UNIPROT:P20366 - FACTA Search

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Query: UNIPROT:P20366 (substance P)
21,176 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The feasibility of extracting and quantifying neuropeptides in bone by radioimmunoassay was investigated in a study including 60 diaphyseal rat femora. Substance P, calcitonin gene-related peptide, neuropeptide Y and vasoactive intestinal polypeptide, previously identified in bone by immunohistochemistry, were extracted from separate homogenates of bone, periosteum and bone marrow in a solution of 4% EDTA and 2 M acetic acid. Measurable amounts of all four neuropeptides in bone, periosteum and bone marrow were obtained by radioimmunoassay in a reproducible manner. The neuropeptide immunoreactivities were characterized by reverse-phase high performance liquid chromatography. Among the four neuropeptides analyzed, neuropeptide Y consistently exhibited the highest concentrations in the different tissues. Overall, cortical bone showed the lowest neuropeptide concentrations. The concentration of vasoactive intestinal polypeptide was higher in periosteum than in bone marrow, whereas that of calcitonin gene-related peptide was uniform in these tissues. The distributional differences observed in bone tissue may be explained by a variety of physiological roles attributed to neuropeptides such as regulation of nociception, vasoactivity, immune function and local bone metabolism. The described methodology offers a new means of investigating a neuropeptidergic involvement in various disorders of the skeleton.
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PMID:Extraction and quantitation of neuropeptides in bone by radioimmunoassay. 752 16

Crude membrane fractions prepared from rabbit gastric fundic muscle degraded vasoactive intestinal polypeptide (VIP) with an average specific activity of 0.96 nmol/min/mg protein at 37 degrees C, pH 7.5, and at [S]o = 0.05 mM. The relative activities towards [Leu5]enkephalin, substance P, VIP, and neurotensin were approximately 7.7, 2.0, 1.0, and 0.54, respectively. The VIP degradation was inhibited by metal chelators EDTA and o-phenanthroline. CaCl2 at 0.3-1.0 mM enhanced VIP degradation up to twofold. Phosphoramidon, captopril, and bestatin, the specific inhibitors for endopeptidase-24.11, angiotensin-converting enzyme, and aminopeptidase M, respectively, did not affect VIP degradation significantly. However, the complex mixtures of VIP fragments generated implicates action of multiple peptidases including the aforementioned three peptidases and other unidentified peptidase(s).
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PMID:Degradation of vasoactive intestinal polypeptide by rabbit gastric smooth muscle membranes. 800 38

Atrial natriuretic peptide (ANP) is a fluid-regulating peptide hormone that promotes vasorelaxation, natriuresis, and diuresis. The mechanisms for the release of ANP and for its clearance from the circulation play important roles in modulating its biological effects. Recently, we have reported that the cell surface of an endothelial cell line, CPA47, could degrade 125I-ANP in the presence of EDTA. In this study, we have characterized this degradation of 125I-ANP. The kinetics of ANP degradation by the surface of CPA47 cells were first order, with a Km of 320 +/- 60 nM and Vmax of 35 +/- 14 pmol of ANP degraded/10 min/10(5) cells at pH 7.4. ANP is degraded by the surface of CPA47 cells over a broad pH range from 7.0-8.5. Potato carboxypeptidase inhibitor and bestatin inhibited 125I-ANP degradation, suggesting that this degradative activity on the surface of CPA47 cells has exopeptidase characteristics. The selectivity of CPA47 cell-surface degradation of ANP was demonstrated when 125I-ANP degradation was inhibited in the presence of neuropeptide Y and angiotensin I and II but not bradykinin, bombesin, endothelin-1, or substance P. The C-terminal amino acids phe26 and tyr28 were deduced to be important for ANP interaction with the cell-surface peptidase(s) based on comparison of the IC50 of various ANP analogues and other natriuretic peptides for the inhibition of ANP degradation. These data suggest that a newly characterized divalent cation-independent exopeptidase(s) that selectively recognizes ANP and some other vasoactive peptides exists on the surface of endothelial cells.
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PMID:Characterization of atrial natriuretic peptide degradation by cell-surface peptidase activity on endothelial cells. 836 38

