RESUMO
The matrix pencil method (MPM) is explored for stable, reproducible data processing in nuclear magnetic resonance (NMR) relaxometry. Data from one-dimensional and two-dimensional relaxometry experiments designed to measure transverse relaxation T2, longitudinal relaxation T1, diffusion coefficient D values, and their correlations in a standard olive oil/water mixture serve as a platform available to any NMR spectroscopist to compare the performance of the MPM to the benchmark inverse Laplace transform (ILT). The data from two practical examples, including the drying of a solvent polymer system and the enzymatic digestion of polysialic acid, were also explored with the MPM and ILT. In the cases considered here, the MPM appears to outperform the ILT in terms of resolution and stability in the determination of fundamental constants for complex materials and mixtures.
RESUMO
Cytosine-5 DNA methylation occurs in the context of CpG dinucleotides in vertebrates. Aberrant methylation of CpG islands in human tumors has been shown to cause transcriptional silencing of tumor-suppressor genes. Most methods used to analyze cytosine-5 methylation patterns require cumbersome manual techniques that employ gel electrophoresis, restriction enzyme digestion, radiolabeled dNTPs or hybridization probes. The development of high-throughput technology for the analysis of DNA methylation would significantly expand our ability to derive molecular information from clinical specimens. This study describes a high-throughput quantitative methylation assay that utilizes fluorescence-based real-time PCR (TaqMan) technology that requires no further manipulations after the PCR step. MethyLight is a highly sensitive assay, capable of detecting methylated alleles in the presence of a 10,000-fold excess of unmethylated alleles. The assay is also highly quantitative and can very accurately determine the relative prevalence of a particular pattern of DNA methylation. We show that MethyLight can distinguish between mono-allelic and bi-allelic methylation of the MLH1 mismatch repair gene in human colorectal tumor specimens. The development of this technique should considerably enhance our ability to rapidly and accurately generate epigenetic profiles of tumor samples.
Assuntos
Adenocarcinoma/genética , Neoplasias Colorretais/genética , Metilação de DNA , Proteínas de Neoplasias/genética , Reação em Cadeia da Polimerase , Proteínas Adaptadoras de Transdução de Sinal , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Proteínas de Transporte , Ilhas de CpG , Reparo do DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL , Proteínas Nucleares , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , SulfitosRESUMO
BACKGROUND: The adenomatous polyposis coli (APC) locus on chromosome 5q21-22 shows frequent loss of heterozygosity (LOH) in esophageal carcinomas. However, the prevalence of truncating mutations in the APC gene in esophageal carcinomas is low. Because hypermethylation of promoter regions is known to affect several other tumor suppressor genes, we investigated whether the APC promoter region is hypermethylated in esophageal cancer patients and whether this abnormality could serve as a prognostic plasma biomarker. METHODS: We assayed DNA from tumor tissue and matched plasma from esophageal cancer patients for hypermethylation of the promoter region of the APC gene. We used the maximal chi-square statistic to identify a discriminatory cutoff value for hypermethylated APC DNA levels in plasma and used bootstrap-like simulations to determine the P: value to test for the strength of this association. This cutoff value was used to generate Kaplan-Meier survival curves. All P values were based on two-sided tests. RESULTS: Hypermethylation of the promoter region of the APC gene occurred in abnormal esophageal tissue in 48 (92%) of 52 patients with esophageal adenocarcinoma, in 16 (50%) of 32 patients with esophageal squamous cell carcinoma, and in 17 (39.5%) of 43 patients with Barrett's metaplasia but not in matching normal esophageal tissues. Hypermethylated APC DNA was observed in the plasma of 13 (25%) of 52 adenocarcinoma patients and in two (6.3%) of 32 squamous carcinoma patients. High plasma levels of methylated APC DNA were statistically significantly associated with reduced patient survival (P =.016). CONCLUSION: The APC promoter region was hypermethylated in tumors of the majority of patients with primary esophageal adenocarcinomas. Levels of hypermethylated APC gene DNA in the plasma may be a useful biomarker of biologically aggressive disease in esophageal adenocarcinoma patients and should be evaluated as a potential biomarker in additional tumor types.
