 x 10(?7)M), poly-l-lysine (30 ?g/ml) or poly-l-glutamate (30 ?g/ml).
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PMID:A search for endogenous modulators of the GABA receptor in different regions of the rat CNS. 2048 56


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