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1.
ACS Infect Dis ; 5(12): 2029-2038, 2019 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-31583882

RESUMO

Naegleria fowleri is a free-living amoeba causing primary amoebic meningoencephalitis, a rapid-onset brain infection in humans with over 97% mortality rate. Despite some progress in the treatment of the disease, there is no single, proven, evidence-based treatment with a high probability of cure. Here we report the chemical library screening and experimental identification of four new compounds with amoebicidal effects against N. fowleri. The chemical library was screened by molecular docking against a homology model of sterol Δ8-Δ7 isomerase (NfERG2). Thirty top-ranking hits were then tested in a cell-based assay for antiproliferative/amoebicidal activities. Eight chemicals exhibited nearly 100% inhibition of N. fowleri at 50 µM, with the EC50 values ranging from 6 to 25 µM. A cell toxicity assay using human HEK-293 cells was also performed. Four of the compounds preferentially kill amoeba cells with no apparent human cell toxicities. These compounds fall into two distinct chemical scaffolds with druglike properties.


Assuntos
Amebicidas/farmacologia , Isomerases/química , Naegleria fowleri/enzimologia , Bibliotecas de Moléculas Pequenas/farmacologia , Amebicidas/química , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Isomerases/efeitos dos fármacos , Isomerases/genética , Modelos Moleculares , Simulação de Acoplamento Molecular , Naegleria fowleri/efeitos dos fármacos , Naegleria fowleri/genética , Fenótipo , Conformação Proteica , Homologia de Sequência , Bibliotecas de Moléculas Pequenas/química
2.
Endocrinology ; 153(4): 1925-35, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22374975

RESUMO

Oviductal disease is a primary cause of infertility, a problem that largely stems from excessive inflammation of this key reproductive organ. Our poor understanding of the mechanisms regulating oviductal inflammation restricts our ability to diagnose, treat, and/or prevent oviductal disease. Using mice, our objective was to determine the spatial localization, regulatory mechanism, and functional attributes of a hypothesized regulator of oviductal inflammation, the hematopoietic form of prostaglandin D synthase (HPGDS). Immunohistochemistry revealed specific localization of HPGDS to the oviduct's epithelium. In the isthmus, expression of HPGDS was consistent. In the ampulla, expression of HPGDS appeared dependent upon stage of the estrous cycle. HPGDS was expressed in the epithelium of immature and cycling mice but not in the oviducts of estrogen receptor α knockouts. Two receptor subtypes bind PGD2: PGD2 receptor and G protein-coupled receptor 44. Expression of mRNA for Ptgdr was higher in the epithelial cells (EPI) than in the stroma (P < 0.05), whereas mRNA for Gpr44 was higher in the stroma than epithelium (P < 0.05). Treatment of human oviductal EPI with HQL-79, an inhibitor of HPGDS, decreased cell viability (P < 0.05). Treatment of mice with HQL-79 increased mRNA for chemokine (C-C motif) ligands 3, 4, and 19; chemokine (C-X-C motif) ligands 11 and 12; IL-13 and IL-17B; and TNF receptor superfamily, member 1b (P < 0.02 for each mRNA). Overall, these results suggest that HPGDS may play a role in the regulation of inflammation and EPI health within the oviduct.


Assuntos
Células Epiteliais/enzimologia , Receptor alfa de Estrogênio/metabolismo , Inflamação/metabolismo , Isomerases/metabolismo , Oviductos/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/citologia , Receptor alfa de Estrogênio/deficiência , Receptor alfa de Estrogênio/genética , Ciclo Estral/metabolismo , Feminino , Técnicas In Vitro , Inflamação/fisiopatologia , Oxirredutases Intramoleculares , Isomerases/antagonistas & inibidores , Isomerases/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Camundongos Transgênicos , Oviductos/citologia , Piperidinas/farmacologia , RNA Mensageiro/metabolismo , Receptores Imunológicos/metabolismo , Receptores de Prostaglandina/metabolismo
3.
Biotechnol Prog ; 19(1): 179-84, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12573023