Assuntos
Adenocarcinoma/metabolismo , Polipose Adenomatosa do Colo/genética , Biomarcadores Tumorais/sangue , Cromossomos Humanos Par 5/genética , DNA de Neoplasias/sangue , Neoplasias Esofágicas/metabolismo , Adenocarcinoma/genética , Esôfago de Barrett/metabolismo , Biomarcadores Tumorais/isolamento & purificação , Carcinoma de Células Escamosas/metabolismo , Distribuição de Qui-Quadrado , DNA de Neoplasias/isolamento & purificação , Neoplasias Esofágicas/genética , Mucosa Gástrica/metabolismo , Humanos , Perda de Heterozigosidade , Metilação , Reação em Cadeia da Polimerase/métodos , Lesões Pré-Cancerosas/metabolismo , Prognóstico , Regiões Promotoras Genéticas , Análise de SobrevidaRESUMO
The molecular basis of aberrant hypermethylation of CpG islands observed in a subset of human colorectal tumors is unknown. One potential mechanism is the up-regulation of DNA (cytosine-5)-methyltransferases. Recently, two new mammalian DNA methyltransferase genes have been identified, which are referred to as DNMT3A and DNMT3B. The encoded proteins differ from the predominant mammalian DNA methyltransferase DNMT1 in that they have a substantially higher ratio of de novo to maintenance methyltransferase activity. We have used a highly quantitative 5' nuclease fluorogenic reverse transcription-PCR method (TaqMan) to analyze the expression of all three DNA methyltransferase genes in 25 individual colorectal adenocarcinoma specimens and matched normal mucosa samples. In addition, we examined the methylation patterns of four CpG islands [APC, ESR1 (estrogen receptor), CDKN2A (p16), and MLH1] to determine whether individual tumors show a positive correlation between the level of DNA methyltransferase expression and the frequency of CpG island hypermethylation. All three methyltransferases appear to be up-regulated in tumors when RNA levels are normalized using either ACTB (beta-actin) or POLR2A (RNA pol II large subunit), but not when RNA levels are normalized with proliferation-associated genes, such as H4F2 (histone H4) or PCNA. The frequency or extent of CpG island hypermethylation in individual tumors did not correlate with the expression of any of the three DNA methyltransferases. Our results suggest that deregulation of DNA methyltransferase gene expression does not play a role in establishing tumor-specific abnormal DNA methylation patterns in human colorectal cancer.
Assuntos
Adenocarcinoma/genética , Neoplasias Colorretais/genética , Ilhas de CpG , DNA (Citosina-5-)-Metiltransferases/biossíntese , Metilação de DNA , DNA de Neoplasias/química , Proteínas de Neoplasias/biossíntese , Adenocarcinoma/enzimologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/enzimologia , DNA (Citosina-5-)-Metiltransferases/genética , Indução Enzimática , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Mucosa Intestinal/enzimologia , Isoenzimas/biossíntese , Isoenzimas/genética , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Esophageal adenocarcinoma (EAC) is thought to develop through a multistage process in which Barrett's metaplasia progresses through low- and high-grade dysplasia to invasive cancer. Transcriptional silencing of tumor suppressor genes by promoter CpG island hypermethylation has been observed in many types of human cancer. Analysis of CpG island hypermethylation in EAC has thus far been limited to the CDKN2A (p16) gene. In this study, we extend the methylation analysis of EAC to include three other genes, APC, CDH1 (E-cadherin), and ESR1 (ER, estrogen receptor alpha), in addition to CDKN2A. Molecular analysis can provide insight into the complex relationships between tissues with different histologies in Barrett's esophagus and associated adenocarcinoma. Therefore, we have mapped the spatial distribution of methylation patterns in six esophagectomy cases in detail. Hypermethylation of the four CpG islands was analyzed by the MethyLight technique in 107 biopsies derived from these six patients for a total of 428 methylation analyses. Our results show that normal esophageal squamous epithelium is unmethylated at all four CpG islands. CDH1 is unmethylated in most other tissue types as well. Hypermethylation of ESR1 is seen at high frequency in inflammatory reflux esophagitis and at all subsequent stages, whereas APC and CDKN2A hypermethylation is found in Barrett's metaplasia, dysplasia, and EAC. When it occurs, hypermethylation of APC, CDKN2A, and ESR1 is usually found in a large contiguous field, suggesting either a concerted methylation change associated with metaplasia or a clonal expansion of cells with abnormal hypermethylation.
