UNIPROT:P20366 - FACTA Search

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)

The data presented in this paper demonstrate a new substance P (SP) binding site that is expressed on human monocytes. The apparent dissociation constant (Kd) for binding of 125I-labeled Bolton Hunter-SP (125I-BH-SP) to the receptor on monocyte membranes is 2.24 +/- 0.9 x 10(-7) M and the maximum binding capacity (Bmax) is 4.7 +/- 0.5 pmol/mg membrane protein. It could be excluded that this receptor is one of the known neurokinin (NK) type of receptors on the basis of binding characteristics for NK1, NK2, and NK3 agonists. Moreover, we demonstrate that the binding site is neither the bombesin receptor nor the serpin enzyme complex receptor nor the FMLP receptor. The order of potency for inhibition of 125I-BH-SP binding to the receptor on monocyte membranes is NK1 antagonist [D-Pro2,D-Trp7,9]SP > SP > NK3 agonist [MePhe7]SP > bombesin. Cross-linking studies with disuccinimidylsuberate, followed by SDS-PAGE analysis, revealed that 125I-BH-SP is specifically bound to a membrane protein with an apparent molecular mass of 47 kDa. At a functional level, SP induces the activation of MAP kinase in human monocytes. The ED50 for activation of MAP kinase positively correlated (r = 0.999, p < 0.0005) with the apparent affinity of the ligands applied in the 125I-BH-SP displacement studies. From these results, we conclude that this SP binding site on monocytes is a non-NK receptor protein that is functionally linked to the activation of MAP kinase.
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PMID:Monocytes express a non-neurokinin substance P receptor that is functionally coupled to MAP kinase. 751 35

To better understand the structural basis of the biological activity of the neuropeptide substance P SP; (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2), two-dimensional nmr spectroscopy experiments and simulated annealing calculations were used to investigate the conformation adopted in the presence of the membrane model system sodium dodecyl sulfate. It was determined that SP in the presence of SDS micelles undergoes a conformational equilibrium between an alpha- and a 3(10)-helix involving the midregion (Pro4-Gln5-Gln6-Phe7-Phe8) of the peptide. The C-terminus adopts an extended conformation while the N-terminus remains quite flexible. The conformation adopted by SP in the presence of SDS micelles yields a structure that is consistent with the model of a neurokinin-1 selective ligand proposed by Convert.
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PMID:NMR and molecular modeling investigations of the neuropeptide substance P in the presence of 15 mM sodium dodecyl sulfate micelles. 753 57

In a guinea-pig model of asthma, active immunization against substance P (SP) prevented the development of airways' hyperresponsiveness and reduced bronchospastic responses to SP (i.v.). The rat-mouse heterohybridoma NC1/34 secretes a specific, rat IgG1, anti-substance P antibody (alpha-SP Ab) which was isolated and purified by passing supernatant from cultures through thiophilic gel. Purity of antibody was about 50% (SDS-PAGE). The relative affinities of the alpha-SP Ab for SP, neurokinin A (NKA) and calcitonin gene-related peptide (CGRP) were estimated by ELISA using a constant amount of SP coupled (glutaraldehyde) to bovine serum albumin (BSA) to capture the antibody, alone and in the presence of increasing concentrations of the neuropeptides. At alpha-SP Ab dilutions of 1 in 5,000 to 1 in 32,000, CGRP did not prevent antibody binding to SP-BSA conjugate bound to the plates, but both SP and NKA prevented binding. In this system, the relative affinity of the alpha-SP Ab, at dilutions of 1 in 5,000 and 1 in 10,000, was about 50 times greater for SP than NKA. Whether passive immunization with alpha-SP Ab prevented bronchospastic responses to SP and NKA (i.v.), in vivo, was determined in groups of anesthetized guinea-pigs by recording pulmonary flow resistance (RL) and dynamic pulmonary elastance (EL). Injection of alpha-SP Ab (i.v., 5:1 molar ratio: alpha-SP Ab:SP total dose) did not alter baseline values of RL and EL, but markedly inhibited increases in RL and EL induced by SP and NKA (i.v.) without affecting responses to methacholine (i.v.). A control, "irrelevant" rat IgG-type antibody at a similar concentration had no effect on responses to SP or NKA. These findings indicate that passive immunization with a monoclonal alpha-SP Ab can prevent the bronchospastic effects of exogenous SP and NKA in guinea-pigs.
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PMID:Passive immunization with an anti-substance P antibody prevents substance P- and neurokinin A-induced bronchospasm in anesthetized guinea-pigs. 754

