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1.
Mutat Res ; 686(1-2): 84-9, 2010 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-20138895

RESUMO

Previous work in our laboratory established that a spontaneous mutagenesis process operating in stationary-phase Escherichia coli cells undergoing selection is subject to regulation by the global regulatory mechanism known as catabolite repression (formerly also called glucose-repression). Here, we set out to determine the identity of this hitherto unknown catabolite-repressible spontaneous mutation generation mechanism(s). We used two different spontaneous mutation detection assays, reversion of a Lac(-) (lacI33OmegalacZ) frameshift marker and forward mutation to valine-resistance, and tested the effects of varying the nature of the carbon source(s) present in the selective plating medium on the mutability of bacterial cells carrying known defects in the recA, umuDC and dinB genes, three well-known SOS response genes, whose products are important for mutagenesis in E. coli. Consistent with the results of our previous Lac(-)-->Lac(+) assay using otherwise SOS-proficient bacterial cells, we found that the overall numbers of spontaneous Lac(+)E. coli revertants were highest when the selective medium contained lactose and lowest when it contained lactose plus the non-utilizable but strongly catabolite-repressing glucose analogue, methyl-alpha-d-glucopyranoside (alphaMG). In contrast, we found that the numbers of Lac(+) revertants appearing on the lactose and lactose+alphaMG selection plates were greatly diminished and not significantly different when the bacterial cells concerned carried either a DeltarecA or DeltadinB mutation. Furthermore, introducing the DeltadinB mutant allele into bacterial cells over-expressing the recA gene reduced the numbers of Lac(+) mutations to those being recovered with the DeltadinB cells. These results appear to suggest that (i) the DinB-dependent mutation generation pathway is alone responsible for spontaneous reversion of the lacI33OmegalacZ frameshift marker, and (ii) the varying numbers of Lac(+) colonies that we recover on the lactose and lactose+alphaMG plates provide a direct measure of the differential effects of these particular carbon compounds on the overall expression of the dinB gene. Interestingly, the yields of spontaneous Val mutations arising in wild-type, DeltarecA, DeltadinB and DeltaumuDC cells were found to be similar, but always tended to be highest when the medium contained only a non-repressing carbon source (glycerol) and lowest when it had been supplemented with a strong catabolite repressor such as glucose or alphaMG. Together, our results would seem to establish that stationary-phase E. coli cells exposed to strong selection pressures can accumulate spontaneous mutations via SOS-dependent and SOS-independent mutation generation pathways whose levels of expression are regulated by catabolite repression.


Assuntos
Proteínas de Bactérias/farmacologia , Escherichia coli/genética , Glucose/análogos & derivados , Mutagênese , Proteínas Repressoras/farmacologia , Resposta SOS em Genética , Lactose/antagonistas & inibidores , Lactose/farmacologia
2.
J Mol Biol ; 376(2): 466-81, 2008 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-18164724

RESUMO

We present here the results of a series of small-angle X-ray scattering studies aimed at understanding the role of conformational changes and structural flexibility in DNA binding and allosteric signaling in a bacterial transcription regulator, lactose repressor protein (LacI). Experiments were designed to detect possible conformational changes that occur when LacI binds either DNA or the inducer IPTG, or both. Our studies included the native LacI dimer of homodimers and a dimeric variant (R3), enabling us to probe conformational changes within the homodimers and distinguish them from those involving changes in the homodimer-homodimer relationships. The scattering data indicate that removal of operator DNA (oDNA) from R3 results in an unfolding and extension of the hinge helix that connects the LacI regulatory and DNA-binding domains. In contrast, only very subtle conformational changes occur in the R3 dimer-oDNA complex upon IPTG binding, indicative of small adjustments in the orientations of domains and/or subdomains within the structure. The binding of IPTG to native (tetrameric) LacI-oDNA complexes also appears to facilitate a modest change in the average homodimer-homodimer disposition. Notably, the crystal structure of the native LacI-oDNA complex differs significantly from the average solution conformation. The solution scattering data are best fit by an ensemble of structures that includes (1) approximately 60% of the V-shaped dimer of homodimers observed in the crystal structure and (2) approximately 40% of molecules with more "open" forms, such as those generated when the homodimers move with respect to each other about the tetramerization domain. In gene regulation, such a flexible LacI would be beneficial for the interaction of its two DNA-binding domains, positioned at the tips of the V, with the required two of three LacI operators needed for full repression.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Lactose/antagonistas & inibidores , Conformação Proteica , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Regulação Alostérica , Sítios de Ligação , DNA/metabolismo , Dimerização , Isopropiltiogalactosídeo/metabolismo , Ligantes , Modelos Biológicos , Modelos Moleculares , Peso Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Espalhamento a Baixo Ângulo , Termodinâmica
3.
PLoS Biol ; 5(10): e246, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17880262