Assuntos
Adenocarcinoma/genética , Esôfago de Barrett/genética , Ilhas de CpG/genética , Metilação de DNA , Neoplasias Esofágicas/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Idoso , Idoso de 80 Anos ou mais , Esôfago de Barrett/metabolismo , Esôfago de Barrett/patologia , Biópsia , Caderinas/genética , DNA/genética , DNA/metabolismo , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Receptor alfa de Estrogênio , Feminino , Genes APC/genética , Genes p16/genética , Humanos , Masculino , Pessoa de Meia-Idade , Receptores de Estrogênio/genéticaRESUMO
Esophageal adenocarcinoma (EAC) arises after normal squamous mucosa undergoes metaplasia to specialized columnar epithelium (intestinal metaplasia or Barrett's esophagus), which can then ultimately progress to dysplasia and subsequent malignancy. Epigenetic studies of this model have thus far been limited to the DNA methylation analysis of a few genes. In this study, we analyzed a panel of 20 genes using a quantitative, high-throughput methylation assay, METHYLIGHT: We used this broader approach to gain insight into concordant methylation behavior between genes and to generate epigenomic fingerprints for the different histological stages of EAC. Our study included a total of 104 tissue specimens from 51 patients with different stages of Barrett's esophagus and/or associated adenocarcinoma. We screened 84 of these samples with the full panel of 20 genes and found distinct classes of methylation patterns in the different types of tissue. The most informative genes were those with an intermediate frequency of significant hypermethylation [ranging from 15% (CDKN2A) to 60% (MGMT) of the samples]. This group could be further subdivided into three classes, according to the absence (CDKN2A, ESR1, and MYOD1) or presence (CALCA, MGMT, and TIMP3) of methylation in normal esophageal mucosa and stomach, or the infrequent methylation of normal esophageal mucosa accompanied by methylation in all normal stomach samples (APC). The other genes were less informative, because the frequency of hypermethylation was below 5% (ARF, CDH1, CDKN2B, GSTP1, MLH1, PTGS2, and THBS1), completely absent (CTNNB1, RB1, TGFBR2, and TYMS1), or ubiquitous (HIC1 and MTHFR), regardless of tissue type. Each class undergoes unique epigenetic changes at different steps of disease progression of EAC, suggesting a step-wise loss of multiple protective barriers against CpG island hypermethylation. The aberrant hypermethylation occurs at many different loci in the same tissues, suggestive of an overall deregulation of methylation control in EAC tumorigenesis. However, we did not find evidence for a distinct group of tumors with a CpG island methylator phenotype. Finally, we found that normal and metaplastic tissues from patients with evidence of associated dysplasia or cancer had a significantly higher incidence of hypermethylation than similar tissues from patients with no further progression of their disease. The fact that the samples from these two groups of patients were histologically indistinguishable, yet molecularly distinct, suggests that the occurrence of such hypermethylation may provide a clinical tool to identify patients with premalignant Barrett's who are at risk for further progression.