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

The angiotensin-converting enzymes (ACE) are involved in the regulation of the specific maturation or degradation of a number of mammalian bioactive peptides. A carboxydipeptidase similar to mammalian ACE has now been identified in the adult stage of the haematophagous fly, Haematobia irritans exigua (buffalo fly), a close relative of the horn fly of North America. The enzyme was purified by lectin-affinity chromatography and ion-exchange chromatography and migrated as a doublet of 70 kDa upon reducing SDS/PAGE. Unlike mammalian ACE, the fly carboxydipeptidase (HieACE) is not membrane bound. The amino acid sequence of an internal peptide from HieACE and a conserved amino acid region present in all mammalian ACE were used to design degenerate oligonucleotide primers suitable for PCR. A DNA fragment amplified from adult buffalo fly cDNA was used to identify a cDNA clone that encoded the enzyme. The cDNA sequence encodes a carboxydipeptidase with 41-42% amino acid identity to the mammalian testicular ACE. The active-site regions of mammalian ACE are conserved in the deduced amino acid sequence of HieACE. Enzymatically, HieACE is very similar to its mammalian counterparts, with comparable Km and V(max) values for the synthetic substrate, benzoylglycylglycylglycine, and similar patterns of inhibition by EDTA, ACE inhibitor peptide and captopril. HieACE also specifically activates angiotensin I to angiotensin II and degrades other mammalian ACE substrates such as bradykinin, substance P and cholecystokinin-8. In the adult fly, HieACE is expressed in the compound ganglion and in the posterior region of the midgut. Similar to the mammalian system, expression of this enzyme is induced in the maturing male reproductive system, which suggests conservation of ACE function in these species.
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PMID:Cloning and characterisation of angiotensin-converting enzyme from the dipteran species, Haematobia irritans exigua, and its expression in the maturing male reproductive system. 864 80

Pig kidney aminopeptidase P (AP-P; EC 3.4.11.9) has been purified to homogeneity after its solubilisation from brush border membranes by phosphatidylinositol-specific phospholipase C. The effects of various activators and inhibitors of AP-P activity have been examined with a number of different substrates for the enzyme. The hydrolysis of bradykinin and ArgProPro is inhibited at Mn2+ concentrations above 10(-5) M, whereas the hydrolysis of other substrates (GlyProHyp, beta-casomorphin, substance P) is substantially activated, with 4-10 mM Mn2+ being optimal. The thiol reagent, p-chloromercuriphenylsulphonic acid, inhibits the hydrolysis of GlyProHyp but markedly activates the hydrolysis of bradykinin. A number of inhibitors of angiotensin converting enzyme (ACE; EC 3.4.15.1), previously reported to inhibit the hydrolysis of GlyProHyp, have no effect on the hydrolysis of bradykinin except in the presence of Mn2+. Differences were also observed in the degree of inhibition of GlyProHyp and bradykinin hydrolysis by EDTA and their reactivation by divalent cations. The hydrolysis of GlyProHyp follows Michaelis-Menten kinetics with a Km value of 2.7 mM. Bradykinin inhibits GlyProHyp hydrolysis with an I50 of 1.4 microM. The hydrolysis of bradykinin by AP-P reveals anomalous nonlinear kinetics indicative of negative cooperativity or the presence of more than one active site for this substrate. These results indicate that substrates for AP-P can be divided into 2 groups based on their responses to inhibitors and cation activators.
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PMID:Inhibition and metal ion activation of pig kidney aminopeptidase P. Dependence on nature of substrate. 869 47

When using radioimmunoassay to measure the levels of peptides from small pieces of nerve tissue it is crucial to maximize the amount of peptides extracted. Here we report on the value of including protease inhibitors in the extraction buffer when extracting substance P (SP) from short lengths of rat saphenous nerve tissue. Nerve segments were removed from terminally anesthetized 13-week-old rats and directly added to acid buffer (including EDTA) either with or without 1 mM 4-(2-amino-ethyl)-benzesulfonyl fluoride-HCl, 2 micrograms/ml aprotinin, 100 microM leupeptin, 1 microgram/ml cystatin, and 1 mM benzamidine. These "direct" samples were then boiled for 10 min. With additional groups of pieces of saphenous nerve tissue the effects of leaving the samples for 10 min in both buffers at room temperature either intact ("delayed") or after mincing the tissue ("minced") were investigated. Addition of protease inhibitors increased the amount of SP extracted in both direct and delayed procedures, although the increase was only significant for the delayed situation (P < or = 0.05). "Delay" in the absence of protease inhibitors resulted in a significantly decreased amount of SP being extracted compared to the direct and minced situations (P < or = 0.05). We recommend use of protease inhibitors be included as part of the standard procedure for extracting neuropeptides from small specimens of nerve tissue for radioimmunoassay.
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PMID:Use of protease inhibitors increases the amounts of substance P extracted from small specimens of nerve tissue. 878 13

