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)

The orientation of cytochrome b6 in the thylakoid membrane and the question of whether the number of membrane spanning helices is an even or odd number was tested through the relative trypsin susceptibility of epitopes (Asp-5 to Gln-14) and (Ile-205 to Leu-214) at the NH2 and COOH termini, respectively, of the 214-residue cytochrome b6 polypeptide. A structure of the cytochrome with an even number of helices and the NH2 and COOH termini on the stromal side of the membrane was inferred from the following: 1) cleavage of cytochrome b6 by trypsin added to thylakoids occurs by removal of both of the exposed NH2- and COOH-terminal epitopes. The epitopes at the termini were more sensitive to trypsin after prior treatment of thylakoids with carboxypeptidase A, indicating that these epitopes are shielded on the stromal side of the membrane by the COOH termini of other proteins. 2) Both epitopes were more trypsin-sensitive in thylakoid membranes than was cytochrome f that is only sensitive to trypsin acting on the lumen side of the membrane. 3) The NH2- and COOH-terminal epitopes of cytochrome b6 were also more sensitive to trypsin added to thylakoid membranes than were the oxygen-evolving complex 16- and 33-kDa proteins that are completely located on the lumen side. 4) The order of trypsin susceptibility was reversed in inside-out membranes, where the cytochrome NH2- and COOH-terminal epitopes were less sensitive than the 16- and 33-kDa proteins. The decreased relative sensitivity of the cytochrome b6 epitopes occurs in spite of a greater absolute sensitivity of these epitopes to trypsin in inside-out membranes. 5) The greater absolute sensitivity can be explained by a 4-helix model that includes trypsin-sensitive sites on the lumen side.
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PMID:Thylakoid membrane protein topography. Location of the termini of the chloroplast cytochrome b6 on the stromal side of the membrane. 169 78

A novel plasminogen-binding protein has been isolated from human plasma utilizing plasminogen-Sepharose affinity chromatography. This protein copurified with alpha 2 antiplasmin when the plasminogen affinity column was eluted with high concentrations of epsilon-aminocaproic acid (greater than 20 mM). Analysis by sodium dodecyl sulfate suggests this protein has an apparent Mr of 60,000. The amino-terminal amino acid sequence showed no similarity to other protein sequences. Based on the amino-terminal amino acid sequence, oligonucleotide probes were designed for polymerase chain reaction primers, and an approximately 1,800 base pair cDNA was isolated that encodes this Mr 60,000 protein. The deduced amino acid sequence reveals a primary translation product of 423 amino acids that is very similar to carboxypeptidase A and B and consists of a 22-amino acid signal peptide, a 92-amino acid activation peptide, and a 309-amino acid catalytic domain. This protein shows 44 and 40% similarity to rat procarboxypeptidase B and human mast cell procarboxypeptidase A, respectively. The residues critical for catalysis and zinc and substrate binding of carboxypeptidase A and B are conserved in the Mr 60,000 plasminogen-binding protein. The presence of aspartic acid at position 257 of the catalytic domain suggests that this protein is a basic carboxypeptidase. When activated by trypsin, it hydrolyzes carboxypeptidase B substrates, hippuryl-Arg and hippuryl-Lys, but not carboxypeptidase A substrates, and it is inhibited by the specific carboxypeptidase B inhibitor (DL-5-guanidinoethyl)mercaptosuccinic acid. We propose that the Mr 60,000 plasminogen-binding protein isolated here is a novel human plasma carboxypeptidase B and that it be designated pCPB.
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PMID:Isolation, molecular cloning, and partial characterization of a novel carboxypeptidase B from human plasma. 193 7

An identical neuropeptide was isolated from the corpora cardiaca of two beetle species, Melolontha melolontha and Geotrupes stercorosus. Its primary structure was determined by pulsed-liquid-phase sequencing employing Edman chemistry after enzymically deblocking the N-terminal pyroglutamate residue. The C-terminus was also blocked, as indicated by the lack of digestion when the peptide was incubated with carboxypeptidase A. The sequence of this peptide, which is designated Mem-CC, is pGlu-Leu-Asn-Tyr-Ser-Pro-Asp-Trp-NH2. It is a new member of the adipokinetic hormone/red-pigment-concentrating hormone (AKH/RPCH) family of peptides with two unusual structural features: it is charged and contains a tyrosine residue at position 4, where all other family members have a phenylalanine residue. Structure-activity studies in the migratory locust (Locusta migratoria) and the American cockroach (Periplaneta americana) revealed that the peptide was poorly active, owing to its structural uniqueness.
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PMID:A unique charged tyrosine-containing member of the adipokinetic hormone/red-pigment-concentrating hormone peptide family isolated and sequenced from two beetle species. 203 45