Assuntos
Adenocarcinoma/genética , Metilação de DNA , Neoplasias Esofágicas/genética , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Ilhas de CpG/genética , Progressão da Doença , Neoplasias Esofágicas/patologia , Perfilação da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Lesões Pré-Cancerosas/genéticaRESUMO
Structural perturbations due to a series of mutations at the 30-51 disulfide bond of bovine pancreatic trypsin inhibitor have been explored using NMR. The mutants replaced cysteines at positions 30 and 51 by alanine at position 51 and alanine, threonine, or valine at position 30. Chemical shift changes occur in residues proximate to the site of mutation. NOE assignments were made using an automated procedure, NASIGN, which used information from the wild-type crystal structure. Intensity information was utilized by a distance geometry algorithm, VEMBED, to generate a series of structures for each protein. Statistical analyses of these structures indicated larger averaged structural perturbations than would be expected from crystallographic and other information. Constrained molecular dynamics refinement using AMBER at 900 K was useful in eliminating structural movements that were not a necessary consequence of the NMR data. In most cases, statistically significant movements are shown to be those greater than approximately 1 A. Such movements do not appear to occur between wild type and A30A51, a result confirmed by crystallography (Eigenbrot, C., Randal, M., & Kossiakoff, A.A., 1990, Protein Eng. 3, 591-598). Structural alterations in the T30A51 or V30A51 mutant proteins near the limits of detection occur in the beta-loop (residues 25-28) or C-terminal alpha-helix, respectively.
Assuntos
Aprotinina/química , Aprotinina/genética , Sequência de Aminoácidos , Animais , Bovinos , Cisteína/química , Cisteína/genética , Dissulfetos/química , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Soluções/químicaRESUMO
PURPOSE: To identify a dose of the demethylating agent 5-aza-2'-deoxycytidine (DAC) with acceptable side effects, and to study its effect on the methylation patterns of relevant genes in tumor biopsies before and after treatment with a novel methylation assay using real-time PCR. METHODS: A group of 19 patients with metastatic solid tumors were treated with DAC by continuous intravenous infusion over 72 h, days 1-3 of a 28-day cycle. Tumor biopsies were taken before and 7 days after starting DAC. RESULTS: The dose levels studied were 20, 30 and 40 mg/m(2). Grade 4 neutropenia was found in two of five patients at 40 mg/m(2) and one of six patients at 30 mg/m(2). No objective responses were seen in this study. Steady-state DAC levels of 0.1 to 0.2 microM were achieved in the 30 and 40 mg/m(2) cohorts. Changes in methylation were observed, but no single gene consistently demonstrated evidence of demethylation. CONCLUSIONS: DAC was tolerated at a dose of 30 mg/m(2) per day for a 72-h intravenous infusion. Changes in gene methylation were observed.
Assuntos
Antimetabólitos Antineoplásicos/efeitos adversos , Azacitidina/análogos & derivados , Azacitidina/efeitos adversos , Metilação de DNA/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Adulto , Idoso , Antimetabólitos Antineoplásicos/administração & dosagem , Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/administração & dosagem , Azacitidina/farmacologia , Biópsia , DNA de Neoplasias , Decitabina , Feminino , Humanos , Infusões Intravenosas , Masculino , Pessoa de Meia-Idade , Neoplasias/genética , Reação em Cadeia da PolimeraseRESUMO
Large-scale medical sequencing provides a focal point around which to reorganize health care and health care research. Mobile health (mHealth) is also currently undergoing explosive growth and could be another innovation that will change the face of future health care. We are employing primary ovarian insufficiency (POI) as a model rare condition to explore the intersection of these potentials. As both sequencing capabilities and our ability to intepret this information improve, sequencing for medical purposes will play an increasing role in health care beyond basic research: it will help guide the delivery of care to patients. POI is a serious chronic disorder and syndrome characterized by hypergonadotrophic hypogonadism before the age of 40 years and most commonly presents with amenorrhea. It may have adverse health effects that become fully evident years after the initial diagnosis. The condition is most commonly viewed as one of infertility, however, it may also be associated with adverse long-term outcomes related to inadequate bone mineral density, increased risk of cardiovascular disease, adrenal insufficiency, hypothyroidism and, if pregnancy ensues, having a child with Fragile X Syndrome. There may also be adverse outcomes related to increased rates of anxiety and depression. POI is also a rare disease, and accordingly, presents special challenges. Too often advances in research are not effectively integrated into community care at the point of service for those with rare diseases. There is a need to connect community health providers in real time with investigators who have the requisite knowledge and expertise to help manage the rare disease and to conduct ongoing research. Here we review the pathophysiology and management of POI and propose the development of an international Clinical Research Integration Special Program (CRISP) for the condition.
