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[PMID]: 25028518 [Au] Autor: Favrot L; Lajiness DH; Ronning DR [Ad] Endereço: From the Department of Chemistry and Biochemistry, University of Toledo, Toledo, Ohio 43606-3390. [Ti] Título: Inactivation of the Mycobacterium tuberculosis antigen 85 complex by covalent, allosteric inhibitors. [So] Source: J Biol Chem;289(36):25031-40, 2014 Sep 05. [Is] ISSN: 1083-351X [Cp] País de publicação: United States [La] Idioma: eng [Ab] Resumo: The rise of multidrug-resistant and totally drug-resistant tuberculosis and the association with an increasing number of HIV-positive patients developing tuberculosis emphasize the necessity to find new antitubercular targets and drugs. The antigen 85 (Ag85) complex from Mycobacterium tuberculosis plays important roles in the biosynthesis of major components of the mycobacterial cell envelope. For this reason, Ag85 has emerged as an attractive drug target. Recently, ebselen was identified as an effective inhibitor of the Ag85 complex through covalent modification of a cysteine residue proximal to the Ag85 active site and is therefore a covalent, allosteric inhibitor. To expand the understanding of this process, we have solved the x-ray crystal structures of Ag85C covalently modified with ebselen and other thiol-reactive compounds, p-chloromercuribenzoic acid and iodoacetamide, as well as the structure of a cysteine to glycine mutant. All four structures confirm that chemical modification or mutation at this particular cysteine residue leads to the disruption of the active site hydrogen-bonded network essential for Ag85 catalysis. We also describe x-ray crystal structures of Ag85C single mutants within the catalytic triad and show that a mutation of any one of these three residues promotes the same conformational change observed in the cysteine-modified forms. These results provide evidence for active site dynamics that may afford new strategies for the development of selective and potent Ag85 inhibitors. [Mh] Termos MeSH primário: Aciltransferases/química Antígenos de Bactérias/química Cisteína/química Inibidores Enzimáticos/química [Mh] Termos MeSH secundário: Aciltransferases/genética Aciltransferases/metabolismo Regulação Alostérica Anti-Inflamatórios não Esteroides/química Anti-Inflamatórios não Esteroides/farmacologia Antígenos de Bactérias/genética Antígenos de Bactérias/metabolismo Azóis/química Azóis/farmacologia Biocatálise/efeitos dos fármacos Domínio Catalítico Cloromercurobenzoatos/química Cloromercurobenzoatos/farmacologia Cristalografia por Raios X Cisteína/genética Cisteína/metabolismo Ativação Enzimática/efeitos dos fármacos Inibidores Enzimáticos/farmacologia Ligações de Hidrogênio/efeitos dos fármacos Iodoacetamida/química Iodoacetamida/farmacologia Modelos Moleculares Estrutura Molecular Mutação Compostos Organosselênicos/química Compostos Organosselênicos/farmacologia Conformação Proteica Estrutura Secundária de Proteína [Pt] Tipo de publicação: JOURNAL ARTICLE; RESEARCH SUPPORT, N.I.H., EXTRAMURAL; RESEARCH SUPPORT, NON-U.S. GOV'T; RESEARCH SUPPORT, U.S. GOV'T, NON-P.H.S. [Nm] Nome de substância: 0 (Anti-Inflammatory Agents, Non-Steroidal); 0 (Antigens, Bacterial); 0 (Azoles); 0 (Chloromercuribenzoates); 0 (Enzyme Inhibitors); 0 (Organoselenium Compounds); 40X2P7DPGH (ebselen); EC 2.3.- (Acyltransferases); EC 2.3.1.- (antigen 85C, Mycobacterium tuberculosis); K848JZ4886 (Cysteine); ZRH8M27S79 (Iodoacetamide) [Em] Mês de entrada: 1501 [Cu] Atualização por classe: 161202 [Lr] Data última revisão: 161202 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 140717 [St] Status: MEDLINE [do] DOI: 10.1074/jbc.M114.582445