RESUMO

In an attempt to increase the specific thrombopoietin (TPO) productivity (q(TPO)) of recombinant Chinese hamster ovary (rCHO) cells (TPO-33), the effect of expression level of ERp57, an isoform of protein disulfide isomerase, on q(TPO) was investigated. To regulate ERp57 expression level, the Tet-Off system was first introduced in TPO-33 cells and stable Tet-Off cells (TPO-33-Tet-Off) were screened by the luciferase assay. The rCHO cells with a doxycycline-regulated ERp57 expression system (TPO-33-ERp57) were obtained by cotransfection of pTRE-ERp57 and pTK-Hyg expression vectors into TPO-33-Tet-Off cells and subsequent screening by Western blot analysis of ERp57 and an enzyme-linked immunosorbent assay of secreted TPO. Western blot analysis showed that ERp57 expression level in TPO-33-ERp57 cells could be regulated tightly by the addition of different concentrations of doxycycline to a culture medium. A doxycycline concentration of 1 microg/mL, which did not influence cell growth and TPO production of TPO-33-Tet-Off cells, was high enough to suppress the ERp57 expression to a basal level. Compared with the basal level, a 1.7-fold increase in ERp57 expression level was obtained in the absence of doxycycline. This increased expression level of ERp57 resulted in a 2.1-fold increase in q(TPO) without growth inhibition, probably as a result of the chaperone-like activity of ERp57 in CHO cells. Taken together, the results obtained here demonstrate that q(TPO) of rCHO cells can be increased by elevating the expression level of ERp57.


Assuntos
Células CHO/efeitos dos fármacos , Células CHO/metabolismo , Doxiciclina/farmacologia , Proteínas de Choque Térmico/metabolismo , Isomerases/metabolismo , Trombopoetina/biossíntese , Animais , Células CHO/citologia , Contagem de Células , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Clonagem Molecular , Cricetinae , Meios de Cultura/farmacologia , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/efeitos dos fármacos , Isomerases/efeitos dos fármacos , Controle de Qualidade , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Trombopoetina/genética
4.
Eur J Immunol ; 27(7): 1685-90, 1997 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-9247578

RESUMO

Peptides derived from cytosolic protein degradation are translocated into the lumen of the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP). In the ER, class I molecules bind the peptides fitting to their respective motifs and present them on the cell surface to CD8+ T lymphocytes. However, most TAP-translocated peptides are not expected to bind to the class I molecules present in a particular cell. Recently, we have demonstrated that TAP-translocated peptides containing a photoreactive phenylalanine analogue can be cross-linked to two luminal ER-resident proteins: with low efficiency to the stress protein gp96 and with high efficiency to a 60-kDa protein (Lammert, E. et al., Eur. J. Immunol. 1997. 27: 923). Both proteins have also been labeled specifically by TAP-translocated peptides conjugated to a different photoreactive group (Marusina, K. et al., Biochemistry 1997. 36: 856). Here, we show that the 60-kDa peptide-binding protein is identical to the multifunctional protein disulfide isomerase (PDI). Since PDI is the only luminal ER-resident protein that is labeled by the photoreactive peptides with high efficiency, it might represent the dominant acceptor for TAP-translocated peptides.


Assuntos
Retículo Endoplasmático/enzimologia , Isomerases/metabolismo , Oligopeptídeos/metabolismo , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Marcadores de Afinidade , Ligação Competitiva , Transporte Biológico/imunologia , Linhagem Celular , Retículo Endoplasmático/metabolismo , Estradiol/farmacologia , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Células Híbridas , Isomerases/efeitos dos fármacos , Peso Molecular , Oligopeptídeos/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/imunologia , Isomerases de Dissulfetos de Proteínas
5.
Protein Expr Purif ; 9(2): 279-87, 1997 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-9056493