RESUMO

Homology-directed repair is a powerful mechanism for maintaining and altering genomic structure. We asked how chromatin structure contributes to the use of homologous sequences as donors for repair using the chicken B cell line DT40 as a model. In DT40, immunoglobulin genes undergo regulated sequence diversification by gene conversion templated by pseudogene donors. We found that the immunoglobulin Vlambda pseudogene array is characterized by histone modifications associated with active chromatin. We directly demonstrated the importance of chromatin structure for gene conversion, using a regulatable experimental system in which the heterochromatin protein HP1 (Drosophila melanogaster Su[var]205), expressed as a fusion to Escherichia coli lactose repressor, is tethered to polymerized lactose operators integrated within the pseudo-Vlambda donor array. Tethered HP1 diminished histone acetylation within the pseudo-Vlambda array, and altered the outcome of Vlambda diversification, so that nontemplated mutations rather than templated mutations predominated. Thus, chromatin structure regulates homology-directed repair. These results suggest that histone modifications may contribute to maintaining genomic stability by preventing recombination between repetitive sequences.


Assuntos
Linfócitos B/citologia , Galinhas/fisiologia , Cromatina/metabolismo , Reparo do DNA , Conversão Gênica , Animais , Linfócitos B/metabolismo , Células Cultivadas , Cromatina/química , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Rearranjo Gênico do Linfócito B , Instabilidade Genômica , Histonas/genética , Histonas/metabolismo , Lactose/antagonistas & inibidores , Mutação , Moldes Genéticos
4.
Glycobiology ; 17(6): 663-76, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17339281

RESUMO

Galectin-8 has two different carbohydrate recognition domains (CRDs), the N-terminal Gal-8N and the C-terminal Gal-8C linked by a peptide, and has various effects on cell adhesion and signaling. To understand the mechanism for these effects further, we compared the binding activities of galectin-8 in solution with its binding and activation of cells. We used glycan array analysis to broaden the specificity profile of the two galectin-8 CRDs, as well as intact galectin-8s (short and long linker), confirming the unique preference for sulfated and sialylated glycans of Gal-8N. Using a fluorescence anisotropy assay, we examined the solution affinities for a subset of these glycans, the highest being 50 nM for NeuAcalpha2,3Lac by Gal-8N. Thus, carbohydrate-protein interactions can be of high affinity without requiring multivalency. More importantly, using fluorescence polarization, we also gained information on how the affinity is built by multiple weak interactions between different fragments of the glycan and its carrier molecule and the galectin CRD subsites (A-E). In intact galectin-8 proteins, the two domains act independently of each other in solution, whereas at a surface they act together. Ligands with moderate or weak affinity for the isolated CRDs on the array are bound strongly by intact galectin-8s. Also galectin-8 binding and signaling at cell surfaces can be explained by combined binding of the two CRDs to low or medium affinity ligands, and their highest affinity ligands, such as sialylated galactosides, are not required.


Assuntos
Membrana Celular/metabolismo , Galectinas/química , Galectinas/metabolismo , Membrana Celular/química , Membrana Celular/genética , Relação Dose-Resposta a Droga , Fluoresceína , Polarização de Fluorescência , Corantes Fluorescentes , Galactosídeos/química , Galactosídeos/metabolismo , Galectinas/genética , Galectinas/farmacologia , Humanos , Cinética , Lactose/antagonistas & inibidores , Ligantes , Modelos Químicos , Modelos Moleculares , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Soluções/química , Células U937 , Vibrio cholerae/metabolismo
5.
Mol Cell Biol ; 26(20): 7682-95, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17015481

RESUMO

Human artificial chromosomes (HACs) are promising reagents for the analysis of chromosome function. While HACs are maintained stably, the segregation mechanisms of HACs have not been investigated in detail. To analyze HACs in living cells, we integrated 256 copies of the Lac operator into a precursor yeast artificial chromosome (YAC) containing alpha-satellite DNA and generated green fluorescent protein (GFP)-tagged HACs in HT1080 cells expressing a GFP-Lac repressor fusion protein. Time-lapse analyses of GFP-HACs and host centromeres in living mitotic cells indicated that the HAC was properly aligned at the spindle midzone and that sister chromatids of the HAC separated with the same timing as host chromosomes and moved to the spindle poles with mobility similar to that of the host centromeres. These results indicate that a HAC composed of a multimer of input alpha-satellite YACs retains most of the functions of the centromeres on natural chromosomes. The only difference between the HAC and the host chromosome was that the HAC oscillated more frequently, at higher velocity, across the spindle midzone during metaphase. However, this provides important evidence that an individual HAC has the capacity to maintain tensional balance in the pole-to-pole direction, thereby stabilizing its position around the spindle midzone.


