Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: UNIPROT:P15088 (mast cell)
14,925 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Oxidation of the Met residues of human interleukin 6 (IL-6) molecule has been performed. Reactivity of Met for the oxidation reaction was found to decrease in the order of Met50, Met118, Met185, Met162, and Met68. Chemical modifications involving oxidation and carboxypeptidase A digestion of IL-6 have led to the assignments of the methyl proton resonances of Met162 and Met185, respectively. The hydroxynitrobenzyl chromophore attached to Trp158 in the IL-6 molecule showed a different absorption spectrum when the labeled IL-6 was bound to the soluble IL-6 receptor. This result indicates that Trp158 is near the receptor-binding region in IL-6. On the basis of the 1H-NMR and chemical modification data, it has been concluded that Trp158 is in spatial proximity to Met162, His165 and Met185. The receptor-binding activity decreased with an increase in the number of oxidized Met residues. Of these five Met residues, Met162 was the residue in which the receptor-binding activity decreased in the most parallel degree with that of the oxidation reaction.
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PMID:Chemical modification and 1H-NMR studies on the receptor-binding region of human interleukin 6. 190 Oct 38

Cross-linking of 125I-IL-4 to the surface of cells expressing IL-4R yields as the major IL-4-binding molecules, polypeptide chains with inferred m.w. of approximately 70,000 (p70) and approximately 120,000 to 140,000 (p120-p140). The demonstration that the functional product of the IL-4R cDNA clone has m.w. of approximately 140,000 and that no p70 product is detected in transfected COS-7 cells has led to an uncertainty regarding the nature of p70. To study this issue, we examined the relationship of the IL-4-binding molecules p120 and p70 and, in parallel, attempted to immunoprecipitate p70 from surface and internally labeled cells using IL-4 and two anti-IL-4R antibodies (M1 and M2), bound to Affigel 10, as ligands. Cross-linked complexes containing 125I-IL-4 and p70 or p120 were isolated and digested with chymotrypsin or with V8 protease. Three distinct IL-4-binding peptides could be compared; these were indistinguishable for cross-linked p70 and p120, strongly implying that p70 and p120 were structurally related. Furthermore, immunoprecipitates made with IL-4 or anti-IL-4R-Affigel did not contain p70. This led us to conclude that p70 is a breakdown product of p120. A second IL-4-binding molecule of 40,000 Da (p40) expressing the M1 and M2 epitopes of the IL-4R was detected and appears to be the product of an mRNA coding for the soluble form of the receptor. mRNA for p40 was detected in both the T cell line CT.4R and the mast cell line CFTL.12 using polymerase chain reaction primers unique to this species of message. Pulse-chase studies of IL-4R in [35S] methionine-labeled cells indicates that p40 is derived from a 42,000-Da precursor that is detectable at the end of the pulse period, and thus, further argue that p40 is an independently translated molecule and not a degradation product of p120. Although p40 has been previously shown to be a soluble, truncated form of the receptor, we failed to observe secretion of p40 into the medium by internally labeled CT.4R cells.
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PMID:The IL-4 receptor: biochemical characterization of IL-4-binding molecules in a T cell line expressing large numbers of receptors. 200 96

A high-performance liquid chromatographic method involving fluorescence derivatization followed by separation on a reversed-phase polymer (octadecylated polyvinylalcohol copolymer gel) column is described for the determination of opioid peptides in rat brain tissues. The peptides extracted from brain tissues were converted into fluorescent derivatives by reaction with hydroxylamine, cobalt(II) ion and borate. The derivatives were separated on an Asahipak ODP-50 column by gradient elution of acetonitrile in the mobile phase containing borate buffer (pH 9.5). The detection limits (S/N = 3) for the peptides were 0.33-1.21 pmol per 100 microliters injected. The method actually permit the determination of leucine enkephalin, methionine enkephalin, methionine enkephalin-Arg-Phe and methionine enkephalin-Arg-Gly-Leu in the tissues. The method is also applied to the characterization of the peptides in the tissues by means of enzymatic degradations with carboxypeptidase A and trypsin.
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PMID:Pre-column fluorescence derivatization high-performance liquid chromatography of opioid peptides in rat brain and its use for enzymatic peptide characterization. 204 96

