RESUMEN
Encoding protein 3D structures into 1D string using short structural prototypes or structural alphabets opens a new front for structure comparison and analysis. Using the well-documented 16 motifs of Protein Blocks (PBs) as structural alphabet, we have developed a methodology to compare protein structures that are encoded as sequences of PBs by aligning them using dynamic programming which uses a substitution matrix for PBs. This methodology is implemented in the applications available in Protein Block Expert (PBE) server. PBE addresses common issues in the field of protein structure analysis such as comparison of proteins structures and identification of protein structures in structural databanks that resemble a given structure. PBE-T provides facility to transform any PDB file into sequences of PBs. PBE-ALIGNc performs comparison of two protein structures based on the alignment of their corresponding PB sequences. PBE-ALIGNm is a facility for mining SCOP database for similar structures based on the alignment of PBs. Besides, PBE provides an interface to a database (PBE-SAdb) of preprocessed PB sequences from SCOP culled at 95% and of all-against-all pairwise PB alignments at family and superfamily levels. PBE server is freely available at http://bioinformatics.univ-reunion.fr/PBE/.
Asunto(s)
Secuencias de Aminoácidos , Conformación Proteica , Alineación de Secuencia/métodos , Análisis de Secuencia de Proteína/métodos , Programas Informáticos , Bases de Datos de Proteínas , Internet , Pliegue de Proteína , Interfaz Usuario-ComputadorRESUMEN
An alternate method for enzyme study is proposed. Multidimensional statistical analysis applied on mid-infrared attenuated total reflectance spectra (Cadet et al. (1991) Appl. Spectrosc. 42, 166-172) collected during a kinetic allows a direct and fast quantification of the remaining substrate, as well as a one step enzymatic assay. Furthermore, the combination of these techniques may be used as a structural tool. The method applied to the study of beta-fructosidase is developed in this paper as an example. With appropriate calibration, the method may be extend to any enzyme.
Asunto(s)
Disacáridos/química , Glicósido Hidrolasas/química , Cinética , Espectrofotometría Infrarroja , Sacarosa/química , beta-FructofuranosidasaRESUMEN
The insulin resistance syndrome, consisting of resistance to insulin and several metabolic abnormalities, is associated with an increased risk of symptomatic coronary artery disease. Asymptomatic persons with increased coronary calcification have increased coronary plaque and an increased likelihood of future cardiovascular events. Electron-beam computed tomography-derived coronary artery calcium scores, metabolic and anthropometric parameters, and fasting and stimulated concentrations of glucose and insulin were measured in 1160 asymptomatic men and women. Coronary artery calcium scores were positively correlated with glucose, insulin, and homeostasis model assessment (HOMA) insulin resistance. Calcium scores were positively correlated with intra-abdominal adiposity, age, total cholesterol/high density lipoprotein (HDL) ratio, low density lipoprotein, triglycerides, blood pressure, and HOMA beta cell function and inversely correlated with HDL and peripheral fat. These correlations, except for 2-hour glucose, remained significant for all subjects with fasting serum glucose <126 mg/dL or all subjects with fasting serum glucose 110 mg/dL. In a multivariate analysis, age, sex, family history of premature coronary artery disease, intra-abdominal adiposity, low density lipoprotein, and smoking independently predicted calcium scores. Blood pressure, HDL, triglycerides, glucose, insulin, and HOMA insulin resistance or beta cell function were not independently correlated with coronary artery calcium scores. Asymptomatic individuals with insulin resistance have elevated coronary calcium scores. The association between insulin resistance and coronary calcification persists with impaired glucose tolerance and normal fasting serum glucose. Central/visceral adiposity may be a determinant of insulin resistance and atherosclerosis even in asymptomatic nondiabetic persons.