Assuntos
Anáfase/genética , Cromossomos Artificiais Humanos/genética , Metáfase/genética , Sítios de Ligação , Linhagem Celular Tumoral , Centrômero/genética , Humanos , Lactose/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Tempo
6.
Org Biomol Chem ; 1(2): 338-49, 2003 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-12929429

RESUMO

Dipyranones, such as 1,2-bis[(2R,3S,6S)-3-hydroxy-6-methoxy-3-oxo-6H-pyran-2-yl]ethane, were exploited as templates for the synthesis of some novel C-linked disaccharide analogues. Efficient methods, such as stereoselective reduction and dihydroxylation, were developed for two-directional functionalisation of these templates. Peracetylated derivatives of ten stereoisomeric disaccharide analogues [acetic acid 4,5-diacetoxy-6-methoxy-[(3',4',5'-triacetoxy-6'-methoxytetrahydropyran- 2'-yl)ethyl]tetrahydropyran-3-yl esters] were synthesised from a virtual library of 136 compounds; furthermore, an additional eight stereoisomers could have been synthesised simply by using the enantiomeric ligand in the enantioselective step. The ability of (2S,3S,4R,5R,6R)-6-methoxy-2-[2'-((2'R,3'R,4'S, 5'R,6'S)-3',4',5'-trihydroxy-6'-methoxytetrahydropyran-2'-yl) ethyl]tetrahydropyran-3,4,5-triol to bind to the repressor protein, LacI, was estimated to be similar to that of isopropyl-beta-thiogalactoside. The disaccharide mimetics were concluded to be a new and interesting class of C-linked disaccharide mimetics with promising, though largely unstudied, biological activity.


Assuntos
Materiais Biomiméticos/síntese química , Técnicas de Química Combinatória , Dissacarídeos/síntese química , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Materiais Biomiméticos/química , Materiais Biomiméticos/metabolismo , Materiais Biomiméticos/farmacologia , Configuração de Carboidratos , Dissacarídeos/química , Dissacarídeos/metabolismo , Dissacarídeos/farmacologia , Hidroxilação , Repressores Lac , Lactose/análogos & derivados , Lactose/antagonistas & inibidores , Espectroscopia de Ressonância Magnética , Oxirredução , Piranos/química , Proteínas Repressoras/efeitos dos fármacos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Estereoisomerismo , Tiogalactosídeos/química , Tiogalactosídeos/metabolismo
7.
Biochemistry ; 42(7): 2202-17, 2003 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-12590610

RESUMO

Thermodynamic and structural evidence indicates that the DNA binding domains of lac repressor (lacI) exhibit significant conformational adaptability in operator binding, and that the marginally stable helix-turn-helix (HTH) recognition element is greatly stabilized by operator binding. Here we use circular dichroism at 222 nm to quantify the thermodynamics of the urea- and thermally induced unfolding of the marginally stable lacI HTH. Van't Hoff analysis of the two-state unfolding data, highly accurate because of the large transition breadth and experimental access to the temperature of maximum stability (T(S); 6-10 degrees C), yields standard-state thermodynamic functions (deltaG(o)(obs), deltaH(o)(obs), deltaS(o)(obs), deltaC(o)(P,obs)) over the temperature range 4-40 degrees C and urea concentration range 0

Assuntos
Proteínas de Bactérias , Proteínas de Ligação a DNA/química , Proteínas de Escherichia coli/química , Modelos Químicos , Dobramento de Proteína , Proteínas Repressoras/química , Termodinâmica , Ureia , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Entropia , Proteínas de Escherichia coli/metabolismo , Sequências Hélice-Volta-Hélice , Repressores Lac , Lactose/antagonistas & inibidores , Modelos Biológicos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas Repressoras/metabolismo , Temperatura , Ureia/química
8.
Biophys J ; 82(5): 2373-82, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-11964227