Assuntos
Pesquisa Biomédica/organização & administração , Insuficiência Ovariana Primária/terapia , Adulto , Suscetibilidade a Doenças/imunologia , Feminino , Predisposição Genética para Doença , Humanos , Gravidez , Insuficiência Ovariana Primária/etiologia , Insuficiência Ovariana Primária/fisiopatologia , Desenvolvimento de ProgramasRESUMO
The interaction of Mn2+ with the substrate glutamate and several transition state analog inhibitors of glutamine synthetase has been studied. With Mn2+ bound to the tight binding site, the frequency and temperature dependence of the paramagnetic contribution to solvent water proton relaxation rates demonstrate changes in the structure of the metal ion environment induced by substrate or inhibitor binding. The water proton relaxation rate data also show differences in the metal ion environment in the presence of glutamate compared to methionine sulfoximine, a structural analog of an intermediate in the reaction mechanism. Additionally, the distance between the metal ion and the phosphorus atom of an inhibitor, 2-amino-4-phosphonobutyric acid, was estimated (approximately 5 A) using NMR measurements. These data are in accord with our recent hypothesis that the role of the metal ion is to stabilize the tetrahedral adduct formed on the reaction pathway.
Assuntos
Glutamato-Amônia Ligase/metabolismo , Sítios de Ligação , Glutamato-Amônia Ligase/antagonistas & inibidores , Glutamatos/metabolismo , Cinética , Espectroscopia de Ressonância Magnética , Manganês/metabolismo , Temperatura , ÁguaRESUMO
Thirty minutes following an intragastric dose of [59]Fe, rats subjected to short-term and long-term iron depletion showed a similar increase in [59]Fe in plasma and a similar decrease in the retention of [59]Fe in mucosal cytosol compared to controls. With both low-iron groups, a two-fold increase in [59]Fe uptake by brush-border membrane vesicles and a six-fold reduction in the [59]Fe incorporated into the ferritin of the mucosal cytosol were observed. These studies indicate that short-term exposure to a low-iron diet triggers changes in both the uptake of iron by the brush-border membrane and the processing of iron within the mucosal cell prior to major changes in body iron status.
Assuntos
Mucosa Intestinal/metabolismo , Ferro/metabolismo , Microvilosidades/metabolismo , Animais , Membrana Celular/metabolismo , Citosol/metabolismo , Ferritinas/metabolismo , Ferro/sangue , Deficiências de Ferro , Cinética , Fígado/metabolismo , Masculino , Ratos , Ratos Endogâmicos F344 , Transferrina/metabolismo , Vacúolos/metabolismoRESUMO
Uptake, transport, and metabolism of tripeptide thyrotropin-releasing hormone were examined in the human intestinal epithelial cell line, Caco-2. A linear relationship between rate and concentration was observed for both the uptake and the transport of thyrotropin-releasing hormone across Caco-2 cell monolayers. Transport of thyrotropin-releasing hormone was not affected by the presence of dipeptide glycylsarcosine, amino acid glycine, tripeptide thyrotropin-releasing hormone free acid as well as active transport inhibitors 2,4-dinitrophenol, sodium azide, ouabain, and amiloride. There was no formation of metabolites during the course of thyrotropin-releasing hormone transport across Caco-2 cells. Incubation of Caco-2 cell homogenate with thyrotropin-releasing hormone, however, showed a time-dependent hydrolysis of thyrotropin-releasing hormone and the formation of thyrotropin-releasing hormone free acid. Increased rate of transport in the presence of EDTA indicates a paracellular passive diffusion as the major route for the transport of TRH. The hydrolytic enzyme present in Caco-2 cells appeared to have little or no access to TRH during the transcellular transport across Caco-2 cell monolayers.