Asunto(s)
Enfermedad de la Arteria Coronaria/diagnóstico por imagen , Enfermedad de la Arteria Coronaria/epidemiología , Diabetes Mellitus Tipo 2/epidemiología , Tejido Adiposo/metabolismo , Comorbilidad , Diabetes Mellitus/epidemiología , Femenino , Humanos , Resistencia a la Insulina , Masculino , Persona de Mediana Edad , Análisis Multivariante , Ciudad de Nueva York/epidemiología , Obesidad , Prevalencia , Factores de Riesgo , Grosor de los Pliegues Cutáneos , Tomografía Computarizada por Rayos XRESUMEN
C(4) phosphoenolpyruvate carboxylase (PEPC) is a key enzyme in the C(4) photosynthetic pathway. To analyze the diversity of the corresponding gene in grasses, we designed PCR primers to specifically amplify C(4) PEPC cDNA fragments. Using RT-PCR, we generated partial PEPC cDNA sequences in several grasses displaying a C(4) photosynthetic pathway. All these sequences displayed a high homology (78-99%) with known grass C(4) PEPCs. PCR amplification did not occur in two grasses that display the C(3) photosynthetic pathway, and therefore we assumed that all generated sequences corresponded to C(4) PEPC transcripts. Based on one large cDNA segment, phylogenetic reconstruction enabled us to assess the relationships between 22 grass species belonging to the subfamilies Panicoideae, Arundinoideae and Chloridoideae. The phylogenetic relationships between species deduced from C(4) PEPC sequences were similar to those deduced from other molecular data. The sequence evolution of the C(4) PEPC isoform was faster than in the other PEPC isoforms. Finally, the utility of the C(4) PEPC gene phylogeny to study the evolution of C(4) photosynthesis in grasses is discussed.
RESUMEN
The advent of more and more powerful micro-computers has allowed the introduction of multidimensional analysis in research laboratories. Complex mathematical treatments are now possible within a few seconds. Prediction equations that linked sucrose, fructose, glucose, total sugars and reducing sugars concentrations to the spectral data, were established by regression on the principal components. Very high correlation coefficient values between the first ten axes and the chemical values were obtained. The bias and standard deviation (S.D.) values obtained between reference and predicted values were good. From such aqueous biological samples containing a ternary mixture of sucrose, fructose and glucose it was possible to (i) identify the characteristic IR bands of these different sugars (and their combination: reducing sugars, total sugars)-using spectral pattern; and (ii) to specifically measure their concentrations with good accuracy.
RESUMEN
In this paper we study activation by dithiothreitol and reduced thioredoxins and deactivation by oxidized thioredoxins f of sedoheptulose-1,7-bisphosphatase. The behaviour of the enzyme when chromatographed on a thioredoxin-Sepharose column is also described. The enzyme is autoxidizable upon removal of reducing agents, and is activated when reduced by any of the thioredoxins. This mechanism may allow the regulation of the Calvin cycle upon light-dark and dark-light transitions. The formation of a stable complex between enzyme and thioredoxin could explain the inhibitory effect of high thioredoxin concentrations. The use of immunological techniques shows that sedoheptulose-1,7-bisphosphatase and fructose-1,6-bisphosphatase are poorly related immunologically.
Asunto(s)
Cloroplastos/enzimología , Monoéster Fosfórico Hidrolasas/metabolismo , Aminoácidos/análisis , Cromatografía de Afinidad , Activación Enzimática/efectos de los fármacos , Fructosa-Bifosfatasa/inmunología , Inmunodifusión , Cinética , Monoéster Fosfórico Hidrolasas/inmunología , Plantas/enzimología , Tiorredoxinas/farmacologíaRESUMEN
The aim of this paper is to study some steady-state kinetic properties of sedoheptulose-1,7-bisphosphatase, its pH-dependence and the effect of a substrate analogue, fructose 2,6-bisphosphate. Studies were carried out with sedoheptulose 1,7-bisphosphate and with fructose 1,6-bisphosphate, an alternative substrate. The pK values are identical for both substrates, and fructose 2,6-bisphosphate behaves like a competitive inhibitor. These results suggest that there exists a unique active site for either sedoheptulose 1,7-bisphosphate or fructose 1,6-bisphosphate on the enzyme molecule. Increasing Mg2+ concentrations shifted the optimum pH. As for fructose-1,6-bisphosphatase, we believe that this shift is due to the neutralization of negative charges near the active centre [Cadet, Meunier & Ferté (1987) Eur. J. Biochem. 162, 393-398]. The free species of sedoheptulose 1,7-bisphosphate and fructose 1,6-bisphosphate are not the usual substrates of enzyme, nor is Mg2+. But the kinetics relative to the (Mg2+-substrate4-)2- complex is not consistent with this complex being the substrate. An explanation of this discrepancy is proposed, involving both the negative charges near the active centre and the positive charges of Mg2+. The observed Vmax. of the reduced enzyme is 65% of the theoretical Vmax. for both substrates, but the observed Vmax. relative to sedoheptulose 1,7-bisphosphate is 3 times the one relative to fructose 1,6-bisphosphate. The specificity constant (kcat./Km), 1.62 x 10(6) M-1.s-1 with respect to sedoheptulose 1,7-bisphosphate compared with 5.5 x 10(4) M-1.s-1 with respect to fructose 1,6-bisphosphate, indicates that the enzyme specificity towards sedoheptulose 1,7-bisphosphate is high but not absolute.