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[PMID]: 15096051 [Au] Autor: Qiu W; Kohen-Avramoglu R; Rashid-Kolvear F; Au CS; Chong TM; Lewis GF; Trinh DK; Austin RC; Urade R; Adeli K [Ad] Endereço: Division of Clinical Biochemistry, Department of Laboratory Medicine and Pathobiology, Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada M5G 1X8. [Ti] Título: Overexpression of the endoplasmic reticulum 60 protein ER-60 downregulates apoB100 secretion by inducing its intracellular degradation via a nonproteasomal pathway: evidence for an ER-60-mediated and pCMB-sensitive intracellular degradative pathway. [So] Source: Biochemistry;43(16):4819-31, 2004 Apr 27. [Is] ISSN: 0006-2960 [Cp] País de publicação: United States [La] Idioma: eng [Ab] Resumo: Co- and posttranslational regulation of apolipoprotein B (apoB) has been postulated to involve degradation by both proteasomal and nonproteasomal pathways; however, nonproteasomal mechanisms of apoB degradation are currently unknown. We have previously demonstrated an intracellular association of newly synthesized apoB with endoplasmic reticulum (ER)-60, an ER-localized protein, possessing both proteolytic and chaperone activities. In the present paper, adenoviral expression vectors containing rat ER-60 cDNA were used to achieve dose- and time-dependent overexpression of ER-60 to investigate its role in apoB100 turnover. Overexpressed ER-60 accumulated in the microsomal lumen of HepG2 cells and was associated with apoB100 in dense lipoprotein particles. Overexpression of ER-60 in HepG2 cells significantly reduced both intracellular and secreted apoB100, with no effect on the secretion of a control protein, albumin. Similar results were obtained in McA-RH7777 rat hepatoma cells. ER-60-stimulated apoB100 degradation and inhibition of apoB100 secretion were sensitive to the protease inhibitor, p-chloromercuribenzoate (pCMB), in a dose-dependent manner but were unaffected by the proteasomal or lysosomal protease inhibitors, N-acetyl-leucinyl-leucinyl-nor-leucinal, E64, and leupeptin. Interestingly, enhanced expression of ER-60 induced apoB100 fragmentation in permeabilized HepG2 cells and resulted in detection of a unique 50 kDa degradation intermediate, a process that could be inhibited by pCMB. Intracellular stability and secretion of apoB100 in primary hamster hepatocytes were also found to be sensitive to pCMB. When taken together, the data suggest an important role for ER-60 in promoting apoB100 degradation via a pCMB-sensitive process in the ER. ER-60 may act directly as a protease or may be involved indirectly as a chaperone/protein factor targeting apoB100 to this nonproteasomal and pCMB-sensitive degradative pathway. [Mh] Termos MeSH primário: Apolipoproteínas B/antagonistas & inibidores Apolipoproteínas B/secreção Cloromercurobenzoatos/farmacologia Cisteína Endopeptidases/fisiologia Regulação para Baixo Retículo Endoplasmático/enzimologia Líquido Intracelular/metabolismo Transdução de Sinais/fisiologia [Mh] Termos MeSH secundário: Adenoviridae/genética Animais Apolipoproteína B-100 Apolipoproteínas B/metabolismo Linhagem Celular Linhagem Celular Tumoral Permeabilidade da Membrana Celular/efeitos dos fármacos Cricetinae Cisteína Endopeptidases/biossíntese Cisteína Endopeptidases/genética Cisteína Endopeptidases/metabolismo Inibidores de Cisteína Proteinase/farmacologia Regulação para Baixo/efeitos dos fármacos Regulação para Baixo/genética Retículo Endoplasmático/genética Vetores Genéticos Hepatócitos/efeitos dos fármacos Hepatócitos/enzimologia Hepatócitos/secreção Seres Humanos Líquido Intracelular/enzimologia Líquido Intracelular/secreção Microssomos/enzimologia Ratos Transdução de Sinais/efeitos dos fármacos Transdução Genética [Pt] Tipo de publicação: JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T [Nm] Nome de substância: 0 (Apolipoprotein B-100); 0 (Apolipoproteins B); 0 (Chloromercuribenzoates); 0 (Cysteine Proteinase Inhibitors); EC 3.4.22.- (Cysteine Endopeptidases); EC 3.4.22.- (ER-60 protease) [Em] Mês de entrada: 0408 [Cu] Atualização por classe: 061115 [Lr] Data última revisão: 061115 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 040421 [St] Status: MEDLINE

