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1.
Protein Expr Purif ; 170: 105574, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31978534

RESUMO

BACKGROUND: Lectins are known to possess interesting biological properties such as anti microbial, nematicidal, anti tumor and anti viral activities. Lantana camara from verbenaceae family is a medicinal plant known for possessing anti oxidant and anticancer activities. Since anticancer activity is reported in plant lectins, leaves of Lantana camara was used to check the presence of lectin. METHODS AND RESULTS: Here we report the purification, characterization and biological properties of a lectin from Lantana camara (LCL) leaves. LCL was purified by ion exchange chromatography on CM-cellulose column followed by affinity chromatography on mucin coupled Sepharose 4B column and gel filtration chromatography on Superdex G75 column. LCL is a glycoprotein with 10% of the carbohydrate and is blood group non specific. SDS-PAGE analysis of affinity purified LCL showed two proteins with apparent molecular weight of 14.49 kDa and 17.4 kDa which were subsequently separated by Gel filtration chromatography on Superdex G75 column. Hapten inhibition studies of LCL revealed its highest affinity for Chitin, Milibiose, α-D-Methyl galactopyranoside and glycoproteins like mucin, asialomucin. LCL showed strong binding to human colon adenocarcinoma HT29 cells with MFI of 242 which was effectively blocked by 68.1 and 62.5% by both mucin and milibiose. LCL showed dose and time dependent growth inhibitory effects on HT29 cells with IC50 of 3.75  µg/ml at 48 h. LCL has potent antibacterial and anti fungal activity. CONCLUSION: LCL can be explored for its clinical potential.


Assuntos
Antibacterianos/farmacologia , Antifúngicos/farmacologia , Antineoplásicos/farmacologia , Lantana/química , Lectinas de Plantas/farmacologia , Antibacterianos/isolamento & purificação , Antifúngicos/isolamento & purificação , Antineoplásicos/isolamento & purificação , Quitina/química , Quitina/metabolismo , Cromatografia de Afinidade , Cromatografia em Gel , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Fungos/efeitos dos fármacos , Fungos/crescimento & desenvolvimento , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/crescimento & desenvolvimento , Células HT29 , Humanos , Melibiose/química , Melibiose/metabolismo , Metilgalactosídeos/química , Metilgalactosídeos/metabolismo , Testes de Sensibilidade Microbiana , Mucinas/química , Mucinas/metabolismo , Extratos Vegetais/química , Folhas de Planta/química , Lectinas de Plantas/isolamento & purificação , Plantas Medicinais , Ligação Proteica
2.
Mol Biotechnol ; 61(9): 633-649, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31177409

RESUMO

Galactose oxidase catalyzes a two-electron oxidation, mainly from the C6 hydroxyl group of D-galactose, with the concomitant reduction of water to hydrogen peroxide. This enzyme is secreted by Fusarium species and has several biotechnological applications. In this study, a screening of galactose oxidase production among species of the Fusarium fujikuroi species complex demonstrated Fusarium subglutinans to be the main producer. The truncated F. subglutinans gaoA gene coding for the mature galactose oxidase was expressed from the prokaryotic vector pTrcHis2B in the E. coli Rosetta™ (DE3) strain. The purified recombinant enzyme presented temperature and pH optima of 30 °C and 7.0, respectively, KM of 132.6 ± 18.18 mM, Vmax of 3.2 ± 0.18 µmol of H2O2/min, kcat of 12,243 s-1, and a catalytic efficiency (kcat/KM) of 9.2 × 104 M-1 s-1. In the presence of 50% glycerol, the enzyme showed a T50 of 59.77 °C and was stable for several hours at pH 8.0 and 4 °C. Besides D-(+)-galactose, the purified enzyme also acted against D-(+)-raffinose, α-D-(+)-melibiose, and methyl-α-D-galactopyranoside, and was strongly inhibited by SDS. Although the F. subglutinans gaoA gene was successfully expressed in E. coli, its endogenous transcription was not confirmed by RT-PCR.