RESUMO

Human carbonic anhydrase IV (CA IV) expressed in Escherichia coli was refolded and activated in cell extracts with the help of endogenous periplasmic protein disulfide isomerase, DsbA, in the presence of oxidized glutathione. The refolding and activation were inhibited by bacitracin but not affected by known cofactors or activators of other chaperones. Although the yield of the purified CA IV recovered from cell extracts was maximal when activated at 4 degrees C in the presence of 2 mM oxidized glutathione, the rate of refolding and activation was much more rapid at 25 and 37 degrees C. The enzyme purified from the E. coli cell extracts following activation in vitro showed similar structural stability and functional properties as CA IV purified from secretion medium from a stably transfected CHO cell line. These studies suggest that the soluble truncated form of human CA IV expressed in E. coli, which is disulfide-bonded zinc metalloenzyme, can provide a useful model enzyme for studies of protein folding and enzyme activation in vitro. Furthermore, the procedure described for recovery of CA IV following expression in E. coli may be useful for in vitro activation and subsequent purification of other disulfide-containing proteins.


Assuntos
Anidrases Carbônicas/genética , Anidrases Carbônicas/isolamento & purificação , Dissulfetos/química , Bacitracina/farmacologia , Anidrases Carbônicas/biossíntese , Anidrases Carbônicas/química , Clonagem Molecular , Ativação Enzimática/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Glutationa/análogos & derivados , Glutationa/metabolismo , Glutationa/farmacologia , Dissulfeto de Glutationa , Glicosilfosfatidilinositóis/metabolismo , Humanos , Isomerases/efeitos dos fármacos , Isomerases/metabolismo , Chaperonas Moleculares/metabolismo , Isomerases de Dissulfetos de Proteínas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Temperatura
6.
Pigment Cell Res ; 9(6): 311-6, 1996 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-9125755

RESUMO

We have found that a melanization inhibitory factor (MIF) extracted from the ventral skin of Rana forreri has a slight inhibitory effect on the activity levels of tyrosinase and dopachrome tautomerase in B16/F10 and Cloudman S-91 murine melanoma cell lines. Furthermore, this factor appears to block the effects of alpha-MSH on these enzymatic activities. However, MIF treatment does not affect the melanogenic action of theophylline on the same cells, suggesting that MIF acts proximal to MSH-mediated cAMP formation, possibly by interaction with the MSH receptor. In this way, we show that this amphibian factor has biological activity on mammalian melanocytes. This suggests the existence of mammalian counterparts of amphibian MIF in the mouse integument that might regulate epidermal melanocytes. These peptides might be related to the agouti protein, as they share similar mechanisms of action. The interaction of different peptides with the MSH receptor would be a complex but general mechanism responsible for many mammalian coat color variants.


Assuntos
Oxirredutases Intramoleculares , Hormônio Inibidor da Liberação de MSH/farmacologia , Melaninas/metabolismo , Melanócitos/patologia , Ranidae , alfa-MSH/farmacologia , Animais , Relação Dose-Resposta a Droga , Isomerases/efeitos dos fármacos , Isomerases/metabolismo , Melaninas/química , Melanócitos/efeitos dos fármacos , Melanócitos/metabolismo , Camundongos , Monofenol Mono-Oxigenase/efeitos dos fármacos , Monofenol Mono-Oxigenase/metabolismo , Teofilina/farmacologia , Tirosina 3-Mono-Oxigenase/efeitos dos fármacos , Tirosina 3-Mono-Oxigenase/metabolismo , Vasodilatadores/farmacologia
7.
Biochem Pharmacol ; 52(4): 669-76, 1996 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-8759040

