UNIPROT:P15088 - FACTA Search

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

The structures of the complexes of carboxypeptidase A (CPA) with two tight-binding phosphonate inhibitors have been determined by X-ray crystallography. The inhibitors, Cbz-Phe-ValP-(O)-Phe[ZFVP(O)F] and Cbz-Ala-GlyP-(O)-Phe[ZAGP(O)F], bind noncovalently to CPA with dissociation constants (Ki's) of 11 fM and 710 pM, respectively. The CPA-ZFVP(O)F complex crystallizes in the space group P2(1)2(1)2(1) with unit cell parameters a = 65.3 A, b = 63.4 A, and c = 76.0 A, and the CPA-ZAGP(O)F complex crystallizes in the space group P2(1)2(1)2(1) with unit cell parameters a = 63.4 A, b = 65.9 A, and c = 74.4 A. Both structures were determined by molecular replacement to a resolution of 2.0 A. The final crystallographic residuals are 0.189 for the CPA-ZFVP(O)F complex and 0.191 for the CPA-ZAGP(O)F complex. The CPA-ZFVP(O)F complex exhibits the lowest Ki yet determined for an enzyme-inhibitor interaction. Comparison of the CPA-ZFVP(O)F structure with that of the CPA-ZAAP(O)F complex [Kim, H., & Lipscomb, W.N. (1990) Biochemistry 29, 5546-5555] indicates the likely important contributions of hydrophobic and weakly polar enzyme-inhibitor interactions to the exceptional stability of the CPA-ZFVP(O)F complex. Among these interactions is a network of four aromatic rings of CPA and ZFVP(O)F in a configuration that allows stabilizing aromatic-aromatic edge-to-face interactions from one ring to the next. A comparison of the structures of the CPA-ZFVP(O)F, CPA-ZAAP(O)F and CPA-ZAGP(O)F complexes shows that all three phosphonates assume a similar binding mode in the active-site binding groove of CPA. For ZAGP(O)F, the glycyl P1 residue does not lead to an anomalous or a partially disordered binding mode as seen in some previous complexes of CPA involving dipeptide analogue inhibitors with glycyl P1 residues. The additional enzyme-inhibitor interactions for these tripeptide phosphonates secure a binding mode in which a Pi portion of the inhibitor is clearly bound by the corresponding Si binding subsite. These three phosphonates have been implicated as transition-state analogues of the CPA-catalyzed reaction. The phosphinyl groups of these phosphonates coordinate to the active-site zinc in a manner that has been proposed as a characteristic feature of the general-base (Zn-hydroxyl or Zn-water) mechanism for the CPA-catalyzed reaction. Further mechanistic proposals are made for Arg-127, whose probable role in binding substrates is apparent in these CPA-phosphonate complexes.
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PMID:Comparison of the structures of three carboxypeptidase A-phosphonate complexes determined by X-ray crystallography. 186 92

The specificity of metal ion inhibition of bovine carboxypeptidase A ([(CPD)Zn]) catalysis is examined under stopped-flow conditions with use of the fluorescent peptide substrate Dns-Gly-Ala-Phe. The enzyme is inhibited competitively by Zn(II), Pb(II), and Cd(II) with apparent KI values of 2.4 x 10(-5), 4.8 x 10(-5), and 1.1 x 10(-2) M in 0.5 M NaCl at pH 7.5 and 25 degrees C. The kcat/Km value, 7.3 x 10(6) M-1 s-1, is affected less than 10% at 1 x 10(-4) M Mn(II) or Cu(II) and at 1 x 10(-2) M Co(II), Ni(II), Hg(II), or Pt(IV). Zn(II) and Pb(II) are mutually exclusive inhibitors. Previous studies of the pH dependence of Zn(II) inhibition [Larsen, K. S., & Auld, D. S. (1989) Biochemistry 28, 9620] indicated that [(CPD)Zn] is selectively inhibited by a zinc monohydroxide complex, ZnOH+, and that ionization of a ligand, LH, in the enzyme's inhibitory site (pKLH 5.8) is obligatory for its binding. The present study allows further definition of this inhibitory zinc site. The ionizable ligand (LH) is assigned to Glu-270, since specific chemical modification of this residue decreases the binding affinity of [(CPD)Zn] for Zn(II) and Pb(II) by more than 60- and 200-fold, respectively. A bridging interaction between the Glu-270-coordinated metal hydroxide and the catalytic metal ion is implicated from the ability of Zn(II) and Pb(II) to induce a perturbation in the electronic absorption spectrum of cobalt carboxypeptidase A ([(CPD)Co]).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of an inhibitory metal binding site in carboxypeptidase A. 200 51