Here we present the cloning of three novel mouse mast cell-specific serine proteases, MMCP-1, MMCP-4 and MMCP-5. A region of approximately 4 kb covering the five exons and 930 bp 5' and 280 bp 3' flanking sequences of the gene for MMCP-1 was characterized by nucleotide sequence analysis. A comparison with the corresponding region of the rat mucosal mast cell-specific protease RMCP-II is presented. cDNA clones for the mast cell proteases MMCP-4 (950 bp) and MMCP-5 (1098 bp) were isolated from a cDNA library of a connective tissue mast cell-like mouse mastocytoma cell line. All three proteases were found to belong to the family of chymotrypic serine proteases as deduced from the absence of the Asp 189 which is characteristic for all serine proteases having cleavage specificities similar to pancreatic trypsin. The active polypeptides, excluding possible post-translational glycosylations, have an Mr of 25-26 kDa. Analysis of the amino acid composition reveals a positive net charge for all three proteases MMCP-1 +3, MMCP-4 +18 and MMCP-5 +12). Based on their high sequence identity (88%) and high positive net charges (+18 and +18, respectively) we assume that the MMCP-4 is the mouse homolog to rat RMCP-I. Probes specific for each of these three highly homologous protease genes have been generated by subcloning of fragments of approximately 100 bp in length, originating from the 3' ends of the mRNA into plasmid vectors. Northern blot analysis of mRNA from a number of murine cell lines shows gene expression of these proteases to be specific for the differentiation stage of the mast cell. The MMCP-1 is expressed only at the mucosal mast cell stage and 5 only in mast cells of the connective tissue mast cell stage. These serine proteases may serve as highly specific markers in the analysis of mast cell heterogeneity, differentiation and function.
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PMID:Cloning and structural analysis of MMCP-1, MMCP-4 and MMCP-5, three mouse mast cell-specific serine proteases. 206 May 76

Sonicates of mouse bone marrow-derived mast cells (BMMC) differentiated in vitro and of mouse serosal mast cells differentiated in vivo contained small but approximately equal amounts of aminopeptidase activity, as determined by cleavage of leucine-beta-naphthylamide and resolution of the reaction products by reverse-phase high-performance liquid chromatography. Aminopeptidase activity was exocytosed from antigen-activated, IgE-sensitized BMMC in proportion to the secretory granule enzyme beta-hexosaminidase, thereby localizing approximately 60% of the total cell-associated aminopeptidase activity to the secretory granules of the mast cells. A prominent secretory granule location for aminopeptidase was confirmed by activity measurement in subcellular fractions of disrupted BMMC. The secretory granule aminopeptidase had a pH optimum of 6.0-8.0 and a Km of 0.36 +/- 0.06 mM (mean +/- SD; n = 3) for leucine-beta-naphthylamide. When various amino acid beta-naphthylamides were used as substrates, the preference of the secretory granule enzyme was Ala greater than Leu greater than Phe much greater than Arg much greater than Asp = Tyr. Most of the aminopeptidase activity that was exocytosed from calcium ionophore-activated BMMC was bound to 35S-labeled proteoglycans in complexes of greater than 1 x 10(7) kDa as defined by exclusion during Sepharose CL-2B gel-filtration chromatography. We postulate that the amino-peptidase in the mast cell protease/proteoglycan complexes allows the removal of N-terminal amino acids from peptides that are generated by the action of mast cell endopeptidases.
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PMID:Identification of aminopeptidase activity in the secretory granules of mouse mast cells. 206 74

