RESUMO
The class I(A) phosphoinositide 3-kinases (PI3Ks) consist of a 110-kDa catalytic domain and a regulatory subunit encoded by the p85alpha, p85beta, or p55gamma genes. We have determined the effects of disrupting the p85alpha gene on the responses of mast cells stimulated by the cross-linking of Kit and FcepsilonRI, receptors that reflect innate and adaptive responses, respectively. The absence of p85alpha gene products partially inhibited Kit ligand/stem cell factor-induced secretory granule exocytosis, proliferation, and phosphorylation of the serine/threonine kinase Akt. In contrast, p85alpha gene products were not required for FcepsilonRI-initiated exocytosis and phosphorylation of Akt. LY294002, which inhibits all classes of PI3Ks, strongly suppressed Kit- and FcepsilonRI-induced responses in p85alpha -/- mast cells, revealing the contribution of another PI3K family member(s). In contrast to B lymphocytes, mast cell proliferation was not dependent on Bruton's tyrosine kinase, a downstream effector of PI3K, revealing a distinct pathway of PI3K-dependent proliferation in mast cells. Our findings represent the first example of receptor-specific usage of different PI3K family members in a single cell type. In addition, because Kit- but not FcepsilonRI-initiated signaling is associated with mast cell proliferation, the results provide evidence that distinct biologic functions signaled by these two receptors may reflect differential usage of PI3Ks.
Assuntos
Mastócitos/enzimologia , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptores de IgE/metabolismo , Animais , Apoptose , Ciclo Celular , Divisão Celular , Exocitose , Citometria de Fluxo , Interleucina-3/metabolismo , Fígado/embriologia , Fígado/enzimologia , Mastócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Fatores de Tempo , Distribuição TecidualRESUMO
Congenital adrenal hyperplasia (CAH) is a common autosomal recessive disorder mainly caused by defects in the steroid 21-hydroxylase (CYP21) gene. We screened 1,000 healthy people, using a previously developed differential PCR method combined with single-strand conformation polymorphism and amplification-created restriction site methods for the carrier detection of the CYP21 gene deficiency. Our results indicated that the rate of occurrence of the heterozygous CAH carrier was about 12 in 1,000, with a gene frequency of 0.0060 and an incidence frequency of 1 in 28,000 in the Chinese population. In addition, 9 cases of CAH families were performed with prenatal diagnosis. Among them, 3 cases were diagnosed as the severe form, 4 cases carried the heterozygous mutation, and 2 were normal. This is the first report of carrier frequency analysis and prenatal diagnosis of 21-hydroxylase deficiency in Chinese.
Assuntos
Hiperplasia Suprarrenal Congênita , Hiperplasia Suprarrenal Congênita/diagnóstico , Hiperplasia Suprarrenal Congênita/etiologia , Povo Asiático/genética , Triagem de Portadores Genéticos , Diagnóstico Pré-Natal , Hiperplasia Suprarrenal Congênita/etnologia , Hiperplasia Suprarrenal Congênita/genética , China , Feminino , Frequência do Gene , Testes Genéticos/métodos , Humanos , Masculino , Mutação , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Esteroide 21-Hidroxilase/genéticaRESUMO
The objective of the present study is to develop a rapid and convenient method to determine antioxidative activity. It was determined by the inhibition capacity on the hemoglobin-catalyzed peroxidation of linoleic acid. The appropriate conditions for reaction of 4 mM linoleic acid were 0.002% hemoglobin at 37 degrees C for 10 min. Adding methanol to the reaction mixture at <20% showed no significant effect on the peroxidation of linoleic acid. Products formed from hemoglobin-catalyzed peroxidation of linoleic acid were 9- and 13-hydroperoxyoctadecadienoic acid at a ratio of approximately 50:50. Eight synthetic antioxidants were assayed for their antioxidative activity; all of them showed linear response to the logarithm of their concentration. Antioxidative activity from different plant samples was also examined. Tea, ginger, chrysanthemum, and roselle showed higher antioxidative activity. Either hydrophobic or hydrophilic antioxidants were able to be assayed with this method within 15 min.