Assuntos
Fusarium/enzimologia , Galactose Oxidase/metabolismo , Galactose/química , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Fusarium/química , Galactose/metabolismo , Galactose Oxidase/química , Galactose Oxidase/genética , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Concentração de Íons de Hidrogênio , Melibiose/química , Melibiose/metabolismo , Metilgalactosídeos/química , Metilgalactosídeos/metabolismo , Modelos Moleculares , Oxirredução , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Rafinose/química , Rafinose/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Temperatura
3.
Glycobiology ; 25(5): 492-501, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25504801

RESUMO

The crystal structure of the α-galactosyl binding Lyophyllum decastes lectin (LDL) was determined to 1.0 Å resolution by sulfur single-wavelength anomalous diffraction (SAD). The 10 kDa protein exhibits no sequence similarity to any protein with known structure and adopts a unique lectin fold, where a core of two antiparallel ß-sheets at the heart of the homodimer is connected to the periphery of the structure by intramolecular disulfide bridges. This fold suggests that LDL is secreted, which sets it apart from other mushroom lectins. Structures of complexes between LDL and the ligands α-methylgalactoside and globotriose shed light on the binding specificity. Sequence comparison suggests a location and function of LDL and homologous proteins in or at the fungal cell wall. Structural comparison allows the identification of a superfamily of secreted proteins with the LDL fold, which may play a role at the interface between fungi and their environment.


Assuntos
Agaricales/química , Proteínas Fúngicas/química , Lectinas de Plantas/química , Sequência de Aminoácidos , Proteínas Fúngicas/metabolismo , Metilgalactosídeos/metabolismo , Dados de Sequência Molecular , Lectinas de Plantas/metabolismo , Ligação Proteica
4.
Methods Mol Biol ; 1200: 501-9, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25117260

RESUMO

One of the most commonly used ligand-based NMR methods for detecting ligand binding is saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy. The STD NMR method is an invaluable technique for assessing carbohydrate-lectin interactions in solution, because STD NMR can be used to detect weak ligand binding (Kd ca. 10(-3)-10(-8) M). STD NMR spectra identify the binding epitope of a carbohydrate ligand when bound to lectin. Further, the STD NMR method uses 1H-detected NMR spectra of only the carbohydrate, and so only small quantities of non-labeled lectin are required. In this chapter, I describe a protocol for the STD NMR method, including the experimental procedures used to acquire, process, and analyze STD NMR data, using STD NMR studies for methyl-ß-D-galactopyranoside (ß-Me-Gal) binding to the C-terminal domain of an R-type lectin from earthworm (EW29Ch) as an example.


Assuntos
Lectinas/química , Lectinas/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Metilgalactosídeos/química , Metilgalactosídeos/metabolismo , Métodos Analíticos de Preparação de Amostras , Animais , Ligantes , Oligoquetos , Ligação Proteica , Estrutura Terciária de Proteína , Soluções
5.
Carbohydr Polym ; 110: 113-20, 2014 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-24906736

RESUMO

A novel crosslinked hydrogel film was prepared from carboxymethylchitosan (CMCS) and carboxymethylcellulose (CMC) by ionical and covalent crosslinking with CaSO4 and genipin, respectively. The swelling ratio of the crosslinked CMCS/CMC hydrogel films was investigated at different pH solutions (1-9), and the results indicated that the crosslinked hydrogels had the swelling-deswelling properties with two primary peaks of swelling ratio at pH 3 and 7. The surface morphologies of the crosslinked hydrogels at different pH values provided evidences of the swelling-deswelling properties. The mechanical properties of the hydrogel films were also examined. The ionical and covalent crosslinking were found to have the primary impact on the toughness and max load, respectively, of the crosslinked hydrogels. The cells comparatively cultured on the crosslinked hydrogels and the negative and positive controls suggested the biocompatibility of the crosslinked CMCS/CMC films. This kind of hydrogel films have potential application in drug delivery vehicles and skin tissue engineering.


Assuntos
Carboximetilcelulose Sódica/síntese química , Quitosana/análogos & derivados , Reagentes de Ligações Cruzadas/síntese química , Metilgalactosídeos/síntese química , Carboximetilcelulose Sódica/metabolismo , Quitosana/síntese química , Quitosana/metabolismo , Reagentes de Ligações Cruzadas/metabolismo , Metilgalactosídeos/metabolismo
6.
J Theor Biol ; 256(1): 14-28, 2009 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-18848570

RESUMO

In the presence of gratuitous inducers, the lac operon of Escherichia coli exhibits bistability. Most models in the literature assume that the inducer enters the cell via the carrier (permease), and exits by a diffusion-like process. The diffusive influx and carrier efflux are neglected. However, analysis of the data shows that in non-induced cells, the diffusive influx is comparable to the carrier influx, and in induced cells, the carrier efflux is comparable to the diffusive efflux. Since bistability entails the coexistence of steady states corresponding to both non-induced and induced cells, neither one of these fluxes can be ignored. We present a model accounting for both fluxes, and show that: (1) The thresholds (i.e., the extracellular inducer levels at which transcription turns on or off) are profoundly affected by both fluxes. The diffusive influx reduces the on threshold, and eliminates irreversible bistability, a phenomenon that is inconsistent with data. The carrier efflux increases the off threshold, and abolishes bistability at large permease activities, a conclusion that can be tested experimentally. (2) The thresholds are well approximated by simple analytical expressions obtained by considering two limiting cases (no carrier efflux and no diffusive influx). (3) The simulations are in good agreement with the data for isopropyl thiogalactoside (IPTG), but somewhat discrepant with respect to the data for thiomethyl galactoside (TMG). We discuss the potential sources of the discrepancy.