Assuntos
Hormônio Liberador de Tireotropina/metabolismo , 2,4-Dinitrofenol , Adenocarcinoma , Amilorida/farmacologia , Azidas/farmacologia , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Neoplasias do Colo , Dinitrofenóis/farmacologia , Dipeptídeos/farmacologia , Ácido Edético/farmacologia , Epitélio/metabolismo , Glicina/farmacologia , Humanos , Intestinos , Cinética , Ouabaína/farmacologia , Azida Sódica , Trítio , Células Tumorais CultivadasRESUMO
The transfer of control rats to a low-iron diet for only 24 h resulted in a 2-fold increase in iron uptake by brush-border membrane vesicles. Extension of the low-iron feeding period to 72 h or 2 weeks resulted in only small additional increases in iron uptake by vesicle preparations. In contrast, the transfer of iron-deficient rats to a control diet resulted in a progressive decrease in iron uptake by vesicles that reached a level equivalent to that of control rats in 2 weeks. 59Fe labelling of detergent extracts of these vesicle preparations provided evidence for the presence of an iron-binding protein composed of subunits of 52,000 Da. The changes in the 59Fe labelling of this protein component were consistent with the changes observed in iron uptake by intact brush-border membrane vesicles. The 59Fe-labelling profiles of mucosal ferritin and transferrin from a test dose also were changed substantially in response to very-short-term alterations in dietary iron. Even though changes in dietary iron rapidly altered iron uptake by brush-border membrane vesicles and the incorporation of 59Fe from the test dose into mucosal transferrin, changes in the incorporation of 59Fe into mucosal ferritin best reflected the actual changes in the transfer of iron from dose to plasma.
Assuntos
Mucosa Intestinal/metabolismo , Ferro/metabolismo , Fígado/metabolismo , Microvilosidades/metabolismo , Animais , Transporte Biológico , Citosol/metabolismo , Dieta , Ferritinas/isolamento & purificação , Ferritinas/metabolismo , Hemoglobinas/metabolismo , Absorção Intestinal , Ferro/farmacologia , Deficiências de Ferro , Masculino , Microvilosidades/efeitos dos fármacos , Ratos , Ratos Endogâmicos F344 , Fatores de Tempo , Transferrina/isolamento & purificação , Transferrina/metabolismoRESUMO
TNS, 2-p-toluidinylnaphthalene-6-sulfonate, has been used as a fluorescent probe to determine the binding constants of metal ions to the two binding sites of Escherichia coli glutamine synthetase (GS). TNS fluorescence is enhanced dramatically when bound to proteins due to its high quantum yield resulting from its interactions with hydrophobic regions in proteins. The fluorescence energy transfer from a hydrophobic tryptophan residue of GS to TNS has been detected as an excitation band centered at 280 nm. Therefore, TNS is believed to be bound to a hydrophobic site on the GS surface other than the active site and is located near a hydrophobic Trp residue of GS. GS binds lanthanide ions [Ln(III)] more tightly than either Mn(II) or Mg(II), and the binding constants of several lanthanide ions were determined to be in the range (2.1-4.6) x 10(10) and (1.4-3.0) x 10(8) M-1 to the two metal binding sites of GS, respectively. The intermetal distances between the two metal binding sites of GS were also determined by measuring the efficiencies of energy transfer from Tb(III) to other Ln(III) ions. The intermetal distances of Tb(III)-Ho(III) and Tb(III)-Nd(III) were 7.9 and 6.8 A, respectively.