A neutral endoprotease was isolated from porcine antral mucosa and purified to homogeneity as examined by SDS-polyacrylamide gel electrophoresis (PAGE). Throughout the purification, t-butyloxycarbonyl-Arg-Val-Arg-Arg-4- methylcoumaryl-7-amide (MCA) was used as a substrate, which was found to be hydrolyzed specifically by the enzyme at the Arg-Arg bond. Unexpectedly, however, the enzyme was also found to hydrolyze vasoactive intestinal polypeptide (VIP) fairly specifically and more efficiently when various neuropeptides and related peptides were examined as substrates. It could degrade VIP by cleaving three peptide bonds not containing an arginine residue(s) with Km = 7.7 x 10(-6) M and kcat/Km = 7.4 x 10(6) M-1 s-1 (at pH 7.6 in the presence of 0.1% Lubrol PX), whereas only secretin, substance P, and a few others were hydrolyzed at much slower rates among the various peptides examined. Both activities toward the MCA substrate and VIP behaved in parallel throughout the purification procedures and showed essentially the same pH optimum and susceptibility toward various inhibitors and detergents. Therefore, both activities are thought to be due to the same enzyme. This endoprotease required 0.001% or a higher concentration of a detergent such as Lubrol PX or Triton X-100 for its maximal activity. Its optimum pH was about 7.5 and the molecular weight was estimated to be approximately 37,000 by SDS-PAGE. This enzyme was strongly inhibited by serine protease inhibitors such as diisopropyl-fluorophosphate and phenylmethanesulfonyl fluoride. It was also inhibited by p-chloromercuribenzoic acid, but not by some other cysteine protease inhibitors. Therefore, the enzyme appears to be most likely a kind of serine protease although its possibility as a cysteine protease cannot be completely excluded. Analysis of its cleavage specificity toward various oligopeptides indicated the possibility that the protease might recognize a specific amino acid sequence(s) and/or conformation in the vicinity of the cleavage site of the target peptide. Various characteristics of the endoprotease suggest that it is a novel membrane-bound neuropeptide-degrading endoprotease fairly specific for VIP.
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PMID:Purification and characterization of a vasoactive intestinal polypeptide-degrading endoprotease from porcine antral mucosal membranes. 771 70

Sixty-four kinds of cell lines were examined as to their ability to degrade glucagon using conditioned-media obtained from their protein-free cultures. Two human tumor cell lines were shown to produce this activity, and the cell line, HPC-YO, established from a human pancreatic carcinoma was shown to produce the highest level of activity. The glucagon-degrading enzyme (GDE) was purified from HPC-YO conditioned-medium by a combination of ion-exchange, gel filtration, and hydroxylapatite column chromatographies. The purified GDE also degraded vasoactive intestinal polypeptide (VIP) and secretin, however, it did not cleave EGF, gastrin, insulin, somatostatin, substance P, neurotensin, or growth hormone. The molecular weight of GDE is 83,000, as determined on SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of GDE was blocked, and the five partial amino acid sequences obtained on lysyl-endopeptidase digestion were determined to be N-L-T-E-E-Y-D-V-S-D-G-E-I-E-L-L-Y-E-K, V-E-T-Y-Y-D-L-L-F-E-K, L-Y-W-F-L-D-E-A-K, S-N-S-T-S-Y-V-K, and Y-Y-A-S-T-S-Y-D-D-T-Y-K. The same or homologous amino acid sequences have not been found in known proteins, demonstrating that GDE is a novel peptidase that degrades the secretin family: glucagon, VIP, and secretin.
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PMID:A novel proteinase, glucagon-degrading enzyme, secreted by a human pancreatic cancer cell line, HPC-YO. 777 1