Assuntos
Antioxidantes/análise , Hemoglobinas/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Plantas Comestíveis/química , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Cromatografia Líquida de Alta Pressão/métodos , Cinética , Ácido Linoleico , Magnoliopsida/química , Extratos Vegetais/química , Espectrofotometria/métodos , Especiarias , Chá/química , Verduras/químicaRESUMO
SLP-76 is an adapter protein expressed in T cells and myeloid cells that is a substrate for ZAP-70 and Syk. SLP-76-deficient mice exhibit a profound block in T-cell development. We found that although SLP-76 is expressed in mouse mast cells, SLP-76(-/-) mice have normal numbers of mast cells in their skin and bronchi. SLP-76(-/-) mice are resistant to IgE-mediated passive anaphylaxis. SLP-76(-/-) mice sensitized with IgE anti-dinitrophenyl (DNP) and then challenged with DNP-HSA developed only mild and transient tachycardia, failed to increase their plasma histamine level, and all survived the antigen challenge. Bone marrow-derived mast cells (BMMCs) from SLP76(-/-) mice failed to release beta-hexosaminidase and to secrete IL-6 after FcepsilonRI cross-linking. Tyrosine phosphorylation of phospholipase C-gamma1 (but not of Syk) and calcium mobilization in response to IgE cross-linking were reduced in SLP-76-deficient BMMCs. These results suggest that SLP-76 plays an important role in FcepsilonRI-mediated signaling in mast cells.
Assuntos
Mastócitos/metabolismo , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Receptores de IgE/fisiologia , Transdução de Sinais/genética , Proteínas Adaptadoras de Transdução de Sinal , Transferência Adotiva , Animais , Sítios de Ligação/genética , Células da Medula Óssea/metabolismo , Células da Medula Óssea/fisiologia , Degranulação Celular/genética , Diferenciação Celular/genética , Células Cultivadas , Citocinas/metabolismo , Imunoglobulina E/administração & dosagem , Imunoglobulina E/fisiologia , Mastócitos/fisiologia , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Anafilaxia Cutânea Passiva , Fosfoproteínas/biossíntese , Receptores de IgE/genética , Receptores de IgE/imunologiaRESUMO
We define by molecular, pharmacologic, and physiologic approaches the proximal mechanism by which the immunoglobulin superfamily member gp49B1 inhibits mast cell activation mediated by the high affinity Fc receptor for IgE (FcepsilonRI). In rat basophilic leukemia-2H3 cells expressing transfected mouse gp49B1, mutation of tyrosine to phenylalanine in either of the two immunoreceptor tyrosine-based inhibitory motifs of the gp49B1 cytoplasmic domain partially suppressed gp49B1-mediated inhibition of exocytosis, whereas mutation of both abolished inhibitory capacity. Sodium pervanadate elicited tyrosine phosphorylation of native gp49B1 and association of the tyrosine phosphatases src homology 2 domain-containing phosphatase-1 (SHP-1) and SHP-2 in mouse bone marrow-derived mast cells (mBMMCs). SHP-1 associated transiently with gp49B1 within 1 min after coligation of gp49B1 with cross-linked FcepsilonRI in mBMMCs. SHP-1-deficient mBMMCs exhibited a partial loss of gp49B1-mediated inhibition of FcepsilonRI-induced exocytosis at concentrations of IgE providing optimal exocytosis, revealing a central, but not exclusive, SHP-1 requirement in the counter-regulatory pathway. Coligation of gp49B1 with cross-linked FcepsilonRI on mBMMCs inhibited early release of calcium from intracellular stores and subsequent influx of extracellular calcium, consistent with SHP-1 participation. Because exocytosis is complete within 2 min in mBMMCs, our studies establish a role for SHP-1 in the initial counter-regulatory cellular responses whereby gp49B1 immunoreceptor tyrosine-based inhibition motifs rapidly transmit inhibition of FcepsilonRI-mediated exocytosis.