Catalase is a characteristic enzyme of peroxisomes. To study the molecular mechanisms of the biogenesis of peroxisomes and catalase in a less complex system than rat liver cells, we expressed recombinant rat catalase in Escherichia coli, which has no peroxisomes. The concentration of recombinant catalase produced in E. coli transformed with the expression vector carrying the complete coding region of rat catalase cDNA was about 0.1% of the total soluble protein. The recombinant catalase was purified by DEAE-cellulose column chromatography followed by acidic ethanol precipitations. The properties of rat liver catalase and those of the recombinant were similar with respect to molecular mass, catalytic properties, profiles of absorption spectra, and iron contents. The NH2-terminal amino acid sequence of the purified recombinant catalase, as determined by Edman degradation, was in complete agreement with the amino acid sequence predicted from the nucleotide sequence of rat catalase cDNA, except that the first initiator methionine was not detected. The COOH-terminal amino acid sequence was determined by carboxypeptidase A digestion and the sequence, -Ala-Asn-Leu-OH, matched the predicted COOH-terminal amino acid sequence of rat catalase. Recombinant rat catalase gave almost the same multiple protein bands on native polyacrylamide gel isoelectric focusing as observed with authentic rat liver catalase.
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PMID:Purification and properties of recombinant rat catalase produced in Escherichia coli. 220 16

Crystallographic studies suggest that Arg-127 is a key amino acid in the hydrolysis of peptides and esters by carboxypeptidase A. The guanidinium group of Arg-127 is hypothesized to stabilize the oxyanion of the tetrahedral intermediate formed by the attack of water on the scissile carbonyl bond. We have replaced this amino acid in rat carboxypeptidase A1 with lysine (R127K), methionine (R127M), and alanine (R127A), in order to define the role of Arg-127 in carboxypeptidase catalyzed hydrolysis. The wild-type and mutant enzymes were expressed in yeast and purified. Kinetic studies show that Arg-127 substitution decreases kcat for both ester and amide substrates, whereas Km is relatively unchanged; for R127M and R127A this corresponds to a 6 kcal/mol decrease in transition state stabilization of the rate-limiting step. The binding affinity for the phosphonate transition state analog, Cbz-Phe-Ala(P)-OAla, was decreased by 5.4 kcal/mol, whereas binding affinity for the ground state inhibitor, DL-benzylsuccinic acid, was decreased by only 1.7 kcal/mol for R127M. Electrostatic calculations employing a finite difference solution to the Poisson-Boltzmann equation predict that the positive charge of Arg-127 should stabilize the transition state by 6-8 kcal/mol. Therefore, the experimental and theoretical data suggest that the primary role of Arg-127 is stabilization of the transition state through electrostatic interaction with the oxyanion.
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PMID:Arginine 127 stabilizes the transition state in carboxypeptidase. 224 16

The protease inhibitor alpha-1-antichymotrypsin, which binds to chymotrypsin-like enzymes in a sodium dodecyl sulfate-resistant manner, has been shown recently to be both a normal constituent of brain and an integral component of the neuritic plaques that form in Down's syndrome and Alzheimer's disease. We have now identified in rat brain a Mr 25,000 alpha-1-antichymotrypsin-binding protein classified as a chymotrypsin-like protease by its inhibitor profile and substrate specificity. Release of 125I-labeled breakdown products from bands containing the protease in substrate-linked polyacrylamide gels was examined in parallel with hydrolysis of tetrapeptide chromogenic substrates in vitro to establish conditions under which the Mr 25,000 protease was the only activity being measured in vitro. The protease was completely membrane associated but was extractable using 1 M MgCl2; prior extraction of detergent- and low ionic strength-soluble proteins from membranes was used to increase its specific activity. The formation of sodium dodecyl sulfate-resistant bonds between human alpha-1-antichymotrypsin and the protease (kassoc = 2.9 X 10(6) M-1 s-1) was used to titrate the concentration of free protease solubilized from membranes. The protease cleaved both succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, and methoxy-succinyl-Ala-Ala-Pro-Met-p-nitroanilide, the latter being of interest because cleavage after a methionine residue is predicted to generate the amino terminus of the neuritic plaque component beta-amyloid from its precursor protein. In fact, the solubilized protease degraded 90% of membrane-associated beta-amyloid precursor protein detected by Western blot analysis. The protease was kinetically distinct from both chymotrypsin and cathepsin G in direct comparisons and did not match kinetic values published for the rat mast cell proteases against comparable substrates; we therefore refer to the protease with the descriptive acronym clipsin (for chymotrypsin-like protease). Proteases similar to and potentially identical to clipsin were detected by enzymography in other organs from rat (most notably spleen and adult lung). The enzyme in brain was distinguished by a narrow window of elevated activity surrounding postnatal day 5, which was 12-14-fold higher than levels in day 1 or adult brain. Because independent lines of evidence suggest that a brain chymotrypsin-like protease may be involved in the etiology of Down's syndrome and Alzheimer's disease, clipsin is discussed as a candidate for such a role.
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PMID:Clipsin, a chymotrypsin-like protease in rat brain which is irreversibly inhibited by alpha-1-antichymotrypsin. 230 81