Asunto(s)
Cloroplastos/enzimología , Monoéster Fosfórico Hidrolasas/metabolismo , Plantas/enzimología , Fructosa-Bifosfatasa/metabolismo , Fructosadifosfatos/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Sustancias Macromoleculares , Magnesio/farmacología , Oxidación-ReducciónRESUMEN
The inhibitory properties of halide salts on palmito polyphenoloxidase (PPO) are described. Halide salts have the same inhibitory effect on the two forms of palmito PPO separated by hydrophobic chromatography. Fluoride and chloride ions showed a non-competitive, mixed type inhibition while bromide and iodide ions were found to be non-competitive inhibitors. A study of the Ki for the different halide salts showed that the smaller F- ion is a stronger inhibitor than I- and Br- and that Cl- has the highest Ki value. This suggests that the active site of the palmito PPO is not easily accessible. The inhibition by chloride and fluoride ion was found to be pH-dependent. The inhibitory effects of these ions increased with a decrease in pH. It is suggested that halide ions (X) could bind to either the protonated enzyme (EH) or the protonated substrate-enzyme complex (EHS) to yield inactive forms EHX and EHSX, respectively.
Asunto(s)
Catecol Oxidasa/antagonistas & inhibidores , Cloruros/farmacología , Inhibidores Enzimáticos/farmacología , Fluoruros/farmacología , Unión Competitiva , Bromuros/farmacología , Catecol Oxidasa/metabolismo , Concentración de Iones de Hidrógeno , Yoduros/farmacología , Cloruro de Sodio/farmacología , Fluoruro de Sodio/farmacologíaRESUMEN
An alternate method for enzyme study is proposed. This technique allows enzymatic reactions by a one step assay, and visualisation of variations in FTIR spectral data of substrate during the reaction. Hydrolysis of sucrose by beta-fructosidase is carried out as an example.
Asunto(s)
Interpretación Estadística de Datos , Enzimas/farmacocinética , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Glicósido Hidrolasas/farmacocinética , Hidrólisis , Técnicas In Vitro , Sacarosa/metabolismo , beta-FructofuranosidasaRESUMEN
This report describes the effects of pH and fructose 2,6-bisphosphate (an analog of fructose 1,6-bisphosphate) on the activity of oxidized and reduced fructose-1,6-bisphosphatase from spinach chloroplasts. Studies were carried out with either fructose 1,6-bisphosphate, the usual substrate, or sedoheptulose 1,7-bisphosphate, an alternative substrate. The reduction of the oxidized enzyme is achieved by a thiol/disulfide interchange. The pK values relative to each redox form for the same substrate (either fructose 1,6-bisphosphate or sedoheptulose 1,7-bisphosphate) are identical, suggesting the same site for both substrates on the active molecule. The finding that the analog (fructose 2,6-bisphosphate) behaves like a competitive inhibitor for both substrates also favours this hypothesis. The inhibitory effect of this sugar is more important when the enzyme is reduced than when it is oxidized. The shift in the optimum pH observed when [Mg2+] was raised is interpreted as a conformational change of oxidized enzyme demonstrated by a change in fluorescence. The reduced and oxidized forms have the same theoretical rates relative to both substrates, but the reduced form has an observed Vmax which is 60% of the theoretical Vmax while that of the oxidized form is only 37% of the theoretical Vmax. The reduced enzyme appears more efficient than the oxidized one in catalysis.
Asunto(s)
Cloroplastos/enzimología , Fructosa-Bifosfatasa/metabolismo , Fructosadifosfatos/farmacología , Hexosadifosfatos/farmacología , Plantas/enzimología , Tiorredoxinas , Tiorredoxinas en Cloroplasto , Fructosa-Bifosfatasa/aislamiento & purificación , Concentración de Iones de Hidrógeno , Cinética , Oxidación-Reducción , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/metabolismo , Especificidad por SustratoRESUMEN
Higher-plant sedoheptulose-1,7-bisphosphatase was isolated and purified over 200-fold from spinach (Spinacia oleracea) chloroplast stromal extracts to apparent electrophoretic homogeneity by DEAE-Fractogel, molecular sieving on Sephadex G-200 and Blue B dye-matrix affinity chromatography. It is a protein of Mr 66,000, made up of two apparently identical subunits (Mr 35,000). The enzyme is activated by reduced thioredoxin fb in the presence of dithiothreitol. Its specificity towards sedoheptulose 1,7-bisphosphate versus fructose 1,6-bisphosphate is high, but not absolute.