RESUMO

Upon gamma-ray or argon ion irradiation of the lac repressor protein, its peptide chain is cleaved and the protein loses its lac operator-binding activity, as shown respectively by polyacrylamide gel electrophoresis and retardation gel electrophoresis. We developed phenomenological models that satisfactorily account for the experimental results: the peptide chain cleavage model considers that the average number of chain breaks per protomer is proportional to the irradiation dose and that the distribution of the number of breaks per protomer obeys Poisson's law. The repressor inactivation model takes into account the quaternary structure (a dimer of dimer) and the organization of the repressor in domains (two DNA binding sites, one per dimer). A protomer is inactivated by at least two different radiation-induced damages. A dimer is inactivated when at least one of the two protomers is inactivated. A tetramer is inactivated when both dimers are inactivated. From the combination of both models, we can deduce that chain cleavage cannot account for the protein inactivation, which should mainly result from oxidation of amino acid side chains. Indeed, particularly oxidizable and accessible amino acids (Tyr, His) are involved in the DNA binding process.


Assuntos
Argônio , Proteínas de Bactérias/efeitos da radiação , Proteínas de Escherichia coli , Raios gama , Lactose/antagonistas & inibidores , Proteínas Repressoras/efeitos da radiação , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Dimerização , Escherichia coli/fisiologia , Escherichia coli/efeitos da radiação , Repressores Lac , Substâncias Macromoleculares , Modelos Biológicos , Modelos Moleculares , Peptídeos/química , Subunidades Proteicas , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/química
9.
Protein Sci ; 11(4): 778-94, 2002 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11910022

RESUMO

LacI and PurR are highly homologous proteins. Their functional units are homodimers, with an N-terminal DNA binding domain that comprises the helix-turn-helix (HTH), N-linker, and hinge regions from both monomers. Hinge structural changes are known to occur upon DNA dissociation but are difficult to monitor experimentally. The initial steps of hinge unfolding were therefore examined using molecular dynamics simulations, utilizing a truncated, chimeric protein comprising the LacI HTH/N-linker and PurR hinge. A terminal Gly-Cys-Gly was added to allow "dimerization" through disulfide bond formation. Simulations indicate that differences in LacI and PurR hinge primary sequence affect the quaternary structure of the hinge x hinge' interface. However, these alternate hinge orientations would be sterically restricted by the core domain. These results prompted detailed comparison of recently available DNA-bound structures for LacI and truncated LacI(1-62) with the PurR structure. Examination revealed that different N-linker and hinge contacts to the core domain of the partner monomer (which binds effector molecule) affect the juxtapositions of the HTH, N-linker, and hinge regions in the DNA binding domain. In addition, the two full-length repressors exhibit significant differences in the interactions between the core and the C-linker connection to the DNA binding domain. Both linkers and the hinge have been implicated in the allosteric response of these repressors. Intriguingly, one functional difference between these two proteins is that they exhibit opposite allosteric response to effector. Simulations and observed structural distinctions are correlated with mutational analysis and sequence information from the LacI/GalR family to formulate a mechanism for fine-tuning individual repressor function.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , DNA/metabolismo , Proteínas de Escherichia coli , Estrutura Terciária de Proteína/fisiologia , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Sítios de Ligação , Simulação por Computador , DNA/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Ligação de Hidrogênio , Repressores Lac , Lactose/antagonistas & inibidores , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Estrutura Secundária de Proteína
10.
Genome Res ; 11(5): 863-74, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-11337480

RESUMO

Many missense substitutions are identified in single nucleotide polymorphism (SNP) data and large-scale random mutagenesis projects. Each amino acid substitution potentially affects protein function. We have constructed a tool that uses sequence homology to predict whether a substitution affects protein function. SIFT, which sorts intolerant from tolerant substitutions, classifies substitutions as tolerated or deleterious. A higher proportion of substitutions predicted to be deleterious by SIFT gives an affected phenotype than substitutions predicted to be deleterious by substitution scoring matrices in three test cases. Using SIFT before mutagenesis studies could reduce the number of functional assays required and yield a higher proportion of affected phenotypes. may be used to identify plausible disease candidates among the SNPs that cause missense substitutions.