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[PMID]: 14714473 [Au] Autor: Zherebtsov NA; Abramova IN; Shelamova SA; Popova TN [Ad] Endereço: Voronezh State Technological Academy, Voronezh, 394017 Russia. [Ti] Título: [Identification of catalytically active groups in inulinase from Bacillus polymyxa 722]. [Ti] Título: Identifikatsiia kataliticheski aktivnykh grupp inulinazy Bacillus polymyxa 772.. [So] Source: Prikl Biokhim Mikrobiol;39(6):619-24, 2003 Nov-Dec. [Is] ISSN: 0555-1099 [Cp] País de publicação: Russia (Federation) [La] Idioma: rus [Ab] Resumo: Inulinase from Bacillus polymyxa 722 hydrolyzing a polyfructosan inulin was studied. The dependence of inulinase activity on pH, measurements of pK value, calculation of ionization heat, photoinactivation with methylene blue, and inhibition with p-chloromercuribenzoate suggest that the active center of this enzyme contains imidazole and sulfhydryl groups. A possible mechanism underlying cleavage of beta-2,1-fructoside bonds in the inulin molecule with inulinase is considered. [Mh] Termos MeSH primário: Bacillus/enzimologia Glicosídeo Hidrolases/metabolismo [Mh] Termos MeSH secundário: Sítios de Ligação Cloromercurobenzoatos/farmacologia Inibidores Enzimáticos/farmacologia Glicosídeo Hidrolases/química Concentração de Íons de Hidrogênio Imidazóis/química Inulina/metabolismo Cinética Azul de Metileno/farmacologia [Pt] Tipo de publicação: ENGLISH ABSTRACT; JOURNAL ARTICLE [Nm] Nome de substância: 0 (Chloromercuribenzoates); 0 (Enzyme Inhibitors); 0 (Imidazoles); 7GBN705NH1 (imidazole); 9005-80-5 (Inulin); EC 3.2.1.- (Glycoside Hydrolases); EC 3.2.1.7 (inulinase); T42P99266K (Methylene Blue) [Em] Mês de entrada: 0406 [Cu] Atualização por classe: 131121 [Lr] Data última revisão: 131121 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 040113 [St] Status: MEDLINE

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[PMID]: 11180979 [Au] Autor: Senthilkumar R; Reddy PN; Sharma KK [Ad] Endereço: Mason Eye Institute, University of Missouri, One Hospital Drive, Columbia, MO 65212, USA. [Ti] Título: Studies on trypsin-modified bovine and human lens acylpeptide hydrolase. [So] Source: Exp Eye Res;72(3):301-10, 2001 Mar. [Is] ISSN: 0014-4835 [Cp] País de publicação: England [La] Idioma: eng [Ab] Resumo: Acylpeptide hydrolase removes the N -acetylated amino acids from the peptide substrates but not from intact proteins. Cleavage between amino acid residues 203--204 of the native acylpeptide hydrolase results in the formation of a 55 kDa truncated active enzyme in the bovine lens, in vivo. In this study we explored the hydrolytic properties of the truncated enzyme using lens beta- and gamma-crystallins as substrates. SDS--PAGE analysis indicated that the beta B2-crystallin was cleaved by truncated acylpeptide hydrolase into several protein fragments (10--26 kDa). No cleavage of the gamma-crystallins was observed under similar conditions. Both the acylpeptide hydrolase activity and the protease activity of the 55 kDa enzyme were completely inhibited by diisopropylfluorophosphate, p -chloromercuribenzoate and ebelactone, and moderately inhibited by N -tosyl phenylalanine chloromethyl ketone. SDS--PAGE analysis followed by fluorography of ((3)H) diisopropylfluorophosphate labeled human lens acylpeptide hydrolase preparation showed the presence of the 55 kDa truncated form of the enzyme, as observed in the bovine lens. The peptide (d)-AIKGDQFL-NH(2)--the amino acid sequence 200--207 of the native bovine acylpeptide