Hirudin, a thrombin-specific inhibitor, comprises a compact amino-terminal core domain (residues 1-52) and a disordered acidic carboxyl-terminal tail (residues 53-65). An array of core fragments were prepared from intact recombinant hirudin by deletion of various lengths of its carboxyl-terminal tail on selective enzymatic cleavage. Hir1-56 and Hir1-53 were produced by pepsin digestion at Phe56-Glu57 and Asp53-Gly54. Hir1-52 was generated by Asp-N cleavage at Asn52-Asp53. Hir1-49 was prepared by cleavage of Gln49-Ser50 by chymotrypsin, elastase, and thermolysin. In addition, Hir1-62 (containing part of the carboxyl-terminal tail) was derived from Hir1-65 by selective removal of the three carboxyl-terminal amino acids using carboxypeptidase A. Hirudin amino-terminal core fragments were stable at extreme pH (1.47 and 12.6), high temperature (95 degrees C), and resistant to attack by various proteinases. For instance, following 24-h incubation with an equal weight of pepsin, the covalent structure of Hir1-52 remained intact and its anticoagulant activity unaffected. Unlike intact hirudin (Hir1-65) the inhibitory potency of which is a consequence of concerted binding of its amino-terminal and carboxyl-terminal domains to the active site and the fibrinogen recognition site of thrombin, the core fragments block only the active site of thrombin with binding constants of 19 nM (Hir1-56), 35 nM (Hir1-52), and 72 nM (Hir1-49). As an anticoagulant Hir1-56 is about 2-, 4-, and 30-fold more potent (on a molar basis) than Hir1-52, Hir1-49, and Hir1-43, respectively. Hir1-56 was also about 15-fold more effective than the most potent carboxyl-terminal fragment of hirudin, sulfated-Hir54-65, although they bind to independent sites on thrombin. The potential advantages of hirudin core fragments as antithrombotic agents are discussed in this report.
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PMID:Production, properties, and thrombin inhibitory mechanism of hirudin amino-terminal core fragments. 226 19

A hypertrehalosaemic neuropeptide from the corpora cardiaca of the blowfly Phormia terraenovae has been isolated by reversed-phase h.p.l.c., and its primary structure was determined by pulsed-liquid phase sequencing employing Edman chemistry after enzymically deblocking the N-terminal pyroglutamate residue. The C-terminus was also blocked, as indicated by the lack of digestion when the peptide was incubated with carboxypeptidase A. The octapeptide has the sequence pGlu-Leu-Thr-Phe-Ser-Pro-Asp-Trp-NH2 and is clearly defined as a novel member of the RPCH/AKH (red-pigment-concentrating hormone/adipokinetic hormone) family of peptides. It is the first charged member of this family to be found. The synthetic peptide causes an increase in the haemolymph carbohydrate concentration in a dose-dependent fashion in blowflies and therefore is named 'Phormia terraenovae hypertrehalosaemic hormone' (Pht-HrTH). In addition, receptors in the fat-body of the American cockroach (Periplaneta americana) recognize the peptide, resulting in carbohydrate elevation in the blood. However, fat-body receptors of the migratory locust (Locusta migratoria) do not recognize this charged molecule, and thus no lipid mobilization is observed in this species.
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PMID:Isolation and structure of a novel charged member of the red-pigment-concentrating hormone-adipokinetic hormone family of peptides isolated from the corpora cardiaca of the blowfly Phormia terraenovae (Diptera). 238 78

Mast cell tryptase is a secretory granule associated serine protease with trypsin-like specificity released extracellularly during mast cell degranulation. To determine the full primary structure of the catalytic domain and precursor forms of tryptase and to gain insight into its mode of activation, we cloned cDNAs coding for the complete amino acid sequence of dog mast cell tryptase and a second, possibly related, serine protease. Using RNA from dog mastocytoma cells, we constructed a cDNA library in lambda gt 10. Screening of the library with an oligonucleotide probe based on the N-terminal sequence of tryptase purified from the same cell source allowed us to isolate and sequence overlapping clones coding for dog mast cell tryptase. The tryptase sequence includes the essential residues of the catalytic triad and an aspartic acid at the base of the putative substrate binding pocket that confers P1 Arg and Lys specificity on tryptic serine proteases. The apparent N-terminal signal/activation peptide terminates in a glycine. A glycine in this position has not been observed previously in serine proteases and suggests a novel mode of activation. Additional screening of the library with a trypsinogen cDNA led to the isolation and sequencing of a full-length clone apparently coding for the complete sequence of a second tryptic serine protease (DMP) which is only 53.4% identical with the dog tryptase sequence but which contains an apparent signal/activation peptide also terminating in a glycine. Thus, the proteases encoded by these cloned cDNAs may share a common mode of activation from N-terminally extended precursors.
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PMID:Molecular cloning of dog mast cell tryptase and a related protease: structural evidence of a unique mode of serine protease activation. 250 77