Assuntos
Escherichia coli/enzimologia , Corantes Fluorescentes , Glutamato-Amônia Ligase/química , Metais/química , Naftalenossulfonatos , Ligação Competitiva , Transferência de Energia , Glutamato-Amônia Ligase/metabolismo , Lantânio/química , Lantânio/metabolismo , Metais/metabolismo , Espectrometria de Fluorescência , Térbio/química , Térbio/metabolismoRESUMO
Electron paramagnetic resonance (EPR) was used to study the environment of Mn2+ bound to the tight (n1) metal ion binding site of glutamine synthetase in the presence of analogues of the tetrahedral adduct, L-methionine (S)-sulfoximine [Met(O)(NH)-S] and L-methionine (R)-sulfoximine [Met(O)(NH)-R]. The Mn2+ EPR spectrum in the presence of Met(O)(NH)-S is identical with the previously published spectrum obtained from a mixture of isomers [Met(O)(NH)-RS] [Villafranca, J. J., Ash, D. E., & Wedler, F. C. (1976) Biochemistry 15, 544] and is characteristic of a highly octahedral metal ion environment with a small zero field splitting. The presence of Met(O)(NH)-R produces an EPR spectrum that appears characteristic of a more distorted metal ion environment, with a larger zero field splitting. These data demonstrate that the two isomers interact differently with the enzyme-bound Mn2+. Broadening of the Mn2+ EPR spectrum in the presence of Met(O)(NH) is observed in 17O-enriched water due to superhyperfine coupling of water to the metal ion. Deconvolution of the spectrum demonstrates the presence of at least a single water molecule in the inner coordination sphere of the metal ion. Superhyperfine coupling due to the 14N nucleus of the imine nitrogen of the sulfoximine moiety of Met(O)(NH)-S but not of Met(O)(NH)-R has been detected by electron spin-echo envelope modulation spectroscopy. Two intense peaks are evident in the presence of Met(O)(NH)-S with frequencies at 1.7 and 3.3 MHz. These peaks are absent when [15N]imine-labeled Met(O)(NH) is used, indicating the presence of the sulfoximine nitrogen of Met(O)(NH)-S in the inner coordination sphere of the metal ion.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Glutamato-Amônia Ligase/metabolismo , Manganês/metabolismo , Sítios de Ligação , Espectroscopia de Ressonância de Spin Eletrônica , Cinética , Ligantes , Metionina Sulfoximina/metabolismo , Ligação ProteicaRESUMO
Several spectroscopic techniques are used to investigate the stoichiometry and properties of ATP complexes with lanthanide(III) (Ln(III)], ions. The ATP2-lanthanide(III) complex predominates at millimolar ATP levels and dissociates to the 1:1 complex with a Kd of 300 +/- 50 microM for the Eu(III) case. Two independent techniques, viz. field-dependent water proton relaxation for the Gd(III) complex and metal ion luminescence lifetime measurements for the Eu(III) complex, yield a value of approximately 2 for the number of water molecules coordinated to the metal ion. The latter technique yields an approximate metal-ion hydration number of 4 for the 1:1 complex. Dynamic properties of the Gd(III) X ATP2 complex including the temperature dependence of correlation times describing rotation of the complex and ATP exchange have been studied by field-dependent water-proton relaxation and by temperature-dependent 31P NMR relaxation studies. These data are consistent with formation of a 2:1 ATP-lanthanide complex at millimolar ATP concentrations. Other types of complexes are detected under conditions in which there is insufficient ATP to satisfy the 1:2 metal:nucleotide stoichiometry.