hydrolase with an in vivo cleavage site of native protein--was hydrolysed by the lens protease(s) suggesting that the in vivo generation of the 55 kDa acylpeptide hydrolase may be mediated through a proteolytic processing. The protease(s) responsible for the cleavage of this peptide was inhibited by diisopropylfluorophosphate and p -chloromercuribenzoate. [Mh] Termos MeSH primário: Cristalino/enzimologia Peptídeo Hidrolases/química Tripsina/química [Mh] Termos MeSH secundário: Adolescente Adulto Idoso Idoso de 80 Anos ou mais Animais Bovinos Cloromercurobenzoatos/farmacologia Cristalinas/metabolismo Eletroforese em Gel de Poliacrilamida Inibidores Enzimáticos/farmacologia Seres Humanos Hidrólise Isoflurofato/farmacologia Lactonas/farmacologia Meia-Idade Fragmentos de Peptídeos/química [Pt] Tipo de publicação: JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T; RESEARCH SUPPORT, U.S. GOV'T, P.H.S. [Nm] Nome de substância: 0 (Chloromercuribenzoates); 0 (Crystallins); 0 (Enzyme Inhibitors); 0 (Lactones); 0 (Peptide Fragments); 12UHW9R67N (Isoflurophate); 76808-16-7 (ebelactone A); EC 3.4.- (Peptide Hydrolases); EC 3.4.21.4 (Trypsin) [Em] Mês de entrada: 0105 [Cu] Atualização por classe: 131121 [Lr] Data última revisão: 131121 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 010222 [St] Status: MEDLINE

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[PMID]: 10647703 [Au] Autor: Yang SJ; An JY; Shim JO; Park CH; Huh IH; Sohn UD [Ad] Endereço: Department of Pharmacology, College of Pharmacy, Chung Ang University, Seoul, Republic of Korea. [Ti] Título: The mechanism of contraction by 2-chloroadenosine in cat detrusor muscle cells. [So] Source: J Urol;163(2):652-8, 2000 Feb. [Is] ISSN: 0022-5347 [Cp] País de publicação: United States [La] Idioma: eng [Ab] Resumo: PURPOSE: Four adenosine receptors (ARs), designated A1AR (A1 adenosine receptor), A2aAR (A2a adenosine receptor), A2bAR (A2b adenosine receptor), and A3AR (A3 adenosine receptor), have been cloned from various species, but the contraction mechanism via A1ARs in cat detrusor muscle cell is not well known. MATERIALS AND METHODS: We examined the cellular mechanism using an A1AR agonist 2-chloroadenosine (2-CA) in cat detrusor cell isolated by enzymatic digestion. To examine which phospholipase mediates the contraction, we used phospholipase inhibitors. RESULTS: The adenosine analog potency order is R-N6-phenylisopropyladenosine (R-PIA) > 5'-N-ethylcarbosamine adenosine (NECA) > 2-chloroadenosine (2-CA) > S-N6-phenylisopropyladenosine (S-PIA). The ratio of equi-effective concentrations of R-PIA/S-PIA was 58.2. 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 300 nM) shifted to the right the concentration-response curves of 2-CA. These results indicate A1ARs mediate 2-CA induced contraction in cat detrusor muscle. G proteins (Gi1, Gi2, Gi3, Go, Gs, and Gq) in cat detrusor muscle were detected by immunoblot analysis. Pertussis toxin (PTX) inhibited 2-CA induced contraction. In permeabilized cells, antibodies against Galphai3 antagonized 2-CA induced contraction, suggesting that the contraction is mediated by Gi3 protein. A phosphatidylinositol-specific phospholipase C (PLC) inhibitor, neomycin, reduced 2-CA induced contraction, but a phospholipase D (PLD) inhibitor, p-chloromercuribenzoic acid, and a phospholipase A2 (PLA2) inhibitor, dimethyl-eicosa-dienoic acid (DEDA), had no effect. We found the presence of the main PLC isozymes, PLC-beta1, PLC-beta3, and PLC-gamma1. 