Asunto(s)
Cloroplastos/enzimología , Monoéster Fosfórico Hidrolasas/aislamiento & purificación , Proteínas de Plantas/aislamiento & purificación , Tiorredoxinas , Tiorredoxinas en Cloroplasto , Cromatografía Liquida , Electroforesis en Gel de Poliacrilamida , Activación Enzimática/efectos de los fármacos , Peso Molecular , Proteínas de Plantas/farmacologíaRESUMEN
pH studies of palmito polyphenol oxidase are carried out with either 4-methylcatechol or pyrogallol as substrates. The pH profile is independent of the nature of the substrates tested. The symmetrical behaviour and the very slight differences between the values obtained suggest the existence of only one site on the molecule for o-diphenol substrates.
Asunto(s)
Sitios de Unión , Catecol Oxidasa/farmacocinética , Árboles , Antioxidantes/metabolismo , Catálisis , Catecoles/metabolismo , Concentración de Iones de Hidrógeno , Pirogalol/metabolismoRESUMEN
Regulation of HLA-DQ gene transcription is a complex phenomenon because the allelic polymorphism associated with these genes and their promoters is a putative source of differential allele expression. Both transcriptional and post-transcriptional regulation could account for the density of the molecules expressed at the cell surface and then for the specificity of the immune response. Different methods have been developed to evaluate the functional consequences of this polymorphism, but at present no universal method allows measurement of either the steady-state level or the half-life time of mRNA species of both DQA1 and DQB1 polymorphic genes in heterozygous cell lines. Here, we propose a potent method, based on relative quantification of reverse transcriptase-polymerase chain reaction products, which analyzes the differential expression of all DQA1 or DQB1 allele combinations. This method is used to analyze the differential expression of HLA-DQB1*020110402 alleles in the human heterozygous lymphoblastoid B-cell line. Nucleotidic sequences of the proximal upstream regulatory region of these alleles exhibit significant differences. We show that the DQB1*0402 promoter is able to mediate a transcription strength twice as efficiently as *0201. In addition, the *0402 mRNA steady-state level is also governed by a remarkable post-transcriptional regulation. Indeed, an important part (20%) of the *0402 primary transcript is derived by alternative splicing in a short mRNA translated into a nonfunctional protein. Despite their variable sequence and length, no difference in the half-life of DQB1*0201 and both DQB*0402 mRNAs was observed in B-lymphoblastoid cells. The implications of these findings are discussed.
Asunto(s)
Linfocitos B/metabolismo , Genes MHC Clase II/genética , Antígenos HLA-DQ/genética , Alelos , Northern Blotting , Línea Celular , Línea Celular Transformada , Cadenas alfa de HLA-DQ , Cadenas beta de HLA-DQ , Heterocigoto , Humanos , Modelos Genéticos , Ácidos Nucleicos Heterodúplex , Procesamiento Postranscripcional del ARN , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción GenéticaRESUMEN
Phosphoenolpyruvate carboxylases (PEPCs) are encoded by a small multigenic family. In order to characterise this gene family in sugarcane, seven DNA fragments displaying a high homology with grass PEPC genes were isolated using polymerase chain reaction-based cloning. A phylogenetic study revealed the existence of four main PEPC gene lineages in grasses and particularly in sugarcane. Moreover, this analysis suggests that grass C4 PEPC has likely derived from a root pre-existing isoform in an ancestral species. Using the Northern-dot-blot method, we studied the expression of the four PEPC gene classes in sugarcane cv. R570. We confirmed that transcript accumulation of the C4 PEPC gene (ppc-C4) mainly occurs in the green leaves and is light-induced. We also showed that another member of this gene family (ppc-aR) is more highly transcribed in the roots. The constitutive expression for a previously characterised gene (ppc-aL2) was confirmed. Lastly, the transcript accumulation of the fourth PEPC gene class (ppc-aL1) was not revealed. Length polymorphism in non-coding regions for three PEPC gene lineages enabled us to develop sequence-tagged site PEPC markers in sugarcane. We analysed the segregation of PEPC fragments in self-pollinated progenies of cv. R570 and found co-segregating fragments for two PEPC gene lineages. This supports the hypothesis that diversification of the PEPC genes involved duplications, probably in tandem.