RESUMO

Recently, we reported that alterations in topoisomerase II (topo II) activity appear to contribute to mitomycin C (MMC) resistance in HT-29R13 human colon cancer cells under aerobic conditions. In this study, the expression of topo II alpha and topo II beta in parent HT-29 and MMC resistant variant HT-29R13 cells was investigated under aerobic, acute hypoxic (after 4 hr in 95% N2, 5% CO2 < 0.01% O2), and chronic intermittent hypoxic (after 4 hr hypoxia/day x 7 days) conditions. Acute hypoxia induced topo II alpha mRNA and protein, effects that were more pronounced in HT-29 cells. Chronic intermittent hypoxia caused a decrease in topo alpha mRNA and protein, changes that were again more pronounced in HT-29 cells. The observed changes in topo II alpha protein were associated with parallel changes in topo II activity under all conditions tested. Topo II beta mRNA was expressed at a very low level in both cell lines under aerobic and hypoxic conditions. Compared with cells under aerobic conditions, HT-29 cells were more sensitive to MMC under acute hypoxia but more resistant under chronic intermittent hypoxia. In contrast, the senstivity of HT-29R13 cells was unchanged under acute hypoxia, but the cells were more resistant under chronic intermittent hypoxia. Under all conditions tested, the degree of cytotoxicity corresponded to the frequency of MMC-induced DNA cross-links and topo II alpha protein levels and activity. Our results demonstrated that MMC cytotoxicity in hypoxic cells is highly dependent upon the type of hypoxia and the cell type. Hypoxia has significant effects on topo II alpha expression in HT-29 and HT-29R13 cells which correlate with MMC cytotoxicity.


Assuntos
Neoplasias do Colo/tratamento farmacológico , Dano ao DNA/efeitos dos fármacos , DNA/efeitos dos fármacos , Hipóxia/metabolismo , Mitomicina/farmacologia , Relação Dose-Resposta a Droga , Humanos , Isomerases/efeitos dos fármacos , RNA Mensageiro/biossíntese
8.
RNA ; 1(4): 437-48, 1995 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-7493321

RESUMO

An Escherichia coli pseudouridine (psi) synthase, which forms both psi 746 in E. coli 23S ribosomal RNA and psi 32 in tRNA(Phe), has been isolated and cloned. The enzyme contains 219 amino acids and has a calculated MW of 24,432 Da. Amino acid sequence comparison with the three other psi synthases that have been cloned to date, two for tRNA and one for 16S RNA, did not reveal any common sequence motifs, despite the catalysis of a common reaction. The gene was cloned behind a (His)6 leader for affinity purification. Upon overexpression, most of the enzyme remained soluble in the cell cytoplasm and could be purified to homogeneity on a Ni(2+)-containing resin. The enzyme reacted with both full-length 23S RNA or a fragment from residues 1-847, forming 1 mol psi/mol RNA at position 746, a normal site for psi. The enzyme has no dependence on Mg2+. The same yield was obtained in 1 mM EDTA as in 10 mM Mg2+, and the rate was faster in EDTA than in Mg2+. Full-length 16S RNA or fragments 1-526 or 1-678, as well as tRNA(Val) transcripts, were not modified in either EDTA or Mg2+. tRNA(Phe) transcripts, however, were modified with a yield of 1 mol psi/mol transcript at a rate in EDTA like that of 23S RNA. Sequencing showed all of the psi to be at position 32, a normal site for psi in this tRNA. Both 23S rRNA psi 746 and tRNA psi 32 occur in single-stranded segments of the same sequence, psi UGAAAA, closed by a stem. Therefore, this synthase may require for recognition only a short stretch of primary sequence 3' to the site of pseudouridylation. This is the first example of a dual-specificity modifying enzyme for RNA, that is, one which is specific for a single site in one RNA, and equally site-specific in a second class of RNA. The essentiality of these psi residues can now be assessed by disruption of the synthase gene.


Assuntos
Escherichia coli/enzimologia , Transferases Intramoleculares , Isomerases/metabolismo , Pseudouridina/biossíntese , RNA Ribossômico 23S/metabolismo , RNA de Transferência de Fenilalanina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Escherichia coli/genética , Genes Bacterianos , Isomerases/efeitos dos fármacos , Isomerases/genética , Isomerases/isolamento & purificação , Magnésio/farmacologia , Dados de Sequência Molecular , Processamento Pós-Transcricional do RNA , Proteínas Recombinantes/metabolismo , Análise de Sequência de RNA , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
9.
Lipids ; 30(3): 231-4, 1995 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-7791531