Assuntos
Substituição de Aminoácidos/genética , Biologia Computacional/métodos , Proteínas de Escherichia coli , Sequência de Aminoácidos , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriófago T4/enzimologia , Bacteriófago T4/genética , Sequência Conservada , Doenças Genéticas Inatas/genética , Protease de HIV/genética , HIV-1/enzimologia , HIV-1/genética , Humanos , Repressores Lac , Lactose/antagonistas & inibidores , Dados de Sequência Molecular , Muramidase , Mutação de Sentido Incorreto/genética , Fenótipo , Probabilidade , Proteínas Repressoras/classificação , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Alinhamento de Sequência , Software
11.
Chembiochem ; 2(11): 822-30, 2001 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-11948868

RESUMO

Galectins are mammalian carbohydrate-binding proteins that are involved in cell-cell and cell-matrix adhesion, cell migration, and growth regulation with relevance to inflammation and tumor spread. These important functions account for the interest to design suitable low molecular weight inhibitors that match the distinct modes of presentation of the carbohydrate recognition domains of the different galectin subfamilies. Using 3,5-di-(2-aminoethoxy)benzoic acid as the branching unit, wedgelike glycodendrimers with two, four, and eight lactose moieties (G1-G3) were synthesized. They were tested in solid-phase competition assays with lactose maxiclusters and various N-glycan branching profiles (miniclusters) as the matrix and also in cell assays. Prototype galectins-1 and -7, chimera-type galectin-3, a plant (AB)(2) toxin, and a lactose-binding immunoglobulin G fraction from human serum were the carbohydrate-binding targets. Potent inhibition and remarkable cluster effects were seen for the homodimeric galectin-1, especially in combination with biantennary N-glycans as the matrix. Remarkably, for the tetravalent G2 glycodendrimer, the inhibitory potency of each lactose unit reached a maximum value of 1667 relative to free lactose. In haemagglutination experiments as a model for cell adhesion, galectin-3 was markedly sensitive to increased sugar valency and a relative potency per lactose of 150 was reached. The spatial orientation of the carbohydrate recognition domains of the endogenous lectins and the branching pattern of the carbohydrates of the glycoprotein matrices used are both important factors in the design and synthesis of glycodendrimers with galectin-selective properties.


Assuntos
Biopolímeros/química , Biopolímeros/farmacologia , Glicoconjugados/antagonistas & inibidores , Glicoproteínas/antagonistas & inibidores , Hemaglutininas/metabolismo , Lactose/antagonistas & inibidores , Glicoproteínas de Membrana/antagonistas & inibidores , Animais , Biopolímeros/metabolismo , Configuração de Carboidratos , Bovinos , Moléculas de Adesão Celular/antagonistas & inibidores , Moléculas de Adesão Celular/metabolismo , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Galectina 1 , Glicoconjugados/metabolismo , Glicoproteínas/metabolismo , Hemaglutinação/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Lactose/metabolismo , Lectinas/antagonistas & inibidores , Lectinas/metabolismo , Espectroscopia de Ressonância Magnética , Glicoproteínas de Membrana/metabolismo , Camundongos , Ligação Proteica/efeitos dos fármacos , Coelhos
12.
Biochemistry ; 39(36): 11074-83, 2000 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-10998245

RESUMO

The mechanism by which genetic regulatory proteins discern specific target DNA sequences remains a major area of inquiry. To explore in more detail the interplay between DNA and protein sequence, we have examined binding of variant lac operator DNA sequences to a series of mutant lactose repressor proteins (LacI). These proteins were altered in the C-terminus of the hinge region that links the N-terminal DNA binding and core sugar binding domains. Variant operators differed from the wild-type operator, O(1), in spacing and/or symmetry of the half-sites that contact the LacI N-terminal DNA binding domain. Binding of wild-type and mutant proteins was affected differentially by variations in operator sequence and symmetry. While the mutant series exhibits a 10(4)-fold range in binding affinity for O(1) operator, only a approximately 20-fold difference in affinity is observed for a completely symmetric operator, O(sym), used widely in studies of the LacI protein. Further, DNA sequence influenced allosteric response for these proteins. Binding of this LacI mutant series to other variant operator DNA sequences indicated the importance of symmetry-related bases, spacing, and the central base pair sequence in high affinity complex formation. Conformational flexibility in the DNA and other aspects of the structure influenced by the sequence may establish the binding environment for protein and determine both affinity and potential for allostery.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Variação Genética/genética , Mutagênese Sítio-Dirigida , Regiões Operadoras Genéticas/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas de Bactérias/química , Pareamento de Bases/genética , DNA Bacteriano/metabolismo , Escherichia coli/genética , Glicina/genética , Glicina/metabolismo , Repressores Lac , Lactose/antagonistas & inibidores , Ligação Proteica/genética , Estrutura Secundária de Proteína/genética , Proteínas Repressoras/química , Análise de Sequência de DNA , Temperatura
13.
Biochemistry ; 38(21): 6715-22, 1999 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-10346891