Asunto(s)
Familia de Multigenes/genética , Fosfoenolpiruvato Carboxilasa/genética , Filogenia , Saccharum/genética , Secuencia de Bases , Northern Blotting , Análisis por Conglomerados , Cartilla de ADN , Datos de Secuencia Molecular , Especificidad de Órganos , Polimorfismo de Longitud del Fragmento de Restricción , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
Regulation of the human leucocyte antigen (HLA) class II genes expression is an important field in immunology, because these molecules play a crucial role in the function of the immune system. HLA DQ genes expression is a complex phenomenon regulated at both transcriptional and post-transcriptional levels. In this study, we have investigated the post-transcriptional mechanisms accounting for alleles-dependent length polymorphism of DQA1 mRNA. We have first sequenced the genomic DNA encoding the 3' untranslated region (UTR) of DQA1 *0101, *0102, *0103, *0201, *0301, *0401, and *0501 alleles. We have identified two competing splicing sites: a unique splicing donor site AG/GTA located 20 nucleotides downstream from the stop codon associated to two spliced acceptor sequences, approximately 165 and approximately 370 nucleotides downstream. In addition, three polyadenylation signals have been identified, respectively, at approximately 475, approximately 795, and approximately 855 nucleotides downstream from the stop codon. Subsequently, we have analyzed mRNAs derived from DQA1 alleles in homozygous B lymphoblastoid cell lines by reverse transcriptase-polymerase chain reaction. We show that allele-dependent length polymorphism of DQA1 mRNA-3' UTR results from a combination of differential splicing and alternative polyadenylations. Four mRNA isoforms (two spliced variant cleaved at two distinct polyadenylation sites) were detected in DQA1 *0101, *0102, and *0103 homozygous cell lines, and six mRNA species (three spliced variant cleaved at two polyadenylation-sequence signal) were generated by the other four alleles. Possible advantages for cells to generate multiple transcripts previously undetected are discussed.
Asunto(s)
Alelos , Empalme Alternativo/genética , Antígenos HLA-DQ/genética , Poliadenilación/genética , Regiones no Traducidas/genética , Linfocitos B , Secuencia de Bases , Clonación Molecular , Cartilla de ADN/genética , Cadenas alfa de HLA-DQ , Humanos , Datos de Secuencia Molecular , Poli A/genética , Polimorfismo Genético/genética , Sitios de Empalme de ARN/genética , Células Tumorales CultivadasRESUMEN
We have discovered a multienzymatic complex in fresh young sugarcane leaves. This complex is constituted of three enzymes: PEPcase, NADP-MDH and malic enzyme. After successive molecular sieving chromatography, we have obtained a highly purified sample of the complex which has a molecular weight of 711 kDa. Its functional interest has been evaluated by comparing the kinetic properties of the enzymes in their free forms to those in their complexed form. We show that the association of the three enzymes leads to important changes in their respective kinetic properties.
Asunto(s)
Malato Deshidrogenasa/metabolismo , Complejos Multienzimáticos/metabolismo , Fosfoenolpiruvato Carboxilasa/metabolismo , Plantas Comestibles/enzimología , Cromatografía , Cromatografía DEAE-Celulosa , Cromatografía en Gel , Durapatita , Cinética , Malato Deshidrogenasa/aislamiento & purificación , Malato-Deshidrogenasa (NADP+) , Peso Molecular , Complejos Multienzimáticos/aislamiento & purificación , Fosfoenolpiruvato Carboxilasa/aislamiento & purificación , Hojas de la PlantaRESUMEN
In order to determine the ethnic origin of the transporter associated with antigen processing 2 (TAP2) G allele, initially discovered by us in a group of type 1 diabetes (insulin-dependent diabetes mellitus) patients living on Reunion Island, HLA TAP2 typing was performed using the polymerase chain reaction-amplification refractory mutation system (PCR-ARMS) method in type 1 diabetes patients and unrelated healthy controls of three different ethnic groups (Caucasians, Indians and black Africans from Senegal and Mauritius). The comparison of TAP2 allele frequencies in controls showed significant racial (ethnic) differences. The TAP2*0101 and TAP2 C alleles were increased, respectively, in the Caucasian (50% in Caucasians vs. 40% in other groups) and Senegalese (27% in Senegalese vs. 10% in other groups) populations. In comparison with Caucasians, the TAP2*0201 variant was significantly increased in the Indian population and decreased in the Senegalese black population. In addition, the TAP2 G allele was observed in the two African populations studied but not in the Caucasian or Indian population. This observation is consistent with the view that this allele is restricted to populations of African origin. In addition, we have determined the large extended haplotype DQA1-DQB1-DRB1 associated with TAP2 G. We found that this allele is preferentially associated with the large conserved haplotype HLA DQA1*0501-DQB1*0201-DRB1*0301.