2-CA induced contraction in permeabilized cells was inhibited by PLC-beta3 but not by PLC-beta1 or PLC-gamma1 antibody. These results imply that A1ARs are coupled to PLC-beta3 via PTX-sensitive Gi3 protein. Sr2+ medium and thapsigargin, which replaces intracellular Ca2+ and deplete intracellular calcium stores respectively, inhibited 2-CA induced contraction. CONCLUSIONS: These results suggest that A1ARs mediating 2-CA induced contraction exist in cat detrusor muscle and the contraction depends on a PTX-sensitive Gi3 protein, PLC-beta3 and the release of intracellular Ca2+. [Mh] Termos MeSH primário: 2-Cloroadenosina/farmacologia Contração Muscular/efeitos dos fármacos Músculo Liso/efeitos dos fármacos Músculo Liso/fisiologia Bexiga Urinária/efeitos dos fármacos Bexiga Urinária/fisiologia [Mh] Termos MeSH secundário: Animais Gatos Cloromercurobenzoatos/farmacologia Proteínas de Ligação ao GTP/fisiologia Toxina Pertussis Antagonistas de Receptores Purinérgicos P1 Fatores de Virulência de Bordetella/farmacologia Xantinas/farmacologia [Pt] Tipo de publicação: JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T [Nm] Nome de substância: 0 (Chloromercuribenzoates); 0 (Purinergic P1 Receptor Antagonists); 0 (Virulence Factors, Bordetella); 0 (Xanthines); 146-77-0 (2-Chloroadenosine); 9PTP4FOI9E (1,3-dipropyl-8-cyclopentylxanthine); EC 2.4.2.31 (Pertussis Toxin); EC 3.6.1.- (GTP-Binding Proteins) [Em] Mês de entrada: 0002 [Cu] Atualização por classe: 161124 [Lr] Data última revisão: 161124 [Sb] Subgrupo de revista: AIM; IM [Da] Data de entrada para processamento: 000127 [St] Status: MEDLINE

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[PMID]: 9720226 [Au] Autor: Shin HH; Choi HS [Ad] Endereço: Department of Microbiology, University of Ulsan, Korea. [Ti] Título: Purification and characterization of cysteine protease from Pleurotus ostreatus. [So] Source: Biosci Biotechnol Biochem;62(7):1416-8, 1998 Jul. [Is] ISSN: 0916-8451 [Cp] País de publicação: England [La] Idioma: eng [Ab] Resumo: Cysteine protease activity in mycelial culture increased 7.7-fold after fruit body formation in Pleurotus ostreatus, using the Leu pNA (LPNA) cleavage assay. The enzyme was purified from fruit bodies and its M(r) was 97,000 by gel filtration and 48,500 by SDS-PAGE, indicating that it is a dimer. The enzyme was sensitive to iodoacetic acid, p-chloromercuribenzoate, N-ethylmaleimide, and HgCl2. The sequence of the first 9 N-terminal amino acids of cysteine protease was ASGLXXAIL. [Mh] Termos MeSH primário: Basidiomycota/enzimologia Cisteína Endopeptidases/isolamento & purificação [Mh] Termos MeSH secundário: Cloromercurobenzoatos/química Cromatografia em Gel Cisteína Endopeptidases/química Dimerização Eletroforese em Gel de Poliacrilamida Etilmaleimida/química Concentração de Íons de Hidrogênio Iodoacetatos/química Ácido Iodoacético Cloreto de Mercúrio/química Peso Molecular [Pt] Tipo de publicação: JOURNAL ARTICLE [Nm] Nome de substância: 0 (Chloromercuribenzoates); 0 (Iodoacetates); 53GH7MZT1R (Mercuric Chloride); EC 3.4.22.- (Cysteine Endopeptidases); O3C74ACM9V (Ethylmaleimide); WF5188V710 (Iodoacetic Acid) [Em] Mês de entrada: 9809 [Cu] Atualização por classe: 131121 [Lr] Data última revisão: 131121 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 980828 [St] Status: MEDLINE

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[PMID]: 9671510 [Au] Autor: Behrens W; Alexiev U; Mollaaghababa R; Khorana HG; Heyn MP [Ad] Endereço: Biophysics Group, Department of Physics, Freie Universität Berlin, Germany. [Ti] Título: Structure of the interhelical loops and carboxyl terminus of bacteriorhodopsin by X-ray diffraction using site-directed heavy-atom labeling. [So] Source: Biochemistry;37(29):10411-9, 1998 Jul 21. [Is] ISSN: 0006-2960 [Cp] País de publicação: United States [La] Idioma: eng [Ab] Resumo: The positions of single amino acids in the interhelical loop regions and the C-terminal tail of bacteriorhodopsin (bR) were investigated by X-ray diffraction using site-directed heavy-atom labeling. Since wild-type bR does not contain any cysteines, appropriate