Assuntos
Simulação por Computador , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Óperon Lac , Modelos Genéticos , Ativação Transcricional/fisiologia , Transporte Biológico , Expressão Gênica , Isopropiltiogalactosídeo/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Metilgalactosídeos/metabolismo , Tiogalactosídeos/metabolismo
7.
Epilepsy Res ; 80(1): 18-22, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18455367

RESUMO

Glucose transporter 1 (GLUT1) deficiency syndrome is caused by a deficit in glucose transport to the brain during the pre- and postnatal periods. Here, we report two cases of GLUT1 deficiency syndrome diagnosed on the basis of clinical features, reduced GLUT1 activities, and mutations in the GLUT1 gene. Patient 1 had a novel heterozygous 1bp insertion in exon 7 that resulted in a shift of the reading frame and the introduction of a premature stop codon at amino acid position 380. His clinical phenotype appeared to be more severe than that of Patient 2 who had a missense mutation in exon 8 resulting in an arginine-to-tryptophan substitution at amino acid position 333. Patient 1 had no meaningful words and could not walk unassisted, while Patient 2 could speak and walk unassisted. Both the patients developed seizures of various types that have been successfully treated with zonisamide. Although several antiepileptic drugs, including barbiturates, diazepam, chloralhydrate, and valproic acid, have been shown to inhibit GLUT1 function, the present study demonstrated no inhibitory effect of zonisamide on GLUT1-mediated glucose transport. Our data suggested that zonisamide might be preferable if add-on anticonvulsant therapy is required to control the seizures in patients with this disorder.


Assuntos
Anticonvulsivantes/uso terapêutico , Transportador de Glucose Tipo 1/deficiência , Isoxazóis/uso terapêutico , Convulsões/tratamento farmacológico , Convulsões/genética , Transporte Biológico/efeitos dos fármacos , Criança , Análise Mutacional de DNA , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Feminino , Glucose/metabolismo , Humanos , Masculino , Metilgalactosídeos/metabolismo , Zonisamida
8.
Bull Math Biol ; 70(4): 1032-64, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18246403

RESUMO

The lac operon of Escherichia coli can exhibit bistability. Early studies showed that bistability occurs during growth on TMG/succinate and lactose+glucose, but not during growth on lactose. More recently, studies with lacGFP-transfected cells show bistability during growth on TMG/succinate, but not during growth on lactose and lactose+glucose. In the literature, these results are invariably attributed to variations in the destabilizing effect of the positive feedback generated by induction. Specifically, during growth on TMG/succinate, lac induction generates strong positive feedback because the permease stimulates the accumulation of intracellular TMG, which in turn, promotes the synthesis of even more permease. This positive feedback is attenuated during growth on lactose because hydrolysis of intracellular lactose by beta-galactosidase suppresses the stimulatory effect of the permease. It is attenuated even more during growth on lactose + glucose because glucose inhibits the uptake of lactose. But it is clear that the stabilizing effect of dilution also changes dramatically as a function of the medium composition. For instance, during growth on TMG/succinate, the dilution rate of lac permease is proportional to its activity, e, because the specific growth rate is independent of e (it is completely determined by the concentration of succinate). However, during growth on lactose, the dilution rate of the permease is proportional to e2 because the specific growth rate is proportional to the specific lactose uptake rate, which in turn, proportional to e. We show that: (a) This dependence on e2 creates such a strong stabilizing effect that bistability is virtually impossible during growth on lactose, even in the face of the intense positive feedback generated by induction. (b) This stabilizing effect is weakened during growth on lactose+glucose because the specific growth rate on glucose is independent of e, so that the dilution rate once again contains a term that is proportional to e. These results imply that the lac operon is much more prone to bistability if the medium contains carbon sources that cannot be metabolized by the lac enzymes, e.g., succinate during growth on TMG/succinate and glucose during growth on lactose+glucose. We discuss the experimental data in the light of these results.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Óperon Lac , Modelos Biológicos , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Glucose/metabolismo , Lactose/metabolismo , Matemática , Metilgalactosídeos/metabolismo , Ácido Succínico/metabolismo , Tiogalactosídeos/metabolismo
9.
Cell Stress Chaperones ; 11(2): 135-47, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16817319