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 two main pathogenetic characteristics of atopic dermatitis (AD) are: (i) antigen-dependent 'specific' reactivity, and (ii) altered non-immunological 'non-specific' reactivity. Our understanding of the role of non-specific reactivity is hampered by the fact that methods available for its quantification are limited. The aim of the present study was to assess the usefulness of two parameters as quantitative measures of non-specific skin reactivity in AD: (i) susceptibility to repeated epicutaneous exposure to an irritant (sodium lauryl sulphate, SLS), assessed by visual scoring and transepidermal water loss (TEWL) measurement, and (ii) reactivity to intracutaneously injected bioactive agents (codeine, FMLP, histamine, methacholine, substance P, trypsin), assessed by measurement of weal and flare size. These two parameters were tested in a group of AD patients, subdivided according to the severity of their dermatitis, and a control group. The visual score and TEWL after SLS exposure tended to be higher in the AD group than in the control group. Furthermore, visual score and post-exposure TEWL were positively correlated with the dermatitis severity score. Weal size following injection of codeine, histamine and substance P, and flare size following injection of all agents, except methacholine, were significantly lower in the AD group than in the control group. Negative correlations were found between weal and flare sizes and the dermatitis severity score. These findings can be explained by down-regulation of structures involved in weal and flare reactions. In conclusion, we propose that epicutaneous irritant susceptibility and reactivity to intracutaneous bioactive agents may be useful indicators of non-specific skin reactivity in AD.
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PMID:Irritant susceptibility and weal and flare reactions to bioactive agents in atopic dermatitis. I. Influence of disease severity. 854 88

Many atopic dermatitis (AD) patients have exacerbations of their skin disease in winter. These exacerbations may be caused by non-immunological 'non-specific' factors, such as low sun exposure and low temperature. To date, the influence of season on non-specific skin reactivity in AD has not been studied. The aim of the present investigation was to assess the influence of season on two skin parameters which may be used as quantitative measures of non-specific skin reactivity in AD: (i) susceptibility to repeated epicutaneous irritant (sodium lauryl sulphate, SLS) exposure, and (ii) weal and flare responses to intracutaneous injection of bioactive agents (codeine, FMLP, histamine, methacholine, substance P, trypsin). Four of 16 AD patients had dermatitis which was more severe in November than in July. Susceptibility to SLS was increased in November, both in AD patients and in control subjects. AD patients were more susceptible to SLS than control subjects in both July and November. Pre-exposure barrier function and skin hydration were reduced in November. The increased irritant susceptibility in November may be attributed to reduced barrier function, reduced skin hydration, and/or absence of the beneficial effects of ultraviolet light on cellular targets beneath the stratum corneum. Flare responses to codeine, methacholine, substance P and trypsin were also increased in November compared with July, especially in AD patients. However, smaller flares were observed in AD patients than in control subjects, in both July and November. Flare values were negatively correlated with dermatitis severity, probably because of down-regulation. Weal responses did not show a clear seasonal variation. Hence, susceptibility to epicutaneous irritants and reactivity to intracutaneously injected bioactive agents are parameters which may be used to monitor season-dependent changes in non-specific skin reactivity.
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PMID:Irritant susceptibility and weal and flare reactions to bioactive agents in atopic dermatitis. II. Influence of season. 854 89