cysteine mutants were produced with a unique sulfhydryl group at specific positions. These sites were then labeled with mercury using the sulfhydryl specific reagent p-chloromercuribenzoate (p-CMB). The cysteine mutants D96A/V101C, V130C, A160C, and G231C were derivatized with labeling stoichiometries of 0.93 +/- 5%, 0.85 +/- 5%, 0.79 +/- 7%, and 0.77 +/- 8%, respectively (Hg per bR). No incorporation was observed with wild-type bR under the same conditions. All mutants and heavy-atom derivatives were fully active as judged by the kinetics of the photocycle and of the proton release and uptake. Moreover, the unit cell dimensions of the two-dimensional P3 lattice were unchanged by the mutations and the derivatization. This allowed the position of the mercury atoms, projected onto the plane of the membrane, to be calculated from the intensity differences in the X-ray diffraction pattern between labeled and unlabeled samples using Fourier difference methods. The X-ray diffraction data were collected at room temperature from oriented purple membrane films at 100% relative humidity without the use of dehydrating solvents. These native conditions of temperature, humidity, and solvent are expected to preserve the structure of the surface-exposed loops. Sharp maxima corresponding to a single mercury atom were found in the difference density maps for D96A/V101C and V130C. Residues 101 and 130 are in the short loops connecting helices C/D and D/E, respectively. No localized difference density was found for A160C and G231C. Residue 160 is in the longer loop connecting helices E and F, whereas residue 231 is in the C-terminal tail. Residues 160 and 231 are apparently in a more disordered and mobile part of the structure. [Mh] Termos MeSH primário: Bacteriorodopsinas/química Cloromercurobenzoatos/metabolismo Estrutura Secundária de Proteína [Mh] Termos MeSH secundário: Alanina/genética Sequência de Aminoácidos Ácido Aspártico/genética Bacteriorodopsinas/genética Bacteriorodopsinas/metabolismo Cisteína/genética Glicina/genética Marcação por Isótopo/métodos Dados de Sequência Molecular Mutagênese Sítio-Dirigida Fotoquímica Bombas de Próton/química Espectrofotometria Ultravioleta Valina/genética Difração de Raios X [Pt] Tipo de publicação: JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T; RESEARCH SUPPORT, U.S. GOV'T, P.H.S. [Nm] Nome de substância: 0 (Chloromercuribenzoates); 0 (Proton Pumps); 30KYC7MIAI (Aspartic Acid); 53026-44-1 (Bacteriorhodopsins); HG18B9YRS7 (Valine); K848JZ4886 (Cysteine); OF5P57N2ZX (Alanine); TE7660XO1C (Glycine) [Em] Mês de entrada: 9808 [Cu] Atualização por classe: 131121 [Lr] Data última revisão: 131121 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 980722 [St] Status: MEDLINE

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[PMID]: 9597757 [Au] Autor: Cunningham DF; O'Connor B [Ad] Endereço: School of Biological Sciences, Dublin City University, Ireland. [Ti] Título: A study of prolyl endopeptidase in bovine serum and its relevance to the tissue enzyme. [So] Source: Int J Biochem Cell Biol;30(1):99-114, 1998 Jan. [Is] ISSN: 1357-2725 [Cp] País de publicação: Netherlands [La] Idioma: eng [Ab] Resumo: Prolyl endopeptidase (PE) belongs to a group of enzymes that specifically recognise the imino acid proline. The characterisation of bovine serum PE was undertaken so that its relationship to its tissue counterparts could be considered. Using various chromatographic methods, PE was partially purified from bovine serum. This preparation was deemed to be enzymatically pure, based on its failure to hydrolyse a wide range of fluorimetric substrates. A native molecular mass of 69.7 kDa was estimated for the enzyme. PE was optimally active at pH 8.0-8.5, demonstrated a preference for phosphate buffer and