RESUMO

We have examined the A431 (human epidermoid carcinoma) and HT29 (human colorectal carcinoma) cellular responses evoked by lectins of dietary origin, Jacalin of Artocarpus integrifolia (native jacalin; nJacalin), peanut agglutinin (PNA) of Arachis hypogea, and recombinant single-chain jacalin (rJacalin), which has the same protein backbone but approximately 100-fold less affinity for carbohydrates than nJacalin. All three lectins (nJacalin, rJacalin, and PNA) are cycotoxic inhibitors of proliferation of A431 cells. However, cells recover once jacalin but not PNA have been removed from the growth medium. Treatment of nJacalin results in morphologically visible cell rounding while retaining the membrane integrity when treated at 40 microg ml(-1), but treatment with PNA did not induce such changes. The observed cell rounding was found to be due to stress as the phosphorylation of caveolin-1 (at tyr14), p38 but not c-Jun N-terminal kinase were up-regulated, while PNA did not up-regulate the phosphorylation of the same. Jacalin also down-regulated the phosphorylation of the epidermal growth factor receptor and extracellular signal regulated kinase in contrast to PNA, which failed to down-regulate the same. Confocal microscopic studies reveal that jacalin is not internalized, unlike the lectin of Agaricus bisporous. Analysis of the proteins that bind to an nJacalin-sepharose column revealed the binding of six to eight proteins, and significant among them is a protein at approximately 110 kDa, which appears to be oxygen-regulated protein 150 (ORP150) (endoplasmic reticulum chaperone) as identified by its isoelectric point, two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometric analysis. This 110-kDa band is detectable with anti-Hsp70 antibody because ORP150 has homology with Hsp70. Confocal microscopic studies reveal the presence of Hsp70-like proteins on the surface of A431 cells as revealed by immunostaining with anti-Hsp70 antibody. Moreover, overexpression of ORP150 in A431 cells has resulted in a dramatic protection of A431 cells against jacalin-induced toxicity, confirming that the jacalin-induced cytotoxicity is mediated through ORP150, and impairment of ORP150 functions with the help of jacalin makes the cells more susceptible to death due to stress. Our studies suggest that the cellular responses, as a consequence of lectin binding, may not be exclusively mediated by carbohydrate binding property alone, but other factors such as protein-protein interactions may also contribute to the observed cellular responses.


Assuntos
Caveolina 1/metabolismo , Fator de Crescimento Epidérmico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Lectinas de Plantas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Agregação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Imunofluorescência , Células HT29 , Humanos , Metilgalactosídeos/metabolismo , Microscopia Confocal , Fosforilação/efeitos dos fármacos
10.
Science ; 307(5711): 922-5, 2005 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-15705852

RESUMO

The development of pest resistance threatens the effectiveness of Bacillus thuringiensis (Bt) toxins used in transgenic and organic farming. Here, we demonstrate that (i) the major mechanism for Bt toxin resistance in Caenorhabditis elegans entails a loss of glycolipid carbohydrates; (ii) Bt toxin directly and specifically binds glycolipids; and (iii) this binding is carbohydrate-dependent and relevant for toxin action in vivo. These carbohydrates contain the arthroseries core conserved in insects and nematodes but lacking in vertebrates. We present evidence that insect glycolipids are also receptors for Bt toxin.


Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Caenorhabditis elegans/metabolismo , Endotoxinas/metabolismo , Glicoesfingolipídeos/metabolismo , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Sítios de Ligação , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Endotoxinas/toxicidade , Galactose/farmacologia , Glicolipídeos/química , Glicolipídeos/isolamento & purificação , Glicolipídeos/metabolismo , Glicoesfingolipídeos/química , Glicoesfingolipídeos/isolamento & purificação , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Proteínas Hemolisinas , Resistência a Inseticidas , Bicamadas Lipídicas , Metilgalactosídeos/metabolismo , Metilgalactosídeos/farmacologia , Metilglucosídeos/metabolismo , Metilglucosídeos/farmacologia , Monossacarídeos/análise , Mutação
11.
Protein Sci ; 13(12): 3264-73, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15557267