A new 28 amino acid peptide, we recently isolated from the venom of the bumblebee Megabombus pennsylvanicus. has been characterized. The peptide, Met-Cys-Ile-Cys-Lys-Asn-Gly-Lys-Pro-Leu-Pro-Gly-Phe-Ile-Gly-Lys-Ile-Cys- Arg-Lys-Ile-Cys-Met-Met-Gln-Gln-Thr-His(NH2), has been named bumblebee mast cell degranulating (MCD) peptide due to its ability to degranulate rat peritoneal mast cells, and its resemblance to the bee venom MCD peptide. Bumblebee MCD peptide, unlike bombolitins, the other mast cell degranulating heptadecapeptides of bumblebee venom, is not lytic and releases histamine at a dose as low as 0.05 micrograms/ml (1.6 X 10(-8) M).
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PMID:Amino acid sequence of bumblebee MCD peptide: a new mast cell degranulating peptide from the venom of the bumblebee Megabombus pennsylvanicus. 242 Dec 65

The expression of the antigenic determinant identified by the B54.2 rat monoclonal antibody on four populations of mouse mast cells has been quantified, and the epitope-bearing surface antigen and its biosynthesis have been characterized. As assessed by indirect immunofluorescence staining and flow cytometric analysis, B54.2 antibody bound to serosal mast cells (S-MC), bone marrow culture-derived mast cells (BM-MC), fetal liver culture-derived mast cells (FTL-MC), and Abelson murine leukemia virus-transformed FTL-MC (ABFTL-MC). However, the intensity of cell surface fluorescence exhibited by ABFTL-MC was approximately eightfold less per cell compared with nontransformed, culture-derived mast cells. Immunoprecipitation of B54.2 antibody-binding molecules from each population of mast cells labeled intrinsically with [35S]methionine and analysis by SDS-PAGE demonstrated that the B54.2 epitope was expressed in each case on two noncovalently associated proteins of 110,000 Mr and approximately 130,000 Mr, but that the percentage of radiolabel in the latter species was approximately threefold less in ABFTL-MC than in BM-MC. As assessed by pulse-chase analysis with [35S]methionine, the 110,000 Mr protein was a precursor of the 130,000 Mr molecule ("B54.2 antigen") synthesized by BM-MC. Labeling of BM-MC with [35S]methionine in the presence of tunicamycin followed by immunoprecipitation and SDS-PAGE of B54.2 antibody-binding material revealed a single species of 93,000 Mr, indicating that the native molecules contained N-linked carbohydrate. Endoglycosidase H treatment of the glycoproteins precipitated by B54.2 antibody from BM-MC reduced the Mr of the 110,000-Mr molecule to 93,000 Mr without an appreciable change in the 130,000-Mr species. These data indicate that the 110,000-Mr precursor form is a "high mannose" type glycoprotein and the 130,000-Mr membrane surface B54.2 antigen is a "complex" type glycoprotein, and that the epitope recognized by the B54.2 antibody on the surface of the mouse mast cell populations is located on the 93,000-Mr peptide core.
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PMID:Biochemical characterization of a mast cell plasma membrane antigen shared by mouse serosal, culture-derived, and virally transformed mast cells. 243 44