Assuntos
Antígenos de Superfície/fisiologia , Cálcio/metabolismo , Imunoglobulina E/fisiologia , Mastócitos/imunologia , Glicoproteínas de Membrana/fisiologia , Proteínas Tirosina Fosfatases/metabolismo , Receptores de IgE/antagonistas & inibidores , Receptores Imunológicos , Tirosina/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Antígenos de Superfície/genética , Sítios de Ligação , Células da Medula Óssea/imunologia , Células Cultivadas , Exocitose , Peptídeos e Proteínas de Sinalização Intracelular , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteína Fosfatase 1 , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/química , Receptores de IgE/química , Proteínas Tirosina Fosfatases Contendo o Domínio SH2 , Homologia de Sequência de Aminoácidos , Domínios de Homologia de srcRESUMO
Congenital adrenal hyperplasia (CAH) is a common autosomal recessive disorder mainly caused by defects in the steroid 21-hydroxylase (CYP21) gene. We have analyzed CYP21 gene sequences in 65 CAH families in Taiwan. All ten exons of the CYP21 gene were analyzed by differential polymerase chain reaction followed by single-strand conformation polymorphism electrophoresis and the amplification-created restriction site method. About 95% (123 chromosomes) contain mutations due to conversion of DNA sequences into its neighboring homologous pseudogene, CYP21P. Four novel mutations representing 5% of the total chromosomes have also been identified. The mutations were confirmed by sequencing an aberrant DNA fragment. These four mutations included a base change of the splicing donor site at intron 2 from GT to AT, a base substitution of C to T at codon 316, deletion of ten bases (TCCAGCTCCC) at codons 330-333 of exon 8, and duplication of 16 bases (CCTGGATGACACGGTC) at codons 393-397 of exon 9. The loss of the splicing donor site at intron 2 and the premature stop at codon 316 may result in aberrant splicing to reduce enzyme activity and a truncated protein with no enzyme activity, respectively. Likewise, both the duplication and the deletion forms create a frameshift and premature stop during translation. The resulting proteins lack the heme-binding domain and hence are expected to lose enzymatic activity. Since these mutations are not found in the neighboring CYP21P pseudogene, gene conversion should not be the cause of these novel mutations.
Assuntos
Hiperplasia Suprarrenal Congênita/genética , Esteroide 21-Hidroxilase/genética , Alelos , Pré-Escolar , Análise Mutacional de DNA , Feminino , Humanos , Recém-Nascido , Masculino , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Splicing de RNA/genética , Análise de Sequência de DNA , TaiwanRESUMO
To examine the role of Arg38 in the peroxidative and peroxygenative activity of horseradish peroxidase (HRP), we have expressed the R38A, R38H, and R38H/H42V mutants. The R38A HRP mutant gives a normal compound I species with H2O2 that is reduced by ferrocyanide to the ferric state without the detectable formation of a compound II species. In the case of the R38H and R38H/H42V mutants, compound I itself is only detected by stopped flow methods. The rates of compound I formation at 4 degrees C are 8.0 x 10(4), 1.3 x 10(6), and 1.6 x 10(3) M-1 s-1 for the R38A, R38H, and R38H/H42V mutants, respectively. The R38A, R38H, and R38H/H42V mutants oxidize guaiacol 10-, 2-, and 55-fold, respectively, more slowly than the wild-type enzyme and oxidize ABTS 6-, 3-, and 32-fold more slowly than the wild-type enzyme. The apparent kcat/K(m) values for thioanisole sulfoxidation and styrene epoxidation indicate that the reaction efficiencies of the R38H and wild-type enzymes are comparable. However, the R38A and R38H/H42V mutants are 190- and 1400-fold more efficient as sulfoxidation catalysts, and 25- and 26-fold more efficient as styrene epoxidation catalysts, respectively, than the wild-type enzyme. Thus, even though Arg38 plays a role in the formation and stabilization of compounds I and II, its replacement by other residues can be used to improve peroxygenative catalysis.