RESUMO

Sugar binding studies, inactivation, unfolding, and refolding of native Jacalin (nJacalin) from Artocarpus integrifolia and recombinant single-chain Jacalin (rJacalin) expressed in Escherichia coli were studied by intrinsic fluorescence and thermal and chemical denaturation approaches. Interestingly, rJacalin does not undergo any proteolytic processing in an E. coli environment. It has 100fold less affinity for methyl-alpha-galactose (Ka: 2.48 x 10(2)) in comparison to nJacalin (Ka: 1.58 x 10(4)), and it also binds Thomsen-Friedenreich (TF) disaccharide (Galbeta1-3GalNAc) with less affinity. Overall sugar binding characteristics of rJacalin are qualitatively similar to that of nJacalin (Gal

Assuntos
Metabolismo dos Carboidratos , Lectinas de Plantas/química , Lectinas de Plantas/metabolismo , Sequência de Bases , Dicroísmo Circular , Glicosilação , Concentração de Íons de Hidrogênio , Metilgalactosídeos/metabolismo , Dados de Sequência Molecular , Lectinas de Plantas/genética , Desnaturação Proteica , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
12.
Acta Crystallogr D Biol Crystallogr ; 60(Pt 8): 1404-12, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15272163

RESUMO

Based on their carbohydrate specificity, the jacalin family of lectins can be divided into two groups: galactose-specific and mannose-specific. The former are cytoplasmic proteins, whereas the latter are localized in the storage vacuoles of cells. It has been proposed that the post-translational modification in some of the lectins that splits their polypeptide chains into two may be crucial for galactose specificity. The mannose-specific members of the family are single-chain proteins that lack the above modification. Although the galactose-specific and the mannose-specific jacalin-type lectins differ in their sequences, they share a common fold: the beta-prism I fold, which is characteristic of Moraceae plant lectins. Here, two crystal structures of a jacalin-related lectin from Artocarpus hirsuta, which is specific for galactose, in complex with methyl-alpha-D-galactose are reported. The lectin crystallized in two orthorhombic forms and one hexagonal form under similar conditions. The crystals had an unusually high solvent content. The structure was solved using the molecular-replacement method using the jacalin structure as a search model. The two orthorhombic forms were refined using data to 2.5 and 3.0 A resolution, respectively. The structures of the A. hirsuta lectin and jacalin are identical. In orthorhombic form I the crystal packing provides three different micro-environments for sugar binding in the same crystal. The observed difference in the specificity for oligosaccharides between the A. hirsuta lectin and jacalin could only be explained based on differences in the molecular associations in the packing and variation of the C-terminal length of the beta-chain. The observed insecticidal activity of A. hirsuta lectin may arise from its similar fold to domain II of the unrelated delta-endotoxin from Bacillus thuringiensis.


Assuntos
Artocarpus/química , Metilgalactosídeos/metabolismo , Lectinas de Plantas/química , Lectinas de Plantas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Bases de Dados de Proteínas , Metilgalactosídeos/química , Modelos Moleculares , Dados de Sequência Molecular , Dobramento de Proteína , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Alinhamento de Sequência , Solventes/química , Especificidade por Substrato
13.
Biochemistry ; 41(43): 13039-45, 2002 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-12390031

RESUMO

Binding of alpha- and beta-D-galactopyranosides with different hydrophobic aglycons was compared using substrate protection against N-ethylmaleimide alkylation of single-Cys148 lactose permease. As demonstrated previously, methyl- or allyl-substituted alpha-D-galactopyranosides exhibit a 60-fold increase in binding affinity (K(D) = 0.5 mM), relative to galactose (K(D) = 30 mM), while methyl beta-D-galactopyranoside binds only 3-fold better. In the present study, galactopyranosides with cyclohexyl or phenyl substitutions, both in alpha and beta anomeric configurations, were synthesized. Surprisingly, relative to methyl alpha-D-galactopyranoside, binding of cyclohexyl alpha-D-galactopyranoside to lactose permease is essentially unchanged (K(D) = 0.4 mM), and phenyl alpha-D-galactopyranoside exhibits only a modest increase in binding affinity (K(D) = 0.15 mM). Nitro- or methyl-substituted phenyl alpha-D-galactopyranosides bind with significantly higher affinities (K(D) = 0.014-0.067 mM), and the strongest binding is observed with analogues containing para substituents. In contrast, D-galactopyranosides with a variety of large hydrophobic substituents (isopropyl, cyclohexyl, phenyl, o- or p-nitrophenyl) in beta anomeric configuration exhibit uniformly weak binding (K(D) = 1.0-2.3 mM). The results confirm and extend previous observations that hydrophobic aglycons of D-galactopyranosides increase binding affinity, with a clear predilection toward alpha-substituted sugars. In addition, the data suggest that the primary interaction between the permease and hydrophobic aglycons is directed toward the carbon atom bonded to the anomeric oxygen. The different positioning of this carbon atom in alpha- or beta-D-galactopyranosides thus may provide a rationale for the characteristic binding preference of the permease for alpha anomers.