RESUMO

Active site mapping of rat liver oxidosqualene cyclase (OSC), a 78 kDa membrane-bound enzyme, was carried out using the mechanism-based irreversible inhibitor, [3H]29-methylidene-2,3-oxidosqualene. The amino acid sequence of the radiolabeled CNBr peptide fragment showed unexpectedly high similarity to the yeast OSC, plant OSC, and bacterial squalene cyclases. Further, radio analysis established that the two adjacent Asp residues in the highly conserved region (Asp-Asp-Thr-Ala-Glu-Ala, or DDTAEA) were equally labeled by the irreversible inhibitor. This result provided the first information on the structural details of the active site of OSC, and showed for the first time the ancient lineage of this vertebrate enzyme to ancestral eukaryotic and prokaryotic cyclases. Interestingly, the covalently-modified DDXX(D/E) sequence of rat liver OSC showed surprising similarity to the putative allylic diphosphate binding site sequence of other terpene cyclases and prenyl transferases. The Asp-rich motif may act as a point charge to stabilize incipient cationic charge.


Assuntos
Transferases Intramoleculares , Isomerases/metabolismo , Lanosterol/biossíntese , Esqualeno/análogos & derivados , Sequência de Aminoácidos , Animais , Sítios de Ligação , Isomerases/química , Isomerases/efeitos dos fármacos , Fígado/enzimologia , Modelos Químicos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Ratos , Análise de Sequência , Homologia de Sequência de Aminoácidos , Esqualeno/metabolismo , Esqualeno/farmacologia
10.
Arch Biochem Biophys ; 312(1): 203-9, 1994 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-7913310

RESUMO

In the biosynthetic conversion of glutamate to the tetrapyrrole precursor, delta-aminolevulinic acid (ALA), glutamate is activated at C-1 by glutamyl-tRNA synthetase-catalyzed ligation to tRNAGlu. Glutamyl-tRNA reductase next catalyzes reduction of the activated glutamate to glutamate-1-semialdehyde (GSA), which is then converted to ALA by GSA aminotransferase. Glutamyl-tRNA synthetase is known to require a divalent metal (usually Mg2+) for activity, but it has not been established whether Mg2+ or another metal ion is also required for glutamyl-tRNA reductase or GSA aminotransferase, because these enzymes have previously been assayed in combined incubations containing all factors required for conversion of glutamate to ALA. We now report the metal requirements individually for each of the three enzyme reactions. Glutamyl-tRNA reductase activity in extracts from both Chlorella vulgaris and Synechocystis sp. PCC 6803 was stimulated by Mg2+ and inhibited by EDTA. EDTA-pretreated Chlorella glutamyl-tRNA reductase-containing fraction had very little activity in the absence of added Mg2+, but recovered full activity in incubations containing added Mg2+. The divalent metal requirement could be met by Mg2+, Mn2+, or Ca2+. Maximum activity was reached at approximately 15 mM concentration of each of these metals, and higher concentrations were inhibitory. Zn2+ was inhibitory at micromolar concentrations. Chlorella glutamyl-tRNA synthetase showed a metal requirement that could be met by Mg2+ or Mn2+, but not Ca2+. Maximum activity was reached at approximately 15 mM Mg2+ or Mn2+. Although the presence of 10 mM Ca2+ did not affect the Mg2+ concentration optimum, Ca2+ increased the effectiveness of low concentrations of Mg2+. In contrast to glutamyl-tRNA synthetase and glutamyl-tRNA reductase, Chlorella GSA aminotransferase did not show a metal requirement or inhibition by EDTA. However, EDTA decreased nonenzymatic transformation of GSA to ALA.


Assuntos
Aldeído Oxirredutases/efeitos dos fármacos , Cátions Bivalentes/farmacologia , Eucariotos/enzimologia , Glutamato-tRNA Ligase/efeitos dos fármacos , Transferases Intramoleculares , Isomerases/efeitos dos fármacos , Ácido Aminolevulínico/metabolismo , Sistema Livre de Células , Chlorella/enzimologia , Cianobactérias/enzimologia , Glutamatos/metabolismo , Ácido Glutâmico , Pirróis/metabolismo , Tetrapirróis
11.
Ecotoxicol Environ Saf ; 26(2): 127-32, 1993 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-7504609