Assuntos
Histidina/genética , Histidina/metabolismo , Peroxidase do Rábano Silvestre/genética , Peroxidase do Rábano Silvestre/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Alanina/genética , Animais , Arginina/genética , Benzotiazóis , Sítios de Ligação/genética , Catálise , Células Cultivadas , Ativação Enzimática/genética , Guaiacol/metabolismo , Peroxidase do Rábano Silvestre/química , Peróxido de Hidrogênio/metabolismo , Cinética , Oxigenases de Função Mista/química , Mutagênese Insercional , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/síntese química , Proteínas Recombinantes/isolamento & purificação , Spodoptera/enzimologia , Spodoptera/genética , Ácidos Sulfônicos/metabolismo , Valina/genéticaRESUMO
Several plano-convex aluminum thin films, ~30 nm thick in the center and ~2 mm in diameter, were deposited on microscope cover slides to function as inverse-Gaussian transmittive filters. By placing one of these filters in front of the Gaussian He-Ne laser, we can modify the beam intensity profile in the downstream direction. To yield a uniform beam, the position of the filter must be aligned in the transverse plane for maximum intensity at the output of the filter. These filters are easy to fabricate and are inexpensive. Most important, they can help produce collimated phase-coherent uniform beams, which are useful in high-precision fringe-analysis techniques.
RESUMO
Phosphotriesterase (PTE) is a zinc metalloenzyme that catalyzes the hydrolysis of an extensive array of organophosphate pesticides and mammalian acetylcholinesterase nerve agents. Although the three-dimensional crystal structure of PTE has been solved (M. M. Benning et al., Biochemistry 34:7973-7978, 1995), the precise functions of the individual amino acid residues that interact directly with the substrate at the active site are largely unknown. To construct mutants of PTE with altered specificities for particular target substrates, a simple methodology for generating a library of mutants at specific sites was developed. In this investigation, four of the six protein ligands to the binuclear metal site (His-55, His-57, His-201, and His-230) were targeted for further characterization and investigation. Using the polymerase chain reaction (PCR) protocols, a library of modified PTE genes was generated by simultaneously creating random combinations of histidine and cysteine codons at these four positions. The 16 possible DNA sequences were isolated and confirmed by dideoxy-DNA sequencing. The 16 mutant proteins were expressed in Escherichia coli and grown with the presence or absence of 1 mM CoCl2, ZnSO4, or CdSO4 in the growth medium. When grown in the presence of CoCl2, the H57C protein cell lysate showed greater activity for the hydrolysis of paraoxon than the wild type PTE cell lysate. H201C and H230C exhibited up to 15% of the wild-type activity, while H55C, a green protein, was inactive under all assay conditions. All other mutants had < 10(-5) of wild-type activity. None of the purified mutants that exhibited catalytic activity had a significantly altered Km for paraoxon.
Assuntos
Proteínas de Bactérias/química , Esterases/química , Biblioteca Gênica , Metais/metabolismo , Arildialquilfosfatase , Proteínas de Bactérias/genética , Sítios de Ligação , Catálise , Ativação Enzimática , Esterases/genética , Cinética , Metais/química , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase , PseudomonasRESUMO
The covalently bound prosthetic group of lactoperoxidase (LPO) has been obtained by hydrolysis of the protein and identified as a dihydroxylated heme. A baculovirus expression system has been developed for LPO and used to obtain protein in which the heme is only partially covalently bound. Reaction of the purified heme. apoLPO complex with H2O2 results in both autocatalytic modification of the heme and covalent attachment to the protein. Hydrolytic experiments establish that the autocatalytically incorporated heme is bound normally. Two monohydroxylated heme intermediates have been detected. The peroxidative activity of LPO increases in proportion to the extent of covalently bound heme. The LPO results provide a paradigm for autocatalytic incorporation of heme groups into the mammalian peroxidases, including myeloperoxidase and eosinophil peroxidase, all of which exhibit strong sequence similarity with LPO and have covalently-bound heme groups.