Assuntos
Proteínas de Ligação ao Cálcio , Proteínas de Escherichia coli , Escherichia coli/enzimologia , Galactose/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Simportadores , Sítios de Ligação , Fenômenos Químicos , Físico-Química , Escherichia coli/metabolismo , Galactose/química , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana Transportadoras/química , Metilgalactosídeos/química , Metilgalactosídeos/metabolismo , Proteínas de Transporte de Monossacarídeos/química , Proteínas de Transporte de Monossacarídeos/metabolismo , Nitrofenilgalactosídeos/síntese química , Nitrofenilgalactosídeos/metabolismo , Proteínas Periplásmicas de Ligação/química , Proteínas Periplásmicas de Ligação/metabolismo , Tiogalactosídeos/química , Tiogalactosídeos/metabolismo
14.
Cell Biochem Funct ; 20(3): 257-62, 2002 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12125103

RESUMO

Trypanosomes do not inhabit or grow in anopheles mosquitoes, the vector for the transmission of Plasmodium parasites the causative agent for malaria. The possession of lytic factors by the anopheline mosquito was thus considered. Head and midgut sections prepared in phosphate buffered saline were tested for trypanocidal action against T. congolense. While the head section was inactive towards the trypanosomes, the midgut extract at 0.2 mg ml(-1) diminished the motility of the parasites within 2 min of incubation; killing 50% of the population after 5 min. At 0.5 mg ml(-1) of the extract, about 90% of the parasites were killed within 2 min of incubation. The midgut fraction was subjected to a purification protocol involving successive chromatography on: octyl-sepharose, reactive brown agarose and fetuin-agarose columns. A final trypanocidal active fraction (gp45), which moved homogeneously during electrophoresis as a 45-kDa protein, was recovered from the fetuin-agarose column. The protein reacted positively with thiobarbituric acid, which suggests it is a sialoglycoprotein. Desialylation of the glycoprotein nullified its trypanocidal activity on T. congolense. Similarly, when the saccharides, lactose, methyl-beta-galactoside, lactulose, methyl-umbelliferyl-beta-galactoside (MU-Gal), were included in the culture medium, they inhibited the gp45 trypanocidal activity. Asialo-fetuin and asialo-RBC also inhibited the gp45-induced killing of T. congolense cells. The potential use of anopheline 45 kDa protein in the production of transgenic tsetse flies (Glossina spp.) in the control of trypanosomiasis is discussed.


Assuntos
Anopheles/metabolismo , Glicoproteínas/metabolismo , Himecromona/análogos & derivados , Animais , Culicidae , Meios de Cultura/farmacologia , Eritrócitos/metabolismo , Galactosídeos/metabolismo , Glicoproteínas/química , Himecromona/metabolismo , Lactose/metabolismo , Lactulose/metabolismo , Malária/metabolismo , Metilgalactosídeos/metabolismo , Ácido N-Acetilneuramínico/farmacologia , Neuraminidase/metabolismo , Polissacarídeos/farmacologia , Substâncias Reativas com Ácido Tiobarbitúrico , Fatores de Tempo , Tripanossomicidas/farmacologia , Trypanosoma
15.
Protein Expr Purif ; 25(1): 16-22, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12071694

RESUMO

BfpA, the structural repeating protein subunit A of the bundle-forming pilus and EspB, a type-III-secreted pore-forming protein of enteropathogenic Escherichia coli (EPEC), both virulence factors central for EPEC pathogenesis, were overexpressed in E. coli DH5alpha and M15 laboratory strains, respectively, using the pQE-30 cloning expression system, as chimeric fusions to a NH(2)-terminal histidine hexapeptide (His(6)-tag) sequence. After isopropyl beta-d-thiogalactoside induction, the expression levels achieved were 11 and 40% of total soluble protein for BfpA and EspB, respectively. The His(6)-tagged recombinant proteins were purified (up to 98% homogeneity) by Ni-agarose affinity chromatography and produced yields varying from 0.65 to 3.1 mg of recombinant protein per gram of wet weight cells. The immunogenicity and antigenicity of the final products were tested in rabbits and using fecal specimens obtained from children suffering from acute watery diarrhea, respectively. The recombinant products correspond to antigenically authentic protein standards, useful in future epidemiological and neonatal vaccinology studies.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Vacinas Bacterianas/genética , Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Proteínas de Fímbrias/genética , Animais , Humanos , Imunoglobulina A/metabolismo , Metilgalactosídeos/metabolismo , Plasmídeos/metabolismo , Coelhos , Proteínas Recombinantes/metabolismo , Salmonella/metabolismo , Tiogalactosídeos/metabolismo
16.
Carbohydr Res ; 337(7): 635-42, 2002 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-11909597