Assuntos
Trifosfato de Adenosina/metabolismo , Lantânio/metabolismo , Európio/metabolismo , Gadolínio/metabolismo , Cinética , Substâncias Macromoleculares , Espectroscopia de Ressonância Magnética , Matemática , Praseodímio/metabolismo , TemperaturaRESUMO
Changes in the intrinsic fluorescence intensity of glutamine synthetase induced by lanthanide(III) ion binding demonstrate the existence of three types of sites for these ions. The sites are populated sequentially during titrations of the enzyme, and the first two have a stoichiometry of 1 per enzyme subunit. The number of water molecules coordinated to Eu(III) bound to the first site was determined by luminescence lifetime techniques to be 4.1 +/- 0.5. The hydration of Gd(III) bound to the same site was studied by magnetic field dependent water proton longitudinal relaxation rate measurements, and by water proton and deuteron relaxation measurements of one sample at single magnetic fields. The magnetic resonance techniques also yield a value of 4 for the hydration number.
Assuntos
Európio/metabolismo , Gadolínio/metabolismo , Glutamato-Amônia Ligase/metabolismo , Sítios de Ligação , Escherichia coli/enzimologia , Cinética , Medições Luminescentes , Espectroscopia de Ressonância Magnética/métodos , Matemática , Ligação Proteica , TermodinâmicaRESUMO
The interaction of Escherichia coli glutamine synthetase with beta, gamma-Cr(III)(H2O)4ATP (CrATP) has been studied. This substitution inert nucleotide functioned as an active site directed irreversible inhibitor of glutamine synthetase in solutions containing 15 mM MgCl2, 100 mM KCl, and 10 mM Pipes (pH 6.6). The inactivation reaction followed pseudo-first order saturation kinetics which demonstrated reversible binding of CrATP prior to the formation of inactive enzyme. CrATP was shown to be a competitive inhibitor versus MgATP. Also, significant protection was afforded by MgATP indicating that CrATP inactivates at the active site. Partial protection was afforded by glutamate or inorganic phosphate while inactivation was enhanced by Mn(II). The stoichiometry of CrATP incorporation was approximately one molecule per enzyme subunit, determined spectrophotometrically. Both the delta and lambda isomers of CrATP bound to glutamine synthetase, but only the lambda isomer was an active site directed irreversible inhibitor.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Marcadores de Afinidade , Cromo , Glutamato-Amônia Ligase/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Sítios de Ligação , Ligação Competitiva , Escherichia coli/enzimologia , Isomerismo , Cinética , MetaisRESUMO
The luminescence of Tb(III) was used to explore the topography of the metal ion sites of Escherichia coli glutamine synthetase and the relationship between these sites and tryptophan residues of the enzyme. By irradiation of tryptophan residues at 295 nm and measurement of the resulting Tb(III) luminescence at 544 nm, a biphasic curve was obtained upon titrating apoenzyme with Tb(III) indicating sequential binding of Tb(III) ions to the two binding sites of glutamine synthetase. The luminescence intensity was greater in the second region of the titration curve which is mostly due to energy transfer from Trp-158 to the second Tb(III) binding site of the enzyme. By use of the Förster equation for energy transfer from donor Trp to acceptor Tb(III), distances from Trp-57 to Tb(III) at the n1 and n2 sites were calculated, by using a mutant enzyme in which Trp-158 was replaced by Ser, to be 16.4 and 15.7 A, respectively; distances from Trp-158 to Tb(III) at the n1 and n2 sites were calculated, by using a mutant enzyme in which Trp-57 was replaced by Leu, to be 16.8 and 9.5 A, respectively. All the distances are in reasonably good agreement with the crystal structure distances from Salmonella typhimurium glutamine synthetase except the distance from Trp-158 to the second Tb(III) binding site. The discrepancies may result from a slightly different conformation of glutamine synthetase in solution and in the crystal and/or a slightly different conformation for trivalent Ln(III) binding compared to divalent Mn(II) binding.