Assuntos
Heme/metabolismo , Lactoperoxidase/metabolismo , Animais , Catálise , Bovinos , Cromatografia Líquida de Alta Pressão , DNA Complementar/química , Peróxido de Hidrogênio/metabolismo , Hidrólise , Modelos Químicos , Peroxidase/metabolismo , Pronase/metabolismo , Proteínas Recombinantes/análiseRESUMO
Phosphotriesterase catalyzes the hydrolysis of organophosphate nerve agents. Four amino acid residues, located within the active site pocket, were mutated in an effort to ascertain the roles that these groups play in the structure and function of this enzyme. Tryptophan-131 is located at the entrance to the binuclear metal center, and the indole ring is positioned to suggest that it could provide a hydrophobic site for interaction of the aromatic leaving group with optimized substrates. The W131F mutant displays catalytic constants for the hydrolysis of paraoxon that are essentially the same as those of the wild type enzyme. However, the Km value for the W131A mutant is elevated by a factor of 6, consistent with a role for this residue in substrate binding. Aspartate-253 is hydrogen bonded to His-230 which, in turn, is directly ligated to the more solvent-exposed metal ion. The D253N mutant possesses catalytic constants that are virtually the same as those of the wild type enzyme, while the D253A mutant is reduced in activity by 500-fold. These results are consistent with a model where this residue is required to orientate the imidazole side chain of His-230 for proper interaction with the binuclear metal center. Aspartate-301 is a primary ligand to the more buried metal ion. Mutation of this residue to histidine, asparagine, alanine, and cysteine reduces the catalytic activity by factors of 2.6 x 10(4), 2.7 x 10(3), 5.6 x 10(2), and 1.5 x 10(2), respectively. These results indicate that alterations to the direct metal ligands, even with residues that can strongly coordinate divalent cations, cause a severe disruption to the proper functioning of the active site. In the wild type enzyme, the side chain of Lys-169 is carbamylated and also acts as a bridge between the two divalent cations. Significant losses in catalytic activity are obtained upon mutation of this residue to either alanine, glutamate, arginine, or methionine. The loss in activity can partially be restored upon inclusion in the assay mixture of short-chain carboxylic acids. A 25-fold enhancement in k(cat) is observed for the K169A mutant in the presence of 100 mM propionic acid.
Assuntos
Esterases/química , Aminoácidos/química , Aminoácidos/genética , Aminoácidos/metabolismo , Arildialquilfosfatase , Sítios de Ligação/genética , Esterases/genética , Esterases/metabolismo , Mutação , Relação Estrutura-AtividadeRESUMO
We demostrate that a specific combination of cytokines elicits high levels of interleukin (IL)-6 gene expression in mast cells and define the cellular mechanisms of the exogenous cytokine action. The addition of c-kit ligand (KL) and IL-10 to IL-3-derived mouse bone marrow mast cells (BMMC) elicited an approximately 2-fold increase in steady-state IL-6 mRNA levels that peaked after 0.5 h and was followed by the release of approximately 0.2 ng of IL-6/10(6) cells by 5-7 h. The addition of IL-1beta to KL + IL-10 elicited a prolonged approximately 12-fold increase in the level of IL-6 mRNA by 3-5 h and an approximately 50-fold increase in the level of IL-6 protein released by 7 h. As determined by nuclear run-on analysis, KL + IL-10 stimulated IL-6 gene transcription within 0.5 h, and the addition of IL-1beta did not increase transcription. Instead, IL-1beta slowed by approximately 8-fold the decay of IL-6 mRNA as compared to its decay in BMMC stimulated with KL + IL-10 alone. The exposure of BMMC to cycloheximide 0.5 h before the addition of the three exogenous cytokines inhibited by approximately 50% the level of IL-6 mRNA generated but did not inhibit the effects of KL + IL-10, indicating that IL-1beta induces the synthesis of a protein that stabilizes IL-6 mRNA. The stabilization of IL-6 mRNA was inhibited by the addition of actinomycin D at 0.5 but not 3 h after BMMC were stimulated with IL-1beta in combination with KL + IL-10, suggesting that once transcribed, the stabilizing protein is long-lived. The addition of cycloheximide to BMMC after stimulation with KL + IL-10 with or without IL-1beta increased the levels of steady-state IL-6 mRNA compared to levels in cells without drug, indicating that in addition to stimulating IL-6 transcription, KL + IL-10 induces a protein factor that destabilizes IL-6 mRNA. Thus, there exists a novel Fcepsilon receptor type I-independent mechanism by which a mast cell can provide substantial amounts of IL-6 protein in response to the synergistic action of KL and IL-10 to induce IL-6 gene transcription, and IL-1beta to stabilize otherwise short-lived IL-6 transcripts.
