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)

Cellular membrane receptors for the immunostimulatory neuropeptide substance P have been previously identified on the cultured lymphoblast cell line, IM-9. The regulation of this receptor by ligand and the contribution to its molecular weight by N-linked sugars was studied by incubating IM-9 cells for 14 hr in the presence of [35S]met with or without substance P and tunicamycin, respectively. Cells were lysed and the receptor proteins were immunoprecipitated with an anti-receptor monoclonal antibody. SDS-PAGE analysis of untreated cellular lysates revealed specifically precipitated proteins of 38 kD and 33 kD, which were down-regulated by substance P. In tunicamycin-treated cells, whose substance P binding was not affected, the major immunoprecipitated protein had an apparent Mr of 29 kD. The time course of receptor processing was studied by pulse chase analysis. Three proteins of molecular weights 38 kD (mature receptor), 36 kD and 33 kD (receptor precursors) were identified for time periods of 30 min to 4 hr. The half life of the mature receptor and its precursors was approximately 1 hr and 0.5 hr, respectively. Results from the present studies suggest that the lymphocyte substance P receptor is translated as a precursor protein that is glycosylated.
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PMID:Processing of the human IM-9 lymphoblast substance P receptor. Biosynthetic and degradation studies using a monoclonal anti-receptor antibody. 245 May 42

A novel substance P-degrading endopeptidase has been solubilized with Brij 35 from a membrane fraction of rat brain and purified by a procedure involving DEAE-cellulose chromatography, hydroxyapatite chromatography, Sephadex G-100 gel filtration, and Mono-Q HPLC. The activity of the degrading enzyme was monitored by measuring the disappearance of substance P by means of a bioassay and HPLC. SDS-polyacrylamide gel electrophoresis under reducing conditions of the enzyme gave a single band corresponding to a molecular weight of 58,000. The molecular weight of the enzyme was estimated to be 55,000 by gel filtration and the optimum pH for its activity was 7.5.. The purified enzyme cleaved substance P at three bonds, Pro4-Gln5, Gln5-Gln6, and Gln6-Phe7, in the ratio of 2:2:3. EDTA, o-phenanthroline, and p-chloromercuribenzenesulfonic acid strongly inhibited the enzyme, while diisopropyl fluorophosphate, E-64, Z-Gly-ProCH2Cl, phosphoramidon, and captopril had little or no inhibitory effect on it. The cleavage of substance P by the rat brain synaptic membrane was also analyzed under the conditions with or without these inhibitors. The inhibitor-susceptibility of the cleavage sites suggests that the present enzyme, together with endopeptidase-24.11, is involved in the degradation of substance P in the synaptic region.
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PMID:Purification and characterization of a substance P-degrading endopeptidase from rat brain. 246 57

The biologic activity of substance P has been demonstrated to be limited both in in vivo and in vitro by a membrane-bound protease, neutral endopeptidase (EC 3.4.24.11). The interaction of substance P with its receptor on guinea pig lung tissues was studied in the presence of an inhibitor of neutral endopeptidase under conditions that protect the peptide from degradation. Uptake of 0.1 nM [125I]-BH-substance P in lung membrane preparations was rapid at 4 degrees C, reaching equilibrium in 30 to 40 min, and binding was stable for at least 30 min thereafter. Binding was reversible and saturable. Scatchard analyses of saturation binding data are consistent with a single class of receptor molecules in both lung parenchymal and airway membranes, with a Kd of 2 to 3 nM and a receptor density of 4,000 to 5,000 fmol/g wet wt of tissue. In competitive binding experiments, neurokinin A and substance P methyl ester were equipotent and required approximately 100-fold higher concentrations to effect equivalent displacement than unlabeled substance P. Eledoisin also competed for [125I]-BH-substance P binding, but was less effective than the other analogs. The spasmogenically inactive derivative, substance P 1-9, did not compete for substance P binding at concentrations as high as 1 microM. Binding of [125I]-BH-substance P was rapidly and completely reversed by addition of 0.1 mM GTP, suggesting that association with a GTP binding protein is required for high affinity binding of substance P to its receptor in lung. The substance P receptor molecule was further characterized by covalently crosslinking [125I]-BH-substance P to membrane preparations followed by SDS-PAGE of the solubilized material.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of the substance P receptor in guinea pig lung tissues. 248 20