RESUMO

1-O-Acetyl-beta-D-galactopyranose (AcGal), a new substrate for beta-galactosidase, was synthesized in a stereoselective manner by the trichloroacetimidate procedure. Kinetic parameters (K(M) and k(cat)) for the hydrolysis of 1-O-acetyl-beta-D-galactopyranose catalyzed by the beta-D-galactosidase from Penicillium sp. were compared with similar characteristics for a number of natural and synthetic substrates. The value for k(cat) in the hydrolysis of AcGal was three orders of magnitude greater than for other known substrates. The beta-galactosidase hydrolyzes AcGal with retention of anomeric configuration. The transglycosylation activity of the beta-D-galactosidase in the reaction of AcGal and methyl beta-D-galactopyranoside (1) as substrates was investigated by 1H NMR spectroscopy and HPLC techniques. The transglycosylation product using AcGal as a substrate was beta-D-galactopyranosyl-(1-->6)-1-O-acetyl-beta-D-galactopyranose (with a yield of approximately 70%). In the case of 1 as a substrate, the main transglycosylation product was methyl beta-D-galactopyranosyl-(1-->6)-beta-D-galactopyranoside. Methyl beta-D-galactopyranosyl-(1-->3)-beta-D-galactopyranoside was found to be minor product in the latter reaction.


Assuntos
Galactosídeos/metabolismo , Penicillium/enzimologia , beta-Galactosidase/metabolismo , Galactosídeos/síntese química , Galactosídeos/química , Cinética , Lactose/metabolismo , Metilgalactosídeos/metabolismo , Nitrofenilgalactosídeos/metabolismo , Especificidade por Substrato
17.
Protein Eng ; 14(4): 261-7, 2001 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11391018

RESUMO

Directed evolution has become an important enabling technology for the development of new enzymes in the chemical and pharmaceutical industries. Some of the most interesting substrates for these enzymes, such as polymers, have poor solubility or form highly viscous solutions and are therefore refractory to traditional high-throughput screens used in directed evolution. We combined digital imaging spectroscopy and a new solid-phase screening method to screen enzyme variants on problematic substrates highly efficiently and show here that the specific activity of the enzyme galactose oxidase can be improved using this technology. One of the variants we isolated, containing the mutation C383S, showed a 16-fold increase in activity, due in part to a 3-fold improvement in K(m). The present methodology should be applicable to the evolution of numerous other enzymes, including polysaccharide-modifying enzymes that could be used for the large-scale synthesis of modified polymers with novel chemical properties.


Assuntos
Evolução Molecular Direcionada/métodos , Galactose Oxidase/genética , Processamento de Imagem Assistida por Computador , Galactose Oxidase/metabolismo , Biblioteca Genômica , Cinética , Metilgalactosídeos/metabolismo , Mutação
18.
FEBS Lett ; 473(2): 199-202, 2000 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-10812074

RESUMO

The selective binding of serum amyloid P component (SAP) to proteins in the pathological amyloid cross-beta fold suggests a possible chaperone role. Here we show that human SAP enhances the refolding yield of denatured lactate dehydrogenase and protects against enzyme inactivation during agitation of dilute solutions. These effects are independent of calcium ions and are not inhibited by compounds that block the amyloid recognition site on the B face of SAP, implicating the A face and/or the edges of the SAP pentamer. We discuss the possibility that the chaperone property of SAP, or its failure, may contribute to the pathogenesis of amyloidosis.


Assuntos
Chaperonas Moleculares/farmacologia , Componente Amiloide P Sérico/farmacologia , Sítios de Ligação , Ligação Competitiva , Proteína C-Reativa/farmacologia , Cálcio/metabolismo , Cálcio/farmacologia , Nucleotídeos de Desoxiadenina/farmacologia , Reativadores Enzimáticos/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Humanos , L-Lactato Desidrogenase/química , L-Lactato Desidrogenase/efeitos dos fármacos , L-Lactato Desidrogenase/metabolismo , Metilgalactosídeos/metabolismo , Metilgalactosídeos/farmacologia , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Desnaturação Proteica , Dobramento de Proteína , Componente Amiloide P Sérico/química , Componente Amiloide P Sérico/metabolismo
19.
J Bacteriol ; 182(9): 2582-90, 2000 May.
Artigo em Inglês | MEDLINE | ID: mdl-10762262