We have investigated the viscosity of carboxypeptidase A catalyzed Bz-Gly-Phe hydrolysis at pH 7.5 (Tris) and 0.5 mol.l-1 NaCl over the range 10-100 mp, varied by addition of glycerol or sucrose. In contrast to previous reports of strong viscosity effects on the corresponding Cbz-Ala-Ala-Ala hydrolysis, both the catalytic constant and the Michaelis constant are virtually independent of viscosity over the 10-fold range investigated. Furthermore, the CD spectra of carboxypeptidase A in the high-viscosity media point to no change in the alpha-helix and beta-sheet structure in these media. The data are compatible either with a compacter, more rigid enzyme-substrate structure or with a more prominent role of intramolecular nuclear reorganization compared to protein reorganization for Bz-Gly-Phe than for Cbz-Ala-Ala-Ala. These views can be given a preciser frame in terms of stochastic chemical rate theory.
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PMID:Substrate specificity of solvent viscosity effects in carboxypeptidase A catalyzed peptide hydrolysis. 200 84

N-(Cyanoacetyl)-L-phenylalanine (compound 1) and N-(3-chloropropionyl)-L-phenylalanine (compound 2) were studied as the first peptidic mechanism-based inactivators (suicide substrates) for the zinc protease carboxypeptidase A (CPA). A crucial deprotonation on the methylene alpha to the amide carbonyl of 1 and 2 has been suggested to lead to the transient formation of a ketenimine and an alpha, beta-unsaturated amide, respectively. Subsequently, it is proposed that these key intermediates trap an active site nucleophile, resulting in covalent modification of the protein. In competition with the inactivation process, the enzyme hydrolyzes the amide bonds in these molecules. Partition ratios of 1180 +/- 40 and 1680 +/- 60 were determined for 1 and 2, respectively. N-Acrolyl-L-phenylalanine (compound 4), the putative intermediate from 2, was independently studied to test the validity of the mechanistic scheme and was observed to be an active site-directed inactivator of CPA. A solvent deuterium isotope effect of 1.39 +/- 0.02 was noted for inactivation by 2 and one of 1.31 +/- 0.01 for its hydrolysis, in keeping with a proposed promoted water hydrolytic pathway for peptide hydrolysis by CPA (Christanson, D. W., and Lipscomb, W. N. (1989) Acc. Chem. Res. 22, 62-69). Details of the kinetic analysis and design concepts are discussed.
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PMID:Peptidic mechanism-based inactivators for carboxypeptidase A. 202 92

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

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

A neuropeptide with adipokinetic activity in Locusta migratoria and the mantid Empusa pennata, and hypertrehalosaemic activity in Periplaneta americana, was isolated by reversed-phase high performance liquid chromatography from corpora cardiaca of the mantids E. pennata and Sphodromantis sp. After brief enzymatic digestion by 5-oxoprolylpeptidase the primary structure of the peptide of each species was determined by pulsed-liquid phase sequencing employing Edman degradation. The C-terminus of both peptides was blocked, as indicated by the lack of digestion with carboxypeptidase A. The peptides of both species were identical: a blocked, uncharged octapeptide with the sequence L-Glu-Val-Asn-Phe-Thr-Pro-Asn-Trp-NH2. The peptide is now called mantid adipokinetic hormone (Emp-AKH). The synthetic peptide was chromatographically indistinguishable from the natural compound and increased blood lipids in locusts and blood carbohydrates in cockroaches when administered in low doses. The structural features clearly define the peptide as a novel member of the large AKH/RPCH-family of peptides. Seven amino-acid residues are at identical positions in Emp-AKH when compared with the adipokinetic hormone of a dragonfly (Lia-AKH) and the hypertrehalosaemic hormone I from the American cockroach (Pea-CAH-I). Evolutionary relationships to other insect orders are discussed.
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PMID:The adipokinetic neuropeptide of Mantodea. Sequence elucidation and evolutionary relationships. 205 98