The recombinant rat testes metallo-endooligopeptidase (EC 3.4.24.15) and the rabbit brain endooligopeptidase A (formerly EC 3.4.22.19) were compared, side-by-side, in view of their striking similarities in both the physicochemical features and the specificities for oligopeptides. Concerning the tissue distribution in rat and rabbit, no relation between the levels of enzyme activity in cytosol and the levels of metallo-endooligopeptidase 24.15 mRNA could be established. The results suggest that the predominant neuropeptide-metabolizing activity attributed to the metallo-endooligopeptidase 24.15 is performed by, at least, two distinct cytosolic enzymes, one predominant in rat testes and the other in rabbit brain and testes, and possibly also in rat brain. Both enzymes are activated by dithiothreitol and irreversibly inhibited by a SH-affinity labeling dynorphin-related compound, but they are not inhibited by EDTA in a concentration dependent manner. Both enzymes exhibit the same specificity toward several bioactive peptides, except for LH-RH and substance P, which are only hydrolysed by the rat testes enzyme. Taken together, these results lead us to conclude that it is unlikely that the recombinant rat testes metallo-endooligopeptidase 24.15 and the rabbit brain endooligopeptidase A are the same molecule although they might belong to the same family of oligopeptidases.
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PMID:Species specificity of thimet oligopeptidase (EC 3.4.24.15). 882 19

An intracellular oligopeptidase from Lactobacillus paracasei Lc-01 has been purified to homogeneity by Fast Flow Q Sepharose, hydroxyapatite, and Mono Q chromatography. The molecular mass of the enzyme was determined to be 140 kDa by gel filtration and approximately 30 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and SDS-capillary electrophoresis. The pI of the enzyme was at pH 4.5. The enzyme expressed maximum activity at pH 8.0 and 40 degrees C. Oligopeptidase activity on bradykinin was inhibited strongly by 1,10-phenantroline and EDTA and partly by p-chloromercuribenzoic acid but not by phosphoramidon or phenylmethylsulfonyl fluoride. Marked inhibition by beta-casein fragment 58 to 72 was demonstrated. The enzyme showed neither general aminopeptidase nor caseinolytic activity, and it degraded only oligopeptides between 8 and 13 amino acids. The enzyme readily hydrolyzed the Phe-Ser and Pro-Phe bonds of bradykinin; the Phe-His bond of angiotensin I; the Pro-Gln, Gln-Phe, and Phe-Gly bonds of substance P; and the Pro-Tyr bond of neurotensin. Weak activity toward the Ala-Tyr and Pro-Ser bonds of alpha(s1)-casein fragment 157 to 164, was observed. The N-terminal amino acid sequence of the oligopeptidase showed a high degree of homology to the lactacin B inducer from Lactobacillus acidophilus.
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PMID:Characterization of an intracellular oligopeptidase from Lactobacillus paracasei. 909 25

Capsaicin depolarizes primary afferent C-fibers releasing substance P (SP) whose N-terminal metabolites appear to play a role in the development of antinociception. Because some effects of SP(1-7) are similar to those of zinc, we tested the hypothesis that zinc in the extracellular area plays a role in capsaicin-induced antinociception, as measured using the abdominal stretch (writhing) assay. Decreases in zinc were achieved by intrathecal (i.t.) injection of membrane-impermeable compounds: ethylenediaminetetraacetic acid disodium-calcium salt (Ca++ EDTA), a calcium-saturated chelator of divalent cations, or dipicolinic acid, a zinc chelator. Ten nanomoles of Ca++ EDTA had no effect on writhing at either 90 min or 24 h after injection, yet pretreatment with Ca++ EDTA prevented the development of antinociception 24 h after i.t. injection of either 2. 8 nmol of capsaicin or 10 nmol of SP(1-7). One nanomole of dipicolinic acid injected i.t. also blocked capsaicin- and SP(1-7)-induced antinociception. When injected 24 h after SP(1-7), Ca++ EDTA failed to reverse antinociception. Acute antinociception produced 30 min after injection of SP(1-7) was also blocked when Ca++ EDTA was injected 24 h, but not 60 min, before SP(1-7). Thus, the optimal time of Ca++ EDTA-induced hyperalgesia (90 min), described previously, did not correspond to that of its inhibitory effect on antinociception (24 h). In contrast, we found that the previously described antinociception after an i.t. injection of zinc (90 min) is greatly attenuated by 24 h. Thus, zinc appears to be necessary, but may not be sufficient, for the long-term antinociceptive effect of capsaicin, acting downstream from the action of substance P N-terminal metabolites.
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PMID:Chelation of zinc in the extracellular area of the spinal cord, using ethylenediaminetetraacetic acid disodium-calcium salt or dipicolinic acid, inhibits the antinociceptive effect of capsaicin in adult mice. 991 86

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