Substance P (SP) is a neuropeptide that mediates multiple physiological responses including transmission of painful stimuli and inflammation via an interaction with a receptor of known primary sequence. To identify the regions of the SP receptor, also termed the NK-1 receptor, involved in peptide recognition, we are using analogues of SP containing the photoreactive amino acid p-benzoyl-L-phenylalanine (Bpa). In the present study, we used radioiodinated Bpa8-SP to covalently label with high efficiency the rat SP receptor expressed in a transfected mammalian cell line. To identify the amino acid residue that serves as the site of covalent attachment, a membrane preparation of labeled receptor was subjected to partial enzymatic cleavage by trypsin. A major digestion product of 22 kDa was identified. Upon reduction with 2-mercaptoethanol the mass of this product decreased to 14 kDa. The 22-kDa tryptic fragment was purified in excellent yield by preparative SDS/PAGE under nonreducing conditions. Subcleavage with Staphylococcus aureus V8 protease and endoproteinase ArgC yielded fragments of 8.2 and 9.0 kDa, respectively. Upon reductive cleavage, the V8 protease fragment decreased to 3.0 kDa while the endoproteinase ArgC fragment decreased to 3.2 kDa. Taking into consideration enzyme specificity, molecular size, determination of the presence or absence of N-glycosylation sites, and recognition by antibodies to specific sequences of the SP receptor, the V8 protease fragment is Thr-173 to Glu-183, while the endoproteinase ArgC fragment is Val-178 to Arg-190. These two fragments share the common sequence Val-Val-Cys-Met-Ile-Glu (residues 178-183). The site of covalent attachment of radioiodinated Bpa8-SP is thus restricted to a residue within this overlap sequence. The data presented here also establish that the cysteine residue in this sequence Cys-180, which is positioned in the middle of the second extracellular loop, participates in a disulfide bond that links the first and second extracellular loops of the receptor.
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PMID:The peptide binding site of the substance P (NK-1) receptor localized by a photoreactive analogue of substance P: presence of a disulfide bond. 855 54

Three new analogues of the neuropeptide substance P (SP) were synthesized. The C-terminal message segment was made more hydrophilic in (Arg9)SP or more hydrophobic in (Nle9)SP. In (AcPro2, Arg9)SP the charge at the N-terminal address segment was reduced, while that of the message segment was increased. The rationale underlying these substitutions was to correlate the physical-chemical properties of the SP-analogues, in particular their lipid-induced conformation and membrane-binding affinity, with receptor binding and functional activity. In solution, all three analogues exhibited random coil conformations as evidenced by circular dichroism spectroscopy. Addition of SDS micelles induced partially alpha-helical structures. The same structure was also produced by negatively charged lipid vesicles for (AcPro2, Arg9)SP and (Arg9)SP whereas both alpha-helix-like structures and beta-sheet structures were observed for SP and (Nle9)SP. The measurement of the Gibbs adsorption isotherms and monolayer expansion studies provided quantitative data on the surface area requirement and on the membrane penetration area of the SP analogues. The thermodynamic parameters for lipid binding were determined with monolayer expansion for measurements and high-sensitivity titration calorimetry. The apparent binding constants, Kapp, for membranes containing 100% POPG were of the order of 10(3)- 10(5) M(-1). The binding was due to electrostatic attraction of the cationic peptides to the negatively charged membrane surface. The intrinsic (hydrophobic) binding constants, obtained after correcting for electrostatic effects, were much smaller with Kp=10+/- 1 M(-1) for (Arg9)SP, 9 +/- 1 M(-1) for (AcPro2, Arg9)SP, and 39 +/- 3 M(-1) for (Nle9)SP. The measurement of the binding affinities to the NK-1 receptor and of the in vitro activities showed that all three peptides behaved as agonists. Their binding affinity to the neurokinin-1 receptor decreased with the size of the side chains at position 9 of the amino acid sequence but was independent of the cationic charge of the peptides. The fact that even the highly charged (Arg9)SP has agonistic activity provides evidence that the binding epitope at the receptor is in a rather hydrophilic environment. This finding is in agreement with the low hydrophobic binding constants and the weak penetration of the three peptides into negatively charged membranes. It argues against a membrane mediated receptor mechanism and suggests that the agonist approaches the receptor binding, site from the aqueous phase.
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PMID:Binding of substance P agonists to lipid membranes and to the neurokinin-1 receptor. 860 85

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