Five structurally related heptadecapeptides rich in hydrophobic amino acids have been discovered in the venom of the bumblebee Megabombus pennsylvanicus. We have named them bombolitin I (Ile-Lys-Ile-Thr-Thr-Met-Leu-Ala-Lys-Leu-Gly-Lys-Val-Leu-Ala-His-Val-NH2 ), bombolitin II (Ser-Lys-Ile-Thr-Asp-Ile-Leu-Ala-Lys-Leu-Gly-Lys-Val-Leu-Ala-His-Val-NH2 ), bombolitin III (Ile-Lys-Ile-Met-Asp-Ile-Leu-Ala-Lys-Leu-Gly-Lys-Val-Leu-Ala-His-Val-NH2 ), bombolitin IV (Ile-Asn-Ile-Lys-Asp-Ile-Leu-Ala-Lys-Leu-Val-Lys-Val-Leu-Gly-His-Val-NH2 ), and bombolitin V (Ile-Asn-Val-Leu-Gly-Ile-Leu-Gly-Leu-Leu-Gly-Lys-Ala-Leu-Ser-His-Leu-NH2 ). Bombolitins are structurally and functionally very similar. They lyse erythrocytes and liposomes, release histamine from rat peritoneal mast cells, and stimulate phospholipase A2 from different sources. The threshold dose is 0.5-2.5 micrograms/ml depending on the peptide and the bioassay. Bombolitin V is as potent as the well-known melittin in lysing guinea pig erythrocytes (ED50 = 0.7 microgram/ml = 4 X 10(-7) M) and is 5 times more potent than mastoparan in causing mast cell degranulation, making it one of the most potent degranulating peptides discovered so far (ED50 = 2 micrograms/ml = 1.2 X 10(-6) M). The bombolitins represent a unique structural class of peptides but they have the same biological properties as melittin (from honeybees), mastoparan (wasps, hornets, and yellow jackets), and crabrolin (European hornets). This unusual circumstance (peptides with different amino acid sequences having the same biological properties) may be a manifestion of their amphiphilic nature, a property these peptides have in common.
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PMID:Bombolitins, a new class of mast cell degranulating peptides from the venom of the bumblebee Megabombus pennsylvanicus. 257 59

Mast cell carboxypeptidase A has been isolated from the secretory granules of mouse peritoneal connective tissue mast cells (CTMC) and from a mouse Kirsten sarcoma virus-immortalized mast cell line (KiSV-MC), and a cDNA that encodes this exopeptidase has been cloned from a KiSV-MC-derived cDNA library. KiSV-MC-derived mast cell carboxypeptidase A was purified with a potato-derived carboxypeptidase-inhibitor affinity column and was found by analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be a Mr 36,000 protein. Secretory granule proteins from KiSV-MC and from mouse peritoneal CTMC were then resolved by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transblotted to polyvinylidine difluoride membranes. Identical aminoterminal amino acid sequences were obtained for the prominent Mr 36,000 protein present in the granules of both cell types. Based on the amino-terminal sequence, an oligonucleotide probe was synthesized and used to isolate a 1,470-base pair cDNA that encodes this mouse exopeptidase. The deduced amino acid sequence revealed that, after cleavage of a 15-amino acid hydrophobic signal peptide and a 94-amino acid activation peptide from a 417-amino acid preproenzyme, the mature mast cell carboxypeptidase A protein core has a predicted Mr of 35,780 and a high positive charge [Lys + Arg) - (Asp + Glu) = 17) at neutral pH. Although critical zinc-binding amino acids (His67, Glu70, His195), substrate-binding amino acids (Arg69, Asn142, Arg143, Tyr197, Asp255, Phe278), and cysteine residues that participate in intrachain disulfide bonds (Cys64-Cys77, Cys136-Cys159) of pancreatic carboxypeptidases were also present in mast cell carboxypeptidase A, the overall amino acid sequence identities for mouse mast cell carboxypeptidase A relative to rat pancreatic carboxypeptidases A1, A2, and B were only 43, 41, and 53%, respectively. RNA and DNA blot analyses revealed that mouse peritoneal CTMC, KiSV-MC, and bone marrow-derived mast cells all express a prominent 1.5-kilobase mast cell carboxypeptidase A mRNA which is transcribed from a single gene. We conclude that mouse mast cell carboxypeptidase A is a prominent secretory granule enzyme of mast cells of the CTMC subclass and represents a novel addition to the carboxypeptidase gene family.
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PMID:Isolation and molecular cloning of mast cell carboxypeptidase A. A novel member of the carboxypeptidase gene family. 258 8


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