remained stable over a pH range of 5.0-9.0. A narrowly focused optimal assay temperature of 37 degrees C was evident. Functional reagent studies indicated that this enzyme was a serine protease with a cysteine residue located near or at the active site. The enzyme was also sensitive to heavy metal inhibition. Substrate specificity investigations revealed that the bioactive peptides angiotensin II, bradykinin, luliberin and substance P were hydrolysed by the enzyme preparation, but lower specificities were evident towards these peptides in comparison with the enzyme's tissue counterparts. Specific inhibitor studies, using a range of compounds previously untested against a single PE source, indicated that alpha-ketobenzothiazole was the most effective PE inhibitor, with an IC50 value of 41 pM. In conclusion, the results presented in this paper indicate that bovine serum PE shares many of the characteristics associated with its tissue counterparts, with the exception of its specificity towards certain bioactive peptides. [Mh] Termos MeSH primário: Inibidores Enzimáticos/farmacologia Metais/farmacologia Serina Endopeptidases/sangue Serina Endopeptidases/efeitos dos fármacos [Mh] Termos MeSH secundário: Angiotensina II/metabolismo Animais Ligação Competitiva Bradicinina/metabolismo Bovinos Cloromercurobenzoatos/farmacologia Cumarínicos/química Cumarínicos/farmacologia Estabilidade Enzimática/efeitos dos fármacos Estabilidade Enzimática/fisiologia Fluorometria Hormônio Liberador de Gonadotropina/metabolismo Concentração de Íons de Hidrogênio Peso Molecular Neuropeptídeos/metabolismo Serina Endopeptidases/química Substância P/metabolismo Especificidade por Substrato Ácido p-Cloromercurobenzoico [Pt] Tipo de publicação: JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T [Nm] Nome de substância: 0 (Chloromercuribenzoates); 0 (Coumarins); 0 (Enzyme Inhibitors); 0 (Metals); 0 (Neuropeptides); 11128-99-7 (Angiotensin II); 33507-63-0 (Substance P); 33515-09-2 (Gonadotropin-Releasing Hormone); 59-85-8 (p-Chloromercuribenzoic Acid); 68542-93-8 (N-carbobenzoxyglycyl-prolyl-4-methylcoumarinyl amide); EC 3.4.21.- (Serine Endopeptidases); EC 3.4.21.26 (prolyl oligopeptidase); S8TIM42R2W (Bradykinin) [Em] Mês de entrada: 9807 [Cu] Atualização por classe: 131121 [Lr] Data última revisão: 131121 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 980523 [St] Status: MEDLINE

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[PMID]: 9576484 [Au] Autor: Park JH; Edwards MR; Schofield PJ [Ad] Endereço: School of Biochemistry and Molecular Genetics, University of New South Wales, Sydney, Australia. parkjh@u.washington.edu [Ti] Título: Swelling detection for volume regulation in the primitive eukaryote Giardia intestinalis: a common feature of volume detection in present-day eukaryotes. [So] Source: FASEB J;12(7):571-9, 1998 May. [Is] ISSN: 0892-6638 [Cp] País de publicação: United States [La] Idioma: eng [Ab] Resumo: It is increasingly evident that cell swelling is associated with the triggering of many biological processes, including progression of the cell cycle, hormonal response, and gene expression. However, the mechanism by which cell swelling is initially sensed and converted into intracellular signals is still ill-defined. We report here an early event in the detection of cell swelling and initiation of the volume regulatory response in Giardia intestinalis, an ancient representative of the eukaryotic kingdom. Giardial cell swelling, irrespective of the extent, was sensed at a cell volume of 1.06 x isosmotic volume (the threshold volume), at which the transition of the volume regulatory transport system from the 'resting' to the 'open' state occurred. Irreversible modification by p-chloromercuribenzoate (pCMB) and N-ethylmaleimide (NEM) of reduced