The role of the C-terminal Phe882-Ala883 residues of bacteriophage T7 RNA polymerase in specific transcription has been investigated by means of site-directed mutagenesis. A mutant enzyme that lacks the C-terminal Phe882-Ala883 residues, denoted the "foot" mutant, has been cloned and overproduced, and the effects of the deletion on promoter recognition, initiation, and elongation have been determined. Gel retardation assays and DNase I footprinting show that the foot mutant specifically recognizes and binds to T7 promoters, although this binding appears to be approximately 30-fold weaker than that of the wild-type enzyme. Transcription assays using oligonucleotide templates that contain the consensus T7 promoter show a dramatic decrease in transcriptional activity for the foot mutant. With templates whose coding region begins CCC..., the mutant synthesizes poly(G) products even in the presence of all four nucleotides. The synthesis of poly(G) products from such templates has previously been observed for the wild-type enzyme when GTP is the sole nucleotide present in the reaction and is thought to occur by a novel mechanism involving slippage of the RNA chain 3' to 5' relative to the template [Martin, C.T., Muller, D.K., & Coleman, J.E. (1988) Biochemistry 27, 3966-3974]. These data suggest that the loss in transcriptional activity by the foot mutant results from a severe decrease in processivity as well as catalytic efficiency of the enzyme. Removal of the C-terminal Phe and Ala residues from the wild-type enzyme with carboxypeptidase A generates the phenotype of the mutant precisely, proving that all of the properties of the foot mutant derive from the loss of the Phe-Ala-COOH moiety.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Processivity of T7 RNA polymerase requires the C-terminal Phe882-Ala883-COO- or "foot". 205 36

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

We have recently found presence of a high concentration of a novel type of kinin, hydroxyprolyl3-bradykinin (Hyp3-BK) in human tumor ascites in addition to conventional bradykinin (BK). Because of their potential physiological activity, it is of interest to know how these bradykinins can be degraded in ascites. Degradation of two synthetic kinins, BK and Hyp3-BK, added to the ascitic fluid from patients with ovarian carcinoma and hepatoma, were analyzed by reversed phase HPLC. Both kinins were degraded into their desArg9-BK or -Hyp3-BK and desPhe8-Arg-9-BK or -Hyp3-BK products following incubation with the ascitic fluid. The rate of the degradation of BK and Hyp3-BK was the same. The formation of desArg9-BK was completely inhibited by kininase I inhibitor, while the formation of desPhe8-Arg9-BK was not completely inhibited by a kininase II inhibitor. The degradation of both kinins was inhibited completely by EDTA. The results indicate the presence of other metalloprotease(s) which cleaves kinins in the ascitic fluid, in addition to kininase I and kininase II. The carboxypeptidase A and carboxypeptidase B inhibitor, benzyl malic acid, failed to block degradation of both kinins. A rapid cleave of Phe-Arg into Phe and Arg was also found in the ascitic fluid. Thus, the major degradation products of kinins in the ascitic fluid were demonstrated to be either desArg9-BK or Hyp3-BK, desPhe8-Arg9-BK or -Hyp3-BK, phenylalanine and arginine. Lysyl-BK and lysylhydroxyprolyl3-BK were rapidly converted into BK and hydroxyprolyl3-BK by the ascitic fluid.
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PMID:Degradation pathway of kinins in tumor ascites and inhibition by kininase inhibitors: analysis by HPLC. 216 Jan 86

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