RESUMO

We have cloned and sequenced the Lactobacillus casei hprK gene encoding the bifunctional enzyme HPr kinase/P-Ser-HPr phosphatase (HprK/P). Purified recombinant L. casei HprK/P catalyzes the ATP-dependent phosphorylation of HPr, a phosphocarrier protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system at the regulatory Ser-46 as well as the dephosphorylation of seryl-phosphorylated HPr (P-Ser-HPr). The two opposing activities of HprK/P were regulated by fructose-1,6-bisphosphate, which stimulated HPr phosphorylation, and by inorganic phosphate, which stimulated the P-Ser-HPr phosphatase activity. A mutant producing truncated HprK/P was found to be devoid of both HPr kinase and P-Ser-HPr phosphatase activities. When hprK was inactivated, carbon catabolite repression of N-acetylglucosaminidase disappeared, and the lag phase observed during diauxic growth of the wild-type strain on media containing glucose plus either lactose or maltose was strongly diminished. In addition, inducer exclusion exerted by the presence of glucose on maltose transport in the wild-type strain was abolished in the hprK mutant. However, inducer expulsion of methyl beta-D-thiogalactoside triggered by rapidly metabolizable carbon sources was still operative in ptsH mutants altered at Ser-46 of HPr and the hprK mutant, suggesting that, in contrast to the model proposed for inducer expulsion in gram-positive bacteria, P-Ser-HPr might not be involved in this regulatory process.


Assuntos
Proteínas de Bactérias , Lacticaseibacillus casei/enzimologia , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Acetilglucosaminidase/metabolismo , Sequência de Bases , Clonagem Molecular , DNA Bacteriano , Proteínas de Ligação a DNA/metabolismo , Glucose/metabolismo , Lacticaseibacillus casei/genética , Maltose/metabolismo , Metilgalactosídeos/metabolismo , Dados de Sequência Molecular , Mutagênese , Fosfatos/metabolismo , Fosfoproteínas Fosfatases/genética , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Repressoras/metabolismo , Elementos de Resposta , Tiogalactosídeos/metabolismo , Transferases/genética
20.
Biochemistry ; 39(15): 4493-9, 2000 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-10757998

RESUMO

To improve the structural organization model of melibiose permease, we assessed the individual contributions of the N-terminal tryptophans to the transporter fluorescence variations induced by the binding of cations and beta-configured sugars, by replacement of the six N-terminal tryptophans by phenylalanines and the study of the signal changes. Only two mutations, W116F located in helix IV and W128F located in the cytoplasmic loop 4-5, impair permease activity. The intrinsic fluorescence spectroscopy analysis of the other mutants suggests that W54, located in helix II, W116, and W128 are mostly responsible for the cation-induced fluorescence variations. These tryptophans, W116 and W128, would also be responsible for the beta-galactoside-induced fluorescence changes observed in the N-terminal domain of the transporter. The implication of W116 and W128 in both the cation- and beta-galactoside-induced fluorescence variations led us to investigate in detail the effects of their mutations on the functional properties of the permease. The results obtained suggest that the domains harboring the two tryptophans, or the residues themselves, play a critical role in the mechanism of Na(+)/sugar symport. Taken together, the results presented in this paper and previous results are consistent with a fundamental role of helix IV in connecting cation- and sugar-binding sites of the melibiose permease.


Assuntos
Metabolismo dos Carboidratos , Cátions Monovalentes/metabolismo , Escherichia coli/enzimologia , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Simportadores , Substituição de Aminoácidos/genética , Sítios de Ligação , Transporte Biológico/efeitos dos fármacos , Carboidratos/farmacologia , Cátions Monovalentes/farmacologia , Cinética , Melibiose/metabolismo , Proteínas de Membrana Transportadoras/genética , Metilgalactosídeos/metabolismo , Metilgalactosídeos/farmacologia , Modelos Moleculares , Mutação/genética , Nitrofenilgalactosídeos/metabolismo , Concentração Osmolar , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteolipídeos/efeitos dos fármacos , Proteolipídeos/metabolismo , Rafinose/metabolismo , Rafinose/farmacologia , Sódio/metabolismo , Sódio/farmacologia , Espectrometria de Fluorescência , Especificidade por Substrato , Tiogalactosídeos/metabolismo , Tiogalactosídeos/farmacologia , Triptofano/genética , Triptofano/metabolismo , Tirosina/genética , Tirosina/metabolismo
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