Cathepsin D was purified about 1000-fold from human brain cortex by a procedure involving ammonium sulfate fractionation (30-70%), Sephadex G-75 chromatography, affinity chromatography on pepstatin-Sepharose and isoelectric focusing. The enzyme was assayed fluorometrically at pH 3.2, the substrates used were globin or haemoglobin modified with pyridoxal-5'-phosphate. 6 multiple forms of cathepsin D were resolved in the isoelectric focusing step with pI values 4.4, 4.8, 5.3, 6.2, 6.5 and 6.8. Km of pyridoxal-globin and pyridoxal-haemoglobin for all 6 multiple forms is 1.8-2.0 X 10(-5) M and 1.3 to 4 X 10(-6) M, respectively, and Ki of pepstatin is 2-4 X 10(-9) M. Gel filtration of the multiple forms on Sephadex G-100 column showed that each has a molecular weight of about 50 000. Human brain cathepsin D has a pH optimum of 3.2 with a smaller second optimum at pH 4.0 (pyridoxal-haemoglobin being used as substrate). All the multiple forms have the same pH-dependence curve. On SDS-polyacrylamide gel electrophoresis the purified enzyme produced 3 bands approximately corresponding to Mr 50 000, 35 000 and 15 000. Study of the breakdown of substance P and its C-terminal heptapeptide by cathepsin D shows that cleavage occurs at the Phe-Phe linkages of both substrates tested.
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PMID:Cathepsin D from human brain: purification and multiple forms. 667 69

Cathepsin B was purified about 11,000-fold from monkey skeletal muscle by ammonium sulfate fractionation and sequential column chromatographies monitored by assaying of Z-Phe-Arg-MCA hydrolase activity. The purified enzyme gave a single protein band on SDS-polyacrylamide gel electrophoresis, and its molecular weight was estimated to be 24,000 by gel filtration. It had a pH optimum of 6.5, required a thiol reducing agent for activation, and was inhibited by various thiol protease inhibitors. These properties were similar to those reported for cathepsins B from other sources. Although the enzyme scarcely hydrolyzed ordinary proteins, such as casein, hemoglobin, and bovine serum albumin, it degraded myosin and actin among various myofibrillar proteins. These results strongly suggested that skeletal muscle cathepsin B may participate in the degradation of muscle proteins in vivo. In addition, cathepsin B was shown to hydrolyze various neuropeptides such as Leu-enkephalin, beta-neoendorphin, alpha-neoendorphin, dynorphin(1-13), and substance P. It appeared to act on these peptides mainly as a dipeptidyl carboxypeptidase, although not so rigorously, presumably due to its endopeptidase activity.
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PMID:Purification and characterization of cathepsin B from monkey skeletal muscle. 672 39

A highly purified preparation of a cation-sensitive neutral endopeptidase was obtained from bovine pituitaries. The enzyme constitutes almost 0.1% of the protein in bovine pituitary homogenates. Polyacrylamide gel electrophoresis of the enzyme showed a single protein band, and in gel filtration experiments on calibrated Sepharose 6B columns the enzyme eluted slightly ahead of thyroglobulin, suggesting an apparent molecular weight of about 700,000. Polyacrylamide gel electrophoresis in SDS-containing buffers indicated the presence of three major components with molecular weights ranging from about 24,000 to 28,000. The enzyme hydrolyzes bonds between hydrophobic and small neutral amino acids in both model synthetic substrates and biologically active peptides such as substance P, LH-RH, and bradykinin. Peptide bonds in which the carbonyl group is contributed by a glutamyl or arginyl residue are also hydrolyzed, especially if they are preceded in the sequence by hydrophobic amino acids. Leupeptin exclusively inhibited enzymatic activity toward the arginine-containing substrates. This observation, together with the high molecular weight and broad specificity of the enzyme, raised the possibility that the isolated enzyme represents a proteolytic complex composed of units with distinctly different activities. Preliminary attempts to dissociate the enzyme into catalytic units of lower molecular weight were not successful and led to loss of activity.
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PMID:Cation-sensitive neutral endopeptidase: isolation and specificity of the bovine pituitary enzyme. 677 72

By acid extraction, ethanol precipitation, affinity chromatography on 4-phenylbutylamine-Sepharose 4B and gel filtration on Sephadex G-100, calf liver neutral proteinase was purified. The purified enzyme was electrophoretically homogeneous and over 2000 times more active than the starting homogenate. The molecular weight, determined by SDS electrophoresis, was calculated as 27000. The pH optimum of the enzyme for whole calf thymus histones and N-benzoyltyrosine, ethyl ester (BTEE) was at 7.0 and 7.0-7.5. The Km value for histones was 2% and for BTEE 1.66 mM. The enzyme was strongly inhibited by soya-bean trypsin inhibitor and leucocyte intracellular I-1A inhibitor and less by alpha 1-antitrypsin and leucocyte inhibitor I-1B. The enzyme hydrolyzed only selected protein substrates, such as total thymus histones, Lys-rich histones, nucleoprotein and substance P, but not Arg-rich histones, hemoglobin and casein. The enzyme showed chymotrypsin-like properties by cleavage of substance P at the carboxyl groups of phenylalanine and leucine.
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PMID:The isolation of liver serine proteinase by affinity chromatography on 4-phenylbutylamine-sepharose 4 B. 705 3

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


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