Assuntos
Interleucina-10/metabolismo , Interleucina-1/metabolismo , Interleucina-6/genética , Mastócitos/metabolismo , Processamento de Proteína Pós-Traducional , RNA Mensageiro/metabolismo , Fator de Células-Tronco/metabolismo , Animais , Células da Medula Óssea , Células Cultivadas , Cicloeximida/farmacologia , Dactinomicina/farmacologia , CamundongosRESUMO
Phosphotriesterase, as isolated from Pseudomonas diminuta, is capable of detoxifying widely used pesticides such as paraoxon and parathion and various mammalian acetylcholinesterase inhibitors. The enzyme requires a binuclear metal center for activity. Recently, the three-dimensional structure of the apoenzyme was solved (Benning et al., 1994) and shown to consist of an alpha/beta-barrel. Here we describe the three-dimensional structure of the holoenzyme, reconstituted with cadmium, as determined by X-ray crystallographic analysis to 2.0-A resolution. Crystals employed in the investigation belonged to the space group C2 with unit cell dimensions of a = 129.5 A, b = 91.4 A, c = 69.4 A, beta = 91.9 degrees, and two subunits in the asymmetric unit. There are significant differences in the three-dimensional architecture of the apo and holo forms of the enzyme such that their alpha-carbon positions superimpose with a root-mean-square deviation of 3.4 A. The binuclear metal center is located at the C-terminus of the beta-barrel with the cadmiums separated by 3.8 A. There are two bridging ligands to the metals: a water molecule (or possibly a hydroxide ion) and a carbamylated lysine residue (Lys 169). The more buried cadmium is surrounded by His 55, His 57, Lys 169, Asp 301, and the bridging water in a trigonal bipyramidal arrangement. The second metal is coordinated in a distorted octahedral geometry by His 201, His 230, Lys 169, the bridging water molecule, and two additional solvents.
Assuntos
Cádmio/química , Esterases/química , Flavobacterium/enzimologia , Metais/química , Pseudomonas/enzimologia , Apoenzimas/química , Arildialquilfosfatase , Sítios de Ligação , Cádmio/metabolismo , Simulação por Computador , Cristalização , Cristalografia por Raios X , Esterases/metabolismo , Concentração de Íons de Hidrogênio , Modelos Moleculares , Estrutura Molecular , Estrutura Secundária de ProteínaRESUMO
Five alkynyl phosphate esters have been synthesized as probes of the active site structure of phosphotriesterase. These compounds have the potential to be converted by the enzyme to a highly reactive ketene intermediate which can then react with an active site nucleophile causing irreversible inhibition of the enzyme by formation of an inactive covalent adduct. All five compounds completely inactivate enzyme function in less than 15 s at pH 7.0. The partition rations of 1-hexynyl diethyl phosphate (I), 1-propynyl diethyl phosphate (II), 1-hexynyl diphenyl phosphate (III), 1-hexynyl dimethyl phosphate (IV), and ethynyl diethyl phosphate (V) fall in the range between 480 and 1700; thus, all five alkynyl phosphate esters work equally well as inactivators despite the differences in their structures. The rate constants for enzyme inactivation, kinact, are 1.7 s-1 with I, 1.3 s-1 with II, and 0.12 s-1 with IV. They compare well with the kcat for the Co-substituted phosphotriesterase; hence these compounds are good substrates. The stoichiometry of inhibitor bound to protein is 1:1, as determined by inactivation of the enzyme using the radiolabeled compound [3-14C]-1-propynyl diethyl phosphate. Addition of an exogenous nucleophile, azide, did not protect phosphotriesterase from being inactivated by the alkynyl phosphate esters, suggesting that the reactive intermediate produced from the inhibitor is not released from the enzyme surface prior to covalent labeling of the protein. Chemical and spectroscopic evidence suggests that a histidine residue is modified in the inactivation reaction. The inactivated phosphotriesterase can be reactivated by increasing the pH of the protein solution. N-Acylimidazoles are known to be easily hydrolyzed at alkaline pH values.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Esterases/antagonistas & inibidores , Etilenos/química , Histidina/química , Cetonas/química , Alcinos/química , Arildialquilfosfatase , Concentração de Íons de Hidrogênio , Cinética , Fosfatos/química , Pseudomonas/enzimologia , Espectrofotometria UltravioletaRESUMO