RESUMO

Primary cultures of hepatocytes were established from sexually mature male rainbow trout (Oncorhyncus mykiss) and treated with the hypolipidemic drugs gemfibrozil (0.25-1.25 mM), clofibric acid (2.25-3.00 mM), or ciprofibrate (0.25-1.00 mM). Significant dose-related increases in peroxisomal fatty acyl-CoA oxidase (FACO) were seen after exposure for 48 hr to clofibric acid (P < 0.01) and ciprofibrate (P < 0.05) but not gemfibrozil (P = 0.08). Strong correlation was obtained between increased acyl-CoA oxidase activity and the relative amount of peroxisomal bifunctional enzyme (PBE), further supporting evidence of a proliferative effect. These preliminary studies demonstrate that peroxisomal beta-oxidation can be induced in vitro in a primary rainbow trout hepatocyte system.


Assuntos
Hipolipemiantes/farmacologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Microcorpos/efeitos dos fármacos , Microcorpos/enzimologia , Oncorhynchus mykiss/metabolismo , 3-Hidroxiacil-CoA Desidrogenases/efeitos dos fármacos , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Acil-CoA Oxidase , Animais , Células Cultivadas , Clofibrato/farmacologia , Ácido Clofíbrico/análogos & derivados , Ácido Clofíbrico/farmacologia , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/metabolismo , Enoil-CoA Hidratase/efeitos dos fármacos , Enoil-CoA Hidratase/metabolismo , Ativação Enzimática , Ácidos Fíbricos , Genfibrozila/farmacologia , Isomerases/efeitos dos fármacos , Isomerases/metabolismo , Fígado/citologia , Masculino , Complexos Multienzimáticos/efeitos dos fármacos , Complexos Multienzimáticos/metabolismo , Oxirredução , Oxirredutases/efeitos dos fármacos , Oxirredutases/metabolismo , Enzima Bifuncional do Peroxissomo
12.
Protein Sci ; 2(5): 717-26, 1993 May.
Artigo em Inglês | MEDLINE | ID: mdl-8495194

RESUMO

The redox properties of periplasmic protein disulfide isomerase (DsbA) from Escherichia coli were analyzed by measuring the equilibrium constant of the oxidation of reduced DsbA by oxidized glutathione. The experiments are based on the finding that the intrinsic tryptophan fluorescence of DsbA increases about threefold upon reduction of the enzyme, which can be explained by the catalytic disulfide bridge quenching the fluorescence of a neighboring tryptophan residue. From the specific fluorescence of DsbA equilibrated in the presence of different ratios of reduced and oxidized glutathione at pH 7, an equilibrium constant of 1.2 x 10(-4) M was determined, corresponding to a standard redox potential (E'0) of DsbA of -0.089 V. Thus, DsbA is a significantly stronger oxidant than cytoplasmic thioredoxins and its redox properties are similar to those of eukaryotic protein disulfide isomerase. The equilibrium constants for the DsbA/glutathione equilibrium were found to be strongly dependent on pH and varied from 2.5 x 10(-3) M to 3.9 x 10(-5) M between pH 4 and 8.5. The redox state-dependent fluorescence properties of DsbA should allow detailed physicochemical studies of the enzyme as well as the quantitative determination of the oxidized protein by fluorescence titration with dithiothreitol and open the possibility to observe bacterial protein disulfide isomerase "at work" during catalysis of oxidative protein folding.


Assuntos
Dissulfetos/metabolismo , Escherichia coli/enzimologia , Isomerases/metabolismo , Ditiotreitol/farmacologia , Escherichia coli/genética , Fluorescência , Glutationa/análogos & derivados , Glutationa/farmacologia , Dissulfeto de Glutationa , Isomerases/efeitos dos fármacos , Isomerases/genética , Oxirredução , Isomerases de Dissulfetos de Proteínas , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Triptofano/química
13.
J Bacteriol ; 174(21): 6857-61, 1992 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-1400236