thiols affected the threshold volume, but in opposing manners: pCMB increased the threshold volume to 1.14 x and NEM decreased to 0.85 x isosmotic volume. The simple modification of the threshold volume by NEM caused a drastic reduction of giardial cell volume under isosmotic conditions, with a process strikingly similar to the opening of mitochondrial permeability transition pore, a causative event in stress-induced programmed cell death. Substantial evidence supports the hypothesis that modulation of the membrane thiol moieties at the threshold volume, causing the 'all-or-nothing' type of swelling detection, represents the event linking cell swelling to the second messenger systems for volume regulation in present eukaryotes. Pathophysiological implications of alteration of the threshold volume are discussed. [Mh] Termos MeSH primário: Tamanho Celular/fisiologia Giardia lamblia/fisiologia [Mh] Termos MeSH secundário: Alanina/metabolismo Aminoácidos/metabolismo Animais Apoptose Tamanho Celular/efeitos dos fármacos Cloromercurobenzoatos/farmacologia Etilmaleimida/farmacologia Células Eucarióticas Giardia lamblia/efeitos dos fármacos Cinética Concentração Osmolar Estresse Fisiológico Fatores de Tempo Ácido p-Cloromercurobenzoico [Pt] Tipo de publicação: JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T [Nm] Nome de substância: 0 (Amino Acids); 0 (Chloromercuribenzoates); 59-85-8 (p-Chloromercuribenzoic Acid); O3C74ACM9V (Ethylmaleimide); OF5P57N2ZX (Alanine) [Em] Mês de entrada: 9805 [Cu] Atualização por classe: 131121 [Lr] Data última revisão: 131121 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 980512 [St] Status: MEDLINE

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MEDLINE

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[PMID]: 9540790 [Au] Autor: Huang HS; Kabashima T; Ito K; Yin CH; Nishiya Y; Kawamura Y; Yoshimoto T [Ad] Endereço: School of Pharmaceutical Sciences, Nagasaki University, Japan. [Ti] Título: Thermostable glycerol kinase from Thermus flavus: cloning, sequencing, and expression of the enzyme gene. [So] Source: Biochim Biophys Acta;1382(2):186-90, 1998 Feb 17. [Is] ISSN: 0006-3002 [Cp] País de publicação: Netherlands [La] Idioma: eng [Ab] Resumo: The thermostable glycerol kinase (EC 2.7.1.30) gene from Thermus flavus was cloned and expressed in Escherichia coli DH5 alpha. An open reading frame of 1488 bp for the glycerol kinase gene (glpK) starting with an ATG methionine codon was found, which encodes a protein of 496 amino acid residues whose calculated molecular weight is 54,835. The amino acid sequence of T. flavus glycerol kinase is 80.6% and 64.1% identical with those of Bacillus subtilis and E. coli. Transformants of E. coli DH5 alpha harboring plasmid pGYK12 with a 1505 bp chromosomal DNA fragment containing the T. flavus glycerol kinase gene showed about 23.8-fold higher glycerol kinase activity than T. flavus. [Mh] Termos MeSH primário: Glicerol Quinase/química Thermus/enzimologia [Mh] Termos MeSH secundário: Sequência de Aminoácidos Proteínas Arqueais/química Sequência de Bases Cloromercurobenzoatos/farmacologia Clonagem Molecular Sequência Consenso/genética Estabilidade Enzimática Expressão Gênica/genética Dados de Sequência Molecular Proteínas Recombinantes Análise de Sequência de DNA Homologia de Sequência de Aminoácidos Ácido p-Cloromercurobenzoico [Pt] Tipo de publicação: JOURNAL ARTICLE [Nm] Nome de substância: 0 (Archaeal Proteins); 0 (Chloromercuribenzoates); 0 (Recombinant Proteins); 59-85-8 (p-Chloromercuribenzoic Acid); EC 2.7.1.30 (Glycerol Kinase) [Em] Mês de entrada: 9805 [Cu] Atualização por classe: 161126 [Lr] Data última revisão: 161126 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 980416 [St] Status: MEDLINE

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