RESUMO

Phosphorescence and optically detected magnetic resonance (ODMR) measurements are reported on four single-tryptophan mutants of lac repressor protein from Escherichia coli: H74W/Wless, W201Y, Y273W/Wless, and F293W/Wless, where Wless represents a protein background containing the double mutation W201Y/W220Y. The single-tryptophan residues are located in the protein core region, either in the monomer-monomer interface of the tetrameric protein or in the region of the inducer binding cleft. Inducer binding elicits large changes in the energy (0,0-band wavelength shifts) and zero-field splitting energies (ZFS) of the triplet states for each of the mutant proteins except W201Y which exhibits more modest effects. F293W/Wless exists in two distinguishable conformations, only one of which appears to be sensitive to the presence of inducer. These effects of inducer binding can be attributed to a conformational change that alters specific polar interactions that occur at each affected tryptophan site. Changes in the tryptophan triplet state indicator depend on the existence of specific polar interactions that are altered by local atomic relocations.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Escherichia coli , Mutagênese Sítio-Dirigida , Proteínas Repressoras/química , Proteínas Repressoras/genética , Triptofano/genética , Substituição de Aminoácidos/genética , Escherichia coli/química , Escherichia coli/genética , Histidina/genética , Isopropiltiogalactosídeo/química , Repressores Lac , Lactose/antagonistas & inibidores , Ligantes , Medições Luminescentes , Substâncias Macromoleculares , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Fenilalanina/genética , Conformação Proteica , Tirosina/genética
14.
Mikrobiologiia ; 46(3): 428-32, 1977.
Artigo em Russo | MEDLINE | ID: mdl-19684

RESUMO

The induction of cellulase synthesis by lactose was studied in the resting cells of Trichoderma lignorum OM 534. The effect depended on the concentration of lactose, pH, and the age of the mycelium. The induction of the enzyme synthesis by lactose is supressed by glucose and its metabolites. The repression by glucose is partly eliminated by Cyk 3'-5'-AMP, theophylline, and coffeine. The induction of cellulase by lactose is regarded as a derepression of the synthesis of this enzyme as a result of slow assimilation of the disaccharide. The synthesis of cellulase in T. lignorum is presumed to be constitutive.


Assuntos
Celulase/biossíntese , Lactose/farmacologia , Fungos Mitospóricos/enzimologia , Trichoderma/enzimologia , Cafeína/farmacologia , AMP Cíclico/farmacologia , Indução Enzimática , Glucose/farmacologia , Concentração de Íons de Hidrogênio , Lactose/antagonistas & inibidores , Teofilina/farmacologia , Trichoderma/crescimento & desenvolvimento
15.
Am J Obstet Gynecol ; 126(7): 920-30, 1976 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-998678

RESUMO

Comparative development of rough endoplasmic reticulum (RER) in mammary glands of rats was studied in relation to levels of progesterone and adrenocorticosteroids. The tranquilizer perphenazine was administered to stimulate secretion of prolactin; serum glucocorticoids also increased. When perphenazine was administered after ovulation, serum progesterone was increased 18-fold, but when treatment was begun on proestrus, ovulation was inhibited and serum progesterone increased only 4-fold. Concentrations of progesterone in the breast were approximately 10-fold greater than, but parallel to, serum concentrations. In ovulating rats the RER was poorly developed. In nonovulating rats extensive development of RER occurred. Cortisol acetate given to ovulating, perphenazine-treated rats did not alter levels of serum progesterone, but tissue progesterone was reduced by 50 per cent and development of RER approached that in nonovulating rats. The development of secretory capacity (formation of RER) in the breast appears to depend on the relation between stimulation by glucocorticoid and inhibition by progesterone.


Assuntos
Corticosteroides/fisiologia , Retículo Endoplasmático/ultraestrutura , Glândulas Mamárias Animais/ultraestrutura , Progesterona/fisiologia , Animais , Feminino , Lactose/antagonistas & inibidores , Glândulas Mamárias Animais/metabolismo , Ovulação , Perfenazina/farmacologia , Gravidez , Progesterona/sangue , Ratos
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