RESUMO

Phosphoenolpyruvate phosphomutase (PEPPM) catalyzes C-P bond formation by intramolecular rearrangement of phosphoenolpyruvate to phosphonopyruvate (PnPy). We purified PEPPM from a gram-negative bacterium, Pseudomonas gladioli B-1 isolated as a C-P compound producer. The equilibrium of this reaction favors the formation of the phosphate ester by cleaving the C-P bond of PnPy, but the C-P bond-forming reaction is physiologically significant. The C-P bond-forming activity of PEPPM was confirmed with a purified protein. The molecular mass of the native enzyme was estimated to be 263 and 220 kDa by gel filtration and polyacrylamide gel electrophoresis, respectively. A subunit molecular mass of 61 kDa was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the native protein was a tetramer. The optimum pH and temperature were 7.5 to 8.0 and 40 degrees C, respectively. The Km value for PnPy was 19 +/- 3.5 microM, and the maximum initial velocity of the conversion of PnPy to phosphoenolpyruvate was 200 microM/s/mg. PEPPM was activated by the presence of the divalent metal ion, and the Km values were 3.5 +/- 1.4 microM for Mg2+, 16 +/- 5 nM for Mn2+, 3.0 +/- 1.5 microM for Zn2+, and 1.2 +/- 0.2 microM for Co2+.


Assuntos
Isomerases/isolamento & purificação , Fosfotransferases (Fosfomutases) , Pseudomonas/enzimologia , Carbono/química , Concentração de Íons de Hidrogênio , Isomerases/efeitos dos fármacos , Cinética , Magnésio/farmacologia , Peso Molecular , Fósforo/química
14.
J Invest Dermatol ; 99(4): 435-9, 1992 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-1328399

RESUMO

alpha-MSH was found to decrease the recently characterized dopachrome tautomerase activity in cultures of B16/F10 mouse melanoma cells. Other stimulating agents of melanogenesis, like dibutyryl cyclic AMP, 3-isobutyl-1-methylxanthine, theophylline, retinol, and retinoic acid, caused the same effect. The grade of inhibition depended on the nature of the agent and the time of exposure. In all cases, both melanin production and tyrosinase activity were activated by these treatments, although the grade of tyrosine hydroxylase and dopa oxidase stimulation was different. Moreover, no correlation among the intensities of dopachrome tautomerase inhibition and tyrosinase activation by the tested agents could be obtained. The significance of these results in the regulation of mammalian melanogenesis is discussed.


Assuntos
Oxirredutases Intramoleculares , Isomerases/efeitos dos fármacos , Isomerases/metabolismo , Melanoma Experimental/enzimologia , Melanoma Experimental/patologia , Monofenol Mono-Oxigenase/efeitos dos fármacos , Monofenol Mono-Oxigenase/metabolismo , alfa-MSH/farmacologia , Animais , Melaninas/biossíntese , Camundongos
15.
Biochem Biophys Res Commun ; 185(1): 211-6, 1992 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-1599458

RESUMO

Rat hepatic t protein that is negatively regulated by thyroid hormone in nuclear globulin extract was characterized by the antibodies. The following evidence indicated that t protein is a peroxisomal enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme (bifunctional enzyme). 1. Both proteins had an identical molecular size, and were immunologically indistinguishable from each other. 2. The t protein was abundant in mitochondrial fraction which contained abundant peroxisomes. 3. The amount of the t protein was increased by a peroxisomal proliferator. 4. The activity of the peroxisomal bifunctional enzyme corresponded to the t protein in CM-Sephadex column chromatography. The amount of peroxisomal bifunctional enzyme was increased by thyroidectomy and decreased by 3,5,3'- triiodo-L-thyronine treatment in the whole homogenate of rat liver. These results indicate that the levels of peroxisomal bifunctional enzyme were regulated by thyroid hormone in vivo.