As assessed by immunoprecipitation analyses, expression of the epitope recognized by the rat mAb B23.1 is approximately sevenfold greater on the surface of mouse IL-3-dependent bone marrow culture-derived mast cells (BMMC) than on serosal mast cells (SMC) obtained directly from the peritoneal cavity. Immunoprecipitation of B23.1 antibody-binding molecules from Na[125I] surface-labeled BMMC and SMC followed by sizing on SDS-polyacrylamide gels under reducing conditions demonstrated that the epitope is located on molecules of 49,000 and 47,500 Mr, respectively. An additional immunoprecipitated molecule of 42,000 Mr was detected from BMMC intrinsically radiolabeled with [35S]methionine, and pulse-chase analyses revealed that this species was a biosynthetic precursor of the 49,000 Mr cell surface form of the Ag. Treatment of the immunoprecipitated 42,000 and 49,000 Mr forms with endoglycosidase F reduced the Mr of both to 37,000, as did intrinsic radiolabeling of BMMC in the presence of tunicamycin, indicating that both the 42,000 Mr precursor form and the 49,000 Mr cell surface molecule (gp49) contained N-linked carbohydrate. Activation of [32P]orthophosphate-labeled BMMC by sensitization with mouse monoclonal IgE anti-TNP and challenge with TNP-BSA or by exposure to the calcium ionophore A23187 elicited the rapid phosphorylation of gp49 but not of its precursor forms, as did treatment of the cells with PMA. Elution of phosphorylated and immunoprecipitated gp49 from SDS-polyacrylamide gels followed by partial acid hydrolysis of the protein and phosphoamino acid analysis by high voltage thin-layer electrophoresis on cellulose plates indicated that serine, but not threonine or tyrosine, was phosphorylated upon stimulation of BMMC with IgE/Ag, calcium ionophore, or PMA. Cholera toxin did not elicit phosphorylation of gp49. These data suggest that gp49, a plasma membrane glycoprotein preferentially expressed by mouse BMMC, may be either directly or indirectly phosphorylated via protein kinase C during mast cell activation-secretion.
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PMID:Activation- and phorbol ester-stimulated phosphorylation of a plasma membrane glycoprotein antigen expressed on mouse IL-3-dependent mast cells and serosal mast cells. 246 32

Active tumor promoters such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or membrane-diffusible synthetic diacylglycerols such as 1,2-dioctanoyl-sn-glycerol (DiC8), which specifically activate protein kinase C (PKC), inhibited the agonist-mediated rise in cytosolic calcium [(Ca2+)i] in a mast cell line (PB-3c) and human platelets. TPA inhibition of agonist-mediated calcium transient in platelets was readily reversed by the PKC inhibitor staurosporine. In contrast to DiCs, only active tumor promoters induced a time- and dose-dependent translocation of cytosolic PKC to membranes as determined both enzymatically or by immunoblotting. However, the concentration of TPA required to induce a half-maximal subcellular redistribution of immunodetectable PKC activity was an order of magnitude greater than the half-maximal dose required to inhibit the intracellular rise in (Ca2+)i. Thus, activation of PKC seems not to be exclusively coupled to its translocation to membranes, suggesting that translocation of PKC is mainly involved in the down-regulation of PKC. Down-regulation of immunoprecipitable PKC was studied in various human breast cancer cell lines that display differential growth inhibitory responses toward the tumor promoter. TPA induced translocation of [35S]methionine-prelabeled cytosolic 80 kDa PKC to membranes followed by complete degradation of the enzyme (t1/2 = 2 h) without affecting PKC synthesis. During prolonged TPA exposure, 20-80% of total 80 kDa PKC of control cells was still synthetized as a membrane-bound 74/80 kDa PKC doublet. Although both proteins lacked PKC activity and phorbol ester binding, they revealed structural similarity with the active 80 kDa PKC form of untreated cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Role of protein kinase C (PKC) in short- and long-term cellular responses: inhibition of agonist-mediated calcium transients and down-regulation of PKC. 246 82


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