The alkynyl phosphate ester, 1-hexynyl diethyl phosphate (I), is a mechanism-based inhibitor of phosphotriesterase. It has been previously determined that a histidine residue in the wild-type phosphotriesterase is covalently modified by this compound. In order to identify which of the seven histidine residues in the native enzyme are required for inactivation, the kinetic properties of phosphotriesterase mutants with this suicide substrate were examined in detail. Six of the seven mutants (histidine to asparagine) were rapidly inactivated by I. The mutants H55N, H57N, and H230N also showed partition ratios that were lower than for the wild-type enzyme. The rate of inactivation of H201N was significantly slower than that of wild-type phosphotriesterase. The H254N mutant could not be inactivated; no more than 60% of the initial activity was lost, even at I/E0 ratios of 4000:1. These results suggest that His-254 is essential for the inactivation of phosphotriesterase and is likely to be the primary target in the wild-type enzyme for modification by I. The inactivation of wild-type phosphotriesterase and the seven mutants was also studied using diethyl pyrocarbonate, a histidine selective reagent. The second-order rate constant for the inactivation of wild-type phosphotriesterase was determined to be 1.3 M-1 min-1. The rate constants for the inactivation of the H55N, H57N, H201N, and H230N mutants were larger than for the wild-type enzyme. Thus, it appears that when these histidine residues are replaced by asparagine, other histidine residues in the active site become more susceptible to modification, resulting in a faster rate of inactivation. The mutant H254N was not inactivated in the presence of DEPC.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Alcinos/química , Dietil Pirocarbonato/química , Esterases/antagonistas & inibidores , Histidina/química , Fosfatos/química , Pseudomonas/enzimologia , Arildialquilfosfatase , Sítios de Ligação , Esterases/química , Cinética , Mutagênese Sítio-Dirigida , Espectrofotometria Ultravioleta , Relação Estrutura-AtividadeRESUMO
Organophosphates, such as parathion and paraoxon, constitute the largest class of insecticides currently used in industrialized nations. In addition, many of these compounds are known to inhibit mammalian acetylcholinesterases thereby acting as nerve agents. Consequently, organophosphate-degrading enzymes are of considerable interest in light of their ability to detoxify such compounds. Here we report the three-dimensional structure of such an enzyme, namely, phosphotriesterase, as determined by single crystal X-ray diffraction analysis to 2.1-A resolution. Crystals employed in this investigation belonged to the space group P2(1)2(1)2 with unit cell dimensions of a = 80.3 A, b = 93.4 A, and c = 44.8 A and one molecule per asymmetric unit. The structure was solved by multiple isomorphous replacement with two heavy-atom derivatives and refined to a crystallographic R factor of 18.0%. As observed in various other enzymes, the overall fold of the molecule consists of an alpha/beta barrel with eight strands of parallel beta-pleated sheet. In addition, there are two antiparallel beta-strands at the N-terminus. The molecular model of phosphotriesterase presented here provides the initial structural framework necessary toward understanding the enzyme's broad substrate specificities and its catalytic mechanism.
Assuntos
Esterases/química , Inseticidas/metabolismo , Compostos Organofosforados , Arildialquilfosfatase , Biotransformação , Cátions Bivalentes , Cristalização , Cristalografia por Raios X , Esterases/metabolismo , Substâncias Macromoleculares , Modelos Moleculares , Estrutura Secundária de Proteína , Pseudomonas/enzimologiaRESUMO
We have mapped five genes encoding protein tyrosine kinases (PTKs) to the pericentromeric region of human chromosome 4. PTK4 and TYRO4, which encode nonreceptor intracellular PTKs, are located at 4p12 and 4q13, respectively. The other three genes, PDGFRA, KIT, and KDR, encode type III transmembrane receptor PTKs for known ligands. We have developed a contig of 29 yeast artificial chromosomes (YACs) spanning approximately 2 Mb of DNA at 4q12 that includes PDGFRA, KIT, and KDR, and we have used this YAC contig to map 12 different sequence-tagged sites in this region. PDGFRA, KIT, and KDR thus constitute a cluster of genes at 4q12 encoding closely related type III receptor PTKs. Mutations of the human KIT gene result in piebaldism, an autosomal dominant disorder of melanocyte development.