Assuntos
3-Hidroxiacil-CoA Desidrogenases/metabolismo , Enoil-CoA Hidratase/metabolismo , Isomerases/metabolismo , Fígado/enzimologia , Microcorpos/enzimologia , Complexos Multienzimáticos/metabolismo , Tri-Iodotironina/farmacologia , 3-Hidroxiacil-CoA Desidrogenases/efeitos dos fármacos , Animais , Enoil-CoA Hidratase/efeitos dos fármacos , Indução Enzimática , Regulação Enzimológica da Expressão Gênica , Isomerases/efeitos dos fármacos , Fígado/efeitos dos fármacos , Masculino , Microcorpos/efeitos dos fármacos , Complexos Multienzimáticos/efeitos dos fármacos , Enzima Bifuncional do Peroxissomo , Ratos , Ratos Endogâmicos
16.
Biochem Biophys Res Commun ; 185(1): 60-8, 1992 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-1599490

RESUMO

Although it is recognized that 4,5-diaminovaleric acid, formed from glutamate 1-semialdehyde, functions as the intermediate in the last step of delta-aminolevulinic acid formation from glutamate, the enantioselectivity of the participating glutamate 1-semialdehyde aminotransferase for 4,5-diaminovaleric acid has remained unknown. In the present work the involvement of (S)- and (R)-4,5-diaminovaleric acids, newly available by organic synthesis, was investigated, using glutamate 1-semialdehyde aminotransferase from Synechococcus. The preferred enantiomer was (S)-4,5-diaminovalerate. In experiments on the transformation of (S)-4,5-diaminovalerate to delta-aminolevulinate it was found that glutamate 1-semialdehyde aminotransferase was unusual among aminotransferases in that the common amino acceptors pyruvate, oxaloacetate, alpha-ketoglutarate were inactive, while 4,5-dioxovaleric acid could be utilized as a sluggish amino acceptor in place of glutamate 1-semialdehyde. In conclusion, glutamate 1-semialdehyde aminotransferase is highly but not absolutely enantioselective for (S)-4,5-diaminovaleric acid, and 4,5-dioxovaleric acid can function as amino acceptor not because of a physiological role in the C5 pathway of delta-aminolevulinic acid formation, but because of its structural resemblance to glutamate 1-semialdehyde.


Assuntos
Diamino Aminoácidos/metabolismo , Ácido Aminolevulínico/metabolismo , Cianobactérias/metabolismo , Transferases Intramoleculares , Isomerases/metabolismo , Cianobactérias/enzimologia , Isomerases/efeitos dos fármacos , Ácidos Cetoglutáricos/farmacologia , Oxaloacetatos/farmacologia , Piruvatos/farmacologia , Ácido Pirúvico , Estereoisomerismo
17.
Biochem J ; 284 ( Pt 1): 109-13, 1992 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-1599391

RESUMO

It is shown that dopachrome (2-carboxy-2,3-dihydroindole-5,6-quinone) tautomerase (DCT) is a glycoprotein containing N-linked oligosaccharides. The enzymic activity can be stimulated by partial deglycosylation with a number of glycosylases such as neuraminidase, beta-mannosidase and beta-galactosidase. However, the stability of the enzyme after the hydrolytic treatment becomes lower. Thus total deglycosylation with peptide N-glycosidase F directly provokes an inactivation of DCT. The native enzyme also shows a strong affinity for concanavalin A-Sepharose. This affinity decreases after treatment with neuraminidase and/or beta-mannosidase. The DCT associated with coated vesicles seems to be mostly glycosylated, since the action of glycosylases on the enzyme obtained from these vesicles produced a similar stimulation to that with the melanosomal enzyme. Treatment of cultured melanocytes with tunicamycin elicited a decrease in the amount of active DCT inside the cells. All data suggest that the structure of the carbohydrate moiety of DCT should be very similar to, if not identical with, the structure proposed for tyrosinase by Ohkura, Yamashita, Mishima & Kobata (1984) Arch. Biochem. Biophys. 235, 63-77.


Assuntos
Glicosídeo Hidrolases/farmacologia , Oxirredutases Intramoleculares , Isomerases/efeitos dos fármacos , Animais , Sequência de Carboidratos , Carboidratos/fisiologia , Cromatografia de Afinidade , Estabilidade Enzimática , Feminino , Glicosídeo Hidrolases/metabolismo , Glicosídeo Hidrolases/fisiologia , Glicosilação , Isomerases/metabolismo , Cinética , Masculino , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Dados de Sequência Molecular , Sefarose/análogos & derivados , Células Tumorais Cultivadas
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