Assuntos
Cromossomos Artificiais de Levedura , Cromossomos Humanos Par 4 , Genes , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Receptores de Fator Estimulador de Colônias/genética , Receptores do Fator de Crescimento Derivado de Plaquetas/genética , Sequência de Bases , Passeio de Cromossomo , Humanos , Dados de Sequência Molecular , Família Multigênica , Reação em Cadeia da Polimerase , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-kitRESUMO
In order to identify which of the seven histidines in phosphotriesterase participate at the active site/binuclear metal center of the enzyme, site-directed mutagenesis has been employed to change, individually, each of the seven histidine residues to asparagine. In addition, the gene for the wild-type enzyme has been subcloned without its leader sequence behind a modified ribosomal binding site, leading to a 5-fold increase in protein expression. The seven mutants, H55N, H57N, H123N, H201N, H230N, H254N, and H257N, exhibit varying degrees of activity compared to the wild-type enzyme. The H123N and H257N mutants are as active as the wild-type enzyme, but all of the other mutant enzymes have 10% or less activity. The metal content of the cobalt-purified mutant enzymes has been determined to be less than that of the wild-type enzyme in all cases. Each of the mutant enzymes has been converted to apoenzyme and reconstituted with 2 equiv of zinc(II), cadmium(II), or cobalt(II). The kinetic parameters, Vmax and V/Km, and apparent pKa's have been determined for each of the reconstituted enzyme derivatives. In almost all cases, the apparent pKa's have shifted toward higher values. The pH-rate profiles for some of the reconstituted mutant enzymes are significantly different from those for the wild-type enzyme, indicating that other groups may become involved in the reaction mechanism upon mutation of the histidine residue to asparagine. His-123 is the only histidine residue that appears to have no involvement in the catalytic activity of phosphotriesterase.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Esterases/metabolismo , Histidina/metabolismo , Metais/metabolismo , Arildialquilfosfatase , Cádmio/metabolismo , Dicroísmo Circular , Cobalto/metabolismo , Ativação Enzimática , Esterases/química , Esterases/genética , Concentração de Íons de Hidrogênio , Cinética , Ligantes , Mutagênese Sítio-Dirigida , Pseudomonas/enzimologia , Pseudomonas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Zinco/metabolismoRESUMO
Presents a vector space framework to study short-term memory filters in dynamic neural networks. The authors define parameters to quantify the function of feedforward and recursive linear memory filters. They show, using vector spaces, what is the optimization problem solved by the PEs of the first hidden layer of the single input focused network architecture. Due to the special properties of the gamma bases, recursion brings an extra parameter lambda (the time constant of the leaky integrator) that displaces the memory manifold towards the desired signal when the mean square error is minimized. In contrast, for the feedforward memory filter the angle between the desired signal and the memory manifold is fixed for a given memory order. The adaptation of the feedback parameter can be done using gradient descent, but the optimization is nonconvex.
RESUMO
We have constructed a YAC contig containing 54 clones and a minimum of 7 Mbp of human DNA, that maps to bands q34-35 on chromosome 5. The contig was nucleated using FISH mapped cosmid clones shown to flank the t(2;5)(p23;q35) translocation breakpoint in a CD30-positive large cell lymphoma cell line. Thirty of the 54 YAC clones are non-chimeric and six span the translocation breakpoint, as determined by FISH analysis. A total of 28 YAC clone end fragments, 14 non-polymorphic YAC end STS probes and 13 polymorphic microsatellite STS markers have been used to order clones within the contig. The most distal genetic markers (D5S498 and D5S619) are separated by 15 cM based on multipoint linkage analysis. This map of overlapping clones and the set of densely spaced physical markers will promote our understanding of the 5q34-35 region and its associated genes.